ASEAN Cooperation in Food, Agriculture and Forestry

MANUAL

PRACTICAL GUIDELINES FOR THE DEVELOPMENT OF HIGH HEALTH Penaeus monodon BROODSTOCK

F i s h e r i e s P u b l i c a t i o n S e r i e s No.2

FORWARD

The Manual on Practical Guidelines for the Development of High Health Penaeus monodon Broodstock has been prepared by the ASEAN Fisheries Sub Working Group which is under the assignment of the ASEAN Sectoral Working Group on Fisheries since 1996. Sincere thanks to the Fisheries Agencies of ASEAN Countries which have supported the work in the preparation of the guidelines. It is hoped that this guidelines would benefit ASEANs shrimp culture industry in terms of its sustainability. For the improvement of the Guidelines, comments and suggestions are welcomed. The comments and suggestions could be submitted to the ASEAN Sectoral Working Group on Fisheries through the ASEAN Secretariat.

CONTENTS
Part A: GUIDELINE ON HOW BROODSTOCK 1 Background and Rationale Selection of Wild Broodstock Breeding Plan Production Process of Primary Stock Facilities Requirement References Part B: EVALUATION OF BROODSTOCK AND POST-LARVAL QUALITY I. A. Viruses 1. Monodon baculovirus (MBV) Disease 2. Hepatopancreatic parvovirus (HPV) 3. White Spot Disease B. Bacteria 1. Vibriosis II. POST-LARVAE What is considered as good quality Postlarval Shrimp? Pathogen Detection Proper Management References Part C: CRITERIA FOR SELECTION OF OFFSPRING I. MORPHOLOGY II. STRESS TEST Part D: IDENTIFICATION OF DISEASE I. C. D. E. VIRUS A. Wet mounts B. Histology Polymerase Chain Reaction In situ Hybridization Immunochemistry BROODSTOCK TO PRODUCE HIGH-HEALTH Penaeus monodon

II. BACTERIA A. B. by Classical Method and Rapid Test Kits C. D. E.

Gram Staining Standard Culture Method, Identification Culture and General Tests Polymerase Chain Reaction Immunochemistry

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A GUIDELINE ON HOW TO PRODUCE HIGH-HEALTH Penaeus monodon BROODSTOCK

BACKGROUND AND RATIONALE The production of certified high-health domesticated Penaeus monodon shrimp broodstock is important to sustain the shrimp culture industry in ASEAN countries. Hence, the development of a guideline on selective breeding program of domesticated P. monodon was proposed and discussed. Based on the last meeting held in Indonesia on 25 27 March 1998, the participants from ASEAN countries had agreed that it is more viable to produce a manual on the general guidelines in producing high-health (HH) broodstock instead of the original plan of producing disease-free broodstock which is difficult to be achieved. The purpose of this guideline is to develop a husbandry protocol for a sustainable supply of high-health domesticated shrimp broodstock. HH shrimp in this guideline is defined as those with improved survival and growth rates and specific pathogen free or SPR broodstock. Improvement of survival and growth rates to marketable size is the top priority to reduce production cost. Likewise, better-feed conversion efficiency and tolerance to various types of environment are important criteria to be considered. Once a high-health shrimp breeding plan is established, selective breeding can be initiated to converse genetic integrity of the HH shrimp and avoid inbreeding. Current breeding procedures permit accurate identification of the maternal parent a cross but not the paternal parent. Therefore, pedigrees based on maternal families will be maintained for HH population. The breeding program developed for P. vannamei was adopted in this guideline with modifications. The major activities for the establishment of high-health stock are : quarantine and screening protocols, breeding strategies, facility requirements for each step, and management of breeding centers and multiplication stations. SELECTION OF WILD BROODSTOCK A genetic survey to assess the genetic diversity of different shrimp populations in each country will be done. A large amount of genetic variability is needed to ensure the success of any genetic improvement program. Allozyme electrophoresis and the more advanced DNA analysis will be used in the screening and genetic evaluation of the different populations. At least 3 populations should be selected and 10 to 12 individual families will be used in the breeding plan. The microchip, bird band and plastic home-made eye tags can also be used for tagging the broodstock. Wild female broodstock with a minimum weight of about 120 g and male weight 70g up will be selected. These brooders should have as perfect phenotype as possible with no signs of infectious cuticle and gonads. The unmated female is preferable. All selected broodstock have to pass the screening for important specific diseases such as SEMBV and MBV using protocol included here. A summary of the different production activities of high health (HH) Penaeus monodon broodstock for each population is shown in Table 1.

Table 1.

Production scheme for high health Penaeus monodon broodstock Working group 2 1) Set up nuclear breeding centers (NBC) 2) Preparation of facilities for spawners and offspring in NBC 3) Assignment of multiplication centers (MC) for grow out of improved HH- P15 4) Pond preparation for grow out of HH- P15 (HH-F0) 5) Rearing of HH-F0 to 120 days (size sampling every 30 days, growth rate determination) 6) Rearing of HH-F0 (mixed and individual family) 120 days to 7 months female : male = 1:2

Working group 1 1). Selection of wild broodstock from many locations based on the population genetic evaluation. 2). Selection of larvae HH-P15 for grows out. Before the first grow-out the high environmental variance in growth rate should be minimized. Screening protocol for PL15 should also be incorporated. 3). Selection of 120 days HH-F0 (large size and photogenic screening) 4). Selection of 7 months HH-F0 (large size and photogenic screening)

5). Selection of 10 months (or 1 year) HHF0 (HH-F0 broodstock or the primary stock) for production of HH-F1 larvae 6). Repeating step 2) to 5) for production of HH-F2 larvae 7). Repeating step 2) to 5) for production of HH-F3 larvae 8). Unselected control line will be simultaneously developed for the proper comparison of selected offsprings after three generations of breeding

