<title>Aquaculture and schistosomiasis</title>
Proceedings of a network meeting
held in Manila, Phillipines
August 6-10, 1991
Board on Science and Technology for International Development
National Research Council
<section>Preface</section>
Inland aquaculture has been vastly underdeveloped in the tropics and subtropics and now there are high hopes for its future expansion to benefit farmers and consumers of aquatic products. The prospects of generating food and profit from well-managed waters are exciting and responsive to the normal developmental trends of high population growth, overstretched natural resources, and environmental degradation. There are, however, serious biotechnical, socioeconomic, and environmental constraints to aquaculture development. Aquaculture, like agriculture, has environmental costs and risks, including the possibility of increased transmission of waterborne diseases to those who work on fish farms and to others who use, or live near, the waters used for, or influenced by, aquaculture. Where fishponds and associated watercourses provide good habitats for the aquatic snail intermediate hosts of schistosomes, the risks of increased transmission can be great, especially in Africa.
Therefore, there is a need for increased interaction among researchers working in support of aquaculture development and among those working in public health, disease control, sanitation, and environmental conservation. These proceedings describe an attempt at such interaction by the juxtaposition of researchers in aquaculture and schistosomiasis in a network meeting that provided an opportunity for presentations, discussion, and interaction of mutual benefit to the attendees.
The aquaculture contributions show the diversity of current research in progress. This reflects the rather backward status of aquaculture science compared with agricultural science. Aquaculturists have yet to develop a well-founded science akin to agronomy and will need many years to build that science from a combination of disciplinary, specific problem-oriented, and broader systems research, including intersectoral studies with such groups as agriculturalists and foresters. The contributions of schistosomiasis research are also wide ranging and reflect the many imaginative approaches toward lessening or eliminating its continuing toll on the human population.
It is hoped that these papers and the accompanying recommendations on future work and research priorities will be seen as modest but useful steps toward supporting the safe and sustainable expansion of aquaculture in developing countries and the effective control of schistosomiasis. Much more research is needed and this will require an increased commitment of resources.
The International Center for Living Aquatic Resources Management (ICLARM), as the newest center to be accepted for membership in the Consultative Group on International Agricultural Research (CGIAR), welcomed the opportunity to host this network meeting and looks forward to being of further service to assist similar efforts involving researchers for development who have diverse backgrounds but work toward interdependent objectives.
Roger S.V. Pullin
Director, Aquaculture Program
ICLARM
<section>Acknowledgments</section>
The staff of the Board on Science and Technology for International Development deeply appreciates the assistance of Kenneth T. MacKay, Jay L. Maclean, Roger S. V. Pullin, and Belen Acosta, International Center for Living Aquatic Resources Management (ICLARM), Manila, for hosting the network meeting and for the arrangements that made this meeting a success.
We also extend special thanks to Patrick Carney, George T. Curlin, James McVey, and Gary Pruder for providing important contributions through their role as external expert reviewers for this proceedings.
The Office of Research, U.S. Agency for International Development (USAID), is gratefully acknowledged for their support of the network meeting and the publication of this proceedings.
National Research Council Staff
Bruce A. Harrison, Senior Program Officer and Scientific Editor
E. Griffin Shay, Senior Program Officer and Scientific Editor
F. R. Ruskin, Editor
Maria Griener, Assistant Editor
Yauthary Keo, Program Assistant
Mary Frances Schlichter, Program Assistant
How to cite this report:
National Research Council. 1992. Aquaculture and Schistosomiasis: Proceedings of a network meeting held in Manila, Philippines, August 6-10, 1991. National Academy Press. Washington, D.C.
<section>Introduction</section>
The idea of mixing aquaculture and schistosomiasis researchers in a single meeting was prompted by the need to emphasize the importance of the interface between these two fields. A network meeting was held in Manila, Philippines, from August 6 to 10, 1991, and involved U.S. Agency for International Development (USAID) grantees working in aquaculture or schistosomiasis. The meeting was organized and sponsored jointly by the Board on Science and Technology for International Development (BOSTID), National Research Council, Washington, D.C., and the International Center for Living Aquatic Resources Management (ICLARM), Manila, and was funded by the Office of Research at USAID. The participants' grants were funded through USAID's Program in Science and Technology Cooperation (PSTC) or the U.S.-Israel Cooperative Development Research (CDR) Program. The meeting consisted of two days of grantee presentations of individual research projects and two days of visits to field research
sites in nearby areas.
The 25 papers presented during the meeting are included here. One additional paper is included from a grantee who could not attend the meeting. The 15 aquaculture papers cover a wide field of topics, ranging from three on technology, four on propagation, two on nutrition, and four on DNA and genetics, to two on ecology and the environment. Likewise, the 11 papers on schistosomiasis cover diverse topics, including four on immunology, two on epidemiology or biology, and four on biocontrol. None of the papers address both aquaculture and schistosomiasis (except for Loker et al., in passing), because the grants did not overlap both study areas. However, the participants formulated and compiled a list of 35 recommendations relevant to future research, including four that specifically propose work involving both study areas.
Aquaculture, an ancient human practice, has become an increasingly important farmer-oriented industry for protein production in many countries, both developing and developed. The technologies required for large- and small-scale farm aquaculture of major or supplemental crops of various fish species, frogs, crayfish and prawns, and mollusks are now in place and are being widely distributed. The development of these technologies has not come about spontaneously, but as a result of three basic trends: (1) the need for more protein production to feed the growing human population; (2) increased pollution in, or destruction of, many of the world's natural fishery habitats and the subsequent decline in fish populations; and (3) poorly managed or unmanaged harvest systems that have permitted the overexploitation of natural fisheries. Realistically, a slowing and reversal of these trends will require considerable education, policy and managerial changes, effort, and time. Thus,
aquaculture technology will continue to grow at a rapid rate and it should be anticipated that many other aquatic species will be "domesticated" and disseminated for mass production of protein.
One of the main organizations involved in the enhancement of fisheries techniques and aquaculture is the International Center for Living Aquatic Resources Management. Currently, there are several ICLARM regional centers in the Eastern Hemisphere that have been very successful in initiating small-farmer-oriented aquaculture programs based primarily on various cichlid species. One of these centers is located in Manila. Accordingly, the participants of this network meeting had the opportunity to visit the Freshwater Aquaculture Center and the Freshwater Fish Hatchery and Extension Training Center at Central Luzon State University and to learn about the project in Genetic Improvement of Farmed Tilapias.
The parasitism of humans by schistosomes is almost certainly more ancient than the development of aquaculture and is still a major public health problem in tropical and subtropical areas of the world. Schistosomiasis is one of the six priority vector-borne diseases targeted for control by the World Health Organization. The disease (also called bilharziasis) is caused by any one of five species of the genus Schistosoma that parasitize humans through direct contact in water. At present, it affects an estimated 200-250 million people in approximately 75 countries, primarily in Africa, Asia, Latin America, and the Middle East. Schistosomiasis normally has a low mortality rate; however, it is typically chronic, extremely debilitating, and causes a very significant loss in work productivity. Thus, the annual impact of lost work in some developing countries with high prevalence rates can amount to hundreds of millions of U.S. dollars.
Human schistosomes develop through a complex life cycle involving freshwater snail intermediate hosts. The stage infecting humans develops in the snail and is released into the water, where it swims until it dies or comes in contact with, and penetrates, human skin. Once in the human the parasite migrates, matures, and develops large numbers of eggs, some of which pass out in the urine and/or feces. If the eggs are deposited in fresh water, another stage of the parasite hatches and swims around until it locates and infects the snail intermediate host. Only a select few species of snails actually function in the intermediate host role, and most are specifically susceptible to only one or two of the schistosome species. Thus, the schistosome cycles are dependent on: (1) an infected human (in Asia other animals can also serve as vertebrate hosts for the parasite, and these enhance the transmission of parasites to humans); (2) the depositing of eggs into fresh water and their
hatching; (3) the location and infection of the correct snail species and further development of the parasite for release into the water; and (4) human skin exposure in water to the infective stage of the parasite that came from the infected snails, and a subsequent infection.
Why the concern about schistosomiasis when efforts to develop a vaccine are progressing well and a medication, praziquantel, is now available for treatment of the disease? Although these developments are promising, and in the case of praziquantel very successful, the disease is continuing to spread and case numbers are increasing annually. There is also some evidence of praziquantel resistance developing in the parasite. Furthermore, there is no realistic expectation that the eventual vaccine will be available, costwise, to many infected rural populations in tropical countries. It is imperative, therefore, that non-vaccine control methods and programs be continued and public awareness and education enhanced through local sources.
As mentioned earlier, schistosomiasis is a tropical and subtropical disease that primarily affects rural populations. In this scenario the focus for infection is continued contact with water through activities such as fishing, farming (for example, rice culture), bathing, washing clothes and cooking utensils, personal hygiene, and recreation. Thus, water development projects in developing countries where the disease occurs offer likely sources for new foci or epidemics of the disease. The need for increased education and local awareness about schistosomiasis is particularly important when considering the current water resources situation. Many of the countries with high prevalence rates of this disease are the same countries experiencing serious water shortages. And, wherever water occurs there are usually large concentrations of humans, which encourages the transmission of waterborne disease. Recently, a number of the countries experiencing such water shortages have
embarked on large-scale dam projects and irrigation schemes. Sadly, most have resulted in large-scale schistosomiasis epidemics in the settlers who come to work the newly habitable lands. These water development schemes are also the very same areas where aquaculture efforts are likely to be initiated; often the persons involved in these projects, like the other settlers, are unaware of the risk of schistosomiasis.
When considering this problem, there are at least four future trends that seem certain: (1) human populations in most developing countries will continue to increase at a rapid rate; (2) freshwater resources will become increasingly scarce, which will result in more water development projects in developing countries; (3) aquaculture will become increasingly important in developing countries as a source for protein and will be linked to the water development projects; and (4) waterborne diseases such as schistosomiasis will continue to spread so that large-scale epidemics will be a common public health problem in humans associated with water development projects. Therefore, the meshing of aquaculture and schistosomiasis researchers in a network meeting seems timely and has resulted in an increased awareness on both sides of the interrelatedness of the two fields. Hopefully, this meeting will inspire future research efforts that encompass both aquaculture and the study of
schistosomes and schistosomiasis.
<section>Recommendations</section>
During the four-day network meeting, the 25 USAID aquaculture and schistosomiasis grantees intermingled well and participated in a vigorous exchange of information and ideas. Their wide range of expertise enhanced the development of a list of recommendations. On the last day they formulated and compiled a total of 35 recommendations for future research in both fields. These recommendations are listed below and are divided into priorities for three categories: (1) the aquaculture and schistosomiasis interface, (2) aquaculture; and (3) schistosomes and schistosomiasis. Of particular importance are the four recommendations involving the aquaculture and schistosomiasis interface. These recommendations stress the need for future coordinated efforts that will consider both nutrition and waterborne diseases in promoting human health.
I. Priorities At The Aquaculture And Schistosomiasis Interface
1.Develop common goals and interaction among aquaculture, public health, engineering,ecology, and environmental workers in planning, constructing, and monitoring water development projects such as aquaculture, potable water sources, dams, irrigation, and chemical and organic waste effluent ponds.
2.Develop and use preliminary public health risk assessment methods before initiating projects that involve water development, such as aquaculture, potable water sources,dams, irrigation, and chemical and organic waste effluent ponds.
3.Ensure that those involved in aquaculture projects are educated in the risks and dangers of schistosomiasis and other waterborne diseases, as well as in the methods for preventing their transmission.
4.Promote and conduct the search for and use of molluscivorous fish and crustacean species that could play an important role in snail control, and potentially be of aquaculture value.
II. Priorities For Aquaculture
Resource Allocation
1.Resource allocation in aquaculture should be studied through modeling of various systems including integrated farming. Subsistence farming could be examined with and without an aquaculture component to identify and predict inputs and outputs, and to project expenses and income. Protocols could be developed for individual farmers based on their resources.
2.More information is needed on micronutrients in fish nutrition. This would be useful to commercial producers in optimizing their yields and to subsistence aquaculturists in allowing the most effective use of available feeds.
Conservation of Genetic Resources
3.Increase research on indigenous species that have been neglected in research and introductions. Research is also needed on remediation in aquaculture as, for example, in the elimination of harmful introduced species. The reintroduction of species that were displaced by unsuitable introductions should also be examined. The reproductive biology of crustaceans, mountain stream fishes (e.g., mahseer), and slow maturers are all worthy of study.
Aquaculture and the Environment
4.The interrelationships of aquaculture and the environment need to be quantified. Although there are aquaculture systems that are environmentally benign or beneficial, the potential exists for negative impacts from disease vectors, pesticide residues, and the like. These risks should be assessed and minimized. The effects of industrial and agricultural pollutants on aquaculture can be severe. More information is needed on the tolerances of various species to pollutants.
Miscellaneous
5.The suitability of marine fish species for farming should be investigated. Reduced profitability in the shrimp farming industry seems likely and substitute crops should be identified. Integrated culture of fish with shrimp should also be investigated.
6.Increase understanding and predictability of ecological processes in eutrophicated lakes and reservoirs to enhance water quality and fish protein production.
III. Priorities For Schistosomes And Schistosomiasis
Control
1.Promote and increase efforts to control schistosomes in a sustainable way using integrated pest management techniques directed at the host snail. Such techniques could combine a number of disciplines (e.g., herbal extracts, molluscivorous fish and crustaceans, pond and stream modification, chemicals, competitive or predator snails, competitive trematodes, snail repellent aquatic plants).
2.Increase the search for, and research efforts directed toward, new biological control agents or organisms of the host snails (e.g., microbial pathogens, Endod, Marisa, Thiara, Helisoma, Echinostoma).
3.Develop cheap, deliverable community-based biocontrol methods.
4.Monitor the resistance of snail hosts to chemical control compounds.
5. Develop slow release compounds targeting cercariae and miracidia that have little or no effect on non-target organisms.
6. Develop methods to destabilize snail habitats and eliminate or reduce host snail populations.
7. Conduct ecotoxicological studies of molluscicidal plants to ascertain their safety and toxicity.
8. Develop practical sanitation techniques and safe water supply systems for rural populations.
Treatment and Disease Prevention
9. Understand the basic aspects of granuloma formation and regulation.
10. Understand the basic aspects of fibrogenesis, resorption, and fibrosis.
11. Develop an anti-pathology vaccine.
12. Develop quantitative non-invasive methods for assessing liver function and pathology.
13. Develop quantitative specific tests for antigens in humans (field ready, rapid, for use in rural areas where microscopes are not available).
14. Increase studies of patient treatment failure and incipient drug resistance of the parasites.
15. Develop alternative drugs for treating schistosomiasis.
Basic Parasitology
16. Support more research gathering information about the taxonomy and biology of schistosomes and their snail hosts (in Africa, South America, and Southeast Asia) and how they might be influenced by massive deforestation and the construction of new dams and irrigation schemes.
Design and Planning
17. Develop strategies separating control and prevention, based on the premise that "control" = treatment + snail control + containment of spread through environmental modification, whereas "prevention" = vaccines, antipenetrants, protective clothing, prophylactic drugs, and behavior modification.
18. Determine rate-limiting points in disease transmission.
19. Develop rapid techniques for surveying water sites (including aquaculture sites) to determine if they are transmission foci (determine if snails or people are infected).
20. Develop methods for determining the cost/benefits of community-level schistosomiasis control, and determining how the benefits of control programs can continue after the formal intervention has concluded.
Vaccine Development
21. Identify relevant conformational antigens.
22. Develop non-invasive measures of resistance.
23. Understand mechanisms of immune regulation.
24. Determine influence of genetics and gene usage.
25. Address how the vaccine will be delivered and what will be the optimum mode of
vaccination.
<section>Presentation: Aquaculture</section>
<section>Technology</section>
<section>Aquaculture Technology Research For Smallholder Farmers In Rural Malawi</section>
R.P. Noble And B.A. Costa-Pierce
International Center for Living Aquatic Resources
Management (ICLARM)-GTZ Africa Project
Zomba, Malawi
Abstract
In order to develop aquaculture systems and technologies relevant to the maize-based farming system in rural Africa, the International Center for Living Aquatic Resources Management (ICLARM) is conducting farmer-participatory research in Malawi. This involves farmer-researcher interaction throughout the aquaculture program, thus enabling researchers to: (1) assess farmers' agroecological resources and the extent of their traditional agricultural knowledge to provide information on the constraints to adoption of aquaculture; (2) develop on-station research to test technologies relevant to rural farmers and their farming systems; and (3) monitor and evaluate, in collaboration with farmers, the performance and impact of new technologies and integrated agriculture-aquaculture models on farming systems.
The research process involves a continuing transfer of information and ideas between farmers and researchers so that research agendas can be adjusted to meet changing needs as indigenous aquaculture systems evolve.
Malawian aquaculturalists generally have one pond of approximately 300-400 mē in which they polyculture Oreochromis shiranus and Tilapia rendalli, sometimes with Cyprinus carpio. Yields from fish ponds are low (1 t/ha/yr). The major pond input is maize bran, which is used for human and animal food and is scarce during the rainy season.
On-farm bioresource assessments have shown that there are agricultural residues such as maize stover (2.5 t/ha/yr), grasses (>4 t/ha measured at the end of the wet season) and wood ash (400 kg/farm/yr), which could be used as pond inputs. Experiments have demonstrated that application of napier grass and maize bran together can improve fish yields up to 3 t/ha/yr. Wood ash from household cooking fires can improve water fertility and raise pH.
Low-cost technology transfer is accomplished by showing farmers a "basket" of technologies at the research station where they are encouraged to critically assess the available technologies. Farmers who have seen and critically evaluated these technologies are more likely to adopt them. In a farmer survey, 76% of those who had visited the research station were using more than one technology compared with only 32% of a control group. Using the "shopping basket" approach to technology transfer has resulted in rapid adoption of rice-fish culture. This has never been practiced in Malawi before. Farmers achieved rice yields of 2-2.4 t/ha/yr and fish yields of 1.5-2.4 t/ha/yr from rice-fish ponds. Farmers who usually grow only one crop of rice per year are now starting second crops during the dry season.
Introduction
Aquaculture has not been adopted widely in rural Africa, primarily because the technology has no traditional base in smallholder agriculture. Most rural households operate outside the cash economy, and thus have little expendable income to purchase the feeds and fertilizer that make aquaculture economically viable.
Malawi's population is 8.2 million (NSO 1987), of which 80% are directly involved in agricultural production. The International Service for National Agricultural Research (ISNAR 1982) stated that smallholder farming accounted for 84% of the agricultural GDP. However, only 25% of that contribution entered the cash economy in the early 1980s. Current figures are not available, but demographic and economic indicators suggest there has been little change since ISNAR's report.
Malawi government policy has favored estate development rather than smallholder agriculture, resulting in further limitations on participation in the cash economy (Kydd and Christiansen 1982, Peters and Herrera 1989). Malawi's high population growth (3.7% per annum, NSO 1987) further exacerbates this situation and is leading to a shortfall in food supply among rural communities.
Production of smallholder food crops has been falling since the mid-1970s. This reduction has been due to increasing shortage of arable land. Average per capita landholdings are expected to decline to 0.26 ha by the year 2000 in Malawi (World Bank 1989). With such severe land constraints it will be necessary to use marginal lands and improve the utilization of existing land.
Fish is the major source of animal protein for human consumption (60-70%, Satia 1989) but is quickly becoming a scarce and expensive food commodity in Malawi. Msiska (1985) noted that between 1972 and 1984, per capita consumption of fish fell by 53 %, from 17.9 kg to 9.5 kg per year. Malawi's lake fisheries have reached their productive limit in terms of fish supply (60,000-70,000 t/yr) and cannot meet the demands of a rapidly growing population (Msiska 1985). The Malawi government is now attempting to expand smallholder aquaculture to help relieve the shortfall in fish supplies and raise rural incomes.
The introduction of aquaculture to farmers in Malawi started in the early 1950s but has not been particularly successful. This is reflected in the low production figures for the 1980s of approximately 100 t/yr for the whole country, just 0.1% of capture fisheries production (Balarin 1987).
Kalinga (1991) suggests that lack of capital, suitable fish species and feeds, appropriate management techniques, and extension capacity all contribute to Malawi's poor performance in aquaculture. Balarin (1987) suggests that the lack of an integrated development approach is also a major problem.
Aquaculture projects often fail to address the problems and needs of smallholder and subsistence farmers and too often treat aquaculture as a stand-alone enterprise in the farming system. For example, many aquaculture projects in Malawi have presented farmers with technological packages designed to operate only as an independent commercial operation (GOPA 1987).
With farm sizes of usually less than 1 hectare, many Malawian farmers are operating at, or close to, subsistence and have very low cash incomes. Under these circumstances, they cannot afford
to purchase the formulated feeds and chemical fertilizers that are demanded by commercial aquaculture. Therefore, such technology packages will only be appropriate for a very small, restricted group of households with relatively high incomes.
If aquaculture is to have a wide impact on nutrition, farm incomes, and rehabilitation of resource systems in rural Malawi, it is necessary to have flexible aquaculture technologies that fit into a wide range of traditional farming practices and farm resources.
Aquaculture Research
In 1987, ICLARM, funded by Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ), initiated a farmer-participatory research project with the Malawi Department of Fisheries (FD), the objective being to develop aquaculture technology appropriate for rural Africa (ICLARM and GTZ 1991). The target group of research efforts are rural smallholder farmers with access to, or tenure rights over, water resources.
Studies are directed toward integrating aquaculture technologies into the smallholder maize-based farming systems. This is achieved by continual farmer-researcher interaction throughout the research program, thus enabling researchers to:
- Assess the agroecological resources of farmers and the extent of their traditional agricultural knowledge, thus providing information on the constraints to adoption of aquaculture;
- Develop on-station research to test technologies relevant to rural farmers and their farming systems; and
- Monitor and evaluate, in collaboration with farmers, the performance and impact of new technologies and integrated agriculture-aquaculture models on farming systems.
A report of the ICLARM-GTZ/FD collaborative research program is presented in Costa-Pierce et al. (1991).
On-Farm Bioresources Assessments and Current Status of Aquaculture
A large survey of farmers practicing aquaculture in Zomba District, Southern Malawi, was conducted to establish baseline data on pond sizes, number of ponds per farm, species of fish reared, etc. Table I summarizes this information.
A smaller survey of farmers was carried out to establish cropping and land use patterns. Biomass and production of crop and weed residues were measured wherever possible. Livestock wastes were estimated for some farms, as well as inputs of agricultural residues into fish ponds. Tables 2, 3, and 4 show land use patterns and availability of crop residues.
Data from bioresource assessments established that certain materials were often underutilized and had the potential for use in ponds. These were maize stovers, fallow land grasses, and wood ash from household cooking fires.
TABLE 1 Vital Statistics of 209 Smallholder Fish Ponds on 128 Farms in Zomba District, Malawi
Quartiles
Mean
Median
Pond area (mē)
338 (SE=34)
Total area of land under ponds on each farm (mē)
537 (SE=80)
Pond number/farm
1.8 (SE=O. 1)
Total number of hatchery ponds: 36 (17%)
Total number of production ponds: 173 (83%)
For example, maize stover production was 2.5 t/ha/yr, all of which was composted directly into the ground. There were enough stovers on farms for a proportion to be converted to high-quality compost as a pond input. Therefore, one potential research study was to look at the suitability of converting maize stover into a high-quality compost for use as a pond input.
Fallow land grasses and weeds were in relative abundance. The values in Table 4 are end-ofseason biomass, so turnover rates and grass production could be increased by cropping grass regularly for use as a pond input. Edwards et al. (1988) has shown that fish yields can reach as high as 5-6 t/ha/yr with vegetation as a sole pond input.
Availability of grass and its use by most farmers as a pond input for growing Tilapia rendalli (macrophytic feeder) led to studies of grass as a direct feed (Chikafumbwa 1990). The most common food input for fish ponds is "madeya" (maize bran), which is seasonally scarce. Maize bran is also sometimes used as food for humans in periods when maize meal is scarce. Therefore, grass has the potential to be a cheap substitute and possibly a more suitable food source for a macrophytic feeding fish such as T. rendalli.
TABLE 2 Pattern of Land Use Areas (ha) on 10 Smallholdings with Fish Ponds in Zomba District, Malawi
Mean
Standard Deviation
Range
Holding size
Crop land
Fallow land
Total pond area
TABLE 3 Average Production of Maize Residues on Farms with Fish Ponds, April-May 1989
Local Maize
Hybrid Maizeē
kg/ha/yr
kg/farm
kg/ha/yr
kg/farm
Stovers
Sheaths
Cobs
Bran
'Sample of 17 farms
ēSample of 3 farms
Farmers' ponds are nutrient poor in the Zomba district of Malawi. The main cause is the low nutrient status of the underlying ferrous soils and the resulting acidic waters, which lead to very low fertility (alkalinities 5-10 mg/1). Wood ash is a resource that is discarded in most households and has been demonstrated to improve the pH of pond waters and act as a phosphorus source (Jamu 1990). More than 400 kg wood ash/farm/yr was produced on the few farms that were sampled, and most was unutilized.
In the examples just noted, it is obvious that there are materials on farms that could be used as potential feeds and fertilizers in ponds. These on-farm assessments provide researchers with information that enables them to rank research objectives and also establish personal contact with farmers. This interaction helps sensitize farmers to the research program and the importance of their role in helping researchers to adjust their study objectives.
Farm Management and Integration
Coupled with the resource assessment above, farmers were asked to explain their calendar of agricultural activities and why these activities were organized in a particular way. Figure 1 shows a seasonal calendar for a farmer with a moderate level of integrated farm enterprises, and Figure 2 shows a calendar for a farmer with a high diversity of integration. These calendars enable researchers to see seasonal changes in bioresource management.
TABLE 4 Average Terrestrial Weed Biomass on Farms with Fish Ponds, June-July 1989
Herbaceous Plants
Grasses
kg/ha
kg/farm
kg/ha
kg/farm
Maize fields'
Fallow landē
'Sample of 6 farms
ēSample of 5 farms
A further step in this process is for researchers to encourage farmers to draw pictorial models of their farm systems (Lightfoot and Tuan 1990). These models show the bioresource flows between farm enterprises, thus providing a picture of the dynamics of the system and its level of integration. These pictures allow farmers to visualize their whole farm and see where new enterprises and linkages can improve farm integration, efficiency, and productivity. The process of pictorial modeling is described in a booklet and accompanying video by Lightfoot et al. (1991).
Smallholder Fish Production
Harvest yields of smallholder ponds were assessed to provide baseline data for comparisons with on-station experimental results. Table 5 shows the harvest yields for farmers practicing polyculture, and Table 6 shows the sizes of fish obtained from harvests (Noble and Chimatiro, unpublished). Yields are generally poor and fish small.
Results of On-Station Research
Results of on-station experimentation with on-farm bioresources and other low-cost materials are shown in Table 7. What is clear is that fish yields can be increased significantly using resources already available on most smallholder farms. In addition, farmers could mix resources, depending on their seasonal availability, and raise production well above that of maize bran input alone.
For example, a mix of grass and maize bran raised mean yields from ponds by a factor of 3 (approximately 3 t/ha/yr) compared with farmers ponds (approximately 1 t/ha/yr) using maize bran. Fish yields from ponds receiving pumpkin leaves (1 t/ha/yr) (Chimatiro and Costa-Pierce 1991) are similar to farmers' ponds. Pumpkin leaves are available when maize bran is not, so it could prove a valuable substitute.
TABLE 5 Harvest Summary for Polyculture of Oreochromis shiranus, Tilapia rendalli, and Cyprinus carpio in 14 Ponds in Zomba District (June-August 1989)
Oreochromis shiranus
Tilapia rendalli
Cyprinus carpio
Harvest biomass (kg/pond, kg/ha)
Mean
Median
Range
Harvest production (kg/halyr)
Mean
Median
Range
Mean number of days between harvests: 345 (range: 177-448); mean pond size: 565 mē (range: 70-1,564 mē).
Identification of locally available materials as pond inputs has proved an important and successful element of the research program. Examples shown above indicate how sustainable aquaculture at smallholder levels could develop.
TABLE 6 Growth Summary for Oreochromis shiranus, Tilapia rendalli, and Cyprinus carpio in Polyculture Ponds in Zomba District (June-August 1989)
Oreochromis shiranus
Tilapia rendalli
Cyprinus carpio
Fish sold (g)
Mean weight
Median weight
Range
Fish not sold (g)
Mean weight
Median weight
Range
All fish (g)
Mean weight
Median weight
Range
Note. The mean weights of each fish species in 16 ponds were averaged to get the overall means, medians, and ranges above. Hence, these figures are showing between-pond variations.
TABLE 7 Mean and Ranges of Net Yields of Fish Ponds, Costs, and Incomes of Fish Farmers in
Malawi Using On-Farm Resources (Chikafumbwa et al., Unpublished)
Mean Yields
Cost of Input
Income
Inputs
Input Rates
(kg/hatyr)
Range
(US$)
(US$)
Napier grass (NG)
100 kg DM/ha/day
Maize bran (MB)
3% MBWD
NO/MB
As above
Waste pumpkin leaves*
50 kg DM/ha/day
Maize stover compost/FWA**
3% MBWD; 2.5 t/ha
Smallholder farmers using MB
When available
NA
Smallholder farmers using MB
When available
*Cost of waste pumpkin leaves based on labor input to harvest waste leaf **FWA= Fuelwood ash and agricultural limestone combination
NA=Data not available
MBWD= Mean body weight per day
[1] Cost of fresh fish, 1991 retail prices @ US $1.21
[2] Cost of maize bran @1 US $0.04/kg dry matter @10% moisture
[3] AL= Agricultural limestone @ US $0.04/lcg
[4] FWA (Fuelwood ash) = No cost: a waste resource from household cooking fires
[5] Cost of maize compost based on labor input @US $0.81/day to construct compost heap; purchase of bamboos for pile aeration @US $0.13/bamboo
[6] Napier grass cost based on labor input to cut grass @US $0.81/day
[7] Costs of inputs are per kg/yr per 200 mē pond (2 fish crops/yr; 1 ha pond)
[8] Income is per 200 mē pond (2 fish crops/yr; 1 ha pond)
Other Potential Research Areas
In the current situation of high population densities and land shortage, more marginal land will have to be brought into production by using it for enterprises such as aquaculture. Lightfoot (1990) points out that new ways of utilizing land to help regenerate environments is urgently needed. He suggests that biological diversification of farms and improved nutrient cycling by incorporating aquaculture could help achieve this objective.
Integration of aquaculture on smallholder farms might help to create sustainable, regenerative farming systems (Lightfoot 1990). Most farmers practicing aquaculture in Malawi have vegetable gardens and rice fields adjacent to ponds. It would require very little effort to interlink these enterprises for mutual benefit.
On-station research and demonstrations have been directed to look at the potential of integrating fish with crops (rice, vegetables, maize) and animals (goats, chickens). Malawian farmers also have difficulty harvesting fish. Appropriate harvesting technologies must be suitable to the income and labor resources available to farmers. One research area for focus was harvesting tools and techniques such as reed seine, basket traps, plunge baskets, and recruit removal (Kaunda 1991).
Another important aspect that has led to low yields is the poor growth performance of the two major cultivated species, O. shiranus and T. rendalli. Research studies have started on utilization of the large Lake Malawi tilapias, particularly O. karongae ("chambo") and catfishes, such as Bathyclarius sp. (Msiska 1991).
The research program is attempting to develop new indigenous systems of aquaculture based on the existing farming system and resource base of the Malawian rural farmer. The combination of new inputs, new fish species, and new systems of integration based on local resources has the potential to produce a more productive, indigenous, and sustainable aquaculture for smallholder farmers.
Farmer Participatory Research
A major problem facing any research project attempting to improve smallholder farming systems is encouraging farmers to adopt new enterprises and modify and integrate existing ones. The ICLARM/FD project has avoided presenting technology packages to farmers. African agroecosystems are highly complex and no one enterprise or technology is going to be applicable over a wide range of farming systems. To ensure that research priorities meet the farmer's agenda, the farmer must be able to assess the technologies and systems being designed by researchers.
Exposure of Farmers to New Technologies
Part of the ICLARM/FD program has been focused on the methodology required to engender farmer participation in the research. One approach has been to encourage farmers to visit and assess on-station experiments and comment on the range of aquaculture options on offer. Farmers are given freedom to express their feelings about the technologies and make suggestions for new lines of research, or modifications to existing research. Farmers are able to explain to project personnel which technologies are most appropriate for their farming system.
On-station open days with farmers are organized with a workshop where farmers lead the discussion and help researchers to reformulate research objectives. This occurs after the farmers have viewed a "basket" of technologies on offer. Showing respect for farmers' opinions and knowledge helps to win their confidence and makes it easier for both researchers and farmers to work together to develop appropriate aquaculture technologies and systems.
The effect of open days on technology research and transfer has been documented by Noble and Rashidi (1990). In May 1990, the first open day was held at the National Aquaculture Center (NAC) with 29 farmers. Six months later, 54 farmers were interviewed: 29 who had been to the open day, and 25 who had not, and 76% of the former were operating more than one technology in their ponds, but only 32% of the latter. Exposure to technologies and a variety of pond management strategies enables farmers to pick options that suit their local circumstances.
However, the open days are only one aspect of farmer-researcher interaction. Farm visits to evaluate bioresources, technology performance, and impact are all part of an on-going dynamic, which has led farmers to adopt new aquaculture technologies more readily and new entrants coming into aquaculture.
Rice-fish Integration
A particularly effective example of this type of process and farmer-researcher interaction is with rice-fsh integration. In December 1990, farmers were shown an experimental rice-fish pond at the NAC. Until that time, farmers had never seen rice and fish grown together. They were shown harvests of both crops on the same day. At a workshop to discuss rice-fish, the farmers were excited by the idea, but heavily critical of the experimental setup. The researchers encouraged them to draw their own designs (Figures 3a, 3b, and 4). These were very sophisticated and demonstrated that farmers were capable of contributing effective ideas even in an area where they had no experience.
Farmers quickly realized that the most effective arrangement was to be able to easily decouple rice and fish and have an efficient means of concentrating fish in deeper waters away from rice. They had come, in one day of exposure, to the same conclusions reported by dela Cruz (1990) in a large collaborative research program by IRRI and ICLARM in the Philippines.
In response to farmers' criticisms and suggestions, a second rice-fsh pond has been built to their design at NAC. This incorporation of farmers' ideas in the on station research program has helped to forge a strong collaborative link between researchers and farmers.
In February 1991, a field survey showed that of the 17 farmers that came to the rice-fish day, eight (47%) had started rice-fsh ponds. Yields were good (ranges: 2.4-4 t/ha/yr for rice, 1.5-2.4 t/ha/yr for fish) and farmers stated they were higher than normally expected. Nutrient-rich pond muds and reduction of water and weed constraints probably contributed to the high rice yields. Four farmers were so impressed that they have started a dry season crop of rice.
We are seeing the evolution of an indigenous rice-fish farming system. Farmers have adopted a technology that is new, but very relevant to their farming environment. They have modified the technology to make it more efficient and started to operate new management strategies that have never before been tried (i.e., two crops of rice per year).
It is particularly exciting that farmers who have not been to open days at the NAC are starting to adopt indigenous technologies that have been developed by farmer-researcher interaction. Some of the new rice-fish farmers are in a large rice-growing area where farmer-to-farmer diffusion of technology can occur rapidly. This is resulting in new entrants to aquaculture through rice-fish integration as well as existing fish farmers adopting this new system for their ponds.
What is evident is that the success of the research program with regard to rice-fish has been aided by the sensitivity and understanding researchers have shown for what is feasible in agriculture-aquaculture integration on Malawian farms. The researchers presented these ideas to farmers and allowed them to decide the applicability of rice-fish integration to their farming situation.
Monitoring and impact assessment of rice-fish and use of other technologies by farmers is now being carried out. Farmers are modifying technologies they have seen at the NAC to suit their own circumstances. Part of the on-farm research program will be to determine if such changes are effective in leading to sustainable aquaculture systems and whether they have wider applicability across the broad spectrum of Malawian farming systems.
Conclusion
The core of the ICLARM/FD research program is farmer-participation, emphasizing on-station research and implementing on-farm experiments. Lightfoot (1990, 1991) points out that this is essential if sustainable aquaculture systems are to be developed.
As most farmers in Malawi operate their smallholdings at, or close to, subsistence level, farmer participation is essential for aquaculture development to be fully integrated into the farming system. The diversity of farming strategies and agroecosystems in Malawi precludes the use of general aquaculture packages. Success in aquaculture development will be achieved by taking a flexible, evolutionary approach to production of appropriate technology for smallholders. This can be achieved only by farmer collaboration in modifying new and existing aquaculture systems to suit a variety of local farming conditions.
Farmer participation in the research program has many facets ranging from farmer visits to on-station experiments, to farmer workshops both on-station and on-farm, and farmer-researcher experiments on farm. This dynamic interchange helps to ensure that research objectives are in line with the needs of farmers.
It is hoped that this farmer-first approach in the research program will lead to indigenous sustainable aquaculture systems for Africa that not only produce fish, but enhance the productivity of the whole farming system.
Acknowledgments
The authors thank the ICLARM research staff and technicians who assisted in field research and with the farmer open days, particularly Sloans Chimatiro, Fredson Chikatumbwa, and Daniel Jamu; Brian Rashidi and his staff of the National Aquaculture Center, Malawi Department of Fisheries, for their collaboration in the research program; and Clive Lightfoot, Roger Pullin, and Jay Maclean for their helpful comments on the manuscript. This research program is funded by the Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) GmbH, Eschborn, Federal Republic of Germany. The authors also thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
References Cited
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<section>Low-Input Technologies For Rural Aquaculture Development In Bangladesh</section>
M.V. Gupta
International Center for Living Aquatic Resources Management (ICLARM)
Makati, Manila, Philippines
Abstract
Fish is the main animal protein source for the people of Bangladesh. In spite of vast water resources, fish production is in decline, resulting in protein-energy malnutrition. Farmers participating in on-farm research developed low-input sustainable aquaculture practices that benefit the poor farmers, who constitute the bulk of the population.
Farmer-oriented studies have confirmed the viability of culturing silver barb (Puntius gonionotus) and nile tilapia (Oreochromis niloticus) in seasonal ponds. Productions of 1,2052,156 kg of P. gonionotus per ha were obtained in 3-6 months using rice bran as supplementary feed. While a production of 2,138-3,554 kg/ha/6 months was obtained in on-station experiments with cultures of O. niloticus, using various supplementary feeds and fertilizers, studies rearing O. niloticus were undertaken in farmers' seasonal ponds, resulting in production of 1,441-2,343 kg/ha in 4-6 months, using rice bran as supplementary feed and fertilizers. Results of a survey conducted to study the socioeconomic impact and farmers' assessment of culturing nile tilapia in seasonal ponds revealed that 70 % of the fish produced were consumed on-farm, and only 23 % of the fish sold was enough to meet operational costs. The overall return on investment was 334%.
Integration of poultry rearing (500 chicken/ha) with carp culture in perennial ponds proved to be economically feasible and resulted in the production of 5,044 kg of fish and 6,676 kg of chicken (live weight) per ha in one year.
Introduction
Fish is the main and cheapest animal protein source for the 110 million people of Bangladesh. Besides nutritional value, fisheries play an important role in the economy of Bangladesh in terms of employment, income generation, and foreign exchange earnings. It is estimated that about 8% of the population depend on fisheries for their livelihood (Planning Commission 1978). The number of households engaged in subsistence fishing is about 10.8 million (DOF 1990).
Despite its importance in nutrition, per capita consumption of fish is low -- about 7.9 kg/yr at present (World Bank 1990). In recent years, there has been a decline in per capita availability of fish, resulting in protein-energy malnutrition because of the increasing human population and decreasing yields from wild capture fisheries due to overexploitation of stocks and environmental degradation. Rural people, who depend on fish catches from the wild, have been most affected. Moreover, average fish consumption figures do not reflect the situation in rural areas: per capita consumption among the rural poor is about 4.4 kg/yr, and for the urban elite is about 22.1 kg/yr (World Bank 1990).
Against this backdrop of declining fish availability, the country has vast water resources: for example, ponds, oxbow lakes, floodplains, rivers, and reservoirs. There are more than 1.3 million ponds covering 146,000 hectares. In addition to these official figures, there exist vast numbers of small ponds and ditches (< 600 mē in area). A majority of rural households have backyard ponds or ditches that have high potential for aquaculture.
Realizing the need for increased fish production and the limitations from marine and inland capture fisheries, the government of Bangladesh is stressing increased production through freshwater aquaculture. The government has set a target production of 308,000 tons by 1994-1995 (an annual growth rate of 10.9%), as compared to a production of 153,000 tons from pond aquaculture during 1988-1989.
The International Center for Living Aquatic Resources Management (ICLARM), with funding from the United States Agency for International Development (USAID), is assisting the Bangladesh Agricultural Research Council (BARC) and the Fisheries Research Institute (FRI) in developing low-input aquaculture technologies. Resource-poor small-scale farmers constitute the bulk of the population in Bangladesh, and hence the major emphasis in project activities is on farmer-participatory on-farm research for developing aquaculture practices that would optimize resource use and maximize production. The project actively involves non-governmental organizations (NGOs) in the on-farm research activities and undertakes impact studies. Some of the activities undertaken and the results obtained are presented here.
Short-Cycle Species Culture in Seasonal Ponds
Many seasonal ponds, ditches, burrow pits, and roadside canals exist in the country. Most of these are lying fallow, covered with obnoxious aquatic weeds, and represent health hazards. At present, the yield from these waters is only 100-200 kg/ha of fish. Two of the reasons for their underutilization are: (1) farmers believe that seasonal waters are not suitable for aquaculture; and (2) the traditional culture species, viz., Indian carp (Catla catla, Labeo rohita, and Cirrhinus mrigala) do not grow well in these seasonal waters. Nile tilapia (Oreochromis niloticus), a hardy fish that can survive under poor water conditions, is resistant to disease, is a good converter of organic wastes into high quality protein (Stickney et. al. 1979, Balarin and Haller 1982, Pullin and Lowe-McConnell 1982), and is suitable for culture in derelict ponds of Bangladesh. Puntius sp. is in much demand in Bangladesh, but the locally available P. sarana is not a good species for culture because of
its poor growth. Puntius gonionotus, with faster growth, would be suitable for culture in seasonal waters. Hence, the need for introducing short-cycle species into the culture system was identified. Oreochromis niloticus and P. gonionotus were introduced into Bangladesh in 1974 and 1977, respectively, but have not been established as cultured species until recently, since there were no developed management practices. Research undertaken in the last two years has revealed that these species could give high production with low-value inputs (mostly agricultural residues and by-products) and are suitable for culture in seasonal waters.
Culture of P. gonionotus in Seasonal Ponds
On-station studies were undertaken for culture of P. gonionotus in six ponds of 360 mē each. Pond preparation included draining, application of lime at the rate of 200 kg/ha, filling with water, and releasing fingerlings of average size 8-10 g, at a density of 16,000/ha. The ponds were divided into two groups, each with three ponds. The first three ponds were fertilized with cattle dung at the rate of 1,500 kg/ha, and triple super phosphate (TSP), with 46% N and urea with 42% P (2:1 ratio) at the rate of 50 kg/ha/fortnight. Organic manure was alternated with the inorganic fertilizers. No supplementary feed was given. The second group of ponds did not receive fertilization, but the fish were fed daily with rice bran at the rate of 5% of the standing fish biomass. The fish were harvested from the ponds after five months of culture. While an average production of 1,953 kg/ha was obtained from ponds where supplementary feed without fertilizers was given, ponds that received
fertilizers, but no supplementary feed, only produced an average of 689 kg/ha.
During these on-station studies, discussions were held with farmers and local NGOs who showed keen interest in participating in research. They felt that their near-zero return from underutilized water resources could be improved with on-farm resources. Eight farmers having homestead ponds of 160-600 mē in size, and with an average depth of 0.9 m, were selected as cooperators for the culture of P. gonionotus.
Pond preparation included liming at the rate of 200 kg/ha, three days after which cattle dung was applied at the rate of 1,500 kg/ha. Five days after application of cattle dung, the ponds were stocked with P. gonionotus fingerlings of 7.5 - 10 g size, at a density of 15,000/ha. The farmers were asked to feed the fish daily with rice bran at the rate of 5% of standing crop and fertilize the ponds at fortnightly intervals with cattle dung at the rate of 1,500 kg/ha. However, it was observed during the culture period that fertilization of ponds was irregular, and on certain days they could not feed the fish due to lack of rice bran in the household. The fish were sampled at monthly intervals and the ponds were harvested after 4-6 months of rearing, depending on the water availability in the ponds. The production for 4-6 months rearing ranged from 1.2 to 2.1 t/ha (Table 1). The cost of production worked out to Tk.13/kg (US $ 1 = Tk. 36) of fish against a market price of Tk.
50/kg. These studies have shown that production can be increased by as much as 10 times with very low investment, and that the system can be managed easily by poor farmers.
TABLE 1 Culture and Production Details of Puntius gonionotus in Farmers' Seasonal Ponds
Pond no.
Pond Size (mē)
Stocking Density
Size at Stocking (g)
Size atHarvesting (g)
Culture Period (month)
Gross Production(kg/ha)
Culture of O. niloticus in Seasonal Ponds
Studies were undertaken at the FRI, Mymensingh, in ponds of 280 mē each, to evaluate the production potential of O. niloticus under different feeding and fertilization regimes. Ponds were stocked with fingerlings of 10-11 g size, at a density of 20,000/ha. Production of 2,739 kg/ha/6 months was obtained with rice bran as supplementary feed but without pond fertilization, while production was 2,128 kg/ha/6 months when ponds were fertilized with 1,500 kg/ha/fortnight of cattle dung, alternating with 50 kg of TSP and urea (2:1 ratio)/ha/fortnight. However, all fish were undersized (average size 52 g + 19) when raised only with fertilization (Table 2). When 40% of the rice bran in supplementary feed was substituted by mustard oil cake, the production increased to 3,554 kg/ha/6 months, with 11.2% undersized fish (Table 2).
TABLE 2 Details of Oreochromis niloticus Production under Different Management Systems
Stocking Density
Size at Stocking
Feed
Fertilization
Production(kg/ha/6 months)
Total
(fingerlings/ha)
(g)
Undersize
Market
Fish
Size Fish
(<80 g)
(>80 g)
Rice bran TSP and urea
Cattle dung,
Rice bran 60%
+ mustard oil
cake 40%
These on-station studies indicate the potential for culturing O. niloticus in seasonal ponds, but studies under farmers' conditions were also felt necessary. For this purpose, six derelict seasonal ponds were selected that were adjacent to homesteads. These ponds had not been used for fish culture previous to this study. The size of the ponds ranged from 80 to 320 mē, with a maximum water depth of 1 m. The ponds were cleared of weeds before the onset of rains and lime was applied at the rate of 200 kg/ha. After filling with rainwater, the ponds were stocked with fingerlings of 5-10 g size at a density of 20,000/ha. The farmers were advised to feed the fish daily with rice bran at the rate of 5% of the standing crop. Three farmers were asked to fertilize the ponds with cattle dung at the rate of 1,500 kg/ha at fortnightly intervals and the other three farmers were asked to fertilize the ponds with TSP and urea (2:1 ratio) at the rate of 50 kg/ha/fortnight. However, the
farmers could not adhere to these feeding and fertilization regimes due to lack of resources and inputs during certain days. The culture period ranged from 4 to 6 months, depending on the availability of water in the ponds.
The ponds were harvested when the water level went below 30 cm. Gross production ranged from 1,500 to 2,343 kg/ha in 4-6 months from ponds that received inorganic fertilizers, while production ranged from 1,441 to 1,925 kg/ha in 4-6 months from the ponds that received organic manure (Table 3). It is difficult to assess the effect of organic and inorganic fertilizers on production, as the farmers did not adhere strictly to the suggested fertilization schedules. However, the study indicated an average net benefit ranging from Tk. 38,250 to 72,750/ha (US $1,062 - 2,020/ha) in 4
months.
TABLE 3 Culture and Production Details of Oreochromis niloticus in Farmers' Seasonal Ponds
Pond Size
Fertilization
Stocking Density
Size at Stocking
Size at Harvesting
Culture Period
Gross Production
(mē)
(g)
(g)
(month)
(kg/ha!)
TSP + urea
TSP + urea
TSP + urea
Cattle dung
Cattle dung
Cattle dung
These results created wide interest among farmers and extension agents. An NGO, Bangladesh Rural Advancement Committee (BRAC), with technical assistance from the project, extended the technology to 309 farmers (32% women) in one district. Subsequent to implementation of the program, the project undertook a survey of 113 farmers to assess the production and economics of the operation. The study revealed Table 4) that the inputs (feeds and fertilizers) applied by the farmers were much lower than had been suggested and even then, they obtained a gross average production of 1,391 kg/ha/6.5 months. While cost of production amounted to Tk. 9,223/ha (US $256), average net return was Tk. 30,860/ha (US $875.23/ha), showing the economic viability and high returns from resources that formerly gave near-zero returns.
Integrated Livestock-Fish Farming in Perennial Ponds
In rural Bangladesh, a majority of the households raise chicken or ducks for either meat or eggs. In recent years the government of Bangladesh has taken up programs to introduce high yielding varieties of poultry to replace low productive native varieties. Studies were undertaken at the FRI to study the economic viability of integrating poultry raising with fish farming under Bangladesh conditions.
Experiments were conducted in ponds of 1,000 mē in area. Broiler chickens were raised over ponds at a density of 500 chickens/ha. Three species combinations of carps, catla (Catla catla), rohu (Labeo rohita), mrigal (Cirrhinus mrigala), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyagodon idella), were stocked in the ponds, at a density of 6,000/ha, each treatment with three replications (Table 5). The ponds were neither fertilized nor given supplementary feed, except for the chicken manure and some spilled chicken feed falling into the ponds. The chickens reached a marketable size of 1.4-1.8 kg each (live weight) in 7-8 weeks. It was possible to raise seven batches of chicken and one crop of fish in one year. Fish production ranged from 4,265 - 4,893 kg/ha/year. While gross biomass production was higher with 40% silver carp in treatment 1 (Table 5), gross economic returns were higher with 30% catla and 10% silver carp in treatment 2, due to the higher
market price for catla in Bangladesh.
TABLE 4 Details of Cost of Production and Benefit of Oreochromis niloticus Culture in Seasonal Ponds by 113 Farmers
Average per Pond (169.38 mē)
Per Hectare
Average Water Depth (m)
Inputs:
Fingerlings (number)
Lime (kg)
Urea (kg)
TSP (kg)
Cattle dung (kg)
Rice bran (kg)
Costs:
Fingerlings (Tk.)
Lime (Tk.)
Urea (Tk.)
TSP (Tk.)
Cattle dung (Tk.)
Rice bran (Tk.)
Total: (Tk.)
Production:
Fish (kg)
Gross return (Tk.)
Net return (Tk.)
1 US$ = Tk. 36.00
These on-station studies have proved the economic viability of integrated chicken-fish farming, but have also raised some issues regarding its adoption by farmers. Will raising poultry over ponds be socially acceptable? Will the farmers be able to manage the high-yielding varieties of chicken? Will there be marketing problems for the chickens? Will the system prove economically viable under farm conditions? Will the financial resources of farmers restrict the purchase of chicken feeds?
To find answers to these questions, studies were initiated with three farmers to whom BRAC provided credit. Details of costs and returns of one of the farmers are presented in Table 6. As can be seen, a farmer could get a net benefit (excluding interest on working capital), of Tk. 12,519 (US $348) from a pond of 680 mē. This study indicates that raising chickens over ponds is socially acceptable; farmers would be able to manage high-yielding varieties of chicken; the practice has proved economical; and extension agencies are willing to provide credit to farmers.
TABLE 5 Fish Production under Different Species Combinations in Integrated Poultry-Fish Farming
Species Combination
Stocking Density
Culture
Production
(fingerlings)
Period
(kg/ha)
(month)
Silver carp 40 %, rohu 20 %, mrigal 30%, and grass carp 10%
Silver carp 10 %, catla 30 %, rohu 20%, mrigal 30%, and grass carp 10%
Silver carp 30 %, catla 10 %, rohu 25 %, mrigal 25 %, and grass carp 10%
Involvement of Extension Agencies in Farmer-Participated Research
One of the constraints for aquaculture development in the past has been the poor links among farmers, extensionists, and researchers. The technology packages developed through on-station research are technically feasible and economically viable, but often fail to make an impact on the farmers because on-station research has failed to consider the resources of the smallholder farmers for whom these technologies are being developed. Therefore, extensionists also showed little interest in on-station research results. Hence, the project has been trying to involve farmers and extensionists (mostly NGOs, who play an active role in Bangladesh) in the process of problem identification and implementation of programs. This has many advantages: (1) it creates confidence among extensionists since they have witnessed successful adoption by farmers; (2) it reduces the time gap between technology development and dissemination; and (3) having been convinced of the economic viability of the
operation, extension agencies are more willing to extend credit and other inputs to farmers. One example of such successful collaboration could be cited here. BRAC has been involved in farmer participatory research for culture of P. gonionotus and O. niloticus. Having been convinced of the viability of the culture operations, BRAC now extends the technology to a large number of smallholder farmers. During 1991, more than 2,000 rural women were involved in the culture of P. gonionotus alone. The project, in collaboration with extension agencies, also organizes Farmer's Days, when the operations and results of the research are demonstrated to farmers in the area.
Impact Studies
After the development of a culture system and its transfer to farmers by the extension agencies, the project undertakes studies to assess socioeconomic impact and farmers' assessment of the technology. These surveys are revealing: (1) the benefits the farmers are getting through the implementation of the technology; (2) the constraints, if any, in practicing the culture system; (3) refinements and improvements needed in the technology; and (4) the policy issues involved.
TABLE 6 Costs and Returns of One Year's Production in Integrated Broiler-Fish Farming, from a
Pond of 0.068 Hectares.
Costs
A. Fish Culture
Inputs
Quantity
Costs(Tk.)
Pond lease value
Fingerlings
408 no.
Lime
15 kg
Labor costs for harvesting
Total
B. Chicken
Chicken shed (total cost
Tk. 1,200; longevity 2 yrs)
Chicks (8 batches)
325 no.
Feed
1,224 kg
Vaccines
Fuel
Labor
Total
TOTAL COSTS
Returns
A. Fish
343 kg
B. Chicken
454 kg
NET PROFIT
*Total costs for 8 batches of broiler. cost per batch is only 1/8 of total.
**Excluding interest on working capital.
A survey of 113 farmers of the total 309 farmers who have taken to O. niloticus culture in their homestead ponds and ditches has revealed that: (1) a pond of 170 mē (average size of tilapia ponds) can produce on an average 23.5 kg of fish, which is almost equivalent to the national annual consumption of low-income rural households with six family members; (2) 70% of the fish produced is consumed on-farm, thus improving the nutrition of farming families; (3) revenue from 23 % of fish sold was enough to meet the operational costs; and (4) return on investment was 334% indicating economic viability of the operation. Ninety percent of the farmers surveyed indicated that they were happy with the technology and wanted to continue, while 10% favored discontinuing. The farmers pointed out several economic, technical, and social benefits as encouragement factors Figure 1). One common observation made by all of the farmers was the small average size of O. niloticus at harvest. They
would like to know ways to control breeding in O. niloticus and have larger fish. As a consequence, studies are in progress to control fry production through introduction of a carnivore, Gariepinus lazera, into the production system.
Acknowledgments
The author thanks the Office of Research, USAID, for funding the network meeting and publication of this paper.
References Cited
Balarin, J.D. and R.D. Haller. 1982. The intensive culture of tilapia in tanks, raceways and cages. pp. 265-355. in Muir, J.F. and J.J. Roberts. (ed.). Recent advances in aquaculture. Westview Press.
Department of Fisheries (DOF). 1990. "Fish Catch Statistics of Bangladesh," 1987-88.
Planning Commission. 1978. The Two-Year Plan: 1978-80. Planning Commission, Government of the People's Republic of Bangladesh, Dhaka.
Pullin, R.S.V. and R.H. Lowe-McConnell. 1982. The biology and culture of tilapias. Proceedings of the International Conference on the Biology and Culture of Tilapias. Bellagio, Italy. International Center for Living Aquatic Resources Management, Manila, Philippines.
Stickney, R.R., J.H. Hesby, R.B. McGeachin, and W.A. Isbell. 1979. Growth of Tilapia nilotica in ponds with differing histories of organic fertilization. Aquaculture 17: 189-194.
World Bank. 1990. Bangladesh Fisheries Sector Review. Report no. 8830-BD.
<section>Hungarian Integrated Aquaculture Practices</section>
Z. Jeney
Fish Culture Research Institute
Szarvas, Hungary
Abstract
Increased production costs, environmental impact problems, and utilization of poor quality soils were the main reasons for the development of integrated aquaculture in Hungary. Fish-cum-duck culture, aquacultural rotation, and the use of different sources of manure in aquaculture systems are the typical forms applied.
In monoculture, 300-500 ducks/ha can increase common carp production by 140-175 kg/ha. In polyculture utilizing silver and bighead carps, a fish yield of 2 t/ha can be achieved without supplemental feeding, and 1,000-1,200 kg of ducks (2.0 to 2.4 kg each) can be produced simultaneously.
A three-phase aquacultural rotation has been developed for areas with poor quality sodic soils. The first phase, lasting 2-3 years, is the double meat production. It is a kind of fish-cum-duck culture with a polyculture of fishes, and results in an approximately 300% increase in total meat production (duck and fish). The second phase is the "forage-crop production on pond-bottoms," lasting 4-5 years. A mixture of alfalfa and red clover proved to be the most economical when raised on the dried bottoms of the same ponds. In the third phase, rice is produced on pond bottoms, resulting in 50-100% higher yields than the average for the country. After three years of rice culture the rotation system starts again.
The use of manure in fish ponds has the "lowest level" of integration with fish culture. Solid and liquid pig manure, fermented chicken manure, as well as domestic sewage water have been tested and are partly applied in Hungary.
Introduction
Asia has been the cradle of different forms of integrated crop-livestock-ftsh farming systems (I:)elmendo 1980, Dela Cruz 1980, Sinha 1986). Although there have been attempts to utilize these systems around the world (North America -- Buck et al. 1979, Latin America -- Pretto 1985, Western Europe -- Muir 1986), probably only the Israeli (Schroeder 1980, Hepher and Pruginin 1981, Sinha 1986) and the Eastern European practices (Muller 1978; Woynarovich 1979, 1980a,b; Kintzly and Olah 1981; Kintzly et al. 1983; Olah 1986; Varadi 1990) are comparable to the Asian version.
The aim of this review is to characterize Hungarian integrated aquaculture practices and briefly to compare them With the Asian practices.
Integrated Aquaculture Systems
In general, integrated aquaculture systems have several advantages and disadvantages, as listed in Table 1.
TABLE 1 Integrated Aquaculture: Advantages and Disadvantages (Muir 1986)
Advantages
Disadvantages
Shared use of resources
Mismatch of production cycles
Elimination or reduction of waste problems
Possible overloading or undersupply
Reduced cost of production
More complex, less definable systems
Improved operation of components
Divided management goals
Wider opportunities
Limitations in potential sites
Low-cost heating and growth
Public health problems
Use of chemicals
The different systems show great variety in size and complexity. Several attempts have been made to classify the integrated aquaculture systems. Muir (1986) grouped them into three main types, depending on where the aquaculture is integrated (Table 2).
TABLE 2 Integrated Aquaculture Systems (Muir 1986)
Agricultural
Industrial
Sanitation
Vegetable
Space
Sewage
Rice, cassava, wheat, crop leaves, palm, rubber, ipil-ipil
Use of land, shelter, security
Nutrients, floc, organic matter
Animal
Water
Waterways
Pigs, ducks, chickens, sheep, goats, cows, buffalo, rabbits, geese
Reservoirs, process water irrigation channels
Aquatic vegetation
Heat
Power stations, furnaces and ovens, breweries, distilleries
By-products
Breweries, distilleries, agricultural processors
Further divisions have been made according to the level of integration (Barash et al. 1982), the basic components of integration (Dela Cruz 1980), and whether integration is direct or indirect (i.e., if the components are some distance apart, or if intermediate processing is required to integrate them), or whether the integration is parallel or sequential (i.e., if components run at the same time or if, e.g., fish crops run alternately with agricultural crops). Sinha (1986) grouped these according to the site of integration (Table 3).
