W RLD WORLD AQUACULTURE •aquaponics • bath sponges • striped trumpeter • freshwater pearl culture • ponds in drought • MARCH 2009 March 2009 Volume 40 No. 1 aquaculture Longlining for mussels along the Black Sea
World Aquaculture 1 WORLD AQUACULTURE Magazine World Aquaculture magazine is published by the World Aquaculture Society. The home office address is: World Aquaculture Society, 143 J.M. Parker Coliseum, Louisiana State University, Baton Rouge, Louisiana 70803, USA. Tel: +1-225-578-3137; Fax: +1-225-578-3493; e-mail: carolm@was.org. World Aquaculture Society Home Page: http://www.was.org WORLD AQUACULTURE SOCIETY OFFICERS, 2008-2009 President, Lorenzo Juarez President-elect, Dr. Jeffrey Hinshaw Past President, Sungchul (Charles) Bai Secretary, Julie Delabbio Treasurer, Jay Parsons DIRECTORS M.C. Nandeesha William Daniels Barry Costa-Pierce Marco Saroglia Graham Mair Junda Lin CHAPTER REPRESENTATIVES Hoang Tung (Asian Pacific) Doug Drennan (USAS) Rodrigo Roubach (Latin America and Caribbean) Kwang-Sik (Albert) Choi (Korea) Hiroshi Fushimi (Japan) HOME OFFICE STAFF Carol Mendoza, Director, carolm@was.org Judy E. Andrasko, Assistant Director, JudyA@was.org WORLD AQUACULTURE EDITORIAL STAFF Robert R. Stickney, Editor-in-Chief Mary Nickum, Editor Amy Broussard, Layout Editor WAS CONFERENCES AND SALES John Cooksey, Director of Conferences and Sales World Aquaculture Conference Management P.O. Box 2302 Valley Center, CA 92082 Tel: +1-760-751-5005; Fax: +1-760-751-5003 e-mail: worldaqua@aol.com Manuscripts and Correspondence: Submit two (2) copies of all manuscripts and one (1) copy of correspondence to Mary Nickum, Editor, World Aquaculture Magazine, 16201 E. Keymar Drive, Fountain Hills, AZ 85268 USA. E-mail: mjnickum@ gmail.com. Letters to the Editor or other comments should be addressed to Editor-in-Chief Robert R. Stickney, 2700 Earl Rudder Freeway South, Suite 1800, College Station, TX 77845 USA. World Aquaculture (ISSN Number 1041-5602) is published quarterly by the World Aquaculture Society, 143 J.M. Parker Coliseum, Louisiana State University, Baton Rouge, Louisiana 70803 USA. Library subscriptions are $50 annually for United States addresses, and $65 annually for addresses outside the United States. Individual subscriptions are a benefit of membership in the World Aquaculture Society. Annual membership dues: Students, $40; Individuals, $60; Corporations (for-profit), $250; Sustaining, $100 (individuals or non-profits); Lifetime (individuals), $1,000; E-Membership, $10 (no publications, meeting discounts and not an active member in last five years). Periodicals Postage paid at Baton Rouge, Louisiana and additional mailing offices. Twenty-five percent of dues is designated for a subscription to World Aquaculture magazine. Postmaster: Send address changes to the World Aquaculture Society, 143 J.M. Parker Coliseum, Louisiana State University, Baton Rouge, Louisiana 70803 USA. ©2009, The World Aquaculture Society. ■ W RLD AQUACULTURE Vol. 40 No. 1 March 2009 Cover photo: Fishermen from the village of Gonio, Ajaria along the Black Sea coast of Georgia tend a longline of mussels. (Photo by Maria Lavnevski [www. photography.ge] and used with permission.) See page 26. (Continued on page 2) 4 Continuous photoperiod can be used to get higher growth performance in juvenile red sea bream (Pagrus major) Amal K. Biswas, Manabu Seoka, Yoshimasa Tanaka, Kiyotaka Ueno, Kenji Takii, Hidemi Kumai 8 Demonstrations and laboratory exercises in aquaculture: VI. Fish pond inventory and feed budgets Matthew Landau and John Scarpa 12 Observing external clinical signs of the idiopathic Myonecrosis (IMN) during production of Pacific Wwhite shrimp (Litopenaeus vannamei) in Brazil Gustavo Dominguez and Juan José Alava 16 Effects of feeding practical diets containing different protein levels to Australian red claw (Cherax quadricarinatus) Linda S. Metts, Kenneth R. Thompson, Laura A. Muzinic and Carl D. Webster 20 Farming bath sponges in tropical Australia Alan Duckworth, Carsten Wolff and Elizabeth Evans-Illidge 23 Aquaponics: The integration of recirculating aquaculture and hydroponics Wilson Lennard 26 Aquaculture in the Republic of Georgia Michael A. Rice 30 Identification and control of parasites in a new species for aquaculture: A case study with striped trumpeter, Latris lineata M. Andrews, B. Nowak, J.M. Cobcroft and S.C. Battaglene 35 Development of integrated prawn-fish-rice farming for sustainable livelihoods of the rural poor in Southwest Bangladesh Nesar Ahmed 45 New immune systems: Disease-specific immune responses in invertebrates, and their potential applications in aquaculture David A. Raftos and Sham V. Nair 51 Stock densities, growth and survival for pacu (Piaractus mesopotamicus) Gustavo Wicki, L. Luchini, L. Romano and S. Panné Huidobro
2 March 2009 Editor’s note In the December 2008 issue of World Aquaculture, I indicated that letters in response to the article entitled “An Interview With a Fish” by James Rose would be printed if space were available. Well, space seems to be available, so the accompanying letter represents what has come in thus far. We’ll keep the offer open through the deadline for the June issue (May 15) in case anyone else would like to weigh in on the issues raised in the Rose article. — RSS Dear Editor: I read with interest the interview with James Rose titled “Interview with a fish” in your magazine. Professor Rose ignored a plethora of scientific research showing that fish are sentient beings and feel pain. This information has been summarized in one of my review essays that can be downloaded online at http://www.int-res.com/articles/ dao_oa/d075p087.pdf and in my book The Emotional Lives of Animals, and there is also updated information in another essay available at http://arbs.biblioteca.unesp.br/viewissue.php (Why “Good Welfare” Isn’t “Good Enough”). Information is continually being collected that contradict the view that fish do not feel pain. Readers can also look at the recent research by Professor Ian Duncan and his colleagues at the University of Guelph (http://www.uoguelph.ca/ abw/iduncan/index.shtml). — Marc Bekoff http://literati.net/Bekoff www.ethologicalethics.org Professor Emeritus Ecology and Evolutionary Biology University of Colorado, Boulder 57 Improved feed management strategy for Litopenaeus vannamei in limited exchange culture systems Susmita Patnaik and Tzachi M. Samocha 60 Aquaculture in China — Freshwater pearl culture Jiale Li and Yingsen Li 61 Ponds in drought Forrest Wynne 64 Ethics in aquaculture Luis Vinatea Departments 19 Editor’s note 42 Scenes from Seattle 63 WAS student members excel in Busan, Korea 72 Calendar 72 WAS Membership Application Contents (continued) Advertisers’ Index AQUACULTURE AMERICA 2010..................55 Aquaculture Association of Canada..................29 Aquaculture Systems Technology..........Inside Back Cover Aquaculture Without Frontiers..........................39 Aquafauna Biomarine..........................................7 Aquatic Eco-Systems, Inc...................................25 AREA..................................................Back Cover Argent Laboratories.................Inside Front Cover aova Technologies..............................................44 Fish Breeding Association Taiwan.....................50 GTC Nutrition...................................................41 Northern Aquaculture........................................47 USAS Sponsored Publications...........................65 WAS Online Store..............................................56 WAS Future Meetings........................................71 WORLD AQUACULTURE 2009......................34 World Fishing Exhibition...................................33
