Domestication and Selective Breeding of Penaeus monodon in Thailand

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1 Domestication and Selective Breeding of Penaeus monodon in Thailand 1 Boonsirm Withyachumnarnkul, 2 Vichai Boonsaeng, 3 T.W. Flegel 2 Sakol Panyim, 4 Chainarong Wongteerasupaya Departments of 1 Anatomy, 2 Biochemistry and 3 Biotechnology, Faculty of Science Mahidol University, Rama 6 Rd.. Bangkok 14, Thailand 4 Department of Biochemistry, Sri Nakarintaraviroj University, Sukumvit Rd., Bangkok, Thailand ABSTRACT: This report describes progress in an ongoing project, Domestication and Selective Breeding of Penaeus monodon in Thailand. Fast-growing and white-spot syndrome virus (WSSV)-free shrimp were selected from commercial grow-out ponds. They were reared for one year to become F pond-reared broodstock which were then bred to produce F 1 offspring. These F 1 offspring were screened to be free of WSSV before being reared onward for another year to become F 1 broodstock. From these broodstock the F 2 offspring presently comprise nine full-sibling families. Fecundity of the F 1 broodstock was lower than, but approaching that of wild, captured broodstock. The improvement was sufficient to make commercial scale production of postlarvae from these broodstock foreseeable. KEY WORDS: Penaeus monodon, breeding, domestication INTRODUCTION Farmed production of P. monodon in Thailand steadily increased for eight years prior to the years during which time there was a leveling-off and even a slight drop in production. Crop loss has been due to several factors, but the most prominent cause has been outbreaks of yellow-head disease (YHD) and white spot syndrome (WSS) disease (Flegel et al. 1997). To a lesser degree, luminescent bacterial infections have contributed to the declining production. White-spot syndrome virus (WSSV), commonly called systemic ectodermal and mesodermal baculovirus (SEMBV) (Wongteerasapaya, et al. 1995) in Thailand, is the infectious agent of WSS, and it can be detected using polymerase chain reaction (PCR) assays (Wongwisansri 1996; Lo et al. 1996). Checking for sub-clinical WSSV infections of by PCR has been a common practice in Thailand to help farmers in screening out WSSV +ve PL before stocking. PLs that are positive by the PCR assay generally appear healthy until one or two months after stocking in earthen ponds, at which time 8% of the ponds develop clinical whitespot disease (Withyachumnarnkul, unpublished data). This finding, and that by Lo et al. (1997) who found WSSV in the ovaries of captured broodstock, leaves little doubt that the disease can be vertically transmitted. A survey currently underway in Thailand by the Shrimp Culture Research Center, Charoen Pokphand Group of Companies and the Thai Department of Fisheries (DOF), suggests that WSSV infections are widespread among wild broodstock. In addition to the disease problem, a decline in the growth rate of shrimp produced from currently available wild broodstock has also been observed. Obviously, the use of wild broodstock as the only source of PL is not wise. As in farming of other agricultural species, domestication is a better alternative; better because it allows for stricter disease prevention and control, and better because genetic selection may also be achieved. In most cases as time passes, domestication also appears to enhance animal fecundity automatically. In shrimp farming, genetic selection for fast-growing and disease-resistant traits would be most desirable, as both would decrease the risk of loss due to infections. A good example is that of P. vannamei domestication. Domesticated stocks of this economic species of South, Central and North America have been available for more than five years. They are free of the major viral diseases that affect the species and especially infectious hypodermal and haematopoietic necrotic virus (IHHNV) which was formerly a serious threat to the industry. A selective breeding program for improved growth and disease resistance is underway. The advent of specific pathogen-free (SPF) or high-health stocks of P. vannamei could be considered a breakthrough, since farmers using them enjoyed more than doubled production during the interval (Wyban et al. 1992). This SPF stock, however, turned out to be more sensitive than wild shrimp stocks to Taura syndrome virus (TSV)when it struck several shrimp farms during (Wyban 1997). Thus, development of TSV-resistant, as well as fast-growing stocks of P. vannamei is being attempted (Carr et al. 1997). This report describes progress to date in a 6 year-project aimed at the domestication of P. monodon for the economical production of SPF broodstock and PL. The project will Withyachumnarnkul B, Boonsaeng V, Flegel TW Panyim S, Wongteerasupaya C (1998) Domestication and selective breeding of Penaeus monodon in Thailand. In Flegel TW (ed) Advances in shrimp biotechnology. National Center for Genetic Engineering and Biotechnology, Bangkok.