7) Rearing of 7 months HH-F0 to 10 (or 12) months, female : male = 1:2 (obtain HH-F0 broodstock) 8) Repeat step 2) to 7) for HH-F1, HHF2 and HH-F3 broodstock to be cultured in the MC

BREEDING PLAN The breeding plan must consider several genetic parameters such as breeding value of individuals, parent-offspring relationships, and genetics gain from progeny testing of full sib and half sib. The following mating plan should be applied for each generation: 1. 2. Stock four females from each of 10 to 12 families and four males from each of 10 to 12 families into maturation tanks (recommended 3 tons). After 7-10 days period, collect all females mated (to eliminate multiple mating by a single male). In families with unsuccessful spawning, artificial insemination will be used with randomly chosen males. Spawn at least one female from each family and stock the offspring in individual larval rearing systems. The reproductive performance of each female such as spawn size, fertilization and hatching rates, and nauplii count and quality should be recorded. Nauplii from each family will be reared to PL 15-20 in separate 1-3 ton tanks. The PL 15-20 from each family will then be reared separately in outdoor tanks or earthen ponds up to 4 months or 120 days. The 120 days shrimp 5-20% of fast growth male and female in each family will be selected and tagged, smaller shrimp will be discarded.

3. 4. 5.

6.

PRODUCTION PROCESS OF PRIMARY STOCK P. monodon (PL 15-20) from pond-reared healthy broodstock F0 (wild parent) are cultured for 120 days in earthen pond at 10 15 pcs/m2. The fast growers are selected and transferred to other ponds while the densities are reduced twice until the final density is 0.5 1.0 ind/m2. They are selected as breeders for the production of F1 by natural mating and artificial insemination technique. Forty pairs of broodstock collected from different localities are acclimated in separate maturation indoor tanks for 7 days. During this period, broodstock are fed with formulated and fresh feeds. The feeds are monitored for the presence of pathogens. Females are unilaterally eyestalk ablated. When gravid females are obtained they are allowed to spawn separately in 500-L black cylindrical tanks. Spawners are taken out the following morning after monitoring the number of females that spawned. Eggs are washed with chlorinated seawater and transferred to the hatching tank of the same size with disinfected water at 28-30 ppt. Parameters (fecundity, % fertilization and hatching rates, and nauplii count and quality) for reproductive performance of each female are taken. Nauplii from each female from each family are reared in 3 - 6 ton tank at 50 ind/L until PL 15-20 Survival rate from each batch

are recorded. Post larvae are transferred to other 6 10 tons concrete ponds or tanks for future observation of growth until they can be tagged. Then the tagged juveniles are pooled in concrete or earthen ponds for 120 days. Body weight (BW) are measured to the nearest 0.01 g and 0.1 cm for body length (BL) from post orbital edge to tip of telson. Samples are taken for morphometric and non-morphometric characters on the 30th , 60th and 120th day. Survival rate, feed conversion ratio, coefficient of variation in size, gonad and mating conditions, and male and female ration of offspring from each family and population are determined on the 120th day. Significant growth differences among shrimp progeny derived from different broodstock will be monitored. The best growing stock is used as the primary stock for the subsequent production of the next generation of domesticated. P. monodon (F2) (Fig. 1). Pathogenic screening for non-carrier viruses is done from each culture phase (broodstock, hatchery and grow-out). Fig. 1. Protocol for Production of High-Health Broodstock F0 Wild Broodstock Pop 1 Random Family 1 2 3 ..10 females Family 1 2 3 10 males Offspring F1 offspring
Culture separately to PL15 or big enough for tagging

Phatogenic screening for Selection for growth Non-carrier of viruses and survival rate
Culture together in same pond with tags* to 120 days at 10-15 i

Photogenic screening for non-carrier of viruses HH-F1 Brookstock

Selection of the top 5-20%

Culture to 10-12 months at 0.5 ind/m2

Photogenic screening for Non-carrier of viruses

Spawn gravid females

Repeat all steps for next generation evolution for genetics gain in growth and survival rate from each family after the third generation

* In case individual tagging cannot be done, the progeny from the mating of each maternal line are reared in separate tanks until marketable size or until 120 days to ensure continuity of the maternal pedigrees. The families are then be mass reared in broodstock ponds after tagging with bird band at the eyestalk. FACILITIES REQUIREMENT Implementation of the HH broodstock shrimp-breeding program requires a network of facilities for successful operation. Facilities for maturation, spawning, larval rearing, nursing, growout, quarantine and pathogenic screening are required. The most important is the nucleus breeding centers (NBC) where the selective breeding activities are conducted under the strict quarantine procedures. Post larvae from the best strains obtained from the selection process are distributed to multiplication centers (MC). MC will produce HH post larvae to HH broodstock. Hence, the center must have full quarantine capabilities to maintain HH status and to exclude pathogens throughout 10 months or one-year broodstock cycle. Each facility should be capable of maintaining at least 40 families from each four populations. The resulting HH broodstock are then transferred for testing at government and private commercial hatcheries for seed production (Fig.2). Fig.2. Facilities Required for this Programme Population 1,2, 3,4
Primary Quarantine Area (PQA) Nuclear Breeding Center (NBC) I, II, III, IV

If possible more than PQA for each Family ..10 (or

Population More)

More than 1 NBC for Each population

Improved genetics selection families More than 1 MC for Each population Multiplication Center (MC)