World Aquaculture 3 President’s column Dear fellow members, In this, my fourth and last column as president, I would like to comment on the recent activities of the board of directors and also to present a preview of the upcoming World Aquaculture convention. Board issues were discussed at the midyear meeting that took place last November in Miami and convention issues at the steering committee meeting that took place in January in Veracruz, Mexico. I am happy to report that most tasks and assignments are on track, thanks to the great efforts and diligent work of the board and steering committee members. At the Miami meeting the board approved the state of candidates for the upcoming election. The ballot will be out early in 2009. This year we will be electing a new President, a Treasurer and two new directors. I urge all members to vote, either electronically, using the link provided in the website, or by returning the ballot through regular mail. In the last elections we have seen low voter participation. This is your chance to influence the way the society is run, please devote a few minutes to make your opinion count. The affiliations committee is organizing a session on promoting responsible aquaculture with the professional societies affiliated to WAS. The first meeting of affiliates during the Busan convention was a success and I am sure this second one scheduled for the meeting in Veracruz will be an even better one. The board continues to work on plans for a fellowship program patterned like that of other professional societies. Discussions have centered on designing a program that is distinct from other honors awarded by the society and that includes the academic, industrial, and regulatory components of our society. I am very excited about this program and I hope the current concept evolves to a point where it can receive final board approval for implementation this year. The Industry Relations committee has organized two review talks specifically requested by industry members for our upcoming Veracruz conference. Thanks to Drs. Albert Tacon and Eric Hallerman for taking on the important topics of Contaminants in Aquaculture Feeds and Genetically-Modified Fish in Aquaculture. I am sure these talks will be highly attended in Veracruz. No pressure to Albert and Eric, but I hope both talks evolve into review papers for our Journal. The board also made the final decisions for Honorary Life Membership awards for this year. Breaking with tradition it was decided to notify the awardees in advance, rather than make a surprise announcement at the yearly convention. The board reasoned that knowing of this honor in advance would allow awardees a better chance to attend the meeting. This year’s honorary life member recipients are Harry Cook, John Halver and Modadugu Gupta. The warmest of congratulations go to them. I can also say that we still have a big surprise reserved for this year’s awards ceremony, but my lips are sealed on that one… World Aquaculture 2009, our yearly WAS conference will take place May 25-29 at the World Trade Center in Veracruz, Mexico. We are happy to see that in spite of the recent global economic slowdown the inflow of presentations, posters, individual registrations, and sales of trade show booths are all ahead of schedule. The Steering Committee has been busy planning this meeting, which is shaping to be one of the most successful ever. We thank the State government of Veracruz and the Mexican Federal Government (CONAPESCA and INAPESCA) for their support and sponsorship of this event. I am excited about several aspects of the meeting, including a very special shrimp session with a book of proceedings, scheduled to be ready at the convention under the able direction of Craig Browdy and Darryl Jory. Shrimp constitutes a large proportion of Mexican aquaculture production, and this session and book are sure to attract local interest and participation. I want to thank Karl-Heinz Holtschmit and Ernesto Garmendia for writing a great historical introduction for the book, covering 40 years of shrimp aquaculture in Mexico. Veracruz is a very attractive destination for meeting participants, not only due to its excellent convention facilities (see www.wtcveracruz.com.mx), but also because it offers a wide range of possibilities for tourism and relaxation. In Veracruz, you will find a taste of the real Mexico, both the modern and the traditional. The city’s beaches, folklore and historical sites make it a common destination for Mexicans, but one yet mostly undiscovered by international tourists. Veracruz is Mexico’s first and most important port. The historic downtown has a distinct Spanish colonial flavor similar to that of old Havana. There are Spanish forts dating from the 1500s, nearby pre-Hispanic archeological zones, and the largest and most modern aquarium in Latin America. There are also good places and facilities for scuba diving and sport fishing. For a taste of Veracruz please visit: http://www.mexconnect.com/mex_/veracruz/veracruzindex.html Finally, please note that the general WAS membership meeting will take place during the upcoming World Aquaculture 2009 convention and is scheduled for Monday, May 25 at 4 p.m. at the World Trade Center in Veracruz, Mexico. I would like to thank members and especially the board of directors for their support and great efforts during my presidency. It has been a true pleasure and an honor to serve the society this year. —Lorenzo Juarez President
4 March 2009 Continuous photoperiod can be used to get higher growth performance in juvenile red sea bream (Pagrus major) Amal K. Biswas*1, Manabu Seoka1, Yoshimasa Tanaka2, Kiyotaka Ueno2, Kenji Takii1, Hidemi Kumai1 This report is one of a series of articles devoted to establishing a light regime that will promote optimal growth for a complete production cycle of red sea bream, Pagrus major (Figure 1). This is one of the most important fish in Japan because of its multipurpose uses as sashimi, sushi or presented in ceremonies such as weddings, where it is seasoned with salt and grilled. There is a growing concern as to how the production of this commercially important fish can be enhanced. Photoperiod manipulation has proven to be a more economic way of stimulating growth performance in this species without adverse affecting its physiology when reared from 1 to 30 g (Biswas et al. 2006a,b; Biswas et al. in press), and has also been effective when used with other species (Boeuf and Le Bail 1999). This article shows a positive effect of photoperiod manipulation on the growth performance of red sea bream