2 74 Withyachumnarnkul et al. lead into the development of SPR stocks and improved growth performance through selective breeding. MATERIALS AND METHODS Selection of F stocks In successful P. monodon culture in normal Thai earthen ponds, shrimp production is in the order of 6 tons per hectare. The survival rate is usually 5% or more, and the standard target body weight (BW) at harvest after 4 months of cultivation is approximately 3 g average per piece. However, in many harvests, it is quite usual to find shrimp of ~65 g BW or higher. It is not yet known whether these fast-growing individuals result from genetic variation or other factors (an area of some interest in itself). Whatever the reason for their existence, these shrimp were considered to be good candidates for fast-growing stock in a selective breeding program. Thus, they were selected from a number of different ponds to constitute the F generation. These fast-growing shrimp were screened for the presence of pathogens by histological examination and PCR. One percent (~2 pcs) of the shrimp was sacrificed for routine histological examination by light microscopy and WSSV- PCR. Any evidence for the presence of WSSV infection by either histology or PCR in any of the tested shrimp from a pond was deemed sufficient grounds to reject all individuals from that pond for selection. The fast-growing shrimp that passed these screening tests were stocked in 16 m 2 ponds (i.e., one rai in Thai land measure) in a quarantine area. Stocking density was 4-8 pc/ m 2 with males and females at the ratio of 1:3. Study in P. japonicus suggests that male shrimp produce pheromones that induce sexual maturation in females (Yano 1993). The quarantine system was designed and constructed to prevent the introduction of diseases, especially of viral origin. Stocking of F 1 and F 2 When the female F shrimp reached 15 g in BW and male 1 g in BW, they were transferred to maturation tanks in the hatchery. Breeding was performed by artificial insemination. The offspring (F 1 ) were reared for one year and then bred to produce F 2 offspring in the same manner. Spawning rate, fertilization rate, number of eggs per spawner and hatching rate were determined. The larvae from individual females were reared separately from nauplius to PL15. Each crop of PL15 was again screened for the presence of WSSV, by PCR and routine histology. If negative by this screen, they were stocked at a density of pcs/m 2 in another separate quarantine area. RESULTS The 1% top-sized shrimp taken from commercial ponds weighed around 5-7 g. These were fast-growing individuals, a typical size distribution curve is shown in Figure 1. After being reared for 8 months in quarantine, they grew to the range of g BW for females and 8-11 g BW for males. Survival rate during this 8 month rearing period was only 3%. After eyestalk ablation, 2% achieved ovarian maturation, reached Stage IV, and spawned. The remaining females either did not show any ovarian development or developed to Stage II or Stage III, but then regressed. From the spawners, the average number of eggs was approximately 3, per individual and the hatching rate was approximately 3%. The nauplii were subsequently reared in the hatchery to PL15 with an average survival rate of 3%. Therefore, PL15 were produced at the rate of approximately 3, pcs per spawner. The fry that passed the WSSV screen were stocked in four 16 m 2 earthen ponds (one family per pond) where they were reared for four months before being harvested. The ADG was , which was better than that of the Number of Shrimp Growth Distribution Curve Penaeus monodon Farm PondNo. Age... days Source of Fry... Number of Parents... Date of Survey... % SR... Total Number Sampled 463 Means 37 g Standard Deviation 13 Take 65 g+. grdist in D Body Weight (g) Figure 1. A typical histogram showing distribution of body weights of P. monodon at harvest.