Commercial Hatcheries and Farms For testing of HH-Broodstock

REFERENCES Gall, G.A.E. 1990. Basis for Evaluating Plans. Aquaculture. 85:125-142. Gjeddrem, T. 1985. Improvement of Productivity through Breeding Schemes. Geojournal. 10(3): 233-241 Gjeddrem, T. and E. Fimland. 1995. Potential Benefit from High-Health and Genetically Improve Shrimp Stocks. Proceeding of Special Session on Shrimp Farming Baton Range. Lousiana USA Kasornchandra, J.,S. Boonyaratpalin, R. Khongpradit and U. Aekpanithanpong. 1995. Mass Mortality Caused by Systematic Bacilliform Virus in Cultured Penaeid Shrimp, Penaeus monodon, in Thailand. Asian Shrimp News. 5:2-3 Lannan, J.E. and J. Wyban. 1990. Broodstock Management Program for SPF Shrimp. Unpublished manuscript. The Oceanic Institute. 24p. Lightner, D.V., R.M. Williams, L.L. Mohney, J.P.M. Clerx, T.A. Bell and J.A. Block. 1985. Recent Advances in Spenaeid Virus Disease Investigations. J. World Mariculture Soc. 16:267-274. Menasveta, P., A.W. Fast, S. Piyatirativorakul, and S. Rungsupa, 1991. An Improved, Closed Seawater Recirculation Maturation System for Giant Tiger Prawn (Penaeus monodon Fabricius). Aquaculture Engineering 10:173-181 Millamena, O.M., R.A. Pudadera and M.R. Catacutan. 1985. Effect of Diet on Reproductive Performance of Wild Ablated Penaeus monodon Broodstock. In Proceedings of the First International Conference on the culture of Penaeid Prawns/Shrimps. Aquaculture Department, Southeast Asian Fisheries Forum. Asian Fisheries Society, pp 178-179. Y. Taki, J.H. Primavera and J.A. Llobrera (eds.), Manila, the Philippines. Quinition, E.T., R.M. Caballers and L. Gustilo. 1993 Ovarian Development in Relation to Changes in the External Genitalia in captice Penaeus monodon. Aquaculture 114-:71-81 Refstie, T,.1990. Application of Breeding Schemes. Aquaculture. 85:163-169 Sano, T,.T. Nishimura, H. Fukuda and T. Hayashida, 1984. Baculovirus Mid-gut Gland Necrosis (BMN) of Kuruma Shrimp (Penaeus japonicus) larvae in intensive culture systems. Helgolander Meeresunters, 37:225-264. Sugama. K. Haryanti and Samuel, L. 1998. Breeding and Genetic Study in Tiger Shrimp, Penaeus monodon. P. 33-40 In Report of the Workshop on Formulation and Assignment of Activities for the Development of Disease Free Penaeus monodon Broodstock in ASEAN Countries. 23-25 March 1998, Bumi Wiyata Hotel and Training Center Depok, West Java, Indonesia.

Sunden, S.L.F. and S.K. Davis. 1991. Evaluation of Genetic Variation in a Domestic Population of Penaeus vannamei (Boone): A Comparison with Three Natural Populations. Aquaculture. 97:131-142. Wyban, J.A., J.N. Sweeney and R.A. Kanna. 1998. Shrimp Yields and Economic Potential of Intensive Round Pond System. J. World Aquaculture Soc. 19(4): 210-217. Wyban, J.A., J.S. Swingle, J.N. Sweeney and G.D. Pruder. 1992. Development and Commercial Performance of High-Health Shrimp using Pathogen-free (SPF) broodstock Penaeus vannamei. In Proceeding of the Special Session of Shrimp farming. World Aquaculture society, Baton rouge, L.A., USA. Pp 254-260. Yano, I. 1993 Ultraintensive Culture and Maturation in Capacity of Penaeid Shrimp. In CRC Handbook of Mariculture, 2nd Edition, Volume 1: Crustacean Aquaculture, J.P. Mc Vey (ed), pp 289-313. CRC Press, Boca Raton, Ann Arbor, London Tokyo Yashiro, R. and S. Songtawontawe. 1996. Cultivation of Black Tiger Shrimp, Penaeus monodon Broodstock by Reducing Stocking Density and Transferring Pond Tech. Paper No. 14/1996. National Institute of Coastal Aquaculture, Songkhla, Thailand. 9pp. Yashiro, R. and P. Na-anant. 1997. Reproductive Performances, Moulting Interval and Artificial Insemination of Penaeus monodon from the Andaman Sea. Tech. Paper No. 7/1997. National Institute of Coastal Aquaculture, Songkhla, Thailand. 17pp.

EVALUATION OF BROODSTOCK AND POST-LARVAE QUALITY I. BROODSTOCK In the last several years, shrimp diseases have had a devasting effect on world shrimp farming. Diseases increase risk, deterring investment and commercial development. Most of these diseaseassociated problems are caused by viruses, for which there are no therapeutic cures. Like the livestock and poultry industries, the shrimp aquaculture industry will require certified high health broodstock in order to overcome these problems. Thus, shrimp broodstock quality is considered as one of the key factors in influencing successful shrimp culture. Shrimp pathogens The main reason for detecting and identifying pathogens in shrimp broodstock is to be able to detect pathogens in the carrier state before the onset of disease. Carrier detection is most useful for diseases associated with obligate pathogens such as viruses that may be transmitted vertically or horizontally. Viruses are intracellular pathogens that may be transmitted vertically through the egg or ovarian fluid during spawning, or they can be transmitted horizontally from host to host. Therefore, viral detection in shrimp is important in controlling the spread of the disease in evaluating the health status of a population or culture facility. At present, there are three (3) viruses considered significance for shrimp broodstock. Techniques currently used to demonstrate viral infections in shrimp include examination of epidemiological features and gross structural changes, microscopy of stained and wet mount preparations, histopathology, electron microscopy, immunological methods and DNA-based protocols. A. 1. Monodon baculovirus (MBV) disease MBV is the most common virus of Penaeus monodon. It has been reported that the disease is latent until the shrimp are faced with stressors, leading to the development of disease. MBV has been observed in all life stages of P. monodon. Shrimp larvae seem to be more susceptible to MBV than postlarvae and adults. Despite massive MBV infections in cultured shrimp populations, no serious impact has been reported. MBV infections may significantly reduce growth, crop value, and over-all health status of the shrimp. MBV, however, may pre-dispose infected shrimp to other pathogens resulting in the increase of the mortality rates. Gross signs accompanying MBV infection may include reduced feeding and growth rates and increased gill and surface fouling organisms. Whitish midgut line through the abdomen has been found in severely affected larvae and postlarvae shrimp. The principal clinical signs of MBV Viruses