TABLE 3 Main Types of the Integrated Aquaculture (Sinha 1986)
A.
Integration in the water body:
B.
Integration on the water:
1. Paddy-cum-carp culture
1. Pig-cum-fish
2. Integration with irrigation
2. Duck-cum-fish
C.
Integration near the water
D.
Fish and sewage
Varadi (1990) differentiated integrated aquaculture based on the size and complexity of integrated animal husbandry:
1. The small-scale, Asian type of integrated fish farming means the aquaculture activity is directly linked to one or more activities (Figure 1). The possible advantages of integrated aquaculture as cited above (Muir 1986), can really be recognized in these systems. Small-scale integration, however, does not necessarily mean complex integration. In many of the small farms, livestock are raised separately from the fish ponds and the manure is transported to the ponds.
2. In large-scale integrated fish farming, integration usually means only the use of manure (liquid manure) in aquaculture systems. The simple integration scheme of a fish and an animal production farm is shown in Figure 2.
Aquaculture In Hungary
Aquaculture in natural waters is one of the oldest activities in Hungary. Pond fish farming became more common with the regulation of rivers in the nineteenth century. About 140,000 ha of water currently are under fishery production; 23,000 ha of that are pond surfaces. Aquaculture in
Hungary is based exclusively on freshwater, and is somewhat unusual in that freshwater fish production per capita is one of the largest in Europe (34,000 tons in 1990), while fish consumption in Hungary is only 4.2 % of the total meat consumption, and is one of the lowest in Europe (3.2 kg/per capita in 1989).
Fish farms in Hungary are using semi-intensive methods on poor soils (not suitable for other agricultural activities). The typical culture system is the carp (Cyprinus carpio L.)-dominated polyculture, with "herbivorous" fishes, such as silver carp (Hypophthalmichthys molitrix Val.), bighead (Aristichthys nobilis Rich.), and grass carp (Ctenopharyagodon idella Val.). In such systems the average total yield is 1 ton/ha.
The more valuable fishes cultured in Hungary are the European catfish or sheatfish (Silurus glands L.), the pike-perch (Stizosteidon lucioperca L.), pike (Esox lucius L.), rainbow trout (Oncorhynchus mykiss Walb.), and the eel (Anquilla anguilla L.). For pike, pike-perch, and sheatfish, special Hungarian methods of propagation and culture have been developed. The Siberian sturgeon (Acipenser baeri Brandt) and hybrids of crosses of the Siberian sturgeon with the sterlet (Acipenser ruthenus L.) are the newest and most promising fish for intensive systems. Development of culture technologies for endemic crayfishes (Astacus astacus L. and Astacus leptodactylus L.) also is a new direction in aquaculture. Meanwhile, the largest challenge met by Hungarian aquaculture is privatization. The domination of state ownership is changing. In 1989, 70% of pond surface belonged to state farms, 28% belonged to agricultural and fishery cooperatives, and less than 1% was private. Among the
"users of natural waters" the ratio of the private sector is even lower. However, in spite of a general recession in Hungarian aquaculture in the last 2-3 years, in 1990, the private sector increased its production five times.
Integrated Aquaculture In Hungary
Increased production costs (primarily water and energy), environmental impact problems, and utilization of poor quality soils were the main reasons for the development of integrated aquaculture in Hungary. Fish-cum-duck culture, aquacultural rotation, and the use of manure from different sources in aquaculture systems are the typical programs.
Thus, Hungarian fish culture was integrated mainly with agricultural activities, namely vegetable culture (fish-duck-rice-alfalfa-red clover), and animal husbandry (fish-cum-duck) to include the use of manure (pig, chicken, sheep). In addition, experiments with industrial integration were initiated to utilize the heated effluents of power stations for fish culture, as well as the utilization of by-products of slaughterhouses and pharmaceutical factories for feeding fish. As an example of the integration of aquaculture with sanitation, the utilization of domestic sewage waters in fish culture was also attempted.
Fish-Cum-Duck Culture
Raising ducks on fish ponds in Europe was developed as a large-scale integration system after the Second World War when there was a serious protein shortage, and the lack of mineral fertilizers became a bottleneck to further development of pond fish culture (Woynarovich 1980b). Hungary initiated such large-scale experiments as early as 1952. The main advantages of fish-cum-duck culture compared to integration with other animals (Barash et al. 1982) include the following:
- Duck culture can be introduced easily without any substantial changes to the environment
and the facilities.
- The nutritional value of the manure is preserved because losses of N and energy due to
fermentation, evaporation, and non-reversible coagulation are eliminated.
- Feed residues are eaten directly by fish.
- Costs of collecting, storing, and transporting the manure are eliminated.
- Land area, otherwise needed for manure-producing livestock is saved.
- A solution is provided to problems of environmental pollution by animal wastes.
- The environment for manure-producing livestock is improved.
- Ducks eat natural feeds that develop in the pond.
Three major duck rearing methods can be differentiated:
o Embankment rearing;
o Platform rearing; and
o Enclosure rearing, or their combination. The schemes of these systems are shown in Figure 3 (after Varadi 1990).
In Hungary, 300-500 ducks can be raised on 1 ha of water during one summer. The vegetation season (when the water temperature is higher than 15° C) is about 150 days. According to the estimation of Woynarovich (1980b) 100 kg of duck manure distributed continuously in pond water increased common carp production in monoculture by 4-S kg/ha. That means 500 ducks can increase common carp production by 140-175 kg/ha. If polyculture of fishes is applied, 1,000-1,200 kg of ducks are produced, in addition to the yields of marketable fish (common carp -- 1,000-1,200 kg/ha; silver carp -- 500-600 kg/ha; bighead -- 150-200 kg/ha). With a decrease in ratio of common carp and an increase in ratio of herbivorous fishes (silver carp and bighead) fish yields of 2 t/ha can be achieved without supplemental feeding (Woynarovich 1980b).
Trials to introduce fish-cum-goose aquaculture into Hungary were limited.
Aquacultural Rotation
Aquacultural rotation was developed for unproductive sodic soils in Hungary by Muller (1978). Aquacultural rotation is a kind of sequential integration and may be adapted for any flat area where the soil is suitable for fish and rice culture. The optimal size of ponds is 30-50 hectares. Inner sides of the dams are to be constructed with a moderate slope (1 :5, 1 :4), thus assuring their protection. The filling and drainage system of each pond must be independent, using irrigation water and a drainage system by gravity. The pond bottom should be large enough for rice fields of 2-3 hectares. Within the rice fields the bottom-level differences should not exceed + 5 cm.
Three Phases of Aquaculture Rotation
1. Double meat production. This is similar to fish-cum-duck culture. On a fish pond of 30-50 ha, 10,000-12,000 ducks may be raised at the same time, and this can be repeated 3-4 times a year. Average weight of marketable ducks 48-50 days old is between 2,600 and 3,000 g. Natural fish yields doubled where duck rearing had been practiced for 2-3 years Table 4, after Muller 1978). Total meat production (duck and fish) of a pond with fish-cum-duck culture was 3.2 times higher than a traditional pond using a polyculture of carp and silver carp.
2. Forage-crop production on pond-bottoms. Organic matter and mud deposited during the first phase of the aquacultural rotation for 4-5 years offers an opportunity for agricultural crop production on the dry pond bottom. Muller (1978) found that leguminous plants and a mixture of alfalfa and red clover gave yields up to 85.05 t/ha of green weight. With irrigation, polyploid red clovers provided the highest yields. The soil is enriched in nitrogen and calcium by the alfalfa.
TABLE 4 Increase in Weight (kg/ha) of Fish and Ducks Raised Together (After Muller 1978)
Year 1
Year
2Year 3
Year 4
Increase in weight of fish
Natural yields
Increase in weight of ducks
Total increase in weight
3. Rice production on pond bottoms. Pond bottoms are considerably improved during the first two phases of aquacultural rotation, significantly increasing organic matter and nitrogen level. This nutrient enrichment can be favorably utilized for rice production. The third phase lasts for three years. Highest yields of rice were obtained following production of alfalfa, with mixtures of red clover. Yields were 0.7-1.0 tons higher using this combination, than those following sunflower, sorghum, and maize production. Muller (1978) achieved 50-100% higher yields using this technique than the average yields of rice in Hungary, with a maximum at the level of 5 t/ha. After three years of rice production, the dams of the rice fields are levered and fish-cum-duck production starts again.
Use Of Manure In Fish Ponds
In this case integration strictly means the application to fish ponds of manure produced elsewhere. Animals like chickens, pigs, and cows are raised in special cultural units that are not integrated into the fish farm. Since Hungary is a large pig producer, the application of pig wastes in fish ponds could be the best example of this type of integration. The utilization of pig manure in fish ponds started in the 1950s, when only common carp were stocked. At that time, a disagreeable side effect of heavy manuring often occurred, in the form of algae blooms. Since the 1970s, Chinese carp have been stocked in polyculture with the common carp (Table 5), and this problem was eliminated.
In early studies, a critical question was how much pig waste could be utilized per unit of fish pond without running the risk of a fish kill from oxygen depletion. Experience has shown that usable quantities of pig wastes in fish ponds depend on the delivery and distribution methods: 1,500-2,000 kg/ha/yr can be used when pig manure is placed in the pond in localized heaps. When a carbon manuring method is applied, however, it is possible to distribute 300-600 kg/ha of manure, 1,000-1,500 kg/ha of the thick liquid phase of the manure, or 1.2-2.5 mē/ha of commercial pig waste, over the pond surface, on a daily basis. The maximum possible waste loading in fish pond was determined to be two to three times higher than the above quantities. The total manure loading per hectare of pond surface was calculated between 40 and 80 pigs/ha (Woynarovich 1980a).
Later studies (Kintzly and Olah 1981, Kintzly et al. 1983) addressed the optimal level of the used liquid pig manure as well as the optimal polyculture structure. Biculture of silver carp with common carp (2,500 p/ha + 1,000 p/ha) gave higher yields than both the monoculture and polyculture, with a maximum yield of 2.06 t/ha/year. In monoculture the fish did not reach the 1 kg body weight that is the marketable size in Hungary. Good results were obtained when tench (Tinca tinca L.) were introduced into the polyculture.
Advantages of the methods used in Hungary are:
- Pig waste can be placed into the fish ponds during the vegetation period, while on the fields the application is possible only during late autumn.
- Fish had low levels of fat.
- The discharged water from these fish ponds met the requirements of environmental protection standards.
- Investigations of fish meat quality gave positive results.
Disadvantages of the methods used in Hungary are:
- The bottom of the ponds accumulate organic materials.
- Transport costs of liquid manure became the limiting factor for the application of this method.
- When using the drainage system, only the gravitation method was economically reasonable under Hungarian conditions (Kintzly 1991, personal communication).
TABLE 5 Typical Hungarian Polyculture Technology
Stocking Density
Harvesting
Yield
(p/ha*)
weight/g
t/ha/year
Year 1
Common carp
Chinese carp
Year 2
Common carp
Chinese carp
same (20-30)
Year 3
Common carp
Chinese carp
same
* p/ha = piece/hectare
Some Basic Differences Between Asian And Hungarian Integrated Aquaculture
Reasons for integration. Economic as well as environmental aspects dominate in Hungary. In Asia, a dominant reason is the increased production of animal protein for the improvement of the economic condition of farmers.
Forms of integration. In Asia, integration has a higher level of complexity, and is scattered among small-scale agricultural activities.
Future of integration. There is less prospect for integration in the near future in Hungary than in
Asia, due to current economic changes. After the ongoing changes in ownership in Hungary,
environmental issues will probably impact strongly on integrated aquaculture.
Acknowledgments
The author thanks the Office of Research, USAID, for funding the network meeting and
publication of this paper.
References Cited
Barash, H., I. Plavnik, and R. Moav. 1982. Integration of duck and fish farming: experimental results. Aquaculture 27: 129-140.
Buck, O.H., R.J. Baur, and S.R. Rose. 1979. Experiments in Recycling Swine Manure in Fishponds. pp. 489-492. in Pillay, T.V.R. and W.A. Dill. (ed.). Advances in Aquaculture, Fishing. News Book, London.
Dela Cruz, G.R. 1980. Integrated farming with fish as major enterprise. pp.22-33. in Integrated Crop-Livestock-Fish Farming Food and Fertilizer Technology Centre Taiwan, Republic of China, May, 1980.
Delmendo, M.N. 1980. A review of integrated livestock-fowl-fish farming systems. pp.59-71. in Pullin, R.S.V. and Z.H. Shehadeh (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARM-SEARCA Conference 4.
Hepher, B. and Y. Pruginin. 1981. Commercial Fish Farming with Special Reference to Fish Culture in Israel. John Wiley and Sons. New York. 261 pp.
Kintzly, A. and J. Olah. 1981. Utilization of liquid pig manure in fish ponds. 6th Symposium of Fish-farming. pp. 37-38. Szarvas, Hungary. (in Hungarian.)
Kintzly, A., Gy. Kovacs, J. Kepenyes, and I. Toth. 1983. Environment saving technology of large-scale pig production. Fish Culture Research Institute. pp. 1-28. Szarvas, Hungary. (in Hungarian.)
Muir, J.F. 1986. Integrated carp farming in Western Europe. pp. 392-399. in Billard, R. and J. Marcel. (ed.). Aquaculture of Cyprinids, INRA, Paris.
Muller, F. 1978. The aquacultural rotation. Aquacultura Hungarica (Szarvas) 1: 73-79.
Olah, J. 1986. Carp production in manured ponds. pp. 295-303. in Billard, R. and J. Marcel. (ed.). Aquaculture of Cyprinids. INRA, Paris.
Pretto, L. (ed.). 1985. Manual de fertilizzacion organica pare extangues de piscicultura. Cuaderno de Acuicultura No. 6. pp. 1-16. Direction Nacional de Acuicultura - Panama.
Schroeder, G.L. 1980. Fish farming in manure-loaded ponds. pp.73-86. in Pullin, R.S.V. and Z.H. Shehadeh. (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARMSEARCA Conference 4.
Sinha, V.R.P. 1986. Integrated carp farming in Asian country. pp. 377-390. in Billard R. and J. Marcel. (ed.). Aquaculture Cyprinids, INRA Paris.
Varadi, L. 1990. Integrated animal husbandry, EIFAC/FAO Symposium on Production Enhancement in Still Water Pond Culture. pp. 1-17. Prague, Czechoslovakia.
Woynarovich, E. 1979. The Feasibility of Combining Animal Husbandry with Fish Farming, with Special Reference to Duck and Pig Production. pp. 203-208. in Pillay, T.V.R. and W.A. Dill. (ed.). Advances in Aquaculture, Fishing. News Book, London.
Woynarovich, E. 1980a. Utilization of Piggery Wastes in Fish Ponds. pp. 125-128. in Pullin, R.S.V. and Z.H. Shehadeh. (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARM-SEARCA Conference 4.
Woynarovich, E. 1980b. Raising Ducks on Fish Ponds. pp. 129-134. in Pullin, R.S.V. and Z.H. Shehadeh. (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARM-SEARCA Conference 4.
<section>Propagation</section>
Gonad Stimulation In Crustaceans: An Interspecies Approach
A. Sagi, E. Snir, D. Cohen, H. Laufer, And Y. Milner
Abstract
Macrobrachium rosenbergii, a prawn that is continuously reproductive, was used as a source for factors to stimulate gonad activity in females of the reproductively stringent shrimp, Penaeus vannamei.
An in vitro bioassay was developed to test the activity of the prospective factors prior to their administration into the recipient. An organ culture of pre-vitellogenic, early-vitellogenic and late-vitellogenic ovaries of M. rosenbergii females was established. The ovaries were cultured in the presence of both ģH-Thymidine and 35S-Cysteine, which showed a linear incorporation (up to 8 hr) into DNA and proteins, respectively. The ratios of ģH to 35S incorporated decreased in the order: pre-vitellogenic > early-vitellogenic > late-vitellogenic.
One of the factors tested during the in vitro bioassay was methyl farnesoate known to be synthesized by the mandibular organ of crustaceans. This molecule is the unepoxidated form of the insect juvenile hormone III, which is a gonadotropin in insects. To study the presence and synthesis of methyl farnesoate in M. rosenbergii, the mandibular organ was identified and cultured in the presence of methyl-ģH-methionine. Radiolabelled methyl farnesoate was extracted and quantitated from cultured glands and a culture media made of females. The concentration of methyl farnesoate in the hemolymph was determined using normal phase HPLC. Hemolymph from females contained 24 + 13.1 ng/ml of methyl farnesoate.
Introduction
In recent years the mariculture industry has experienced an increasing shortage in gravid females of several panaeidae shrimp species (NOAA, NMFS 1988). This shortage is noticeable in particular species of importance to the industry, such as Penaeus vannamei (Liao 1990). This problem could be solved by the establishment of a method for the stimulation of gonad maturation in captive females. Several sources for crustacean gonad stimulating factors are suggested in the literature. The aim of this study is to establish a primary in vitro bioassay that will enable the extraction of active substances from the continuously reproductive prawn, Macrobrachium rosenbergii, to be later administrated into the stringently controlled shrimp, P. vannamei.
In vitro gonad culture has been established for many crustacean species (Lui and O'Connor 1976; Berreur-Bonnenfant and Lawrence 1984; Sagi et al. 1988; Quackenbush 1989 a,b; Rankin et al. 1989; Browdy et al. 1990). Most of the researchers used a short-term incubation and studied de novo protein and DNA synthesis. The kinetics of the incorporation of ģH-Leucine into the crab Pachygrapsus crassipes lipovitellin (Lui and O'Connor 1976) and the incorporation intensity in different developmental stages of the ovaries of P. semisulcatus (Browdy et al. 1990) were described. Ovary culture was used as a bioassay for the effects of active substances on macromolecular synthesis in crustacean gonads (Berreur-Bonnenfant and Lawrence 1984). Incubations of early and full-vitellogenic ovaries of the crab, Carcinus maenas, and the amphipod, Orchestia gammarellus, served as a bioassay for the effects of farnesylacetone on protein synthesis. Quackenbush (1989 a,b) demonstrated that extracts of
P. vannamei eyestalk inhibited protein synthesis in ovary culture. An in vitro incubation system was established also for M. rosenbergii testes by Sagi et al. (1988).
Vitellogenin synthesis in crustacea is performed either in the ovary or in different sites such as the hepatopancreas and adipose tissue. Vitellogenin is found in the developing oocytes in the form of egg yolk, and vitellin (Quackenbush 1986). During vitellogenesis, the oocyte diameter constantly increases until ripened eggs are formed. The diameter of the oocytes can serve as a parameter for the vitellogenetic stage (O'Donovan et al. 1984, Browdy et al. 1990).
Various hormones were found to effect vitellogenesis. Gonad Inhibiting Hormone (GIH) is a small neuropeptide found in shrimps and crabs that inhibits gonad maturation (Fingerman 1987, Quackenbush 1986). Another gonad-stimulating factor was extracted from the thoracic ganglions of vitellogenic U. pugilator females (Eastman-Reks and Fingerman 1984). In certain amphipods, ecdysteroids are present which are essential for vitellogenesis, although their role is not yet known (Quackenbush 1986).
The first substance to be tested using the above bioassay was methyl farnesoate, a secretory product of the mandibular organ of crustaceans identified by Laufer et al. (1987) and suggested to be a gonadotropin. Methyl farnesoate is the unepoxidated form of the insect juvenile hormone (JH III). In insects, juvenile hormones play several regulatory roles, including that of gonadotropins (Herman and Bennett 1975). In crustaceans, mandibular organ implants into immature females L. emarginata caused noticeable enlargement of the ovaries accompanied by several morphological and tiltrastructural signs of active vitellogenesis (Hirsch 1980). When injected into reproductively active crab females, methyl farnesoate caused a small, but significant, increase in blood vitellogenin levels (Vogel and Borst 1990). The identification of the mandibular organ and its product, methyl farnesoate, in reproductive females M. rosenbergii are described here.
Materials And Methods
Animals
Adult females of M. rosenbergii were obtained from earthen ponds in Ginossar Research Station, Israel, and from Aquaculture Enterprises in Sabana Grande, Puerto Rico. The prawns were acclimated for at least one week before experiments were undertaken. Acclimation was done in a 500 gal freshwater tank. The water was recirculated through a gravel biofilter and temperature was kept between 25-28°C. The prawns were fed frozen Daphnia or squid and commercial prawn pellets daily. Females were selected as ovary donors according to their reproductive state. Females possessing mature ovaries (late-vitellogenic) were distinguished by their orange carapace coloration (Sagi and Ra'anan 1985). Pre- to early-vitellogenic ovaries were selected according to their dark carapace coloration or the color of the eggs carried by the females (O'Donovan et al. 1984). Molt stages (Peebles 1977) and oocyte diameter (O'Donovan et al. 1984) were determined for each dissected female. The latter was
measured by means of an objective micrometer.
Gonad Culture In Vitro
The ovaries were dissected into a sterile petri dish containing phosphate buffered saline (PBS), adjusted to the osmolarity of M rosenbergii hemolymph at pH = 7.4 (Stern 1985). Both lobes of the ovary were sliced into fragments of 2-4 mm in length. The fragments were incubated in Dulbecco's Modification of Eagle's Medium (DMEM) adjusted to M. rosenbergii osmolarity (Sagi et al. 1991a), supplemented with 40,000 units penicillin per 100 ml of 1 % BSA and 35S-Cysteine and ģH-Thymidine (3 uci/ml, each isotope). Incubations were carried out at 30°C with gentle shaking under an oxygen enriched atmosphere. At the end of the incubation period the tissue fragments were removed and homogenized on ice with 0.05 m Tris-HCI buffer at pH 7.2, supplemented with ethylenediamine tetra acetic acid (EDTA) 4 mM, Benzamidine 1 mM, Epsylon Amino Caproic Acid 1 mM, and the proteases inhibitors: Leupeptine 10 ~g/ml, PMSF 0.2 mM (in dimethyl sulfoxide [DMSO]) and Pepstatin 10 ~g/ml (in DMSO). The
homogenate was centrifuged at 14,000 RPM for 10 min at 4°C. The supernatent was separated and kept at -20°C. Total protein precipitation was performed by 59 trichloroacetic acid (TCA) on Whatman No. 3 paper discs. The discs were washed with 500 ml 5% TCA (twice), 500 ml acetone and 500 ml of petrol ether, dried and submerged in scintillation fluid (Toluene) and counted. The level of incorporation was expressed in CPM/~g total protein. Total protein in each sample was determined by a mini assay based on the Lowry method (Lowry et al. 1951) in a 96 well-plate, using an ELISA reader. Samples were also taken from the incubated medium at each time. These samples went through the same treatment as described above for the tissue fragments. The level of radioactivity incorporated into protein in the medium was expressed in CPM/,ul medium, and was then compared with control wells containing medium but no tissue fragments.
Mandibular Organ Culture In Vitro
Mandibular organs were dissected and cultured attached to the mandibular tendon. Similarly sized fragments of muscle, connective tissue, and androgenic glands were cultured as controls. Each tissue was cultured in 400 ~l of media for 2 hr at 30°C with slight agitation. Culture medium was M. rosenbergii saline (Nagamine et al. 1980) containing 20 mM HEPES (pH= 7.6), 3.8 mM dextrose and 0.2 % BSA. Methyl-3H-methionine (ICN) served as a radiolabelled methyl group donor for methyl farnesoate synthesis. The specific activity was 200 mci/mmole, and total activity in the culture media was 40 uci/ml (Laufer et al. 1987). Following incubation, the tissues were processed as described in Sagi et al. (1991b) and the hexane phase loaded onto a Waters HPLC equipped with a 5 ~ silica column, and the percentage of radioactivity which co-eluted with the methyl farnesoate standard was determined.
Methyl Farnesoate in the Hemolymph
Hemolymph samples (2 ml) were taken from the cephalothorax using a 3 cc syringe and 18 ga needle prerinsed with 0.019 EDTA. The samples were transferred to 15 ml Kimax culture tubes, on ice, containing 5 ml acetonitrile and 2 ml 4% NaCl. Twenty-five ng of the cistrans (non-biological)
isomer of methyl farnesoate was added to each tube as an internal standard. Hemolymph titers of methyl farnesoate were determined according to the method developed by Laufer et al. (1987) and modified by Borst and Tsukimura (1991) using a Waters HPLC equipped with a 5 ~ silica gel column. Peak areas were calculated using omega software (Version 1.4 Perkin Elmer/Nelson) and hemolymph methyl farnesoate titers were determined with reference to the known amount of internal standard in the sample.
Results
Long-term incubations (Figure 1) were attempted to establish a reliable bioassay using cultured ovary fragments from M. rosenbergii. The levels of incorporation of both labeled compounds were higher in early-vitellogenic ovaries. After 13 hr of incubation, the incorporation rate of 35S-Cysteine was 45 and 10 cpm/~g protein in early and late-vitellogenic ovaries, respectively. Incorporation of ģH-Thymidine at the same time was 14 and 4 cpm/~g protein in early and late-vitellogenic ovaries, respectively. The level of 35S-Cysteine incorporation into both early and late-vitellogenic ovaries was higher than the ģH-Thymidine incorporation. Pre-vitellogenic ovaries incorporated both 35S-Cysteine and ģH-Thymidine in rates close to, but somewhat below, early-vitellogenic rates. All curves show a saturation pattern occurring after approximately 10 hr of incubation. The linear phase of incorporation seems to be approximately 6 hours. Based on these results, further experiments of
short-term incubations (up to 8 hr) were performed and showed a linear pattern similar to that of the long-term incubation (Figure 2).
Figure 3 illustrates the ratio of 35S-Cysteine/ģH-Thymidine that was incorporated into pre-, early-, and late-vitellogenic ovaries. This ratio is the highest (14) in late-vitellogenic ovaries and the lowest (8.4) in pre-vitellogenic ovaries. Early-vitellogenic ovary shows an intermediate value (12).
A comparison of medium from wells that contained ovary fragments with control wells that did not contain tissue fragment shows that the level of radioactivity does not differ significantly. 35SCysteine and ģH-Thymidine levels in high molecular weight material in both incubation medium and medium without a tissue fragment remained close to the level of a zero-time medium sample, showing that no licks of biomolecules occur in these organ cultures.
The mandibular organ of M. rosenbergii females was identified in dissected animals. In vitro culture of this structure resulted in the synthesis of a radiolabelled product that was extracted by hexane and upon HPLC analysis proved to be predominantly methyl farnesoate. The mandibular organ from a reproductive female produced as much as 175.8 pmol methyl farnesoate within a 2 hr incubation period (n=7 average production rate for females 57.8 + 60.6 pmol). The amounts of methyl farnesoate in the hemolymph were calculated using relative peak areas and were as high as 40 ng/ml in reproductive females (n=6 average concentration 24 + 13.1 ng/ml).
Discussion
Testing ovaries from different vitellogenic stages proved that pre- or early-vitellogenic ovaries will be the best organs exhibiting maximal synthetic activity for the bioassay. Early-vitellogenic ovaries were also big enough to provide several fragments per tissue. Similar results were reported by Berreur-Bonnenfant and Lawrence (1984) in which farnesylacetone had a stronger inhibitory effect on immature ovaries than on mature ovaries of Carcinus maenas. On the other hand, our results are not in agreement with those of Browdy et al. (1990). These last authors reported that P. semisulcatus ovaries, with small-diameter oocytes, show low levels of protein synthesis, while ovaries with oocytes at the last stage of vitellogenesis show a higher rate of synthesis. A possible explanation for this contradiction could be that at the crypt formation stage, the proteins synthesized in the ovary are those forming the egg jelly precursor (Browdy et al. 1990). The eggs of Palaemonidae
prawns may not contain similar egg jelly, due to differences in maternal care patterns between the two families.
Our results show a saturation-pattern of the incorporation of the labelled compounds, with a linear phase of up to 8 hr of incubation. To establish a bioassay for active substances the linear phase may be preferred. Eastman-Reks and Fingerman (1985) showed 24-hr linear-kinetics for ģH-Leucine incorporation into total TCA-precipitable ovarian proteins of Fiddler Crab. Browdy et al. (1990)
incubated P. semisulcatus ovaries as long as 8 hours. An oxygen-enriched atmosphere caused a 30% increase in organ activity (Dr. M. Tom, personal communication).
The low incorporation ratio in pre-vitellogenic ovaries, in comparison with late-vitellogenic ovaries, suggests that the de novo synthesis of nucleic acids is more intensive in the first stages. One should note that even though total protein synthesis is obviously lower in late-vitellogenic ovaries, its incorporation ratio is the highest, suggesting a sharp reduction in nucleic acid synthesis as vitellogenesis proceeds. Using the double-labeling of both proteins and nucleic acids seems to widen the ability of the bioassay system to detect the distinct effects of hormones and growth factors.
The role of JH as a gonadotropin in insects (Herman and Bennett 1975) and the supporting evidence for such a role for methyl farnesoate in crustaceans (Hirsch 1980, Vogel and Borst 1990) suggested that such a substance should be abundant in the hemolymph of the continuously fecund M. rosenbergii female. Synthetic rates in M. rosenbergii were lower than the rates reported for L. emarginata (Homola 1989), and slightly higher than those reported for crayfish (Landau et al. 1989) and several other crustacean species (Laufer and Borst 1988). However, the last researchers were measuring only the amounts secreted into the incubation media. Methyl farnesoate secretion by L. emarginata was estimated to be 10% of the total synthetic methyl farnesoate produced in vitro in a 2 hr period by a mandibular organ (Homola 1989).
The wide differences that were found between females in both synthesis rates and hemolymph titers of methyl farnesoate could reflect differences in reproductive states, as was shown for female spider crabs (Laufer et al. 1987). The effect of methyl farnesoate on M rosenbergii ovaries in the culture system described above is currently being investigated in our laboratory. The effect of methyl farnesoate on our target organism, P. vannamei, will be addressed in future studies.
Acknowledgments
The authors thank the Office of Research, USAID, for funding grant number DHR-5544-G-000075, the network meeting, and publication of this paper.
References Cited
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Borst, D.W. and B. Tsukimura. 1991. Quantification of methyl farnesoate levels in hemolymph by high-performance liquid chromatography. J. Chromat. 545:71-78.
Browdy C.L., M. Fainzilber, M. Tom, Y. Loya and E. Lubzens. 1990. Vitellin synthesis in relation to oogenesis in vitro-incubated ovaries of Penaeus semisulcatus (Crustacea, Decapoda, Penaeidae). J. Exp. Zool. 255:205-215.
Eastman-Reks, S.B. and M. Fingerman. 1984. Effects of neuroendocrine tissue and cyclic AMP on ovarian growth in vivo and in vitro in the fiddler crab, Uca pugilator. Comp. Biochem. Physiol. 79A:684-697.
Eastman-Reks, S.B. and M. Fingerman. 1985. In vitro synthesis of vitellin by the ovary of the fiddler crab, Uca pugilator. J. Exp. Zool. 233:111-116.
Fingerman, M. 1987. The endocrine mechanisms of crustaceans. J. Crust. Biol. 7:1-24.
Herman, W.S. and D.C. Bennett. 1975. Regulation of oogenesis, female specific protein production, and male and female reproductive gland development by juvenile hormone in the butterfly, Nymphalis antiopa. J. Comp. Physiol. 99:321-338.
Hinsch, G.W. l980. Effect of mandibular organ implant upon the spider crab ovary. Trans. Am. Microsc. Soc. 99:317-322.
Homola, E.M., 1989. Regulation of methyl farnesoate synthesis in the spider crab, Libinia emarginata. M. Sc. Thesis. University of Connecticut. 85 pp.
Landau, M., H. Laufer and E. Homola. 1989. Control of methyl farnesoate synthesis in the mandibular organ of the crayfish Procambarus clarkii: Evidence for peptide neurohormones with dual functions. Invertebr. Reprod. Dev. 16:165-168.
Laufer, H., D. Borst, F.C. Baker, C. Carrasco, M. Sinkus, C.C. Reuter, L.W. Tsai and D.A. Schooley. 1987. The identification of a crustacean juvenile hormone. Science 235:202-205.
Laufer, H. and D.W. Borst. 1988. Juvenile hormone in crustacea. pp. 305-313. in Endocrinology of selected invertebrate types. Alan R. Liss, Inc., New York.
Liao, I.C. 1990. The world marine prawn culture industry: Today and tomorrow. pp.11-27. in Hirano, R. and I. Hanyu. (ed.). The second Asian fisheries forum. Asian Pisheries Society, Manila, Philippines.
Lowry O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. Protein measurement with folin-phenol reagent. J. Biol. Chem. 193:265-275.
Lui, C.W. and J.D. O'Connor. 1976. Biosynthesis of crustacean lipovitellin. 3. The incorporation of labeled amino acids into the purified lipovitellin of the crab Pachigrapsus crassipes. J. Exp. Zool. 199:105-108.
Nagamine, C.M., A.W. Knight, A. Maggenti and G. Paxman. 1980. Masculinization of female Macrobrachium rosenbergii (De Man) (Decapoda, Palaemonidae) by androgenic gland implantation. Gen. Comp. Endocrinol. 41:442-457.
NOAA, NMFS. 1988. Aquaculture and captive fisheries. US Dept. of Commerce, April 1988. pp. 99-198.
O'Donovan, P., M. Abraham, and D. Cohen. 1984. The ovarian cycle during the intermolt in ovigerous Macrobrachium rosenbergii. Aquaculture 38:347-358.
Peebles, J.B. 1977. A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture 12:173-180.
Quackenbush, L.S. 1986. Crustacean endocrinology, a review. Can. J. Fish. Aquat. Sci. 43:22712282.
Quackenbush, L.S. 1989a. Yolk protein production in the marine shrimp Penaeus vannamei. J. Crustacean Biol. 9:509-516.
Quackenbush, L.S. 1989b. Vitellogenesis in the shrimp, Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary. Comp. Biochem. Physiol. 94B:253-261.
Rankin, S.M., J.Y. Bradfield and L.L. Keeley. 1989. Ovarian protein synthesis in the South American white shrimp, Penaeus vannamei, during the reproductive cycle. Invertebr. Reprod. Dev. 15:27-33.
Sagi, A. and Z. Ra'anan. 1985. Rapid identification of reproductive state and the receptive period of females in pond populations of Macrobrachium rosenbergii - a new technique. Aquaculture 48:361-367.
Sagi, A., Y. Milner and D. Cohen. 1988. Spermatogenesis and sperm storage in the testes of behaviourly distinctive male morphotypes of Macrobrachium rosenbergii (Decapoda, Palaemonidae). Biol. Bull., 174:330-336.
Sagi, A., L. Karp, Y. Milner, D. Cohen, A.M. Kuris and E. Chang. 1991a. Testicular thymidine incorporation in the prawn Machrobrachium rosenbergii: molt cycle variation and ecdysteroid effect in vitro. J. Exp. Zool. 259:229-237.
Sagi, A., E. Homola and H. Laufer. 1991b. Methyl farnesoate in the Prawn Macrobrachium rosenbergii: In vitro synthesis by the mandibular organ and titer in the hemolymph. Comp. Biochem. Physiol. 99B:879-882.
Stern, S. 1985. Osmoregulatory mechanisms in the euryhaline crustacean Macrobrachium rosenbergii (De Man). Ph.D. thesis, Life Sciences Institute, the Hebrew University of Jerusalem, Israel. 74 pp
Vogel, J.M. and D.W. Borst. 1990. Spider crab yolk protein: molecular characterization and the effect of methyl farnesoate (MF) on its hemolymph levels. Am. Zool. 30:49A.
Woynarovich, E. 1979. The Feasibility of Combining Animal Husbandry with Fish Farming, with Special Reference to Duck and Pig Production. pp. 203-208. in Pillay, T.V.R. and W.A. Dill. (ed.). Advances in Aquaculture, Fishing. News Book, London.
Woynarovich, E. 1980a. Utilization of Piggery Wastes in Fish Ponds. pp. 125-128. in Pullin R.S.V. and Z.H. Shehadeh. (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARM-SEARCA Conference 4.
Woynarovich, E. 1980b. Raising Ducks on Fish Ponds. pp. 129-134. in Pullin, R.S.V. and Z.H. Shehadeh. (ed.). Integrated agriculture-aquaculture farming systems. Proc. ICLARM-SEARCA Conference 4.
<section>Cryopreservation Of Sperm Of The Mekong Giant Catfish, Pangasianodon Gigas Chevey</section>
K. Mongkonpunya, T. Pupipat, S. Pholprasith, M. Chantasut,
R. Rittaporn, S. Pimolboot, S. Wiwatcharakoses, And M. Chaengkij
Abstract
Spermatozoa of an individual male giant catfish, Pangasianodon gigas Chevey, were successfully cryopreserved in liquid nitrogen (LN), retaining fertilizing capacity for up to 18 months. Spermatozoa were extended ( 1:3 milt:medium ratio) in extender "Sl6 " (1.25 g KHCO3 8.55 g sucrose, and 0.30 g reduced glutathione in 100 g distilled water combined with dimethylsulfoxide [8 % final concentration]) within two types of containers (1 ml glass ampules, and in half broken artificial insemination [AI] catheters) and then frozen by suspending the containers of extended milt for 15 minutes in the vapor above LN before immersion. After 2, 360, and 540 days of storage, samples of cryopreserved sperm were thawed (20 seconds in 70°C-80°C water bath) and tested with fresh eggs of Pangasianodon gigas, Pangasius sp. (Mekong origin), and Pangasius sutchi to determine fertilization rates. Mean fertilization rates of sperm cryopreserved in AI catheters were comparable to those of controls,
fresh sperm, but significantly greater than those preserved in ampules. Fertilization rates (%) using sperm preserved in AI catheters were (mean _ SE), 67.7 _ 7.1, 67.7 + 0.8 and 34.7 + 5.7 (controls were 79.0 :: 1.4, 64.6 + 0.6 and 59.2 + 9.8) for eggs of Pangasianodon gigas, Pangasius sp., and Pangasius sutchi, respectively).
Introduction
Cryopreservation techniques offer considerable potential for fish culture and have been explored for a variety of species (Horton and Ott 1976, Mounib 1978, Erdahl et al. 1984, Kerby 1983). However, most of the work was done on a small-scale experimental basis. Recently, Kerby et al. (1985) designed their experiments to expand the scope of investigation. They examined the growth, survival, and health of larval striped bass hatched from eggs fertilized with cryopreserved sperm. They produced and harvested thousands of normal fish 43-47 days after stocking. In Thailand, a monsoon country, spermatozoa preservation has not been practiced in fish hatcheries because high quality milt is available almost year-round. However, there has been an increasing awareness of the benefits of cryopreserved sperm, which would contribute to the controlled breeding and husbandry of some uncultivated fishes, e.g., Pangasius snithwongsei and Pangasianodon gigas. Hatchery production of P. gigas has
been obstructed by the death of males and/or females while being kept alive waiting for the opposite sex. Preservation of spermatozoa with relatively high viability should be an excellent solution for this problem. Thus, techniques of chilled storage (0-5°C) and cryopreservation have been developed in Puntius gonionotus (Markman 1984) and in Pangasius sutchi (Pupipat 1988, unpublished data). It was found that by using medium "S16" (Table 1) with 0.25 ml straws and 1-ml glass ampules, fertility of the cryopreserved spermatozoa was maintained. However, 0.25 ml straws are too small for large-scale production. We also found that frozen glass ampules containing cryopreserved sperm often explode dangerously during the thawing process.
TABLE 1 Ingredients of Extenders
EXT
KHCO3
SUC
GLUT
MP
DMSO
CH3OH
S16
S17
S18
S19
S20
Notes: SUC= sucrose, GLUT= reduced glutathione, MP= milk powder.
In this study, we examined the use of 1 ml sterile AI catheters (by cutting them into two pieces of equal length), in comparison with 1 ml glass ampules. We also used fresh ova of Pangasius sp. for testing of fertilizing capacity of P. gigas cryopreserved spermatozoa.
Materials And Methods
Fresh milt (about 100 ml) used for cryopreservation in this study was collected from an individual male Mekong giant catfish, which weighed 173 kg and was caught in the 1989 spawning season (April-May). Abdominal stripping was done 20 hr after an injection of Domperidone (5 mg/kg), plus Ovaprin (5 mg/kg). Only creamy white semen was collected into a 250 ml beaker. Motility or mass movement of the whole sperm was examined immediately after activation with an equal volume of distilled water. It was found that motility of the milt sample was excellent. Then, every 100 ml of semen was diluted in a milt:medium ratio of 1:3 with an extender (Table 1) of 1.25 g KHC03, 8.55 g sucrose, and 0.30 g reduced glutathione in 100 g distilled water. The final concentration of dimethylsulfoxide (DMSO) in extended milt was 8%. This extender was chosen based on our previous work, which indicated that it provided the best result.
Approximately 0.8 ml of extended semen was placed into 1 ml glass ampules or into half-broken AI catheters. Then, all samples were frozen by suspending them in the vapor above liquid nitrogen for 15 minutes before immersion.
After varying periods of storage (2, 360, and 540 days), samples of cryopreserved sperm were thawed rapidly by swirling for 20 sec in a 70-80°C water bath and tested with fresh eggs to determine fertilization rates.
For fertilization testing, fresh eggs from a female P. gigas were available two days after sperm preservation and were used for this purpose. Since fresh eggs of P. gigas were not available during the 1990 spawning season, or for about 360 days after the sperm preservation, we used fresh eggs of a female Pangasius sp. of Mekong origin for the fertilization test. Another subsequent fertilization test using the same batch of cryopreserved sperm was attempted with fresh eggs from six female Pangasius sutchi at 540 days after storage. It must be mentioned that hybridization was not a purpose of this study. The Pangasius sp. eggs were used only because P. gigas eggs were not available.
The females were induced to spawn by two hormonal injections using human chorionic gonadotropin (hCG) (100 + 100 i.u./kg) for Pangasius sp., and Domperidone plus Ovaprim (7.5 mg/kg + 25 ug/kg) for P. gigas. These treatments resulted in spawning 6-20 hr after the second injection. Details of the investigation on hormonal-induced spawning will be reported separately.
When a female was stripped, the eggs were split into two containers. One, used for normal production, was also used as the control and received fresh sperm from a male. The second container was further split into 0.5 ml aliquots (ca. 500 eggs) and each aliquot was fertilized by pouring 0.8 ml of nearly thawed semen into the containers, adding hatchery water, and stirring the mixture gently. The step of rinsing was repeated a few times to remove debris and excess sperm. After that, the presumptive fertilized eggs were placed in one lifer plastic bowls full of water to incubate. About 18 hr post-fertilization, the percentages of fertilization were determined. This assay of semen fertility was based on the percentage of embryos that developed to the eyed stage.
An analysis of variance was used to test differences between control (fresh semen) and frozen semen, and also between the two types of containers. Eggs from a single female were used to compare fertility of semen between the treatment groups.
Results And Discussion
Spermatozoa activity (mass movement) was checked for the diluted semen left in the beaker. This was done to ensure that the sperm were still alive at the end of the 3-fur freezing procedure. Sperm motility was found to be as good as fresh whole semen.
The motility of post-thawed sperm was also determined for those left in the ampules and catheters. However, this motility determination was done 5-10 min after thawing. Probably due to time delay, sperm motility was generally poor or unobservable. This indicated that the post-thaw quality of the sperm was deteriorating rapidly. From our experience, sperm activity lasted about 30-60 sec after being activated. Thus, in doing fertilization tests, time is a critical factor. It was our practice to swirl the ampule or the catheter containing cryopreserved sperm in hot water (70-80°C) for 20 sec and then the neck of the ampule or the end of the catheter was cut off and sperm were shaken out and mixed with fresh eggs. The time elapse between thawing and adding hatchery water was a matter of 1-2 min or less.
Fertilization rates examined at 18 hr are shown in Table 2. Due to a limited amount of available semen in Pangasius sp. (Mekong origin), the sample was preserved only in half-broken AI catheters, and a comparison between container types was not possible.
Fertilization percentages of cryopreserved sperm under these experimental conditions were quite inconsistent. They ranged from 8 to 14% (Table 2) for glass ampules and 26 to 77% for halfbroken AI catheters. Thus, our results are comparable to those reported by many others. However, the later set of data showed no significant difference from those of the controls (fresh semen of the same species). Of the containers tested, glass ampules provided sperm with a significantly lower fertility percentage. Besides, quite often the glass ampules exploded or broke while thawing. Therefore, using glass ampules for sperm cryopreservation is not recommended. In addition, both types of containers held approximately 0.8 ml of diluted semen. There was, however, a difference in the diameters of the containers, with the glass ampules having a greater diameter. Thus, larger diameters of glass ampules prevented an even rate of freezing and thawing of semen samples within the ampules, and
resulted in lower sperm fertility. Half-broken AI catheters were smaller in diameter and provided higher sperm fertility. The capacity and configuration of the catheters are similar to those of 1 ml French straws. We preferred 1 ml French straws over the catheters, but the straws were not available. Since the spawning season of 1991 (Apr-May) we have been using 4.5 ml cryotubes (Nunc, Inc.) for holding 4 ml diluted semen or about 1 ml fresh semen with a dilution ratio of 1:3. This container is believed to be practical and applicable for hatchery production, since its capacity is large. However, we found that the rates of cooling and thawing used in this experiment were not suitable for the cryotubes, as the fertility of the preserved sperm could not be maintained (data not shown).
TABLE 2 Fertilization Percentagesđ of Sperm Cryopreserved in Medium S16 Examined 18 hrs after Insemination
Ova Sourceē
Container
Control
Ampule
AI Catheter
Pangasius gigas
79.0 i 1.4
Pangasius sp.
Pangasius sutchi
đFertilization percentages were based on the percentage of embryos which developed to eyed stage. ēEggs from one each of P. gigas and Pangasius sp. were used, but from six of Pangasius sutchi.
ģEggs from a single female were used to compare fertility of semen between treatment groups. Within each treatment, there were three incubations of about 500 eggs.
Since P. gigas is an uncultivated and endangered species, the eggs and semen, if available, are seriously needed for hatchery production. The harvesting season for this species has a very short duration of about two weeks between the end of April and May. During our study period it was not possible to obtain P. gigas eggs for intraspecies fertilization trials. Thus, interspecies fertilization, using eggs of Pangasius sp., was considered the best option for testing the fertility of the cryopreserved sperm of Pangasianodon gigas.
The fertilizing capability of the cryopreserved sperm with Pangasius eggs was comparatively lower than those with P. gigas eggs (Table 2). However, this difference probably was not due to incompatibility of the eggs and sperm, because the control data (fresh eggs x fresh sperm of the same species) also showed the same trend. Therefore, these differences may be a reflection of poor egg quality or other unknown factors.
The large variability in the fertilizing capability of the cryopreserved sperm in our study seems
to be a phenomenon common to this type of research. Apparently, small alterations in the thawing process can cause highly variable results. If the potential for cryopreservation is to be realized, precise standards must be established and used consistently.
Results from this study clearly indicated that, when working with scarce and endangered
species of fishes, cryopreserved sperm represent a valuable tool, and interspecies/intergeneric fertilization may be a useful tool for testing the fertility of the cryopreserved spermatozoa.
Acknowledgments
Support for this work was provided, in part, by the Office of the Science and Technology
Development Board offered through the Division of Inland Fisheries, and, in part, by Kasetsart University. The work was conducted, in part, at the Chainat Inland Fisheries Station and special thanks are due to Mr. Sontipun Pasukdee for his cooperation and assistance. The authors also thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.
References Cited
Erdahl, A. W., D. A. Erdahl, and E. F. Grahm. 1984. Some factors affecting the preservation of salmonid spermatozoa. Aquaculture 43: 348-350.
Horton, H. F. and A. G. Ott. 1976. Cryopreservation of fish spermatozoa and ova. J. Fish. Res. Board Can. 33: 995-1000.
Kerby, J. H. 1983. Cryogenic preservation of sperm from striped bass. Trans. Am. Fish. Soc. 112: 86-94.
Kerby, J. H., J. D. Bayless, and R. M. Harrell. 1985. Growth, survival, and harvest of striped bass produced with cryopreserved spermatozoa. Trans. Am. Fish. Soc. 114: 761-765.
Markman, N. 1984. A preliminary study on cryopreservation of fish spermatozoa. Faculty of Science, Kasetsart University, Bangkok. M.Sc. Thesis. (in Thai.)
Mounib, M. S. 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil. 53: 13-18.
<section>Propagation Of Mahseer In The Himalayan Waters Of Nepal</section>
T.K. Shrestha
Department of Zoology, Tribhuvan University
Kathmandu, Nepal
Abstract
The number of migratory mahseer (for putitora) is dwindling because of overexploitation, environmental pollution, habitat modification, and power dams. Conservation and management of this superior game fish requires special propagation practices. Spawning runs of the mahseer start in early September, terminate in late October, and occur at night in gravel-bottomed creeks, pools, and rapids. Eggs from the spawning beds were collected and successfully cultured in egg trays. The complete incubation period (fertilized egg to free swimming fry) was about 25 days at mean temperature of 35°C. Sexually mature migratory spawners were also caught for artificial breeding experiments. Spawners injected with hormones were stripped and the fertilized eggs were incubated in trays and aquaria. Fry were reared in several rearing ponds within 60 days and 50 % survived. Early 8-day-old sac fry were also reared in ponds, but they did not survive well. Fry were successfully raised in cloth bags
(happa) by providing artificial food (egg yolk). Critical stages of fertilized spawn were monitored in the laboratory and field and a life table for the mahseer was constructed for use in transplantation of mahseers into new habitats.
Introduction
In recent years, various investigators have given considerable attention to the propagation of the seriously endangered mahseer in Himalayan waters (Badola and Singh 1984, Shrestha 1986, Nautiyal and Lal 1984, Das 1990). They have been prompted by several considerations: (1) loss of natural habitat, endangering the breeding population; (2) overfishing in natural waters and pollution, which is destroying the spawning grounds and broodfish; (3) scarcity of healthy broodfish for artificial breeding by hormone injection; (4) recognition by cold water fish culturists of the need for propagating mahseer in lakes and reservoirs to boost recreational fisheries; and (5) the realization that the mahseer can be farmed and that wild populations can be restored in natural waters.
In developed countries, particularly in the United States, methods have been developed for the successful propagation of fish (McFarland 1960, Collins and Hulsey 1964, Anderson 1968, Webster et al. 1978, Mauri et al. 1979).
TABLE 1. Observations on the Spawning of Mahseer in Gadkhar Creek
Date
Weather
Temperature
Interstream Migratory
Downstream Movement
Spawning Dens
Spawning
Lunar
Movement
Assembly
Cycle
(°C)
Sun and clouds.
Vigorous movement started
Spawners moved
Gravel, sand
Possibly 3
Two
Rain for whole day.
around 1 a.m. at creek
downstream at 1 a.m.
bar,intergravel
males, 1
days
Barometric pressure 385.
confluence and upstream in
Fifty (38 males, 12
shallow water
female.Low
before
net across confl.
creek. Preliminary courtship
females) captured in gill
current speed to
turbidity
full
acts and false spawning in the
0.5 m/sec
gravel beds
moon
Temp. 31°C
clearly vis.
Spawners moved
SSpawners moved
Cloudy, heavy rain
Mass movement of mahseer at
downstream at 5 p.m.
Gravel,
Possibly 53
Full
(for about 6 hr.)
about 12 p.m. Spawners
Six females, six males
sandbar,
males,18
moon relatively clear
and wind. Sky
travelled lOO meters upstream
were captured in gill
intergravel
females. High
first at day. night. Full moon
from confluence site. Male
nets.
water with
turbidity,
chased the female, brief
depth 0.5 to 1
gravel beds not
visible. Barometric
courtship took place.
m. Current
visible.
pressure 360.
Spermatic fluid released over
velocity 0.5 to
the eggs deposited in the
2 m/sec. Temp
gravel. Creek water at certain
32°C.
spots appeared milky.
Spawners moved
Cloudy, drizzling
Movement of mahseer at the
downstream. Two
About 500
Full moon. with wind
Full
creek. Jumping, courtship and
females, two males
spawners
moon. Barometric
spawning.
captured in gill nets.All
Edge of deep
congregated,
pressure 380.
had running eggs or
pool, with
peak spawning
sperm.
mixed stones,
period reached
pebbles and
Gravel bed
and current
turbidity low.
Spent spawners moved
velocity as
Cloudy, drizzling.
Usual spawning movement
downstream. Twenty
above. Temp.
Number of
New
New moon.
followed by courtship action
females and sixty males
35°C.
spawners
moon..
Barometric pressure
No eggs or sperm released.
captured in gill net. All
decreasing
False spawning occurred.
were in spent condition.
Spawners very
sleeply. Few
active. No
ovulation or
sperm
observed
Temp. 28°C
As a contribution to this problem, studies were initiated in 1985 on the propagation of the mahseer, Tor putitora (Hamilton). Although experiments are still in progress, sufficient data have been obtained to warrant the publication of results for the four-year period 1985-1989.
A few accounts of induced breeding of the species of mahseer have been published (Tripathi 1978, Ogale and Kulkarni 1987). Methods for artificial propagation have yet to be described in detail (Shrestha 1985). This is the first attempt at large-scale breeding and propagation of mahseer species in the ponds, lakes, and reservoirs of Nepal.
Materials and Methods
For the study of natural spawning and behavior, both direct-sensing and remote-sensing methods were used. Direct sensing of the mahseer habitat and behavior was made visually. Spawners were dazzled with light to facilitate the study of spawning performance. Florescent-tube lamps were also used as a light source in remote and inaccessible places. Occasionally, underwater torch lights were used to observe spawning activity and to sample freshly deposited spawn or fertilized eggs. Plankton nets of various sizes were used to collect the spawn. The physico-chemical features of the spawning grounds were recorded in situ (Tables 1 and 2). Remote sensing of the habitat was done with binoculars and a camera fitted with a telephoto lense and a video recorder. The shape and size of the spawning ground and other physical characteristics were recorded. Freshly deposited eggs were removed from the spawning beds and successfully incubated in hatchery eggs trays. Both early and advanced fry
were reared in trays or cloth bags suspended in pond water. For stocking purposes, fry of different stages were transported to different places.
For artificial breeding, adult fish (Figure 1) were collected from the spawning creeks by using gill and trammel nets. Captured fish were tranquilized using tricaine methanesulfonate (MS-222). All adults were tagged with Floy tags on 15-16 September 1987. Mahseer were transported to the laboratory by truck and jeep. At the laboratory they were acclimatized, by sex, in separate plastic pools or cloth bags suspended in river water. The conditioned fishes in the plastic pools were fed with soybean and rice bran in a 1:1 proportion. Spawning was induced by injecting extracts of pituitary glands from carp, the dosage was four glands per kg body weight of fish. Females spawned after 18 hr with only one intramuscular injection. Artificial fertilization was accomplished by stripping the male and female brood fish by conventional wet and dry methods. Early fry produced as a result of artificial breeding were reared in incubation trays. Advanced fry were reared in cloth bags suspended
in pond water. The developmental stages of fry were monitored for three years (1986-1989) and a life table was constructed.
To study the survival of mahseer in new habitats, experimental releases were made in Trisuli reservoir and Kulekhani reservoir (Indrasarovar). About 1,044 semi-adult fish were released in the Trisuli and Tadi river confluence site. One year later, 580 of these fish were recovered.
TABLE 2. Physical and chemical Parameters of the Tisuli River Water (1986)
Month
Mean
Discharge
Temp.
Depth
Color
Odor
Tur-
pH
Dis-
Free
Total
Spec.
Current
Mean cu
(0°C)
(m)
bidity
solved
CO2
Alkalinity
Conductivity Velocity
m/sec
Mean
Average
(cm)
O2
(ppm)
m/sec
(ppm)
January
Clear green
Fishy
February 0.5
Clear green
Fishy,
methane
March
Unstable
Methane
green
April
Silty brown
Fishy,
methane
May
Silty brown
Strong,
methane
June
Red silty
Methane
brown
July
Reddish silty
Weak
brown
methane
August
Red brown,
Weak
silty
methane
September 3.0
Red silty
Fishy,
brown
methane
October
Dark green
Fishy,
methane
November 0.8
Clear green
Fishy
December 0.7
Clear green
Fishy
Results
Natural Spawning and Physical Features of Spawning Sites
Gadkhar Creek is located in Chokedovan, near His Majesty's Government (HMG) Fish Farm (Figure 2). The creek becomes flooded during early May and dries to a trickle during February and March. Its bed consists of gravel, pebbles, cobbles, and boulders. There is a wide area where the creek enters the Tadi River. Three spawning beds, A, B, and C, were identified in the creek. Bed A, consisting of pebbles less than 10 cm in diameter, is the cleanest among the three. Bed B consists of rocks 15-40 cm in diameter, and Bed C consists of rocks from 50 to more than 80 cm in diameter. Water quality of the spawning ground is given in Table 2.
At midnight, mature mahseers came to the creek-river confluence a few at a time. They gathered into a group numbering 10-30. While swimming together, several males would actively pursue the females, nuzzling and biting. After midnight, during the full moon period, courtship behavior was intensified and suddenly the mahseers swam rapidly in shallow zones of the creek near the bank, where they vigorously flopped and trembled. Eggs and sperm were released and fertilization took place. The fertilized eggs came to rest on, and adhering firmly to, stones and pebbles. After an interval of two hours, ejection of eggs and sperm were again noticeable when the water of the outgoing current turned milky. The majority of eggs 2.5-3.5 mm in diameter were washed away gradually by the current and the number of eggs adhering to the pebbles was very small. Hydrological and biological observations of the mahseer and its spawning habitat are given in Table 1.
Observations of mahseer spawning activity commenced in August 1986, and active spawning was observed from August to September 1986. As evident in Table 1, there is a strong correlation between spawning days and weather conditions. Both daytime and nighttime observations were made. During the breeding season (September to October), spawning took place every evening on billowy days or stormy days with heavy rainfall. Commencement of spawning behavior also seemed to be related to the hours of high flood in the parent river and medium flood in the feeder creek. Spawning took place from midnight to early dawn. There also was a significant relationship between the hours of onset of spawning and the weather (high rainfall and low barometric pressure). Spawners selected rocks, stones, pebble, gravel, sand, and log debris as spawning substrata (Table 1). Spawning was not noticed among algae or discarded plastic bags that had drifted to the creek bank.
Spawning of mahseer was initially observed on five evenings, during the period September 2 to October 5 (full moon September 2 and October 5). Spawning was noted to be related to flood conditions and usually began within 30 minutes after heavy flooding. The time of first spawning occurred after midnight on September 2, when there was a full moon. On November 15 no spawning was seen. During this last period, there was no moon, flood intensity was too low, and the water was relatively clear (Table 1). Mass spawning during and after floods showed that flood levels may provide a mechanism whereby fertilized eggs (Figure 3) are transported to the river bank to find suitable shallow nursery habitat to settle and develop into fry.
High temperature accelerated hatching time when eggs were reared in incubation trays and aquaria, but caused a decrease in oxygen content. This situation was rectified in aquaria by supplying fresh river water and maintaining aeration. Laboratory observations revealed that a continuous freshwater supply, proper aeration, right start food, and regular removal of dead eggs and waste material are the best ways to reduce mortality and shorten the time required for hatching. The mahseer sac fry undergo latency for a period of two days until the yolk is fully absorbed; this phase was considered suitable for long distance transfer (Figure 4).
Collecting Naturally Spawned Egg Fry
The collection net (3-7 m long) devised for this purpose was conical or funnel shaped and made of coarse cloth to allow easy filtering (Figure 5). The tapered end, fitted with a ring 9-12 inches in diameter, was made of reeds. A small pocket net (0.5 m long and 2.5 cm deep) that looked like a monk's hood was attached to the tapering end. Water flow through the net was often enhanced by adding two wings at the mouth.
The net was stretched with the mouth facing upstream in shallow bends of the creek where the flow is gentle, 1.2-2.5 m/sec. The downstream portion of the net would drift in the direction of the current and was tied in this position to poles just below the surface. The upper edge of the mouth of the net and the upper edge of the tail piece was just above the water. The collected spawn and fry were acclimatized separately in cloth bags (Figure 6) and were reared to fingerling stage. Fully grown fingerlings were released later in adult fish ponds at the Gadkhar Fish Farm (Figure 7). Hatchery bred and naturally spawned fry were transplanted to different water bodies.