without a stress response when reared from 20 to 100g. Photoperiod Design Four different light regimes were established (Figure ２): 6 h light:6 h dark (6L:6D), 12 h light:12 h dark (12L:12D), 16 h light:8 h dark (16L:8D) and continuous light (24L:0D). A programmed time controller3 was used to maintain the periods of light and dark, including dimming over 30 minute periods. Three tanks in each treatment were illuminated with one 40 W fluorescent tube suspended 45 cm above the water surface. Light intensity was maintained at 1500 lux Fig. 1. Red sea bream (Pagrus major) on the water surface throughout the experiment. Each set of three replicates was isolated from the other set and from stray light by shielding with an opaque partition. Fish and Experimental Design Juvenile red sea bream (body weight 10-30 g) of the Kinki University strain (Taniguchi et al. 1995, Murata et al. 1996) were obtained from the Fish Nursery Center of Kinki University, Uragami, Japan and acclimated to the new rearing environment. During the acclimation period, photoperiod in all tanks was set at 12L:12D. The tanks were supplied with filtered seawater and aerated to maintain the oxygen level near saturation. The water flow was 5 L/min and the temperature was maintained at 21±1°C throughout the rearing period. After conditioning for one week the fish were exposed to the test photoperiods at a density of 32 fish in each of three replicates (200 L) for each treatment. The initial mean body weight was approximately 20 g. Fish were fed a commercial diet to apparent satiation for eight weeks according to the feeding schedule presented in Figure 2. Individual body length and weight were taken at the end of the trial to calculate the growth performance. Blood samples were also taken to investigate the levels of stress indicators in the plasma. To investigate the effect of photoperiod manipulation on the digestive performance of red sea bream, fish were reared for another three weeks and fed a diet containing 0.5 pecent chromic oxide (Cr2O3) using to the feeding schedule in Figure 2. Fig. 2. Feeding schedule in different photoperiods. Arrow indicates the time of feeding and the black bar shows the dark phase of the photoperiod.
World Aquaculture 5 Before fecal collection, all possible care was taken during feeding so that no uneaten feed settled to the tank bottom. The fecal collectors were removed from the tanks and the tanks were thoroughly cleaned 30 min after feeding. After collection, fecal samples were freeze-dried and analyzed to estimate the digestibility of protein, lipid and energy. Results and Discussion Red sea bream exposed to a 24L:0D photoperiod showed the highest total weight gain and specific growth rate [SGR (percent) = 100 × (lnW2-lnW1)/time (days), where, W1 and W2 indicate the initial and final weight (g)] compared with fish exposed to other photoperiods (Figure 3). Weight gain in fish exposed to 24L:0D was 44.4 percent higher than that of fish exposed to 12L:12D. Similarly, feed intake in fish reared under 24L:0D photoperiod was 41.0 percent higher than those reared under 12L:12D (Figure 4). Feed conversion efficiency [FCE ( percent) = 100 × {wet weight gain (g) / dry feed intake (g)], was higher in fish exposed to 24L:0D and 16L:8D (Figure 4). The higher food intake in continuous photoperiod is a result of diurnal fishes being more active under continuous photoperiods and having greater foraging activity when food is delivered. It is also related to a positive effect of growth hormone on appetite (Johnsson and Björnsson 1994). Feeding time is also one of the important factors causing variation in feed intake among the treatments. It is generally assumed that the fish take more feed when the feeding time coincides with the time of maximum appetite. Therefore, the remarkable higher food intake and FCE in 24L:0D suggested that the feeding strategy in fish exposed to that photoperiod reflected most closely the times of maximum appetite. Fig. 3. Variation in weight gain and specific growth rate (SGR) among photoperiods. [SGR ( percent) = 100 × (lnW2-lnW1)/time (days), where, W1 and W2 indicate the initial and final weight (g), respectively]. Fig. 4. Variation in feed intake and feed conversion efficiency (FCE) among photoperiods. [FCE ( percent) = 100 × {wet weight gain (g) / dry feed intake (g)}]. The digestibility of protein, lipid and energy was higher in fish exposed to 16L:8D and 24L:0D (Table 1). This was the result of a longer time interval between feeding times in fish exposed to 16L:8D and 24L:0D that have allowed the most efficient digestive process. This might have improved digestion and retention efficiency in both treatments. This resulted in a significantly higher FCE in fish exposed to 16L:8D and 24L:0D. These results suggest that growth was influenced by photoperiod through better food conversion efficiency and not just through stimulated food intake (Boeuf and LeBail 1999). The lower growth performance in 6L:6D, in spite of a longer time interval between feeding times, may be attributed to the dissipation of energy for other purposes. In the aquaculture industry, fish stress, which is simply defined as any threat to or disturbance of homeostasis, is a growing concern inasmuch as it has caused reduced growth rate, disease resistance and food intake and increased mortality. Therefore, although higher growth performance was observed in fish reared under 24L:0D photoperiod, it would be premature to propose that photoperiod as optimal for rearing fish without careful analysis of how it photoperiod affects stress level. To clarify whether or not 24L:0D caused Table 1. Apparent digestibility coefficient (ADC) of protein, lipid and energy in fish exposed to different photoperiods. ADC (%)1 6L:6D 12L:12D 16L:8D 24L:8D Protein 94.6 94.4 96.2 95.4 Lipid 91.5 91.5 93.6 93.5 Energy 87.2 86.3 87.1 88.2 1ADC of nutrients or energy (%) = 100 × [1 – {(dietary Cr 2O3 / fecal Cr2O3) × (fecal nutrient or energy / dietary nutrient or energy)}]
6 March 2009 Table 2. Comparison of different stress indicators in fish exposed to different photoperiods with levels observed in stressed fish Parameters Control Stressed Values from different photoperiods values values1 6L:6D 12L:12D 16L:8D 24L:0D Cortisol (ng/mL) 6.7 190.1 7.7 7.1 7.4 7.7 Glucose (mg/100 mL) 69.2 109.5 76.4 73.6 70.8 72.9 Protein (g/100 mL) 3.8 3.0 4.8 4.3 4.6 4.3 Cholesterol (mg/100 mL) 237 180 246 244 238 248 1Biswas et al. (2006a) stress, a number of stress indicators were investigated. The results are summarized in Table 2, where the values of different stress indicators observed in this study are compared with stress-induced levels (Biswas et al. 2006a). The results demonstrate that the levels of different stress indicators in fish exposed to the 24L:0D were far lower than the stress-induced levels. Although stress has been demonstrated to reduce food intake and growth rate in different fish, red sea bream exposed to 24L:0D showed neither a decreased growth rate nor reduced food intake compared to those exposed to 12L:12D. Therefore, photoperiod manipulation did not cause a noticeable stress response in red sea bream when