Aquaculture (2012) Contents lists available at SciVerse ScienceDirect. Aquaculture

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1 Aquaculture (2012) 7 13 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: Triploid Penaeus monodon: Sex ratio and growth rate Pattira Pongtippatee a,, Karemah Laburee a, Pinij Thaweethamsewee c, Ratana Hiranphan c, Somluk Asuvapongpatana d, Wattana Weerachatyanukul d, Theera Srisawat b, Boonsirm Withyachumnarnkul d,e,f, a Aquatic Animal Biotechnology Research Center, Surat Thani Campus, Surat Thani 84100, Thailand b Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, Surat Thani 84100, Thailand c Faculty of Science, Prince of Songkla University, Hat-Yai, Songkla 90112, Thailand d Department of Anatomy, Mahidol University, Bangkok, 10400, Thailand e Center of Excellence for Shrimp Biotechnology and Molecular Biology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, 10400, Thailand f Shrimp Genetic Improvement Center, Center of Genetic Engineering and Biotechnology (Biotec), Surat Thani 84100, Thailand article info abstract Article history: Received 13 October 2011 Received in revised form 20 May 2012 Accepted 8 June 2012 Available online 17 June 2012 Keywords: Penaeus monodon Chromosome manipulation Triploid Sex ratio Cold shock PBII triploidy induction This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon. Chemical (cytochalasine-b and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8 C, for 10 min at 8 min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2±3.2 g) and triploid males (31.5±3.5 g) was significantly higher (Pb ) than that of their diploid counterparts (24.5±0.5 g for females and 23.1±3.8 g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production Elsevier B.V. All rights reserved. 1. Introduction The purpose of inducing polyploidy in aquaculture species is to achieve high growth rates of individual polyploid animals. This induction leads to higher production per crop, when compared to raising normal diploid ones. Additionally, these findings may also help deter extended breeding programs exploited by non-patent breeders, since polyploid animals are likely to be sterile (Han et al., 2010). There are serious environmental concerns regarding any cultured species that escape and find their way into natural waterways. Polyploid cultured species would not risk spreading hatchery-born species into the wild, and thus would not upset any ecological balance. This risk is particularly of concern whenever exotic species are introduced into countries where the species have not existed naturally. Commercial production of polyploid fish and oysters is currently taking place mainly because of their Corresponding author. Tel./fax: Correspondence to: B. Withyachumnarnkul, Department of Anatomy, Mahidol University, Bangkok, 10400, Thailand. Tel.: ; fax: addresses: pattirataweepreda@yahoo.com (P. Pongtippatee), wboonsirm@yahoo.com (B. Withyachumnarnkul). larger size and their growth rates are not periodically interrupted by their reproductive activities, resulting in higher productions (Allen and Downing, 1986; Arai, 2001; Guo and Allen, 1994; Thorgaard, 1983). Although triploid induction has been successful in several fish species, its impact on commercial production has been limited (Hulata, 2001). This is probably due to the lack of a clear-cut advantage of triploids over diploids on a commercial scale. In shrimp, triploid induction has been carried out in red-legged banana shrimp Penaeus (Fenneropenaeus) indicus, white-legged shrimp Penaeus (Litopenaeus) vannamei, Chinese shrimp Penaeus (Fenneropenaeus) chinensis, Kuruma shrimp Penaeus (Marsupenaeus) japonicus, and ridgeback shrimp Sicyonia ingentis (see review by Sellars et al., 2010). However, not all polyploid shrimp have advantages over diploid ones. Some species such as P. vannamei, do not survive well, while others such as P. japonicus, grow at the same rate as their diploid counterparts. Therefore, any possible commercial application for triploid induction involving these species is problematic at best. The most extensively studied shrimp species that have had triploid induction is P. chinensis, in which its triploid individuals grow faster than the diploid ones. Unfortunately, this increase in growth rate occurs only during the adult stage. Thus, its impact on commercial production is also still questionable /$ see front matter 2012 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 8 P. Pongtippatee et al. / Aquaculture (2012) 7 13 In order to have a significant impact on commercial scale production, triploid shrimp must grow faster than their diploid counterparts during a period