Artificial Spawning
Induced spawning experiments during 1986-1989 utilized injected extracts of pituitary from carp, with the dosage being four glands per kg body weight of the target fish. The pituitary extracts were injected (with 0.3% saline water as carrier) into male and female breeders. Normally, they spawned after 18 hours with only one intramuscular injection. No death was caused by a higher dosage.
Artificial fertilization, by stripping the male and female breeders, was done by both conventional dry and wet methods. These brood fish ranged from 80 to 160 cm in length and weighed 2,500-3,800 grams. The dry method was far more successful than the wet one.
The larvae obtained by induced breeding as well as the artificial stripping process resulted in 80,000 fry. They were reared in incubation trays and aquaria for a fortnight. About 40% mortality was recorded. Dead fry were immediately preserved for development and life history studies, and live ones were reared to adulthood in the Gadkhar Fish Ponds over a three-year period (Table 3). Various stages such as sac fry, swim-up fry, and fingerlings were transferred from the Gadkhar Fish Farm to the Balaju Fish Pond, Gokarna Fish Ponds and the Kulekhani Reservoir, where the survival of young fish in new habitats was monitored.
During the embryonic development of T. putitora, a characteristic latency period was noticed in which the sac fry became quite inactive, with only feeble motion, and congregated in dark corners of the incubator. This period lasted about 8-12 hours. The hatching period appeared to depend on temperature, with temperatures of 32-34°C accelerating the hatching process. In these experiments, hatching occurred when oxygen concentrations were 8-15 ppm. Although warm, overcast rainy days triggered spawning in T. putitora, hot and sunny days were harmful for the survival of hatchlings.
TABLE 3 Biometric Data on Stages of the Mahseer during a Three-Year Period
Life History Stage
Time
Weight
Length
(gm)
(mm)
Egg or spawn
0 hrs
Fertilized egg
30 minutes
Hatchlings
55 hours
Sac fry
72 hours
Swim-up fry
110 hours
Jumping fry
180 hours
Fingerlings
3 months
Young fish
6 months
Young fish
1 year1
Adolescent fish
1.5 years
Maturing fish
2 years
Mature fish
2.5 years
Spawning fish
3 years
Transfer of Hatchery Bred Fry
During the 1986 studies it was found that 4- to 8-day-old sac fry could be handled safely and transported long distances. On 21 September of that year, 10,000 sac fry (volumetric measurement, 150 fry per cmģ) were carried for a distance of 76 km without significant loss. This successful operation was repeated again on September 15, 1987, at which time 50,000 fry were moved the same distance. Each of these trips required about three-and-a-half hours to complete and the water was not changed en route. The sac fry were not advanced enough for free-swimming and settled to the bottom when placed in cans where they remained during transit. The sac fry were raised to fingerling stage in the cloth bags suspended in pond water.
Some transfers of advanced fry (free swimming for 60 days) were made from the Gadkhar Fish Ponds to ponds at Balaju. These fry were collected by net from incubators and transported in fish cans. A total of 22,000 fry of this age were transported by truck in 10-gallon cans for a distance of 70 km without significant loss. It was concluded from these results that advanced fry can survive handling and transportation over long distances.
Stocking of Advanced Fry
There is little information on the relationship between the rate of stocking and the age of stocked fry, and equally little information on the rate of survival and growth. Two of the Gadkhar Fish Ponds were used for stocking 60-day-old fry in 1986. One year later, in September 1987, these two ponds were drained and the fish removed with representative samples collected and recorded. After a 360-day growing period, the fish had attained an average length of 25 cm and weight of 135 gm (Table 3).
Two ponds in Gadkhar were also stocked with 30-day-old fry, which after 360 days had a much lower rate of survival than the two ponds originally stocked with 60-day-old fry. After 360 days, the fish originally stocked as 30-day-old fry had attained an average length of 18-22 cm and a weight of 120-130 am.
Tagging Mahseer
To study the migration and dispersion of mahseer, 1,044 fully grown mahseer and 2,500 young mahseer were tagged (Figure 8). Thirty percent of adults and 10% of young mahseer were recaptured. The young fish were often captured 5-10 km from the release site, but adult fish were usually captured 70-200 km downstream from the release site. This clearly showed that adult fish moved slowly to warm water feeding zones. The younger fish also converged in feeding zones downstream. The release sites in parent creeks often dried up as soon as normal floods were over. Therefore, most of the fish stayed in lower reaches where there was sufficient water for them to survive dry spells. In any case, the mahseer are very mobile. As noted, long distance movement was previously thought to be spawning migration. It is now established that mahseer need fast and relatively warm rainfed creek water for spawning, so they migrate to headwater streams like the Trisuli and Tadi for spawning. Soon
after spawning is over, exhausted spawners move downstream as the floodwaters in creeks start to recede until they reach slow water in downstream reaches of the Narayani River. They repeat such migratory cycles year after year. The spawning and feeding areas are geographically separated between mountains and often range over great distances. Mahseer tags were recovered at different points of the Trisuli, Tadi, and Narayani rivers. The tags were returned voluntarily by fishermen, for which they received an award of a mahseer T-shirt. Most of the tags were returned during the spawning season. Tagging studies are still in progress.
Transfer Of Adults
Adult mahseer frequently suffer heavy mortality during transportation. This was counteracted, to some extent, when salt was added to the transport water and, in some species, by adding an anesthetic. The present study was conducted to determine the methods, concentrations, and kinds of anesthetics appropriate for use in the successful transport of mahseer for as long as 10 hours. The 20 adult males used in these experiments had mean total lengths of 50-60 cm and weights of 8-10 kg and the fingerlings were 18-20 cm in length and weighed 45-50 am.
Tricaine methanesulfonate (MS-222) was used as the experimental anesthetic. The anesthetized fish were subjected to a series of tests to evaluate this anesthetic at various concentrations; the stocking density was 2 fish/10 l of water. Concentrations of MS-222 were tested at 5 mg/l with increases up to 35 mg/l. At concentrations up to 15 mg/l the fish exhibited loss of equilibrium. Those exposed to 20-25 mg/l were lightly anesthetized and those exposed to 30-35 mg/l exhibited deep anesthesia. All fish quickly revived. Water quality was better at higher levels of MS-222. The deep anesthesia at 30-35 mg/l indicated the approach of lethal concentrations. Fish were also held without an anesthetic (control) in transport water containing salt at concentrations of 0.05 or 10%. The tests were made in double-lined plastic bags (Figure 9) held in boxes. Air was forced out by deflating the bags to water level; oxygen was then added and the bags were sealed and covered. Boxes were left
unattended for 10 hours, after which the bags were opened and fish behavior (Table 4) evaluated by the criteria described by McFarland (1960), with modifications. Fish were acclimatized to 21-28°C and then released together in troughs for observation 10 hours later to evaluate their behavior after being anesthetized (Table 4).
TABLE 4 Classification of the Behavioral Changes of Mahseer under Anesthesia
Stage
Anesthesia
Fish Behavior
Classification
Normal or natural
Highly excited, frantic reaction to external stimuli when caught from riverine habitat
Drugged
Decreased reaction to external stimuli, but swimming ability retained
Loss of equilibrium
Able to stay afloat, but unable to maintain body equilibrium
Light anesthesia
Total loss of swimming power, but able to move on the bottom (with tail movement)
Deep anesthesia
No movement on bottom, gill movement greatly reduced
Death
Gill movement ceased, fish float belly upwards
Hauling Mahseer By Truck
Three additional tests were conducted to determine the best method for hauling the mahseer in trucks. Anesthetized fish were put into a rectangular tank containing 5,0Q0 1 of water with the anesthetic and salt. Oxygen (3-51/min) was added through a porous pipe. Fish were hauled in the tank for 10 hours, then removed to a holding trough for a 10-hour observation period.
Hauling Mahseer By Jeep
The transportation of mahseer to Kathmandu in a jeep was also evaluated, using MS-222. Two fish were put into each bag containing water (25°C; 10% salinity) and anesthetic, (25 mg/l). Bags were inflated with oxygen and ice was added to the styrofoam boxes. Seventeen boxes of fish were brought to Kathmandu. Fish were acclimatized by floating the bags in a pond at the Balaju Fish Pond following the trip. Water temperature in the bags was about 18°C, whereas in the pond it was 16°C. When the fish were released into the pond they appeared to be in good condition. After one month, two mortalities were noted.
The effectiveness of MS-222 in different species of fish is variable. The threadfin shad (Dorosoma petenese) and gizzard shad (D. copedianum) were hauled successfully in 22 mg/l of MS-222 (Collins and Hulsey 1964, Anderson 1968), whereas the American shad (Alosa sapidissima) was intolerant of a 10-mg/1 dose of MS-222 (Mauri et al. 1979).
Adult mahseer were also transported in big earthen pots filled with water. Natural red clay rich in hematite and collected from river banks was added at the rate of 200 gm in 40 1 of water. Common salt concentration was maintained at 0.5%. Three changes of water were made during the transit, with each change made after a one-hour interval. Fresh red soil and salt were also added at each interval. The dissolved oxygen in the water was found sufficient for the transported fish. Splashing water provided good aeration while the fish were being carried in the earthen pots. This method is useful in places where manpower is cheap and access by road is poor.
Further studies may show that stocking densities in both bags and tanks could be increased. Results of these present tests, however, confirm that every effort should be made to reduce stress and scale loss during the handling and transport of mahseer.
The present work demonstrates the possibility of stocking mahseer in the tailwaters of dams and reservoirs in Nepal to rebuild the declining populations of this valuable fish. Only fingerlings of mahseer are recommended for transplanting and restocking using anesthetics and therapeutic oxygen-inflated bags. In this study, it was possible to transport and release mahseer over a long distance (more than 180 km), from the Trisuli River to the Kulelchani Reservoir.
Discussion
The ranges of mahseer within Nepalese drainage are primarily restricted to the headwaters. Poor habitat caused by man-induced disturbances have eliminated or reduced mahseer and benthic macroinvertebrate communities. The main disturbance in river and creek drainages has been surface stone-mining and siltation. Abandoned stone mines and eroded gullies in stream banks have also contributed to the overall decline in environmental quality. Other factors reported to have an impact on fish communities in these drainage systems include agricultural runoff, drilling runoff, domestic and industrial pollution, and low water discharge during dry periods. All these factors are reported to be of greater magnitude in river tributary watersheds (Shrestha 1988, 1990a,b).
Increased environmental degradation in these drainages in the future could eliminate other allied mahseer species such as Acrossocheilus hexagonolepis, Barbus chillinoids, and B. dukai,
especially those with rigid habitat requirements. Reduced fish communities in tributaries already heavily polluted indicate that this is a serious problem. Additional man-induced disturbances can only escalate the likelihood of the extinction of pristine golden mahseer habitat. Entire watershed control or management and faster propagation may be essential if endangered mahseer populations with limited spawning habitats are to survive. The present study provides some clues for the long-term preservation of Tor putitora by adopting ranching practices or modifying existing fish farming practices.
It was noted that the spawning of mahseer in the Tadi River was always associated with lunar periodicity, water level, flood cycle and heavy monsoon rain, thunderstorms, some physico-chemical features, and undetected biological factors. Majumdar (1940) noted the influence of thunderstorms, rains, and cloudy weather on the spawning of carp. Patra and Azadi (1984, 1985) noted the dates of spawning in the Halda River and found that spawning occurred about the time of the full moon or new moon. The lunar periodicity in T. putitora Is similar to that of many fishes (Ross 1983).
Bandola and Singh (1984) observed that floodwaters and the current of running water act as stimulating factors for carp spawning. It appears from the present study that the water current provided aeration, dispersion, and defense from pathogenic infections of the developing eggs.
The function of flood in the reproductive activities of fish is not clearly known (Shrestha 1986). The turbid waters of flooded streams are apparently not a deterrent to spawning, although great turbulence due to rapid flooding, as in July and August, is unfavorable and mahseer breed with decreasing intensity at high flood periods. It appears that well-oxygenated, gently advancing floodwater over the gravel beds provides the necessary rheotactic stimulus, evoking sexual play in T. putitora. A newly inundated streambed is less likely to be inhabited by enemies and predators such as fry-eating insects and amphibians, and there is less chance of fungal and bacterial infections of the developing egg and fry. Therefore, mahseer leave their parent river for spawning grounds in feeder streams to breed. After the onset of the flood is over, they move downriver to rejoin the main river channel.
It may be concluded from this study that the pattern of spawning behavior of the cold water game fish T. putitora appears to be adapted to reproduction in shallow water creeks and streams. The most distinctive feature is the spawning run, which serves to disperse the pelagic eggs across the current, which are then carried downstream.
Management Consideration
Because mahseer travel extensively, management-oriented ranching research must be conducted. The management of mahseer in relatively closed systems of ponds and reservoirs in Nepal requires very specific strategies. However, research in open water ranching and release systems must also be conducted on a long-term basis. In open river systems, a well-coordinated research effort needs to be directed toward assessing the status of mahseer populations and determining the geographic areas over which uniform management can be exerted.
Acknowledgments
This study (grant number 3.E30) was supported and financed by the U.S. Agency for International Development, Washington, D.C. I am grateful to Mr. B.B. Shah, Deputy Secretary,Department of Wildlife Conservation, Royal Palace, Kathmandu, for his cooperation. I am also grateful to Mr. R.B. Thapa, Additional Secretary, Ministry of Agriculture, for encouragement and support. My thanks also to Professor Douglas A. James and Mr. Robert Jenkins, University of Arkansas, for reading of the manuscript and for providing valuable suggestions. I also want to thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
References
Anderson,R.O. 1968. Transport of gizzard shad. Prog.Fish-Cult.30:184.
Bandola, S.P. and H.R. Singh. 1984. Spawning of some important coldwater fish of the Garhwal Himalaya. J. Bombay Nat. Hist. Soc. 81:54-58.
Collins, J.L. and A.H. Hulsey. 1964. Reduction of threadfin shad hauling mortality by and use of MS-222 and common salt. Proc. Southeast Assoc. Game Fish Comm. 18:522-524.
Das, S.M. 1990. The mahseer fishes of India: bioecology and the problem of their rapid decline in central Himalaya. in Shaha, N.K., S.D. Batt and R.K. Pandey. (ed.). Himalaya Environment, Resources and Development, Shree Almora Book Depot. Almora, India.
Mauri, T., J.W. Andrews and J.W. Muller. 1979. Effect of valium, MS-222 and sodium chloride on handling mortality. Prog. Fish-Cult. 41:27-29.
Majumdar, C.H. 1940. Spawning grounds and hatcheries in the district Chittagong, Bangladesh, Science and Culture. 5:735-739.
McFarland, W.M. 1960. The use of anesthetics for the handling and the transport of fishes. Calif. Fish Game 46:407-431.
Nautiyal, P. and M.S. Lal. 1984. Natural history of Garhwal mahseer: racial composition. Indian J. Anim. Sci. 58:283-294.
Ogale, S.N. and C.V. Kulkarni. 1987. Breeding of pond-raised hybrids of mahseer, Tor kudree (Sykes) and T. tor (Ham.). J. Bombay Nat. Hist. Soc. 84:332-334.
Patra, R.W.R. and M.A. Azadi. 1984. Collection and hatching of fertilized eggs of major carps. Chittagong Univ. Stud., Vol. 8.
Patra, R.W.R. and M.A. Azadi. 1985. Hydrobiological conditions influencing the spawning of the major carps in the Halada river, Chittagong, Bangladesh. Bangladesh J. Zool. 13:63-72.
Ross, R.M. 1983. Annual, semilunar and diel reproductivity rhythms in Hawaii in Labrid Thalassoma duperry. Mar. Biol. 72:311-318.
Shrestha, T.K. 1985. Induced Spawning of the Himalayan mahseer T. putitora (Hamilton) by pituitary hormone stimulation. Unpublished Report. USAID, Kathmandu.
Shrestha, T.K. 1986. Spawning ecology and behaviour of Himalayan mahseer Tor putitora (Hamilton) in the Himalayan waters of Nepal. pp. 689-692. in Maclean, J.L., L.B. Dizon and L.V. Hoslillos. (ed.). First Asian Forum, Asian Fishery Society, Philippines.
Shrestha, T.K. 1988. Artificial Himalayan mahseer spawning.~Department of Zoology, Tribhuvan University, Kathmandu, Nepal. 25 pp.
Shrestha, T.K. 1990a. Behaviour of Golden Mahseer Tor putitora (Hamilton) in nature and captivity. J. Freshwater Biol. (India) 2:209-219.
Shrestha, T.K. 1990b. Rare fishes of Himalayan waters of Nepal. J. Fish Biol. 37:213-216. Tripathi, Y.R. 1978. Artificial breeding of Tor putitora (Hamilton). J. Inl. Fish. Soc. India 9:161.
Webster, J., A. Trandahl and J. Leonard. 1978. Historical perspective of propagation and management of cold water fishes of the United States. Am. Soc. Spec. Publ. 11:161-166.
<section>Integrated Aquaculture System: Tilapia, Crocodiles, And Rice Culture</section>
R.D. Haller And S. Baer
Baobab Farm Limited
Mombasa, Kenya
Abstract
In its efforts to rehabilitate and utilize a coral limestone quarry excavated for cement production, Baobab Farm has developed an economically self-sustaining ecosystem. One of its main components is an aquaculture system, where tilapia production is integrated with crocodile rearing and rice culture.
The slightly saline water used for tilapia production is pumped into the fish farm, where it is oxygenated in a cascade system. Before entering the circular, concrete production tanks, it is utilized in a double set of raceways. The production capacity of the tilapia farm is 25-30 tons per year, which are marketed locally.
The waste-loaded water from the fish farm is recirculated through a biofilter unit, consisting of a rice field, and a subsequent pond that is densely covered with the floating water plant Pistia stratiotes. Four rice harvests can be achieved per year, with a potential yield of 24 tons per hectare per year. The Pistia plants are removed regularly and used as green manure in fruit and vegetable culture. In order to balance the nutrient supply for the biofilter unit, the nitrogen-rich water from the fish farm is supplemented with phosphate-rich water from the crocodile-rearing pens.
At present, Baobab Farm holds 800 crocodiles of all sizes, which convert waste fish and meat from the farm into valuable skins and meat. The aim is to produce 1,000 crocodiles of marketable size per year. The crocodiles also play their role as a tourist attraction. Tourists contribute significantly to the revenue of the system.
Introduction
The Baobab integrated aquaculture system is located in the exploited coral limestone quarry of the Bamburi Portland Cement Company, 10 km north of Mombasa, on the Kenya coast. It is part of a large-scale land-reclamation project that was initiated by the Cement Company in 1971, and planned and carried out by Baobab Farm Ltd, Mombasa. The project's main aim is to establish a biologically and economically self-sustaining ecosystem.
Most of the 2.5 kmē of the partially to fully excavated quarry area has been planted with the pioneer tree species Casuarina equisetifolia, to serve as a primary vegetation cover and to give a fast financial return. The humus buildup from fallen needles and the improved microclimate allowed other plant and animal species to colonize the disused quarry (Hailer 1974).
An abundance of groundwater led to the excavation of fish ponds and to the development of aquaculture systems. More recently, the reclaimed site has been developed into a major tourist attraction, visited by 75,000 local and foreign tourists per year.
Materials and Methods
Site Description
Coral limestone was excavated as deep down as 50-20 cm above the groundwater level; therefore, water is abundant. The quarry is situated only about 500 m from the shore of the Indian Ocean, and the groundwater body under the quarry floor is connected to the sea. Its level rises and falls with the tides in the sea, the water is slightly saline (2-3 ppt in undisturbed ponds, but up to 8-10 ppt where water is pumped intensively). The undisturbed groundwater has a year-round near-constant temperature of 26°C (+ 1°C) and a pH of 7.5-7.8.
Figure l shows the general layout of the integrated aquaculture system. The fish farm is the central and original part, to which the other parts of the system were connected.
Water Recirculation
The water for the fish farm is pumped from a sump, where the recirculated water from the two biofilters is collected and supplemented with groundwater from a reservoir pond. From 200 to 300 mģ of water are pumped per hour; it is lifted to a height of 4 m and dropped in a cascade into a distribution channel. From there it runs by gravity through the whole aquaculture system back to the pump site.
In the fish farm the water is utilized three times, in two sets of 12 on-growing raceways and a set of circular fattening tanks. Before entering any new step the water is re-aerated in a small cascade.
The fish farm effluents flow into a faecal settlement pond, where heavy waste particles are removed and can be collected for conversion into biogas and fertilizer. The water leaving the settlement pond is diverted and led into either of the two biofilter units.
The original Biofilter 1 consists of a swamp forest, which acts as a second settlement area, and a set of deeper ponds overgrown with the floating water plant, Pistia stratiotes (Nile cabbage). The settlement swamp is a forest of Conocarpus lancifolius, fig trees (mainly Ficus lutea), and some mangrove trees (Rhizophora mucronata, Xylocarpus moluccensis, and Heritiera littoralis), as well as mangrove ferns (Acrostichus aureus). The swamp forest supports a rich bird life and is part of the nature walk through the rehabilitated quarry.
In the subsequent Pistia ponds, the water surface is covered with P. stratiotes. Its feathery roots filter out fine insoluble waste and are also a substrate for numerous microorganisms, which mineralize the trapped organic substance. The released nutrients, together with the dissolved waste from the fish and crocodiles, are partly taken up by the Pistia plants. The large amount of biomass produced by the fast-growing Nile cabbage plants is utilized as mulch in the banana plantation (Beer 1990a).
Three years ago a set of rice fields was installed, which act, together with a subsequent Pistia pond, as a second biofilter.
Part of the nitrogen-rich drainage water from the fish farm flows through the crocodile rearing unit, where it is enriched with dissolved phosphates excreted by the crocodiles before it enters the rice field (Beer l990b).
The rice paddies act mainly as a mechanical filter by sedimentation, whereas in the Pistia pond, fine insoluble waste as well as dissolved waste products are eliminated by the Nile cabbage plants and the associated microorganisms. The channel connecting the rice fields with the Pistia pond is planted with submersed water plants (Elodea sp., Ceratophyllum sp.) to oxygenate the water by photosynthesis during the daytime.
The water recirculated through the two biofilters collects again at the sump. There, water loss through evaporation and seepage (approx. 25 mģ per day) is replaced with groundwater. The total water surface area of the two biofilters at present is approximately 4,000 mē (Hailer 1990a).
The Tilapia Farm
The fish farm started production in 1982 and has been described in detail (Balarin 1983, Balarin and Haller 1983, 1986).
The farm consists of 24 on-growing raceways (10 x 1.5 x 0.5 m in size; volume 7.5 mģ) and 12 circular, self-cleaning fattening tanks (diem. 6 m, depth 0.9 m, volume 25 mģ). The tilapia, mainly Oreochromis niloticus and O. spilurus, are bred in the Baobab arena hatchery. They are brought into the raceway unit at a size of 4-5 g, and are graded several times before they are finally stocked into the fattening tanks at a size of 30-50 g. There they are kept for 120 days, until they reach marketable size (150-350 g).
The fingerlings from the hatchery (Hailer and Parker 1981) are supplemented with fish caught from the surrounding channels and ponds. A special trapping system has been developed to catch fingerlings from an artificial lake where a pair of hippopotamus live. By opening the flood gates to flush the drainage water from the fish farm into the lake, the fish follow their urge to swim upstream toward the water source, and virtually swim into the fish farm, where they are trapped easily.
Two types of food are used to feed the tilapia: (1) commercially available tilapia pellets with a total protein content of about 25%; and (2) farm-manufactured moist food consisting of meat and bone meal, fresh minced fish, wheat pollard, cotton seed cake, and a vitamin-mix.
Every 10 days a tank is harvested and restocked. Growing time in the fattening tanks is 120 days, allowing three production cycles per year for every tank. The tilapia are marketed locally to private buyers as well as to restaurants and hotels. Slow-growing fish and mortalities make up an important part of the food for the crocodiles.
Crocodiles
Crocodiles were introduced originally to fill a gap in the ecosystem by making economic use of animal waste protein from the farm. At present, the farm holds 800 crocodiles, ranging in size from 40 cm to 3.8 m. The crocodile eggs are collected from the breeding pens and incubated at approximately 32 °C in styrofoam boxes in an incubator house. The hatchlings are placed in an indoor brooder tank for the first four weeks until they have hardened. Then they are moved into outside nursery tanks with controlled water temperatures (30-32°C), where the space available per animal is 650 cmē. The crocodiles are graded into groups according to size to avoid food competition. The hatchlings are fed on prawns, tilapia, and red meat. At a length of 70 cm they are placed into ongrowing pens, graded every six months, and then moved on according to their size (no more than 10 cm length variation is allowed in a pen) (Hailer 1988).
All crocodiles are numbered and individual data are collected (total length, head length, belly width, weight). The crocodiles are fed between 2% and 10% (wet weight) of their body weight per day. The food consists of waste fish from the fish farm, and mortalities from Baobab Farm's livestock section (cattle, sheep, goats, poultry). The number of crocodiles held is determined by the amount of animal protein available on the farm.
The farm holds its own captive-bred parent stock in various specially designed breeding lakes and pens. The breeders will produce sufficient eggs to supply the farm with all the hatchlings required for the system (Hailer 1990b).
Rice
The rice paddy attached to the fish farm has an area of 1,200 mē and is divided into three fields. In each field the water flow is guided in a zigzag way by small, vegetable-carrying intermittent dams, in order to utilize the area more efficiently.
The nitrogen-rich effluents from the fish farm, supplemented with phosphate from the crocodiles, flow slowly through the dense stems of rice plants. The waste particles settling between the plant stems build up sediment, which provides nutrients for the rice plants. The sediment also supports the benthic fauna, which itself provides food for predaceous organisms (Beer 1990b).
Four salt-tolerant rice varieties (two wild and two hybrids) were obtained from the International Rice Research Institute (IRRI) in the Philippines. The original seeds were packed in bulk so that the whole paddy area could be planted with the same variety. Best results so far were obtained with Nona Bokra, one of the wild varieties. With proper management, four harvests can be obtained per year. The seeds are sown in a nursery and then after 4-6 weeks, transplanted with a spacing of 25-30 cm, when the plants reach a size of about 30 cm. The time from transplanting to harvest takes about three months.
Results And Discussion
Water Recirculation
Biofilter I, the original water treatment unit through the swamp forest and Pistia-ponds, has been operating without major problems for eight years. Accumulated sediment had to be removed once from one of the Pistia stratiotes ponds to maintain the functioning of the system. Careful monitoring of the waterflow is important to avoid shortcuts of the water through the swamp, which reduces its retention time in the filter and the filtration efficiency. An unexpected problem occurred with an increasing population of catfish (Clarias gariepinus). During times when they have no access to the rice paddies, they mainly feed on the Pistia plants and, periodically, this causes severe setbacks in its growth. We assume that this affects the water quality, as the filtration capacity of a badly damaged Pistia population must be low, and an excess of decaying plant material adds an additional load of organic matter into the filter system. After reducing the feeding pressure of catfish on
the Pistia, the plant's population recovers rapidly.
The rice field biofilter has been functional for about three years and has markedly improved the filtration efficiency of the water recirculation system. However, the excess of nutrients deposited between the rice plants has an adverse effect on the performance of the rice. We suppose that the excessive nitrogen in the substrate is the cause of the lodging problem encountered with all of the four rice varieties. The amount of nitrogen coming out of the fish and crocodile farm could fertilize a much bigger paddy field. Baer (1990b) indicated that if all released nitrogen was channelled into the
rice field and supplemented with other nutrients necessary for optimal rice growth, an area more than 10 times the present size could be maintained and give a potential yield of 39 tons of rice per year.
At present, little is known of the water quality changes during the flow through the recirculation systems. This is due to lack of equipment and an infrastructure for water analysis. The buildup of fish pathogens in the system (e.g., Aeromonas sp.) has been countered by improved management, such as the removal of excessive sludge from the sump and supply channels, as well as increased water aeration.
The Tilapia Farm
The production capacity of the tilapia farm is 25-30 t/yr. Under good management each tank is harvested three times per year, giving a yield of 700-800 kg of fish per tank and harvest.
At present, the tanks and raceways are aerated by gravity only; to install an energy-dependent aeration system is not economically feasible. A promising new way of improving the oxygen supply to the fish farm, i.e., by utilizing the oxygen produced during photosynthesis by planktonic algae, is being investigated. Controlled quantities of algae-rich water from the crocodile ponds are pumped into the fish farm during maximum sunshine hours to increase the oxygen levels in the tanks during peak feeding hours. Due to the short retention time of the water in the fish farm, the algae will be removed before nightfall and thus prevent depletion of oxygen.
A second constraint on the fish farm is the food quality. Any imported food, such as fish meal, is expensive and difficult to obtain, as are food ingredients that compete with human food requirements. The quality of manufactured fish food is unreliable.
Crocodiles
Only recently, Baobab Farm has been registered as a captive breeding operation by the Convention on the International Trade in Endangered Species (CITES), which allows them to trade internationally in crocodile skins and its by-products. The crocodile farm is laid out to incubate 1,2001,500 eggs per year, and to sell 1,000 skins per year. The markets for crocodile skins and meat are still being investigated. Therefore, the crocodile farm is now becoming an important economic part of the aquaculture system.
Baobab Farm provides a considerable amount of waste meat through livestock mortalities and slaughter by-products (approximately 18 t/yr), and the fish farm can supply 6,000 kg of waste fish per year. Therefore, 24,000 kg of waste meat, not suitable for human consumption, are available from the farm to feed to the crocodiles.
Crocodiles kept under optimal temperature (30-32°C), feeding and management condition, attain growth rates of approximately 1.4 mm per day and can produce a marketable skin of 35 cm belly-width within 2.5-3 years. Many of our crocodiles were growing much more slowly in the beginning, as they were not fed intensively (as long as we could not sell skins). Also, the animals displayed as a tourist attraction seemed to be stressed constantly. Even though they feed well, they only attain growth rates of 0.4 mm per day.
No serious disease problems have been encountered.
Rice
The maximum yield attained so far from the 1,200 mē area amounted to 720 kg of rice, giving a potential annual production of 24 t/ha (four harvests).
In addition to the above-mentioned problem of nutrient lodging, another problem in the paddy fields is plant damage and grain losses through birds. Minor outbreaks of rice pests (leafhoppers, rice stem borers) have occurred, but have been kept under control in the small area by biological pest control methods. An invasion by leafhoppers was kept under control by boosting the resident spider population with a variety of local spider species. As no agrochemicals are used in the integrated aquaculture system, nature's own regulation mechanisms are still functional. An increasing stem borer infestation was eventually controlled by an ichneumon wasp whose larvae were found to be parasites of the stem borer larvae.
Conclusion
The complexity of the Baobab Farm ecosystem needs a special management approach (Figure 2). Apart from aiming for good performance in the individual sections, the system as a whole has to be monitored constantly. For any change, its benefit for one section must be weighed against its effects on the other parts of the system. This is one of the reasons why agrochemicals and chemotherapy are not used in the system. Our experience so far shows that the described integrated aquaculture system can function economically and is manageable. Thus, a thorough scientific investigation to solve many still unanswered questions now seems feasible and appropriate.
Acknowledgments
The authors thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
References Cited
Baer, S. 1990a. Nile Cabbage - an extraordinary plant. Habari Ya Baobab, Baobab News 57/11, Mombasa.
Baer, S. 1990b. Rice paddy for water treatment. Habari Ya Baobab, Baobab News 58/1O, Mombasa.
Balarin, J.D. 1983. Intensive tilapia farm developed in East Africa. Fish Farming Int. April 1983. Balarin, J.D. and R.D. Haller. 1983. Commercial tank culture of tilapia, pp. 473-483. in Fishelson, L. and Z. Yaron (ed.). Proc. Int. Symp. on Tilapia in Aquaculture.
Balarin, J.D. and R.D. Haller. 1986. Baobab tilapia farm in Kenya (A Case Study). SADCC Workshop. Kariba, Zimbabwe. 29 September-3 October.
Hahn, C. 1990. Baobab Farm: description of an integrated aquaculture system used as one method to rehabilitate a limestone quarry in Kenya's coastal region. Diploma Thesis, Univ. Kassel, Germany.
Haller, R.D. 1974. Rehabilitation of a limestone quarry. Bamburi Portland Cement Company Publication. 32 pp.
Haller, R.D. 1988. Crocodile and tilapia farming as part of an integrated aquaculture system. (A system description). 9th Working Meeting of the Crocodile Specialist Group, I.U.C.N. LAE, Papua New Guinea 19-21 October.
Haller, R.D. 1990a. Integrated aquaculture. Aquaculture News. Jan. 1990. Institute of Aquaculture. Univ. of Stirling, Scotland.
Haller, R.D. 1990b. Integrating crocodile farming into an existing livestock and tilapia farm. Habari Ya Baobab. Baobab Farm News. 59/12, Mombasa.
Haller, R.D. and I.S. Parker. 1981. New tilapia breeding system tested in Kenya. Fish Farming Int. 8:14-18.
<section>Nutrition</section>
<section>Effect Of Varying Levels Of Sulfate Concentration In Saline Waters On Fish Yield</section>
R.D. Fortes, N.R. Fortes, And I.G. Pahila
Institute of Aquaculture,
University of the Philippines in the Visayas
Iloilo, Philippines
Abstract
Milkfish (Chanos chanos) were stocked in 12 90-lifer aquaria to determine the effect of sulfate on fish yield. Saline waters from various sources with known sulfate levels, and different food sources were used as treatments in first and second runs, respectively. The levels of sulfate (first run) decreased from 1,200, 1,380, 1,700, and 1,100 mg/l to 976.53, 840.53, 772.56, and 441.07 mg/l, respectively, after 24 days; and to 477.00, 490.79, 339.78, and 325.05 mg/l after 28 days. The concentrations of sulfide and ammonia increased (0.01140.0286 mg/l and 0.0105-0.1820 mg/l, respectively). Populations of microorganisms adhering to rice bran particles were highest (450, 570, 850/ml) where sulfate and sulfide concentrations (325.05 mg/l and 0.0192 mg/l) were lowest and ammonia concentration (0.08006 mg/l) was highest.
The levels of sulfate and sulfide were not different among the treatments (range of 919.5-970.5 mg sulfate/l, and 0.02252-0.02542 mg sulfide/l) (second run). The levels of ammonia, however, were highest in the rice bran-sugar (II) and commercial-feed (IV) treatments compared to treatments that received fertilizers (I and III). The populations of microorganisms that adhere to substrates (tiles) were fewer in the rice bran-sugar and commercial-feed treatments than in the treatments that received fertilizers (I and III).
In both runs, the effect of sulfate concentration on fish yield could not yet be established due to low survival (40-67 %), poor growth attributable to poor water quality, low feeding rate, and poor food quality (first run). In the second run, fish survival was very high (100%) except one replicate each in Treatments II (88%) and III (93%). The growth of milkfish in all treatments was low (4-6 % per day), apparently caused by foul water and acidity, which could have resulted from high sulfate concentration in the water. An in-depth study using stable isotope technology is in progress to identify important feeding niches and to intensify the productivity of these niches. Success in this study could lead to a new pond management strategy.
Introduction
This paper is an initial attempt to demonstrate the effect of sulfate concentration in saline water on fish yield with the goal of evaluating the role of sulfate-sulfide reaction and the role of sulfide as a toxin that limits the availability of natural foods in brackish and saltwater earthen ponds. The role of sulfate as a cause of mineral acidity in ponds through its reaction in water has been the object of investigations since the 1960s. There has been considerable research to help alleviate the negative influence of acid sulfate soils on fish and shrimp yields (Singh 1980); however, the problem of sulfide accumulation in the sediment interstices still exists. The effect of this accumulation on crude organic matter, which is the main source of nutrition for the target animals, needs to be properly understood. In milkfish (Chanos chanos) and prawn (Penaeus monodon) farming, traditional or extensive, modified extensive, and semi-intensive methods based on animal density,
added food, and other inputs are in use. These provide the necessary organic matter that could be converted into nutritious food for the fish. However, the users have been unable to differentiate these methods (Fortes et al. 1989, Fortes 1991). Tilapia (Oreochromis niloticus) raised in brackish-water ponds that received feed, either alone or in combination with chicken manure and/or fertilizer, exhibited better growth and higher production (Fortes et al. 1986), but the actual sources of growth are still uncertain. In fresh water, despite the presence of full rations of protein-enriched feed pellets, natural food still accounted for more than half of the growth of the target fish (Schroeder 1983).
The goal of this study is to determine food niches in brackish-water or marine ponds that provide target animals with their nutrition. Armed with this information, a management strategy can be designed that will improve the use of fertilizers as locally available replacements for costly imported feeds. This particular component of the project was implemented (initially using milkfish as the test organism) to pursue the following objectives:
- To determine the effect of sulfate on fish yield;
- To determine the influence of sulfate and sulfide ions on the production of natural food; and
- To determine the influence of sulfate on the population of microorganisms that adhere to the added organic or inorganic material in saline water.
Materials and Methods
Location and Facilities The site is located in Barangay Nabitasan, municipality of Leganes, Iloilo Province, Philippines. The municipality of Leganes is located N 10° 8' longitude; E 122° 5.4' latitude. The laboratory facilities included 12 glass aquaria (H= 35 cm; L= 90 cm; W= 35 cm) provided with an airlift system and a bottom filter made of 10 cm sand on perforated marine plywood (0.635 cm thick). Aeration, water delivery, and lighting systems were also installed.
First Run
Collection and Preparation of Water Water used during the first run of the experiment was collected from three points (Guimaras, SM area, and Gui-gui Creek) along Guimaras Strait approximately 7 km from the laboratory. This was necessary to ensure that pure seawater was collected. Seventy-five 60-1 plastic bags filled with seawater to a 40-1 line (total of three tons of seawater) were transported to the laboratory by means of a 4-ton motor boat. Water from each source was used separately or in combination with water from other sources, including freshwater, and of the four, each treatment was replicated three times, as follows:
Treatment
Initial Sulfate Content (ppm)
Source of Water
I
Guimaras
II
Gui-gui Creek
III
SM + Gui-gui (1:1)
IV
SM + freshwater (1:1)
Samples of water were also sent to the service laboratory of the Natural Science Research Institute of the University of the Philippines at Diliman, Quezon City, for the analysis for cations.
Monoammonium phosphate fertilizer was added before fish stocking to permit microbial growth. Concentrations of sulfate, sulfide, and ammonia in the water were measured before and after stocking and every week thereafter using the methods described by Strickland and Parsons (1972). Salinity, pH, temperature, and dissolved oxygen were monitored every other day using an Atago refractometer, digital pH tester, and a YSI D.O. meter (model 51B), respectively.
Fish Stocking and Feeding Sixteen milkfish fingerlings (average weights: 1.27 g, 1.26 g, 1.23 g, and 1.45 g, for Treatments I, II, III, and IV, respectively) were stocked in each of the 12 units of 90-1 aquaria (equivalent to 1 fish/6 1). The fish were fed rice bran mixed with 1% refined cane sugar given at 5% of fish biomass daily and adjusted to 10% after the first sampling. The fish were raised in these aquaria for 28 days and sampled at the midpoint and at the end.
Sulfate, Ammonia, and Microbe Populations Finely ground rice bran was mixed with refined sugar at a ratio of 100:1 (rice bran: refined sugar). Two grams of the mixture were added into a 2-1 plastic jar filled with seawater collected from different sources with known levels of sulfate concentration. The mixtures were analyzed for nitrogen after 1, 2, 12, and 24 hours; and after 7, 14, and 30 days of contact with water. Nitrogen content of the water was determined by the use of a micro-kjeldahl apparatus, which converts the nitrogen from small samples into ammonia, which is then measured volumetrically. The changes in the nitrogen content in the feed substrate during each time of exposure was taken to represent the microbial processing that could take place if rice bran were not immediately consumed by the fish and remained in the water or sediments to serve as food substrate. The water was observed for visual changes, especially the occurrence of detritus in the container.
Water samples and detrital material or organic residues were taken and examined under the microscope. Organisms were counted using a hemacytometer.
Second Run
Due to the difficulty of maintaining the desired level of sulfate concentration in water, the treatments were changed. Instead of using the levels of sulfate concentrations as treatments, different sources of fish food were used as treatments, then the levels of sulfate concentrations in water were monitored. This new experimental design is consistent with the tank experiments of our collaborators in Israel and a tilapia experiment in aquaria. The new treatments with the initial weights of the fish are as follows:
Treatment
Initial Weight (g)
I
Rice bran + cane sugar + fertilizer
II
Rice bran + cane sugar
III
Natural food + fertilizer
IV
Commercial pelleted feed
Each treatment had three replicates (aquaria) that were stocked with 15 milkfish fingerlings each. The rate of feeding for the three different food sources was 10% of the fish biomass given every day. Natural food in the form of a benthic mat composed of lower forms of microflora and microfauna (lab-lab) and filamentous algae were added to the aquaria. The natural food was given at the rate of 20% of fish biomass and was later increased to 40%.
A total of 16 tiles (equivalent to 16 samplings) were scattered over the bottom of each aquarium. One tile was then randomly picked every sampling and samples of the microorganisms were taken by scraping off the brownish-whitish substance adhering to the tiles. The scrapings were then weighed. The wet weight of the organisms was estimated using the following equation:
OW = WTBS--WTAS
where:
OW - weight of organisms (g)
WTBS - weight (g) of tiles before scraping
WTAS - weight (g) of tiles after scraping
The samples were then fixed in formalin and the organisms were enumerated and identified. Population counts of minute organisms were made using a hemacytometer; for larger ones, the Sedgewick rafter counting chamber and cell were used.
Results and Discussion
First Run
The initial sulfate concentrations of seawater from the different sources were 1,200 mg/l, 1,380 mg/l, 1,700 mg/l, and 1,100 mg/l for Treatments I, II, III, and IV, respectively. These were
TABLE 1 Sulfate Concentration of the Different Treatment used in Aquarium Experiment No. l and No. 2
Treatment
Sulfate (ppm)
Salinity (ppt)
I
II
III
IV
significantly lower (P < 0.05) than the theoretical concentration of seawater of 2,657 mg/l (Church 1975). These initial concentrations decreased to 976.98 mg/l, 840.53 mg/l, 772.56 mg/l, and 441.07 mg/l after 24 days of storage, and were the initial sulfate concentrations of the various treatments at the start of the experiment (Table 1). They decreased further to 477.00 mg/l, 490.79 mg/l, 339.778 mg/l, and 325.05 mg/l, respectively, 11 days after the fish were stocked. However, an increase was observed a few days before the experiment was terminated.
The values of different water parameters that were monitored (sulfate, sulfide, ammonia, pH, DO, temperature, and salinity) are discussed as follows and are given in Table 2.
TABLE 2 Physicochemical Properties of Water and Sediment (Milkfish Aquarium Experiment -- First Run)
Sulfate (mg/l)
Sulfide (ml/l)
A. Treatment:
Dec. 27
Jan. 7
Jan. 10
Jan. 17
Jan. 7
Jan. 10
Jan. 17
Jan. 24
I
II
III
IV
Ammonia (mg/l)
Dissolved Oxygen (mg/l)
B. Treatment:
Dec. 27
Jan. 7
Jan. 10
Jan. 17
Jan. 24
Jan. 7
Jan. 10
Jan. 16
Jan. 24
I
II
III
IV
Salinity (ppt.)
pH
C. Treatment:
Jan. 7
Jan. 10
Jan. 16
Jan. 24
Jan. 7
Jan. 10
Jan. 16
Jan. 24
I
II
III
IV
Temperature (C°)
Organic Matter (%)
Soil Sulfate (mg/l)
D. Treatment:
Jan. 7
Jan. 10
Jan. 16
Jan. 24
Dec. 27
Jan. 28
Dec.27
Jan.28
I
II
III
IV
Sulfate An analysis of variance showed significant differences (P < 0.01) in sulfate concentrations in water among the treatments. The sulfate concentration in Treatment IV (mixture of seawater and rainwater) was significantly lower (P < 0.01) than those of Treatments I, II, and III. The significant reduction in the sulfate concentration could be attributed to the anoxic condition at the bottom as detected in the decreasing dissolved oxygen level of the water (Table 2). Sulfate was found to be positively correlated with dissolved oxygen and salinity (P < 0.01). As the dissolved oxygen level
increased, sulfate concentrations subsequently increased in all treatments. Sulfate concentrations were relatively higher in water with higher salinity levels. This observation is corroborated by the fact that seawater with higher salinity levels actually contains higher sulfate ions than freshwater (Church 1975). A significant correlation (P < 0.01) was also found between the concentration of sulfate and ammonia. The presence of small amounts of ammonia in the sediment could have been contributed by the accumulated unconsumed feed (rice bran), which contained 5.7%-10.9% crude protein.
Initially, the washed and dried sand bottom in all the treatments were free of sulfate, but after harvest, significant amounts of sulfate (3,337.0 mg/l-3,983.71 mall) (Table 2) were recorded from the sand bottom. The sulfate concentration of the sediment was higher than the sulfate concentration found in the water. Statistical analysis showed that sulfate in the sediment is highly correlated (P < 0.01) with sediment organic matter. It was also possible that sulfur contained in protein (e.g., methionine) from the accumulated unconsumed food could also have contributed, to some degree, to the level of sulfate in the sediment.
Sulfide Sulfide contents of waters collected from the different treatments were significantly different (P < 0.01) among each other. Hydrogen sulfide was relatively higher during the latter part of the experiment and may have been contributed by decomposing unconsumed food and enhanced by the breakdown of the aeration system. This phenomenon was highly possible because hydrogen sulfide is formed by heterotrophic bacterial metabolism; thus, unionized hydrogen sulfide usually does not occur in well-oxygenated water (Chiu 1988). Sulfide was also found to be positively correlated with salinity, (P < 0.01), pH (P < 0.05), and temperature (P < 0.01).
Ammonia Ammonia concentrations were relatively high three days after stocking. This could be attributed partly to inadequate aeration, as evidenced by the low dissolved oxygen (Table 2). From the sixth day onward, ammonia concentrations decreased, especially after water exchange, but an increase was observed toward the end of the experiment. Again, unconsumed food was noted on the bottom of the aquaria. Correlation analysis showed that ammonia was negatively correlated with temperature (P < 0.01) and dissolved oxygen (P < 0.05), which indicated that ammonia accumulates in the water column in lower dissolved oxygen and temperature.
TABLE 3 Changes in the Total Count of Organisms in Different Treatments (Water Sources) with Rice Bran
Treatment
2 Days
7 Days
14 Days
I (Guimaras)
II (Gui-gui)
III (SM + Gui-gui)
IV (SM + freshwater)
Sediment Organic Matter Initially, the sand bottom had practically no organic matter. After 28 days, an appreciable amount of organic matter was recorded from the samples taken from the sand bottom (0.81%, 0.97%, 0.85%, and 0.85% in Treatments I, II, III, and IV, respectively) (Table 2). This indicated that organic matter was formed from the different inputs, particularly the rice bran, which, even when put in at a relatively low rate, was not completely utilized by the fish.
Sulfate Concentration and Microbe Populations A whitish film developed on the water and spread across the surface after six days. This film was composed mostly of round and filamentous bacteria and protozoans. The total counts of these microorganisms on the second day were 4,210,000, 1,160,000, 6,650,000, and 5,343,000 for Treatments I, II, III, and IV, respectively. These populations continued to increase until day 7 and finally decreased on day 14 (Table 3). There were indications that the populations of microorganisms were higher in the treatments with lower sulfate concentration (Table 4).
TABLE 4 Population of Organisms (org/ml x 10ģ) in Seawater with Different Sulfate Concentrations
January
Treatment
Number of Organisms
I
(Guimaras)
Mean
II
(Gui-gui)
Mean
III
(SM + Gui-gui)
Mean
IV
(SM + fresh water)
Mean
Obviously, the particles of rice bran harbored microorganisms and became food substrates. An organic fraction of the food was apparently converted into an assemblage of microorganisms that could serve as fish food. Schroeder (1978) and Hobbie and Lee (1980) pointed out that the relative contribution of supplied foods and fertilizers to the growth of the fish is attributable to the sunlit pond ecosystem in which minerals and organic fractions of the food and fertilizers are converted into a complex of algae, bacteria, protozoans, and their mucopolysaccharide exudates, which can be used as food for fish growth.
Changes in the nitrogen content of water from the first hour to day 30 are shown in Table 5. A build-up of nitrogen in all treatments was observed between days 2 and 7, and then decreased tremendously between days 14 and 30. Fluctuations in the number of microorganisms followed the increase and decrease of the nitrogen content of the water. Increases in the nitrogen content could be due to the organisms that adhere to the food substrate and enrich the protein source of the substrate within the one-week period of time (observed in this run). After 14 days, however, the nitrogen content abruptly decreased, probably due to the observed decrease in the microorganisms in the water.
TABLE 5 Changes in the Nitrogen Content (mg/l) of Seawater with Varying Sulfate Concentration with Rice Bran as the Organic Residue
Exposure Time
Treatment I
Treatment II
Treatment III
Treatment IV
0 hour
1 hour
2 hours
12 hours
24 hours
7 days
14 days
30 days
Survival and Fish Yield The mean survival of milkfish on a per treatment basis ranged from 40% to 67%, although a general decrease in the average body weight was observed in all treatments (Table 6). An inverse relationship was recorded between the average body weight (ABW) of milkfish at harvest and the levels of sulfate concentration in water. The following are the means of sulfate concentrations and the average body weights of milkfish.
Treatment
ABW(g)
Sulfate (mg/l)
I
II
III
IV
Although there was a slight negative effect of sulfate on the growth and yield of milkfish, other parameters also could have affected the fish. Salinity, pH, and temperature were all within tolerable limits, but the dissolved oxygen contents were all in the lower range of tolerance.
TABLE 6 Sampling Weights of Milkfish Raised in Seawater in Aquaria for 28 Days
Treatment
Weights in Grams Sampling
Survival
Initial
1st
2nd
Final (%)
I
Mean
II
Mean
III
Mean
IV
Mean
Number of milkfish stocked in each aquarium -- 16.
Second Run
The different feeding treatments started on April 10, 1991. Based on our data, the fish in Treatment IV (commercial feed) were observed to have a significantly higher growth rate than the fish in other treatments. Statistical analyses were run on the water and sediment parameters to determine treatment differences, and are summarized below.
Sulfate An analysis of variance showed that the amounts of sulfate in the water of each treatment were significantly different. Treatment IV, which received commercial feed, had the highest range of sulfate concentrations (787.96-1155.99 mg/l) (Table 7). The average sulfate concentrations showed lower sulfate, over time, in the treatments that received rice bran (Treatments I and II). Fluctuation in the sulfate concentrations could be due to the water change and other factors that affect the sulfate
TABLE 7 Physiochemical Properties of Water and Sediment (Milkfish Aquarium Experiment -Second Run)
Sulfide (ml/l)
Treatment
April 10
April 17
April 24
April 29
May 9
I
II
III
IV
Sulfate (mg/l)
Treatment
April 10
April 17
April 24
April 29
May 9
I
II
III
IV
Ammonia (mg/l)
Treatment
April 10
April 17
April 24
April 29
May 9
I
II
III
IV
Phosphorus (mg/l)
Treatment
April24
April 29
May 9
I
II
III
IV
Soil Sulfate (mg/l)
Soil pH
Organic Matter(%)
P(mg/l)
N (%)
Treatment
Feb. 1
May 10
May 10
Feb. 1
May 10
Ma 10
May 10
I
II
III
IV
* Significant based on SYSTATS
levels in water. Correlation analysis showed that sulfate in water is negatively correlated to
phosphorus and temperature (P< 0.01). Also, a slight correlation was observed with soil organic
matter (P < 0.10). However, sulfate levels in soil were not significantly different (P > 0.05) among treatments, when based on analyses made before stocking and after harvest.
Sulfide An analysis of variance showed no differences among the treatments in terms of sulfide concentrations in water. Mean concentration of sulfides ranged from 0.0225 mg/l to 0.0254 mg/l. Sulfide concentrations, in general, tended to increase in time in all of the treatments (Table 7). This could be due to the accumulation of decomposing organic material from unconsumed food on the bottom and an increase in the metabolic wastes of the fish during the latter part of the experiment. Sulfides were found to be highly correlated (P < 0.01) with ammonia, pH, soil organic matter, soil phosphorus, and soil nitrogen.
Ammonia No differences in ammonia concentrations were found among the treatments. The concentration of ammonia in water followed a fluctuating trend from one sampling period to another (Table 7), wherein ammonia was seemingly influenced by certain parameters such as dissolved oxygen, temperature, and organic or nitrogen input. An analysis for this run showed significant correlations of ammonia with dissolved oxygen (P < 0.05), soil nitrogen (P < 0.01), and sulfide (P < 0.01).
Sediment Organic Matter The amounts of organic matter in the sediment increased a*er each experimental run (Table 7), obviously because of the unconsumed food that accumulated as a white precipitate on the sand. The organic matter contents of the sediment ranged from 0.69% to 1.31% (Table 7). Treatment I was supplied with rice bran mixed with 1% cane sugar plus fertilizer, while Treatment III had no food supplement except fertilizer. It seems evident that the unconsumed food contributed significantly to the organic matter in the sediment. Correlation analysis between organic matter and the different parameters showed significant relationship (P < 0.05) with sulfide, soil and water phosphorus, and soil nitrogen. It is a fact that upon decomposition, organic matter will release sulfide and phosphorus as well as inorganic nitrogen.
Phosphorus An analysis of variance for water phosphorus showed significant differences among treatments. The highest mean phosphorus concentration in the water was found in Treatment I (1.59 mg/l P) and the lowest in Treatment IV (1.19 mg/l P), which was not significantly different from Treatment III (Table 7). Phosphorus in water was found to be correlated with sulfate, water pH, sediment organic matter, and soil phosphorus. Sulfates could affect water pH, which in turn determines the solubility of phosphorus in water. Thus, the amounts of organic matter and soil phosphorus are directly related to soluble phosphorus in water.
Sulfate and Microbe Population The total population counts for the microorganisms, mostly protozoans and bacteria, are significant. Highest population counts were obtained in Treatment IV (commercial feed) (Table 7) where the sulfate concentration of seawater was recorded as 523.94 mg/l. Total population counts of organisms were significantly different from each other (P < 0.01). Based on the mean of sulfate concentration (accumulated values in time), total counts of organisms were greater in the treatments with lower sulfate concentrations. There is an indication that the sulfate-sulfide concentrations in seawater negatively affected the total populations of the microorganisms. This is likely, because of the negative effect of acidity on the organisms resulting from high sulfate concentrations in water. The other measured parameters, ammonia (P < 0.05), salinity (P > 0.10), pH (P > 0.10), dissolved oxygen (P > 0.10), and organic matter did not affect the total population.
The density of the microorganisms in terms of the total population counts for the two sampling periods in the aquaria are shown in Table 8. Mean counts show the highest density (26,610,000) in Treatment III (natural food), followed by Treatment IV (commercial feed) and Treatment I (rice bran + refined sugar + fertilizer). In terms of population counts, the treatments were not significantly different from each other, but numerically the treatments that received fertilizers exhibited more microorganisms. Average population counts of 313,000 organisms/1 and 266,150 organisms/1 were recorded from Treatments I and III, respectively, compared to 133,400 organisms/1 and 191,250 organisms/l, respectively, in Treatments II and IV. The positive effect of fertilizer on the development of organisms is consistent with the findings of Hepher (1962) that the primary productivity of chemically fertilized ponds is about 4-5 times greater than ponds that do not receive fertilizer. In terms of
the weight of microorganisms that adhered to the tiles, Treatment I produced significantly greater biomass than the rest of the treatments, possibly due to microbial organisms associated with the organic matter (rice bran). Schroeder (1978) reported that large increases in sediment-related microbial protein are regularly associated with the deposition of organic matter. An aerobic environment rich in coarse organic matter can produce large communities of bacteria and protozoans in small straw-like particles that serve as substrate for microbial growth (Schroeder 1978).
TABLE 8 Total Population Count (org./1 x 104) and Weight (g) of Microorganisms on Tiles (Milkfish Aquarium Experiment Second Run)
April 9
April 26
Treatment/Replicate
Population Count
Weight
Population Count
Weight
Treatment I
Mean
Treatment II
Mean
Treatment III
Mean
Treatment IV
Mean
Fish Survival and Yield The survival of milkfish in all treatments was very high (100%) except in one replicate each of Treatments II (83%) and III (93%) (Table 9). Fish in all treatments registered
a daily growth rate of 4%, 4%, 4%, and 6% for Treatments I, II, III, and IV, respectively. Such growth rates were very low, but they provide information about the indirect effect of sulfate on the growth and yield of fish under the conditions of the experiment. The expectation that Treatment IV would have the highest yield was realized. This is mainly because this treatment used commercially formulated food given at 10% of total fish biomass. The highest yield, however, coincided with the highest level of sulfate, lowest level of sulfide, and a higher level of ammonia (Table 7).
TABLE 9 Sampling Weights of Milkfish Raised in Seawater in Aquaria for 30 Days
Treatment
Weights in Grams Sampling
Survival
Initial
1st
Final
I
Mean
II
Mean
III
Mean
IV
Mean
Treatments: I - Rice bran + cane sugar + fertilizer
II - Rice bran + cane sugar
III - Natural food + fertilizer
IV - Commercial feed
Conclusion
A negative influence of sulfate on milkfish yield and on the production of natural food (phytoplankton and other algal forms, bacteria, and protozoa) in seawater was found in this preliminary study. The duration of the experiment was not sufficient to draw concrete conclusions; however, a trend can be gleaned from the results. This experiment needs to be replicated several times to obtain consistent results.
Several experiments addressing the same objectives and overall goals of the project are in
progress. These are being done in glass aquaria, concrete tanks, and brackish-water earthen ponds.
Identification of important feeding niches with the use of stable isotope technology is also in progress.
The results of these new studies should strengthen the findings of this study.
Acknowledgments
The authors thank the Office of Research, USAID, for funding grant number DHR-5544-G-SS
9068-00, the network meeting, and publication of this paper.
References Cited
Chiu, Y.N. 1988. Water quality management for intensive prawn ponds. pp. 79-92. in Chiu, Y.N., L.M. Santos and R.O. Juliano. (ed.). Technical considerations for the management and operation of intensive prawn farms. Univ. Phil. Aquac. Soc. Coll. Fish., Univ. Phil., Iloilo City, Philippines. 172 pp.
Church, T.M. (ed.). 1975. Marine chemistry in the coastal environment. ACS Symposium Series 18. Amer. Chem. Soc., Washington, DC, U.S.A. 710 pp.
Fortes, R.D. 1991. Utilization of selected prawn farming technologies in Western Visayas. Paper presented during the Regional Symposium on R & D Highlights, WESVARRDEC-Central Phil. Univ., May 28, Iliolo City, Philippines. 11 pp.
Fortes, R.D., V.L. Corre, and E. Pudadera. 1986. Effects of fertilizers and feeds as nutrient sources on Oreochromis niloticus production in Philippine brackishwater ponds. pp. 121-124. in Maclean, J.L., L.B. Dizon, and L.V. Hosillos. (ed.). The First Asian Fisheries Forum. Asian Fish. Soc., Manila, Philippines.
Fortes, R.D., B. Posados, R. Cajilig, L. Baylon, E. Pudadera, and I. Belleza. 1989. Technology assessment of prawn, milkfish and mussels in Western Visayas. Terminal Report.
Brackishwater Aquac. Center, Coll. Fish., Univ. Phil. Visayas, Leganes, Iloilo, Philippines. 128 pp.
Hepher, B. 1962. Primary production in fishponds and its application to fertilization experiments. Limnol. Oceanogr. 7:131-136.
Hobbie, J. and C. Lee. 1980. Microbial production of extracellular material: importance in benthic ecology. in Tenore, K. and B. Coull. (ed.). Marine Dynamics. Bell Baruch Symp. on Benthic Ecology. Carolina Press, U.S.A.
Schroeder, G. 1978. Autotrophic and heterotrophic production of microorganisms in intensely manured fishponds and related fish yields. Aquaculture 14:303-325.
Schroeder, G. 1983. Natural food web contributions to fish growth in manured ponds. J. World Mariculture Soc. 14:505-509.