infection is the presence of single or multiple spherical occlusion bodies in the hepatopancreas and midgut epithelial cells. Histological examination of MBV infections is dependent upon the demonstration of prominent, single or multiple eosinophilic spherical bodies within hypertrophied nuclei of hepatopancreatic tubule or midgut epithelial cells. Vertical and horizontal transmission of MBV are likely to occur. Presumably, MBV is transmitted per os by ingestion of free virus, occlusion bodies and by cannibalism. It has been suggested that wild-caught broodstock may be a source of MBV giving rise to contaminated nauplii to hatcheries and finally to the shrimp ponds. DNA probes are available for MBV diagnosis and primer sequences for PCR amplification have been published recently. Spreading of MBV is possible via horizontal and vertical transmission. 2. Hepatopancreatic parvovirus (HPV)

HPV is spherical, 22-24 nm in diameter and occurs within an intranuclear inclusion body. Signs of HPV disease are not specific, including poor growth rate in appearance, reduced preening activity as evident by increased gill and surface fouling organisms. HPV infects hepatopancreatic epithelium commonly in the distal portions of hepatopancreatic tubule, the E-cells. HPV infection results in the formation of prominent nuclear inclusion bodies showing intense basophilic reaction by H&E staining. These distinctive inclusion bodies are useful for diagnosis of HPV infection. The mode of transmission for HPV is unclear. DNA probes and primer sequences for PCR amplification have been published and available for diagnostic procedure. 3. White Spot Syndrome Virus (WSSV)

The affected shrimp exhibit white patches or spots on the inside surface of the carapace and shell accompanied by reddish to pinkish red discoloration of the body. The disease occurs in ongrowing juvenile shrimp of all ages and sizes. It is caused by a DNA virus of bacilliform to cylindrical morphology. Histological changes observed from diseases shrimp showed widespread cellular degeneration and severe nuclear hypertrophy in most tissue derived from ectodermal and mesodermal origin, especially from subcuticular shell epithelium, gill epithelium, subcuticular stomach epithelium, lymphoid organ, connective tissue, hematopoietic tissue and nervous tissues. Diagnosis procedures for WSV infection are based upon its gross and clinical signs and histological demonstration of eosinophilic to basophilic hypertrophied nuclei. DNA probes and PCR are available for detecting WSV infected shrimp including broodstock, postlarva and reservoir hosts.

B. Bacteria Vibriosis Gross signs of vibriosis are light or dark brown focal lesions and necrosis of appendage tips. The change of color is the result of melanin produces by host hemocytes involved in the inflammatory process. Large number of motile bacteria are visible in the hemocoel and hepatopancreas of moribund shrimp. Affected shrimp exhibit decreased appetite. Some also had a darker, larger or shrunken hepatopancreas. The main cause of this disease are four (4), mostly involve with Gram-negative bacteria belonging to the genus Vibrio. The possible four (4) main species are : V. parahemolyticus, V. vulnificus, V. alginolyticus and V. harveyi. However, V. harveyi, a luminescent species, is considered as the most devastating that causes extreme losses in both the hatcheries and shrimp-rearing farms. Histopathological examination revealed extensive hepatopancreatic tubular necrosis and replacement by bacterial-hymecytic nodules, often melanized., and marked hemolytic enteritis. II. POST-LARVAE

Shrimp larval quality is considered as another key factor in influencing the successful shrimp culture. Proper management in hatchery and nursery systems would ensure good survival and growth rate of shrimp post-larvae before stocking. In recent years, shrimp health management has become the main focus of improving production and minimizing infectious diseases in shrimp ponds. However, the ultimate goal of shrimp health management is to: (1) prevent the disease from occurring; (2) reduce the incidence of infectious diseases when it occurs; and (3) reduce the severity of the disease when it occurs. To accomplish this goal, one should be concerned with the quality of postlarval shrimp especially the selection of high-health postlarval shrimp, before stocking in the pond. What is considered as good quality postlarval shrimp ? Evaluation of postlarval quality may be obtained by observation using naked eye or with the aid of a light microscope. Shrimp larvae of good quality possess certain characteristics listed in Appendix 1. Pathogen Detection The pathogens of concerns to broodstock quality are the same as those that are of concern to larval quality, thus detection methods for these pathogens in both broodstock and post-larvae follow the same protocol. The difference lies in the sampling procedure since for broodstock, a less invasive methods, such as hemolymph collection near the heart, is preferred rather than sacrificing the whole animal.