reared under different artificial photoperiods. In growout farms, photoperiod manipulation can be used to hasten growth rate and has been practiced in many countries in recent years. The main concern is how the different photoperiods could be controlled in outdoor farms. Some possible ways were discussed by Bromage et al. (2001). Generally, this involved the installation of light-proof covers over culture units and the provision of artificial lighting controlled by automatic time clocks. The heavy-duty polythene or butyl linings are suspended over a simple metal, plastic pipe or wooden framework providing a cheap and effective method of blacking-out the desired areas. Tungsten or fluorescent sources of illumination can be used, preferably with a spectrum as close as possible to that of natural light. The lights should provide intensities of at least 100 lux at the water surface in all areas of the enclosures. Intensities less than 20 lux should be avoided inasmuch as they may lead to inconsistent results. To reduce stress from abrupt changes in light at the switch-on and switch-off times, fluorescent lights may require a second system of less bright lights to be installed, which are switched on shortly before and after the main system and, hence, provide the necessary twilight periods. In conclusion, the growth performance of juvenile red sea bream reared from 20 to 100 g can be stimulated remarkably by using a continuous (24L:0D) photoperiod without any adverse effect on physiology. Biswas et al. (2006b) demonstrated that the growth performance of red sea bream, when reared from 1 to 30 g, can also be stimulated. These results together help to establish a light regime giving optimal fish growth for a complete production cycle of red sea bream. It is assumed that more attention will be given to indoor and outdoor culture in the near future. Photoperiod manipulation will definitely be the option of choice to get higher output from those types of systems. Notes 1Fisheries Laboratory, Kinki University, Uragami, Nachikatsuura, Wakayama 649-5145, Japan. Corresponding author e-mail: ns_ akb@nara.kindai.ac.jp; Fax: +81 735 58 1246 2Kansai Electric Power Co. Inc., Nakanoshima 3-6-16, Kita-ku, Osaka 530-8270, Japan 3Matsushita Electric Works Ltd., Osaka, Japan. Acknowledgments This study was financially supported by the 21st Century COE program of the Ministry of Education, Culture, Sport, Science and Technology, Japan. The expenses were defrayed in part by a grant from Kansai Electric Power Corporation, Japan. References Biswas, A. K., M. Seoka, K. Takii, M. Maita and H. Kumai. 2006a. Stress response of red sea bream Pagrus major to acute handling and chronic photoperiod manipulation. Aquaculture 252:566572. Biswas, A. K., M. Seoka, Y. Tanaka, K. Takii and H. Kumai. 2006b. Effect of photoperiod manipulation on the growth performance and stress response of juvenile red sea bream (Pagrus major). Aquaculture 258:350-356. Biswas, A. K., M. Seoka, Y. Tanaka, K. Takii and H. Kumai. In press. Use of photoperiod manipulation to stimulate the growth performance of juvenile red sea bream (Pagrus major). World Aquaculture. Boeuf, G.. and P. Y. Le Bail. 1999. Does light have an influence on fish growth? Aquaculture 177:129-152. Bromage, N. R., M. Porter and C. F. Randall. 2001. The environmental regulation of maturation in farmed finfish with special reference to the role of photoperiod and melatonin. Aquaculture 197:63-98. Johnsson, J. I. and B. T. Björnsson. 1994. Growth hormone increases growth rate, appetite and dominance in juvenile rainbow trout, Oncorhynchus mykiss. Animal Behavior 48:177-186. Murata, O., T. Harada, S. Miyashita, K. Izumi, S. Maeda, K. Kato and H. Kumai. 1996. Selective breeding for growth in red sea bream. Fisheries Science 62:845-849. Taniguchi, N., S. Matsumoto, A. Komatsu and M. Yamanaka. 1995. Difference observed in qualitative and quantitative traits of five red sea bream strains propagated under the same rearing conditions. Nippon Suisan Gakkaishi 61:717-726.
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8 March 2009 Demonstrations and laboratory exercises in aquaculture VI. Fish pond inventory and feed budgets Matthew Landau1 and John Scarpa2 Introduction to Animal Inventory One of the recurring problems in pond aquaculture is estimating the number of fish in a pond. You can’t rely on stocking data, since fish populations decrease over time because of predation, cannibalism and disease. The reasons a farmer would want to determine the number of fish in a pond (i.e., the inventory) are obvious. If the farmer underestimates the number of fish, and puts too little feed in the pond, growth will be suboptimal. However, if the farmer thinks that there are more fish in the pond than there really are, and overfeeds, this not only means that money is wasted on uneaten feed, but water quality will also likely be reduced. So how do you know how many fish are in the pond? Some techniques that have been suggested by researchers involve acoustical measurements or optical fish counters. Other techniques, much more commonly used, require an historical knowledge of how the fish populations change over time in the ponds that are used. That is, if you know what’s happened in the past, that’s a good place to start when trying to figure out what’s going on at the moment. This is certainly important to know, but we’ll take a different approach. In this exercise, we’ll be using tagging/marking. Currently, these are used more in fisheries management than aquaculture, but still can be useful for farmers. Tagging is the term used when an internal or external tag (Figure 1) is applied that can identify a particular individual fish. Tagging is useful for fisheries biologists, but it requires a lot of manpower to tag fish, as well as for record keeping. Marking refers to fin clipping, branding or the use of dyes and stains. Marking is easier to do when you have a large number of animals, or the animals are too small to be easily tagged. Marking allows you to identify a fish as part of the batch that was manipulated, but not the individual fish. Regardless of which method is used, it should not significantly affect the fish in terms of its behavior, growth or survival. The tags/marks that work well for one type of fish will not necessarily work well for another. Ecologists have developed a number of ways to estimate population size using marks and tags. The methods depend on knowing if the ecosystem is open” or closed (a pond would certainly be closed) and if there is replacement after sampling and future samplings. We will use a relatively straight forward procedure called a Petersen-Lincoln estimator: Fig. 1. Examples of tags that are used to identify fish. Photo by M. Landau. In practice, some fish (a) are collected and marked, then released back into the pond to mix with the unmarked fish. After a day or two, a small seine net is dragged through the pond and a sample of fish (n) is collected and counted. While examining the fish in the sample, the farmer counts how many of those are marked (r). Finally (N), the estimate of the number of the fish in the pond is computed.