Singh, V.P. 1980. Management of fishponds with acid-sulphate soils. Asian Aquacult. 3:4-6. (SEAFDEC Aquac. Dept. Tigbauan, Iloilo, Philippines).
Strickland, J.D.H. and T.R. Parsons. 1972. A practical handbookfor seawater analysis. Fish. Res. Board Can., Ottawa.
<section>Feeding Value Of Fresh Perennial Leguminous Shrub Leaves To Nile Tilapia (Oreochromis Niloticus L.)</section>
M.A.G. Castanares,đ D.C. Litile,đ A. Yakupitiyage,đ
P. Edwards,đ And L.L. Lovshinē
đDivision of Agricultural and Food Engineering, Asian Institute of
Technology
Bangkok, Ihailand
ēDepartment of Fisheries and Allied Aquaculture, Auburn University
Auburn, Alabama
Abstract
The feeding value of fresh pigeon pea (Cajanus cajan), leucaena (Leucaena leucocephala), gliricidia (Gliricidia septum), and sesbania (Sesbania grandifloraJ leaves to Nile tilapia (Oreochromis niloticus) was evaluated in an outdoor recirculating water system comprising 15 5 mģ concrete tanks for 56 days. Commercial catfish pelleted feed was used as a control diet. The protein content of all green fodder was within the limits of the required dietary protein level for Nile tilapia. Average green fodder intake of fish fed ad libitum ranged from 0.2 to 0.4% body weight/day, but fish lost weight during the experiment. Results of this experiment indicate that there is no value in feeding the leguminous fodders tested here to fish.
Introduction
Legume fodder is considered cheap nutritious feed for terrestrial farm animals (NRC 1984). Pigeon pea, gliricidia, leucaena, and sesbania are four common perennial forage legumes that are often used by Asian farmers as animal feed or as green manure for conventional crops. Although some perennial legume fodders have been found to have deleterious effect to farm animals, gliricidia, leucaena, and sesbania are frequently used as ruminant feed in the tropics (Williamson and Payne 1978). Pigeon pea, which is also a food crop for humans, is usually cut 50-75 cm from the ground for forage when the pods begin to ripen (Bogdan 1977).
The nutritive value of these fodders as nonconventional feed for livestock suggests that they are a potential source of herbivorous fish feed for small-scale fish farmers. There have been several studies, especially on the inclusion of leucaena leaf meal in the diet of finfish (Jackson et al. 1982, Ferraris et al. 1986, Wee and Wang 1987a) and Penaeus monodon (Vogt et al. 1986, Penaflorida 1989). However, there is no information on the nutritive value of the fresh leaves of these plants as the major nutritional source for fish.
The present study was, therefore, designed to evaluate the potential use of these fresh legume leaves as the main dietary source for Nile tilapia. This species is primarily a microphagous feeder (Colman and Edwards 1985), but it is also known to ingest plant leaves.
Materials and Methods
This study was conducted at the Asian Institute of Technology (AIT), Bangkok, Thailand, from March 28 to May 23, 1991. A completely randomized design (CRD) with five treatments, in triplicate, was utilized. Treatments consisted of fish fed the four different legumes and a control diet. The feeding trial was conducted in a recirculating system that consisted of 15 outdoor concrete tanks (2 x 2 x 1.2 m) and a biofilter. A flow rate of recirculating water was maintained to provide sufficient oxygen and to circulate the total volume of water through the biofilter unit at least once a day. A rotating octahedron plastic net cage was placed in each tank. The cage had a frame of painted steel and wood and the sides of the cage were covered with a black plastic net (2 cm mesh size). Each cage unit was rotated daily to expose the submerged half to the air to minimize fouling. The experimental cages were stocked with sex-reversed male Nile tilapia (Oreochromis niloticus) with an
initial weight of approximately 25 g, at a density of 15 fish per cage.
Pigeon pea, gliricidia, leucaena, and sesbania were cultivated on the AIT campus. Fresh leaves of the four legumes were collected daily from the field and a known weight of fodder was presented to the fish once a day to feed to satiation. A bundle of fodder was tied to a floating PVC frame (0.5 x 0.5 m) in each cage at 0900 hr and the uneaten materials were collected 24 hr after. The dry matter content of offered and uneaten fodder from each tank was determined for two successive days, at biweekly intervals, to obtain the average daily dry matter intake by the fish. A commercial catfish pellet (Charoen Pokphand Company Ltd., Thailand) was the control diet, fed at 3% body weight per day.
Experimental fish were sampled biweekly to record their survival and batch weight, and the feed ration of commercial pellets was adjusted accordingly. All fish were counted and weighed individually at the end of the experiment. Samples of initial and final experimental fish were sacrificed and dried in an oven at 100°C for 24 hours. Green fodder, commercial catfish pellets, and fish carcasses were analyzed for moisture, crude protein, crude lipid, and ash. The crude fiber content of experimental feeds was also determined (AOAC 1975).
Results
The average approximate compositions of the four green fodder types and the commercial pellets are presented in Table 1. The mean body weights of the experimental fish during the feeding trial are shown in Figure 1. The mean final body weights of the fish fed the four legumes decreased compared to their initial mean body weights (Table 2). However, the fish fed commercial pellets grew from approximately 24 g to 93 g during the feeding trial.
More than half (approximately 60%) of the experimental fish fed leucaena fodder developed eye cataracts. Gross morphological disorders were not observed in the other treatments.
TABLE 1 Average Proximate Composition of Green Fodder and Commercial Pelleted Feed
Percent, Dry Matter Basis
Feed
Dry Matter
Crude Protein
Crude Lipid
Crude Fiber
Ash
Nitrogen Free Extract
Pigeon pea
Gliricidia
Leucaena
Sesbania
Pellet
The dry matter intake of fish fed the experimental fodders was markedly low throughout the experimental period Cl able 3). The body lipid contents of experimental fish fed fodder were extremely low compared to their initial body lipid contents and that of the fish fed the control diet (Table 4).
Discussion
Chemical analysis of the tested fodders indicated that their protein contents were within acceptable limits for Nile tilapia (Tacon 1987). Crude protein contents ranged from 24.2 to 32.5% on a dry matter basis, with pigeon pea the lowest and leucaena the highest. In contrast, the crude lipid contents were relatively low. The crude fiber contents of the fodders was relatively high, ranging from 12.1% for sesbania to 19.4% for pigeon pea. The low lipid and high fiber content of these plants may pose a problem for their use as fish feed.
There was a reduction in the body weight of fodder-fed tilapia. Low feed intake indicated the unpalatability of the tested fodders since the highest estimated total dry matter consumption among the fodders was only 5.5 g for gliricidia-fed fish in 56 days. The corresponding protein intake of fish fed gliricidia was therefore only 1.5 g, which may not be sufficient to maintain the nitrogen balance of the fish. However, fish fed commercial pellets containing 36% crude protein at 3% body weight/day grew by 75.1 g within 56 days. This resulted from 27 g of protein ingested over the same period.
It can be concluded, therefore, that fish growth was hindered by poor fodder intake by the fish. Direct feeding on these fodders could not supply sufficient nutrition for normal growth of Nile tilapia. The relatively low body lipid content of the fodder-fed fish reflects the utilization of stored body fat as an energy source for metabolism during the experimental period, which resulted in weight loss.
The observed low dry matter intake of fodder-fed fish may be attributed to the presence of potential anti-nutritional factors in leguminous fodder. However, a gross morphological disorder resulting from an anti-nutritional factor was only detected among leucaena-fed fish, i.e., eye cataracts.
TABLE 2 Growth Performance of Nile Tilapia Fed with Leaves of Four Legume Species and Commercial Catfish Pelleted Feed for 56 Days + 1 Standard Error)
Initial Weight
Final Weight
Weight Gain
Relative Growth
Percent
Feed
(g)
(g)
(g)
Rate (mg/g/day)
Mortality
Pigeon pea
Gliricidia
Leucaena
Sesbania
Pellet
No feed
Sallmann et al. (1959) reported that a reduction of the mitotic index in lens epithelial cells of rats fed leucaena indicated a mimosine induced eye cataract. However, inclusion of leucaena leaf meal (LLM) in the diet of Nile tilapia, with or without pretreatment by soaking, did not cause eye cataracts in previous nutritional studies, although there was growth retardation with an increased inclusion of LLM in the ration (Wee and Wang 1987a, Santiago et al. 1988). The results of the present experiment indicate that it may be necessary to pretreat fresh leucaena leaves to reduce the detrimental effect of mimosine to Nile tilapia. Pretreatment of leucaena fresh leaves by soaking at room temperature for 48 hr has been reported to reduce the mimosine content (Wee and Wang 1987b).
Recirculated water in the tanks had a brown color, probably due to the diffusion of tannins and other pigments from the leaves. Krishnamurthy et al. (1972) and Rao and Mariappan (1972) reported that tannin as low as 6.5 mg/l is lethal to freshwater fish, and that 320 mg/l of tannin was toxic to Catla catla, respectively. However, direct toxicity by water soluble tannins could not be a major influencing factor in the present study because water was recirculated through all five treatments, including the control treatment wherein fish grew at 1.3 g/day. Further research on tannin toxicity is required. It may be possible to reduce the water soluble fraction of tannin in a pretreatment by submerging the fodder in water for a longer duration.
In addition to tannins, pigeon pea fodder was possibly deleterious to fish due to the presence of other toxic factors. Pigeon pea leaves, like the pods, may also contain haemaglutinins, protease inhibitors, cyanogen, and physic acid, which depresses appetite (IDRC/ICAR 1988).
TABLE 3 Feed Utilization Indexes of Nile Tilapia Fed with Four Legume Leaves and Commercial
Catfish Pellet for 56 Days
Feed
Average Total Dry Matter (g)
Average Feeding Consumed Per Fish (% Body Weight) per Day
Pigeon pea
Gliricidia
Leucaena
Sesbania
Pellet
The results of the present experiment suggest that it is not possible to use the four tested leguminous fodders as direct feed for Nile tilapia. If toxic components were the major factors affecting low dry matter intake, it is highly unlikely that other more voracious macrophagous fish such as grass carp (Ctenopharyngodon idella), giant gourami (Osphronemus goramy), and silver barb (Puntius gonionotus) would be able to utilize these fodders efficiently. It is known that non-ruminants have a lower toxicity tolerance to mimosine and other toxic factors than ruminants (IDRC/ICAR 1988).
TABLE 4 Initial and Final Carcass Composition of Experimental Fish
Percent
Percent, Dry Matter Basis
Treatment
Dry Matter
Crude Protein
Crude Lipid
Ash
Initial Fish Sample
Pigeon pea
Gliricidia
Leucaena
Sesbania
Pellet
No feeding
However, before generalizations are made, further research should be conducted to verify the
feeding value of these fodders for other herbivorous species. Identification of the anti-nutritional
factors and the development of a cheap pretreatment for these fodder may be necessary to improve their potential as fish feed. As an alternative, however, the relatively high nitrogen content of legume leaves suggests that they may have potential as green manure and serve as a source of nitrogen for fertilizing fish ponds.
Acknowledgments
The authors thank the Office of Research, USAID, for funding grant number 493-5600-9-44
0075-00, the network meeting, and the publication of this paper. Peter Edwards and David Little are
seconded to AIT by the Overseas Development Administration (ODA), London.
References Cited
Association of Official Analytical Chemists (AOAC). 1975. Official Methods and Analysis. 12th Edition. Washington, D.C.
Bogdan, A.V. 1977. Tropical Pasture and Fodder Plants (Grasses and Legumes). Tropical Agriculture Series. Longman Inc., New York, U.S.A.
Colman, J.A. and P. Edwards. 1985. Feeding pathways and environmental constraints in waste-fed aquaculture: balance and optimization. Paper presented at the Bellagio Symposium on Detritus and Aquaculture. August 1985. Bellagio, Italy. 42 pp.
Ferraris, R.P., M.R. Catacutan, R.L. Mabelin, and A.P. Jazul. 1986. Digestibility in milkfish, Chanos chanos (Forsskal): effects of protein source, fish size and salinity. Aquaculture 59:93-105.
International Development Research Centre/Indian Council of Agricultural Research (IDRC/ICAR). 1988. Non-conventional Feed Resources and Fibrous Agricultural Residues. Strategies for expanded utilization. Devendra, C. (ed.). Proceedings of a consultation. 21-29 March 1988. Hissar, India.
Jackson, A.J., B.S. Capper, and A.J. Matty. 1982. Evaluation of some plant proteins in complete diets for the tilapia Sarotherodon mosambicus. Aquaculture 27:97-109.
Krishnamurthy, V.S., C.A. Sastry, and R. Bhaskaran. (ed.). 1972. Treatment and disposal of tannery and slaughterhouse waste. CLRI, Madras, India.
National Research Council (NRC). 1984. Leucaena: Promising Forage and Tree Crops for the Tropics. Second Edition. National Academy Press, Washington, D.C.
Penaflorida, V.D. 1989. An evaluation of indigenous protein sources as potential component in the diet formulation for firer prawn, Penaeus monodon, using EAAI. Aquaculture 83:319-330.
Rao, A.V.S.P. and M. Mariappan. 1972. Toxicity of tannery waste and their components to fish. in Krishnamurthy, V.S., C.A. Sastry and R. Bhaskaran. (ed.). Treatment and disposal of tannery and slaughterhouse waste. CLRI, Madras, India.
Sallmann, L.V., P. Grimes, and E. Collins. 1959. Mimosine cataract. Am. J. Ophthalmol. 47:107117.
Santiago, C.B., M.B. Aldaba, M.A. Laron, and O.S. Reyes. 1988. Reproductive performance and growth of Nile tilapia (Oreochromis niloticus) broodstock fed diets containing Leucaena leucocephala leaf meal. Aquaculture 70:53-61.
Tacon, G.J. 1987. The Nutrition and Feeding of Finned Fish and Shrimp. A Training Manual. FAO, Brazil.
Vogt, G., E.T. Quinitio, and F.P. Pascual. 1986. Leucaena leucocephala leaves in formulated feed for Penaeus monodon: a concrete example of the application of histology in nutrition research. Aquaculture 59:209-234.
Wee, K.L. and S.S. Wang. 1987a. Nutritive value of leucaena leaf meal in pelleted feed for Nile tilapia. Aquaculture 62:97-108.
Wee, K.L. and S.S. Wang. 1987b. Effect of post-harvest treatment on the degradation of mimosine in Leucaena leucocephala leaves. J. Sci. Food Agric. 39: 195-201.
Williamson, G. and W.J.A. Payne. 1978. An Introduction to Animal Husbandry in the Tropics. Longman, London.
<section>DNA and Genetics</section>
<section>Applications Of Dna Fingerprints In Fish Genetics For Species Determination And Conservation Of Indigenous Tilapiine Genetic Resources In Zimbabwe</section>
J.M. Gopo
Biological Sciences Department, University of
Zimbabwe
Harare, Zimbabwe
Abstract
Tilapiine fish samples were collected from tributaries and rivers of the six independent river basin systems of Zimbabwe. For the DNA fingerprinting experiments, gonadal tissues were removed from fresh fish, frozen in liquid nitrogen, and transported to the laboratory for genetic analysis. High molecular weight genomic DNAs were extracted, isolated, and purified.
The purified genomic DNAs from each suggested species were digested with restriction endonuclease (Meganuclease l/See D, Boehringer Mannheim GMbH, according to the manufacturer's conditions. The digests were fractionated on agarose gel-electrophoresis, using the Bio-Rad Pulsed-Field gel-electrophoresis system, model Cheff-Dr. II. The gels were stained with ethidium bromide, viewed under the Fotodyne transillumination U.V. system, and the DNA fragments were transferred onto nitrocellulose filters by the southern blotting method in preparation for DNA-DNA hybridization. The hybridizations were carried out using appropriate multi-locus fish DNA probes supplies by Cell-Mark diagnostics, United Kingdom. Bacteriophage M.13 - DNA was also used as a probe. Arbitrarily chosen DNA primers in Polymerase Chain Reaction (AP/PCR) will be used to analyze the number and size of the different fragments generated through the use of the endonuclease (meganuclease l/Secl) digestion.
Preliminary results have shown that the isolation, punfication, and restriction digestion of the DNAs are quite successful. Genetic analysis is now in progress.
Introduction
Studies on molecular biology in the last decade have brought about an explosion of new and elegant techniques in research methods that are expected to be of great significance to biomedical sciences in areas such as biology, cell biology, cell physiology, biochemistry, biotechnology, classical genetics, and medical genetics. These developments have, on one hand, led to the cloning of specific genes with which commercial lines can be transformed (Solter and Beckmann 1986, MacLean et al. 1987, Salter et al. 1987). On the other hand, molecular biology techniques can be applied to classical genetic analysis such as plant and animal breeding to increase their efficiency. One of the most elegant molecular biology techniques developed is DNA fingerprinting (DFP). DNA fingerprinting may well revolutionize genetic analysis of population-level variation, particularly the assessment of parentage and paternal settlement. DNA fingerprinting will contribute to studies of sexual selection,
mating behavior, gene introgression and genome selection for breeding programs, population ecology, sire evolution in animal husbandry, molecular taxonomy, and forensic studies. The major policy issue that will affect the application of biotechnology in agriculture is the management of intellectual property. Lack of patent protection is and will continue to be a major disincentive to both local private sector companies and transnational corporations. The use of DNA fingerprints can protect plant and animal breeders' rights. DNA fingerprint methods can be applied in fish genetics in the identification and conservation of indigenous tilapiine genetic resources in Zimbabwe.
The use of cichlid fishes in Africa as an alternative source of cheap protein is very significant. However, the demand to develop the aquaculture industry in Zimbabwe to meet this need had led to introductions of new and exotic species. Many Zimbabwean tilapiine species, previously not sympatric, are now found in sympatry due to these introductions. The tilapiine species represent the best hope for developing strong aquaculture programs based on indigenous fishes. Introductions of new exotic species and the possibility of hybridizations between the indigenous fishes and the newly introduced species may lead to losses of indigenous fishes. This, in turn, will lead to a significant decline in aquatic biodiversity in the region. Our project aims at the use of DNA fingerprints and isozyme gene-loci analysis to: (1) unambiguously identify all tilapiine species native to Zimbabwe and estimate and characterize the genetic variation within and among species; (2) assess the threat to
the integrity of Zimbabwean tilapiine species from hybridization with the introduced new exotic species; and (3) develop a conservation plan to mitigate against potential future losses.
DNA fingerprinting
DNA fingerprinting may be defined as the generation of individually specific DNA-band patterns that are revealed when minisatellite DNA sequences from the hypervariable gene loci of the genomic DNAs of different organisms are used as DNA probes in DNA-DNA hybridization experiments. Such simple reproducible band patterns (fingerprints) may also be generated by using single, arbitrarily chosen DNA primers in the Polymerase Chain Reaction technology (AP-PCR). The AP-PCR can produce both intraspecies and interspecies DNA fingerprints.
What Are DNA Fingerprints?
Previous studies have shown important genetic information about the structural organization of the genomic DNAs of various organisms.
1.The genomes of most eukaryotes possess regions with hypervariable DNA sequences (HS). Each hypervariable DNA sequence is present in these genomes in low-copy number. The HS generally include a series that is itself dispersed throughout each genome (Jeffreys et al. 1985a,b; Jeffreys and Morton 1987).
2.The hypervariable DNA sequences that have been described so far are not well conserved and come from several unrelated minisatellite gene families (Jeffreys et al. 1985 a,b; Jeffreys and Morton 1987; and Nakamura et al. 1987).
3.Some hypervariable loci contain a DNA sequence similar to the one found in the phage M.13 DNA, making the phage M.13 DNA a possible universal DNA probe in DNA fingerprinting (DFP) studies (Vassar" et al. 1987).
4.Epplen (1988) described hypervariable loci (HVL) that were revealed by hybridization to an oligonucleotide probe (GA/CT). Further study demonstrated that the mutation rates at these hypervariable loci vary widely and that these mutation rates may be as high as 10-3 per gamete per generation with numbers of alleles and the heterozygosity approaching the number of individuals and one, respectively (Jeffreys et al. 1988). This extensive variation is mostly due to the differences in copy number of the tandem repeats. This may be generated by a combination of unequal crossing over and replicative slippage (Jeffreys et al. 1985a).
5.The alleles at these extremely polymorphic loci are inherited as codominant Mendelian traits.
6.The most important finding of these studies is that alleles at any such polymorphic loci can be detected simultaneously by minisatellite probes based on the tandem repeats of the core-sequences, shared by many minisatellites. These probes reveal DNA band patterns specific to each individual genome on organisms tested that are called DNA Fingerprints (Jeffreys et al. 1985b).
DNA fingerprints of complex genomes may also be generated by using single arbitrarily chosen DNA primers and the polymerase chain reaction system (Welsh and McClelland 1991). Using the arbitrarily primed PCR (AP-PCR), which involved two cycles of low stringency amplification followed by one PCR at higher stringency, Welsh and McClelland showed intraspecies patterns (fingerprints) of AP-PCR products from six strains of S. aureus. In the same study, they also showed interspecies AP-PCR products when genomic DNA strains of five different species of Staphylococcus were each amplified at two different DNA concentrations. In general, this AP-PCR method may be used to generate both intraspecies and interspecies DNA fingerprints.
Uses Of DNa Fingerprints
DNA fingerprinting will become useful in genetic analysis of population-level variation, particularly the assessment of parentage. It will contribute to studies of sexual selection for both animal and plant breeding, biodiversity strategy programs, species conservation, and molecular taxonomy (Jeffreys et al. 1985a,b; Jeffreys and Morton 1987; Wetton et al. 1987; Dallas 1988; Burke et al. 1989; Burke 1989). It will be particularly useful in protection of breeders' property rights by enabling them to develop particular introgression breeding programs when a general similarity of the recipient line is sought. This similarity would be too general to be measured by the level of resemblance in one of several defined quantitative traits. When such breeding programs are conducted, it is expected that the genome of the organism under investigation is considered in its entirety. It therefore requires an infinitely large number of backcross generations to achieve any meaningful
results. The number of such backcross generations can be reduced by the use of polymorphic markers that are scattered in the genomes of most organisms (Tanksley and Rick 1980, Tanksley et al. 1981, Beckmann and Solter 1986). Therefore, it is reasonable to suggest that the alleles of minisatellite loci be used in genomic selection (G.S.) studies as the best genetic markers suited for tagging the entire genomes. This would substantially reduce the number of backcross generations required in any meaningful breeding program. Genomic selection, employing DNA fingerprinting techniques, can be used in gene introgression breeding programs to hasten the recovery of the recipient line genome from the backcross population with the introgressed trait included in it.
If this method is adopted, the individuals that carry the desired introgressed trait can be selected for the genome (positive G.S.) or against the undesired one (negative G.S.). More analytical and comparative studies can also be made by use of both negative and positive G.S. In all of these studies, the selection criterion is the degree of resemblance between the DNA fingerprint of the selected candidate and that of the desired or undesired genome. The basis of genome selection is the condition that the genomes can be tagged by use of minisatellite tandem repeats of DNA probes to reveal DFP bands that are specific to each individual genome under investigation. The efficiency of the G.S. is proportional to the faithfulness of the tagging. Large numbers of DFP loci have been identified (Jeffreys and Morton 1987, Hillel et al. 1989). Studies have also shown that part of these DFP loci are scattered throughout the genomes (Jeffreys and Morton 1987, Royle et al. 1988, Hillel et
al. 1989). The level of similarity between two individual organisms is measured by band sharing. Band sharing level is defined as the ratio of common bands in two individuals to the sum of all their bands (Hillel et al. 1989).
It seems clear that our attempts to preserve biodiversity must be based on an understanding of the genetic resources before biodiversity is lost, especially in the tropical countries. Genomes of organisms, eukaryotes and prokaryotes, plants, and animals can be better understood through the use of RFLPs and DNA fingerprints. The establishment and development of DNA fingerprints in crop and animal breeding can be used to protect the intellectual property rights of plant and animal breeders by using the fingerprints in the registration of their new hybrids.
Applications Of DNA Fingerprints
The DNA fingerprints may be used in several ways, such as the analysis of genetic variation at population levels, studies of species determination and molecular taxonomy, the assessment of parentage and paternal settlements in civil courts, mating behavior, population ecology, genetic selection for breeding programs by gene introgression, sire evaluation in animal breeding and improvement, forensic studies, and protection of property rights by plant and animal breeders. There are four distinct strategies that may be employed.
1.Use minisatellite probes that reveal variation in a large number of hypervariable gene loci (Jeffreys et al. 1985b). This results in multifragment patterns that are usually unique to each individual organism. This method is extremely powerful in testing parentage, in sire evaluation in animal husbandry, and in genomic selection. Putative parents are lined up next to the individual in question for use in cases of paternity settlements and in forensic cases.
2.Use the phage M.13 DNA as a universal probe. This would provide similar and simpler probe penetration.
3.Assay the hypervariable loci one at a time, using synthetic oligonucleotide probes (Nakamura et al. 1987). This method is more labor intensive but has the major advantage that alleles can be assigned to specific loci and genotypes can be identified.
4.Use arbitrarily primed PCRC (AP-PCR) products that can reveal interspecies and intraspecies DNA fingerprints.
It seems clear that DNA fingerprinting can yield more reliable results than classical genetics in such areas as population, species, and higher level comparisons, and also in the analysis of hybrid zones and molecular taxonomy. DNA fingerprinting has already been used in case studies to demonstrate the application of this new technology (e.g., Palmer et al. 1988, to detect the intron and gene losses during angliosperm cp.DNA evolution).
Applications Of Dfp In Fish Genetics: Identification And Conservation Of Indigenous Tilapiine Genetic Resources In Zimbabwe
The use of cichlid fishes in Africa as a source of relatively cheap protein has grown substantially over the past several decades and the demand for fish protein is expected to increase. The development of aquaculture projects to meet these demands in Zimbabwe has led to accidental and intentional releases of exotic species. Many Zimbabwean species not previously sympatric are now found in sympatry due to these manipulations. Recently, the government of Zimbabwe, through the Department of National Parks and Wildlife, expressed concern over the long-term consequences of these release programs on the native fauna.
Tilapiine fish represent the best hope for developing strong aquaculture programs based on the use of indigenous species. However, many of these species will freely hybridize with introduced species. Introductions of new and exotic species may represent a significant threat to the genetic integrity of many native species. Their loss would not only represent a significant decline in aquatic biodiversity in the region, but would severely hamper future development of aquaculture. In response to this, a project funded by USAID was recently initiated on the identification and conservation of indigenous tilapiine genetic resources in Zimbabwe. Initially, populations of the fish species that are unlikely to have been disturbed by introductions of new and exotic species have been sampled. These fish populations are now being characterized by isozyme analysis DNA fingerprints. The results obtained from each experiment will be compared. Previously, meristic and morphometric analysis
have proved unsuccessful because of the phenotypic plasticity characteristic of each species. This analysis will determine the extent of hybridization among the native Zimbabwean species and enable us to develop a strategy to preserve our species and maintain their genetic integrity.
Specific project objectives are: (1) to unambiguously identify all tilapiine species native to Zimbabwe using enzyme loci and DNA fingerprints as genetic markers, and also to estimate the population level variations; (2) to establish enzyme assays and appropriate DNA probes (M.13 DNA as a universal probe or hypervariable minisatellite-tandem repeat loci-probes) for large-scale sampling in areas where introductions have occurred to assess the degree of hybridization; and (3) to develop a conservation plan based on those areas where genetically intact species still exist.
Acknowledgments
The author thanks the Office of Research, USAID, for funding grant number COM-5542-G-000016-00, the network meeting, and publication of this paper.
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Solter, M. and J.S. Beckmann. 1986. Restriction fragment length polymorphism in poultry breeding. Poult. Sci. 65:147-148.
Tanksley, S.D. and C.M. Rick. 1980. Isozymic gene linkage map of the tomato: applications in genetics and breeding. Theor. Appl. Genet. 57:161-170.
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Wetton, J.H., R.E. Carter, D.T. Parkin, and D. Walters. 1987. Demographic study of a wild house sparrow population by DNA fingerprinting. Nature (London) 327: 147-149.
<section>Genetic Profiles Of Pure Strains Of Cultivable Cichlid Species In Nigeria And The Identification Of Premium Quality Broodstock And Fry</section>
A.O. Anyanwu, T.O. Ajayi, And P.E. Anyanwu
Nigerian Institute for Oceanography and Marine Research 7,agos, Nigeria
Abstract
As in most of Africa, cichlids are the primary cultivable fish in Nigeria, but dependence on fingerlings from the wild is heavy because hatchery production is inadequate. Predictably, wide growth differentials result, and often runts predominate in harvests after 6-12 month grow-out periods, jeopardizing production economics. Hatchery-bred fingerlings are only marginally better because indigenous broodstocks are also "recruited" wholesale from the wild. Screening these wild recruits is mere guesswork. Consequently, the intensification of fish culture to reduce domestic fish production shortfalls is perpetually constrained by a short supply of superior quality fingerlings. Accordingly, research into reliable field and laboratory methods for differentiating tilapia strains with desirable growth characteristics for producing premium quality indigenous hatchery parent stocks is overdue.
Electrophoresis is exceptionally useful in genetic studies for fish production. Its potential contribution to cichlid systematics, genetics, and strain separation in Nigeria have to be explored because some of the cultivable cichlid species in Lake Kainji, Nigeria, exceed a weight of one kilogram.
Progressive hatchery selection of the wild stock to produce cross-breeds ultimately comparable to "red tilapia" in growth rates also appears desirable and inevitable. At the very core of this proposed research, and a prelude to hybridization studies, is the establishment and documentation of the genetic profiles of pure and uncontaminated strains of all local cichlid species, emphasizing intra- and interspecies genetic distances or compatibility.
Introduction
The need to bridge the huge annual deficit (greater than 0.5 million tons) between food fish demand and local production in Nigeria is very urgent. Whereas capture fisheries are approaching full exploitation, Nigeria's 1.75 million hectares of swamp, creek, and brackish-water habitats (Tobor 1984) that are suitable for fish farming lie fallow. Aquaculture, therefore, offers the surest opportunity for the economic expansion of the fisheries industry, and for increases in local fish production in Nigeria.
African cichlids of the genus Tilapia have become cosmopolitan and prevalent in warm water aquaculture throughout the world, but, regrettably, have remained poorly developed in most African countries (Pullin 1988). One of the main constraints to the increased production of these species in Nigeria is an inadequate supply of good quality fingerlings. There is a heavy dependence on fry and fingerlings from the wild, and fish farmers are usually confronted with the problem of identification of good strains of tilapia "seeds" for cultivation. Improvement in the methodologies of screening of cultivar stocks is clearly needed, given the increased interest in fish farming and its potential contribution to the food fish supply in Nigeria.
Electrophoresis is a very useful technique for documenting the "purity" status of wild fish cultivars. It can establish a set of markers to delineate stocks, indicating genetic distances (Cruz et al. 1982). It can also be useful in the identification and selection of potential wild strains of tilapia for culture purposes in Nigeria.
This paper proposes the establishment of a reference collection and strain registry of cultivable cichlids. The long-term goals are the identification of the more important cichlid genetic resources, the preservation of their pristine genetic status by protecting them from introgression and hybridization with introduced species, and the conservation of their natural habitats.
Cichlid Culture In Nigeria
Although aquaculture in Nigeria has been practiced for 40 years, it is still practiced largely at subsistence levels because of a tradition for capture fisheries rather than fish husbandry. However, an awareness of the potential of aquaculture has been growing because of the priority recently placed on attaining self-sufficiency in food production.
Among the indigenous fish groups that are cultivable the following cichlids rank high: Tilapia guineensis, T. zilli, T. mariae, Sarotherodon melanotheron, S. galileaus, Oreochromis niloticus, and O. aureus. In freshwater culture practice, O. niloticus and S. galileaus are preferred, whereas T. guineensis and S. melanotheron are preferred in brackish-water systems; but overall, O. niloticus is the most widespread. In an attempt to improve the yield per hectare, "pure" strains of O. niloticus (broodstock) and "red" tilapia hybrids were introduced into Nigeria in the last 10 years. Unfortunately, most of these transfers were neither recorded nor monitored rigorously, and the performance of only a few of these introduced strains are being evaluated.
Mixed-sex tilapia culture and polyculture in combination with other species, including Lates niloticus, Tarpon atlanticus, Clarias gariepinus, Heterobranchus bidorsalis, Gymnarchus niloticus, and Lutjanus sp., are therefore predominant in Nigeria. These predators control the over-reproduction of tilapia species and their overcrowding in earthen ponds. In many fish farms, these predators become the primary target fish, relegating tilapia to prey status, which has a negative impact on total production.
More often than not, the supply of tilapia fry is heavily dependent upon wild collections and fry quality is not guaranteed. After 6-12 months of extensive or semi-intensive culture, tilapia attain average weights varying from 80 to 200 g. In most cases, 35% or more of the harvested fish may be small (less than 60 g) and of low economic value. Because the few large tilapia are promptly selected for the table in the few fish farms producing their own fingerlings, only smaller stunted fish are unwittingly recruited for hatchery production. Thus, credible selection and breeding programs are rare.
Identification And Cultivation Of Good Quality Cichlid Broodstock
The circumstances in Nigeria notwithstanding, cichlids (tilapias) are the most farmed fish in the tropics and are second only to carps, globally. In Southeast Asia, annual production of tilapia is quite high. In Nigeria, farming is handicapped by poor pond management, resulting in poor production, which seriously hinders the development of aquaculture. The growth of tilapia farming in Nigeria is predicated upon the resolution of the problems of over-reproduction and stunting, partly through all-male monosex tilapia culture or the use of fast-growing hybrids. However, the production of all-male progeny requires that the parents be pure strains.
Excluding Lakes Kainji and Chad, there are about 323 lakes and reservoirs in Nigeria ranging from 0.5 to 29,000 ha in size and totalling 275,534.91 ha (Ita et al. 1985). There is also an extensive network of rivers, including the Niger-Benue river system. Most of these water bodies harbor indigenous tilapia populations. Oreochromis niloticus and O. aureus, which are very important for aquaculture worldwide, are native to the country, but the genetic profiles of the Nigerian populations have not been investigated. This is most important since the Ivory Coast and Ghana strains of O. niloticus are acknowledged as fast growers and were probably derived from the River Niger complex, which also feeds Nigeria. Populations of O. niloticus from Lake Kainji have a high index of growth when compared to other populations in Africa (Moreau et al. 1986). Oreochromis aureus is important for culture in Israel and some other countries but is rarely used in Nigeria, where it has not been
investigated.
In Nigeria, habitat modification through land reclamation for industrial growth, damming of rivers for irrigation purposes and fish transplantation for aquaculture, are on-going and large scale. There is also an upsurge in the number of fish ponds in the country and therefore more fish transfers, especially recruitment of broodstock from the wild. As a result, different populations inhabiting different water bodies or different parts of the same water body that rarely hybridize in nature may cross-breed rather easily when enclosed in the same pond (Pullin 1988). Transplanted fish may also escape from the ponds into local river systems and may interbreed with the resident stocks. This ultimately may result in genetic instability of fish populations through nonbeneficial modifications. There is an urgent need to survey, evaluate, and document tilapia genetic resources in Nigeria as a first step toward conservation before their "purity" is compromised.
Tilapia Farming Problems
Stock management and genetic improvement of fish stocks require some reliable means of identification of different species and the delineation of hybrids. This helps in distinguishing strains of the same species and facilitates selection. To date, there have been no assessments of strain differences in tilapia in Nigeria. Thus, investigations of the genetic similarity and variability of the wild and cultured tilapia stocks in Nigeria are urgently needed to elucidate or document their potential growth status. Biochemical methods, especially electrophoresis, can indicate polymorphism and estimate genetic distances and heterozygosity levels in sympatric and allopatric fish species. Avtalion (1982), Cruz et al. (1982), McAndrew and Majumdar (1983), and Taniguchi et al. (1985) have demonstrated the utility of electrophoresis in cichlid identification. At present, it is being used successfully in the taxonomy of Nigerian marine fish.
Electrophoresis will therefore be used to label different tilapia species native to Nigeria, and premium quality tilapia strains will be identified for subsequent use as broodstock based on the following criteria:
- Ability to produce 100% male F1 progeny
- High fry production
- Growth rate greater than 1.0 kg in a 12-month growth-out
- Ability to adapt to pond conditions and handle stress.
Using these techniques and criteria, the all-male monosex culture and mass selection of tilapia for a faster growth rate and larger size would facilitate the improvement and expansion of the tilapia culture industry and annual fish production in Nigeria.
Conclusion
The location of pristine tilapia populations and their genetic evaluation for culture performance and breeding programs are a matter of urgency in Nigeria. Strains of O. niloticus, O. aureus, T. guineensis, and S. galilaeus shall be screened for production traits such as fast growth rate and high percentage male progeny. During this process, the genetic adequacy, or otherwise, of indigenous strains shall be scientifically proved and documented. The establishment of a cichlid (tilapia) culture reference collection and strain registry resulting from this work would be the scientific basis for discouraging or legislating against introductions of genetically "contaminated" fish strains.
Acknowledgments
The authors thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
References Cited
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<section>Sex Heredity Of Tilapia Hybrids From Stable Female Line Parents</section>
E. Lahav And Z. Raanan
Abstract
Males from various Oreochromis aureus ecotypes were cross-bred with O. niloticus females in Nir David Fish Breeding Farm. One of the test crosses produced the desired 100% F1 male progeny. The parents that yielded the all-male hybrids were repeatedly interbred within their respective families to produce genetically stable O. aureus and O. niloticus lines for use as broodstock for the commercial production of male hybrids.
Following the same methodology, two additional stable female lines (different from the original O. niloticus) were identified, which consistently produced 100% male progeny when crossed with O. aureus males from the stable line. When males and females from the newly identified lines were cross-bred with each other, their F1 progeny included both males and females. However, when F1 females obtained from this cross were mated with O. aureus males, the result was 100% F2 male hybrids.
Until now it was believed that only pure parental lines were capable of producing allmale progeny. Based on our results, however, it should be possible to use selected F1 hybrids (as opposed to pure lines only) as broodstock for the production of all-male progeny. This opens a new avenue for tilapia genetic selection programs by which desired traits can be transferred from pure line parents to their F1 hybrids, that can, in turn, transfer those traits to their all-male progeny.
Since most red tilapia mutants known today are hybrids, the main objective of our research is to identify red tilapia hybrids that can be used for the production of all-male red tilapia.
Introduction
Tilapias are becoming an important component in tropical and subtropical aquaculture, mainly due to the hardiness, rapid growth rate, and adaptability of the fish to a wide range of environmental conditions.
The factor that has most inhibited growth of the tilapia industry, in spite of its great potential, is early sexual maturation, which often results in uncontrolled spawning in the production ponds, overcrowding, and cessation of growth before market size is reached. Another limiting factor is that males usually grow faster than females, especially when the fish approach adulthood and females begin reproductive activity.
Production of all-male tilapia populations is the ideal solution to both problems. All-male populations are obtained either by genetic hybridization (Wohlfarth and Hulata 1983) or by sex-reversal of the females through exposure to male hormones (Berger and Rothbard 1987). The latter method, although well-established, is less favorable for several reasons: (1) the treatment causes temporary growth retardation; (2) the treatment requires special facilities; (3) breeding of generations of fish originating from the same stock causes depression of good traits; and (4) consumers are increasingly wary of hormonally treated foods.
All-male hybrids are obtained from certain interspecific crosses. At least six such crosses of mouth brooding tilapias have been found that produce almost 100% male progeny, i.e., a female Oreochromis mossambicus crossed with a male O. hornorum (Chen 1969) or O. macrochir (Majumdar and McAndrew 1983), a female O. niloticus crossed with a male O. aureus (Pruginin 1967), O. hornorum (Pruginin 1967), O. variabilis (Pruginin 1967), or O. macrochir (Jalabert et al. 1971).
In 1980-1981, pure stocks of O. aureus and O. niloticus were isolated in the Nir David Fish Breeding Farm. The crossing of a male O. aureus with a female O. niloticus from these stocks produces 100% male hybrids repeatedly and consistently (Lahav and Lahav 1990). These stable lines were used to find new stocks that would produce all-male progeny. Females of the new stocks were crossed with males of the stable O. aureus line and males of the new stocks were crossed with females of the stable O. niloticus lines. Any stocks that produced all-male progeny were identified as new male or female lines, according to which sex was used in the cross. In this way, two additional stable lines (different from the O. niloticus line) were isolated, the females of which, when crossed with O. aureus males, produce all-male progeny (Lahav unpublished).
The genetic mechanism of sex determination in tilapias has been the subject of many studies (Wohlfarth and Hulata 1983, McAndrew 1989, Lester et al. 1989). Several models have been proposed in the attempt to explain sex ratios of progeny of different crosses between various "pure" lines. These models are based on either sex chromosomes alone, such as the dual system of sex-determining chromosomes in different species of mouthbreeding tilapias, XX/XY and WZ/ZZ (proposed by Hickling 1960, and by Chen 1969), or on the presence of several sex-determining factors located on autosomes, operating together with the sex chromosomes, to determine the sex of the fish (Majumdar and McAndrew 1983). Other models attribute a significant effect of environmental factors on the determination of sex (Lester et al. 1989).
A distinction is made between "female lines" and "male lines." Female lines are defined as lines whose females, when crossed with males from a male line, produce all-male progeny. Likewise, the males of a male line, when crossed with a female of a female line, produce all-male progeny. Correlated to the simple model presented by Chen (1969), females in the female lines are homogametic (XX) and males in the male lines are homogametic (ZZ). Crosses between females of a female line and males of a male line result in heterogametic (XZ) all-male progeny (McAndrew 1989).
In all cases, all-male hybrids result only in the first generation (F1) cross between stable, pure line parents. This presents severe difficulties to producers because: (1) the number of crosses that yield the desired result (100% male) is very limited; (2) most of these crosses are not suitable for commercial production either because of difficulties in mass producing fry or because of the inferior growth of the hybrids, (3) and it is, so far, impossible to preserve the all-maleness and improve other, yield-associated traits of the hybrids (e.g. red color, McAndrew 1989).
The goal of our study was to examine sex-determining characteristics in first generation female offspring of a female from a female line crossed with a male from another female line. If the F1 female inherited the tendency to produce all-male progeny, then the number of female lines would, in effect, be increased and, along with it, the possibilities of genetically improving yield-associated traits of the hybrids without relinquishing the all-male trait.
Methods And Materials
Males and females from the three female lines isolated at Nir David were crossbred. The three lines include ND2 (the stable O. niloticus population, which was among the first two "stabilized" populations) and ND3 and ND4 (the two lines discovered later). Judging by their phenotypic appearance, neither ND3 nor ND4 is O. niloticus. ND3 has orange skin with dark spots or grey skin and, although ND4 has silver skin similar to that of O. niloticus, both lines lack the typical O. niloticus stripes on the tail fin. Moreover, electrophoretic tests showed clear differences between these three lines (Avtalion, A. 1991, personal communication). We would like to stress that for the purpose of this study species identification is not necessary. The original fish used to establish ND3 and ND4 were purchased from a private collector in 1983 and the lines were stabilized by 1988.
On March 5, 1987, a sexually mature female of one line was placed in a 20 mm net spawning cage (0.8 x 2.5 x 0.8 m) with a sexually mature male of another line. Females weighed more than 200 g and males weighed more than 400 g. Fish were randomly chosen.
The cage was surrounded by mosquito net (3 x 5 m) and placed in a 5 x 20 m earthen pond, protected by net against birds in the summer and by plastic for warmth in the winter. The water exchange rate in the pond was maintained at 1.0 mģ per hour. The parents were not fed while in the spawning cage.
Three combinations were tested: a female of ND4 with a male of ND2, a female of ND3 with a male of ND2, and a female of ND4 with a male of ND3. Table 1 shows the mating combinations and the course of the experiment.
After the female had spawned and released the fry from her mouth, the cage with the parents was removed, leaving the fry in the mosquito net. Fry were fed fishmeal containing 70% protein and remained in the mosquito net for another 40-45 days until they reached approximately 1 gram.
At the size of 1 gram, the fry were transferred to round fiberglass tanks (2.5 m diameter, 0.6 m deep) and stocked at a density of approximately 500 per mģ. Fry from each mating were stocked separately. Clean water flowed through the tanks at a rate of 1 mģ/hour. The water temperature was maintained at 25-28°C. Tanks were aerated by blower and cleaned through an opening in the center of the tank floor. Fry were fed 25% protein pellets according to Nir David's standard feeding table and weighed every 10 days.
When the fingerlings were more than three months old and weighed at least 10 g, sex was identified by microscopic examination of the gonads. At least 50 fingerlings of each spawn were examined. Following the sexing of the hybrids, a random sample of 29 fingerlings were stocked at a density of 10 per mģ in separate tanks for further growout. Sex of sexually mature fish was determined by external examination of the sex organs.
On March 20, 1988, F1 females (averaging 60 g) from each cross were placed with males from the stable O. aureus male line (ND1) into spawning cages. Each cage contained one F1 female and one male (a total of nine cages). Two weeks after spawning was observed, the cages with the parents were removed. The fry remained in the mosquito nets for an additional 65-80 days, at which time samples of at least 50 fingerlings weighing more than 10 g from each group were randomly selected for sex examination.
Results
Results of the first spawning (by fish of the stable lines) are summarized in Tables 1 and 2. The pairs containing an ND3 female and ND2 male spawned approximately 30 days later than the other two combination. In all cases, the sex ratio of the F1 hybrids did not differ significantly from 1:1.
TABLE 1 Mating of Female Tilapia from Stable Female Lines with Males from Stable Female Lines (Parents Stocked in Spawning Cages on March 5)
Cage #
Female
Male
Days until
Parents'
Fry Reached
Parent
Parent
Spawning
Removal
1 gram
ND4
ND2
April 5
May 16
April 5
May 16
April 5
May 16
ND3
ND2
May 5
June 20
May 5
June 20
May 5
June 20
ND4
ND3
April 10
May 20
April 10
May 20
April 10
May 20
Results of the spawning of F1 females with O. aureus males from the stable male line ND1 are summarized in Table 3. There was a significant difference in the number of days until spawning, depending on the female parent. Thus, ND4 x ND2 females spawned after 14-16 days, whereas ND3 x ND2 females and ND4 x ND3 females spawned after 54-59 days and 72-84 days, respectively. In all cases, the F2 progeny were 100% males.
Discussion
The results of our study demonstrate that F1 females obtained by crossing a female from a stable female line with a male from another stable female line maintain the sex heredity characteristics
of their parents. In all cases examined, the F1 females of such crosses produced 100% males when crossed with males from the male line.
These results support the chromosomal sex-determining mechanism proposed by Chen (1969). Assuming that females of the three female lines are homogametic (XX), crossing a female from one female line with a male from another female line (XY) would produce an F1 hybrid population with a sex ratio of 1:1. Our results were indeed in accordance with this expectation. The percentage of males in the F1 populations ranged from 46% to 54%. According to Chen's model, F1 females are also homogametic (XX) and, therefore, when bred with homogametic males (ZZ) from a male line, can be expected to produce an all-male F2 population. Again, our results show that, without exception, all F2 progeny examined were male.
TABLE 2 Growth After ~60 Days and Sex Ratio in Hybrid Progeny
Tank #
Hybrid
Size at
Size at
Number
Definition
Stocking
Harvest
of Males
(g)
(g)
ND4 x ND2
ND3 x ND2
ND4 x ND3
These results do not necessarily prove the model. The involvement of additional autosomal factors in sex determination as proposed by Majumdar and McAndrew (1983), or environmental factors as proposed by Lester et al. (1989), cannot be ruled out. However, our results indicate that hybrids of two parents from stable female lines maintain the sex heredity mechanism of their parents and therefore could serve as mothers for the production of all-male F2 progeny.
This finding may be useful in genetic selection programs that are working on improvement of other economically important traits such as color, growth rate, temperature resistance, and salinity resistance. Fish can be selected for inherited characteristics without losing the all-male trait by breeding F1 hybrid females instead of females from the stable line.
In addition, our method can be used for solving the problem of mass producing all-male hybrids. Such difficulties are often encountered with some of the all-male producing crosses due to reproductive barriers between some of the pure lines (Wohlfarth and Hulata 1983). For example,
whereas breeding ND1 males and ND4 females is often unsuccessful, the F1 female from the ND4 x ND2 cross readily produced fry when crossed with the ND 1 male Table 3). Thus, the F1 female that carries desired traits from the ND4 line may transmit these traits to the F2 all-male hybrids.
TABLE 3 Mating of Female Tilapia Hybrids from Stable Female Lines with Males from a Stable Male Line (Parents Stocked in Spawning Cages on March 20)
Cage #
Female Parent
Male Parent
Days until Spawning
Fry Examined
Percentage of Males
ND4 x ND2
ND1
June 20
June 20
June 20
ND3 x ND2
July 21
July 21
July 21
ND4 x ND3
August 15
August 15
August 15
Our study shows that the sex heredity characteristics of females from the stable female lines examined are inherited by their female F1 progeny. It is still important to examine whether this phenomenon is valid for other crosses between female lines. It is also important to know whether a similar phenomenon occurs when crossing different male lines and testing the cross between an F1 male hybrid and a female from a female line. The prospect of producing F1 male and female lines, capable of producing 100% F2 male progeny, opens a wide range of new possibilities in tilapia genetic selection programs.
Acknowledgments
The authors thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.
References Cited
Berger, A., and S. Rothbard. 1987. Androgen-induced sex-reversal of red tilapia fry stocked in cages within ponds. Isr. J. Aquacult. Bamidgeh 39:49-57.
Chen, F.Y. 1969. Preliminary studies on the sex-determining mechanisms of Tilapia mossambica Peters and T. hornorum Trewavas. Verh. Int. Ver. Theor. Angew. Limnol. 17:719-724.
Hickling, C.F. 1960. The Malacca Tilapia hybrids. J. Genet. 57:1-10.
Jalabert, B., P. Kannacher, and P. Lessent. 1971. Determinisme de sexe chez les hybrides entre Tilapia macrochir et T. nilotica. Etude de la sexe-ratio dans les recroisements des hybrides des premier generation par les especes parentes. Ann. Biol. Anim. Biochim. Biophys. 11:155165.
Lahav, M., and E. Lahav. 1990. The development of all-male tilapia hybrids in Nir David. Isr. J. Aquacult. Bamidgeh 42:58-61.
Lester, L.J., K.S. Lawsom, T.A. Abella, and M.S. Palada. 1989. Estimated heritability of sex ratio and sexual dimorphism in tilapia. Aquacult. Fish. Manage. 20:369-380.
Majumdar, K.C. and B.J. McAndrew. 1983. Sex ratios from interspecific crosses within the tilapias. pp. 261-269. in Fishelson, L. and Z. Yaron. (ed.). Proc. of the Internat. Symp. on Tilapia in Aquacult. in Nazareth. Tel Aviv University, Tel Aviv, Israel.
McAndrew, B. 1989. Sex determination and its manipulation in tilapia. pp. 199-210. in Proc. Satellite Symp. on Applications of Comparative Endocrinology to Fish Culture, Granada, Spain, May 22-23, 1989. Published by Universitat de Barcelona, Spain.
Pruginin, Y. 1967. Report to the government of Uganda on the experimental fish culture project in Uganda, 1965-66. Rep. FAO/UNDP (TA) (2446). 16 pp.
Wohlfarth, G.W. and G. Hulata. 1983. Applied genetics of tilapias. ICLARM Studies and Reviews 6. International Center for Living Aquatic Resources Management, Manila. 26 pp.
<section>Identification And Conservation Of Indigenous Tilapiine Genetic Resources Of Zimbabwe</section>
F. Shonhiwa And J.H. Howard
Department of Biology, Frostburg State University
Frostburg, Maryland
Abstract
Use of tilapiine fishes for aquaculture in Zimbabwe has increased greatly in recent years. Unfortunately, many allopatric species of tilapias will freely hybridize when placed in sympatry. The common practice for fisheries officers has been to collect broodstock from a variety of locations, pool them, allow them to breed, and distribute the fry throughout the country for stock ponds and reservoirs. There is a concern, therefore, that some indigenous tilapias may disappear as a direct result of hybridization.
Previously, the only method of identifying tilapias was morphology. However, that method has proven unreliable, especially for tilapias, due to overlap and variation in the taxonomic characteristics of the fish. Now, the utilization of biochemical techniques provides a means of unambiguous species and hybrid identification. In this study, starch gel electrophoresis was used to detect enzyme loci that are useful as diagnostic characters.
Introduction
Although the use of tilapiine species for aquaculture in Zimbabwe, and Africa as a whole, has greatly increased in recent years, it must continue to grow to keep pace with the demand for fish protein. In Zimbabwe it is estimated that catches should increase from the current 20,000-30,000 t/yr to perhaps as much as 160,000 t/yr by the end of the century. This growth is necessary to offset increased demand for fish protein from a population that is expected to double by the year 2000. Much of the technology necessary for such aquacultural programs is both simple and inexpensive. Imports do not represent a reasonable possibility because of a severe foreign currency problem. While more than 30 exotic fish species have been introduced into Zimbabwe, none have the production potential of the indigenous tilapias.
Programs to culture and release fish into non-native habitats to supplement or replace the harvest of natural populations have no significant parallel in terrestrial systems. A common practice for fisheries officers is to collect broodstock from a variety of locations, pool these stocks, allow them to breed, and distribute the fry throughout the country for release in stock ponds and reservoirs. Unfortunately, many allopatric species of tilapias will freely hybridize when placed together.
Therefore, we are concerned that some indigenous tilapias may disappear as a direct result of hybridization.
Although there is some confusion about the actual number of tilapias indigenous to Zimbabwean waters, the best current estimate is eight (five in the genus Oreochromis and three in the genus Tilapia) (Table 1).
TABLE 1 Distribution of Tilapias in Zimbabwean River Systems (After Bell-Cross and Minshull 1988)
Species
River Systems
UZ
LK
MZ
LZ
N
L
P
B
USR
LSR
O. andersonii
o
O. macrochir
o
i
i
i
i
O. mortimeri
o
o
O.mossambicus
o
o
o
o
o
O. placidus
o
i
o
T. sparrmanii
o
o
o
o
o
o
T. rendall rendalli
o
o
o
o
i
o
T. rendalli swierstrae
o
T. ruweti
o
River system: UZ -- Upper Zambezi, LK -- Lake Kariba, MZ -- Middle Zambezi, LZ -- Lower
Zambezi, N -- Nata, L -- Limpopo, B -- Budzi, P -- Pungwe, USR -- Upper
Save/Runde, LSR -- Lower Save/Runde
Other: o -- collected in the past, i -- fish introduced into the system, * -- new records from
this study.
In addition to occasional disagreements regarding the number of species, hybridization of
existing species makes it even more difficult for fisheries officers to identify fish stocks with certainty. Over the past 30-40 years, extensive introductions and translocations of both exotic and indigenous tilapia species have occurred. According to Bell-Cross and Minshull (1988), Oreochromis andersonii, O. mortimeri, and O. mossambicus may hybridize freely when they occur together. Philippart and Ruwet (1982) report extensive transfers of O. macrochir, O. placidus, O. mossambicus, and O. mortimeri into new reservoirs within Zimbabwe. This complex situation is worsened further by the introductions of exotics from other parts of Africa.
Hybridization between indigenous and introduced fish species can result in local extinctions, and over a broader range, threaten species with extinction. Among introduced populations of O. macrochir and O. niloticus in Madagascar, introgression ultimately resulted in the disappearance of O. macrochir (Moreau 1981). Other studies demonstrating the potential for introgression and such losses in African cichlids are Macaranus et al. (1986), using O. mossambicus and O. niloticus in the Philippines, and Elder et al. (1971), working with O. spiturus and O. leucosticus in Kenya. The difficulty of obtaining pure stocks in Zimbabwe has already been commented on by several investigators (e.g., McAndrew and Majumdar 1984, Beattie 1986, Van der Bank et al. 1989).
Some interspecific hybridizations result in nearly all-male populations. Exploitation of this event in aquacultural practices can mean that most of the biomass consumed by all-male fish will be converted to flesh rather than reproduction, resulting in much higher farm-pond production. Preliminary research has shown that indigenous male O. macrochir and female O. mossambicus produce 100% male hybrid progeny (Thompson 1984). This work has also shown that tilapia species from different river systems do not yield similar results. In addition, some hybrids grow faster than the parental species and are superior in converting food or exhibit greater cold tolerance. These potentials make it imperative that controlled experiments be conducted on indigenous Zimbabwean tilapias and hybrids of known ancestry. Without unambiguous stock identification of these resources at the outset, it is probable that such experiments could not be duplicated. The utilization of biochemical techniques
provides a means for the unambiguous identification of hybrids and species.
With this background in mind, the following two objectives were established:
1. To unambiguously identify all tilapiine species native to Zimbabwe using enzyme loci as species-specific genetic markers and once so identified, develop a key based on morphological characteristics; and
2. To ascertain the degree of hybridization where introductions are known to have occurred.
Materials and Methods
Sample Collection
Samples consisting of 25 fish per sample were collected from the major river systems of Zimbabwe (Figure 1). Collections were made in the native ranges of the various species and in areas where introduction of other species were unlikely. Over a period of one-and-one-half years, the following populations were sampled: 16 Tilapia rendalli, 13 Oreochromis mossambicus, 9 O. mortimeri, 3 O. macrochir, 2 O. andersonii, 3 T. sparrmanii, and 2 O. placidus. In addition, five fish each of O. niloticus and O. aureus were collected from the Edwards and Son Farms located just outside of Harare. These two species are not indigenous to Zimbabwe, but are currently being farmed there. The strains at the Edwards farm were imported from Stirling, U.K., in 1985 and are believed to have come from cultures in Israel. Tilapia ruweti was not collected, probably due to its scarcity in the Upper Zambezi system.
Collecting Techniques
Specimens were collected using seine nets and gill nets. Muscle, liver, eye, heart, and brain tissues were dissected from freshly killed fish, labelled, placed in Eppendoff tubes, and immediately frozen in liquid nitrogen at the collection site. The tissues were then taken to the University of Zimbabwe and were stored in an ultra freezer at 80°C. The tissues were shipped on dry ice to Frostburg State University, Maryland, for analysis. The carcasses were preserved in 10% formalin and stored in 50% alcohol. These were also shipped to the United States.
Sample Preparation
In the laboratory, tissues were prepared for electrophoresis by adding equal amounts of water (1: 1 v/v) to tissue and homogenized with a tissue grinder for one minute. The homogenized tissue was then centrifuged at 12,800 x g for 3 minutes and the supernatant was absorbed onto 1.5 x 0.3 cm strips of Whatman No. 3 filter paper.
TABLE 2 Buffer Systems Used in the Study
System
Reference
Gel Buffer
Electrode Buffer
Gel Composed
RW
Ridgway, Shelborne,
0.03M Tris
0.06m Lithium
of 99% gel
and Lewis (1970)
0.005m Citric acid
hydroxide
buffer, 1 %
pH 8.5
0.3 M Boric acid
electrode
pH 8.1
buffer
AC
Clayton and
1:20 dilution
0.04 M
pH was adjusted
Tretiak (1972)
of electrode: H2O
Citric acid
with N-(3
pH 6.1
pH 6.1
aminopropyl)
morpholine.
MF
Markert and
1:20 dilution of
1.5 dilution
Stock
Faulhaber (1965)
stock: H2O pH 6.1
of stock: H2O
0.9M Tris
0.5M Boric acid
0.02M Na EDTA
pH 8.7
TC
Shaw and Prasad
1:14 dilution of
56.4g Tris
Undiluted stock
electrode H2O
27.0g Citric acid
pH 7.5
3,000 ml H2O
pH 7.5
Gel Preparation
Gel preparation procedures followed those outlined in Selander et al. (1971) and May (1975), employing the use of 9.5 x 20 x 1 cm and 9.5 x 20 x 2 cm molds. Gels consisted of 13% Sigma hydrolyzed starch and one of the buffer solutions listed in Table 2. Two-thirds of the gel buffer was heated in a microwave oven for two minutes. The last third was mixed thoroughly with the starch in a 1,000 ml Erlenmeyer flask. The heated buffer was then added to the starch/buffer mixture and heated for three minutes in a microwave. This mixture was swirled every 15 seconds to ensure a smooth, lump-free gel mixture as the end product.