Molecular biology techniques such as DNA probes and polymerase chain reaction (PCR) provide accurate, sensitive and rapid diagnostic tools for detecting and identifying certain shrimp pathogens especially viral diseases. Recently, various sets of primer sequences have been successfully designed from WSV. These primers have been used in the PCR methods for WSV, both in postlarval shrimp and shrimp broodstock. PCR exploits the capability of amplifying a small amount of genetic material from an organism, thus facilitates the detection of virus especially in carriers which normally contain a small amount of virus particles. The establishment of PCR laboratories, both from government and private sectors, for screening WSV postlarval shrimp in Thailand, not only helps in improving the high health shrimp postlarval production, but also minimizes the spread of WSV in shrimp pond as well. Proper Management Producing high health postlarval shrimp is not only dependent on the absence of shrimp pathogens but also relies on proper hatchery management. The rearing conditions include nutrition aspects, phytoplankton density maintenance, water quality and other environmental conditions. These include seawater supply system, regular disinfection and dry-out of hatchery facilities and adequate aeration in larval tanks. Moreover, the use of some chemicals, antibiotics or additional hormones larval shrimp rearing should be reduced. Application of some antibiotics such as chloramphenicol and oxolinic acid in the hatchery may reduce larval growth and inhibit defense mechanisms of larvae itself since these type of drugs may reduce the activity of acid or alkaline phosphatase which are considered to play a major role in the protein metabolism in shrimp.

REFERENCES Anonymous. 1991. Shrimp culture problems in southern Thailand. Asian Shrimp News Collected Volume 1989 1995: 117-120 Anonymous. 1994. SEMBV-an emerging viral threat to cultured shrimp in Asia. Asian Shrimp News Collected Volume 1989 1995: 170-177 Boonyaratpalin S. 1990. Shrimp larval diseases. In: M.B. New, H. de Saram, and T. Singh, (eds), Technical and Economic Aspects of Shrimp Farming, INFOFISH, Kuala Lumpur, Malaysia. pp. 158-172 Boonyaratpalin S., K. Supamattaya, J. Kasornchandra, S. Direkbusarakom, U. Aekpanithanpong and C. Chantanachooklin. 1993. Non-occuluded baculo-like virus the causative agent of yellowhead disease in the black tiger shrimp Penaeus monodon. Fish. Pathol. 28:103-109. Chang, P.S., C.F. Lo. G.H. Kou, C.C. Lu and S.N. Chen. 1993. Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV). J. Invert. Path. 62:116-120. Flegel, T.W., S. Boonyaratpalin, D.F. Fegan, M. Guerin and S. Sriurairatana. 1992. High mortality of black tiger prawns from cotton shrimp disease in Thailand. In: Mr. Shariff, R.P. Subasinghe and J.R. Arthur (eds). Diseases in Asian Aquaculture I, Fish Health Section, Asian Fisheries Society, Manila, Philippines, pp. 181-197. Flegel, T.W., D.F. Fegan, S. Kongsom, S. Vuthikorudomkit, S. Sriurairatana, S, Boonyaratpalin, S. Chantanachookhlin, J.L. Vickers and O.D. Macdonal. 1992 Occurrence, diagnosis and treatment of shrimp diseases in Thailand.Diseases of penaeid shrimp. In: W. Fulks and K.L. Main (eds), Diseases of cultured penaeid shrimp in Asia and United States, Oceanic Institute, Honolulu, Hawai. Pp. 57-112. Jiravanichpaisal, P., T. Miyazaki and C. Limsuwan. 1994 Histopathology, biochemistry and pathogenicity of Vibrio harveyi infecting black tiger prawn Penaeus monodon. J. Aquat Health. 6:27-35. Kasornchandra, J.S. Boonyaratpalin, R. Khongpradit and U. Akepanitphanpong. 1995. Mass mortality caused by systematic bacilliform virus in cultured penaeid shrimp, Penaeus monodon, in Thailand. Asian Shrimp News. 21(1):2-3. Kou, G-H. and C-F., Lo. 1998. White spot syndrome virus in shrimp broodstocks and their offspring. In: Proceeding of VIIth International Colloquium on Invertebrate Pathology and Microbial Control & IVth International Conference on Bacillus thuringiensis, Supporo, August, 1998, Supporo, Japan. pp116

Lightner, D.V. (1998). Diseases of cultured penaeid shrimp and prawns. In C.J. Sindermann and D.V. Lightner (eds), Disease Diagnosis and Control in North American Marine Aquaculture, 2nd ed Elsevier, New York. Pp. 8-127. Lightner, D.V. and R.M. Redman. 1985. A parvo-like virus disease of the penaeid shrimp. J. Invertebr. Pathol. 45:47-53 Lightner, D.V. and R.M. Redman and T.A Bell. 1983. Observation on the geographic distribution, pathogenesis and morphology of the baculovirus from Penaeus monodon Fabricius. Aquaculture 32:209-233. Nash, G.A., A. Arkarajamon and B. Withyachumnarnkul. 1992. Routine and rapid diagnosis of yellow-head disease in Penaeus monodon. Asian Shrimp News. 12 (4): 2-3. Nash, G.A., C. Nithimathachoke, C. Tungmandi, A. Arkarjamorn, P. Prathanpipat and P. Ruamthaveesub. 1992. Vibriosis and its control in pond-reared Penaeus monodon in Thailand. In: M. shariff, R.P. Subasinghe and J.R. Arthur (eds), Diseases in Asian Aquaculture I, Fish Health Section, Asian Fisheries Society, Manila, Philippines. Pp. 143155. Ruangpan, L. and T. Kitao. (1991). Vibrio bacteria isolated from black tiger shrimp, Penaeus monodon Fabricus. J. Fish Dis. 14:383-388. Wongteerasupaya, C., J.E. Vickers, S. Sriurairatana, G.L. Nash, A. Anutara, V. Boonsaeng, S. Panyim, A. Tassanakajon, B. Withyachumnarnkul and T.W. Flegel. 1995a. A non-occuleded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and cause high mortality in Penaeus monodon. Dis. Aquat. Org. 21:69-77. Wongteerasupaya, C., S. Sriurairatana, J.E. Vickers, A. Anutara, V. Boonsaeng, S. Panyim, A. Tassanakajon, B. Withyachumnarnkul and T.W. Flegel. 1995b. Yellow-head virus of Penaeus monodon is an RNA virus. Dis. Aquat. Org. 22:45-50. Wongteerasupaya, C., S. Wongwisansri, V. Boonsaeng, S. Punyim, P. Pratanpipat, G.L. Nash, B. Withyachumnarnkul and T.W. Flegel. 1996. DNA fragment of Penaeus monodon baculovirus PmNOBII gives positive in situ hybridzation with viral infection in 6 penaeid shrimp specied. Aquaculture 143:23-32.