from postlarva to marketing size. The period usually falls within 3 6 months after hatching when the shrimp reach g size. Survival rates of the triploid shrimp should also be comparable to that of the diploid individuals. Thus far, these two major obstacles have not been overcome in any commercial farmed shrimp production. Another shrimp species that challenges researchers and aquaculturists is the black tiger shrimp Penaeus monodon. Wood et al. (2011) described a successful triploid induction in this species. However, this report provided only its success on the induction methods and studies up to the nauplius, but not to the juvenile, stage. Sellars et al. (2012) extended the study by rearing 6-dimethylaminopurine (6-DMAP)-induced triploid P. monodon to marketing size, or postlarva 184 stage. However, they found that, in terms of growth rate, triploid P. monodon had no advantage over the diploid ones. Triploidy in shrimp species can be induced by inhibiting the extrusion of the first (PBI), or the second polar body (PBII); using temperature, pressure, or chemical shock (see review by Sellars et al., 2010). Success of any particular method seems to be species-dependent. For instance, heat shock was successful in P. indicus and P. chinensis; while chemical shock, using 6-DMAP, was successful in P. japonicus, and cold shock was successful in P. vannamei. Triploid induction rate was in the range of 70 90% in P. chinensis (Li et al., 2003a), P. vannamei (Garnica-Rivera et al., 2004) and P. japonicus (Coman et al., 2008). Another feature of triploid shrimp that is of particular concern is sex ratio, since females grow faster than males and thus having higher ratio of females to males would result in higher production. Most studies revealed that in triploid shrimp the sex skews toward being female; as the triploid P. japonicus populations were almost 100% females (Sellars et al., 2009) and triploid P. chinensis has the ratio of 4 females:1 male (Li et al., 2003b). However, in a study by Sellars et al. (2012), in triploid P. monodon, sex ratio was 1 female:1.625 males. Since there are concerns regarding commercial applications, we have produced triploid P. monodon by using cold shock, after testing its efficiency against other known methods. In addition, we have cultured the shrimp from larvae, through juvenile, and finally to adult stages, comparing its growth rate and sex ratio with their diploid counterparts. Our results suggest that triploid P. monodon is commercially feasible. 2. Materials and methods 2.1. Trials of PBII triploidy induction methods The Shrimp Genetic Improvement Center (SGIC), a Thai government agency, has been rearing domesticated specific pathogen-free P. monodon and operating a selective breeding program since In this study, all the animals were from SGIC and the experiments were performed in SGIC facilities. Female P. monodon broodstocks at SGIC were allowed to spawn in a plastic tank containing 200 l of 28 C clean seawater. After complete spawning (taking 3 7 min), the females were removed from the tank, and the water in the tank was circulated gently by plastic paddle to allow the fertilized eggs to fall into the center of the bottom of the tank. The eggs were then gently collected by siphoning out from the bottom of the tank 8 min after the beginning of spawning activity. Next, the eggs were subjected to shock treatments. The extrusion of the PBI happens 5 min post-spawning, and that of PBII 15 min post-spawning (Pongtippatee-Taweepreda et al., 2004). The reason for allowing 8 min to elapse before applying the shock was to allow as many eggs as possible to receive the shock during the 5 to 15 minute period post-spawning. Thus, the first spawned egg would likely receive the shock at the appropriate time. However, the last egg might not be shocked at the right time if the spawning process exceeded 7 min. If it took 7 min, as described earlier (Pongtippatee-Taweepreda et al., 2004), then the last-spawned egg would still be shocked at the right time. Therefore, this 8 minute post-spawning shock regimen was selected so that 100% of the eggs could be cold shocked at the appropriate time. The broodstocks and their individual sets of eggs were divided into five groups (approximately 10,000 eggs per group), for cytochalasine B (CCB) shock, 6-DMAP shock, cold shock, heat shock and no treatment (control). This procedure was repeated 8 times for 8 separate broodstocks Chemical shocks Cytochalasine-B (CCB, Sigma Inc., St. Louis, MO), 1.0 mg, dissolved in 1.0 ml dimethyl sulfoxide (DMSO) was added to 1-l filtered seawater, giving a final concentration of 1 μg/ml CCB and 0.1% DMSO. The eggs