3 T o t a l L e n g t h ( i n ) Domestication and breeding of Penaeus monodon Hatching Rate (%) 1,, 8, 6, 4, 2, Number of Eggs/Spawner Number of Nauplii/Spawner Fertilization Rate (%) Survival Rate From Nauplius to PL14 (%) Figure 2. Data on the size, fecundity and offspring of the pond-reared F 1 spawners. The values are means ± S.E. parent stock ( ). The 1% top-sized shrimp from these ponds were selected and reared on for a further 8 months. These shrimp were designated the F 1 generation and they were used for breeding as described previously. This time, the spawning rate was 5% (i.e., half of the eyestalk-ablated females became spawners). Figure 2 shows the average size of the F 1 broodstock, and other parameters related to fecundity and survival rate of the F 2 PLs. The length of the F 1 spawners, measured from the base of the eyestalk to the end of the telson, was 1 inches and the average BW was 14 g. This size was comparable to that of wild broodstock. The number of eggs per spawner was higher than for the parental stock (approximately 7, eggs per spawner), and only slightly lower than the average 8, obtained from wild broodstock. Some of the F 2 broodstock produced more than one million eggs, and most spawned at least twice. The fertilization rate was variable, ranging from % to 95% and averaging approximately 45%. The hatching rate was also variable and averaged at 38%. The average number of nauplii produced was 3, pcs per spawner. As the survival rate from nauplius to PL14 was slightly over 3%, the production of PL14 was approximately 1, pcs per spawner. The F 2 generation of pond-reared broodstock, consisting of nine full-sibling families, is now contained in a strict quarantine area. At the time of writing, the days of culture ranged from 6 to 15 days. They have been screened and found to be SEMBV free for two generations. Further screening for MBV, HPV and other viruses is under way. DISCUSSION Table 1 compares the performance of pond-reared P. monodon broodstock for the different generations studied. There is a trend of increasing fecundity, most notably for the spawning rate and the number of eggs per spawner. Millamena et al. (1986) reported approximately 3% spawning rate. The average number of eggs per spawner ranged from 12, to 333, and the hatching rate ranged from 4% to 36%. Menasveta et al. (1991) tried to improve the reproductive performance of pond-reared broodstock in a re-circulating water system and obtained slightly improved fecundity. San Miguel Foods, Inc. in the Philippines recently reported successful breeding of four generations of P. monodon pond-reared broodstock from 1991 to 1995 (Castro et al. 1997). The average number of eggs per spawner was improved up to 587, with a 42.7% hatching rate. The improvement in fecundity seen here was probably due to improvements in nutrition and in the environment in which the broodstock were kept. Fresh, natural feeds containing high amounts of poly-unsaturated fatty acids (PUFAs), asthaxantin and cholesterol, were used. PUFAs like 2:4n6, 2:5n3 and 226n3 are known to be essential for ovarian development (Shimma 1977; Millamena et al. 1985) and codliver oil, blood worms (Pereneresis nunta vallata), squid and other marine species contain high amounts of these fatty acids (Millamena et al. 1986). In addition to these long-chained fatty acids, certain shorter chain fatty acids like linolenic acid (C18) have also been shown to enhance ovarian maturation in P. japonicus (Kanazawa et al. 1979). Table 1. Comparison of the performance of pond-reared and wild broodstock. Wild Pond-Reared Broodstock Broodstock Millamena et al. Menasveta et al. San Miguel SCRD (F1 data) Size of the Female (g) Spawning Rate (%) >8% ~3%? ~5% No. of Eggs/Spawner >8, ~2, ~6, ~7, Hatching Rate (%) ~8% ~2% ~4% ~4% Breeding Generation - F4 F2

4 76 Withyachumnarnkul et al. Water depth, salinity, temperature, photoperiod and light intensity may also play roles in shirmp maturation (Quinitio et al. 1993; Yano 1993). Light-dark cycle is an important factor in controlling reproductive function of several species. In mammals, for example, the environmental light message is conveyed to internal organs via the pineal gland (for review, see Reiter 1991). Crustaceans lack a pineal gland, but such messages might be carried through the optic lobe in the eyestalk, which contains a pineal-equivalent structure. The effects of photoperiod on the optic lobe biochemistry of the freshwater crustacean, Macrobrachium rosenbergii de Man, and the black tiger prawn, P. monodon, have been reported (Withyachumnarnkul et al.199, 1992, 1995). Yano (1993) reported that a light:dark ratio of 14:1 h, with an intensity of 3-5 lux was optimum for the promotion of ovarian growth of P. japonicus in maturation tanks. The finding that the F1 generation grew 1% faster than the parental stock suggests the possibility of inheritance for growth performance of P. monodon. However, more data are needed to draw definite conclusions, as the environment also plays an important role in growth. This work showed an improvement in the performance of the pond-reared broodstock, with an averaged production of 1, PL15 per broodstock individual. This figure is about one-third of that for wild broodstock. Although lower, production at this rate is commercially viable. Moreover, the fry produced from pond-reared broodstock are healthier (i.e., WSSV-free). In the future, it is hoped that genetic improvement and better culture performance of the fry will compensate for lower production numbers. Acknowledgments. This work was supported by the Shrimp Culture Research and Development, Inc. (SCRD), Thailand and the National Fisheries Institute, USA. 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