CRITERIA FOR SELECTION OF OFFSPRING I. MORPHOLOGY 1. Size Uniform size post-larvae coming from single complete spawning (preferably first and second spawning) and large clutch of eggs (250,000 500,000 eggs/spawner) appear to perform well in ponds. Postlarval stage between PL15-18 is preferred for stocking. Selection for postlarval length and depth does not directly appraise for soundness but only indicate state of nutrition of larvae. Number of rostral spines indicates age of the postlarvae (4 to 6 spined-PL15-20). 2. Survival Rate Select postlarvae from batch with good survival rate. AN SR benchmark of should serve as standard. 3. Color This criterion has often been used, as a measure of quality. However, there is very little information available regarding the effect of molt stage and color. The presence of pigment cells in the uropods, which gives the tail an open appearance, is a useful indication of the stage of development. If the upropods are not pigmented which may make the tail appear closed, then the postlarvae are not sufficiently developed for stocking. 4. Sixth Abdominal Segment (Gut: Muscle Ratio) The thickness of the gut should be about one quarter of the thickness of the muscle > 3:1. Consider trunk muscle to fill the carapace. However, this will not be the case in normal shrimp just after molting. 5. Pathognomonic Lesions In assessing the state of health of the postlarvae, consider the following typical external lesions as indicating poor quality: 1. External fouling with protozoa or bacteria. The presence of these organisms indicates poor water quality and larvae have irregular molt cycle. It is important to check both moribund and active animal for the degree of fouling. 2. Cuticle black spot 3. Postlarvae with larval mycosis (demonstration of hyphae, discharge tube and motile zoospores). > 40% or better

4. Appendage or body deformities 5. Opacity of trunk muscle 6. Reddish discoloration 7. Whitish line along the dorsal part of the trunk exoskeleton 8. Monodon Baculovirus occlusion bodies (MBV) 9. Presumptive diagnosis for Vibrio spp. Infection (examined under the microscope) a. bacterial plaques in oral region. b. appearance of white balls in the gut usually made up of sloughed hepatopancrease and midgut caecal cells. c. appearance of black balls in the gut made up of poorly digested algae. d. melanized appendages tips or foci. e. motile rod-shaped bacteria in hemocoel. 6. Normal Appearance 1. 2. 3. 4. II. 1. Formalin Postlarvae before stocking in grow-out pond are selected by exposing animal to 150 ppm, formalin for 30 minutes. This is used to cull weak animal for WSSV. This technique was successful in reducing the number of infected PL stocked into ponds (Limsuwan, 1997a). Active feeding behavior. Full gut larvae. Swim with straight body. Ability to swimp upside down at water surface Consider lipid levels in the hepatopancreas (take average numerical assessment). STRESS TEST

2. Salinity Shock The survival of a shrimp sample following rapid immerson for a 15 minutes period in freshwater is commonly applied stress test to determine postlarval fitness. In this test, postlarvae in good health have high survival after returned into normal seawater for another 15 minutes. 3. Starvation A 50 PL/2 liter starved for a 1 day period and showing good survival at a flat water bowl without aeration. 4. Activity and Behavior Healthy postlarvae swim with straight bodies and respond rapidly to external stimuli. Good PL actively swim when the water is stirred and cling to the sides rather than swept into the center of the container. Unhealthy post larvae appear lethargic and unresponsive to external stimuli. They usually swim with arched bodies. 5. Stress Hormone Test Some authors have tried using stress hormone such as corticosteroid to assess response of animal to induce stress using chemical. The test is experimental and would need further documentation to verify its usefulness.

IDENTIFICATION OF DISEASE I. VIRUS A. Wet mounts To check directly for the presence of occlusion bodies due to MBV in postlarvae, representatives sample of PLs hepatopancreas are teased out from the shrimp head in a clean slide. A drop of 0.01% Malachite green is added on the slide and the tissue is squashed using a coverslip. Examine under the microscope for presence of the globular, single or multiple and stained green Baculovirus occlusion bodies. Also, hypertrophied hepatopancreas cells nuclei indicate presence of the virus. Positive diagnosis can only be confirmed in PLs in 13th and above stage group. For hemolymph samples, looking at fresh hemocytes morphology under the microscope can do presumptive diagnosis for Yellow-head virus (YHV). Using phase contrast microscopy (at 40X-100X objectives) observe for hemocytes showing pyknosis and/or possible perinuclear inclusion bodies. Alternatively, hemolymph smear can be stained with Wright-Giemsa and observed for different stages of cell necrosis. Before staining, collect hemolymph using syringe pre-filled with 0.5 ml. of cool 10% seawater formalin. Infection of White-spot syndrome virus (WSSV) can be presumptively diagnose using stained (Wright-Giemsa) squashes or impression smears of epithelial and connective tissue of the gills or stomach of shrimp showing hypertrophied or vacuolated nuclei. B. Histology 1. Fixation Most shrimp virus infections can be confirmed by looking for typical cellular abnormalities and these can be demonstrated using routine histology. Fixation of shrimp specimen is extremely important, as inadequate or improper fixation will often lead to misinterpretation of the sectioned material. The relatively impervious chitinous exoskeleton of shrimp does not allow for adequate fixative penetration by simple immersion, except in larvae and early postlarvae. Also, certain shrimp tissue autolyze more rapidly than comparable tissue types in other animals. Davidsons AFA fixative best fits the requirement for proper fixation of the shrimp specimen. A new RNA-friendly Davidsons fixative has been formulated to preserve integrity of cellular lesion due to RNA viruses (Hasson et al. 1997). Timing of fixation is of equal important, live shrimp samples should be fixed immediately and placed in adequate amount of the fixative. Transfer fixed postlarvae after 24 hours to 1 : 10 (W/V) ethyl alcohol for storage. As the juvenile shrimp, samples transferred to 1 : 10 (W/V) ethyl alcohol after 24-72 hours depending on the specimen size.