were quickly submerged in prepared CCB seawater for 10 min, and were provided with fresh seawater after that before being placed in a tank containing 200 l clean seawater at 28 C. An identical procedure was carried out for 6-DMAP (Sigma, St. Louis, MO) using a concentration of 100 μm in DMSO (Norris et al., 2005) Temperature shocks Cold shock was applied at 8 C for 10 min, and heat shock was applied at 41 C for 10 min. The shocks were applied by placing eggs taken from the spawning tank (28 C) and placed into a 500 ml beaker containing seawater at a specific temperature. The beaker was then quickly placed in a thermo-regulated shaking water bath, at 60 rpm. After 10 min of treatment, the eggs were quickly poured from the beaker into a tank containing 200 l clean seawater at 28 C. The no-treatment control was processed in the same manner, but at 28 C Observation of PBII extrusion and pronuclei Eggs of all the experimental treatments were sampled immediately after treatment in order to observe PBII extrusion blocking. They were sampled again at min afterward, in order to observe the presence of pronuclei during syngamy. Approximately 500 eggs from each treatment were fixed in 95% ethanol and stained with Hoechst (Pongtippatee et al., 2010). Hoechst (0.005%) solution containing thimerosal (0.1%), was filtered, diluted (1:200), and used to stain fixed eggs (10 min in 98% acetic acid and absolute methanol at 1:3 v/v) at dark room temperature. The slides were airdried, mounted, and observed under a fluorescent microscope. The percentage of prospective triploidy was assessed by counting the number of eggs with PBII extrusion blocking, while having the appearance of 3 pronuclei during syngamy. These eggs were then compared with the total number of eggs Cold shock for triploid induction and raising triploid/diploid shrimp Results from the trials revealed that cold shock treatments achieved the highest percentages of blocked PBII extrusion and the formation of three pronuclei. Therefore, only cold shock was applied for triploid induction. Eggs from each of 24 spawnings were divided into two groups (each of approximately 10,000 per spawning); with one subjected to cold shock at 8 min post-spawning at 8 C, for 10 min, and the other (control) was not. The eggs from each spawning were incubated (28 C) with gentle stirring in 200-l plastic tanks under a stocking density of 100 eggs/l of seawater. Fertilization (FR) and hatching (HR) rates were determined at approximately 6 h and 12 h post-spawning, respectively. After hatching, the nauplii (approximately 5000) from each spawning were raised separately in l plastic tanks, at the stocking density of 50 nauplii/l of seawater, to postlarval 15 (PL 15) stages. Survival rate from nauplius to PL 15 (approximately 23 days) was then determined. After that, all the PLs (approximately 35,000) were

3 P. Pongtippatee et al. / Aquaculture (2012) pooled and 2500 individuals were randomly sampled and stocked in a 113 t circular plastic pond (12 m diameter at 1 m depth), and raised to juveniles for 150 days. The pond was stocked 20 ppt seawater and operated under zero-water exchange or biofloc system (Avnemelech, 2005; Burford et al., 2003) throughout the culture period. The intention for culturing all the shrimp (triploid, cold-shocked diploid and nontreated diploid) together in the same pond was to avoid any difference in growth rate caused by different environment, which would likely occur if the treated and non-treated shrimp were reared in separate ponds. Water quality was monitored throughout the rearing period in order to ensure the healthiest environment for growth of the shrimp (ph, ; alkalinity ppm; total ammonia nitrogen, b0.1 ppm; total nitrite, b0.1 ppm; dissolved oxygen, >5 ppm). The shrimp were fed with commercial feed from a rate of 10% of biomass (during the first month) to 5% of biomass (during the 1st 2nd month), and 3% of biomass thereafter. At days 82, 96, 110, 124 and 138 in culture, shrimp were randomly sampled by net casting, and body weights (BW) individually determined. No attempt was made to separate the sex at these stages. Chromosome number of each animal in the sample was determined as described below. After obtaining the ploidy level results, the BW of the triploid and diploid (cold-shocked diploid+no-treatment diploid) were averaged and statistically compared. At day 150 in culture, 166 shrimp were randomly sampled, individually weighed, identified for sex and their chromosome number was determined Determination of chromosome number Chromosome number of 166 shrimp was determined by Fluorescence Activating Cell Sorting (FACS) (or flow cytometer), as