World Aquaculture 9 Exercise 1 – Pond Inventory Using Marked Simulated Fish This involves counting marked individuals and estimating the size of the population after releasing the marked simulated, in this case, fish back into the pond. To start, we’ll use a 0.45 kg bag of large dry lima beans; these beans will be our fish. Count out 80 beans and mark each of them with an X on both sides (Figure 2). Then, mix them with the unmarked beans in a small bucket (our pond). Use you hand to mix the marked and unmarked beans completely. After the beans are mixed up, reach in and get a handful. Count the number of marked and unmarked beans, and then return all the beans to the bucket. Repeat this procedure three times. Show your answers on Table 1. Table 1. Estimated beans in the bucket using one handful samples. Sample Total number Total number Total number of Estimate of total number of marked of beans in marked beans number of beans beans (a) sample (n) in sample (r) in bucket (N) 1 80 2 80 3 80 Fig. 2. Lima beans in a bucket. Some are marked with X. Photo by M. Landau. Solve for N using the equation above; do this for all three samples. The value of a will be the original number of marked beans (80). Finally, count all the beans in the bucket to find out how good the estimate (N) was of the true total. Student Question – You now know how many beans were really in the bucket. Why did you get different values for N each time you sampled? Answer – Even though the beans were mixed in the bucket, this doesn’t mean that they had a perfectly uniform distribution. That is the reason that one handful is not exactly the same as another. Therefore, since each (n) and (r) were different, each (N) was different. To increase the accuracy of (N) you can either increase the number of marked beans, increase the sample size or both. This results in more marked beans being counted, which should result in less sample variation. To test this, mark 20 more beans and mix them in with the rest of the beans, so now (a) is 100. Now, rather than taking one handful of beans, take two handsful for each sample. Repeat the experiment above to see if (N) is now a better estimator of the true number of beans in the bucket. Put your answers in Table 2. Table 2. Estimated beans in the bucket using two handful samples. Sample Total number Total number Total number of Estimate of total number of marked beans (a) of beans in marked beans number of beans sample (n) in sample (r) in bucket (N) 1 100 2 100 3 100
10 March 2009 Table 3. Feeding chart for hypothetical fish. Values are in percent of body weight fed per day. Mean fish weight (g) 4.4-5.6ºC 6.1-7.2ºC 7.8-8.9ºC 0.9 3.6 4.8 6.9 1.4 3.5 4.7 6.5 2.3 3.4 4.6 6.1 4.5 3.2 4.5 5.7 5.6 2.9 4.3 5.4 8.2 2.7 4 5 11.3 2.5 3.8 4.6 13.6 2.2 3.5 4.3 22.7 1.9 3.1 4 31.8 1.6 2.7 3.7 38.6 1.4 2.4 3.2 45.4 1.3 2 2.9 68.0 1.2 1.8 2.5 79.4 1.1 1.6 2.3 90.7 1 1.5 2.1 181.4 0.9 1.3 2 317.5 0.8 1.2 1.8 454.0 0.7 1.1 1.7 Introduction to Feed Budget As we said, too little or too much feed going into ponds results in both biological and economic problems. When a farmer buys feed from a commercial source, the manufacturer will often include information about feeding rates and schedules. This will include what feed sizes should be for fish of different sizes (small fish are fed small granules, while big fish get large pellets) and the frequency that the fish should be fed (young fish are fed more often than older, larger fish); feed size and frequency are critical, but will not be considered in this exercise. The other information that you need, which again will typically be supplied by the feed manufacturer, is how much feed should be used per day. This is dependent on fish species, size/age and water temperature, all of which are factors determining the food conversion ratio (how much feed is needed to increase the weight of the fish by a given amount). In Table 3, a feeding chart for a hypothetical fish is shown. For any species, there is an ideal temperature for growth, and if growth is optimized, feed intake will be at its maximum to support that growth. Like many animals, commercially cultured fish need proportionately more feed when they are small, and less as they grow. Note, for example, that at 4.4-5.6ºC, fish weighing 0.9 g should get 3.6% of their body weight in feed each day, but large fish of 0.45 kg only need 0.7% of their body weight in food daily. To use Table 3, let’s say that you do a population inventory using marked fish and estimate that a pond has 5,000 fish. Based on the fish sampled you determine that the fish have an average weight of 13.6 kg; you also know that the average water temperature is 6.1ºC. Looking at Table 3, you see that the fish should be fed 3.5% of their body weight each day. Since there are 5,000 fish, with an average weight of 13.6 kg, you calculate that there are 68 kg of fish in the pond, and, therefore, 2.38 kg of feed per day are needed. In practice you can’t do daily inventories, and average fish weights will rarely match the table values exactly. While it is possible to use interpolation to calculate feeding rates, in many instances it is safer and easier to simply use a little less feed, since this will probably increase the digestive efficiency. It’s also worthwhile mentioning that slavish adherence to manufacturer feed charts is probably not in the best interests of the farmer, who should experiment a little and fine tune the feeding regimen for the particular situation.