2.

Selection and Collection of Specimens

Minimize handling to prevent stress induced tissue anomally (i.e. cell hypoxia). Transport live shrimp to laboratory in well-oxygenated container. If the animal will be left for a short period before fixation, provide adequate aeration and water. For the study of presumably diseased shrimp, select those which are moribund, discolored, displaying abnormal behavior and appearance. In disease prevalence estimation, intentional random sampling is preferred. 3. Paraffin Embedding and Sectioning

The paraffin embedding of shrimp tissue differ little from that of vertebrate tissue. General information concerning embedding procedures can be obtained from the manual of standard histological techniques. As for the tissue sectioning, procedure also follows routine vertebrate tissue sectioning. 4. Staining Routine histological preparations employ standard Hematoxylin and Eosin staining (H&E). For special staining to show viral effects in tissue, the following staining techniques can be used: a. b. Wolbachs Giemsa Method to show viral inclusions

Feulgen Method to demonstrate viral DNA C. Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) is a procedure, which amplifies small target amounts of DNA. This is accomplished by using specific primers designed for the target DNA sequence. When DNA sequence information is known for specific penaeid shrimp viruses, primers can be synthesized to target specific viruses. Target shrimp tissues are homogenized and nucleic acid isolated using chemical extraction. The CTAB (2% w/v hexadecyl trimethyl-ammonium bromide, 1.4M NaCl, 20mM EDTA, 100mM TrisHCl pH 7.5 and 0.25% v/v 2-mercaptoethanol) extraction procedure is excellent for isolation of good quality total nucleic acid (TNA), both DNA and RNA. For shrimp, the TNA can be used directly in conjunction with specific probes or primers for the analysis of either DNA or RNA pathogens. The CTAB extract can also be used to obtain a DNA fraction free of RNA if an Rnase digestion is included in the extraction procedure. CTAB extraction is not recommended for the isolation of RNA free of DNA. Use either the TRIZOL (Gibco/BRL) or Qiagen Rneasy spin columns. Likewise, DNA can also be extracted using DNAzol (Gibco/BRL). The commercial nucleic acid extraction reagents come with procedure for proper use of the product.

Shrimp tissues preferred for PCR are as follows (they can prepare fresh, frozen or fixed in 70% ethanol: a. Hemolymph fresh specimen is required. b. Cephalothorax (head) with tail, eyes and carapace removed. For shrimp weighing between 1-5 gm. c. Postlarvae pooled and ground whole. d. Gills, nerve cord, hepatopancreas and lymphoid organ. e. Pleopod biopsy for broodstock (non destructive method). D. In-situ Hybridization

This molecular biological tool may be used to detect viral and other genomic sequences with specific complementary DNA probes. The use of non-radioactive probes labeled with substances like digoxigenin-11-dUTP (DIG) provide a highly specific diagnostic method since any non-specific effects (which may result in false positive diagnosis in a dot blot assay with homogenized tissue samples) can be readily distinguished from specific histological lesions that have reacted with the labelled probe. Tissue samples can either be fixed in Davidsons fixative or gluteraldehyde. The procedure can also be used using frozen tissue section (using cryostat microtome). Tissue section processed for in-situ hybridization procedure is observed under bright field microscopy looking for cellular deposition i.e. dark blue to black precipitate and/or for specific cytopathology labeled by the probe (in situ results description lifted in entirety from Lightner, 1996 A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp): 1. Infectious Hypodermal and Hematopoitec Necrosis Virus (IHHNV) In cells of ectodermal and mesodermal origin (i.e. gills, hematopoietic tissue, nerve cord, etc.), look for Cowdry A intranuclear inclusion bodies (CAIs) which are dark blue or black, as well as other deposits labeled probe associated with cytoplasmic virus masses or infected nuclei that are without pathognostic CAIs. 2. Hepatopancreatic Parvovirus Virus (HPV) In hepatopancreatic tubule epithelial cells, look for generally spherical intranuclear inclusion bodies, which are dark blue or black, as well as other deposits of labeled probe in the target area, which may contain HPV nucleic acid. 3. Monodon baculovirus (MBV) In epithelial cells of the hepatopancreas and midgut, look for labeled nuclei containing pathognostic intranuclear spherical occlusion bodies, as well as other positive areas in the target tissues, which may contain viral nucleic acid.