described by Li et al. (2003b); the shrimp were thereafter divided into diploid (2n) and triploid (3n) individuals. Five shrimp from the 2n and 3n groups were further confirmed for their chromosome numbers by the morphological method described by Lakra et al. (1997). In this method, in order to have a high-quality metaphase spread of the chromosomes, regenerated blastemas of the first pair periopods were produced and used as tissue for processing. Regenerated blastemas were obtained by cutting the first pair of juvenile shrimp periopods, which would regenerate as blastemas to the size of 2 4mm in length within 8 10 days. The blastemas were removed and incubated in a 0.025% colchicine solution (Sigma Chemical Co., St. Louis, MO), and were placed in clean seawater for 8 h at room temperature, in order to block dividing cells at metaphase. Subsequently, the blastemas were treated with hypotonic solution, 0.4% potassium chloride for 27 min at room temperature, in order to separate the metaphase chromosomes. The tissue was then transferred to a Carnoy's fixative (ethanol:acetic acid, 3:1), which were changed every 15 min three times. The fixed tissue measuring 1 mm was then put on a glass slide and stained with carbol fuchsin for 10 min. The slide was then covered with a cover slip and viewed under a light microscope. In the FACS method, 150 μl of hemolymph (approximate hemocytes) was collected from the ventral abdominal sinus using a 25-gauge needle and a 1 ml syringe filled with an equal volume of cold shrimp salt solution (SSS; 450 mm NaCl, 10 mm KCl, 10 mm ethylenediamine-tetra-acetic acid [EDTA], 10 mm N-[2-hydroxyethyl] piperazine-n -2-ethanesulfonic acid [HEPES]); the solution was used as an anticoagulant. The cell suspension was centrifuged at 500 g for 10 min at 4 C. A solution of 290 μl of cold phosphate buffered saline (PBS) was added to the cell pellet and mixed gently. Ten microliters of cold 8% paraformaldehyde was added to make 0.25% final concentration of fixation, and was then incubated for 1 h at 4 C. Cell suspension was centrifuged and a permeabilization solution (Triton X-100) was added to the pellet with a final concentration of 0.1%, which was then incubated for 15 min at 37 C. Cell suspension was centrifuged and cell pellet was resuspended with 150 μl PBS containing 20 μg/ml propidium iodide (PI) and 20 μg/ml ribonuclease A (RNase A) (Sigma Chemical Co., St. Louis, MO), and incubated for 30 min at 4 C. Analysis was performed within 2 h of staining, at which time 150 μl of cell suspension from untreated shrimp was added as an internal standard control, and another 200 μl PBS was added to the sample to make 500 μl. The sample was filtered through a 62 μm nylon mesh to remove any clumps, and was analyzed on a flow cytometer by FACS analysis using a Calibur Flow Cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, California, USA). Shrimp hemocytes were counted at a total of 20,000 cells for each sample. Peak means were obtained by CellQuest software (Becton Dickinson and Company, Franklin Lakes, NJ), and level of ploidy was analyzed by ModFit LT software (Verity Software House, Topsham, Maine, USA) Statistical analysis Numerical data were expressed as a mean±standard deviation. Ploidy levels among differently treated groups and body weights of the shrimp, comparing between both sexes of triploid and diploid shrimp, were analyzed using one-way ANOVA and Student t-test. 3. Results 3.1. Trials of PBII triploidy induction methods The extrusion of PBII was inhibited by both chemical and temperature shocks. In the control eggs stained with Hoechst and then observed under fluorescent microscopes, the PBI and PBII were observed at the periphery of the eggs at 5 and 15 min postspawning, respectively (Fig. 1a, b). In all experimental groups, PBII remained inside the eggs, and two groups of chromosomes were detected in the ooplasm (Fig. 1c f). Following fertilization the control eggs contained two pronuclei (Fig. 2a), while the treatment eggs contained three (Fig. 2b e). In addition, at min post-spawning, the first mitosis had occurred being present in each dividing cell (Fig. 2f). The percentage of eggs with inhibition of PBII extrusion by CCB, 6- DMAP, and heat and cold applications was in the range of 40 60%. There was no statistical difference between them (Table 1). The percentage of triploid induction, as being revealed by the presence of three pronuclei during syngamy, was highest in the cold-shock group (approx. 