The mixture was then aspirated for approximately one minute with constant swirling to remove air bubbles. At the end of this period, the starch solution was poured in the plexiglass mold, formed, and allowed to cool at room temperature. The gels were kept covered with plastic wrap overnight.
Electrophoresis
Horizontal starch gel electrophoresis was carried out following procedures outlined in Selander et al. (1971), May (1975), Allendorf et al. (1977), and Abersold et al. (1987), employing the use of Model 1P-17 Heath Kit power supplies. Voltage was maintained at 300 mv for gels made with RW and MF buffers and those made with TC and AC buffers were maintained at 200 mv (Table 2). Gels were run over a duration of 3-5 hr until the dye marker had moved to a distance of 6 mm. The resulting gel was sliced into 1.5 mm replicates and each was stained with specific histochemical solutions as outlined by Allendorf et al. (1977) and May (1975).
Data Analysis
The electrophoretic data were analyzed using the BIOSYS-I program (Swofford and Selander 1981). This program allows for the computation of allele frequencies and associated measures of genetic variability, determination of conformance to expected genotypic frequencies based on the Hardy-Weinberg equilibrium, and calculation of F-statistics. The F-statistics developed by Wright (1978) were useful in the analysis of population structure. In addition, BIOSYS-I was used to calculate similarity and distance coefficients as developed by Nei (1978). BIOSYS-I was also used in constructing distance Wagner trees (Ferris 1970) and UPGMA phenograms.
Assay
A laboratory enzyme assay for the Zimbabwean tilapias was developed using two fish each from O. mossambicus and T. rendalli populations. Initially, all five tissues, eye, muscle, heart, and brain, were run on the same gel and on each of the four buffer systems.
PRELIMINARY Results
Collections
Bell-Cross and Minshull (1988) did not list the presence of any tilapias in the Nata River system. However, specimens that could be classified as O. mossambicus or O. mortimeri were collected in the Nata system at three sites (Table 1). Tilapia rendalli was also collected in the Budzi system, although no prior tilapia records exist in this system. A search for tilapias in the Pungwe system was unsuccessful. No T. ruweti were collected, probably because they are scarce.
Currently, data have been gathered on the activity and resolution of 64 enzymes on four buffer systems and five different tissues. Of these systems, 28 provide reasonable resolution and activity in three of the tissues: the muscle, liver and eye, and should provide data on over 38 loci (Table 3). Although the assay is essentially complete, we expect it will become refined as we proceed. We have now started the analysis of some experimental populations collected in Zimbabwe. No diagnostic loci were found between O. mossambicus and O. mortimeri from the loci used in the study.
TABLE 3 Enzyme Loci Among Tilapiine Fish Species of Zimbabwe that are Diagnostic Between at Least One Species Pairđ
Enzyme
Tilapia
Oreochromis
Indigenous
Exotics
rendalli
sparrmanii
placidus
mortimeri
mossambicus
macrochir
andersonii
niloticus
aureus
sAAT-2
cē
a
b
b
a
AK-1
a
b
a
a
a
a
a
a
a
CK-B
c
d
b
a
b
a
a
c
EST-1
d
e
a
a
c
a
c
GDA-1
c
c
a
b
PROT-2
a
b
a
b
a
b
b
GPI-B
b
d
b
b
a
c
GPI-A
b
a
a
a
a
a
a
G3P-1
c
d
e
a
a
a
a
a
mIDHP
a
c
a
a
a
a
a
a
b
LDH-C
b
b
a
a
a
a
a
a
a
LDH-B
a
a
a
a
b
a
a
a
LDH-A
a
c
a
a
a
a
a
sMDH-A
a
a
b
a
sMDH-B
b
b
a
a
a
a
a
a
a
mMDH
c
a
a
b
a
a
a
sMEP
a
a
b
a
a
b
a
a
a
mMEP
a
a
a
a
a
a
b
a
a
PEP-B
b
a
a
a
a
a
a
a
a
PEP-D
b
a
a
a
a
a
a
a
a
PGM-1
a
d
a
a
a
a
c
a
sIDDH-1
d
c
c
PGD-1
b
c
a
a
a
a
a
a
a
sSOD-1
b
b
a
b
b
TPI-1
b
a
b
a
a
a
a
* SAAT-1, MAAT-1, ADA-1, FBALD-I, ADH-1, CK-A, FBP-1, GAM-1, GAP-1, PROT-1, MPI-1, PEP-A AND PEP-F did not exhibit diagnostic
mobilities.
2** mobilities are based on "a~ as the fastest, and decreasing as you go down the alphabet.
3 (-) indicates multiple alleles.
Acknowledgments
The authors thank the Office of Research, USAID, for funding grant number COM-5542-G-00
0016-00, the network meeting, and the publication of this paper.
References Cited
Abersold, P.B., G.A. Winans, D.J. Teel, G.B. Milner, and F. Utter. 1987. Manual for starch gel electrophoresis: a method for the detection of genetic variation, National Oceanic and Atmospheric Administration (NOAA) Tech. Rep. National Marine Fisheries Service 61:20 pp.
Allendorf, F.W., N. Mitchell, N. Ryman, and G. Stahl. 1977. Isozyme loci in brown trout (Salmo trutta): detection and interpretation from population data. Hereditas 86: 179-190.
Beattie, I.H. 1986. The Development of Pure Strains. pp. 35-38. in Proc. of Fish Forum held at Henderson Research Station, 21 March, 1986. Department of National Parks and Wildlife, Zimbabwe.
Bell-Cross, G. and J. Minshull. 1988. The Fishes of Zimbabwe. National Museums and Monuments of Zimbabwe, Harare.
Clayton, J.W. and D.N. Tretiak. 1972. Amino-citrate buffers for pH control in starch gel electrophoresis. J. Fish Res. Bd. Can. 29: 1169-1172.
Elder, H.Y., D.J. Garrod, and P.J.P. Whitehead. 1971. Natural hybrids of the African cichlid fishes Tilapia spirulus nigra and Tilapia leucostrica: a case of hybrid introgression. Biol. J. Linn. Soc. 3:103-146.
Farris, J.S. 1970. Methods of computing Wagner trees. Syst. Zoo. 19:83-90.
Macaranus, J.M., N. Taniguchi, M. Pante, J.B. Capili, and R. Pullin. 1986. Electrophoretic evidence for Oreochromis niloticus (L.) stocks in the Philippines. Aquacult. Fish Manag. 17:249-261.
Markert, C.L. and I. Faulhaber. 1965. Lactate dehydrogenase isozyme patterns of fish. J. Exp. Zool. 159:319-332.
May, B. 1975. Electrophoretic variation in the genus Oncorhynchus: the methodology, genetic basis, and practical applications to research and management. Master of Science Thesis, Washington State Univ., Dept. of Fisheries.
McAndrew, B.J. and K.C. Majumdar. 1984. Evolutionary relationships within three Tilapiine genera (Pisces: Cichlidae). Zoo. J. Linn. Soc. 80:421-435.
Moreau, J. 1981. Application trial of a model to study the fisheries in tropical floodplains: the Alaotra Lake (Madagascar). Hydrobiologia 13:83-92.
Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetica 89:583-590.
Philippart, J-CI. and J-CI. Ruwet. 1982. Ecology and distribution of tilapias. pp. 15-59. in Pullin, R.S.V. and R.H. Lowe-McConnell. (ed.). The biology and culture of tilapias. ICLARM Conference Proceedings. Manila, Philippines.
Ridgway, G.J., S.W. Shelborne, and R.D. Lewis. 1970. Polymorphism in the esterases of the Atlantic herring. Trans. Am. Fish Soc. 99:147-151.
Selander, R.K., M.H. Smith, S.Y. Yang, W.E. Johnson, and J.B. Gentry. 1971. Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse Peromyscus polionotus). Studies in Genetics IV. Univ. Tex. Publ. 7103:49-90.
Shaw, C.R. and R. Prasad. 1970. Starch gel electrophoresis of enzymes - a compilation of recipes. Biochem. Gen. 4:297-320.
Swofford, D.L. and R.B. Selander. 1981. BIOSYS-I: a FORTRAN program for the comprehensive analysis of electrophoretic d$a in population genetics and systematics. J. Heredity 72:281283.
Thompson, P.J. 1984. Hybridization of indigenous Oreochromis species in Zimbabwe and the production of all-male offspring. pp. 1-11. in Aquaculture in southern Africa. Joint Symposium of CSIRO and SAAU. Cathedral Peak. 34 May 1984.
Van der Bank, F.H., W.S. Grant, and J.T. Ferreira. 1989. Electrophoretically detectable genetic data for fifteen southern African cichlids. J. Fish Biol. 34:466-483.
Wright, S. 1978. Evolution and the Genetics of Populations. IV. Variability Within and Among Natural Populations. Univ. of Chicago, Illinois.
<section>Ecology and Environment</section>
<section>Ecological Changes In Lake Victoria After The Invasion Of Nile Perch (La Tes Niloticus):The Catchment, Water Quality,And Fisheries Management</section>
P.B.O. Ochumba, M. Goshen, And U. Pollingher
Abstract
Lake Victoria, the second largest lake in the world, has undergone dramatic changes since the 1920s. Some of these include: intensive non-selective fisheries; severe alteration of the drainage basin through agricultural, vegetational, and industrialization changes; introduction and invasion of exotic fish species that has led to the elimination of native species; and a progressive buildup of physico-chemical changes in the lake environment. Current studies of Lake Victoria have identified substantial increases in chlorophyll concentration and primary productivity, as well as decreases in silica compared to values measured 30 years ago. Present sulphate concentrations (0.1 mg/l) are lower than the lowest values reported from other large lakes in the world. There has been a shift in the phytoplankton community toward a dominance of blue-greens. The zooplankton densities are relatively low and the body sizes of the organisms are small. Anoxic waters have recently been found at
shallower depths than previously reported in Lake Victoria, suggesting significant increases of oxygen demand in the seasonally formed hypolimnion. Algal blooms have also become more extensive in Lake Victoria. Fisheries management in Lake Victoria has led to a shift from relying on a multi-species stock (400-500 haplochromids) to one relying on only two major exotic species, Lates niloticus and Oreochromis niloticus and the endemic Rastrineobola argentea. The present practices of lake management have not improved water quality.
Introduction
Lake Victoria (100 m maximum depth), located in the Syrian-African Rift Valley was created after tectonic activity that formed westward-flowing rivers in the lake basin (Talling 1966, Serruya and Pollingher 1983, Bugenyi and Balirwa 1989). The catchment area (193,000/kmē) (Figure 1) and the wet drainage basin is covered by grassland savanna, agricultural crops, and the forested mountains of Rwanda and Burundi. The Kagera and Nzoia are the main inflowing rivers and the outflow is the River Nile. The lake is the second largest in the world and a major source of protein for 10 million people in Kenya (Ochumba 1984). The lake is seasonally stratified (Fish 1957) and wind is the major factor that determines the annual thermal cycle and water column mixing (Newell 1960, Talling 1969).
The long water residence time (23.4 years) increases vulnerability to long-term changes caused by environmental modification in the catchment area.
Lake Victoria has undergone successive disruptions since the early 1920s. Major changes in the ecosystem are: intensive nonselective fisheries, modification of the vegetation in the drainage basin, Nile perch (Lates niloticus) invasion and introduction of other exotic fish species, and the progression of physico-chemical changes in the lake. One of the dramatic changes is the development of a seasonal and lake-wide anaerobic hypolimnion which now threatens the integrity and biodiversity of this ecosystem. The endemic fish community of haplochromids has undergone a reduction in abundance and species diversity. The exotic Nile perch currently dominates the commercial catch together with the exotic Oreochromis niloticus and the small endemic cyprinid Rastrineobola argentea. Periodic fish kills in the lake raised serious concern about the environment of Lake Victoria and the impact of developmental activities in the lake basin (Ochumba 1987). Pollution from industrial,
agricultural, and urban sources has increased significantly and the physical alteration of the lake shores through construction is proposed.
Changes In The Fish Community And Fisheries
The nearshore fisheries have been dramatically altered since 1920 in term of species composition and reduction of fish stocks. At the beginning of the century, fishing efforts were determined by subsistence requirements using traditional gear (Acere 1988), which was subsequently replaced by the introduction of cotton, nylon, and multifilament gill nets. The Lake Victoria fisheries, as presented by Graham (1929) and Fryer and Iles (1972), described Oreochromis esculenta and O. variabilis, over 250 species of haplochromine species, mormyrids, catfish, and cyprinids as major contributors to the commercial landings. The introduction of gill nets resulted in a drop in catch per unit effort and initiated overfishing conditions (Acere 1988). It was accompanied by the introduction of four species of exotic tilapias, Ti1apia zilli, O. niloticus, O. Ieucostictus, and O. mossambicus. The objectives of these introductions were to increase fish production. Nevertheless, since then the
fisheries, in terms of catch per effort, has fluctuated (Garrod 1961).
TABLE 1 Fish Landings in the Kenya Waters of Lake Victoria from 1969 to 1989 (Percents of Total Tons)
Genus
Lates
Haplochromis
Rastrineobola
Tilapia
Clarias
Bagrus
Protopterus
Schilbe
Alestes
Barbus
Labeo
Mormyrus
Synodontis
Small mixed
Prior to the introduction of the Nile perch (early 1960s), haplochromine fishes were the economic basis for the local fishery, as well as the dominant protein resource for human consumption. Detritivore and planktivore fishes were major prey species for the piscivore Nile perch (Fryer 1973, Ogari and Dadzie 1988). In 1976, only 0.5% of the commercial catch in the Kenya waters of Lake Victoria was Nile perch; by 1983 it was up to 67.7%. Haplochromines in the catches varied between 21.6 and 36.8% during 1968-1979 and 0.8% in 1985 (Table 1) (Ogutu-Ohwayo 1985). The landings of other native nearshore species like Tilapia, Protopterus, and Clarias declined as well (Okemwa 1981, FAO 1987). The present drastic reductions in nonpredatory commercial fishes (Table 1) predictively will be accompanied by a decline in Nile perch (Ssentongo and Welcomme 1985) due to its cannibalistic behavior (Barer et al. 1985, Ogari 1985).
Trawl surveys during the 1970s in Lake Victoria (Kudhongania and Cordone 1974, Benda 1981, Muller and Benda 1981) indicated large stocks of haplochromines and several endemic fishes in the nearshore and offshore (Table 2). Trawl fishing during the 1980s (Asila and Ogari 1988, Okemwa 1984) indicated that endemic cyprinids, catfishes, lungfishes, and mormyrids were too scarce to support a significant fishery (Table 2). Consequently, it was concluded that both haplochromines and tilapiines in Lake Victoria were commercially extinct (Balon and Bruton 1986, Ribbink 1987, Les Kaufman, personal communication). The decline of haplochromine and tilapiine fishes resulted not only in a reduction of commercial catch, but also in a disruption of trophic dynamics in the Lake ecosystem. These fishes were consumers of the dominant and bloom-forming algae and detritus and their high feeding capacity prevented water quality deterioration. Their absence was partly responsible for recent algal
blooms (Ochumba and Kibaara 1989) and detritus accumulation in the deep layers, followed by an increase in anoxia.
TABLE 2 Trawl Catches of Dominant Species from 1969 to 1990 (Kg per Haul)
Species
(19 hauls)
(69 hauls)
(167 hauls)
(54 hauls)
(41 hauls)
Bagrus docmac
Clarias gariepinus
Haplochromis spp.
Labeo victorianus
Lates niloticus
Protopterus aethiopicus
Schilbe mystus
Synodontis spp.
Oreochromis variabilis
Oreochromis niloticus
Rastrineobota argentea
x
x
x
x
x -- not recorded, or not available in trawl.
The stocking of the exotic species in Lake Victoria increased the total catch (Welcomme 1966, Fryer 1973), but had negative impacts on the lake ecology. The increase of stocked species populations and catches caused a decline in the haplochromine, tilapiine, and catfish landings Table 1), with which several of the exotic species compete for food resources. We know of no previous comprehensive study of the impact of exotic fish introductions on the ecosystem like that of Lake Victoria Fryer 1973). However, environmental changes caused by the introduction of exotic species have been observed in several lakes after the occurrence of irreversible changes in the food web, as reported from Laurentian Great Lakes (Smith 1968, 1972), various Scandinavian lakes (Svardson 1976), Lake Tahoe (Morgan et al. 1978), and Lake Kinneret (Gopher et al. 1983).
Changes In The Catchment Area
Changes in the Lake Victoria catchment basin (Bugenyi and Balirwa 1989, Kendall 1969) include the construction of a drainage system, vegetation removal, soil erosion, increased livestock, and recreational and industrial developments. The most severe threat is the vegetation removal by tree cutting (Harking 1987) for agricultural land development and for charcoal and firewood production. The Kenyan part of the catchment area is populated by 42% of the country's population and is drained by several rivers. Pollutants and wastes from urban centers, industries, and agricultural farmland (Ochumba 1984) are flowing into Lake Victoria via these rivers and direct runoffs (Allabaster 1981).
Natural conditions in the catchment provide high river discharges. Therefore, the impact of pollutants on the aquatic communities in the rivers and the lake itself are not effectively nullified (LBDA 1984). The degree to which these pollutants might be harmful cannot be adequately assessed because of the absence of long-term water-quality data. There are several localized parts of Lake Victoria where pollution impacts are significant and fish kills, as well as algal blooms, have been recorded (Ochumba and Kibaara 1989, Ochumba, 1990). Chabeda (1982) pointed out that loads of drained pollutants from wheat fields contain higher levels of nutrients compared to those from sugar-producing areas. River studies (LBDA 1984) indicated a general decrease of water quality from the upper catchment areas to downstream sections. The average annual nutrient input from the catchment of the Kenyan pan is 20 kg/kmē/yr for total phosphorus and 400 kg/kmē/yr for total nitrogen.
Present Kenyan water management legislation is administered by several governmental institutions (Moore and Christy 1978) with relatively low levels of coordination:
-The Ministry of Water Development controls water use permits and pollution prevention.
-The Ministry of Health is responsible for traditional nuisance abatement, water supply protection, and pesticide control.
-The Ministry of Agriculture is responsible for soil erosion control, provision of food, and chemical and toxic substances utilization.
-The Chief's Authority coordinates the local administrations to issue instructions for a reforestation program and the prevention of stream pollution.
-The Ministry of Environment and Natural Resources is responsible for watershed management. Efforts are under way at present that focus on pollution control, and on achieving the statutory basis for environmental impact assessments.
The influence of modifications in the catchment area on fish stocks in Lake Victoria is difficult to estimate. Nevertheless, these effects are apparently significant (Marten 1979, Benda 1979, Barel et al. 1985). Early studies show that the potamodromous fish species were more abundant in the rivers, but their densities are very reduced at present. Ancient commercial fisheries in Lake Victoria were based on migratory species that moved upstream to spawn during the rainy season when rivers were flooded (Whitehead 1958, 1959, Corbet 1961). The abundance of these fish has declined dramatically and this fishery has effectively been destroyed (Whitehead 1958, Balirwa and Begenyi 1980). The construction of dams, unrestricted dumping of industrial and agricultural wastes and other pollutants in the catchment area, reduced flows and increased sediment accumulation accompanied by deforestation, and draining of marshes and swamps has brought about the decline of this fishery. Soil
erosion and increased concentrations of suspended matter also reduced algal photosynthesis and consequently fish productivity (Meadows 1980). The stocks of riverine fish populations are now at their lowest levels since the early 1960s (Rabuor 1989). Consequently, fishing potential has declined significantly, accompanied by a direct negative impact on fishermen's families, who rely on fisheries for their livelihood from Lake Victoria. There is an urgent need for effective efforts aimed at conservation of the native fish communities, accompanied by stocking operations of the endemic species (Evans et al. 1988).
Eutrophic Status Of Lake Victoria
Studies on the water quality of Lake Victoria revealed many uncertainties (Ochumba 1987). The lake is acting as a sink for most of the imported contaminants from inflowing waters that accumulate in the ecosystem, by increasing concentrations in the lake water, in the sediments, and in the biota, especially fish. About 85% of inflowing water into Lake Victoria evaporates (Talling 1966). A few comprehensive studies of water quality were carried out 30 years ago focusing on substances, but not on organic matter and heavy metal contamination. The chemical composition of lakes and rivers in Africa is predominantly controlled by atmospheric conditions (Kilham 1990). Studies of Lake Victoria's trophic status are scarce, whereas studies of fishery biology (mostly in bays) are more numerous. Recent fish kills have brought to the attention of the authorities the possible increased eutrophication of Lake Victoria.
TABLE 3 Summary of Comparative Limnological Data from Various Authors on Lake Victoria
(Range Given)
Feature
Author
Talling
Akiyama
Melack
LBDA
Ochumba/
Hecky/
et al. 1977
Kibaara 1989
Mungoma 1990
Phosphate phosphorus(PO4P) ,ug/l
Nitrate nitrogen(NO3N) ~g/l
Sulphate sulphur(SO4S) mg/l
Silicate silica(SiO2Si) mg/l
Secchi disctransparenc y m
Primary productivity mg O2/m3
Chlorophyll concentrations ~ĩg/l
Algal dominance
diatoms
diatoms
diatoms
cyanophytes
cyanophytes
cyanophytes
Zooplankton dominance copepods
Thermocyclops
Recent comparative studies on the limnology of Lake Victoria (Table 3) emphasized the eutrophication processes (Akiyama et al. 1977, Hecky and Mungoma 1990, Ochumba and Kibaara 1989). We identified substantial increases in chlorophyll (3-10) and primary productivity (2-3) and substantial decreases in silica concentration (5-10). Sulphate concentrations are 10 times lower than the lowest concentrations measured in other large lakes in the world (0.1 mg/l). There has been a shift in the phytoplankton community towards nitrogen fixing blue-green species, fewer green algae, and the diatomid Stephanodiscus (Kitaka 1971). The zooplankton community is currently dominated by small-bodied species of copepods and cladocerans with low densities (Gopher et al., unpublished data).
TABLE 4 Dissolved Oxygen Concentrations (ppm) in "Bottom" Waters at Open Lake Locations
Date
Location
Depth
Total
DO2
Reference
Sampled (m)
Depth (m)
(ppm)
Jan. 61
deep north sta
Talling 1966
Feb-May 54
deep north sta
Fish 1957
Feb. 58
fake transect
Newell 1960
Feb. 61
deep north sta
Talling 1966
Feb. 69
N-S lake transect
Kitaka 1971
Mar 61
deep north sta
Talling 1966
Mar 67
N-S lake transect
Kitaka 1971
Mar 85
"open"
Ochumba & Kibaara 1989
Mar 89
Sta 32
NURP 1989
Mar 89
Sta 33
NURP 1989
Mar 89
Sta 100
NURP 1989
Mar 89
Sta 103
NURP 1989
Apr 61
deep north sta
Talling 1966
May 61
deep north sta
Talling 1966
May 86
Sta 32
Ochumba & Kibaara 1989
May 86
Ochumba & Kibaara 1989
Jun61
deep north sta
Talling 1966
Jun-Aug 84
Sta 32
Ochumba 1984
Jun-Aug 84
Sta33
Ochumba 1984
Table 4 represents anoxic cases measured in Lake Victoria (see also Fish 1957). In Kenya, anoxic waters have recently been found at shallower depths than before, suggesting an increase of oxygen demand in the hypolimnion. A eutrophication process, whose causes need to be determined, is now clearly evident. The decimation of the haplochromine fishes and endemic tilapias following the introduction of Nile perch may have profoundly altered the trophic status of the lake.
Conclusion
Lake Victoria exhibits unquestionable symptoms of eutrophication, including decreased water transparency, increased blue-green phytoplankton blooms, elevated nutrient concentrations, and hypolimnetic deoxygenation. Changes in the phytoplankton community altered the availability of food sources to primary consumers, although these grazers were considerably suppressed by higher trophic levels. However, the most important impact on water quality came about through fish introductions that modified the phytoplankton, zooplankton, and fish assemblages in Lake Victoria. Lake Victoria fish species are threatened by the worsening conditions in the lake itself and from the rivers in the catchment basin. Urban, agricultural, and industrial pollution, as well as the introduction of predatory Nile perch and competitive tilapiine species are suggested as the major factors for the deterioration of Lake Victoria's ecosystem. Oreochromis esculentus is almost extinct and many other
haplochromid species are endangered. Current fisheries legislation and efforts aimed at reduction of soil erosion and sedimentation in the inflowing rivers in the catchment basin are insufficient for significant improvement of the present deterioration of the lake's water quality. Also, localized manual harvest of papyrus, shoreline dredging, and sand mining have not improved lake conditions.
Stocking the lake with large-bodied plant-grazing cichlids that are less vulnerable to predation by Nile perch is recommended to increase forage pressure on algae and detritus and reduce their densities. In addition, long-term monitoring of the lake and its tributary rivers will be required to enable administrative agencies to make decisions with regard to protecting the Lake Victoria fisheries resources.
Acknowledgments
We thank Messrs E. Okemwa and J. Ogari who provided facilities for fieldwork. Background information was provided by Les Kaufman and W. Cooper. Funding was provided by USAID Grant No. DPE-5544-G-SS-7075-00. The authors also thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
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Percid Fish As A Tool For Restoration Of Reservoir Ecosystems, Improvement Of Water Quality, And Optimization Of Fishery Yield
M. Zalewski
Department of Applied Ecology, University of Lodz
Lodz, Poland
Abstract
In temperate lowland reservoirs, percid fish are one of the most important components of the ecosystem. They control the density of the large filter-feeders, such as Daphnia, which in turn eliminate algae and thus improve water quality.
Over a six-year period, the density of young of the year (YOY) perch was studied in a lowland reservoir. Yearly densities were established in correlation with late spring water levels when littoral zone resources were available. In years of high YOY perch densities, there were sharp reductions in large zooplankton and increases in phytoplankton. These conditions were detrimental to water quality and reduced the growth of YOY perch and pike-perch, which in turn reduced their winter survival rates. Further, the elimination of YOY pike-perch reduced their predatory control of pelagic plankton-feeding cyprynids and negatively influenced the zooplankton:phytoplankton ratios in the reservoir.
This study demonstrated that water quality and fish yields in eutrophicated lowland reservoirs can be restored, to a large extent, by the control and enhancement of percid fish.
Introduction
From a historical perspective, three overlapping stages can be distinguished in the development of ecology as a science. The first was focused on a description of ecosystems. The second began when a certain amount of knowledge was acquired about the structure of ecological communities and different levels of their organization. This stage focused on analysis of ecosystem dynamics in terms of the density of plants and animals and their biomass and energy content. Progress in the first two stages gave ecologists a certain predictive ability, which allowed them to start attempts at the third stage, i.e., the manipulation of the biotic structure of ecosystems for the purpose of directing the ecological process in ways that enable optimal utilization of the resources of that ecosystem by humans.
An example of such a sequence of events is a biomanipulation theory, pioneered by Hrabacek et al. 1961. This concept is based on the assumption that large filter-feeders (Daphnia) in most situations are able to maintain the biomass of phytoplankton at a low level, thus maintaining water quality. However, the drawback of this process is that Cladocerans are a most attractive food for many fish species.
The potential improvement of the water quality by biomanipulation is especially important in the case of reservoirs, which are the main sources of drinking water for large human communities. In the case of reservoirs, two groups of fish are supposedly the most efficient in eliminating filter-feeders and nektonic zooplanktivores, such as Alburnus alburnus, and juvenile perch and cyprinids. In most temperate reservoirs, the juvenile fish play the prevailing role in this process because there usually exists a strong population of efficient pelagic predatory pike-perch, which efficiently control the density of planktivorous pelagic species. The juvenile fish are less vulnerable to predation at some stages because their preferred habitat is in the shallow littoral zones with dense vegetation (e.g., flooded terrestrial vegetation not easily accessible for pike-perch). Biomanipulation can improve water quality by the "top down effect" (Northcote 1988). This means that through the
introduction of predators, planktivorous fish pressure on large filterfeeders (Cladocerans) is reduced. Until now this has been done mostly in lakes; however, experimental evidence exists that it can be successful in some types of reservoirs. Fish fry may be the most important factor in reducing Daphnia density (Holcik 1977, Zalewski et al. 1990a) and the density of fish fry (reproductive success) is dependent on water level (Martin et al. 1981, Zalewski et al. 1990b), i.e., through shoreline habitat and food resource utilization (Ploskey 1986).
From the trophodynamic point of view, efficiency of biomanipulation increases when the number of trophic levels between the level manipulated and phytoplankton decline (McQueen 1990). Also, biomanipulation has a greater chance of success at low and intermediate levels of eutrophication, and with the physically unstable epilimnion which drives algal communities into the early successional stages (Reynolds 1987). At this stage, algae are usually characterized by small forms such as Chlorococcales and Bacillariophyceae (Puchalski 1990), which are easily filtered by Cladocerans.
All three of the conditions cited above can be fulfilled to a greater extent by reservoirs than by lakes. This is because in reservoirs the density of zooplanktivorous fish fry can be controlled by water level manipulation. Eutrophication can, to a certain extent, be slowed down by release of nutrient-rich hypolimnic waters. In addition, the physical instability of the epilimnion may be enhanced not only by wind action, which can be amplified by reduction of shoreline ecotone complexity, but also by pulsing water release.
PERCID FISH AS POTENTIAL TOOL FOR
RESTORATION OF RESERVOIR ECOSYSTEMS
Reservoirs are traps for nutrients carried down by the river; thus, they possess an inherent tendency to eutrophicate. The biomanipulation technique of restoring water quality by manipulating biotic components increases the density of large efficient filtrators such as Daphnia, thereby reducing manyfold the concentration of phytoplankton. To achieve this effect, the planktivorous fish (cyprinids and perch) densities must be reduced by predators, such as pike-perch or walleye. This process, in terms of energy flow through the trophic structure of an ecosystem, can be described as reconstruction (restoration) (Figure 1).
In oligotrophic systems (good water quality, low production) the largest portion of energy is diverted from primary producers (algae) to subsequent trophic levels (planktivorous and predatory fish). In eutrophic systems, characterized by high primary productivity, most of the phytoplankton production is not consumed by filtrators (Figure 1). This is because large Daphnia are eliminated by dense populations of juvenile percid and cyprynid fishes (Zalewski et al. 1990b).
The dead algae nutrients are recirculated by the "microbial loop" and a continuing high nutrient supply maintains concentrations of algae, resulting in deterioration of water quality. Benthic detritivores may be stimulated in the system as long as the bottom oxygen supply of the reservoir is sufficient. In such a system (Figure 1) the large part of the energy is accumulated in bottom sediments leading to a decrease of the aquatic habitat quality. Figure 1 demonstrates the restoration of a reservoir by enhancing the predatory pike-perch, which reduce pelagic, zooplanktivorous fish populations (in this case perch, Perca fluviatilis). In reservoirs, pike-perch or walleye is the most important biomanipulation tool because the second important predator of temperate zone lakes, the northern pike, Esox lucius, needs a diversified littoral zone as an optimal habitat. This rarely exists in reservoirs due to water level fluctuations.
On the basis of initial research (Zalewski et al. 1990a,b) and other published data (Gulati et al. 1990), it can be hypothesized that, in reservoirs, the most efficient way to restore water quality and fish yield would be to enhance pike-perch populations to control pelagic zooplanktivorous fish. From a dynamic point of view, such restoration can be described in terms of changes of the energy flow pattern from detrital food chain characteristics of eutrophic ecosystems (Figure 1A) to mesotrophic, high water quality and high fish yield ecosystems (Figure 1B). Using pike-perch or walleye as a biomanipulating tool, it may be possible to restore conditions of the ecosystem. If true, the advanced stage of eutrophication in many reservoirs can be reversed, or at least seriously reduced. Restoring aquatic habitat quality should provide time to create strategies for the conservation of the catchment area. These strategies should include sewage treatment plants and the reduction of
aerial pollution.
Perch Recruitment, Water Quality, And Predator Dynamics
Some of the assumptions discussed above were confirmed in practice at a lowland reservoir in central Poland. The Sulejow Reservoir is situated in the middle course of the Pilica River, a tributary of the Vistula River. Its area is approximately 20 kmē and the average depth is 3.3 m.
Galicka and Penczak (1989) evaluated annual nutrient loadings at the beginnings of the eighties at a level of 30 g N (total) and about 5 g total P per mģ. Detrital sedimentation occurs mostly in the upper eutrophic part of the reservoir and is separated from the main part by a kind of bottleneck. The oxygen level is usually high due to water mixing by wind and algal photosynthesis. In 1984, a stable high water level during spring and summer was responsible for the demographic explosion of perch and cyprinids, which are major components of the fish community. Analysis of data on perch fry densities in the littoral zone and in changing water levels during the spawning and post-spawning periods (May, June, July) demonstrated a correlation between an "area stability index" (As) (Zalewski et al. 1990b) and perch fry density (Figure 2).
During the year of the highest and most stable water level (1984), a high density (mean 37 per mē) of perch fry was observed in the shoreline zone. At that time the zooplankton density was drastically reduced compared to former years (1982, 1983) when perch density did not exceed 5 per mē (Zalewski et al. 1990 a,b). This reduction of zooplankton, mostly large Cladocerans, was reflected in the stomach contents of the perch and in their foraging strategy. During the year of low perch density, when Cladocerans were abundant, a distinct feeding peak (stomach contents [weight] measured as a percentage [4%] of perch fry body weight) occurred in the evening when Cladocerans migrate to the surface and to the littoral zone (Figure 3). But during the year of very high perch fry density, they fed continuously, almost 24 hours a day, and drastically reduced the density of Cladocerans (Figure 4b). At that time, the perch fry were eating fewer Copepods and perch stomachs were only
approximately 1% full (Figure 3).
The drastic reduction of Cladoceran biomass was reflected in water quality. During the year of high density of filter feeders (1983), the amount of suspended matter doubled, despite the fact that Cladoceran biomass decreased almost eight times. Consequently, the ratio of zooplankton changed from 2:1 (1983) to 1 :15, because of an intensive algal bloom. Obviously the sharp reduction in the availability of optimal food was reflected in the perch growth rate (Figure 3). In years when average density was below 8 perch specimens per square meter of littoral zone (e.g., 1983), the Cladocera population was not severely reduced (Figure 4a) and served as a main food component, and helped maintain a high juvenile perch growth rate. When the perch fry density exceeds approximately 10 specimens per mē, a sharp decline in the instantaneous coefficient of growth was observed. This phenomenon was a result of a break in the dynamic equilibrium of Cladoceran population density and a shift in
perch fry diet to non-optimal prey, Copepods and benthos. These bad feeding conditions were confirmed by broad size ranges of juvenile perch and cannibalism observed among the O+ age group.
The sharp reduction in large zooplankton density was reflected in a greater pike-perch fry growth than that in perch. In mid-July 1982, they achieved 5.5-8 cm, but in 1984 only 3.3-4.2 cm. In both years the temperature ranges and patterns during late spring and early summer were similar. The fry sizes always decreased upstream in the more eutrophic part of the reservoir. The very poor growth of juveniles in 1984 was due to lack of optimal prey, Cladocerans. As a result, pike-perch were late in achieving the size at which they become piscivorous (3.8 cm). The potential prey-fry of perch and cyprinids were too large at that time; consequently, 43% of the pike-perch had empty stomachs, and all were in poor condition. It is a well-known phenomenon that poor fry growth during summer can lead to low winter survival. Loss of one generation of this easily overexploited, short-lived species seriously reduced its populations for many years. The elimination of predator control in the
fish community of a reservoir, due to intraspecific competition at the early fry stages, may have great effect on the dynamics of pelagic planktivorous fish, which in normal years are at very low densities because of efficient pike-perch predation. Pelagic zooplanktivores may react to a sharp decline in predator numbers by a rapid increase in density and increased feeding pressure on zooplankton. In addition, such-fast growing populations of pelagic fish, in contrast to perch fry communities, will be impossible to control by water level changes. These results are summarized in Figure 5, and clearly show that in a lowland reservoir it is possible to achieve concordant maintenance of water quality and fishery yield by control of water level. However, it should be expected that this regularity will be disturbed when the structure of upper trophic levels is sharply modified (e.g., by the drastic reduction of the main predator) and a shift occurs in the main mechanism regulating
zooplanktivorous fish pressure on filter feeders in the shoreline ecotone and open water areas.
Discussion
Looking at this process in a broader context, one can say that the general mechanisms described can be expressed in terms of general ecological theory. Fish tend to be "r" selected organisms, which means they have an ability to provide large numbers of offspring when environmental conditions are favorable.
Usually, in ecosystems where abiotic control mechanisms prevail (Zalewski and Naiman 1985) biotic structure is simple and feedback control mechanisms, such as predation and intraspecific competition, are not well balanced.
In the case described above, the perch population took advantage of the high water level, and with its opportunistic reproductive style (Baron 1975) it dominated energy flow and nutrient distribution, which put the shoreline ecotone in an early succession stage of an aquatic ecosystem. These data demonstrate that the explosion of one species may occur when even part of an ecosystem is modified. Such a warm, highly diversified, trophically rich ecotone, without specialized predators, is created periodically in reservoirs as a result of water level changes. This is especially true in lowland reservoirs where a relatively small water level increase creates large inundated areas rich in food that are warm and safe from limnetic predators. The above data confirm the hypothesis of Naiman et al. (1989) about the importance of land-water ecotones in landscape process dynamics.
This process also supports the view of Patten and Odum (1981) concerning the cybernetic nature of ecosystems, that is, an advantage achieved by one species in a relatively mature system (10-year-old reservoir) may occur only when abiotic factors are changing the dimensions of the resources. Thus, this would be an ecotone without predatory pressure, and with a diversified and rich food supply (Ploskey 1986). An analogous reaction of species to the extension of resources has also been described for another species, Perca flavescens (Thorpe 1973).
Despite the great reproductive success of perch in 1984, which was a rather catastrophic event from the point of view of the homeostasis of an ecosystem, the ecosystem still possesses a tendency toward equilibrium. This was expressed by a sharp reduction in growth that usually results in a mass winter mortality of any cohort that exceeds the carrying capacity of the given environment. In a stable and trophically limited ecosystem, such as Scottish lakes, the perch population demonstrates a feedback control of its own population density by cannibalism. The juvenile perch form 14-30% of the adult fish diet (Thorpe 1977).
The sharp reduction of basic food resources by one species, described above, that resulted in densities exceeding the carrying capacity of its environment, also impacted on the predators' recruitment and long-term population density. Under natural conditions, pike-perch occur as a multigenerational population and thus, presumably, do not react sharply to a population reduction in one generation. However, in the Sulejow Reservoir, intensive angling pressure seriously reduces the life span of the predator population. Thus, human impact amplifies the ecological effect of the resource's reduction by competing species.
Juvenile pike-perch are especially vulnerable to a decline in food resources, because juvenile
percids consume up to 32% of their body weight in food a day. Thus, a reduction in the density of
crustaceans below 100 per lifer stimulates high percid mortality (Li and Mathias 1982). That is why
stocking of good quality pike-perch, above 4 cm in length, that can feed on cyprinid fry might be of
great importance for enhancement of this species and controlling the density of plankton-eating fishes.
Finally, does the relation described above, involving water level, fry density, and water quality, occur in all types of reservoirs and make the manipulation of the biotic structure of reservoir ecosystems a future management tool for water quality and optimization of fishery yield? Obviously, the answer depends on the type of reservoir. The shape of the reservoir valley, complexity of ecotonal vegetation cover, and eutrophication stage will be the major factors. These will determine the shift in resource dimensions during water level changes that affect fish community dynamics, as well as the "top down" effect resulting from water quality.
Acknowledgments
The author thanks the Office of Research, USAID, for funding the network meeting and the
publication of this paper.
References Cited
Balon, E.K. 1975. Reproductive guilds of fishes: a proposal and definition. J. Fish. Res. Board Can. 32:821-864.
Galicka, W. and T. Penczak. 1989. Total nitrogen and phosphorus budgets in lowland Sulejow Reservoir. Arch. Hydrobiol. 117: 177-190.
Gulati, R.D., E.H.R.R. Lammens, M.-L. Meijer, and E. van Donk. (ed.). 1990. Biomanipulation, tool for water management. Proceedings of an international conference held in Amsterdam. Hydrobiologia 200/201: 1-628.
Holcik, J. 1977. Changes in fish community of Klicava Reservoir with particular reference to Eurasian perch (Perca fluviatilis), 1957-72. J. Fish. Res. Bd. Can. 34:1734-1747.
Hrabacek, J., M. Dvorakova, V. Korinek, and L. Prochazkova. 1961. Demonstration of the effect of the fish stock on the species composition of the zooplankton and the intensity of metabolism of the whole plankton association. Verh. Int. Ver. Theor. Angew. Limnol. 14:192-195.
Li, S. and J.A. Mathias. 1982. Causes of high mortality among cultured larvel walleyes. Trans. Am. Fish. Soc. 111:710-721.
Martin, D.B., L.J. Mengel, J.F. Novotny, and C.H. Walburg. 1981. Spring and summer water levels in a Missouri River reservoir. Trans. Am. Fish. Soc. 110:370-381.
McQueen, D.J. 1990. Manipulating lake community structure. Where do we go from here? Freshwater Biol. 23:613-620.
Naiman, R.J., H. Decamps, and F. Fournier. (ed.). 1989. The role of land/inland water ecotones in landscape management and restoration: a proposal for collaborative research MAB Digest 4. Unesco, Paris. 93 pp.
Northcote, T.G. 1988. Fish in the structure and function of freshwater ecosystem: a "top-down" view. Can. J. Fish. Aquat. Sci. 45:361-379.
Patten, B.C. and E.P. Odum. 1981. The cybernetic nature of ecosystem. Am. Nat. 118:886-895.
Ploskey, G.R. 1986. Effects of water-level changes on reservoir ecosystems with implications for fisheries management. pp. 86-97. in Hall G.E. and M.J. Van Den Avyle. (ed.). Reservoir Fisheries Management: Strategies for the 80s. Reservoir Committee, Southern
division. American Fisheries Society. Bethesda, Maryland.
Puchalski, W. 1990. Eutrophication and stress in physically stable and unstable lakes: Effects on phytoplankton structure and seasonality. Verh. Int. Ver. Theor. Angew. Limnol. 24:909-912.
Reynolds, C.S. 1987. The response of phytoplankton communities to changing lake environments. Schweiz. Z. Hydrol. 49:220-236.
Thorpe, J.E. 1973. Synopsis of biological data on the perches Perca fluviatilis L. and P. .flavescens Mitchill. FAO. U.N., Rome.
Thorpe, J.E. 1977. Daily ratio of adult perch, Perca fluviatilis L. during summer in Loch Laven, Scotland. J. Fish. Biol. 11:55-68.
Zalewski, M. and R.J. Naiman. 1985. The regulation of riverine fish communities by a continuum of abiotic-biotic factors. pp. 3-9. in Alabaster, J.S. (ed.). Habitat modification and freshwater fisheries. Butterworths, London.
Zalewski, M., B. Brewinska-Zaras, and P. Frankiewicz. 1990a. Fry communities as a biomanipulation tool in a temperate lowland reservoir. in Lind, O.T., V. Sladecek and V. Staskrabova. (ed.). Proceedings of the International Conference on Reservoir Limnology, eutrophication, water quality and its prediction. Arch. Hydrobiol. Beih., Ergebn. Limnol. 33:763-774.
Zalewski, M., B. Berwinska-Zaras, P. Frankiewicz, and S. Kalinowski. 1990b. The potential for using fry communities in a lowland reservoir: concordance between water quality and optimal recruitment. pp. 549-556. in Gulati, R.D., E.H.R.R. Lammens, M.-L. Meijer and E. van Donk. (ed.). Biomanipulation, tool for water management. Proceedings of an international conference held in Amsterdam. Hydrobiologia 200/201:1 -628.
<section>Presentation: Schistosomiasis</section>
<section>Immunology</section>
<section>Schistosomiasis: An Immunological Disease</section>
S.M. Phillips
University of Pennsylvania School of Medicine Philadelphia, Pennsylvania
Abstract
Schistosomiasis, a helminth infection, affects an estimated 200 million people. The complex life cycle is reflected by equally complex clinical manifestations. However, in this context, it is reasonable to regard schistosomiasis as an immunological disease. The four major stages of the life cycle: skin penetration; transpulmonary migration; initial mating and egg deposition in the vascular space; and chronic infection, are associated with discrete cutaneous, pulmonary, vascular, and hepatosplenic forms of disease.
Skin penetration is associated with a variety of immuno-inflammatory responses, but the pathology is dominated by acute IgE or Type I immunological reactions. The pulmonary migratory phase is associated with both Type I and Type III immunological reactions involving acute IgE mediated responses as well as interactions contingent on the formation of circulating and in situ immune complexes. Acute gastrointestinal schistosomiasis is primarily a Type III or immune-complex vasculitis. The chronic form of schistosomiasis, or hepatosplenic disease, is characterized by granuloma and chronic fibrosis. This form of the disease, responsible for principal morbidity in schistosomiasis, is primarily contingent on Type IV or cell mediated T cell dependent immune responses. Teleologically, the wide spectrum of immunologic responses to the parasite may be interpreted as representing the body's attempt to optimize protective immunity and immunopathologic responses to the parasite.
Introduction
Schistosomiasis is a disease of tremendous international significance affecting an estimated 200 million individuals. The study of schistosomiasis has addressed many basic immunological questions including immune evasion mechanisms, eosinophil function, and granuloma formation (Phillips and Fox 1984a). It is of interest that, reviewed mechanistically, one may explain the clinical manifestations of disease based on precisely defined immunological phenomena (Phillips and Pox 1984b). Moreover, immunologic techniques have been used to illuminate many of these reactions.
The Cell And Coombs Classification
Classically, immune effector mechanisms have been divided into four categories (Brostoff et al. 1991, Male et al. 1991).
1. Hypersensitivity. The first form of immune reactivity is an acute immunoinflammatory reaction caused by the interaction of IgE antibody, bound to the surface of mast cells, and specific ligands or allergens. As a result of this surface interaction, the mast cell is triggered to release a wide variety of mediators including histamine and products of the arachadonic acid pathways. This, in turn, results in the classic manifestation of the wheel and flare reaction, an acute inflammatory response, which characterizes ailments such as asthma, hay fever, and hives.
2. Cytotoxic antibody reactions. Antibodies possessing cytotoxic or Iytic activity interact with the surface of living cells, which results in their death. A number of mechanisms are postulated; however, common to all of these is an informational bridge between target and effector cells or molecules that is formed by the antibody. Immunologic specificity rests in the Fb fragment and an executive cell binding capability on the Fc fragment.
3. Circulating immune complex reactions. When an immunogen is introduced into the body it results in the production of antibodies. These antibodies combine with the circulating antigen to produce circulating immune complexes. These complexes, in turn, fix vasoactive amplifying proteins (such as complement) and result in the activation of a number of inflammatory reactions of the level of the endothelium. This reaction produces a variety of clinical diseases, loosely classified as vasculitis.
4. Cell mediated immunity. T cells bearing receptors for specific antigens migrate to the site of these antigens and are stimulated to undergo blast transformation, division, and subsequent differentiation. As a consequence of this differentiation the cells produce a number of recruiting Iymphokines or cytokines. These molecules then activate other mononuclear cell populations and result in the generation of a delayed hypersensitivity reaction or granuloma.
Schistosomiasis: Clinical Spectrum
As illustrated in Table 1, the clinical manifestation of the migratory cycle of the schistosome can be divided into four phases: (1) skin and penetration phase; (2) intermediate pulmonary/hepatic migratory phase; (3) acute intermediate phase, associated with ova position; and (4) chronic stage, characterized by tissue proliferation and fibrosis. In light of the classification of immunological effector mechanisms described above, it is useful to look at these stages as forms of immunopathology.
1. Invasion: Schistosome Dermatitis. Schistosomiasis begins initially with penetration of the skin by cercariae. On initial exposure, awareness of the event is usually minimal. However, in the case of multiple exposures, sensations of pruritis and local erythema may occur within 15 minutes to 2-3 hours of penetration. When non-human schistosomes (that is, those which normally complete their life cycles in other animals) enter the skin of humans, a significantly greater dermal reaction is observed. A high percentage of these heterologous cercariae are quickly destroyed in the skin of the host. A local dermatitis that becomes increasingly severe may occur following recurrent exposure to the parasite. Papules appear in sensitized individuals within 5-15 hours post-entry. A wide variety of clinical manifestations, including erythema nodosum, arthus-like reactions, and exfoliation, may occur. Histologically, there is tissue edema with mononuclear and polymorphonuclear cells that
invade the dermis and epidermis. Although the mechanisms of this intense reaction are not clear, a number
TABLE 1 Spectrum of Schistosomiasis.
Stage of Infection
Clinical Manifestations
Pathologic Manifestations
Immunologic Mechanisms
Invasion:
Swimmer's itch
Cutaneous
IgE, IgG,ADCC,
Skin penetration and dermatitis
inflammation
CM
Migration:
PIE, cough, fever
Pulmonary/hepatic inflammation
IgE, IgG,ADCC
Transpulmonary
Maturation:
Initial oviposition
Katayama fever, serum sickness
Intense local and generalized vasculitic reaction
CIC, ADCC
Chronic: Initial
Intense oviposition with maximum egg production and excretion
GI, symptoms, hematuria, vasculitis
Large granulomata
CIC
Initial deposition of matrix proteins
CMI (S.m.) B cell (S j.)
Chronic: Progressive
Prolonged infection with decreased egg production and excretion
Chronic disease, portal hypertension nephropathy CNS cor pulmonale
Modulated granulomata, Symmer's fibrosis hepatosplenomegaly
CMI (S.m.) B cell (S.j.)
'Abbreviations: ADCC, antibody dependent cell-mediated; CIC, circulating immune complexes; CMI, cell-mediated immunity; CNS, central nervous system; GI, gastrointestinal; PIE, pulmonary infiltrates with eosinophilia; S.j., Schistosoma japonica; S.m., Schistosoma mansoni.
of possibilities have been suggested. First, high titers of IgE and IgG antibodies have been described in humans with experimental acute dermatological reactions. These may result in an eosinophil-rich inflammatory response to the subsequent invading cercariae. The ability to block this initial reaction with antihistamines and the observation of mast cell degranulation in situ further support the implication of these mechanisms. However, subsequent skin biopsies have also demonstrated deposits of immune complexes and basophils along the capillary loops of the skin. Arthus-like lesions have also been observed. Cercarial extracts possess the ability to activate, complement directly, and produce chemotactic substances. In addition, the cellular infiltrate, the chronicity of the response, and the clinical response of corticosteroids suggest that there is a cell-mediated component as well.
2. Migration: Acute Pulmonary Schistosomiasis. Schistosome pulmonary infiltrates with eosinophilia (PIE) syndrome: 7-10 days after initial exposure, when the parasites are actively migrating through the lung, transient symptoms of cough, fever, and mild wheezing are sometimes observed. These events usually follow extremely heavy re-exposures to schistosomes. Chest x-rays at this time have demonstrated occasional transient pulmonary infiltrates and the patients manifest a peripheral blood eosinophilia. Thus, this syndrome qualifies as an additional cause of the PIE syndrome. Although extensive studies in humans are not available, studies in primates and mice have indicated that schistosomes migrating through the lung are associated with accumulations of significant numbers of eosinophils, granulocytes, and small numbers of mononuclear cells. A number of parasites are actually trapped in the lung where they are destroyed.
Recent evidence suggests that much of this destructive process is mediated through ADCC mechanisms involving a variety of immunoglobulin isotypes and effector cell subpopulations. Radiobiologic techniques have been used to trace parasite migration, quantitate antigen binding, and assess effector activation.
3. Maturation: Acute Gastrointestinal Schistosomiasis. The next clinical phase of schistosomiasis begins 20-40 days after exposure. This syndrome was originally described as Katayama Fever in Japan in the 19th century. This syndrome was often observed in newly arrived immigrants ("non-immures") to the fertile Katayama valley and indeed was responsible for such significant morbidity that customs on "non-migration" developed to obviate this occurrence. In later years the connection between this condition, "Katayama fever," and Schistosoma japonicum was established. The syndrome can occur with any schistosome infection, but is most common with S. japonicum, and perhaps is related to the higher number of infecting organisms and the greater metabolic activity, egg deposition, and antigen production of this parasite. The condition is occasionally seen with exposure to S. mansoni and rarely with S. haematobium.
Patients develop an acute onset of fever, chills, sweating, anorexia, headache, diarrhea, and cough. Clinically, the acute onset of hepatosplenomegaly, generalized Iymphadenopathy, and urticaria are also observed. A mild leucocytosis, associated with eosinophil counts of up to 50%, is expected. The fever may last for many weeks, at which point it will spontaneously subside. In severe cases, death has been recorded. In those cases, extremely high levels of parasites have been observed.
Although the etiology of Katayama fever is not clear, the overwhelming evidence would suggest that it is related to the sudden production and release of large quantities of parasite antigens at the onset of egg deposition. A number of studies have clearly shown that, although the immune response against schistosomes can begin very early, the levels of antibodies and circulating antigens are low until the eggs are deposited. These studies in humans are analogous with observations in animals. At this time, there is a progressive increase in the level of a number of antibody classes (IgM, IgE, IgG) as well as a circulating antigenaemia. It is postulated that the production of increasing amounts of antigen-antibody complex results in the production of a serum-sickness-like syndrome. The rapid release of antigen may produce immune complexes in slight antigen excess, which is the prerequisite for the generation of immune complex disease. Consistent with this hypothesis is the
observation that serum complement levels may fall during this syndrome. In addition, studies in humans and experimental animals have indicated that a significant number of individuals, infected both acutely and chronically with Schistosoma, had immune complex deposits within the kidney and associated nephropathy, most commonly with membrane-proliferative glomerulonephritis. These complexes are composed of schistosome-specific antigens, IgG, IgA, IgM, IgE, and a variety of complement components. It is of interest that, although the deposition of the circulating immune complexes in the kidney is a nearly ubiquitous phenomenon, the pathological consequences are relatively mild. Only 3-7% of the primates and certainly less than 10% of humans infected with S. mansoni develop clinically significant proliferative glomerulonephritis. In addition, the antibodies deposited in the capillary loops are not often found intimately associated with discrete parasite antigens, perhaps
indicating that the phenomenon is secondary to local membrane activation of the immunoglobulins. Perhaps even more interesting is the evidence for C3b activation, that is, local tissue activation of the complement sequence leading to augmentation of local pathology. The observation of a high incidence of local necrotic, egg-associated lesions is compatible with a mechanism that implicates the local activation of immunoinflammation and/or the release of toxic materials. Finally, the observation of anti-DNA antibodies and reactivity against collagen in schistosomiasis suggests the possibility of secondary "auto-immune" phenomena.
4. Chronic Schistosomiasis. Patients with chronic schistosomiasis experience subjective fatigue, abdominal pain, intermittent diarrhea, and dysentery. Histopathologically, the local deposition of embolized eggs within the vessels and an associated granulomatous reaction are observed. The distribution of the eggs determines the nature of the pathology. For example, S. mansoni tends to inhabit the mesenteric vessels; hence, the vast majority of eggs are embolized to the liver. Schistosoma haematobium inhabits the pudendal vessels; thus, eggs embolize preferentially to the organs of the lower pelvis. There is compelling evidence to suggest that the granulomatous reaction and resulting fibrosis in chronic S. mansoni schistosomiasis is the result of a T cell mediated immune response to the products of the egg. Histologically, the lesions resemble delayed hypersensitivity reactions. Moreover, immunosuppressants in humans and animals show decreased granulomatous hypersensitivity.
Additional studies have indicated that the formation and regulation of granuloma formation is highly contingent upon multiple subpopulations of cells that participate in a delicately orchestrated immunological battle. Although the stimulating epitopes, participants, and modes of interactions of the cells responsible for the generation and regulation of granulomatous hypersensitivity are not yet clear, T cell dependence of the phenomena is firmly established. As a result of this pathologic response, hepatosplenic pulmonary, urinary tract, and CNS pathology will ensue progressively. The chronic phase of schistosomiasis culminates in liver failure, hydronephrosis, and other complications of fibrosis.
Summary
Schistosomiasis represents a broad spectrum of clinical disease. Although many questions still exist as to the basis of the proposed immunopathologic reactions, it is clear that antibodies, antigens, circulating immune complexes, and cellular mechanisms are germane to the development of a wide variety of pathology. Current studies are investigating the subpopulations of cells and humoral factors that can pertubate the expression of immunopathology, and it is hoped that an increased knowledge of the basis of immunopathology will provide a possible source for future medical exploitation. For example, active immunization to produce reduced reactivity against antigens of the parasite has been suggested as a possible approach to limit pathology.
As yet, no specific immunological manipulations have been shown to be of definitive value in humans. Indeed, the tremendous variation of the clinical manifestations of parasitic diseases with pathology resulting from contributions of both the parasite and host makes these approaches difficult.
It would be useful also to view this variable host-parasite relationship and subsequent morbidity in a conceptual mode. In this theoretical construct of disease manifestation, certain hypotheses are required:
1. The total immune response ranges from hyper- to hypoergic poles. The spectrum is determined by the relative dominance of "helper" versus "suppressor" modulatory phenomena. The relative salience of a given parameter is contingent upon unique aspects of the host and parasite.
2. The hyperergic state tends to be dominated by the immunopathological consequences of host-dependent mechanisms with relatively good containment of the parasites. The hypoergic state is associated with relatively uncontrolled parasite growth and the pathological consequences are related to this phenomenon.
3. The total immune response may be divided loosely into two categories: those that control disease and thus minimize parasite-associated pathology, and those that produce pathology per se. These two aspects need not be consonant kinetically or mechanistically. The clinical disease is most salient when these two aspects of the immune response are most dissonant or when effective control of parasite growth is not attained.
Teleologically, the body ought to seek that homeostatic balance of immunological responses that will maximize resistance and minimize morbidity. Unfortunately, the optimal protective immunity and total immune reactivity, which includes immunopathological reactions, may not be consonant, thereby resulting in "unnecessary" immunopathology. Moreover, even if consonant, the protective immunological response may contribute to the spectrum of disease. If an intense immune response is required to contain disease, then this reaction may lead to associated tissue destruction (e.g., an "innocent bystander" effect). For instance, although there is generally good correlation of disease control and Delayed Type Hypersensitivity (DTH) competence in leprosy, large nerve damage will occur in polar Tuberculoid Leprosy (TL) and indeterminate or intermediate forms may actually show less morbidity than either polar form. Antibody titers rise progressively during conversion to the lepromatous
form and are actually negatively correlated with organism-associated pathology. A similar picture emerges in the spectrum of leishmaniasis and schistosomiasis. Conversely, protective variant-specific antibody appears to be more important than competent DTH in controlling African trypanosomiasis. Thus, although the dominant mechanisms of immunological response may vary, the ratio of protective versus pathological potential remains critical in determining diseases (Phillips and Fox 1984a,b).
The mechanisms that determine the relative efficiencies of these seemingly contradictory immunological mechanisms are largely enigmatic at present. They include: (1) unique genetically determined host immune responses; (2) the virulence of the parasite and relative effectiveness of initial host defense mechanisms; and (3) the development of secondary or modulating phenomena.
In summary, the immune response to the schistosome or any other parasite and its subsequent modulation is a two-edged sword. If the immune system reduces the consequences of parasite invasion, the response is beneficial and potentiation of that response would be desirable. However, if the immune response produces pathology, attenuation would be preferable. There is a growing realization that the factors which combine to orchestrate the immune response, ultimately determining both resistance and pathology, display a delicate balance of internal self-modulation. With the growing understanding of these modulating factors, there is every reason to hope that the therapeutic control of parasite-associated morbidity and mortality will be realized.
Acknowledgments
The author thanks the Office of Research, USAID, for funding grant number DHR-5600-G 00
1042 00, the network meeting, and the publication of this paper.
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Phillips, S.M. and E.G. Fox. 1984b. The immunopathology of parasitic diseases. p.421 in Marchalonis, J.J. (ed.). Contemporary Topics in Immunobiology. Vol. 12. Plenum Press, New York.
<section>Anti-Embryonation Immunity As A Granuloma Modulating Mechanism In Schistosomiasis Caused By Schistosoma Japonicum</section>
E.G. Garcia, G.F. Mitchell, W.U. Tiu And P.T. Rivera
Abstract
In a recent hypothesis we proposed that one of the key events in granuloma modulation is the inhibition of maturation or embryonation of the egg and its destruction at an immature (i.e., pre-miracidial) stage of development. One consequence of this anti-embryonation immunity is the failure of the egg to mature and produce immunopathologic antigens responsible for T-cell dependent granuloma formation. This hypothesis focuses on immune effects on the egg rather than on immunoregulation as the central factor in granuloma modulation.