World Aquaculture 11 Exercise 2 – Pond Feeding Budget Suppose we are growing a fish species that can grow from 4.54 g at stocking to 454 g (harvest weight) in 32 weeks. The feed costs $0.55/kg. Use Table 3 above to complete Table 4 (weeks 1-2 are filled in as a guide; remember to convert percent numbers to decimals). Student Question – What is the total feed budget for they year? Answer – Adding up the last column, you should get a total of $3,377.02. Student Question – Assuming that feed makes up 50% of the total budget each year, what would the farmer have to sell the fish for just to break even? Assume that the inventory estimates in Table 4 are the actual numbers of fish. Answer – If $3,377.02 is the feed budget, the total cost of operating the farmer is twice that, or $6,754.04. We know from Table 4 that 8,400 fish averaging 0.5 kg are harvested. To break even, the farmer must sell the fish for $6,754.04/3,818 kg (8,400 x 0.4545 kg) = $0.884/kg. Notes 1Department of Marine Science, Richard Stockton College, Pomona, NJ 08240 USA mlandau@stockton.edu 2Center for Aquaculture and Stock Enhancement, Harbor Branch Oceanographic Institute at Florida Atlantic University, Fort Pierce, FL 34946 USA jscarpa1@hboi.fau.edu Table 4. water mean inventory feed percent kg. feed kg feed weeks temperature weight estimate body weight used/day used/2 weeks cost 1-2 4.4 0.01 10,000 3.2 1.45 44.8 $22.40 3-4 4.4 0.0125 9,500 5-6 5.0 0.018 9,400 7-8 5.6 0.025 9,400 9-10 5.6 0.025 9,350 11-12 6.1 0.03 9,200 13-14 6.7 0.05 9,150 15-16 6.7 0.07 9,100 17-18 7.2 0.1 9,050 19-20 7.2 0.15 9,000 21-22 7.8 0.15 8,800 23-24 8.3 0.2 8,800 25-26 8.9 0.2 8,700 27-28 8.9 0.4 8,600 29-30 8.3 0.7 8,600 31-32 45 1.0 8,400
12 March 2009 Observing external clinical signs of the idiopathic Myonecrosis (IMN) during production of Pacific white shrimp (Litopenaeus vannamei) in Brazil Gustavo Dominguez1 and Juan José Alava2* In Brazil, shrimp aquaculture has been developed in the last decade and shows signs of growth. For example, the output of the shrimp production in northeastern Brazil increased from 40,000 to 60,128 t during the period 2001-2002 and the demand for Pacific white shrimp (Litopenaeus vannamei) postlarvae went from 0.5 to 11.4 billion in 1994 compared with 2002 (Camara et al. 2004). This productive activity is associated with increased demand for land, hatchery stocks and feed. At the same time, shrimp viral diseases have emerged on Brazilian shrimp farms and threaten the country’s outstanding production. The importation of non-native shrimps (L. vannamei and L. stylirostris) during early 1980s introduced several viruses, such as the Infectious Hypodermal and Haematopoietic Necrosis (IHHNV), Taura Syndrome Virus (TSV) and Necrotizing Hepatopancreatitis (NHP) into Brazil (Briggs et al. 2004). Another viral disease that has appeared there and, which is causing severe shrimp mortality, is the Idiopathic Muscle Necrosis, recently renamed Infectious Myonecrosis (IMN; Lightner et al. 2004). Other names used to describe this disease are white muscle disease, muscle necrosis, spontaneous muscle necrosis, muscle opacity, idiopathic myopathy, white syndrome and milky prawn disease (Rigdon and Baxter 1970, Lakshmim et al. 1978, Nash et al. 1987, Tonguthai 1992, Flegel et al.1992). The disease first appeared in a shrimp farm located in the Municipality of Parnaíba, state of Piaui, northeastern Brazil, in September 2002 and it has been identified in other countries where Pacific white shrimp are cultured (Lightner et al. 2004). In Brazil, the economic losses in shrimp production because of the IMN were calculated to be US$20 million in 2003 (Nunes et al. 2004). Infectious myonecrosis has been found elsewhere in the world in other penaeid shrimp species (Penaeus aztecus, P. japonicus and P. monodon; Rigdon and Baxter 1970, Lakshmim et al. 1978, Momoyama and Matsuzato 1987, Flegel et al. 1992), giant freshwater shrimp (Macrobrachium rosenbergii; Nash et al. 1987, Andserson et al.1990), freshwater crayfish (Cherax terminatus; Evans et al. 1999), swamp crayfish (Procambarus clarkii; Lindqvist and Mikkola 1978) and Norway lobsters (Nephrops norvegicus; Stentiford and Neil 2000). Lightner et al. (2004) recently reported that the etiologic pathogen is a spherical RNA-virus 40 nm in diameter. Viral IMN (IMNV) has been cataloged as a new disease for cultured Pacific white shrimp, causing necrosis of the skeletal muscle (Lightner et al. 2004, Tang et al. 2005). This information is similar to the description previously made in black tiger shrimp (Penaeus monodon; Flegel et al. 1992). More recently, bioassay studies have demonstrated that both L. stylirostris and P. monodon are also susceptible to IMNV infection (Tang et al. 2005). Infectious myonecrosis is characterized as a disease with an acute display of gross signs and high mortalities, followed by a chronic phase with persistent low-level mortality, affecting postlarvae, juveniles and subadult cultured stocks of Pacific white shrimp (Lightner et al. 2004). This particular species of shrimp has been found to be the most susceptible to INMV infection when compared to infected L. stylirostris and P. monodon during bioassays (Tang et al. 2005). In this article, we present an original description of a set of several clinical signs found in Pacific white shrimp from ponds on a shrimp farm located in Camocim, a village located 360 km from Fortaleza, which is the capital city of the state of Ceará, northeastern Brazil, as well as notes on the shrimp culture production during the outbreak of this viral disease in 2003 and 2004. Shrimp Production during the Epidemic Before IMN appeared, at the beginning of 2003, shrimp production on the farm was generally considered to range from 2,500-3,000 kg/ha, with a stocking density of about 25-30 individuals/m2. During the period August-December 2003, total shrimp production area was 30.1 ha, divided into five ponds ranging 4.7-8.9 ha, with an average size of 6.0 ha. Routinely, feeding was done four times daily, twice in the morning (0700 and 1030) and in the afternoon (1330 and 1630). In accordance with consumption strategies, feed trays were used to provide food and to monitor feed consumption for feeding rate adjustments and biomass es-