38%), and was significantly (Pb0.001) different from that of other treatments. However, extreme variation from 0% to 82% was observed. In the heat-shock group, the percentage was about 27%, which was significantly (Pb0.01) higher than that of the CCB and 6-DMAP-treated groups Cold shock for PBII triploidy induction Eggs that had received cold shock underwent embryonic development and hatched h after spawning. This period was similar to that of the typical hatching times for P. monodon under normal conditions. In the control group, FR and HR were close to 60%, while those of the cold-shock group were close to 40%; which was significantly lower (Pb0.05) than the control values (Table 2). High variation among batches of eggs was also observed. Survival from nauplius to postlarval 15 (PL15) stage for the untreated control and cold-shock larvae did not differ significantly (66±18.0% and 65±23.6%, respectively). Results of flow cytometry revealed two sets of hemocytes being clearly separated (Fig. 3). The two methods were used to separate the diploid and triploid status of individual shrimp, which revealed 72 diploid (44 males, 28 females) and 94 triploid (31 males, 63 females) individuals. The chromosome counting by morphological method revealed 88 and 132 chromosome numbers per cell at metaphase for all five diploid and five triploid shrimp, respectively. During the raising of the triploid (successful induction) and diploid (unsuccessful induction plus control untreated) shrimp in the

4 10 P. Pongtippatee et al. / Aquaculture (2012) 7 13 Fig. 1. Fluorescent photomicrograph of the eggs stained with Hoechst 33258, showing PBI (a) and PBII (b) extrusion at 5 min and 15 min post-spawning, respectively, in control eggs. At 8 min post-spawning, eggs treated with CCB (c), 6-DMAP (d), heat shock (e), and cold shock (f) showed no PBII extrusion, resulting in having two groups of chromosomes in the ooplasm (arrow). FN, female nucleus. same pond, BW was determined from both groups from days 82 to 138 in culture. Fig. 4a shows the mean BW of the shrimp; the triploid shrimp had a mean BW significantly higher than that of the diploid shrimp of the same age. At day 150, when the shrimp were further separated into males and females, the mean BW of the triploid females and triploid males were significantly higher than that of their diploid counterparts (Fig. 4b). The sex ratio of the triploid shrimp was 2 females: 1 male, whereas that of the diploid shrimp was 2 females: 3 males. These results suggest that the sex ratio was skewed toward being female. The survival rate of the shrimp, regardless of ploidy status, was 60%, from PL 15 to 150 days of age. 4. Discussion In this study, cold shock was found to be the best method to induce triploidy in P. monodon. Heat shock and the two chemical shocks can also block the extrusion of PBII. However, the eggs failed to have three pronuclei in subsequent development. The reason for this failure is not known, but it is not due to the inhibition of fertilization, since sperm fertilize the egg within 45 s after spawning (Pongtippatee-Taweepreda et al., 2004). The finding that the chemical treatment groups, CCB and 6-DMAP, showed the lowest percentage of having three pronuclei features, suggests that these chemicals were toxic to early developed embryo of the shrimp. As the cold shock resulted in about 60% of eggs undergoing PBII extrusion blocking with 40% of eggs manifesting three pronuclei, it can be stated that the success rate of PBII triploidy induction by cold shock was 40% on average, with high variation among spawners. Using the same shrimp species, Wood et al. (2011) also reported an extreme variable result from 0 to 76.7% induction rate for a cold shock of 9 C for 6 min. After comparisons have been made regarding growth and survival of triploid shrimp, this study suggests that triploid P. monodon may have some advantages over other farmed shrimp species such as P. chinensis, P. vannamei and P. japonicus. TriploidP. chinensis and P. japonicus were reported to have reduced survival rates in early larval stages, and triploid P. chinensis did not grow faster than the diploid ones until they had reached the reproductive stage. In addition, the growth rate of P. japonicus was not significantly different from that of the diploid ones (Li et al., 2006; Sellars et al., 2010). Triploid P. vannamei was also reported to have a low survival rate (Dumas and Campos-Ramos, 1999; Garnica-Rivera et al., 2004). It has been determined that the sex of triploid P. monodon, although being skewed toward females (2 females:1 male), did not have ratios that were as high as in P. chinensis, which had a sex ratio of 4 females:1