The more important observations that support anti-embryonation immunity are: (1) maturation of fresh uterine eggs injected into the pulmonary vasculature of chronically eggsensitized mice, with modulated granuloma response, was inhibited; (2) sera taken from humans with chronic schistosomiasis injected into BALB/c mice reduced the rate of maturation of eggs deposited in the liver and intestines and reduced the size of granuloma in mice sensitized for accelerated granuloma formation; (3) BALB/c mice sensitized by repeated injection of viable immature eggs and challenged with few cercariae had at D+42 of infection significant increase of dead eggs and a decrease in the proportion of mature eggs to immature eggs in the liver and intestines; and (4) infected mice sensitized with immature viable eggs prior to the onset of egg-laying had at D+75 of infection lesser and smaller granuloma relative to controls with portal pressure and relative spleen weight comparable to uninfected
controls. We consider these findings as sufficient basis for the development of a vaccine to prevent hepatosplenic disease. An anti-embryonation vaccine will also be anti-transmission.
Introduction
Manifestations of disease from Schistosoma japonicum and S. mansoni result from granulomatous and fibrotic responses to antigens released from eggs entrapped in the liver, intestines, lungs, and other organs (Warren 1982). The more common and more serious clinical manifestations of hepatosplenomegaly, increased portal pressure, ascites, and collateral circulation are readily ascribed to obstructive granuloma formation around eggs with subsequent fibrosis and impedance to blood flow through the liver. In S. japonicum infection, as in S. mansoni infection, it is generally accepted that CD4 T-cells of the delayed hypersensitivity type are the principal initiators of these reactions. It has also been demonstrated that the mature or embryonated egg containing a miracidium is the source of the immunopathologic antigen {Hang et al. 1974, Hamburger et al. 1976, Mitchell et al. 1982). The speed and size of the granulomatous response is dependent on the duration and intensity of
sensitization with egg antigens. After initial sensitization, there is an accelerated formation of larger and more destructive granulomas. However, as the infection becomes chronic, or with prolonged sensitization, the size of granuloma formed is reduced or modulated (Domingo and Warren 1968, Olds et al. 1982, Garcia et al. 1983).
This phenomenon of granuloma modulation, previously called endogenous desensitization (Domingo and Warren 1968), opened the prospects for vaccination against disease. Immunization to promote modulated granuloma response could lead to a reduced likelihood of severe hepatosplenic disease in infections with S. japonicum or S. mansoni. The principal mechanisms involved in granuloma modulation, probably operating singly or in combination, include: (1) reduced efficiency of maturation or embryonation of eggs in tissues, thus preventing them from secreting immunopathologic antigens, i.e., anti-embryonation immunity (Garcia et al. 1983, Mitchell et al. 1991); (2) suppressor T cell mediated inhibition of CD4 T-cells or their products responsible for granuloma formation (Stavitsky 1987, Olds 1989, Boros 1986); and (3) antibody mediated inhibition of anti-egg-response, probably by diversion of antigens from T cells or inhibition of antigen recognition by anti-idiotype antibodies
(Pelley and Warren 1978, Olds et al. 1982, Garcia et al. 1985).
It is not the intent of this brief review to discuss in detail the different postulated mechanisms of granuloma modulation, but to recount the series of observations made in our laboratory on antiembryonation immunity that indicate that it may be the principal mechanism involved in granuloma modulation, at least in S. japonicum. Anti-embryonation immunity focuses on the effects of the immune response on the egg rather than on immunoregulation as the central factor in granuloma modulation.
TABLE 1 Evidence for Maturation Arrest of Eggs of Schistosoma japonicum in BALB/c Mice* Sensitized by Weekly Intraperitoneal Injections of Eggs for 28-33 Weeks
Mice
No. of Mice
No. of Mice with
Percentage of Mature
< 25 Mature Eggs
Eggs in Lungs
Egg-sensitized
p < 0.025**
Age-matched controls
* Mice were injected intravenously with 50-100 uterine eggs from S. japonicum adult worms and lungs examined for mature eggs (as distinct from immature and egg shells) at days 12-16. Mean, numbers of eggs examined in each mouse of the egg-sensitized and control groups were 46.4 + 18.9 and 58.7 + 20.0, respectively. Examination was made by compressing the lungs between glass slides and microscopy.
** P values determined by the Mann-Whitney U statistics (Sokal and Rohlf 1969).
Evidence Of Anti-Embryonation Immunity
Of the postulated mechanisms of granuloma modulation, reduced embryonation of eggs in the tissues or anti-embryonation has more attractive features in terms of disease prevention. If egg maturation can be halted, the release of immunopathologic egg products is curtailed and both the sensitization and expression for immunopathology should be reduced (Garcia and Mitchell 1982, 1985; Garcia et al. 1985, 1987, 1989). The continuous deposition of immature eggs in the tissues and their destruction results in continuous reboosting of anti-embryonation immunity.
The initial suspicion that S. japonicum eggs in the liver are inhibited from maturing in chronic infections was aroused in connection with our attempts to standardize intact eggs as antigen for the circumoval precipitin test (COPT) (Oliver-Gonzales 1954, Garcia et al. 1981). We wanted to determine at what point, during infection, donor rabbits should be killed for eggs (recovered by digestion of livers) so as to have an optimal proportion of mature eggs that are the more suitable antigen for the COPT. (In the course of this study it was also demonstrated that immature and dead eggs are less suitable as antigens for the test.) It was observed that egg batches recovered from livers of infected rabbits at time points earlier or later than 55th to 60th day of infection were less suitable for the performance of COPT. Differences presumably reflected immaturity of eggs at early time points and loss of viability at later time points (after D+60). Thus, the possibility was
entertained that embryonation of maturation of eggs was retarded or inhibited in addition to the previously reported observation of accelerated egg destruction in long-term infected animals (James and Colley 1976, Olds and Mahmoud 1980).
Inhibition Of Embryonation In Modulated Mice
Chronically egg-sensitized mice (28 weekly injections) with modulated granuloma response inhibited the maturation of intravenously injected freshly recovered immature uterine eggs (Table 1). On D+12 after immature egg challenge (based on the fact that the S. japonicum ovum needs 10-12 days to mature), in the chronically egg-sensitized mice, only 25.8% of the eggs were mature compared to 59.8% in the lungs of the unsensitized controls. This led us to infer that reduced embryonation of 5. japonicum eggs results in chronically egg-sensitized mice of infected hosts and anti-embryonation may be a granuloma modulating mechanism (Garcia et al. 1983).
Inhibition Of Maturation Or Embryonation Of S. Japonicum Eggs In Infected Mice By Injection Of Sera From Humans With Chronic Infection
The preceding observations prompted us to determine if anti-embryonation immunity is induced in humans with chronic infections (Garcia et al. 1985).
Sera from humans with clinically defined chronic S. japonicum infections and classified by the nature of precipitates formed when reacted with eggs in the standardized COPT were injected from D+24 to D+38 of infection into BALB/c mice exposed to four cercariae. The start of injection at day 24 coincides with the earliest period that S. japonicum oviposits in mice. Control mice were injected with COPT-negative sera following the same dosage and schedule. The mice were killed at D+41 of infection. At this time point there should be none or very few dead eggs in the control mice
injected with COPT negative sera. Schistosoma japonicum starts ovipositing from day 24 to day 27 after cercarial penetration and the eggs need 10 days to mature or embryonate and survive another 1014 days in the tissues.
TABLE 2 Maturity of Eggs in Livers of BALB/c Mice Injected with Schistosoma japonicum* and Injected with Human Sera Between Days 24 and 38 After Infection
Mouse No.
No. of
No. of
Ratio of Immature
Immature Eggs
Mature Eggs
to Mature
Injected with COPT-positive sera
Injected with COPT-negative sera
*Numbers of adult worms in mice nos. 1-5 were 4,4,4,4, and 2, respectively (mean + SEM = 3.6 + 0.4) and in mice nos. 6-10 were 8,6,4,3, and 4, respectively (mean + SEM = 5.0 + 0.9). ** p < 0.05, Mann-Whitney U-test.
Tables 2 and 3 show the number and maturity of eggs in the livers and intestines of the mice injected with human sera. As these tables demonstrate, there is significant inhibition of maturation of eggs in the infected mice injected with sera from humans with chronic S. japonicum, which form large segmented (LS) precipitates in the standard COPT. This is evidenced by the much higher proportion of immature eggs to mature eggs in the livers and intestines of the mice that received COPT-positive sera. This indicates that some serum factor, probably antibody, inhibited maturation (of the eggs).
Evidence Of Anti-Embryonation Immunity And Egg Destruction In Mice Sensitized With Immature Eggs Of S. Japonicum
The next experiment undertaken demonstrated that anti-embryonation immunity is induced by injections or sensitization with viable immature S. japonicum eggs (Garcia et al. 1987). Mice were
sensitized by injections of viable intact immature eggs recovered by the digestion of livers of infected rabbits killed from D+25 to D+29 of infection. In experiments 1 and 2, five weekly injections of 10,000 eggs were given to mice and infected with four cercariae I and 4 days, respectively, after a second injection of eggs. In experiment 3, the mice had 23 injections of a mixture of mature and immature eggs at 1 to 3-week intervals followed by three weekly injections of immature eggs. They were infected with four cercariae after the last injection.
TABLE 3 Maturity of Egg Clusters in Intestines of BALB/c Mice Infected with Schistosoma japonicum and Injected with Human Sera Between Days 24 and 38 of Infection
Mouse No.
No. of Clusters
No. of Clusters
No. of Clusters
Ratio of
of Immature Eggs
of Mixed Eggs
of Mature Eggs
Immature to Other Egg Clusters
Injected with COPT-positive sera
Injected with COPT-negative sera
* p < 0.01, Mann-Whitney U-test.
The results of the assessment of egg development in the intestines and livers of the eggsensitized mice and unsensitized controls killed at day 40-42 of infection are shown in Figure 1. In the analysis of the data, mice that had no eggs in the liver and intestines (uninfected mice) as well as those with less than 50 eggs in either intestinal wall or in liver samples were excluded. This was done to limit the potential sampling error (in the case of the liver) and distortion brought about by too few eggs. This excluded 9 of 53 mice in the three experiments. The notable aspects of these data follow.
·There is a very obvious increase in dead eggs, particularly in the intestines in most of the immunized mice.
·Ten of the 29 immunized or egg-sensitized mice had no mature eggs either in the liver or intestines, although they had both dead and immature eggs. In contrast, all of the 15 unsensitized mice had mature eggs.
· When expressed as a ratio, the percentage of immature to mature eggs is clearly shifted to a deficiency of mature eggs in tissues of egg-sensitized mice.
The fact that 10 of the infected egg-sensitized mice had no mature eggs strongly suggests that the eggs were killed before maturation, in which case it would be most probable that secretion and excretion of the immature and maturing eggs were responsible for the induction of anti-embryonation immunity. One implication of the data is that anti-embryonation could reduce export of mature eggs with the feces and consequently contribute to reduction of transmission as well as amelioration of intestinal pathology.
Efects Of Induction Of Anti-Embryonation Immunity On Liver Granulomas, Relative Spleen Weight, And Portal Pressure Of Infected Mice
This experiment was undertaken to determine if mice sensitized for the induction of antiembryonation immunity prior to egg deposition will develop hepatosplenic disease (Garcia et al. 1989).
In this study, BALB/c mice received five weekly injections of 10,000 viable immature eggs by subcutaneous and intraperitoneal routes. On the tenth day of the sensitization regimen, the mice were infected with four cercariae in anticipation of egg-laying commencing after the last of the egg injections. The mice were sacrificed on day 75 of infection for determination of number and size of granulomas in the liver, portal vein pressure, and relative spleen weight as well as evidence of antiembryonation immunity. Controls included an equal number of unsensitized infected mice and unsensitized uninfected mice of corresponding ages for determining normal values of portal vein pressure and relative spleen weight. No count of eggs in the liver was made. However, serial sections were made for assessing granuloma formation. Each egg or granuloma was followed up serially to identify the section with maximum dimensions that was used for calculating granuloma volume and amount of
reaction. "Granuloma" were classified as having no reaction (0), minimal reaction (+) (i.e., size 1-3 x 10(-4) mmģ and usually involving 1-2 layers of inflammatory cells) and positive (+) (i.e., size greater than 3 x 10(-4) mmģ). The only error in such classification is likely to be either overestimation or underestimation of granulomas around unusually large or small eggs. The observations on the state of development of eggs in the intestines, assessment of granulomas in the liver, portal vein pressure, and relative spleen weight are presented in Figures 2, 3, and 4, respectively. The notable aspects of these observations follow.
-At D+75 of infection only one of the egg-sensitized mice had mature eggs, with the remaining eight mice having only dead or immature eggs in the intestines. In contrast, there were fewer dead eggs but a greater proportion (40-64%) of mature eggs in the unsensitized group (Figure 2).
-The egg-sensitized mice had a much higher (61%) number of eggs without granulomatous reaction in contrast to the unsensitized controls where only 8% had no reaction (p < 0.001) (Figure 3).
-The mean portal venous pressure (207 mm of H2O + 76.31) of the unsensitized group was significantly higher (p < 0.005) than that (122.3 + 54.19 mm of H2O) of the egg-sensitized group (Figure 4).
-The mean relative spleen weight in the unsensitized group is much greater (p < 0.001) than that of the sensitized group.
-The portal vein pressure and spleen weight of the egg-sensitized group were comparable to normal uninfected mice of similar age (Figure 4).
The observations above demonstrated that the induction of anti-embryonation immunity prior to infection, through sensitization by injection of viable immature eggs, has resulted in reduced number and size of granuloma in the liver of the infected mice with prevention of portal hypertension and its consequences. The number of adult schistosomes from four cercariae, extrapolated for a 60-kg human would mean a worm burden of 3,000 flukes, which is much higher than the usual worm burden in infected humans.
Discussion And Conclusions
The observations reported here provide unequivocal evidence that an anti-embryonation vaccine can prevent the development of hepato-splenic disease, the more serious consequence of schistosomiasis caused by S. japonicum. The fact that S. japonicum, which is deposited in the multicellular stage, requires 10-12 days to embryonate and develop a miracidium, provides ample time for anti-embryonation immunity to act. The observation of no mature eggs in many of the sensitized mice suggests that anti-embryonation immunity may be the principal granuloma modulating mechanism in chronic infections.
An alternative strategy of altering disease susceptibility in cases of schistosomiasis by promoting suppressor T-cell mediated immunoregulation may not only be difficult to achieve but may also be associated with some changes. Defective anti-egg antibody responses or granulomatous encapsulation of eggs and sequestering of egg products may lead to a hepato-toxic effect, at least in schistosomiasis caused by S. mansoni.
We have yet to investigate the molecular basis of anti-embryonation immunity to determine how to: (1) identify the antigen responsible for its induction; (2) isolate and produce said antigen(s); and (3) determine how the immune response inhibits embryonation.
Inhibition of the establishment or persistence of infection is clearly a more desirable consequence of vaccination than inhibition of disease (but consider tetanus and diphtheria immunoprophylaxis). However, if the former is difficult to achieve through "conventional" vaccination (and this seems certain to be the case with regard to schistosomiasis), then vaccination against severe disease may have a place in disease control. This is especially so if the strategy also leads to reduced transmission of infection in locations where nonhuman reservoirs of adult worms are not of epidemiological significance. Anti-embryonation responses directed towards immature eggs in the intestinal wall should lead to a reduction in the transmission of infection. Accelerated destruction of eggs should also reduce the export of eggs to the environment via the feces.
Acknowledgments
The studies on schistosomiasis reported herein were supported principally by a grant from the Program for Science and Technology Cooperation, U.S. Agency for International Development, and also from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases and the Australian Development Assistance Bureau. The authors also wish to thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.
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James, S.L. and D.G. Colley. 1976. Eosinophil mediated destruction of Schistosoma mansoni eggs. J. Reticuloendothelial Soc. 20:359-374.
Mitchell, G.F., E.G. Garcia, K.M. Cruise, W.U. Tiu, and R.E. Hocking. 1982. Lung granulomatous hypersensitivity to eggs of Schistosoma japonicum in mice analyzed by radioisotope assay and effects of hybridoma (idiotype) sensitization. Aust. J. Exp. Biol. Med. Sci. 60:401-416.
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<section>Identification And Localization Of Surface Antigens In Adult Schistosoma Japonicum And Schistosoma Mekongi</section>
P. Sobhon, E.S. Upatham, V. Viyanant, T. Kusamran, V. Mohthong, And S. Anantawara
Faculty of Science, Mahidol University
Bangkok,Thailand
Abstract
When the tegument glycoproteins of adult Schistosoma japonicum, S. mekongi, and S. mansoni are separated by SDS-PAGE and electroblotting and stained with biotinylated lectins, the binding patterns and the numbers of positive bands shown by Con A, WGA, SBA, and UEA I are generally similar, but with slightly different staining intensities: the intensity is strongest with Con A, moderate with SBA and UEA I, and weakest with WGA. This may imply that most tegument glycoproteins contain similar proportions of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, N-acetyl-D-galactosamine, and L-fucose. In contrast, there are significant differences in the binding patterns of RCA I and PNA among the three species of schistosomes. Schistosoma japonicum (Philippine and Chinese strains) appear to be more intensely stained than S. mekongi and S. mansoni, and the binding of RCA I is most pronounced on the bands at MW of 86,000 to 56,000 Daltons and at MW of 42,000 to 32,000 Daltons.
Thus, it can be concluded that both strains of S. japonicum probably contain much larger amounts of D-galactose and D-galactose-N-acetyl-D-galactosamine residues in their tegument glycoproteins than other species, particularly with reference to glycoproteins within the ranges mentioned above.
Treatments of freeze-thawed tegument proteins separated by SDS-PAGE and electroblotting with mouse antisera against S. japonicum (Chinese strain) (ISCH), Philippine strain (ISPH), S. mekongi (ISME), and S. mansoni (ISMA) reveal similar numbers of major immunogenic bands among all three species of adult schistosomes. In both strains of S. japonicum, there are at least 15 major antigens at MW of 205,000, 116,000, 110,000, 97,000, 86,000, 68,000, 64,000, 56,000, 54,000, 47,000, 45,000, 38,000, 28,000, 27,000, and 26,000 Daltons. The immunogenic bands at MW of 28,000, 27,000, and 26,000 Daltons are prominent in S. japonicum and may be abundant antigens in this species. There are 15 major immunogenic bands in S. mekongi at MW of 116,000, 105,000, 97,000, 86,000, 76,000, 68,000, 64,000, 56,000, 52,000, 50,000, 47,000, 45,000, 43,000, 38,000, and 30,000 Daltons. The band of MW 43,000 Dalton is very prominent in S. mekongi even though it is also weakly stained with ISPH. In S.
mansoni, there are 17 major immunogenic bands at MW of 105,000, 97,000, 80,000, 76,000, 68,000, 64,000, 56,000, 52,000, 47,000, 45,000, 38,000, 37,000, 34,000, 32,000, 30,000, 28,000, and 27,000 Daltons, of which the bands at MW of 37,000, 34,000, and 32,000 appear to be specific for this species. In all three species of schistosomes, the strongest staining intensities are shown by bands at MW of 97,000 and 68,000 Daltons, indicating that they are the most prominent of the common antigens. Comparison of immunogenic bands from immunoblotting and lectin-binding bands snows that almost all bands are labeled by at least two lectins, which makes it probable that all of them are glycoproteins.
Immunofluorescence and immuno-electron microscopic studies show that mouse antisera resulting from natural infection principally contain antibodies against antigens residing on the adult surface membrane. Judging from the similarity between the patterns of antibody and pectin binding, these antigens may be mainly glycoproteins and/or other carbohydratebearing glycoconjugates in the surface membrane.
Introduction
The chemistry of the schistosome surface membrane and its glycocalyx coating is increasingly being studied to further understanding of the host-parasite interaction and the mechanisms of the parasite evasion from the hosts' immune responses. Murrell et al. (1978) showed that lectins, concanavalin A (Con A), and wheat germ agglutinin (WGA) bind to the surface of adult S. mansoni, whereas Ulex europeus (WEA) and soy-bean agglutinins (SBA) do not. In a similar study, Simpson and Smithers (1980) found very strong binding of Con A, Ricinus communis agglutinin (RCA), and WGA, limited binding of SBA and peanut agglutinin (PNA), but no binding of UEA. Furthermore, Stein and Lumsden (1973) also showed that Con A binds to similar sites on the surface membrane of fixed worms as colloidal iron, which provides strong evidence that the membrane-bound anions are probably residing in sugar residues on the surface. These sugar residues may be part of glycoproteins and/or glycolipid molecules
that constitute the surface membrane (Simpson and Smithers 1980). Caulfield et al. (1987), using phenol-water extraction and gel chromatography, reported that glycocalyx on the surface of S. mansoni cercaria is composed mostly of carbohydrates and proteins; and Hayunga et al. (1982, 1983) and Hayunga and Sumner (1986b,c) also reported that major Con A binding surface materials from adult S. mansoni are glycoproteins. Biochemical studies have revealed that glycoproteins are the major components of the tegument surface membrane. Employing lectin affinity chromatography, Bennett and Seed (1977) found that immobilized Con A binds to three radio-iodinated surface membrane proteins of S. mansoni, and using a similar technique, Strand et al. (1982) and Aronstein and Strand (1984) have shown up to 35 bands of glycoproteins from the adult tegument. More recently, Hayunga and Sumner (1986a), using column chromatography containing Con A, Lentil lectin, WGA, SBA, and RCA and Helix
promatia agglutinin, found 15 đē5I-labeled glycoproteins with molecular weights of >300,000, 125,000, 168,000, 152,000, 134,000, 122,000, 108,000, 83,000, 58,000, 53,000, 46,000, 41,000, 34,000, 30,000, and 23,500, and many of these glycoproteins react with more than one lectin.
It has also been demonstrated that surface glycoproteins, especially their carbohydrate moieties, are major epitopes of schistosome antigens that can elicit antibody production in the naturally as well as artificially infected hosts (Omer-Ali et al. 1986, Weiss et al. 1986, Grzych et al. 1987). By radio-labeling surface proteins and glycoproteins with đē51 or ģH, most investigators generally agreed that in S. mansoni there are between 7 and 15 bands of surface proteins and glycoproteins, ranging in molecular weights from 12,000 to 200,000 Daltons, that could be immunoprecipitated with antisera from patients and infected animals. Consequently, these may be antigens that are released into the host circulation and stimulate the antibody production.
In contrast to S. mansoni, there is only limited information to date on the surface glycoproteins and antigens of S. japonicum and none on another Oriental schistosome, S. mekongi. Therefore, in this study we have: (1) identified the surface protein and glycoprotein antigens by immunoblotting, and (2) localized these antigens on the surface and in the tegument of adult parasites by immunofluorescence and immunoelectron microscopy.
Materials and Methods
Maintenance of S. japonicum and S. mekongi Life Cycles
Collection of Cercariae Schistosoma japonicum and S. mekongi cycles have been maintained at the Center for Applied Malacology and Entomology, Department of Biology, Mahidol University. Miracidia were obtained by hatching the eggs recovered from the liver of infected Swiss Albino mice. Specific snail hosts that were used consisted of Oncomelania hupensis hupensis (hosts for Chinese strain), O. hupensis quadrasi (hosts for Philippine strain), and Tricula aperta (for S. mekongi). The snails for S. japonicum were collected from fields in Anhui Province, People's Republic of China, and Mindoro Island, Philippines, while those for S. mekongi were collected from the Moon River, northeast Thailand. Each snail was infected with approximately 6-8 miracidia. Eight to nine weeks after the infection, cercariae were shed naturally from the snails by placing them into aquarium water under warm light for 2-3 hours. Cercaria were then concentrated in a small volume of water and used for
further experiments.
Collection of Adult Schistosomes Swiss Albino mice were infected with approximately 20-30 cercariae per mouse by the intraperitoneal route. Eight weeks later, adult worms were obtained by portal perfusion via the right ventricle with 0.15 M sodium chloride and 0.05 M sodium citrate. Before use, the worms were washed 5 times with Minimum Essential Medium (MEM). In addition, adult S. mansoni were collected from mice kept in cycle similar to S. japonicum and used for comparative studies.
Collection Of Antisera Immune mouse antisera were obtained by heart puncture from Swiss Albino mice, each infected with 30 cercariae for 8 weeks. Animals were bled and the blood was allowed to clot at 25°C for 3 hours. The clot was removed and the antisera were centrifuged at 1,000 x g for 5 minutes to obtain a clear supernatant. Normal mouse serum was obtained in a similar manner from uninfected mice. In addition, both pooled and individual human antisera from S. japonicum (Philippine)-infected patients were graciously supplied by Professor Edito Garcia, Institute of Public Health, University of the Philippine System, and Dr. Biyani Blas, Director, Schistosomiasis Control and Service Center, Ministry of Health, Manila, The Philippines.
Identification and Comparison of Tegument Antigens of Adult S. japonicum, S. mekongi, and S. mansoni by Immunoblotting
In this experiment, the tegument antigens of parasites were obtained as follows: the parasites' tegument were extracted by freeze-thawing on dried ice in Tris-buffer for 20 cycles, and the shed pieces of tegument in the supernatant were collected after centrifugation of the mixture at 100 x g for 5 minutes. The supernatant was solubilized in the SDS-PAGE sample buffer and applied onto 12.5% SDS-PAGE gels. Then, electrophoresis was performed at 100 volts for 4 hours, and the proteins on gel were blotted onto nitrocellulose (NC) paper using a blotting apparatus with a setting at 0.5 mA for 1 hour. Antigenic proteins on the nitrocellulose paper were detected by incubating NC strips in mouse immune sera and followed by rabbit anti-mouse Ig labeled with horseradish peroxidase (HRP). The antigen-antibody complexes that bind with anti-mouse Ig-HRP were visualized by incubating the nitrocellulose strips in 3,3-diaminobenzidine (DAB) and H202. In addition, similar nitrocellulose
strips were also stained with biotinylated lectins-avidin-biotinylated HRP, in order to identify glycoprotein bands.
Indirect Immunofluorescence
Freshly obtained adult worms were fixed in 2% paraformaldehyde in 0.1 M PBS, pH 7.4, for 2 hours at 4°C. The fixed worms were washed with the same buffer, then they were frozen and cut into 4-6 m thick sections in a cryostat. These frozen sections were picked up on gelatin-coated slides and allowed to thaw at room temperature for 30 minutes. The sections were covered with 10% normal sheep serum in 0.1 M PBS for 30 minutes and followed with the appropriate dilution of mouse immune sera for 2 hours at room temperature. After being washed with PBS buffer, the sections were incubated with secondary antibody (sheep anti-mouse IgG conjugated with FITC) for 1 hour at room temperature. At the end of the incubation period, the sections were washed with double distilled water 3 times for 10 minutes at each washing. The sections on slides were mounted in buffered glycerol and covered with glass coverslips. The completely stained sections were examined by UV light using the incidence
illumination in the Leitz Orthoplan microscope. The controls were carried out simultaneously by replacing the immune serum with a normal mouse serum.
Immunoelectron Microscopy
Parasite samples were briefly fixed with 2.5% glutaraldehyde in 0.5 M cacodylate buffer at 4°C for 10 minutes. They were then rinsed 3 times in the buffer and incubated in 0.1% NaBH4 for 30 minutes to block free aldehyde groups. The specimens were pre-incubated in normal mouse serum and then overnight in immune mouse sera against S. japonicum (Chinese) and S. mekongi. Subsequently, they were washed thoroughly and incubated in rabbit anti-mouse Ig labeled with horseradish peroxidase (HRP). The antibody binding sites were demonstrated by incubation with DAB and H2O2 for 10 minutes, and specimens were prepared for TEM by the conventional method and examined without electron staining.
Results and Discussion
Tegument Glycoproteins in Adult Schistosoma japonicum (Chinese and Philippine Strains) and S. mekongi Compared with S. mansoni as Detected by Electro-blotting and Lectin Staining.
In S. mansoni, several previous studies have shown the presence of specific carbohydrate moieties in the side chains of tegument glycoproteins. Rumjanek et al. (1979) demonstrated that the major monosaccharide components in the freeze-thaw isolated tegument of adult worms are mannose, glucose, and galactose residues. By using radiolabeled hexose precursors to metabolically labeled glycoproteins, Hayunga and Sumner (1986a) found that the radiolabeled saccharides were incorporated into at least 10 tegumental glycoproteins of S. mansoni with MW ranging from 30,000 to < 300,000. Furthermore, by employing direct radioiodination of adult surface membrane and lectin affinity chromatography with Con A, lentil lectin, WGA, SBA, RCA I and II, and Helix promatia to fractionate membrane proteins, Hayunga and Sumner (1986a) also revealed 14 glycoproteins in the eluated fractions from these lectin columns. From these studies, it was suggested that D-mannose, D-glucose,
N-acetyl-D-glucosamine, D-galactose, D-galactose-N-acetyl-D-galactosamine conjugate, and L-fucose residues are the major carbohydrate moieties of the adult S. mansoni tegument glycoproteins.
In our study, the tegument of adult S. japonicum (Chinese and Philippine strains), S. mekongi, and S. mansoni were obtained by repeated freeze-thawing of the adult parasites. This treatment usually
resulted in a fairly clean separation of the tegument from the body of the parasite, leaving the basement membrane and the underlying connective tissue and muscle layers intact (Figure 1A,B). Hence, it is believed that there is very little leakage of the content from tissues underneath the basement membrane. The shed tegument was then solubilized and its protein and glycoprotein contents were separated in 12.5% SDS-PAGE and stained with Coomassie blue. In general, the number and pattern of protein bands in all three species are quite similar, with molecular weight (MOO) ranging from 300,000 to 14,000 Daltons. There are nine regions of major bands at MW 205,000; 97,000 and 90,000; 68,000, 64,000, 58,000, 56,000, and 54,000; 52,000; 48,000; 46,000, and 45,000; 42,000, 38,000, and 37,000; 33,000, 32,000, 30,000, 29,000, 28,000, and 27,000; and 14,000. Schistosoma japonicum has a total of 42 bands, whereas S. mekongi and S. mansoni have 36 and 29 bands, respectively (Figure
1C,D). It is believed that since the basement membrane is still intact after the freeze-thaw (as revealed under TEM), most of the glycoproteins shown in the gels are probably derived from the parasite tegument with little contamination of proteins from tissues Iying underneath the basement membrane.
TABLE 2 Comparison of Surface Antigens of Adult Schistosomajaponicum (Chinese and Philippine),Schistosoma mekongi, and Schistosoma mansoni as Revealed by Immunoblotting with Corresponding Mouse Immune Sera
S japonicum
S japonicum
S mekongi
S mansoni
(Chinese)
( Philippine)
+ISCH
+ISPH
+ISME
+ISMA
Major bands=15
Major bands = 20
Major bands=15
Major bands=17
The number of resolved tegument proteins in 12.5% SDS-PAGE were further characterized by electrophoretic transfer onto nitrocellulose sheets and then stained with biotinylated lectins (Con A, WGA, RCA I, PNA, SBA, and UEA I). The results show that most bands bind to at least two lectins except bands at MW < 25,000, which only bind weakly to SBA, and therefore most, if not all bands, are glycoproteins. These positive bands are more likely to be glycoproteins than glycolipids because the latter are usually lost very rapidly from the gel due to their fast migration as a result of their much smaller molecular weight.
Based on the staining intensities, the bindings of lectins to tegumental glycoproteins in all three species of adult schistosomes appear to have four different patterns. Con A uniformly stains most bands in all three species with fairly high intensity, except in the regions between MW 42,000-32,000 and 28,000-26,000 where there appear to be more bands in S. japonicum that are intensely stained by Con A than in other species (Figure 2A). WGA and UEA show the weak staining of most bands (Figure 2B,C). RCA I shows moderate staining of most bands, but in S. japonicum it shows two regions of very intense staining at MW of 56,000-86,000 and 32,000-42,000 (Figure 2D). PNA shows moderate staining of most bands, but in S. japonicum it shows very intense staining at MW 56,00064,000 (Figure 2E). SBA also stains most bands with moderate intensity, but in Oriental schistosomes it also shows two regions of fairly intense staining like RCA at MW 32,000-42,000 and 56,000-68,000 (Figure 2F).
These staining patterns imply that the tegument glycoproteins in all three species contain similar basic sugar residues: i.e., D-mannose/D-glucose (Con A), N-acetyl-D-glucosamine/sialic acid (WGA), D-galactose (RCA), D-galactose-N-acetyl-D-galacto-samine (PNA and SBA), and L-fucose (UEA), but probably with slightly different quantity as indicated by the varied staining intensity, the most striking of which are in S. japonicum, which exhibits heavy bindings of RCA I and PNA and SBA to glycoproteins in two regions at MW of 56,000-86,000 and 32,000-42,000, and in S. mekongi, which exhibits heavy bindings to PNA at MW 56,000-64,000 (Figure 2E). When compared with Oriental schistosomes, S. mansoni shows only weak binding to both RCA and PNA in these regions. These results confirm the lectin-labeling studies in TEM, which show that the surface membranes of both Oriental schistosomes are more intensely stained by RCA- and PNA-labeled horseradish peroxidase, and that therefore they
may contain a larger amount of D-galactose and D-galactose-N-acetyl-D-galactosamine residues than S. mansoni. Between the two species, the tegument of S. japonicum may contain a larger amount of galactose-bearing glycoproteins than that of S. mekongi.
Immunogenic Glycoproteins in the Tegument of Adult Sehistosoma japonicum (Chinese and Philippine Strains), S. mekongi, and S. mansoni.
The immunogenic glycoproteins in the tegument of Oriental schistosomes were characterized and compared with those of S. mansoni by immunoblotting technique in which proteins/glycoproteins in the freeze-thawed tegument were separated in 12.5% SDS-PAGE and electroblotted onto nitrocellulose (NC) papers. Then NC strips were incubated sequentially in mouse antisera against S. japonicum (Chinese strain) - ISCH, against S. japonicum (Philippine strain) - ISPH, against S. mekongi - ISME, against S. mansoni - ISMA, and human antisera from patients infected with S. japonicum (Philippine) - IHPH. The immunogenic bands that bind to these antisera were detected with rabbit-antimouse Ig- and goat antihuman Ig-labeled with HRP. To verify which immunogenic bands are glycoproteins, the nitrocellulose strips of electroblotted tegument proteins were also stained with biotinylated lectins (Con A, WGA, RCA I, PNA, SBA, and UEA I) and avidin-HRP.
Immunoblotting by Mouse Antisera
Both strains of S. japonicum showed almost identical patterns of immunogenic bands against all antisera being used, but with slight differences in staining intensity of some bands. Against ISCH and ISPH, there are at least 15 major common antigens in both strains that show comparable intensity; the most intense bands are at MW 205,000, 116,000, 110,000, 97,000, 68,000, 47,00O, 45,000, 28,000, 27,000, and 26,000; the moderately intense bands are at MW 86,000, 64,000, 56,000, 54,000, and 38,000; while major antigens at 105,000, 76,000, 50,000, 46,000, and 25,000 appear to be more intense in ISPH then ISCH treatment (Tables 1 and 2; Figure 3A,B). In addition, there are at least 24 faintly stained minor bands (Table 1). The treatment of S. japonicum antigens with ISME shows intense staining of most major bands except at MW 205,000, 28,000, 27,000, and 26,000-19,000. On the other hand, ISMA reacts weakly with most major bands of S. japonicum except at MW 116,000, 105,000, 97,000,
86,000, 54,000, 45,000, and 38,000, where the cross reactions are strong; furthermore, it shows particularly strong cross reaction to MW 76,000, 37,000, and 32,000, which are only faint bands when treated with ISCH and ISPH (Table 1, Figure 3C,D). When compared to lectin stainings of freeze-thawed tegument proteins, all immunogenic bands except the lower MW at 27,000, 25,000, 19,000, and 14,000, are labeled with at least two lectins. However, another prominent group of immunogenic bands at MW 97,000, 68,000, and 64,000 are stained intensely by RCA I, PNA, and SBA, but not with Con A and WGA. These glycoproteins may contain especially large amounts of galactose residues. In adult S. mekongi, when tegument proteins/glycoproteins react with ISME there appear to be at least 15 major immunogenic bands; the most intense are at MW 97,000, 86,000, 68,000, 64,000, and 43,000, and the moderately intense are at MW 116,000, 105,000, 76,000, 56,000, 52,000, 50,000, 47,000, 45,000,
38,000, and 30,000. Band 43,000 appears to be particularly intense in S. mekongi, while it is only a faint band in S. japonicum and S. mansoni (Tables 1 and 2; Figure 3C). In addition, there are about 21 minor bands that are mostly similar to those of S. japonicum (Table 1). When reacted against ISCH and ISPH the immunogenic bands of adult S. mekongi show similar patterns to those of S. japonicum, except band 43,000, which is particularly intense when treated with ISPH (Table 1; Figure 3A,B). ISMA also reacts with most major immunogenic bands of S. mekongi, except bands 68,000, 64,000, 56,000, 52,000, 50,000, and 47,000, which become very weak (Table 1, Figure 3D). When compared to lectin stainings, all immunogenic bands of S. mekongi are labeled by at least two lectins except the major band at 47,000, which binds to only one lectin (PNA). Like S. japonicum, a group of major antigens at 97,000, 68,000, 64,000, 52,000, and 47,000 in S. mekongi are stained by RCA I, PNA, SBA,
and UEA I, but not Con A and WGA, even though the staining is not as intense as in S. japonicum. Therefore, it can be concluded that all tegument antigens of S. mekongi are glycoproteins, and that the glycoproteins at MW 98,000, 68,000, 64,000, 52,000, and 47,000 may contain especially large amounts of D-galactose, D-galactose-N-acetyl-D-galactosamine, N-acetyl-D-galactosamine, and L-fucose residues.
In S. mansoni the treatment of tegument proteins/glycoproteins with ISMA reveals 17 immunogenic bands: the most intense are at MW 76,000, 68,000, 56,000, 45,000, and 37,000, and the moderately intense bands are at MW 105,000, 97,000, 80,000, 64,000, 52,000, 47,000, 38,000, 34,000, 32,000, 30,000, 28,000, and 27,000. The bands at MW 37,000, 34,000, and 32,000 appear to be particularly intense in S. mansoni, while they are either absent or very faint in S. japonicum and S. mekongi. In addition, bands at MW 28,000 and 27,000 also appear intense though not as much as in S. japonicum, while they are absent in S. mekongi (Table l and 2, Figure 3D). ISCH and ISPH react with most major immunogenic bands of S. mansoni but usually with lower intensity than corresponding bands in S. japonicum; however, a band at MW 56,000 exhibits higher intensity than in S. japonicum (Table 1; Figure 3A,B). ISME reacts with only nine major immunogenic bands while the remaining appear very faint
(Figure 3C, Table 1). Despite a slight shift in molecular weights, most of the major immunogenic bands in S. mansoni appear to correspond to those reported by Hayunga et al. (1979a,b), who used immuno-precipitation to reveal surface antigens at MW 110,000, 78,000, 68,000, 60,000, 43,000, 36,000, 30,000, 26,000, and 12,000. When compared with lectin-binding patterns, almost all immunogenic bands of S. mansoni are also labeled by at least two lectins. Like S. japonicum and S. mekongi, bands at MW 97,000, 86,000, 76,000 68,000, 56,000, 47,000, and 45,000 are stained by RCA I, PNA, SBA, and UEA I, but not Con A and WGA; however, the staining is weaker than in Oriental schistosomes. Even the band at 68,000, which shows the most intense staining with RCA I and PNA, is rather weak when compared to the same band in Oriental schistosomes.
IHPH1 is the first lot of pooled serum; IHPH2 is the compilation of results from individual antisera from 5 patients.
IHPH2 antisera were kindly given by Dr. Bayani Blas, Director, Schistosomiasis Control and Service Center, Ministry of Public Health; IHPH1 by Prof. E. Garcia Institute of Public Health, University of the Philippine System, Manila, Philippines.
Immunoblotting by Human Antisera
In this experiment, both pooled and individual antisera from patients infected with S. japonicum (Philippine) were used. Immunoblotting patterns of tegument antigens of both Chinese and Philippine strains of S. japonicum vary slightly from one antiserum to another (Figure 4A to F). However, when results from all individual antisera (IHPH2) are analyzed together with the pooled antiserum (IHPH1) that was used to detect antigens (Table 3), positive bands correspond in range and molecular weights with those detected by mouse antisera already presented in Tables 1 and 2. Both strains of S. japonicum show similar immunoblot pattern against IHPH1 and individual antisera; when all results are compiled together there appear to be 22 bands that are consistently detected at MW 180,000, 150,000, 116,000, 97,000, 68,000, 64,000, 62,000, 58,000, 54,000, 52,000, 50,000, 48,00Q, 47,000, 45,000, 43,000, 39,000, 38,000, 28,000, 27,000, 25,000, 23,000, and 19,000. Among these the most
prominent bands are 116,000, 97,000, 68,000, 64,000, 62,000, 50,000, 45,000, 28,000, and 27,000 (Table 3, Figure 4A to F). Fewer S. mekongi antigens are detected by IHPH and the prominent ones are at MW 116,000, 97,000, 68,000, 64,000, 62,000, 48,000, 45,000, and 38,000. Schistosoma mansoni generally shows more positive bands against IHPH than S. mekongi, but not as many as those of S. japonicum, and the major bands appear at MW 97,000, 68,000, 64,000, 52,000, 48,000, 39,000, 38,000, 37,000, and 27,000 (Figure 4A to F, Table 3).
Localization of Surface Antigens
Attempts were made to localize antigens of circulating adult Oriental schistosomes by using naturally infected mouse antisera against S. japonicum and S. mekongi by immunofluorescence and immunoelectron microscopic methods.
Indirect Immunofluorescence In control specimens, the autofluorescence is strong in the gut lumen and possibly also in the gut epithelium. The tegument and muscle layers do not fluoresce, but the interior of the parasite bodies show small, scattered weak fluorescent spots (Figure 5A,B). In adult male S. japonicum (Chinese strain) treated with ISCH, apart from the autofluorescence gut, strong fluorescence due to specific binding of antibodies is present over the whole thickness of the tegument of the dorsal and lateral aspects, but only weak fluorescence is observed on the tegument of the gynaecophoral canal (Figure 5C,D). At high magnification, the whole width of the tegument from the ridges down to the level of the basement membrane shows strong fluorescence, while the muscle layer is completely black. However, in some areas underneath the muscle layer a row of fluorescent tegument cells can be observed. Each cell has fluorescent cytoplasm surrounding a completely black
nucleus (Figure 5D inset). In adult male S. mekongi treated with ISME, the pattern of fluorescence is similar to that of S. japonicum; however, in and around bubble-like structures, which may be pleomorphic papillae, the fluorescence appears weaker than the rest of the tegument (Figure 5E,F).
Immunoelectron Microscopy At TEM level, the binding of antibodies in adult S. japonicum appears only on the surface membrane, and no reaction product is detected within the tegument cytoplasm or tegumental bodies (Figure 6). This, however, could be due to the limited penetration of primary and secondary antibodies in the preembedding technique. Therefore, the postembedding technique should also be tried to verify the immunofluorescence study and to determine whether membrane antigens are already present in Mb granules, or whether they become "completed" only at the surface membrane level. The similarity between the binding of lectins and the binding of natural antibodies to the surface of the tegument in adult schistosomes suggests that the receptor sites for these two groups of ligands may be the same. This, together with evidence from immunoblotting, strongly suggests that the lectin receptor sites are antigenic glycoproteins that constitute major portions of the surface
membrane of adult parasites.
Acknowledgments
This work was supported financially by USAID/PSTC grant no. 936-5542-G-00-6076-00 and UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. The authors also thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.
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<section>Human Immune Responses During Infection With Schistosoma Haematobium: Cell Mediated Immunity</section>
P.D. Ndhlovu, S.K. Chandiwana, P. Taylor, S.M. Phillips, P. Mason P.S. Sola, K.K. Kaondera, G. Mandaza, And E. Kubara
Abstract
The role of cell mediated immunity in relation to infection with Schistosoma haematobium in different age groups was investigated in an endemic community in Zimbabwe. Cell mediated immunity was measured in vitro by the granuloma index (GI). Results from the study showed that the mean GI of 1.402 +_ 0.386 in infected people < 20 years old was significantly higher than in infected people > 20 years old (GI = 1.199 +_ 0.237, t = 2.22, p = 0.043.
Introduction
Studies conducted in schistosomiasis endemic areas show an age-related decline in prevalence and intensity of infection that is indicated by a decrease in the number of eggs detected in urine and feces. The age-related decline in infection was ascribed to the development of immunity (Fisher 1934, Clarke 1966, Kloetzel and da Silva 1967, Wilkins et al. 1984, Butterworth et al. 1985). However, no specific part of the immune system has been shown to contribute to the acquired immunity noted in older age groups.
Initial immune-mediated pathologic responses to schistosomiasis occur around the egg The egg-focused pathology is due to a Type IV hypersensitivity reaction, which results in an egg granuloma and is T cell dependent (Colley 1981). The reaction benefits the host in that it insulates the liver cells from toxic products secreted by the worm and eggs (Roitt et al. 1989). These events result in stabilization of the relationship between the host and parasite (Phillips and Colley 1978). However,
if the egg granuloma is not limited, irreversible fibrotic changes may occur that may inhibit egg passage through the bladder wall in S. haematobium infections (Warren 1973). The morbidity resulting from egg granulomas in the affected organs was reported in experimental animals (Murare 1983) and in both in vivo and in vitro studies by Bentley et al. (1985). Neither of the two studies showed whether the capacity to form an egg granuloma contributed to the resistance to S. haematobium observed in older individuals residing in endemic areas. The present study was designed to investigate whether natural resistance to S. haematobium infection can be related to granuloma activity.
The technology to study in vitro granuloma reactions that occur in vivo was pioneered by Doughty et al. (1984) and improved by Bentley et al. (1985), who worked on S. mansoni and designed the bead-coated model. The model uses a soluble egg antigen chemically bonded to a polyacrylamide bead as the nidus. Phillips and Lammie (1986) considered reactions occurring in vitro to be analogous to granuloma formation in vivo. A similar model was used in this study to investigate whether the observed age-related prevalence and intensity patterns in human S. haematobium can be related to changes in cell mediated immunity and to relate morbidity indexes to cell mediated immunity.
Materials and Methods
Study Area and Population
The study population was selected from Chikwaka Communal Lands, which are situated in the Goromonzi district of Mashonaland East Province of Zimbabwe. The area is approximately 200 kmē and has a population of 21,000.
Interventions for schistosomiasis control in this area were limited to mollusciciding that was carried out between 1974 and 1978. Mollusciciding had a marginal effect on the prevalence of urinary schistosomiasis in schoolchildren, which has remained at about 60% (Shiff et al. 1979, Taylor and Makura 1985). Schistosoma mansoni infections occur uncommonly (Shiff et al. 1979).
Initially, urine and stool samples from 600 people of all ages were examined for schistosome eggs. For each individual, samples were collected on three consecutive days. Urine specimens were examined by the filtration technique (Most et al. 1982), while stool samples were examined using Kato smears (Peters et al. 1980). Only persons infected with S. haematobium in the absence of S. mansoni were included in the study population. Controls were those individuals not excreting eggs.
Preparation of Antigen Beads
Polyacrylamide beads of 45 in diameter (Biorad in UK) were swollen in sterile distilled water for 48 hours at room temperature. The swollen beads were washed in sterile bicarbonate buffer using a sintered glass funnel and then suspended in 100 ml of sterile bicarbonate buffer for four hours at 63°C. Beads were washed in distilled sterile water before being mixed with soluble egg anitigen (SEA) and 1-ethyl-3-(3-dimethylamino propyl carbodiimide HCI) (EDAC) in proportions of 20 mg beads, 2 mg antigen 10 mg EDAC and 10 ml slightly acidic distilled sterile water. This mixture was rotated for at least 18 hours at 4°C. Thereafter, the beads were washed with Phosphate Buffered Saline (PBS, pH 7.4) to remove excess antigen, unreacted EDAC, and the urea by-product of the reaction.
Blood Sample Collection and Cell Separation
Twenty ml of blood were collected from each subject into a heparinized syringe and mixed gently. Samples were transported to the laboratory within two hr of collection. The heparinized blood was gently layered onto leucoprep cell separation tubes and centrifuged at 1900 x g for 15 minutes. Peripheral blood mononuclear cells (PBMN) were removed, washed three times using RPMI 1640, with the last wash being in RPMI 1640 containing 10% AB+ human serum.
Cell Bead Cultures
Cell bead cultures were carried out using a modified method of Bentley et al. (1985). Briefly, 200-300 SEA coated beads in 100 ,ul PBS were added to RPMI 1640 containing 10% heat inactivated AB+ human serum, 1.6% L-glutamine, and 3% antibiotic (penicillin and streptomycin) in RPMI 1640. PBMN beads, 1x106 - 5x106 were added to each culture well to bring the final volume to 2 ml. Each test and control group was set up in triplicate and maintained at 37°C in 5% CO2 in air for five days.
Cell bead reactions were observed using a phase contrast microscope and the reaction was scored following the scales of Doughty et al. (1984) and Patrick et al. (1985).
1. No cells adhering to bead
2. One to five cells adhering to bead
3. Six to 20 cells adhering to bead
4. More than 20 cells accompanied by circumoval mononuclear cell migration and blast transformation
5. A complete cell layer attached to bead accompanied by circumoval mononuclear cell migration
6. Multiple cell layers surrounding the bead accompanied by mononuclear cell migration
At least 100 beads were examined and scored per well. The mean reaction score of granuloma index (GI) was computed for each well and data reported as the mean GI + S.D.
Blast Transformation Assays
To determine the viability and functional integrity of cells, a blast transformation assay was set up in parallel to the granuloma index. One ml of RPMI containing 10% heat inactivated AB+ serum, 1.6% L-glutamine, and 3% antibiotic was mixed with 5X106 cells in 50 ul RPMI and 50 u1 phytohaemagglutin (PHA) mitogen. Aliquots (200 ,ul) of the mixture were then dispensed into a microtitre flat-bottomed well plate and cultured at 37°C in 5% CO2 in air. Controls for each test sample comprised the cells from the same subject, but without adding mitogen. On day 3, 1.0 ,uCi tritiated thymidine (Amersham UK) was added and incubation continued for an additional 18 hours. Cells were harvested using a semi-automated cell harvester and the uptake of thymidine was measured in a liquid scintillation spectrophotometer. Data were expressed as mean counts per minute (cpm) which was calculated as follows: mean CPM = reading from cells minus reading from control (unstimulated) cells.
Measurements of Morbidity
Haematuria was determined using the Ames reagent strip. Bladder and kidney fibrosis was determined using ultrasonography following ingestion of about 300 mls of orange drink to ensure a stretched bladder. Results were recorded as normal if no abnormalities were found; abnormalities were recorded as follows: 1 = mild, 2 = moderate, 3 = severe.
Data
Data obtained from each assay were stored on computer and analyzed using the SPSS program. Egg counts were transformed and results expressed as mean log counts. Blastogenesis results were recorded as counts per minute; the log of the mean counts per minute were used in analysis.
Results
Stool and urine specimens were initially collected from 600 people. Of these, 27% had S. haematobium infections, 4% had S. mansoni infections, and 5% had mixed infections. Other helminths detected in the stool samples included H. nana, but these were rare, with low worm loads, and thus were ignored. People excreting eggs of S. mansoni at the beginning of the study were excluded.
The age prevalence and intensity of S. haematobium infections followed a typical pattern with a peak prevalence and intensity in children up to the age of 20 years (Figure 1). In all, 112/272 (41.2%) of people under 20 years were excreting S. haematobium ova, compared with 9/78 (11.5%) of people 20 years or older.
There was significant difference in the mean GI of people excreting ova and controls living in the same endemic area (1.38 i 0.377 versus 1.486 + 0.387, t = 1.99, p = 0. 047) when they were compared as a group (Figure 2). However, comparing infected people and controls in each age group independently showed that the mean GI of the age groups up to 29 years was not significantly different. The mean GI for those over 30 excreting ova (1.144 + 0.182) was significantly lower than that of the controls of that age group (mean GI = 1.572 + 0.491), t = 2.10, p = 0.001. Among the controls, the mean GI remained more or less constant with age while the mean GI of the infected people decreased with age.
The relationship between intensity of infection and the mean GI is shown in Figure 3. Where the intensity of infection was high (mean log egg counts 1.3), the mean GI was 1.480 + 0.312. On the other hand, where the mean log egg count was low (0.671), the mean GI was lower (1.140 + 0.182). Individuals under the age of 20 excreted more eggs and their mean GI of 1.402 + 0.386 was significantly higher than individuals over the age of 20 (mean GI = 1.200 + 0.237, p = 0.043).
Phytohaemagglutin induced blastogenesis of Iymphocytes was not related to either age or infection status (Figure 4). A one-way anova analysis among all the groups showed that there was no significant difference between any two groups. However, infected people tended to have marginally higher blastogenesis than those who were not excreting eggs (Figure 4), and the infected older individuals had greater blastogenesis than the younger individuals.
Bladder wall abnormality was detected in only 5/192 (2.6%) of people excreting S. haematobium ova. For people with no abnormality, the mean GI was 1.453 + 0.401, while the mean GI of those with bladder wall abnormality was slightly higher (mean GI 1.520 + 0.342). The difference, however, was not significant (p > 0.05). No abnormality was noted in any of the kidneys.
Discussion
In this paper we present baseline data on responses to S. haematobium SEA of cells, from those who are infected (people excreting S. haematobium ova) and controls (those not excreting S. haematobium ova) living in an endemic area of Zimbabwe. It should be noted that the chronicity of the infections and degree of exposure were unknown. Future aspects of this study will examine responses to reinfection following treatment.
People with schistosome infections other than S. haematobium were excluded from the study because of extensive cross reactions, which may have confounded our findings. Previous studies on cell mediated immunity in schistosomiasis have focused on S. mansoni infections, and to our knowledge, this is the first time that in vitro granuloma responses have been measured in S. haematobium.
The prevalence and intensity of infection in the study population was typical of an endemic area of Zimbabwe (Chandiwana et al. 1991), where the intensity and prevalence is highest in young adults, and thereafter decreases with increasing age. There was no evidence in the community as a whole that this correlated with changes in in vitro granuloma responses to SEA coated beads at the 95% confidence limit (Figure 2). The interaction between infection and immune response is exceedingly complex, and quite a few factors such as intensity of infection, chronicity of infection, induction of suppressor cell activity, and many others may play a role.
In vitro granulomatous responses to S. mansoni egg antigens have previously been recorded as not correlating with the intensity of infection as measured by egg excretion, but correlated with duration of infection (Doughty et al. 1984). Greater responses were observed in the acute stage of infection. Feldmeier et al. (1981) also recorded that responses to schistosome antigens of people infected with schistosomiasis did not correlate with the intensity of infection. Gazzinelli et al. (1985) reported that in vitro responses to schistosome infections were greater in acutely infected individuals. It is therefore important to know the duration of infection if the responses obtained in this study are to be accurately interpreted. In this study, the duration of infection was not known, but it could be that patients < 20 years old had a recent infection and those > 20 years old had a long-standing infection. This was reflected by the fact that the granulomatous responses of those <
20 years old (1.402 + 0.386) was significantly higher than that of individuals > 20 years old (mean GI = 1.120 + 0.237, p < 0.05).
Although there were no significant differences in the GI of infected versus control groups, the age-related GI was different in the two groups. In the infected group, mean GI decreased with age (Figures 2, 3) and this could be explained in terms of suppression of responses to SEA, which reduce activity in chronic infection (Rocklin et al. 1981), perhaps resulting from activation of specific suppression by CD8+ Iymphocytes (Feldmeier et al. 1985).
The persistence of a high GI in the control population in the older aged individuals is worthy of comment. Since our studies on schistosome antigens (unpublished) have shown that there are many similarities between egg antigens and schistosomular antigens, it may be that those who were able to exert a strong GI response in vitro to SEA were also capable of mounting an effective immune attack to the schistosomula in vivo. Hence the lack of infection in that group, despite having been exposed to infection. The granulomatous immune response of an individual from a non-endemic area (Denmark) was significantly lower (GI = 1.035, p < 0.05) than that of our controls in the endemic area, confirming that the controls had been exposed to schistosome infection. These speculative suggestions will be investigated in future planned studies that will examine responses of people over a long period.
There was no evidence of different responses to mitogens of PBMN cells in infected and uninfected people (Figure 4). Similar results were recorded in S. mansoni infected patients by Colley et al. 1979. Both findings imply that uninfected people living in endemic areas are primed and this priming occurs early in life (Camus et al. 1976).
The level of morbidity (2%) in the study area was low and the mean GI of these few individuals was slightly higher than that of those without morbidity. There is need for further studies in an area with higher levels of morbidity in order to elucidate the possible relationship between CMI and morbidity.
The finding that in vitro granulomatous activity in individuals over 20 is significantly higher in controls than in infected people suggests that people who have lived in an endemic area for at least 20 years have the ability to form granulomas, and may also be able to mount a better immune response against invading schistosomulae, and are therefore less susceptible to schistosome infection. However, the failure to find a very close association between CMI and intensity of infection suggests that the granuloma index on its own may not be sensitive enough to measure CMI responses in humans infected with S. haematobium, but that additional analysis of T cell subpopulations and immunoregulating activities may produce a better picture. Using PBMN cultures, conditions can be better suited to examine immuno regulating activities that may develop during schistosome infections (Todd et al. 1981).
Summary
The role of cell mediated immunity in relation to infection with Schistosoma haematobium in different age groups was investigated in an endemic community in Zimbabwe. Cell mediated immunity was measured by the granuloma index, whereas infection to S. haematobium was measured by excretion of eggs in urine and by morbidity indices, namely haematuria and ultrasound examination. The study showed that the mean granuloma index (GI = 1.402 + 0.386) in infected people < 20 years old was significantly higher than in infected people > 20 years old (GI = 1.1999 0.237, t = 2.22, p = 0.043). The mean GI of 1.573 + 0.494 in controls of individuals > 20 years old, that is, those not excreting S. haematobium ova, was significantly higher than that of the infected individuals, p < 0.05. There was no difference in the mean GI of younger infected and control individuals.
Relating intensity of infection to granulomatous activity showed that where the intensity of infection was high, (mean log egg counts = 1.140), the mean GI of 1.480 + was significantly higher than in individuals with low egg intensity (mean GI = 1.140 + 0.182). Individuals excreting more eggs were under 20 years old.
These results suggest that in an endemic area, young individuals with granuloma forming capacity excrete more eggs, whereas the ability of older individuals to form granulomas in vitro may imply the presence of an immune response that is capable of making the individuals less susceptible to S. haematobium infection.
Individuals with bladder wall abnormality had marginally higher mean GI (1.52 + 0.342) than those without bladder wall abnormality where the mean GI was 1.453 + 0.4013.
Acknowledgments
We are grateful to the Secretary for Health, Zimbabwe, for permission to publish this article. This investigation received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. Schistosoma haematobium soluble egg antigen was kindly donated by Dr. Liang through the World Health Organization. The authors also thank the Office of Research, USAID, for funding grant number DHR-5600-G-00-1042-00, the network meeting, and the publication of this paper.
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<section>Epidemiology and Biology</section>
<section>Epidemiology And Control Of Schistosomiasis In The Philippines: A Review</section>
B.L. Blas, P.F. Velasco, O.B. Alialy, E.S. Erce, J.C. Basas, And E.S. Bautista
Schistosomiasis Control Service, Department of Health
Manila, Philippines
Abstract
Schistosomiasis, or snail fever, is a serious disease problem in the Philippines. Primarily rural in distribution, schistosomiasis has grave socioeconomic ramifications as it largely affects farmers and their families, and thus hampers agricultural productivity.
The success of praziquantel as a chemotherapy for schistosomiasis has revitalized the campaign for case detection and treatment. A logical result of an effective campaign will be fewer human cases and, consequently, the surveillance and maintenance phase will follow. In this phase, the medical, malacological, and public health engineering staff can then intensify their role as coordinators in controlling the snail intermediate hosts and in the alteration of the snail habitats or the environment. Such roles are necessary in maintaining or further enhancing whatever gains have been achieved in the chemotherapy phase of the schistosomiasis control program.