World Aquaculture 13 timations. Water exchange was carried out using a continuous bottom water releasing method. Because of production costs and the lack of appropriate aquaculture fertilizers, urea was used as a nitrogen source and super triple phosphate was used as a phosphorus source. During the last quarter of 2003, shrimp production was 2,000 kg/ha after 138 days of culture, with a FCR of 2.1. By that time, some shrimp showed grey discoloration in muscle tissues of the abdominal region. Mortality appeared two months before harvesting when the shrimp reached a size of 6 g. Final survival was 65 percent. When there were hypoxic conditions in the ponds, some shrimp displayed a reddish tail that was associated, in most of the cases, with necrosis. During the molting period, shrimp mortality increased dramatically. Normally, the number of dead shrimp encountered daily in ponds or feed trays were 6-10 individuals. In contrast, this number increased to as many as 200 dead shrimp daily in a large pond during molting periods and the acute phase of IMN. To enhance production, stocking was decreased from 25 to 16 individuals/m2 in ponds with reduced survival. However, this shrimp pond management strategy did not function as expected and the mortality continued to hamper production. The increased mortality persisted until the final cycle. In the final cycle, the maximum mortality registered was approximately 400 individuals per day in a large pond. In general, mean survival was only 53 percent. Light microscopic examination of shrimp guts revealed the presence of an extreme abundance of bacteria and gregarines, especially during the 2004 rainy season (January-May), as well as broken and evacuated guts. Gregarines were represented by different developmental stages (gamonts, gametocysts and trophozoites) and could have also played a critical role in low shrimp production. IMN Macroscopic Clinical Signs The external appearances and features of the affected shrimp showing the different grades of infection generated by the IMNV are as follows: Initial phase (grade 1 infection). This phase is initiated with a minor, light whitish–pink coloration, opacity, or grey discolorations in focal areas, expanded slightly along the muscle of the abdominal segments from the under parts to the upper parts (Figure 1). Moderate phase (grade 2 infection). During this stage of the disease, the opacity increases in size, reaching other areas of the abdominal region and becoming more whitish than the coloration found in the initial phase (Figure 2). Here, the muscular necrosis extends more into the abdominal area. Severe or Pre-Acute phase (grade 3 infection). In this grade of IMN, the focal whitish opacities are more evident and concentrated, mainly on the under parts and sides of all the abdominal segments, including the base of the pleopods (Figures 3 and 4). The extensive opaque white coloration can appear in the cephalothorax. Acute–Chronic phase (grade 4 infection). This transition of the terminal phase is characterized by complete necrosis of the abdominal striated muscles of the segments. Here, a diffuse milky white opacity can be observed through the entire abdominal area, though it is most evident in segments four through six. The myonecrosis extends from the telson and uropods to the cephalothorax. A reddishpink necrotic coloration is clearly evident on the tail fan (telson and uropods; Figures 4 and 5), and even in the last segments. Mortality occurs during this phase. Fig. 1. Individual showing the IMN–initial phase or grade 1. (Credit picture: ®Gustavo Dominguez). Fig. 2. A shrimp reflecting the IMN–moderated phase or grade 2. (Credit picture: ®Gustavo Dominguez). Fig. 3. Specimens presenting IMN–pre-acute phase or grade 3. (Credit picture: ®Gustavo Dominguez).
14 March 2009 Discussion IMN is a relatively new pathology for L. vannamei, though it has already been described in several other crustacean species. Field clinical identification of IMN is an important preliminary step in the management and control of this disease during production, especially when laboratory diagnostic approaches are not available. Even though we have not shown histological or ultrastructural evidence of this viral disease, a prominent existing body of literature, including presentations of well documented and detailed histopathological and electron microscope slides concerning with IMN, can be found in Nash et al. (1987), Stentiford and Neil (2000), Lightner et al. (2004) and Tang et al. (2005). Both histopathologically and ultrastructurally, microscopic diagnosis is characterized by myofibrillar and sarcoplamic necrosis or fibrosis, with hemocytic infiltration during the chronic phase (Nash et al. 1987, Lightner et al. 2004). Shrimp with acute and chronic IMN showed lesions with coagulative muscle necrosis and coagulative–liquefactive necrosis, respectively Fig. 4. Unhealthy individuals showing pre-acute or grade 3 (indicated by a white arrow) and acute phase or grade 4 (indicated by arrow) during shrimp production. It is noted almost total necrosis of the abdominal segments (white color) and presence of red uropods during the acute phase. (Credit picture: ®Gustavo Dominguez). Fig. 5. A clear distinction of the IMN-acute phase (grade 4), when compared to a healthy Pacific white shrimp. (Credit picture: ®Gustavo Dominguez). (Lightner et al. 2004). The external appearance of infected individuals in advanced stages is similar to the morphological alterations previously found in penaeids, showing the myonecrosis in the distal abdominal segments, mainly from four to six (Rigdon and Baxter 1970, Lakshmim et al. 1978). Nash et al. (1987) pointed out that the myonecrosis is likely to predominate in distal segments inasmuch as this abdominal region presents the highest metabolic activity, most obviously in hypoxic conditions during hyperactive stress. The chronic advanced stage of myonecrosis and septic form of this disease is reached when the distal region of the abdomen turns red, becoming entirely necrotic and generally linked with shrimp mortality (Lightner 1993). Tang et al. (2005) reported that typical IMNV lesions on L. vannamei, injected with purified virions, were exhibited after six days, with a mortality of 20 percent. However, diagnosis of IMN based on only clinical signs and histopathological examinations is not sufficient to confirm the disease, so other methods, such as in situ hybridizations (ISH) to detect shrimp virus have been found to be effective for definitive IMN diagnosis (Tang et al. 2005). As evidenced by recent bioassays conducted in Brazil, the major route of exposure to this disease appears to be ingestion of contaminated food or infected tissues of shrimp when compared to fecal matter of birds fed with contaminated shrimp, horizontal infection using effluents from cultured contaminated shrimps and biomass of adult Artemia fed with contaminated shrimp extracts.3 From those preliminary experiments, the transmission of the IMN virus by direct consumption of contaminated tissues, 1.6 g of infected material caused a maximum mortality of 35 percent in a population of 25 juvenile shrimp ranging 0.2-0.5g (total biomass = 5-12.5 g) after 24 hr exposure under laboratory conditions (salinity = 35, temperature = 25-28° C; feeding was 5 percent of biomass/day with 35 percent protein; water was exchanged at 50 percent daily).3 Additionally, several environmental and intrinsic factors can promote the generation of IMN. For example, dramatic oscillations in salinity and temperature, hypoxia, overstocking, hyperactivity, poor