4. White-spot Syndrome Virus (WSSV) In cuticular epithelial cells and connective tissue cells (as well as in antennal gland epithelium, Lymphoid organ sheath cells and hematopoietic tissue) look for intranuclear inclusion bodies which are dark blue or black and for other deposits of labeled probe associated with cytoplasmic virus masses or infected nuclei without inclusion bodies. E. Immunochemistry

Antigens and/or genomic nucleic acid must be accessible to antibodies in serological diagnostic tests or to genomic probes in diagnostic tests or assays using labeled gene probes. Homogenates of whole shrimp or particular organs are used for this purpose in certain serological and gene probe diagnostic assays as follows: 1. Enzyme Linked Immunosorbant Assay (ELISA) Specific antibodies (monoclonal or poyclonal) may be used as diagnostic reagents to identify specific antigens which are structural (usually protein) components of pathogens or unique proteins produced or induced by them. ELISA method provides a diagnostic strategy which is potentially very rapid, sensitive and adaptable to large numbers of samples in minimal laboratory or field situations. 2. Transfer of Nucleic Acid to Membrane Supports for Hybridization Analysis Involve the adsorption and binding of nucleic acid onto a membrane support prior to hybridization assay. Some of the assays successfully used in diagnosis of specific shrimp viruses are: a. Dot blots-examine unfractionated nucleic acid sample permanently cross-linked to the membrane. It uses small sample volumes as low as 1 microliter ( 1) to test whether the sample contains the target sequence of interest. Crude or purified samples can be examined and the system is applicable for detection of DNA and RNA targets. Dot blot method has the ability to examine large number of samples in a short time and on a small membrane area. This is an ideal method for initial screening of samples and diagnostic screening of samples when the specificity of the probe for the target has been established. Limitation is that it only provide presence/absence information about a sequence. This additional information is gained after blotting nucleic acid fractionated by gel electrophoresis. Southern and Northern blots examine fractionated nucleic acid transferred from a gel after electrophoresis. Southern blots examine DNA, while Northern blots examine RNAs.

b.

To allow the probe to hybridize to the nucleic acid strand during transfer to support membrane, DNA is denatured in alkaline solution to disrupt the H+ bonds, while for RNA, it is transferred in high salt solution buffered to neutrality with denaturing agent.

II. BACTERIA A. Gram Staining This method is invaluable in demonstration of staining characteristic of the bacteria of concern. Gram negative bacteria are the primary concern in shrimp culture, mostly belonging to Vibrio spp. B. Standard Culture Method, Identification by Classical Method and Rapid Test Kits Marine agar (Difco-Zobells) is the preferred general agar medium to obtain the greatest number and most variety of marine organisms present in the sample. Alternatively, the following general purpose media containing 2 to 2.5% NaCl can also be used: a. Tryptic Soya Agar (TSA) b. Beef Heart Infusion Agar (BHIA) c. Nutrient Agar (NA) These culture media will be used for primary isolation and purification of the bacteria. Likewise, Triple Citrate Bile Salts (TCBS) agar selective for Vibrio spp. can be used to make tentative diagnosis to involvement of potentially pathogenic Vibrio spp. to bacterial infection. Obtaining Samples for Microbiological Diagnosis a. Nauplii, larvae and postlarvae

Use the whole animal after rinsing in sterile seawater or 2.5% NaCl saline solution. Pooled animal are homogenize, dilution made and streak on agar plates. Juveniles Do surface disinfection (1% calcium hypochlorite, 1-2% povidone iodine and 70% ethyl alcohol) of the shrimp samples. Rinse in sterile seawater or 2.5% NaCl saline solution. Target tissue excise using flame sterilized dissecting tools and isolation made as follows:

i. ii.

Systemic infections: excise a block of abdominal muscle or the heart, touch it to the surface of the agar plate, steak and incubate Enteric infections: excise the hepatopancreas, midgut and foregut and touch the exposed inner surfaces or contents of the excised organ to the surface of the agar plate, streak and incubate

c.

Sub-adult and adult i. Preferred sample is the hemolymph. This can be removed either by using a syringe or cutting the tail (if animal is to be sacrificed). Place a drop of the hemolymph onto the agar plate and streak with sterile loop. If needed, dilutions can be made. C. Culture and General Tests

1. Check plates at 12 to 18 hours for luminescent colonies as luminescence tends to fade within 24 hours after incubation 2. Purification is made in suitable media and primary identification can be employed in 24-hour culture using the following identification strategy: Rapid Identification Test Kits Biolog, API NFT strips b. Classic methods employing salt tolerance, biochemical reactions, fermentation of selected carbohydrates, growth using selected compounds as sole carbon sources

Antibiotic sensitivity of the bacteria D. Polymerase Chain Reaction PCR-based application in shrimp bacteriology has not fully defined the difference of potential pathogenic bacteria to identical environmental isolates. The presence of the bacterium, even when demonstrated by a technique of high validity, i.e. PCR, does not guarantee association of the organism to precipitation of disease condition. Hiney and Smith (1997) reiterate the importance of establishing first the degree of correlation between the presence of a bacterium and the incidence of disease. This can only be accomplished through predictive validation. However, this approach is large and multi-centered and required long term investigation. It is argued that systematic performance of cheaper and more rapid laboratory-based validation experiments can play an important role in eliminating techniques that have little or no validity for any proposed environmental application.

E. Immunochemistry 1. ELISA based assays is available only for identification of pathogenic Vibrio penicida affecting the Kuruma shrimp, Penaeus japonicus 2. A commercial DIG-labeled DNA probe for Nectrotizing Hepatopancreatitis (NHP) affecting North American cultured penaeid species is available through DiagXotics. This is on in situ or dot blot hybridization assay formats.

REFERENCES Brock JA and KL Main 1994. A Guide to the common problems and diseases of cultured Penaeus vannamei. The Oceanic Institute, Hawaii. Pp. 242 Chanratchakool P, JF Turnbull, S Funge-Smith and C. Limsuwan 1995. Health management in shrimp ponds-Second Edition. Aquatic Animal Research Institute, Thailand. Pp. 111 Hasson KW, J Hasson, H. Aubert, Rm Redman and DV Lightner 1997. A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes. J. Virol. Mthds. 66:227-236 Hiney MP and PR Smith 1998. Validation of polymerase chain reaction-based techniques for proxy detection of bacterial fish pathogens: Framework, problems and possible solutions for environmental applications. Aquaculture 162: 41-68 Lightner DV 1996. A Handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. The World Aquaculture Society.