Distribution And Extent Of The Problem
Schistosomiasis, or snail fever, is one of the more severe disease problems in the Philippines. Primarily rural, schistosomiasis has socioeconomic ramifications because it affects mostly farmers and their families, and thus hampers agricultural productivity.
There are about half-a-million endemic cases of Schistosoma japonicum distributed in 24 endemic provinces including: Oriental, Mindoro; Sorsogon, Luzon; the 3 provinces in Samar; Leyte and Bohol in the Visayas; and all the provinces of Mindanao except Misamis Oriental and Sulu (Figure 1). In these provinces, the human population at risk is 5.1 million and there are 2,987 known snail colonies with an approximate area of 28,731 hectares (Table l). The exposed human population in the 1,160 barangays (villages) is about 1.5 million (Table 2).
Climatic Factors
There is a definite relationship between geographical distribution of the disease and the annual rainfall pattern. This is supported by the observation that the affected provinces are limited to areas that fall under either Type II or IV of the PAGASA classification of climate in the Philippines. The Type II does not have a dry season but has a very pronounced maximum rainfall from November to January, while Type IV has a rainfall more or less evenly distributed throughout the year. The type of rainfall is a contributing factor to the existence of the snail intermediate host (Figure 2).
Health Status
The overall prevalence of schistosomiasis as of 1990 in the 24 affected provinces is 6.6% (Table 3). According to studies in Leyte, the annual mortality rate due to schistosomiasis is 1.78% of the estimated positive cases.
TABLE 1 Schistosomiasis Endemic Places and Snail Areas in the Philippines as of 1990
Province
No. of Endemic
Population
Snail Colonies
Towns and Cities
Number
Areas (ha)
Oriental Mindoro
Sorsogon
Bohol
Leyte
West Samar
North Samar
East Samar
Zamboanga del Norte
Zamboanga del Sur
Bukidnon
Misamis Occidental
Agusan del Norte
Agusan del Sur
Surigao del Norte
Surigao del Sur
Dayao del Norte
Dayao del Sur
Oriental Dayao
South Cotabato
Lanao del Norte
Lanao del Sur
North Cotabato
Sultan Kudarat
Maguindanao*
Total
*Province of Maguindanao was just recently discovered endemic.
Prevalence According to Age, Sex, and Occupation
The prevalence of S. japonicum in the Philippines, with respect to age, sex, occupation, and environment, follows a pattern that can be explained on the basis of contact with parasite infection (Pesigan et al. 1958). It was observed that infection during childhood and adolescence builds up rapidly until adulthood is reached, then a general downward trend follows. As far as age and sex distribution of infected individuals are concerned, there are significant sex differences between age groups past childhood, with the rates being higher for males than for females. Children of both sexes run an equal risk, but the differences begin to show up after 14 years, when males become more active working in the rice fields and run greater chances of acquiring infection.
TABLE 2 Exposed Population in Schistosomiasis Endemic Villages (Barangays) as of 1990
Endemic ProvinceNo. of Endemic Villages (Barangays)
Exposed Population
Oriental Mindoro
Sorsogon
Bohol
Leyte
West Samar
North Samar
East Samar
Zamboanga del Norte
Zamboanga del Sur
Bukidnon
Misamis Occidental
Agusan del Norte
Agusan del Sur
Surigao del Norte
Surigao del Sur
Dayao del Norte
Dayao del Sur
Dayao Oriental
South Cotabato
Lanao del Norte
Lanao del Sur
North Cotabato
Sultan Kudarat
Maguindanao
Total
Farmers, as an occupational class, have the highest infection rate (74.1%). When not planting or harvesting rice, they work as fisherman, unskilled laborers, or tuber gatherers, which would explain the next highest prevalence (more than 60%) among this group of professions. The occupations that most often brings people in contact with infected waters are farming and inland fishing. Seagoing fishermen who live mostly in the coastal division, would naturally be the people less exposed to infection in rivers, swamps, and streams. The class of workers with the lowest rates (exclusive of preschool children) are, for obvious reasons, office workers and the professional group, with an overall infection rate ranging between 16 and 26%. The rest (students, housekeepers, and "jobless" persons) occupy an intermediate position between the above two groups, with a range of approximately 51-58%. There are significant differences in the general prevalence of infection in the three
environments (cities, coastal, and inland), with the highest prevalence rate (61.1%) in the inland division. The differences, as one would expect, are due to the chances of infection and the general sociological makeup of the population (Pesigan et al. 1958).
Age is also a significant factor in schistosomiasis infections, i.e., there is a reduction in infection with increasing age after the peak prevalence is reached. This may be explained on the basis of a host's reaction arising from a humoral response to infection with a possible immunity mechanism coming into play, or from host-cell reactions around infiltrated eggs that tend to wall them off in the intestinal tissues, or from both.
Reservoir Host of Transmission
The disease is not limited to man. It also affects domestic animals (pigs, dogs, goats, cats, carabaos, cows) and even wildlife (field rats and monkeys), which serve as reservoirs of infection. Cross infection studies of the Philippine parasite from man, cow, dog, pig, and rat in the Leyte Pilot Project have shown that only one Schistosoma species is involved. This indicates that transmission of the disease could be attributed to all of these sources. Studies on the relative role of man and the reservoir hosts in transmission have shown that man contributes 75%, whereas animals only contribute 25%.
Socioeconomic Importance
Schistosomiasis should be considered a serious menace to the inhabitants of rural areas, especially farmers, for it causes not only a tremendous reduction in their work efficiency but also suffering and death. In a study carried out by Blas et al. (unpublished) an average of 45.4 man days per year per infected person is lost. Since the farmers are the mainstay of the country, this disease adversely affects the agricultural economy. Infected people cannot produce crops in quantities sufficient for their needs because they are sick.
This disease is identified as one of the major health problems in the Philippines today (Blas et al. 1986). The annual economic loss due to cost of treatment, and the manpower loss due to disability and death, is estimated to be about 375 million pesos, or about 14 million U.S. dollars per year.
Methods Of Control And Prevention
Based on our knowledge of the life cycle of the parasite, the methods of approach to the prevention and control of schistosomiasis are fourfold, and all are targeted at the weak links in the cycle. These are:
·Case finding and treatment with praziquantel.
·Environmental sanitation. This consists of: (1) proper waste or excrete disposal and control of animal reservoirs to prevent schistosome eggs from getting into snail infested areas; (2) a safe source of water for laundering and bathing to prevent exposure to cercaria-laden waters in streams, rivers, or creeks; (3) construction of foot bridges to avoid contact with the cercaria-infested waters; and (4) control of stray animals.
·Snail control. Control of the snail intermediate host includes: (1) drainage of water logged areas by stream channelization, seepage control, diversion, and intercepting channels together with irrigation schemes; (2) ponding; (3) filling; (4) modern methods of farming; and (5) chemical control or mollusciciding as a terminal measure on remaining small pockets of snails remaining after agroengineering measures.
·Health education.
Problems With The Targeted Control And Preventive Measures
In the area of case fading and treatment, there is: (1) insufficient manpower in terms of microscopists; (2) relatively poor community participation in submitting stools and in treatment of cases; and (3) inadequate manpower for rapid treatment of cases discovered during surveys.
Difficulties encountered in the environmental sanitation aspects are: (1) a slow change in personal habits, attitudes, and practices in the use of toilets; (2) low priorities placed on the maintenance of toilet housing; and (3) problems with the costs of providing and maintaining safe water sources and coping with local traditions of laundering among rural women.
Snail control has presented several special problems, for example: (1) it is beyond the Department of Health's capabilities because snail control is considered a non-medical approach. (2) the large number of vehicles needed in snail control requires frequent maintenance; and (3) chemicals are expensive and practically useless without preliminary agroengineering measures.
Public health education has also experienced problems. Educating the public on proper sanitation and health procedures is a slow process and requires persistence and continuous use of information, education, and communication materials.
Present Program Thrust
Considering the huge expense needed for stopping the transmission of schistosomiasis, control measures by the Department of Health are now directed more toward (1) morbidity or disease control mainly through case finding and treatment of cases (following the encouraging results of Santos et al. (1979), and the WHO (1983) publication on the role of chemotherapy in schistosomiasis control), and (2) health education through the use of information, education, and communication materials. Whenever possible, monitoring of activities by other government agencies that will affect transmission control will be carried out. These government agencies include the National Irrigation Administration, the Department of Public Works and Highways, and the Department of Agriculture and Food.
Resources, particularly the number of microscopists, were extremely limited in the Integrated Provincial Health Office through 1990. Instead of spreading the manpower too thinly in all of the endemic municipalities in 24 provinces, only 50% of the endemic villages or municipalities in 16 of the provinces were covered in 1988, and the other 50% were covered in 1989. This alternating pattern of coverage continued through 1990. In the eight endemic provinces with less than four endemic municipalities (Bohol, Zamboanga Norte, Davao Sur, Davao Oriente, South Cotabato, Sultan Kudurat, Lanao Sur, and Maguindanao) 100% coverage was targeted for case finding and treatment.
In 1991, with additional manpower, 100% of the eligible population was covered by mass stool examination and treatment. Where manpower and logistics was, and still is, a problem in provinces with higher endemicity, coverage will be carried out based on the level of prevalence. Areas with the highest endemicity will be covered for a number of years until schistosomiasis ceases to be a major public health problem.
Program Impact On Prevalence Of Disease
The results of the schistosomiasis efforts during the last five years (from 1986-1990) have displayed an encouraging trend, with the prevalence dropping from the weighted 5-year average baseline of 10.4% to 7.4% in 1986, to 6.6% in 1987, to 6.2% in 1988, 7.5% in 1989, and 6.6% in 1990 (Table 3).
Apparently, after obtaining a 28.8% reduction in prevalence in 1986, compared to the 5-year average of 10.4% for 1981-1985, the prevalence levered off at 6-7%, probably due to the 50% coverage strategy. Starting in and throughout 1991, a World Bank loan permitted a full (100%) implementation of the case finding and treatment program. We hope to see the prevalence of disease reduced below the 5% level in all endemic areas by the end of 1994.
TABLE 3 Prevalence of Schistosoma japonicum Infections from 1981 to 1990
year
Number Examined
Number +
*Five-year average.
Using a continuous and effective campaign for case finding and the treatment of cases with praziquantel, it is reasonable to predict that there will be fewer human cases of schistosomiasis and, consequently, a surveillance and maintenance phase will follow. At that time, the medical, malacological, and public health engineering staff could then intensify their role as coordinators in controlling the snail intermediate host, domestic and wild animal reservoirs, and the alteration of the snail habitat or the environment. These roles are necessary for maintaining or enhancing whatever gains have been achieved in the chemotherapy phase of the schistosomiasis control program.
Prospective Research Areas
There are a number of prospective research areas that could significantly enhance the control of schistosomiasis in the Philippines, and elsewhere. They are:
Chemotherapy
· Drug Resistance
- Observation on possible development of drug resistance to praziquantel.
- Search for alternative drug (herbal or synthetic) in case drug resistance develops.
· Drug Delivery System
- Effect of targeted vs. selective mass treatment on the prevalence of disease.
- Cost benefit analysis (targeted vs. selective mass treatment).
- Socioeconomic studies.
· Effect on Morbidity
- Development of more practical procedures for detecting liver size and portal hypertension before and after treatment.
· Drug Metabolism
- Determination of severity or reaction in relation to morbidity, liver and spleen enlargement, and serum drug levels.
Knowledge, Attitude, and Practices
- Development of teaching materials for the elementary and high school levels.
Service Delivery
- Organizational management for cost effectiveness.
Diagnosis
- Antigen detection on body fluids, especially urine.
Vector Control
- Cost analysis of environmental and ecological control measures.
- Biological methods of control.
Prophylaxis
- Use of slow release niclosomide rubberized clothing as cercarial repellent.
- Effects of small doses of praziquantel in the development of schistosomula.
Vaccine Production
- Vaccine for protection against infection.
- Vaccine for disease modulation (reduction of pathology).
Concluding Remarks
In the Philippines, the present program thrust is selective mass treatment with IEC support.
In provinces receiving foreign assistance, coverage includes monitoring of snail control and environmental sanitation, or interagency collaboration.
It is anticipated that interagency collaboration will be given more weight once the prevalence of the disease reaches a point where significant reduction can no longer be achieved. Other policyrelated concerns will be formulated subject to the results of further research activities directed toward more effective control and prevention of the disease.
References Cited
Blas, B.L., B.D. Cabrera, A.T. Santos, Jr., and J.S. Nosenas. 1986. An attempt to study the case fatality rate of Schistosoma japonicum infection in the Philippines. Southeast Asian J. Trop. Med. Public Health 17: 67-70.
Pesigan, T.P., M. Farooq, N.G. Hairston, J.J. Jauregui, E.G. Garcia, A.T. Santos, B.C. Santos, and A.A. Besa. 1958. Studies on Schistosoma japonicum infection in the Philippines. I. General considerations and epidemiology. Bull. WHO 18:345-455.
Santos, A.T., Jr., B.L. Blas, G.P. Portillo, O.M. Ortega, M. Hayashi, and K. Boehme. 1979. Preliminary clinical trials with Praziquantel in Schistosoma japonicum infection in the Philippines. Bull. WHO 57: 793-799.
World Health Organization. 1983. The role of chemotherapy in schistosomiasis control. WHO/Schisto/83.70.
<section>Aspects Of The Epidemiology Of Schistosoma Haematobium In Morocco</section>
K. Khallaayoune, H. Laamrani, And J. Mahjour
Abstract
Urinary schistosomiasis caused by Schistosoma haematobium has been recognized in Morocco for a century. Extension of the irrigation network in the last 30 years has increased the distribution of freshwater snails and snail-transmitted diseases such as schistosomiasis. The irrigation system is composed of cement lined canals and distribution boxes ("puisards" or "regards") that remain filled with water throughout the year. These puisards are snail habitats and transmission sites of schistosomiasis, particularly to schoolchildren who use them for bathing.
In the Attaouia area, where schistosomiasis was recently introduced, the density of Bulinus truncatus, the only intermediate host in Morocco, was monitored for a period of one year in correlation with weather variations. Snails were active throughout the year, with two overlapping generations, and particularly abundant by the end of spring and the summer. The infection rate of B. truncatus reached 3.5% in the summer, when human water contact was frequent.
A survey conducted in the village of Lamyayha showed that the prevalence of infection with S. haematobium was 21.2 % among the local population, who were passing from 10 to 80 eggs per 10 ml of urine.
Introduction
Although urinary bilharziasis has been known in Morocco since the time of ancient trade with the Middle East, the first clinical cases were first reported by Job (1915). The hypothesis of its introduction to Morocco is still controversial, but an Egyptian-Sudanese origin appears the most probable. Until 1959, schistosomiasis was restricted to palm groves in southern Morocco, where traditional irrigation was practiced. The extension of irrigation into arid zones in Morocco has created an increase in freshwater snail populations, corresponding schistosome transmission sites, and a large increase in snail-transmitted infections such as schistosomiasis and fascioliasis. Actually, Schistosoma haematobium occurs in most irrigated agricultural areas where extensive irrigation networks have promoted the development of suitable habitats for the snail intermediate host. The primary transmission sites in Morocco are: (1) natural snail habitats -- temporary swamps (merja), marshy
plains, and residual water along pre-Saharan palm groves in the south; and (2) artificial snail habitats - newly created sites represented by so-called "regards" or "puisards," which are water distribution boxes in the irrigation system that hold water permanently and are commonly used for bathing by schoolchildren, and as a water supply for households.
After the National Schistosome Control Program was created in 1978, the number of reported cases of schistosomiasis decreased in some endemic areas (Figure 1), but remained high in newly irrigated zones (Attaouia and Beni-Mellal) and pre-Saharan regions (Tata and Errachidia). However, from 1982 to 1989, schistosomiasis was reported in 28 provinces (M.S.P. 1989). According to data published by the Ministry of Public Health, schistosomiasis was reported in all age groups and was particularly prevalent among schoolchildren whose ages ranged from 7 to 14 (Figure 2).
In endemic areas, control of the snail intermediate hosts remains a promising means to reduce schistosome transmission. However, despite molluscicide treatments, snail control in Morocco has failed to achieve the desired results because treatments were applied without considering epidemiological data pertaining to the seasonal activity of the snail, which is intimately linked to weather variations and water availability (Babiker et al. 1985, Lwambo 1988, Woolhouse and Chandiwana 1989). Successful snail control can only be achieved if based on detailed data for snail ecology in each area.
During the period January-December 1987, a preliminary study was conducted to evaluate the dynamics of Bulinus truncatus populations and the transmission patterns of S. haematobium to initiate the development of a control strategy for the snail intermediate host that would reduce the prevalence of infection. Since this was only a preliminary study, a more comprehensive study has been planned to obtain an accurate description of seasonal transmission patterns in Morocco. Such patterns require long-term epidemiological investigations over several successive years to reduce the effects of annual weather variations.
Materials and Methods
Study Area
The Attaouia area is about 50 miles northeast of Marrakech. It is located in a large plain, which was recently provided with a large irrigation system. This study site was chosen because of the recent introduction of S. haematobium and the comparatively high prevalence of schistosomiasis. The climate is a sub-arid type with an annual rainfall not exceeding 300 mm. The cold rainy season extends from November to March, followed by the hot and dry period. The 1987 study period was relatively rainy compared to preceding years (Figure 3).
Irrigation System
The irrigation scheme is composed of cement lined open canals. The water is provided from the Moulay Youssef Dam and distributed successively to the primary, secondary, and tertiary canals. The tertiary canals are usually above ground and connected by concrete distribution boxes (puisards), where water remains stagnant throughout the year.
Snail Sampling Method
Four puisards were monitored for snail density (abundance of B. truncatus), closeness to human habitations, and usage for bathing, particularly by children. Snail abundance was monitored monthly for one year using a drag-scoop as a sampling tool for snails of at least 1 mm shell length. The dredge was pulled along the four sides of the puisard from the bottom to the edge. Snails collected were measured along their axis to the nearest millimeter and screened for schistosome infection by exposing each snail to artificial light for 24 hours. The cercariae shed by the snails were exposed to mice to induce infections and for subsequent identification of the schistosome. Snails were then returned to their habitats within 48 hours. Egg packets collected were sorted to species and counted. Snail density was given as the mean number of snails collected from the four puisards.
Survey
In the village of Lamyayha, midday urine specimens of 85 people age 5-48 years were examined for S. haematobium egg counts by the filtration method. This village is known for its high prevalence of schistosomiasis.
Weather Data
Records of daily minimum and maximum air temperatures and monthly rainfall of the closest station to the study sites (Kalaa des Sgharna) were obtained from the National Meteorological Center, Casablanca.
Results
Snail Activity
The snail population data for B. truncatus for the period from January 1987 to December 1987 are shown in Figure 4. Snails were active throughout the year in the Attaouia area. There were two major peaks in snail numbers, one that occurred in July at the onset of summer and another in September, after which a definite drop occurred in the numbers of snails. The first peak represented the first generation and was associated with increased egg production in August (Figure 5). A high mortality rate was observed in snail populations during November-December, with the majority of deaths among aged snails. A low snail recovery rate was recorded throughout the winter of 1987.
In surveyed habitats the freshwater snail fauna was composed of B. truncatus, Lymnaea truncatula, L. peregra, Physa acuta, Ancylus fluviatilis, Melanopsis praemorsa, and Mercuria confusa. Among these snails, B. truncatus was the only snail found infected with S. haematobium.
Snail Infections
Snails infected with S. haematobium were encountered throughout the four seasons of the year. The monthly snail infection rate ranged from 0 to 3.5% (Figure 6).
Prevalence of Infection
Of the 85 urine specimens examined, 18 (21.2%) were positive (Table 1). The prevalence of schistosomiasis was particularly high in schoolchildren age 7-14 years, who were passing from 32 to 80 eggs per 10 ml of urine.
Discussion
This study demonstrated that B. truncatus was active throughout the year. Snail populations followed a cyclical pattern where high density occurred in summer. A similar pattern was observed in the same area in Lymnaea truncatula, the snail intermediate host of Fasciola hepatica (Khallaayoune and Elhari 1991). Apart from water availability, temperature was considered to be the main factor that influenced the fluctuations in snail populations. Because the mean daily temperature was above 10°C throughout the year, snails remained active during the four seasons. These findings are in agreement with those of Moussa and Abou El-Hassan (1972) who found that the development of B. truncatus can only occur within a temperature range of 10-37 °C. Moreover, embryonic development of the snail was shown to be accelerated when the temperature was increased up to a limit of 37 °C. The considerations suggested that the life span of B. truncatus did not exceed one year, depending on when the
eggs hatched, because of the intensive breeding activity resulting from environmental conditions in the habitat. In fact, in the summer, when the prevailing temperature was high, puisards provided an ideal environment for the survival of the snails.
One factor that may have had considerable effect on snail activity was the relative decrease in temperature in winter. This resulted in low egg production and a drop in snail density. The relative resistance of B. truncatus to low temperatures (Appleton 1978) may have contributed to the population of the habitat from early ovipositions that occurred in midwinter. The second peak of egg numbers observed in August and the variations in snail size over the study period led to the assumption that two snail generations overlapped during this year.
Observations on the pattern of snail infection rates showed that maximum snail infection was recorded in the summer when the mean daily air temperature was high and human contact with water was frequent, particularly for children. A similar pattern was described by Woolhouse and Chandiwana (1990) in B. globosus in Zimbabwe. The potential for carry-over infections into the winter may occur because of the relative resistance of B. truncatus to low temperature, as revealed by the occurrence of observed infections in 10 snails during the winter. Schistosome eggs can be excreted by the host throughout the year, but only a few of them will complete their full development within the snail. Shiff et al. (1975) demonstrated that in cold seasons low development of intra-molluscan stages may result in the accumulation of pre-patent infections, which will become patent when adequate temperatures are restored. Moreover, a significant correlation between monthly snail infection rates and
S. haematobium incidence in schoolchildren occurred in endemic areas (Lwambo 1988).
TABLE 1 Prevalence of Schistosomiasis in the Village of Lamyayha, Attaouia
Age (years)
Number examined
Number positives
Prevalence (%)
Mean No. eggs/10 ml
Range
Based on the results of this preliminary study, the main transmission season of urinary schistosomiasis in Attaouia occurs during the summer, so that most clinical cases should be detected in the fall and winter. Although cercariae shedding can occur throughout the year, the risk of infection for schoolchildren is mainly restricted to the summer season when they bathe in the puisards. However, for farmers and young girls assisting in housework there is risk of infection in the Attaouia area throughout the year.
In Attaouia, canals and puisards located near villages remain the major sources of water for households. Therefore, control of the disease can only be achieved if it is based on well-founded snail control and chemotherapy. Molluscicides could be applied in May and June to reduce snail populations before the peak of schistosome transmission occurs. Furthermore, puisards located at the end of tertiary canals are heavily populated with B. truncatus, because of a continual drift of snails and mud from upstream breeding pockets. Locating and treating these snail-breeding pockets are essential to prevent repopulation of these puisards. Dazo et al. (1966) demonstrated that B. truncatus can double its population in 14-16 days when optimum field conditions are established. Similarly, a rapid increase in snail density associated with an increase in temperature (Mousse and Abou El-Hassan 1972) was also observed in our snail habitats during the hot season.
It can be concluded that the cyclical activity of the snail and the seasonal transmission patterns
of schistosomiasis in our study area were entirely dependent upon the occurrence of favorable temperature and presence of water. The great expansion of irrigation systems in Morocco has created ideal biotopes for B. truncatus and other freshwater snails, which may have a serious impact on the health of the exposed populations.
According to the National Agricultural Program, one million hectares will be under irrigation
by the year 2000. This will certainly increase the risk of the spread of schistosomes and introduction of bilharziasis to newly irrigated areas. Prospective physical changes of the landscape may also create favorable biotopes for snail invasion and establishment. It is imperative that appropriate measures be implemented to prevent the spread of schistosomiasis and the possible introduction of other snail-transmitted diseases. A large-scale epidemiological survey using parasitological tests and possibly urine dip-stick tests should be organized to assess precisely the incidence of schistosomiasis in Morocco and to assist the National Control Program in devising a new approach in their control strategy.
Acknowledgments
The authors would like to thank the medical staff of Attaouia and Kalaa des Sgharna (SIAAP)
for their technical assistance. The authors also thank the Office of Research, USAID, for funding the network meeting and publication of this paper.
References Cited
Appleton, C.C. 1978. Review of literature on biotic factors influencing the distribution and life cycles of bilharziasis intermediate host snails. Malacol. Rev. 11: 1-25.
Babiker, A., A. Fenwick, A.A. Daffalla, and M.A. Amin. 1985. Focality and seasonality of Schistosoma mansoni transmission in the Gezira irrigated area, Sudan. J. Trop. Med. Hyg. 88:57-63.
Dazo, B.C., N.G. Hairston, and I.K. Dawood. 1966. The ecology of Bulinus truncatus and Biomphalaria alexandrina and its implications for the control of bilharziasis in the Egypt-49 Project area. Bull. WHO 34:339-356.
Job, F. 1915. Bilharziose au Maroc. Soc. Med. Hopitaux de Paris, 31: 1283-1285.
Khallaayoune, K. and M. Elhari. 1991. Variations saisonnieres de ['infestation par Fasciola hepatica chez la chevre dans la region du Haouz (Maroc). Ann. Rech. Vet. 22:219-226.
Lwambo, N.J.S. 1988. Transmission of urinary schistosomiasis in Sukumaland, Tanzania. 1. Snail infection rates and incidence of infection in school children. J. Helminthol. 62:213-217.
M.S.P. (Ministere de la Sante Publique). Programme de lutte contre la bilharziose, Etat d'avancement, Situation Annee 1989, Rapport No. 9, pp. 27.
Moussa, A.H. and A.A. Abou El-Hassan. 1972. The effect of water temperature on the snail intermediate hosts of schistosomiasis in Egypt. J. Egypt. Med. Assoc. 55: 148-165.
Shiff, C.J., A. Evans, C. Yiannakis, and M. Eardley. 1975. Seasonal influence on the production of Schistosoma haematobium and S. mansoni cercariae in Rhodesia. Int. J. Parasitol. 5:119-123.
Woolhouse, M.E.J. and S.K. Chandiwana. 1989. Spatial and temporal heterogeneity in the population dynamics of Bulinus globosus and Biomphalaria pfeifferi and the epidemiology of their infection with schistosome. Parasitology 98:21-34.
Woolhouse, M.E.J. and S.K. Chandiwana. 1990. Temporal patterns in the epidemiology of schistosome infections of snails: a model for field data. Parasitology 100:247-253.
<section>Biocontrol</section>
<section>Thiara (Tarebia) Granifera (Lamarck): An Agent For Biological Control Of Biomphalaria</section>
W.A. Sodeman, Jr.
Division of Gastroenterology, Department of Medicine, Medical College of Ohio
Toledo, Ohio
Abstract
Thiara (Tarebia) granifera is a Prosobranch snail with a native range from Madagascar to Hawaii, including most countries in Southeast Asia and the Philippines. It is found as far north as the Ryukyu Islands. It has been widely transported and is now widely distributed in the New World. Spontaneous introduction of T. (T.) granifera into Puerto Rico in water containing Biomphalaria glabrata, a Neotropical intermediate host of Schistosoma mansoni, was noted to cause suppression of the intermediate host snail. A reported trial of T. (Melanoides) tuberculata to control B. glabrata in St. Lucia showed similar suppression of this intermediate host snail. The published reports of these studies indicate that the snail used was T. (T.) granifera obtained on the island of Dominica. However, subsequent information suggests that the snail used was T. (M.) tuberculata, not T. (T.) granifera. Studies reported here suggest that this suppression is competitive, not predatory. The
competitive mechanism is unknown. Thiara (T.) granifera is environmentally safe. It is a first intermediate host for Haplorchis taichui, but not for Paragonimus westermani. Early reports identified the association between T. (T.) granifera and P. westermani and this has been incorporated into parasitologic folklore in spite of the subsequent retraction of this association.
Suppression is time limited and has been best demonstrated in partially impounded water. In selected sites this is an effective snail control technique.
Biology
There has been some interest in the use of Thiara (T.) granifera as a biologic agent in the control of intermediate hosts of schistosomiasis. However, little information has been published concerning the field use of this snail.
Diagnosis
Thiara (T.) granifera (Lamarck) is an ovoviviparous, parthenogenetic, melaniid snail native to the Indo-Pacific radiation. The shell is turreted with the body whorl in the adult snail more than half the height of the entire shell. The sculpture of rectangular knobs follows a spiral pattern. Sutures are distinct and the whorls are flat sided. This snail can be distinguished from closely related Thiaridae by the anatomy of the female reproductive tract. The seminal receptacle is lengthened to join the diverticulum and the connecting duct midway between the seminal receptacle and the oviduct. Comparative diagnosis of the Thiaridae has been reviewed by Pace (1973).
Geographic Distribution
Thiara (T.) granifera inhabits the Indo-Pacific radiation. It can be found from Madagascar through India and Southeast Asia. In the Pacific basin it is found in the Philippines and the Society Islands as well as Taiwan, the Ryukyu Islands, and Hawaii. In 1935, T. (T.) granifera was introduced into the United States and sold widely as an aquarium snail. Permanent colonies became established in the United States in Florida and Texas. Both of these were in constant temperature springs, which provided protected overwintering for this cold-sensitive snail. As reported in Ferguson (1977) and Prentice (1980), the snail subsequently spread from Florida to Puerto Rico (1953), Vieques (1964), Dominican Republic (1967), Grenada (1970), Venezuela (1970), Haiti (1972), Antigua (1980), and Costa Rica (1983) (Figure 1). Colonies have also been reported from Guadeloupe and Martinique. The mechanism for most of this spread is unknown.
One study (Sodeman, unpublished) of the spread within Haiti, demonstrated that over two decades the snail was seen to spread widely, eventually involving almost all suitable habitats.
Environmental Impact
Thiara (T.) granifera is a detritus feeder. It browses on algae and diatoms and does not feed on vegetation. Rice and other water-grown plants are not damaged by snail feeding. These snails are adapted to fast-moving water by their streamlined shape and hold their station well during sudden changes in current velocity, a feature that enhances the utility of these snails in environments subject to sudden inundation. In the optimum environment it is prolific. Sand bars in free-running streams are often coated with shoals of Thiara. There have been no reports or field observations of these snails proliferating to the point where they would plug water intakes or irrigation equipment. Stable populations in still water environments are at much lower densities. However, high densities, as a result of temporary overpopulation, can be forced in still water environments.
Parasitology
Thiara (T.) granifera came to medical attention in 1917 when it was identified as a first intermediate host of Paragonimus westermani (Kerbert) by Nakagawa (1917). Nakagawa was working in Taiwan searching for the then unknown intermediate stages of P. westermani. He erroneously identified cercaria shed from T. (T.) granifera as P. westermani. He quickly established that this association was erroneous and published a correction. The initial error was published in English in the Journal of Experimental Medicine and received wide circulation. The subsequent correction appeared in a paper in Tokyo Iji Shinshi No. 2062 (Nakagawa 1918) in Japanese and apparently was overlooked. The error has become imbedded in parasitologic folklore. I can find no authentic reports of T. (T.) granifera identified as a first intermediate host for P. westermani or of successful laboratory infection of this snail by P. westermani.
Thiara (T.) granifera has also been listed (Abbott 1952) as a first intermediate host for Metagonimus yokogawai (Katsurda). However, I cannot find this substantiated by any reported field collection or laboratory report of infection. There is a well-substantiated report of finding T. (T.) granifera infected with the Heterophyid Haplorchis taichui (Nishigori), an occasional parasite of man (Faust and Nishigori 1926), as well as a number of flukes that do not regularly parasitize man (Ito 1964, Malek 1962, and Sodeman 1991). Transfer of T. (T.) granifera to new areas would seem to pose a small, if any, parasitologic risk for man.
Role In Snail Control
Ferguson et al. (1968) in the discussion of a paper on the control of schistosomiasis on Vieques Island, Puerto Rico, mentioned the possibility that the encroachment of T. (T.) granifera might be a factor in the fall in prevalence of schistosomiasis in Puerto Rico, which they had noted at that time. Murray (1970), made an anecdotal observation during a discussion of a paper by Taylor that Elimia comalensis (Pilsbry.) was displaced from much of its habitat in Texas by introduced T. (T.) granifera. Ferguson, working in Puerto Rico with a series of collaborators, noted displacement of B. glabrata (Say) from a number of river, stream, pond, and lake habitats by naturally introduced T. (T.) granifera. He performed a number of ecologic studies of this snail, all of which remain unpublished. A summary of his observations on competitive displacement in Puerto Rico was published in 1977. While B. glabrata was suppressed or displaced from a number of sites, this coincided with other
environmental changes and it was not clear that the introduction of T. (T.) granifera was the responsible factor.
In an attempt to clarify the role that T. (T.) granifera might play in displacing B. glabrata, Prentice (1983) and Prentice in Jordan (1985) initiated controlled trials of T. (T.) granifera in St. Lucia. The published reports of these studies indicate that the snail used was T. (T.) granifera obtained on the island of Dominica. However, subsequent information suggests that T. (M.) tuberculata, not T. (T.) granifera, was the snail used. The snails pictured in the publications by Prentice (1983) and by Jordan (1985) have the shell characteristics of T. (M.) tuberculata rather than T. (T.) granifera. Thiara (M.) tuberculata, but not T. (T.) granifera, is reported from the island of Dominica. On a recent visit to St. Lucia only T. (M.) tuberculata were found at 13 sites examined. Two of these sites are part of the studies described by Prentice (1983). Prentice's studies involved five controlled trials and one uncontrolled trial. Five sites consisted of marshy areas drained by
small streams. The sixth testing site involved banana drains. All sites were successfully colonized by introduced T. (M.) tuberculata. The periods of observation varied from 6 to 36 months. Biomphalaria were eliminated at four of the six sites. Biomphalaria persisted $ one of the marshy sites and in the banana drains, but at lower densities in both areas. Population densities of T. (M.) tuberculata about twice the density of Biomphalaria were necessary to observe an effect.
Several additional observations and qualifications may be made. The observation periods were short and the long-term relationship between T. (M.) tuberculata and B. glabrata was not evaluated. Several sites desiccated then refilled, with a resurgence of Biomphalaria in some and of Thiara in other sites. Desiccation was a feature of both sites where elimination of Biomphalaria was incomplete. Ferguson (1977) made similar observations on the effect of environmental changes on population balance in Puerto Rico. Environmental changes, particularly desiccation, can result in reversals in the balance between these two snails.
Robart et al. (1976) reported the presence of T. (T.) granifera in Haiti with all of the snails found east of 72°15'. Subsequent studies (Sodeman unpublished) show an extension of the range of the snail throughout the country. Many of the sites now colonized by Thiara also contain Biomphalaria, either Biomphalaria havenensis (Pfeifferi), B. helophila (Orbigny) or B. glabrata. Stable colonies of Thiara and Biomphalaria coexist.
A similar coexistence of a Biomphalaria species and T. (T.) granifera was observed at a fish farm outside of San Jose, Costa Rica, in 1983 (Sodeman, unpublished). Here native B. obstructa (Morelet) were abundant in rearing ponds, while T. (T.J granifera, thought to be brought in when Tilapia were introduced to the farm, share ponds and outflow ditches. What is missing is data that could identify whether this coexistence represents a response to a particular environment, a stable longterm balance into which the snails settle, or in fact a much reduced and partially controlled population of Biomphalaria.
Mechanisms
There is no clear understanding of how or why Thiara causes displacement of Biomphalaria. They do compete for food? but do not share the same preferred habitat. Prentice (Jordan 1985) was unable to demonstrate an effect of Thiara conditioned water on the fecundity or development of B.
glabrata. In similar experiments I have been able to demonstrate a short term effect on adult B. glabrata of shared residence with Thiara in small aquaria. I have set up isolated tanks with balanced numbers of Biomphalaria and Thiara (5/5) and find that after thirty days the Biomphalaria had all died. There is no evidence of predation by Thiara on Biomphalaria. There was no apparent explanation for the mortality of the Biomphalaria. Too few trials were performed to permit statistical analysis. All Biomphalaria placed in tanks with Thiara died (100% mortality) while the controls all survived through day 42. Thiara in control and trial tanks all survived through day 42.
A single trial was done with conditioned water. Thiara and Biomphalaria were each placed in separate tanks for 30 days. The snails were then removed and five Biomphalaria were placed in the tank conditioned with Thiara and five Thiara were placed in the tank conditioned with Biomphalaria. The test Biomphalaria had all crawled out of the tank by day four and died. The Thiara remained unaffected and alive.
Prentice (1983) noted that there seemed to be a mass effect. When the density of Thiara about doubles that of Biomphalaria then the effect on the Biomphalaria population becomes apparent.
Thiara (T.) granifera is parthenogenetic and ovoviviparous. Eggs are laid into a brood pouch opening just beneath the edge of the mantle. Abbott (1952) described the release of one or two juvenile snails daily by mature adults. I have been able to confirm that these high reproductive rates are obtainable in a laboratory setting. Four plastic tanks were set up as follows. Each tank was 1 m square and 20 cm deep. Each tank was prepared with a pea gravel (0.5 cm diameter avg.) slant and filled to a depth of 5 cm with conditioned water. Pumps circulated the water from the bottom of the slant to a trough above the top of the slant (Figure 2). Water flow was adjusted to cascade a continuous running stream down the slant. Thiara is best adapted to rapidly running water. Tanks were permitted to run until a visible film of green algae formed on the gravel. Then 10, 1, and 0.5 cm T. (T.) granifera were introduced into each tank. Tanks were harvested at the end of a 30-day interval.
More than 600 juvenile snails were found in each tank.
Over the past five years I have carried out studies on the ability of T. (T.) granifera to colonize various sites on the north coast of Haiti. In the field in Haiti results have been variable. Five sites were selected along the Port Margot River on the north coast of Haiti. This river has a resident population of Thiara and the sites selected were free of colonies of this snail. Adult Thiara (1.5 cm) were seeded into the site. Each seeding involved 1,000 adult snails. These were followed over nine months. At four sites small colonies of Thiara were found on subsequent visits, but there was really no assurance that these snails were related to the seeding. The rainy season caused the river to go into spate and Thiara from other sites in the river could easily have been washed into these locations. Five control sites with established colonies of Thiara were selected in the River Limbe. This river ran parallel to the Port Margot in the adjacent valley. Marked seasonal
fluctuation was seen in the snail populations at these control sites. Snail density fell as the rainy season progressed.
Careful surveys at each visit showed that there were always some areas of the river with shoals of Thiara blanketing a sand bar but the location of these shoals varied seasonally. While Biomphalaria are found in rivers, it is not a favored location. Rivers are a favored location for Thiara. Riverine colonies of Biomphalaria and Thiara have little overlap in their preferred habitats within the river bed, and it seems unlikely that Thiara would be effective in playing any role in competitive displacement in this setting.
Biomphalaria do favor ponds, swamps, irrigation ditches, and flooded fields. Five sites involving irrigated fields on the flood plain of the River Port Margot were selected. Similar control sites were selected on the flood plain of the River Limbe. One thousand adult (1.5 cm) Thiara were released at each site on the Port Margot. At Port Margot two of the sites were irrigated fields that were diked and held standing water with no visible flow. Fewer than 25 Thiara were found at these sites at subsequent visits. Two of the sites were small pools adjacent to the road and to irrigated fields. Each pool was 5 m in diameter and 1 m deep. There was no visible flow of water at either of these sites. The maximum collection of Thiara at these sites on any subsequent visit was four snails. The last Port Margot site was an irrigated field traversed by a slowly flowing stream 2 m wide. Snails were released at the stream edge. The population of Thiara slowly rose and levered off
suggesting the establishment of a resident population. The control sites on the River Limbe included four with established Thiara populations. One of these four became inundated in the rains and the Thiara density fell. The other three sites were small (2 m) sluggish streams, not inundated during the rains. Here the Thiara population remained at a stable density throughout the study.
Prentice (1983) and Jordan (1985) on St. Lucia released Thiara in several environments. He reported excellent results in swampy fields that were drained by small streams. He noted rapid population expansion and subsequent displacement of Biomphalaria. Thiara also did well in semipermanent environments (banana drains), but the effect on Biomphalaria was not clear. These results, in terms of establishing Thiara populations, do not differ from the experience in Haiti.
Incidental observations were also made at a site on the Plaine du Nord, also on the north coast of Haiti. This rice field harbors Biomphalaria glabrata and Thiara (T.) granifera. The snails were first reported in 1977 (Robert et al. 1976) and both have remained, coexistent, through 10 years of observation (Raccurt et al. 1985). This field is alternately flooded and dried with wet rice cultivation. Thiara survive in an adjacent pool and Biomphalaria aestivate in the mud in the rice field during the intervals when standing water is not present. During the wet periods the two populations remain balanced and tolerant of one another.
Conclusions
A great deal remains unknown concerning the interactions of Thiara and Biomphalaria. The most important unknown is the mechanism of this interaction. The long term interactions between these two snails, especially in environments where sites are periodically dry, is unknown. There is little information concerning the response of snails other than B. glabrata to the introduction of Thiara (T.) granifera. With the limited information in hand, several observations can be made.
1. Thiara (T.) granifera is an effective agent for the control of B. glabrata in environments with slowly moving water. Thiara (T.) granifera and B. glabrata do not share optimum environments. Biomphalaria do best in still water, while Thiara do best where there is a substantial current. Where the current flow is light, both snails are capable of establishing substantial populations but they are not capable of sharing this environment. The more Thiara "friendly" the environment, the smaller need be the initial inoculum size. Natural population expansion will reach the size necessary to provoke suppression.
2. Population density seems to be a key factor. Introduced adult Thiara will begin to release young immediately. One thousand snails theoretically are capable of producing a population of 20,000 in 10 days and 60,000 in a month. Thiara are easily harvested in rivers in the tens of thousands. It represents "low tech" snail control. There are limits to the growth that a given environment will support but this does not preclude initial expansion of the population at a site well beyond these limits. Even in less favorable habitats for Thiara if the inoculum is large enough, the snails in the brood pouch at the time of the release will permit the initial expansion of a Thiara population to the point where it could displace Biomphalaria, but at the price of a later fall to the stable density that the site can support. Reintroduction of Biomphalaria or adjustment of density ratios because of seasonal change or drying may allow reemergence of Biomphalaria and balanced survival of
both snails. Effective control at semipermanent sites will need repeated refreshing of the Thiara population.
3. Relatively few Thiara are needed to establish a growing colony in flowing water, however, Thiara are not effective agents for control of Biomphalaria in rivers. In theory, since there are relatively fewer Biomphalaria in rapidly flowing water, critical population densities that can provoke suppression of Biomphalaria are more easily reached. Actually an overlap in the two populations at densities that would provoke suppression of the Biomphalaria is relatively uncommon. If suppression depends on something released by the Thiara, adequate concentrations are less likely in rapidly flowing water. If snail to snail contact is the important feature, then Biomphalaria have the option to just move on in a flowing water environment.
4. Thiara are less effective for control of Biomphalaria in still water, where it probably will take much larger numbers of added Thiara to be effective. It may be that these still water sites will support, only transiently, a density of Thiara sufficient to suppress Biomphalaria.
5. The effect of Thiara on Bulinus species is unknown.
Acknowledgments
The author thanks the Office of Research, USAID, for funding grant number PDC
5542-G-SS-5088 00, the network meeting and the publication of this paper.
References Cited
Abbott, R.T. 1952. A study of an intermediate snail host (Thiara granifera) of the Oriental lung fluke (Paragonimus). Proc. U.S. Nat. Mus. 102:71-116.
Faust, E.C. and M. Nishigori. 1926. The life cycles of two new species of Heterophyidae, parasitic in mammals and birds. J. Parasitol. 13:91-128.
Ferguson, F.F. 1977. The Role of Biological Agents in the Control of Schistosome-Bearing Snails. U.S. Department of HEW/PHS.
Ferguson, F.F., J.R. Palmer, and W.R. Jobin. 1968. Control of schistosomiasis on Vieques Island, Puerto Rico. Am. J. Trop. Med. Hyg. 17:858-863.
Ito, J. 1964. A monograph of cercariae in Japan and adjacent territories. in Morishita, K., Y. Komiya and H. Matsubayashi. (ed.). Progress of Medical Parasitology in Japan. Vol. I. Meguro Parasitological Museum, Tokyo.
Jordan, P. 1985. Schistosomiasis: The St. Lucia Project. Cambridge University Press, Cambridge.
Malek, E.A. 1962. Laboratory Guide and Notes for Medical Malacology. Burgess, Minneapolis.
Murray, H.D. 1970. Discussion of Dr. Taylor's paper. Malacologia 10:33-34.
Nakagawa, K. 1917. Human pulmonary distomiasis caused by Paragonimus westermani. J. Exp. Med. 26:297-323.
Nakagawa, K. 1918. Further notes on the study of the human distome, Paragonimus westermani. Tokyo Iji Shinshi, No. 2062. (in Japanese.)
Pace, G.L. 1973. The freshwater snails of Taiwan (Formosa). Malacol. Rev. Supplement 1:1-118.
Prentice, M.A. 1980. Schistosomiasis and its intermediate hosts in the Lesser Antillan islands of the Caribbean. Bull. Pan Am. Health Organ. 14:258-268.
Prentice, M.A. 1983. Displacement of Biomphalaria glabrata by the snail Thiara granifera in field habitats in St. Lucia, West Indies. Ann. Trop. Med. Parasitol. 77:51-59.
Raccurt, C.P., W.A. Sodeman Jr., G.L. Rodrick, and W.P. Boyd 1985. Biomphalaria glabrata in Haiti. Trans. R. Soc. Trop. Med. Hyg. 79:455-457.
Robart, G., G. Mandahl-Barth, and C. Ripert. 1976. Inventaire, repartition geographique et ecologic des mollusques dulcaquicoles d'haiti (caraibes). Department de L'Agriculture, des Ressources Naturelles et du Developpement Rural 2(4)20:63.
Sodeman, W.A. Jr. 1991. Mollusks involved in disease transmission. pp.963-970. in Strickland, G.T. (ed.). Hunter's Tropical Medicine. 7th edition. W.B. Saunders Company, Philadelphia.
<section>Controlling Transmission Of Schistosomiasis Using Phytolacca Dodecandra (L'herit) Berries In Zimbabwe</section>
J. Ndamba And S.K. Chandiwana
Blair Research Laboratory
Harare, Zimbabwe
Abstract
Molluscicidal efforts using synthetic compounds are very expensive. Accordingly, molluscicidal potency tests on several strains of the plant known as Endod (Phytolacca dodecandra), that grows in Zimbabwe, were performed and the most potent local strain identified. A pilot field trial using domestically produced berries was undertaken. A single application of the berries suppressed snail population numbers for several months. The successful introduction of the Ethiopian type 44 strain, as well as the local strain in various parts of Zimbabwe where the plant does not grow naturally, has paved the way for studies to evaluate the efficacy of Endod in reducing schistosomiasis in the human population. Recently completed Tier I toxicological studies have also facilitated implementation of more elaborate field studies.
Introduction
Due to the high cost of synthetic molluscicides used in controlling schistosomiasis transmitting snails, there is a growing interest in plants with molluscicidal properties. The most extensively studied plant molluscicide is one derived from the berries of Phytolacca dodecandra (commonly known as Endod), a plant indigenous to East, Central, West, and Southern Africa. Its molluscicidal properties were first reported in Ethiopia where the berries were traditionally used as soap and as a medicine (Lemma 1965). The active molluscicidal ingredients of the berries are several derivatives of oleonolic acid of triterpenoid saponin (Parkhurst et al. 1974).
Although the berries are lethal to small fishes at molluscicidal concentrations that are required for snail control (Lemma 1970), unlike chemical molluscicides, the berry extract rapidly biodegrades in water and this reduces the possible accumulation of the toxic residues in the environment (McCullogh et al. 1980).
In Zimbabwe, P. dodecandra is commonly called "gopo" and is a shrub or semi-climber often found growing on termite mounds. Chandiwana et al. (1985) have established a rough distribution for the plant in Zimbabwe. However, no efforts have previously been made to determine the actual
distribution of the plant throughout the country and no studies have been done to evaluate the molluscicidal potency of the local varieties. Further, P. dodecandra has only been tried under field condition in Ethiopia, and because of the recently completed toxicological studies, interested countries should carry out independent laboratory and field trials to determine the most appropriate method of using the plant molluscicide. This paper summarizes studies that have been carried out in Zimbabwe to facilitate the use of P. dodecandra for wide-scale schistosomiasis control.
Materials and Methods
Natural Distribution of P. dodecandra in Zimbabwe
The search for P. dodecandra was conducted in places where the plant had previously been reported (Chandiwana et al. 1985) and on termite mounds and other sites where the plant is likely to grow. To facilitate the search, local people were asked to assist in the location of the plant after they had been shown a specimen. The local name of the plant, its traditional uses (medicinal or otherwise), if any, and its location were recorded.
A kilogram of green berries was collected into pre-labeled transparent plastic bags from plants whose racemes had reached the unripe green fruit stage of development (Lugs 1981). These were brought back to the laboratory where they were oven dried for three days at 30°C. The dried berries were crushed into tiny particles by an electric blender. The particles were standardized by passing them through a 0.5 mm mesh after which they were stored in screw top glass bottles until bioassays were performed.
Molluscicidal Potency Tests
Bioassays to determine the molluscicidal activity of the berries were performed using adult Bulinus (Physopsis) globosus, the most important intermediate host of schistosomiasis in Zimbabwe (Taylor and Makura 1985). A stock solution of 50 parts per million (ppm) was prepared using dechlorinated water. Four replicates of five laboratory-bred snails each were exposed to molluscicide concentrations of 30, 20, 15, 10, 8, and 5 ppm in a total volume of 200 ml for 24 hours. In each experiment, five snails were exposed to 200 ml of dechlorinated water for 24 hours as a control. After exposure, snails were washed, provided with food, and allowed a recovery period of 24 hours, after which mortality was assessed. This was done by probing the snails to elicit typical withdrawal movements. If no withdrawal movements were evident, such snails were considered to be dead.
Pilot Field Trial of P. dodecandra for Snail Control in a Rural Area
A pilot field trial was conducted in the Chiweshe communal lands where snail intermediated hosts of schistosomiasis were found in large numbers in two streams that passed through the village. The snail populations in the streams were monitored by dipping with an aquatic net along the margins of the streams at monthly intervals for 12 months before beginning the snail control exercise (Shiff et al. 1979). The snails recovered were examined for infection and returned to their respective sites on the same day. In addition, land was requested and received from the local people, where 550 plants of high molluscicidal potency were planted.
Application of P. dodecandra Berries
Before application of the molluscicide, the volume, as well as the flow rate, of the study streams were determined. Application was carried out in May when the water levels were low. A stock solution made from ground berries that had been harvested from the plot and sundried was prepared at least eight hours before application. Application was designed to achieve a concentration of 100 ppm. Efficacy of the molluscicide was assessed by exposure of sentinel snails to treated water for 24 hours. Due to the non-ovicidal nature of P. dodecandra berries, molluscicide application was repeated two weeks after the initial treatment. Snail population studies were carried out as before for both the study and the control streams for a further period of 12 months.
Results
Distribution and Molluscicidal Tests
The areas where P. dodecandra berries were collected are shown in Figure 1. Twenty-seven of 103 plants located in the eastern region (E) of the country, 15 of 21 plants in the central region (C), 6 of 13 plants in the northern region (N), 10 of 15 in the southern (S), and 16 of 32 plants in the southeastern region (SE) had berries. Crude extracts from the crushed berries from all the plants killed all or some of the snails at concentrations of 20 ppm or above.
Table 1 shows the mean percentage mortalities that were recorded when B. globosus were exposed to a molluscicide concentration of 15 ppm. For comparative purposes, 15 ppm was preferred, as this was the minimum concentration at which at least one snail died. The table also shows the mean altitude of the places from which P. dodecandra berries were collected.
TABLE 1 Mean Percentage of Snail Mortalities Recorded When Bulinus globosus were Exposed to a Molluscicide Concentration of 15 ppm. The Number of Plants Collected in Each of the Districts (n), the Average Altitudes, Average Annual Mean Temperatures, and Average Annual Rainfalls are also Shown for Each District from Which Berries were Collected. Mean ą SEM.
District
n
Average Altitude
Average Annual Mean Temperature
Average Annual Rainfall
Mean % Snail Mortalities
(m)
(°c)
(mm)
Eastern (E)
Central (c)
Northern (N)
S Eastern (SE)
Southern (S)
n = number of plants from which P. dodecandra berries were collected and bioassay tests performed. Source: Ndamba and Chandiwana 1987, in part.
From 74 crude berry extracts used in the bioassay tests, berries from eight plants were lethal to 83.8 + 5.3% of snails at a molluscicide concentration of 10 ppm. The extracts resulted in markedly high snail mortality even at lower concentrations.
Of the eight plants from which berries of high potency were collected, seven were found growing in places in the southern region (S) of the country at an altitude of 989 + 30 m, with annual rainfall figures of 760 i 1.58 mm and annual mean temperatures of 19.30 + 0.13°C. The only other plant with berries of equally high potency was found growing in the eastern region (E) of the country at an altitude of 960 m, and with an annual rainfall of 700 mm and an annual mean temperature of 19.50°C.
Field Trial
All the sentinel snails died after 24 hours of exposure to the treated water. No snails were recovered from the treated stream one week after treatment. Further, during the monthly snail population studies, snails were not recovered from the study stream until the onset of the rainy season (November). However, snails were recovered from the control stream throughout the study (Figures 2 and 3).
Sociological Evaluation
Knowledge about schistosomiasis was restricted to 78.9% of the respondents. Only 44.2% of the people interviewed had prior knowledge of P. dodecandra, while 55.8% said they had never seen the plant before. People residing in areas where the plant does not grow naturally only knew the plant was used for floor polish. (Tables 2 and 3.)
TABLE 2 Number of People Interviewed and Percentage of Those Having Previous Knowledge of Phytolacca dodecandra
Females
Male
Total
Area of respondents
n per area
% of total
n per area
% of total
Total
% of
responses
total
(A)
(B)
(C)
(D)
n = number of people interviewed. (A) and (D) -- areas where plant is known to grow. (B) and (C) -- areas where plant is not known to grow.
After a brief explanation of the intended use of the plant, 27% of the people interviewed were willing to cultivate the plant for snail control. However, 23% of the respondents had reservations about the idea. The reasons for reservations ranged from scarcity of land (47.7%), lack of knowledge about the plant (21%), lack of confidence in the use of a wild plant to control an important disease like schistosomiasis (11.2%), and lack of interest (20%) (Ndamba et al. 1989b).
TABLE 3. Use of Phytolacca dodecandra in Areas (A) and (D) by Sex
Area (A)
Area (D)
Total % (A + D)
Females
% total
Males
% total
Females
% total
Males l
% tota
Females
Males
(n = 78)
(n = 66)
(n = 45)
(n = 37)
(n=123)
(n=103)
Use of P. dodecandra*
Floor polish(leaves)
Medicine for
unspecified
human ailments
(roots and leaves)
Disinfectant for
domestic
animals (leaves)
Emetic
(roots, berries,
leaves)
Lightning protector
(plant)
Snake repellent
(plant)
Abortifacient
(leaves and berries)
Flea repellent
(leaves)
Don't know
n = total number of people interviewed.
* An additional 2.7% and 10.8% females from areas (B) and (C), respectively, reported P. dodecandra used only as a floor polish.
Discussion
The results of this study show that P. dodecandra is widely distributed in Zimbabwe and that there are marked differences in the molluscicidal potency of plants from different regions of the country (Table 1). The study also showed that a small proportion of plants yielded berries with very high molluscicidal activity. This finding clearly suggests that extensive work on the natural distribution of P. dodecandra plants is essential before proceeding to set up test plots to serve as gene banks for further agrobotanic and breeding requirements. It is desirable that plants with high molluscicidal activity and high berry yield be selected for propagation.
Data from Table 1 suggest that variation in molluscicidal potency of P. dodecandra is associated with altitude, rainfall, and temperature. The most potent berries were generally from low altitude and poor rainfall areas and may be attributable to a high concentration of the active ingredients in the berries due to the limited amount of water available to plants in such areas. In Zimbabwe, the most poisonous plants are found in the lower and drier parts of the country (Shone and Drammond 1965), a factor that might have some bearing on our findings.
As a result of these findings, test plots of selected local varieties of the plant with high molluscicidal potency and high berry yield, as well as the Ethiopian type 44, have been set up in different climatic regions of Zimbabwe. These were initiated to study the effects of soil types, altitude, rainfall, and temperature on molluscicidal activity and hence define regions of the country where the better plants can be grown for snail control purposes. Data on this part of the study are still being analyzed.
It is clear from Figures 2 and 3 that a single application of P. dodecandra berries can significantly reduce the snail population. Shiff et al. (1979) have shown that a single winter application of a molluscicide results in a significant reduction in the transmission of schistosomiasis. Our findings seem to support Shiff's conclusions. These preliminary results suggest the need for further studies to explore ways and means of incorporating P. dodecandra into our national schistosomiasis control programs. Further, unlike synthetic molluscicides, P. dodecandra can be used at a community level in self-help programs.
In view of the recently completed toxicological studies (Lambert et al. 1991), a study has been initiated in Zimbabwe to demonstrate the efficacy of P. dodecandra in reducing schistosomiasis in the human population. Further, laboratory studies to determine the effectiveness of P. dodecandra formulated as a slow release are being undertaken at the Johns Hopkins University. The slow release formulations will be field tested in Zimbabwe.
Acknowledgments
This paper is published with the kind permission of the Secretary for Health, Zimbabwe. The study was supported by funds from "Old Mutual" - Zimbabwe Ltd. The authors are very grateful to Miss G. Mandisodza for her excellent secretarial services. The authors also thank the Office of Research, USAID, for funding grant number DHR-5600-G-SS-0058-00, the network meeting, and the publication of this paper.
References Cited
Chandiwana, S. K., S. Mavi, and J. Ndamba. 1985. A preliminary report on the distribution of P. dodecandra in Zimbabwe. Zimb. Agric. J. 83: 1-2.
Lambert, J.D.H., J.H.M. Temmink, J. Marquis, R.M. Parkhurst, Ch.B. Lugt, E. Lemmich, L. Wolde-Yohannes, and D. De Savigny. 1991. Endod: safety evaluation of a plant molluscicide. Regul. Toxicol. Pharmacol. 14:189-201.
Lemma, A. 1965. Preliminary Report on the Molluscicide Properties of ENDOD (Phytolacca dodecandra). Ethiop. Med. J. 3:187-190.
Lemma, A. 1970. Laboratory and field evaluation of the molluscicidal properties of Phytolacca dodecandra. Bull. WHO 42:597-617.
Lugt, Ch.B. 1981. Phytolacca dodecandra berries as a means of controlling bilharzia-transmitting snails. Institute of Pathobiology, Litho Printers, Addis Ababa University, Ethiopia.
McCullogh, F.S., P. Gayral, J. Ducan, and J. Christie. 1980. Molluscicides in schistosomiasis control. Bull. WHO 58:681-689.
Ndamba, J. and S.K. Chandiwana. 1987. Appendix I. Paper 7. A report on the geographical variation in the molluscicidal potency of Phytolacca dodecandra berries in Zimbabwe. pp. 88-95. in Makhubu, L., A. Lemma and D. Heyneman. (ed.). Endod: Towards the use of Endod as a plant-derived molluscicide for control of schistosomiasis on a community self-help basis. Second International Workshop on Endod, Phytolacca dodecandra, Mbabane, Swaziland, April 1986. Council on International and Public Affairs, New York. 162 pp.
Ndamba, J., S.K. Chandiwana, and N. Makaza. 1989a. The use of Phytolacca dodecandra berries in the control of trematode-transmitting snails in Zimbabwe. Acta Trop. 46:303-309.
Ndamba, J., S.K. Chandiwana, and N. Makaza. 1989b. Knowledge, attitudes and practices among rural communities in Zimbabwe in relation to Phytolacca dodecandra - a plant molluscicide. Soc. Sci. Med. 28:1249-1253.