handling and transfer techniques, collection by cast-net, direct solar radiation and low-quality feeds have been identified as stress factors associated with IMN (Rigdon and Baxter 1970, Lakshmim et al. 1978, Nash et al. 1987, Lightner 1993, Lightner 1988, Lightner et al. 2004). IMN induced by environmental stressors has been reported in marine shrimp, such as P. aztecus, P. japonicus and P. californiensis (Rigdon and Baxter 1970, Lakshmim et al. 1978, Momoyama and Matsuzato 1987). If predisposing environmental stressors are removed preior to development of the advanced grades of infections, IMN signs can be arrested (Rigdon and Baxter 1970, Lakshmim et al. 1978). Nash et al. (1987) reported that reduction of stocking density by transferring Asian freshwater shrimp postlarvae affected by IMN to different and lower density ponds apparently helps mitigate the mortality. Management health strategies, such as total cleaning and aseptic conditions associated with tanks and equipment, avoiding overcrowding and maintaining optimal aeration have been used successfully in prevent-
World Aquaculture 15 ing IMN outbreaks (Nash et al. 1987). During the outbreak described here, shrimp stocking was reduced (from 25 to 16 individuals/m2), but no sign of recovery was noticed. This suggests that in addition to the persistent mortality that characterizes IMN through the culture cycle, the gregarines and bacterial infection have also contributed to the mortality. Although gregarines were not taxonomically identified in this study, it is likely that the species involved was either in the genus Nematopsis or Cephalobus. These two genera of gregarines have global distribution in penaeid shrimp aquaculture (Lotz and Overstreet 1990). Generally, gregarines are linked to impaired shrimp health and bacterial infections, such as hemocytic enteritis, that cause reduced growth and causes mortality in cultured pacific white shrimp in Ecuador (Jiménez et al. 2002) and Mexico (Fajer–Avila et al. 2005). The high abundance of gregarines and intestinal bacteria might have exacerbated the reaction of the shrimp to the viral agent and enhanced mortality. The sporozoites, gamonts and gametocysts of several species of Nematopsis (N. penaeus, N vannamei, N. marinus) have been found infecting L. vannamei cultured in Ecuador (Jiménez 1992, Lightner 1993, Jiménez et al. 2002), where infections have been mitigated by removing such intermediate carriers as the polychaete Polydora cirrhosa, which were common benthic pond dwellers (Lightner 1993). Recently, food medicated with sodium monensin (Elancobank) and sulfachloropyrazine (Avimix-STk) has been shown to remove and control Nematopsis gametocysts from the intestine of naturally infested cultured Pacific white shrimp (Fajer–Avila et al. 2005). Best management practices such as maintaining a high dissolved-oxygen concentration; keeping stable temperature, pH and salinity levels; and controlling shrimp feeding are important strategies in pond management. Moreover, according to Horowitz and Horowitz (2001), minimization of waste, removal of sludge and organic matter and maintaining optimal water quality, including the reduction of excess ammonia and nitrite, the generation of both specific pathogen free and specific pathogen-resistant shrimp stocks, stimulation of shrimp immune systems with stimulants and enhancement of immnotolerance to viruses by using tolerins are priority issues of biosecurity and shrimp health management that will help the manager avoid the occurrence of various shrimp diseases. Notes 1Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, 800 Sumter Street, Columbia, SC 29208, USA. E-mail: gusadoca@gmail.com 2Center for Coastal Environmental Health and Biomolecular Research (CCEHBR)/ National Oceanic and Atmospheric Administration (NOAA)/National Ocean Service (NOS)/NCCOS; 219 Ft. Johnson Road, Charleston, South Carolina 29412-9110, USA. *Current address: Environmental Toxicology Research Group, School of Resource & Environmental Management, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, CANADA. E-mail: jalavasa@sfu.ca 3Graf, Ch., N. Gervais, M.C. Fernandes, and J.C Ayala.2003. Transmissão da Síndrome da Necrose Idiopática Muscular (NIM) em Litopenaeus vannamei. (Technical manuscript unpublished).5p. (Available from the first author). Acknowledgments The author thanks Dr. Victoria Otton for her valuable review and corrections on this contribution. References Anderson, I.G., G. Nash and M. Shariff. 1990. 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Hernandez-Gonzalez. 2005. Effectiveness of oral Elancobank and Avimix-STk against Nematopsis (Apicomplexa: Porosporidae) gametocyts infecting the shrimp Litopenaeus vannamei. Aquaculture 244:11-18. Flegel, T.W., D.F. Fegan, S. Kongsom, S. Vuthikornudomkit, S. Sriurairatana, S. Boonyaratpalin, C. Chantanachookhin, J.E. Vickers and O.D. Macdonald. 1992. Occurrence, diagnosis and treatment of shrimp diseases in Thailand. Pages 57-112 In W. Fulks and K.L. Main, editors. Diseases of culture penaeid shrimp in Asia and the United States. Oceanic Institute, Honolulu, HA. USA Horowitz A. and S.Horowitz. 2001. Biosecurity basics for shrimp aquaculture. Global Aquaculture Advocate 4:12-14. Jiménez, R. 1992. Analisis de gregarinas asociado al detenimiento de crecimiento en camarones P. vannamei (Part II). Acuacultura del Ecuador 17:17-25. Jiménez, R., L. de Barniol and M. Machuca.2002. Nematopsis marinus n.sp., a new septate gregarine from cultured penaeiod shrimp Litopenaeus vannameis (Boone), in Ecuador. Aquaculture Research 33:231-240. Lakshmim, G. J., A. Venkataramiah and H. D. Howse.1978. Effect of salinity and temperature changes on spontaneous muscle necrosis in Penaeus aztecus Ives. Aquaculture 13(1): 35-43 Lightner, D.V.1988. Muscle necrosis of penaeid shrimp. Pages 122–124 In C.J. Sindermann and D.V. Lightner, editors. Disease diagnosis and. control in North America marine aquaculture. Developments in aquaculture and fisheries science, Vol. 17. Elsevier Press, New York, NY. USA Lightner, D.V. 1993. Diseases of cultured penaeid shrimp. Pages 393485 In J.P. McVey, editor. CRC Handbook of Mariculture, 2nd. ed. Crustacean Aquaculture Vol.1. CRC Press, Boca Raton. Lightner, D. V., C. R. Pantoja, B. T. Poulos, K. F. J. Tang, R. M. Redman, T. Pasos-de-Andrade and J. R. Bonami. 2004. Infectious Myonecrosis–New Disease in Pacific White Shrimp. Global Aquaculture Advocate 7(5):85 Lindqvist O.V. and H. Mikkola.1978. On the etiology of the muscle wasting disease in Procambarus clarkii in Kenya. Freshwater Crayfish 4:363-372. Lotz, J.M.E. and R.M.Overstreet. 1990. Parasites and predators. Page 92-121 In J.C. Chavez and N. O. Sosa, editors. The aquaculture of shrimps, prawns, and crawfish in the world: basics and technology. Midori Shobo, Ikebukuro, Toshima-ku, Tokyo. (Continued on page 70)
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