Parkhurst, R.M., D.W. Thomas, W.A. Skinner, and L.W. Cary. 1974. Molluscicidal saponins of Phytolacca dodecandra. Lemmatoxin. Can. J. Chem. 52:702-705.
Shiff, C.J., W.C.C. Coutts, C. Yiannakis, and R.W. Holmes. 1979. Seasonal patterns in the transmission of Schistosoma haematobium in Rhodesia and its control by winter application of molluscicide. Trans. R. Soc. Trop. Med. Hyg. 73:375-380.
Shone, A.K. and R.B. Drammond. 1965. Poisonous plants of Rhodesia. Rhod. Agric. J. 62:4.
Taylor, P. and O. Makura. 1985. Prevalence and distribution of schistosomiasis in Zimbabwe. Ann. Trop. Med. Parasitol. 79:287-299.
<section>Testing Of Echinostoma Liei As A Biocontrol Agent Against Schistosoma Mansoni Under Simulated Natural Conditions In Egypt</section>
F. Yousif, M. El-Emam, N. Haroun, S. Mohamed, And G. Kamel
Abstract
Echinostoma liei was evaluated as a biological control agent of Schistosoma mansoni under simulated natural conditions by obtaining data on the effect E. liei may have on the population density of Biomphalaria alexandrina, the snail host of S. mansoni in Egypt, and the effect E. liei may exhibit on the infection of the snail with S. mansoni. Five experiments, of 22 replicates, were carried out between 1988 and 1990 in similarly controlled ditches at the "Snail Research Station" near Cairo. In each experiment, five treatment groups of snails were introduced into ditches as well as into submerged cages: group (C) without infection, group (S) exposed to Schistosoma, group (E) exposed to Echinostoma, group (S +E) exposed to Schistosoma and less than two weeks later to Echinostoma, and group (S' +E') to Schistosoma and after 2-3 weeks to Echinostoma. Samples of ditch snails and all caged snails were examined twice weekly until all snails died and the infection rate, duration of
cercarial shedding (i.e., period of infection risk), and periodic cercarial output were determined.
Results obtained show that the infection rate of snails with S. mansoni was significantly lower in group (S + E) and was marginally significantly lower in group (S'+E') than in group (S). Including the zero values, duration of cercarial shedding (i.e., infection risk) was significantly shorter in groups (S + E) and (S'+ E') than in group (S). However, no significant difference was found in cercarial output of S. mansoni per shedding snail between various groups. The population density of snails became significantly lower in groups (S + E) and (S'+E') than in the control group (C), even though the original densities of snails were similar in these groups.
Introduction
Echinostoma lied Jeyarasasingam, Heyneman, Lim, and Mansour (1972) utilize Biomphalaria alexandrina, the vector of Schistosoma mansoni in Egypt, as first natural intermediate host. There is considerable evidence demonstrating the value of E. liei as a biocontrol agent against S. mansoni under laboratory conditions. Thus, Heyneman et al. (1972) proved that E. liei is a strong dominant competitor over S. mansoni in B. glabrata. Echinostoma revolutum (possibly the same parasite as E. liei) proved to have marked antagonistic effect against S. mansoni in the shared natural snail host B. alexandrina (Barus et al. 1974). Nevertheless, no field tests have been carried out in Egypt to evaluate E. liei as a biocontrol agent against S. mansoni and its snail vector.
In other countries, field evaluation of echinostomes has been restricted to a few trials in ponds performed in Malaysia, Thailand, and Guadeloupe (Lie and Owyang 1973; Heyneman and Umathevy 1967; Lie et al. 1970, 1971, 1974 a,b, Nassi 1978; Nassi et al. 1979; Pointier 1989). In some of these tests, Echinostoma malayanum, Schistosoma spindale, and the snail host Indoplanorbis exustus were utilized. In Guadeloupe, Ribeiroia marini guadeloupensis, S. mansoni, and B. glabrata were used.
In the present study, E. liei was tested against S. mansoni and its vector, B. alexandrina, in controlled experimental ditches similar to those common in the irrigation system in Egypt. Data on effects of Echinostoma, Schistosoma, cercarial output, and duration of infection risk were obtained. These data should be of considerable value in the final evaluation of E. liei as a biocontrol agent against S. mansoni.
Materials and Methods
Experimental Ditches
Ten experimental ditches (Figures 1-3) in the Snail Research Station of the Theodor Bilharz Research Institute, 25 km north of Cairo, were utilized in this study. These ditches are parallel, with muddy bottoms and sloping banks, each 30 m long, 150 cm wide at water level, and 50 cm wide at the bottom. The water depth was about 50 cm. The ditches are each divided into three sections by special partitions, which allow complete separation of sections and water flow without snail mixing. Two channels at right angles to the ditches served as common feeding and draining channels. At the inner side of either channel there is a base in which a series of levered tubes is installed opposite the ditches. The tubes of the common feeding channel act as water inlets and are located a few centimeters higher than those of the common drainage channel, which act as outlets to allow the flow of water in one direction. All outlet tubes were guarded by plastic sieves to prevent loss of snails.
This system of experimental ditches is supplied with underground water (40 m deep). The ditches have about 60% of water surface shaded equally. Ditches were originally supplied for two years with surface water from a nearby canal and maintained for natural balance before changing to the underground water source. Vegetation was allowed to grow in all ditches, but partial clearing was necessary to maintain almost equal vegetation density.
Ecological Data
The major abiotic and biotic components were examined. The chemical analysis of the underground and ditch water are presented in Table 1. This analysis shows that both types of water were suitable for the snails and the free-living stages of both parasites. The temperature of surface
water at midday and weekly means of temperatures at the middle and the bottom of ditches were determined using a thermograph (Figure 4). The mean water temperature was 16-26°C during the period of experiments (March-November). The period from December to February was avoided because water temperatures are low and the development of S. mansoni and E. liei stops at 14°C and 13°C, respectively (Pfluger 1980, Yousif et al., unpublished). In the cool season, December-February, the mean water temperature at the bottom of the ditches (14-16°C) is higher than at the middle by about 2°C. The reverse occurs during the rest of the year when the mean temperature at
Fauna and Flora in Ditches
Insects
Lethocerus sp.
Dolomedes sp.
Anisops sp.
Anisoptera nymph
Amphihians
Tadpoles of Bufo sp.
Fish
Tilapia nilotica
Aquatic Plants
Eichhornia crassipes
Cyperus dives
Phragmites australis
Phragmites communis
Typha domingensis
Paspalum paspaloides
Ceratophyllum demersum
Chara sp.
Mollusks
Bulinus truncatus
Physa acuta
Lanistes carinatus
Cleopatra bulimoides
Lymnaea caillaudi
Bellamya unicolor
Filamentous algae
TABLE 1 Chemical Analysis of Underground and Ditch Water
Parameter (ppm)
Underground Water
Ditch Water
Sulfides (S)
Dissolved oxygen (O2)
Biochemical oxygen demand(B.O.D.)
Chemical oxygen demand (C.O.D.)
Total dissolved solids (T.D.S.)
Ash of dissolved solids
Suspended solids
Ash of suspended solids
Ammonia (N)
Nitrites (N)
Nitrates (N)
Oils and fats
Nil
Iron (Fe)
Manganese (Mn)
Total alkalinity (CaCO3)
Total hardness (CaCO3)
Calcium hardness (CaCO3)
Magnesium hardness
Calcium (Ca)
Magnesium (Mg)
Chlorides (Cl)
Silicates (SiO2)
Phosphates (P04)
Copper (Cu)
Nil
Lead (Pb)
Fecal coliforms/100 c.c.
the bottom of the ditches is 1-3 °C lower than that at halfway to the bottom. Surface water temperature at midday was about 3°C lower than the mean temperature at the bottom during January. This temperature was higher by up to 5°C than the bottom temperature in December and February. In the warm season, March-November, the temperature of water surface at midday was higher than the mean at the bottom by 7-12°C in June-August. The fauna and flora in various ditches (see below) were generally similar both in species and density.
Specimens required for this research were B. alexandrina snails, E. liei eggs, and S. mansoni eggs. Snails were collected from canals and drains near Cairo, examined successively for natural infection, and shedding snails were discarded. In each experiment, batches of collected snails were randomly distributed in equal numbers between different sections of the ditches. Only medium-sized snails (5-8 mm in diameter) were used.
Echinostoma lied and S. mansoni were maintained in the laboratory for the production of eggs. Both parasites originated from naturally infected B. alexandrina snails collected from the irrigation scheme near Cairo. Echinostoma lied was maintained in albino rats (Rattus norvegicus) as definitive host, and B. alexandrina and Physa acuta as first and second intermediate hosts, respectively. Egg production of both parasites was optimized and a technique was refined for egg extraction by passing a fecal suspension or tissue homogenate through a number of sieves of descending mesh sizes. Eggs were incubated and only fully embryonated eggs ready to hatch were used. Schistosoma mansoni was maintained in golden hamsters (Mesocricetus auratus) and B. alexandrina snails. An extra source for Schistosoma eggs from human origin was also utilized.
Five experiments, of 22 replicates each, were set up during 1988-1990 (Table 2). In each experiment, five treatments were used. These groups are: group (C) snails without infection, group (S) snails exposed to Schistosoma eggs, group (E) snails exposed to Echinostoma eggs, group (S + E) snails exposed to Schistosoma and to Echinostoma after less than two weeks, group (S'+ E') snails exposed to Schistosoma and to Echinostoma after 2-3 weeks. Thus, in each experiment (except those
TABLE 2 Intervention by Echinostoma lied Against Schistosoma mansoni Under Simulated Field
Conditions
Experiment no.
Date
Snails
Number per Ditch Section
Schistosoma Eggs (1,000)
Echinostoma Eggs (1,000)
I
May '88
May '88
May '88
II
Aug. '88
Sept. '88
Sept. '88
Oct. '88
III
Mar. '89
Mar. '89
Apr. '89
Apr. '89
May '89
June '89
IV
Aug. '89
Aug. '89
Sept. '89
V
May '90
May '90
May '90
May '90
June '90
July '90
of experiment 3) five equal groups of snails were introduced into five similarly controlled ditch sections. Simultaneously, 150 or 300 snails were placed in three cages and submerged in each ditch section. A rotation technique for the distribution of groups in various sections was applied (Figure 1). Therefore, the effect of the position of the section within the ditch was eliminated. In the replicates of experiment 3, equal numbers of snails were introduced into the ditch sections during the previous autumn and maintained until the next spring. Before Schistosoma and Echinostoma eggs were introduced, the snail population in each ditch section was assessed by counting the number of snails obtained by 12 dips, using the standard dip net. This assessment showed that the snail populations in ditch sections of replicates 4 and 6 were considerably lower than in replicates 1, 2, 3, and 5. Therefore, 2,000 and 500 snails, respectively, were added to the sections of replicates 4 and
6 (see Table 9).
Eggs were introduced into ditches at midday, using shallow pots hung 5 cm under the water surface and left for five days in the case of Echinostoma, and one day in the case of Schistosoma.
Ditch sections were separated from each other by impermeable screens during periods of exposure to miracidia to prevent miracidial contamination between sections. Samples of ditch snails (12 dips per ditch section and all caged snails) were examined twice weekly until the death of all the introduced snails. These snails were examined individually for cercarial shedding every two hours. Positive snails were marked according to parasite species, and all snails were replaced into their original sites. The infection rate, duration of cercarial shedding in each group (i.e., period of infection risk), and periodic cercarial output per shedding snail were determined in each case.
TABLE 3 Infection of Biomphalaria alexandrina with Echinostoma lied Under Simulated Field Conditions (in Ditches)
Experiment no.
Echinostoma (E) alone
Schistosoma + (E)
Schistosoma + (E)
(< 2 weeks)
(2-3 weeks)
Examined
+ve
Examined
+ve
Examined
+ve
I
Total
II
Total
III
Total
IV
Total
V
Total
Grand total
Statistical Analysis
The primary analysis methods used for this project were multiple logistic regression and multiple regression. Additionally, the non-parametric median test was used for certain comparisons of highly skewed data. All analyses were conducted using the statistical software package "Statistix" for personal computers. In all analyses, evaluating factors related to infection of Schistosoma or Echinostoma in snails, multiple logistic regression was used. When making comparisons across treatment groups (or allowing for differences among experiments, experiment and replicates and/or treatment groups), the appropriate dummy variables were created.
In determining whether there was a correlation between the infection rates of snails in ditches with those in cages in the same treatment group, multiple regression was used; in some cases, the data were skewed, so that a logarithm transformation was first made. (In such cases, zero values were recoded to one-half the lowest positive value.) When the non-parametric median test was employed, it is indicated in the appropriate place of the paper.
Results
Echinostoma Infection
Snails in Ditches (Table 3) After allowing for differences in the shedding rates of Echinostoma in snails, among the five experiments, this rate in group (S'+ E') was significantly lower than in group (S + E) (one-tailed p = 0.006). The probability of infection in group (S + E) was not significantly different from that in group (E). However, the probability for group (S'+ E') was significantly lower than for group (E) (one-tailed p = 0.027).
When data from all three snail groups exposed to Echinostoma were combined, the probability of infection was significantly and positively related to the number of Echinostoma eggs introduced (one-tailed p = 0.006); the number of eggs remained significant (one-tailed p = 0.01) after allowing for differences in infection across the three groups. However, when the three groups were analyzed separately, the number of eggs was a significant factor only for group (E) (one-tailed p = 0.006).
Snails in Cages (Table 4) As with the data from ditch snails, the prevalence of infection (cercarial shedding) in caged snails was significantly lower in group (S'+ E') than in group (S + E) (one-tailed p < 0.0002). The probability of infection for group (S + E) was not significantly different from that in group (E), but was significantly lower in group (S' + E') compared with group (E) (one-tailed p = 0.0004).
For snails in cages, (cf. ditch snails), the numbers of Echinostoma eggs introduced were negatively correlated with the probability of infection, after controlling for differences across groups (p < 0.0001). When the three treatment groups were analyzed separately, these inverse relationships were significant for groups (S + E) and (S'+ E'), but only marginally for group (E) (two-tailed p = 0.13).
There were 17 replicates in which snails from both ditches and cages were examined in sufficient numbers for Echinostoma infection. The infection rates (as well as their logarithms, to account for non-normality) were positively correlated (one-tailed p = 0.026).
TABLE 4 Infection of Biomphalaria alexandrina with Echinostoma lied Under Simulated Field Conditions (in Cages)
Experiment no.
Echinostoma (E) alone
Schistosoma + (E)
Schistosoma + (E)
(< 2 weeks)
(2-3 weeks)
Examined
+ ve
Examined
+ ve
Examined
+ ve
I
Total
II
Total
III
Total
IV
Total
V
Total
Grand total
Schistosoma Infection
Snails in Ditches (Table 5) In the five experiments, the percentage of infected (shedding) snails in group (S'+ E') was statistically higher than in group (S + E). Therefore, the two groups cannot be combined. In these analyses, data from experiment I, replicate 3 were deleted; it was judged that too few snails were examined in group (S) and (S'+ E').
TABLE 5 Effect of Echinostoma lied on Infection of Biomphalaria alexandrina with Schistosoma mansoni Under Simulated Field Conditions (in Ditches)
Experiment no.
Schistosoma (s) alone
(s) + Echinostoma
(S') + Echinostoma
(< 2 weeks)
(2-3 weeks)
Examined
+ ve
Examined
+ ve
Examined
+ve
I
Total
II
Total
III
Total
IV
Total
V
Total
Grand total
1,98s
Differences across the five experiments as well as across replicates were allowed. Results were nearly identical regarding differences across groups. Because there were clear differences across replicates within some of the experiments, differences across experiments and replicates were appropriate. After allowing for these replicate differences, the percentage of infected snails was significantly lower (47%) in group (S + E) than in group S (one-tailed p = 0.0005) and was marginally significantly lower in group (S'+ E') than in group (S) (one-tailed p = 0.65).
When all three groups were combined, the number of eggs introduced was a highly significant factor in the probability of infection in snails (p < 0.0001). When the three treatment groups were analyzed separately, the number of eggs was either significant as in groups (S) and (S'+ E'), one
tailed p-values < 0.01) or marginally significant group as in (S + E), (one-tailed p = 0.056). In all cases, the probability of infection increased with increasing number of eggs. Because of the small number of snails counted in either one or both of the treatment groups, the data from experiment I, replicate 2 and experiment II, replicate 2 were not included in these analyses.
Snails in Cages (Table 6) The same approach was used as with the snails in the ditches; that is, differences in infection rates across experiments and replicates were allowed for before the effect of the treatment group was assessed.
TABLE 6 Effect of Echinostoma lied on Infection of Biomphalaria alexandrina with Schistosoma mansoni Under Simulated Field Conditions (in Cages)
Experiment no.
Schistosoma (S) alone
(S) + Echinostoma
(S') + Echinostoma
( < 2 weeks)
(2-3 weeks)
Examined
+ ve
Examined
+ ve
Examined
+ ve
I
Total
II
Total
III
Total
IV
Total
V
Total
Grand
total
The probability of infection for snails in group (S + E) was significantly lower (32%) than those in group S (one-tailed p < 0.0001), but was not significantly lower in group (S'+ E') compared with group S. In these analyses of snails in the ditches and in cages, the coefficients for group (S + E) were very similar: 0.79 for ditches, and 0.80 for cages. Using the value of 0.80, the ratio of the odds of infection for snails in group (S + E) relative to group (S) is 0.45. Thus, for an odds ratio of 0.45, if the prevalence of infection among group (S) is, for example, 4%, then the prevalence among group (S + E) would be estimated as approximately 1.8%.
When data from all three groups were combined, the probability of infection was significantly and positively related to the number of Schistosoma eggs introduced (one-tailed p = 0.024); the number of eggs remained significant (one-tailed p = 0.036) after allowing for differences in infection across the three groups. However, when the three groups were analyzed separately, the number of eggs was a significant factor only for group (S + E) (one-tailed p = 0.014). There were 17 replicates in which snails from both ditches and cages were examined in sufficient numbers for Schistosoma infection. The infection rates (as well as their logarithms, to account for non-normality) were highly correlated (p <0.0001, r = 0.42).
Effect of Echinostoma on Periodic Cercarial Shedding by Snails: (Table 7)
Because of the very small number of positive snails for some replicates, results within replicates of the same experiment were combined. Data from experiment III were not included in the analyses since only one snail from the treated groups for this experiment were observed to be positive. Multiple regression, both unweighted and weighted by the number of infected snails, was used to compare the numbers of cercariae per snail in the treated groups (S + E) and (S'+ E') with the Schistosoma group (S). Because the means of each experiment had differing precision because of the different numbers of snails contributing to these means, the weighted regression was more appropriate.
For snails in the ditches, when averaged across the four experiments, the mean number of cercariae per shedding snail per 2 hr was lower in group (S'+ E') than for the Schistosoma alone in group (S). However, this difference was not statistically significant; moreover, for two of the experiments the mean was higher in group (S'+ E') than group (S). There was also no significant difference between group (S + E) and group (S).
For snails in cages, the mean numbers of cercariae per shedding snail per 2 hr were significantly lower in both groups (S + E) and (S'+ E') than in group (S) (one-tailed p-values 0.015 and 0.026, respectively) for unweighted regression. However, when the number of snails was considered, using weighted regression, the difference did not approach statistical significance. The great majority of infected snails were observed in experiments IV and V. For these two experiments, the means across treatment groups are similar (with the exception of experiment IV, treatment group 4, which also was based on the smallest number of snails).
Thus, since the unweighted results are considered more reliable, it is concluded that for both ditches and cages there were no differences in the periodic cercarial output per shedding snail in various treatment groups.
TABLE 7 Effect of Echinostoma lied on Cercarial Output of Schistosoma mansoni Under Simulated Field Conditions
Experiment no.
Schistosoma (S)
(S) + Echinostoma
(S') + Echinostoma
alone
< 2 weeks
2-3 weeks
Cercariae/Snail/2hr
Cercariae/Snail/2hr
Cercariae/Snaail/2hr
Ditches
Cages
Ditches
Cages
Ditches
Cages
I
II
III
IV
V
Mean
Duration of Schistosoma Infection Risk (Table 8)
The data concerning days of cercarial shedding for various snail groups (days of infection risk) in ditches and cages were highly skewed, with many zero values. Therefore, in comparing each of the treatment groups (S + E) and (S'+ E') with group (S), the non-parametric median test for paired data was used. In this, the number of times the duration of shedding in group (S) exceeded the other groups (S + E) and (S'+ E') was noted for 20 replicates; based on the binomial distribution, the exact p-value was calculated. (Note: under the null hypothesis of no difference in the number of shedding days, for approximately half the replicates, the number of days for group S should be larger than for the other groups and for half the replicates it should be smaller.)
For both ditches and cages, both groups (S + E) and (S'+ E') were found to have significantly fewer days of cercarial shedding, i.e., shorter period infection risk. In the ditches, the duration of Schistosoma infection risk is 49% in group (S + E) and 57% in group (S'+ E') of that of group (S). The respective one-tailed p-values were: group (S + E) ditches 0.048; group (S'+ E') ditches 0.0004; group (S + E) cages 0.004; group (S'+ E') cages 0.0008.
TABLE 8 Effect of Echinostoma lied on Mean Period in Days of Schistosoma Infection Risk
Exper- iment no.
Schistosoma (S)
(S) + Echinostoma
(S') + Echinostoma
alone
< 2 weeks
2-3 weeks
Ditches
Cages
Ditches
Cages
Ditches
Cages
I
II
III
IV
V
Mean
Effect of Echinostoma on the Population Density of Snails (Table 9)
Data obtained from experiment III is used here since the snail populations were more natural than in other experiments (see Materials and Methods). In order to assess the original conditions for each treatment group (E, S + E, S'+ E'), the original snail densities estimated by the number of snails recovered in 12 dips, were compared with the control group (C). Because of non-normality of the data, the non-parametric median test was used. None of the three treatment groups differed significantly from the control.
TABLE 9. Effect of Echinostoma liei on the Population Density of Biomphalaria alexandrina Under Simulated Field Condition
Exper-
Observation
Control
Echinostoma (E)
Schistosoma + (E)
Schistosoma
iment no.
period
alone
(< 2 weeks)
2-3 weeks
Original
Number
Original
Number
Original
Number
Original
Number
density
of
density
of
density
of
denity
of
(snails/
collected
(snails/
collected
(snails/
collected
(snails/
collected
12 dip)
snails
12 dip)
snails
12 dip)
snails
12 dip)
snails
III
* Number of snails added to each ditch section at start of experiment.
For comparing the population density of snails, two measures were considered: the number of snails collected during the experiment and the ratio of the number of snails collected to the original density. For groups (S + E) and (S'+ E') the results were identical for the two measures. The control group (C) exceeded group (S + E) for all six experiments and exceeded group (S' + E') for all five experiments in which data were collected for both. These differences were statistically significant; one-tailed p = 0.02 and p = 0.03 for groups (S + E) and (S'+ E'), respectively. The numbers collected for group (E) tended to be lower than for group (C); the mean number of snails collected for groups (E) and (C) were, respectively, 166.0 and 235.7, and the number collected in the control group exceeded that for group (E) in five of the six experiments. However, these differences were not significant (p = 0.11). When the ratios of collected snails to original densities were considered,
the values for group (E) again tended to be lower than group (C) (mean ratios of 4.9 and 5.8, respectively with the ratio in the control group exceeding that in group (E) for four of the six experiments, but these differences were not significant.
Discussion
Echinostoma lied was tested here for the first time against S. mansoni and the shared snail host B. alexandrina under simulated natural conditions and in controlled experimental ditches similar to those common in the irrigation schemes in schistosomiasis areas of Egypt. Previous field trials with echinostomes for control of schistosomes are limited to those performed in Malaysia, Thailand, and Guadeloupe using E. malayanum against S. spindale, and the snail host ludoplanorbis exustus and Ribeiroia mariru against S. mansoni and B. glabrata, respectively. Some of these trials proved that echinostomes are generally dominant over schistosomes, are effective in reducing mollusk populations, and will almost eliminate schistosomes from the ponds (Heyneman and Umathevy 1967, Lie et al. 1970, 1971, 1974a,b; Nassi 1978; Lie and Owyang 1973; Nassi et al. 1979; Pointier 1989).
Schistosoma mansoni, E. liei, and the shared snail vector B. alexandrina used in the present study were originally obtained from the same locality. This was meant to avoid any effect of strain difference on susceptibility of the snails to either parasite. Snails used in experiments were always of the same medium size to neutralize the effect of snail size on susceptibility to infection. Echinostoma eggs were incubated before being used in the experiments. Fully embryonated readily hatchable eggs were placed in pots and not simply thrown into ditch water. This avoided the settling of eggs on the bottom of the ditches and their being buried in mud or ingested by other organisms.
Conditions in the experimental ditches were kept as uniform as possible, especially water flow, depth, and quantity; type and density of vegetation; and shade. Meanwhile, the number of replicates were numerous (22), including negative and positive controls, to allow for statistical analyses of the results.
The results show that the infection of snails with Echinostoma was significantly lower in group (S'+ E') than in groups (E) and (S + E) in both ditches and cages. Infection of snails with Echinostoma in the last two groups was not different. This lower infection rate with Echinostoma in group (S'+ E') was expected since Echinostoma eggs were introduced into this group about 10 days later than into the other groups. Infections with Echinostoma in caged snails was found to be significantly negatively correlated to the number of introduced eggs of the parasite, while positively correlated in ditch snails. This may be explained on the basis of the higher exposure of cage snails to echinostome miracidia leading to a higher mortality of infected snails when compared to ditch snails. Such effects have not been observed in the case of Schistosoma because of the much less pathogenic effect of this parasite on vector snails in comparison with Echinostoma.
The results also show that the infection rate of ditch snails with Echinostoma or Schistosoma ranged between 5.7 and 11. 1% (7.5%) and 1.5 and 11. 1% (3.2%) in positive control groups, that is, groups (E) and (S), respectively. These averages are somewhat higher than the averages of natural infection rates for both trematodes in Egypt. Much higher infection rates of snails with these parasites may be obtained under laboratory conditions, but this appears not to be the case under simulated natural conditions.
Intervention with Echinostoma should be by introduction of completely developed eggs into foci where Schistosoma is transmitted. It appears from the results of this study that the timing of this intervention with echinostomes is an important factor to consider. Thus, a period less than two weeks between schistosome infection of snails and echinostome intervention was necessary to demonstrate a significant effect in the present study. The optimization of this factor still needs further studies.
This study has demonstrated that a single application of E. liei eggs can have a significant reducing effect on B. alexandrina populations that were previously exposed to S. mansoni. Moreover, this application can significantly suppress the schistosome parasite in snails and shorten the period of infection risk if it occurs less than two weeks from the exposure of the snails to the schistosome. These effects are additive, and consequently, E. lied can provide a biological method of schistosomiasis control with some advantages over the present chemical methods, especially if applied repeatedly. It could be especially useful in aquacultures where molluscides are not allowed.
Acknowledgments
The authors express their deep thanks and gratitude to Dr. Emile Malek, Professor of Parasitology, Department of Tropical Medicine, School of Public Health, Tulane University, New Orleans, Louisiana, USA for his interest and valuable advice throughout this research work and for reading the manuscript. They are also deeply grateful to Dr. lanes Hughes, Acting Chairman, Department of Biostatistics and Epidemiology, School of Public Health, Tulane University, New Orleans, Louisiana, USA, for the statistical analysis of the results. The authors also thank the Office of Research, USAID, for funding grant number 936-5542, the network meeting, and publication of this paper.
References Cited
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Heyneman, D., H.K. Lim, and U. Jeyarasasingam. 1972. Antagonism of Echinostoma lied (Trematoda: Echinostomatidae) against the trematodes Paryphostomum segregatum and Schistosoma mansoni. Parasitology 65:223-233.
Heyneman. D, and T. Umathevy. 1967. A field experiment to test the possibility of using double infection of host snail as a possible biological control of schistosomiasis. Med. J. Malays. 21:273.
Jeyarasasingam, U., D. Heyneman, H.K. Lim, and N. Mansour. 1972. Life cycle of a new echinostome from Egypt, Echinostoma lied sp. nov. (Trematode: Echinostomatidae). J. Parasitol. 65:203-222.
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Lie, K.J., E.H. Kwo, and C.K. Owyang. 1971. Further field trial to control Schistosoma spindale by trematode antagonism. Southeast Asian J. Trop. Med. Public Health 2:237-243.
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Lie, K.J., C.R. Schneider, S. Sornmani, G.R. Lanza, and P. Impand. 1974b. Biological control by trematode antagonism II. Failure to control Schistosoma spindale in a field trial in Northeast Thailand. Southeast Asian J. Trop. Med. Public Health 5:60-64.
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Pfluger, W. 1980. Experimental epidemiology of schistosomiasis 1 - The prepatent period and cercarial production of Schistosoma mansoni in Biomphalaria snails at various constant temperatures. Z. Parasitenkd. 63:159-169.
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<section>Control Of Natural Populations Of Schistosome-Transmitting Snails By The Crayfish, Procambarus Clarkii In Temporary Man-Made Ponds In Kenya</section>
G.M. Mkoji, B.V. Hofkin, B.N. Mungai,J.H. Kihara, F.K. Mungai, J.H. Ouma, D.K. Koech, And E.S. Loker
Abstract
A study was conducted to assess the ability of the Louisiana red swamp crayfish, Procambarus clarkii, to control the schistosome-transmitting snail Biomphalaria pfeifferi in small, rain-filled quarry pits near Kangundo, Machakos, Kenya. This area is within the known geographic range of crayfish in Kenya and these man-made habitats are far removed from other water bodies. In six control sites where crayfish were not introduced, populations of B. pfeifferi, Bulinus (Physopsis) or Bull forskali persisted and in three of the sites, B. pfeifferi populations remained abundant. Crayfish were introduced into four experimental sites that initially had snail populations. In two of these sites, snails disappeared within one month and have not reappeared, and crayfish populations have persisted. In the remaining two sites, snails also quickly disappeared following crayfish introduction, but the habitats subsequently dried, interfering with long-term establishment of crayfish. In one
additional site originally selected as a control habitat, a sharp reduction in snail abundance was correlated temporally with the unexpected appearance of crayfish. The results are in agreement with earlier studies implicating P. clarkii as a potential biological control agent for schistosome-transmitting snails in selected habitats in Kenya and indicate that additional study of this possibility is warranted.
Introduction
Freshwater habitats play an integral role in the life cycle of the parasitic flatworms responsible for causing schistosomiasis, one of the most common infectious diseases of humanity (Walsh and Warren 1979). Infections are acquired in freshwater when free-swimming schistosome cercariae penetrate human skin. Following the development of adult schistosomes in humans, their eggs are
passed in feces or urine. If deposited in water, the eggs will hatch and release miracidia, which penetrate and initiate larval development in certain species of freshwater pulmonate snails. This culminates in the production and release of cercariae into water. These parasites, therefore, take advantage of the need for people to contact water and are also favored by the contamination of water bodies by human wastes. (For a review of schistosome biology, see Rollinson and Simpson 1987.)
Within major endemic regions, it is very difficult to reduce morbidity and mortality to acceptable levels over sustained intervals of time; except in a few unique circumstances, eradication is not a feasible goal (Jordan 1977, WHO 1985, Mott 1987). The task of schistosomiasis control is rendered more difficult by water-development projects such as dams, irrigation schemes, and aquaculture projects that create new, and often favorable, habitats for snails, and that offer new opportunities for human water contact (Heyneman 1979).
Various strategies for control include chemotherapy to treat infected people, improved sanitation, public health education programs, and snail control. Each of these approaches have advantages and drawbacks (e.g., Combes and Cheng 1986, Mott 1987, Madsen 1990) and it is generally acknowledged that a combination of methods is required to achieve success.
The focus of our work is to investigate alternative methods of snail control that offer increased hope for sustained control and that deemphasize dependence on the use of relatively nonspecific, expensive molluscicides. We have concentrated on elucidating the potential of other freshwater invertebrates to control the pulmonate snails in Kenya that transmit not only the human schistosomes Schistosoma mansoni and S. haematobium, but also schistosomes and liver flukes of domestic animals.
In the early stages of our investigations, our efforts concentrated on species of ampullariid snails indigenous to Kenya that might function as effective competitors and/or predators of disease-transmitting pulmonates (Hofkin et al. 1991a, Stryker et al. 1991). While defining the geographic ranges and patterns of coexistence of ampullariids and pulmonates, we encountered several habitats that contained the North American crayfish Procambarus clarkii (see Loker et al. in these proceedings). Further study indicated that habitats containing crayfish were significantly less likely than crayfish-free habitats to support pulmonate species (Hofkin et al. 1991b). A possible explanation for this phenomenon was provided by laboratory experiments, which showed that crayfish readily consumed pulmonate snails, and by field experiments demonstrating that crayfish were capable of eliminating pulmonates in enclosures and that snails protected by cages were able to survive in crayfish
habitats (Hofkin et al. 1991b). Further field and laboratory studies showed that crayfish could significantly reduce the abundance of macrophytes used by snails for food and oviposition sites (Hofkin et al. 1991c). It was also documented that crayfish could effectively block the build-up of pulmonate populations in laboratory aquaria (Hofkin et al., unpublished observations).
Given the encouraging results obtained in both laboratory and field experiments, we were interested in determining if P. clarkii could displace natural populations of pulmonate snails. This represents an important step in determining the potential utility of P. clarkii as a possible biological control agent of schistosomiasis in Kenya. In conjunction with the Division of Vector Borne Diseases of the Kenya Ministry of Health, small, isolated, man-made habitats containing target snail species were selected for a preliminary trial of this possibility. Although P. clarkii is an introduced species in Kenya, it is now widely distributed in the country and is present in areas immediately adjacent to the sites selected for study. The results of this pilot study are outlined below.
Materials and Methods
Study Sites
This study was conducted near Kangundo, Machakos District, in Kenya. The study sites consisted of man-made pits excavated to obtain stone blocks used in building construction. During the rainy season, these pits fill with water and, at maximum water volume, measure about 60-70 mē in area and 1-1.5 m in depth. Water volumes fluctuate seasonally, and many of the pits dry completely during the dry season. In spite of such fluctuations, many of these sites support thriving populations of pulmonate snails including Biomphalaria pfeifferi and Bulinus (Physopsis) spp., important schistosome-transmitting species in Kenya. The pits contained clear water, had rocky substrates, and some emergent vegetation was found near their margins.
Crayfish were not known to occur here, but the sites are within the known geographic range of crayfish in Kenya, and are far removed (at least 10 km) from other waterbodies. Eleven sites were selected for the present study, seven of which served as controls and received no crayfish, and four of which were designated as experimental sites and received P. clarkii. These particular sites were chosen because they contained populations of pulmonate snails and their shorelines were easily accessible for sampling. In July 1990, just prior to crayfish introductions, snail searches on the shorelines of the sites were conducted (see below), and snails were identified and enumerated. Three control and three experimental habitats contained relatively small numbers of snails (< 20 snails per 5 minutes searching) and were designated as "low-density" habitats; four control and one experimental site typically contained > 50 snails per 5 minutes searching and were designated as "high
density" sites.
Crayfish Introductions
Procambarus clarkii (carapace size 3.5-4.5 cm) were obtained from a dam near Thika, 35 km away, and were transferred the same day into the sites selected to receive crayfish. Each experimental site received 80 crayfish.
Sampling of the Sites Regular sampling was initiated at the time of crayfish introductions. During sampling, a systematic five-minute search for snails was conducted along the shoreline of each site. As the water was relatively shallow and clear, the relative abundance of snails on the surfaces of submerged rocks was enumerated directly, and disturbance to the habitat resulting from the use of other sampling procedures was avoided. Presence and relative abundance of crayfish was assessed by counting crayfish caught in meat-baited minnow traps held in each site overnight.
Results
The three pulmonate species recorded in the 11 study sites were B. pfeifferi, Bulinus (Physopsis) spp., and Bull forskali, with the first species comprising > 95% of all collected snails. To simplify presentation, the counts for all three species are combined in the results. Relative abundance of pulmonates in the three low-density control sites are shown in Figure 1. Snails were found in low numbers in each site, with the number recovered per site varying considerably between sampling dates. In some months no snails were found in some of these habitats; however, snails were found in each control site for up to at least seven months after initiation of sampling. These sites dried by 12 months.
In the first low density experimental site, crayfish clearly became established and exhibited a pattern of increasing abundance for the 11 months of study (Figure 1). Snails were not found after crayfish had been placed in the habitat. In the two remaining low density experimental sites, snails quickly disappeared following the introduction of crayfish. However, crayfish did not become established and small numbers of snails later appeared. Snails could be found in these sites for up to eight months (data not shown).
The three high density control sites supported vigorous snail populations throughout the five-month period of observation (Figure 2), and although sampling was discontinued after this time, these habitats continued to support viable snail populations. In contrast, in the one experimental site, no snails were ever found after crayfish had been introduced (Figure 2). Crayfish persisted in this location and increased in abundance after six months.
An additional high density control site was of particular interest because crayfish appeared there unexpectedly (Figure 3). During the first five months of study, the snail population was
abundant, but by month six had started to decline, and by month nine had disappeared. This decline was correlated with the appearance of crayfish, which were first detected by month six. Crayfish subsequently disappeared, also by month nine of the study.
Discussion
Schistosome-transmitting snails were apparently eliminated within one month from both experimental quarry pits in which crayfish became well established. Snail surveys, conducted monthly at these sites for up to 12 months, were consistently negative, suggesting that snails were unable to recolonize these sites. Snails also rapidly disappeared in the two experimental sites where crayfish did not persist, but subsequently reappeared following the disappearance of crayfish. Snails were also eliminated within four months of the unexpected appearance of crayfish in a control site. In the three sites where crayfish persisted, their numbers began to increase after about 5-6 months, indicating they were capable of successful reproduction in the quarry pits. In the remaining control sites, snail populations persisted for at least four months after the disappearance of snails from crayfish introduction sites.
The unexpected arrival of P. clarkii in one control site, apparently after having migrated from an experimental site approximately 12 miles away, illustrates the dispersal ability of this crayfish species. In conjunction with earlier results (Hofkin et al. 1991b), the results indicate that P. clarkii can rapidly eliminate and/or control B. pfeifferi and other snails of public health significance, at least in certain habitat types.
The Kangundo quarry pits are small and man-made. They are completely dependent on rain water and they experience large seasonal fluctuations in volume. Many of the pits dry completely for up to three months of each year. Because of these considerations, it is unclear if the results obtained here can be extrapolated to larger, more permanent water bodies, or to lotic habitats that serve as important transmission foci. Nevertheless, the results obtained in the quarry pits are encouraging as well as relevant to public health because these sites are important transmission foci. Contact by local people with these sites is frequent, and human feces is commonly observed near the pits. We have also observed large numbers of snails recovered from the pits to be shedding schistosome cercariae (unpublished observations).
It will be of particular interest to learn if crayfish will persist through the dry season in Kangundo, and whether snails will recolonize the introduction sites. Procambarus clarkii is capable of surviving dry periods by constructing burrows and is well adapted to temporary habitats (Huner 1988). Whether it can survive in quarry pits with limited opportunities for burrowing requires further investigation. Also, B. pfeifferi is capable of rapid population growth when conditions become favorable (Teesdale 1962, Woolhouse and Chandiwana 1989), and it remains to be seen if surviving crayfish are able to prevent such population expansion.
The ultimate goal of snail control is to reduce morbidity and mortality in the human population. Should P. clarkii prove capable of long-term snail control, studies should be initiated to determine if the presence of crayfish can be associated with lower disease incidence. The Kangundo quarry pits may be especially suitable for such an epidemiological study. The quarry pits are appropriately isolated and schistosomiasis is an important public health concern in this area. For many local inhabitants, these sites represent the only available source of water. Should such a study indicate that the presence of P. clarkii can be associated with lower disease incidence and prevalence, further study of its potential for achieving control in other habitat types would be warranted.
Acknowledgments
The authors thank Mr. Jonathan K. Koske for technical assistance. This paper is published with the approval of the Director of the Kenya Medical Research Institute. The authors also thank the Office of Research, USAID, for funding grant number DPE-5542-G-55-6018-00, the network meeting, and the publication of this paper.
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<section>Procambarus Clarkii In Kenya: Does It Have A Role To Play In The Control Of Schistosomiasis?</section>
E.S. Loker, B.V. Hofkin, G.M. Mkoji, J.H. Kihara, F.K. Mungai, B.N.Mungai, And D.K. Koech
Abstract
The Louisiana red swamp crayfish, Procambarus clarkii, introduced into Lake Naivasha, Kenya, about 1970, has become well established and now supports a small but thriving aquaculture industry. This crayfish has since dispersed over a wide area in Kenya, giving rise to concerns about its ability to disrupt natural ecosystems both in Lake Naivasha and elsewhere. In addition, P. clarkii may adversely affect fin fisheries in Lake Naivasha, and has been implicated as a pest in rice cultivation schemes outside of Africa. In contrast, our investigations indicate that P. clarkii can effectively control or eliminate certain freshwater snail species, including those involved in the transmission of human schistosomiasis. As such, P. clarkii appears to offer hope as a biological control agent of snail-transmitted diseases in selected habitats in East Africa. In this paper, we present details regarding the currant distribution of P. clarkii in Kenya and discuss possible approaches to the
use of crayfish in snail control operations. Furthermore, concerns regarding the impact of P. clarkii on non-target species and on Kenyan freshwater habitats are discussed, and areas in need of additional investigation are highlighted.
Introduction
The Louisiana red swamp crayfish, Procambarus clarkii, is the most widely distributed of all crayfish species, having been introduced into 24 countries on five continents (Hobbs et al. 1989). In the United States, P. clarkii supports a thriving aquaculture industry. Elsewhere, this species is now harvested commercially in countries as diverse as Spain, Turkey, and Kenya (Hobbs et al. 1989).
Africa represents a unique situation regarding such introductions in that it lacks an indigenous crayfish fauna (Hobbs 1988). Procambarus clarkii is now present in the Sudan, Kenya, Uganda, Zambia, Zimbabwe, and South Africa (Hobbs et al. 1989). Details regarding the original introduction of P. clarkii into Kenya remain unclear, but the first individuals were probably imported from Uganda into small ponds in the Rift Valley in the mid-1960s (Oluoch 1990). In 1970, P.clarkii was introduced into Lake Naivasha, apparently in an attempt to develop a fishery for this species (Lowery and Mendes 1977a, Parker 1974, Oluoch 1990). Actually, there is an unconfirmed report that P. clarkii may have been introduced into the lake as early as 1952 for the purpose of leech control, but this claim needs verification. Since its original introduction, P. clarkii has become well established in Lake Naivasha and has supported a viable aquaculture industry. Crayfish are now considered to be one
of four important commercial species harvested from the lake, with an annual average catch of approximately 60,000 kg (live wet weight) over the past 10 years. More than 80% of this catch is exported, primarily to western European countries (Kenya Fisheries Department, personal communication). The remainder is consumed locally, mostly in restaurants in Nairobi; crayfish have not been readily adopted by most Kenyans as a preferred food item. Other large freshwater habitats in Kenya, for example, the Masinga dam complex to the south and east of Mt. Kenya, may have potential for crayfish aquaculture. In Kenya, crayfish are not harvested commercially from the numerous small dams and ponds they occupy at present, and it seems unlikely that this will change in the future, owing to excessive transportation costs.
Current Distribution Of Procambarus Clarkil
In the estimated 20-30 years since its first introduction into Kenya, P. clarkii has expanded its range and its known distribution now includes a large portion of the country (Figure 1). Besides Lake Naivasha, its presence in the Rift Valley has been confirmed in freshwater habitats near Lake Nakuru (Lowery and Mendes 1977b) and from streams and ponds in the western highlands (near the towns of Eldoret and Kitale), which form part of the Lake Victoria drainage basin. Procambarus clarkii is especially abundant in the Athi River basin in and around Nairobi. It extends through the Muranga District in the central highlands and has been collected as far north as Nanyuki. Its presence has also been confirmed in the upper basin of Kenya's largest river, the Tana. We have also recovered P. clarkii in Machakos District, near the town of Tala. It has apparently not colonized habitats on the tropical coastal plain. The town of Matuu, approximately 300 km from the Indian Ocean,
represents the easternmost site from which we have collected this species. Reports of its presence in Lake Victoria (Lowery and Mendes 1977b) have recently been discounted by local fisheries experts, and our own recent efforts to locate crayfish in Lake Victoria and in inflowing streams have not been successful. Of considerable interest are possible future extensions of its range in East Africa, either through natural or human-assisted dispersal.
Snail-Transmitted Parasites Of Medical And Veterinary Significance
Our interest in P. clarkii stems from our investigations into the biological control of schistosomes and other snail-transmitted parasites in Kenya. Schistosomiasis, a debilitating, chronic infection of the tropics and subtropics, is caused by blood flukes of the genus Schistosoma. It is estimated that between one and two million Kenyans are infected with either the intestinal or urinary forms of the disease, or both (Highton 1974, Diesfeld and Hecklau 1978, Iarotski and Davis 1981, Butterworth et al. 1984, Ouma et al. 1985, King et al. 1988). Pathology, especially in children, can be severe (Wilkins 1987). The life cycle of schistosomes is complex and involves humans (and potentially other mammals) and particular species of freshwater snails as hosts for adult and larval worms, respectively (see review by Rollinson and Simpson 1987). Humans are exposed to infection by entering bodies of water containing infected snails. Infective stages, termed cercariae, released from the
snails penetrate human skin to initiate infection. Infected humans facilitate transmission by urinating or defecating in water, thereby ensuring passage of parasite eggs into the environment of the snail intermediate hosts.
Snails of the genus Biomphalaria serve as intermediate hosts for Schistosoma mansoni, the causative agent of intestinal schistosomiasis. Snails within the genus Bulinus fulfill this function for S. haematobium, the parasite responsible for urinary schistosomiasis. Other snail-transmitted trematode parasites commonly infect domestic ruminants in Kenya and include the blood flukes S. bovis and S. mattheei, the common liver fluke Fasciola gigantica, and paramphistome flukes (see Brown 1980 for a comprehensive review of freshwater snails and associated trematode parasites in Africa).
The distribution of human schistosomes in Kenya is widespread (Figure 2) and is dictated ultimately by the distributions of the associated snail species (Brown et al. 1981, Loker et al. unpublished data). It is noteworthy that the present geographic ranges of the relevant snails and of P. clarkii are broadly, but not completely, overlapping (Figures 1 and 2). Owing to a rapidly increasing human population, unabated pollution of freshwater habitats by human excrement, construction of dams and irrigation schemes that favor the growth of snail populations, and the lack of a comprehensive national control program, it is likely that the prevalence of human schistosomiasis will remain high, or even increase. Although effective chemotherapeutic agents are available and are capable of reducing morbidity and mortality resulting from infection, they are expensive, and unless repeatedly applied to infected individuals are unlikely to diminish transmission significantly. A schistosome
vaccine is a laudable goal, but remains elusive (Skier et al. 1989). Snail control, in conjunction with other control measures, offers hope for interrupting the transmission cycle, but traditional methods for controlling snails have primarily relied on molluscicidal chemicals that are also expensive. Moreover, because of their lack of selectivity, molluscicides pose environmental concerns and can only be condoned for use in focal applications (Klumpp and Chu 1987). New approaches to the control of schistosomes are needed (Most 1987, Madsen 1990).
Crayfish-Snail Interactions
In the course of snail survey studies of Kenyan freshwater habitats, it was observed that schistosome-transmitting snail species rarely, if ever, were found in habitats containing P. clarkii. A more formal analysis of the extent to which crayfish and medically important gastropods occurred at the same time revealed a highly significant (p < 0.001) negative association between the presence of snails and P. clarkii (Hofkin et al. 1991). Of the 53 sites examined, snail-crayfish co-occurrence was recorded in only four; the snail species recovered from these four sites do not transmit human schistosomes. All 53 sites were within the known distribution of both medically important snails and crayfish and were considered, a priori, as suitable snail and crayfish habitats.
Subsequent experiments indicated that both juvenile and adult P. clarkii were able to significantly reduce Biomphalaria abundance in both laboratory aquaria and field enclosures in as few as five days (Hofkin et al., unpublished data). Crayfish consumed large numbers of snails even when alternative foods were available and frequently eradicated Biomphalaria populations within the five-day experimental period.
Furthermore, P. clarkii was able to significantly reduce the abundance of the aquatic plant, Nymphaea caerulea, in experimental aquaria and field enclosure cages (Hofkin et al., unpublished data). The presence of this common water lily has been positively correlated with the presence of both Biomphalaria and Bulinus snails (Woolhouse and Chandiwana 1989), which use them for food, oviposition sites, and refuge. By feeding on, and reducing the abundance of such plants, P. clarkii may also serve as a competitor of snails of medical and veterinary significance.
These research results have led us to believe that P. clarkii is effective in controlling and/or eliminating certain snails of relevance to human public health. As such, this crayfish may represent a viable option as a biological control agent of schistosomes and other snail-borne diseases. Some of the potential advantages offered by P. clarkii as a control agent are: 1) its impact on target species is likely to be long-lasting and, unlike chemically based methods of control, would not have to be repeatedly applied; 2) the expense involved in its use would be minimal, 3) it could simultaneously reduce transmission of all snail-transmitted parasites, including species relevant to both humans and domestic animals, and 4) because P. clarkii is able to withstand habitat drying, it could be used in both permanent and seasonal transmission sites. Enthusiasm for the use of P. clarkii in snail control operations must, however, be tempered by the realization that biological control,
in and of itself, is probably only rarely sufficient for bringing about a complete cessation of transmission. Moreover, the use of P. clarkii in particular poses certain environmental risks (see below) that must be carefully considered before their widespread use as putative control agents can be advocated.
Environmental Concerns
Perhaps the most immediate concern regarding the presence of P. clarkii in Kenya is its impact on fin fisheries in Lake Naivasha and, potentially, in other waterbodies with commercially viable fisheries. Because of its voracious feeding habits, P. clarkii is thought to pose a risk to nest-breeding, commercially important species such as Tilapia (Lowery and Mendes 1977a). For example, crayfish might interfere with the breeding of Tilapia zillii (itself an introduced species in Lake Naivasha) either by consuming its eggs or by disturbing its nest sites. Also, because of its omnivorous nature, P. clarkii can reduce the abundance of aquatic plants (Lowery and Mendes 1977a, Feminella and Resh 1989, Hofkin et al., unpublished) and therefore might restrict the available nursery areas for young fish. For example, crayfish are thought to be at least partially responsible for the dramatic decline of indigenous water plants in Lake Naivasha. Tilapia zillii catches have declined in the
lake since the introduction of P. clarkii, to the point that fewer than 3,100 kg of this species have been harvested since 1980 (Kenya Fisheries Department, personal communication).
Crayfish might adversely affect commercial fisheries in Lake Naivasha in other ways. Fish taken in gill nets are often attacked and partially consumed by crayfish before the nets are recovered, resulting in a lower market price for the damaged catch. Crayfish also become entangled in the nets, necessitating both their removal and repair of the nets. It is interesting to note, however, that fishermen in Lake Naivasha are legally required to place their nets at least 100 m from the lakeshore. The vast majority of P. clarkii in the lake are found in shallow water near the shore, meaning that most net fishermen who suffer crayfish-related damage are actually fishing illegally (Kenya Fisheries Department, personal communications).
The exact role that crayfish may have played in the decline of lake fisheries is complex and unclear, owing in part to simultaneous man-made habitat modifications, natural climatic fluctuations, and introductions of several other exotic species. For example, a section of Lake Naivasha known as "Small Lake" was cut off from the main lake basin when a causeway was constructed in 1982. This now isolated part of the lake was regarded as a prime breeding area of T. zillii, so the decline in catches for this species may, in part, reflect this loss of breeding habitat. In addition, fish populations in the lake tend to fluctuate naturally as increasing or decreasing water levels either add to, or subtract from, the amount of suitable inshore breeding habitat. Lake Naivasha has experienced relatively dry conditions in recent years, which partially explains reduced T. zillii catches (Kenya Fisheries Department, personal communications).
The effect of P. clarkii on the other two important fisheries species in Lake Naivasha, largemouth bass (Micropterus salmoides) and a second tilapia species (Oreochromis leucostictus), seems more likely to be neutral or even beneficial. Oreochromis leucostictus is a mouth breeder and is therefore not subject to egg predation by crayfish. Largemouth bass nest along rocky shorelines where crayfish are uncommon. Additionally, P. clarkii comprise more than 70% of the largemouth bass diet, which, in turn, relieves predation pressure by bass on other potential prey, including Tilapia (Kenya Fisheries Department, personal communications).
A second concern regarding the further introduction of P. clarkii in Kenya involves its potential as a pest in rice cultivation schemes. Rice is grown in Kenya both for domestic consumption and for export. Procambarus clarkii is known to consume rice seedlings and to damage dikes as a consequence of its burrowing activities (Miltner and Avault 1980, Huner 1988). To date, P. clarkii has not been considered a problem for Kenyan rice farmers (Kenya National Irrigation Board, personal communication). In all major rice growing schemes in Kenya, seedlings are cultivated in nurseries for one month prior to transplantation into the paddies. At the time of transplantation, FuradanŪ, a carbamate insecticide and nematicide, is applied to the paddies at a concentration of 4 kg/acre. The effects of FuradanŪ, as used in Kenya, on crayfish are not known, but its use may explain the apparent absence of P. clarkii from rice schemes. For example, crayfish are absent from the Mwea scheme but
are present in the Makuyu area, approximately 15 km away.
Thus, the potential impact of P. clarkii on rice cultivation remains unclear and deserves additional consideration. If crayfish are indeed excluded from rice fields through the routine use of FuradanŪ, it is encouraging that a widely used, commercially available pesticide may have value in preventing the spread of crayfish into habitats where their use in snail control might be contraindicated.
Another concern pertaining to P. clarkii that deserves consideration relates to its possible involvement in transmission of parasites of medical significance. Crayfish can serve as second intermediate hosts for lung flukes of the genus Paragonimus, including species known to infect humans (Sogandares-Bernal 1965). Paragonimus uterobilateralis and P. africanus are known from West Africa but have not been reported in Kenya or other parts of East Africa, presumably because the appropriate species of snail first intermediate host is absent (Brown 1980). In any case, it seems unlikely that lung fluke transmission in Africa would be substantially altered by the introduction of P. clarkii as several species of freshwater decapods are already present and are probably as likely as P. clarkii to serve as efficient second intermediate hosts. Because P. clarkii are sometimes frozen and usually cooked thoroughly before consumption, it is unlikely that they would serve as a significant
source of parasitic infection for humans.
In addition to its possible impact on species of direct relevance to human commerce and health, the effect of P. clarkii on other non-target organisms and on natural ecosystems in general should be considered as well. Crayfish are voracious omnivores and are often able to significantly alter the structure of natural aquatic communities (Carpenter and Lodge 1986, Lodge and Lorman 1986). This might be achieved by direct depredation of indigenous aquatic invertebrates or vertebrates, or by vigorous consumption of macrophytes and detritus used by other organisms as food sources or as cover from predators. Its escape into large natural habitats such as Lake Victoria, or the other African great lakes, with their large numbers of endemic animal species, might have particularly drastic consequences.
Possible Sites Appropriate For Use Of P. Clarkii In Biological Control
Thousands of small man-made impoundments or rain-filled pools exist throughout rural Kenya and are heavily used as a source of water for domestic purposes and for watering livestock. These habitats are often heavily polluted by human or domestic animal wastes, and are frequently populated with disease-transmitting snails and/or mosquitoes (Ripert and Raccurt 1987, Jewsberry and Imbevore 1988). Insofar as such habitats are already extensively modified by intense usage, often in ways that promote disease transmission, they may be ideal candidates for receiving crayfish in schistosomiasis control operations. Another category of habitat that should be considered as possible introduction sites are polluted streams that are located within drainage basins in which crayfish are already abundant (for example, the upper basin of the Athi River).
Additional research addressing the points raised below should be undertaken before any introductions are contemplated, with both appropriate local and national authorities involved in the decision-making process. In general, experience should be gathered regarding the desirability of crayfish introductions in candidate habitats in regions within Kenya in which crayfish already are present. Particular attention should be given to the effects of crayfish activities such as burrowing on the structural integrity of dams and on the suitability of water for human consumption. Based on these experiences, decisions should then be made regarding possible introduction in suitable habitats within areas such as the coastal lowlands where crayfish are not known to be present. We presently do not advocate the introduction of crayfish for snail control into countries where they are not already found.
Conclusion And Priorities For Future Study
The potential environmental ramifications of further P. clarkii introductions that have been highlighted here underscore the need for additional study. Current concerns regarding the protection of natural ecosystems, the maintenance of biodiversity, and the protection of rare endemic species should give pause to anyone contemplating additional introductions of this exotic species into East Africa. These very real concerns must, however, be balanced by the realization that a species such as P. clarkii offers the potential of increased economic opportunity and improved public health. Furthermore, P. clarkii is, for better or worse, already firmly established in Kenya and may well increase its present range, irrespective of human activity. Bearing this in mind, further studies should focus on how to best manage this resource, and how to take maximum advantage of its positive attributes while keeping its environmental effects to a minimum.
Potential Avenues For Further Study
1. The ability of P. clarkii to reduce actual schistosome transmission. Although P. clarkii can control schistosome-transmitting snails in the laboratory and in field enclosures, this by no means ensures that it can actually reduce schistosomiasis transmission in complex natural foci of infection. That P. clarkii can actually reduce incidence and prevalence of both S. mansoni and S. haematobium in human populations in an endemic area should be clearly demonstrated before any decision to use P. clarkii in large-scale control programs. Such field trials might best be conducted in small man-made impoundments or quarry pits, for reasons specified above.
2. Effects of P. clarkii on the abundance and species composition of mosquito larvae. It is intriguing to consider what effect crayfish, used to control schistosomes, might have on the transmission of mosquito-borne diseases such as malaria, filariasis, and arboviral infections. Because it has the capacity to alter aquatic habitats by consuming macrophytes, P. clarkii has been suggested as a means of controlling mosquitoes in California marshes (Feminella and Resh 1986). In tropical Africa, the effect on mosquito breeding might be positive or negative, depending on the tendency of a particular mosquito species to breed in open or shaded water.
3. Are commercially available pesticides used in rice cultivation effective in controlling the spread of P. clarkii? Crayfish are absent from major Kenyan rice growing schemes, possibly due to the use of certain pesticides such as FuradanŪ to control insect pests. Even if P. clarkii should prove highly successful as a means of controlling schistosomes, its presence in rice fields would probably be counterproductive, owing to its ability to consume rice seedlings. Studies should be initiated to determine whether FuradanŪ or other pesticides that are routinely used for agricultural purposes can be used to eliminate crayfish from areas where they pose economic or environmental risks.
4. Assessment of the effects of P. clarkii on other components of the aquatic environment. Careful studies are required to ascertain how P. clarkii introductions might effect non-target species. With such information available, decisions about the use of crayfish for snail control can be better evaluated. Special care should be taken to elucidate how crayfish introductions might affect endangered aquatic species.
5. What factors control the present distribution of P. clarkii in Kenya? Is its distribution likely to expand? Procambarus clarkii is absent from large areas of Kenya, and it is unclear to what degree various biotic and abiotic factors interact to explain this distribution. A clearer understanding of these factors would ensure a greater likelihood of success in snail control operations and would make predictions about its capacity for environmental damage more valid.
Acknowledgments
The authors thank Dr. John H. Ouma, Director, Division of Vector Borne Diseases for his valuable comments. We also thank Mr. N. Odero, Director, Mr. R. Kundu, Mr. J.W. Kariuki, Mr. J. Arunga, Mr. B. Ogilo, and Mr. H. Murunga, all of the Kenya Fisheries Department, and Mr. A.A. Mohdhar, Kenya National Irrigation Board, for providing helpful insights. Dr. Rene Haller and Ms. Sabine Baer of Baobab Farms Ltd., in Bamburi, also provided helpful comments. This paper is published with the approval of the Director of the Kenya Medical Research Institute. The research was supported under Grant No. DPE-5542-G-SS-6018-00, Program in Science and Technology Cooperation, Office of Research, U.S. Agency for International Development. The authors also wish to thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.
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<section>Attendees And Contributors</section>
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