5 P. Pongtippatee et al. / Aquaculture (2012) Fig. 2. Fluorescent photomicrograph of the eggs at min post-spawning, with two pronuclei in control group (a) and three pronuclei following CCB (b), 6-DMAP (c), heat (d), and cold (e) shocks, and at 45 min showing the first mitosis of an egg. N, nucleus; PB, polar body; PBI, first polar body; PN, pronuclei. male (Li et al., 2003a). Nor were they as high as in P. japonicus, where 100% females was reported (Coman et al., 2008; Preston et al., 2004). This suggests that triploid induction in P. monodon did not have a strong effect on sex ratio when compared to these other shrimp species. It is more commercially feasible to have a high proportion of female P. monodon, as females are about 20% larger than the males at marketing size (Withyachumnarnkul, unpublished). It should be mentioned that Table 1 Percentage of the eggs with inhibition of the second polar body (PBII) extrusion and with three pronuclei following chemical and temperature shocks. CCB, cytochalasine- B; 6-DMAP, 6-dimethylaminopurine. Treatments Eggs without PBII extrusion Eggs with three pronuclei CCB 58.0± ±1.4 6-DMAP 41.3± ±4.3 Heat (41 C) 53.6± ±7.0 Cold (8 C) 61.4± ±14.6 P b0.01, compared to the CCB and 6-DMAP-treated groups. P b0.001, compared to other groups. not all species that were made triploid have skewed sex ratios. For instance, in the oyster Crassostrea gigas, triploidy did not result in skewed sex ratios (Allen and Downing, 1986). Additionally, in the mussel Mytilus galloprovincialis, triploidy led to ratios that were skewed toward being male (Kiyomoto et al., 1996). The results from our study are quite different from those of Sellars et al. (2012). In their study, triploid induction was by employing 6-DMAP and the shrimp were reared in tanks from PL 10 to PL 184 in controlled environment clear-water tank systems. At PL 184 (equivalent to 169 days of culture in our system), the growth rate of triploid females was the same as that of the triploid male and diploid Table 2 Fertilization and hatching rate (%) of the control and cold-shocked eggs, obtained from 24 spawnings. Fertilization rate Hatching rate Control 57.9 ± ±22.1 Cold shock 43.3 ± ±22.0 Pb0.05, compared to the control value.

6 12 P. Pongtippatee et al. / Aquaculture (2012) 7 13 Fig. 3. Fluorescence Activating Cell Sorting analysis of hemocytes from an individual Penaeus monodon at 150 days in culture that received no treatment (control) showing 2n peak (a) and that received cold shock at its early embryonic stage, with hemocytes of the control shrimp as internal standard showing both 2n and 3n peaks (b). Histological pictures showed chromosome at metaphase from blastema of diploid (2n) and triploid (3n) shrimp. males, and that of the diploid female was significantly higher than that of other groups. The sex ratio in their study was also different from ours, as they showed 1 female:1.625 males. The difference in results may be due to different method of induction and different rearing system from postlarval to harvesting size. This discrepancy should be further investigated. 5. Conclusions The best induction method for obtaining triploid P. monodon was found to be the application of cold shock at 8 min post-spawning at 8 C, for 10 min. Fertilization and hatching rates were lower than that of the control, which was left untreated. In addition, the PBII triploidy induction rate was 38% on average. The nauplii were reared to PL 15 and both the cold-shocked and control nauplii had the same survival rate. The shrimp were further reared from PL 15 to juvenile stage and the triploid shrimp grew faster than the diploid ones, and the sex ratio of the triploid shrimp was found to be 2 females:1 male. Due to the fact that they perform better in culture than normal diploid shrimp, production of triploid P. monodon is commercially feasible. It appears likely that more experimentation will lead to increased fertilization, hatching, and triploidy induction rates. Acknowledgment This study was supported by the Center for Molecular Biology and Biotechnology of the National Science and Technology Development Agency (NSTDA), Thailand.

7 P. Pongtippatee et al. / Aquaculture (2012) Fig. 4. Mean body weights without sex separation, from day 82 to 138 in culture (a) and with sex separation, at day 150 in culture (b) of diploid (2n) and triploid (3n) Penaeus monodon reared in a plastic tank (round, 12 m in diameter, 1 m water depth). In (a), the numbers of samples for diploid shrimp were 15 and for triploid shrimp 10, in all values. In (b), the numbers in parentheses are the numbers of shrimp samples. *Pb0.05, **P b0.01, compared to their 2n counterparts. Appendix A. Supplementary data Supplementary data to this article can be found online at dx.doi.org/ /j.aquaculture References Allen Jr., S.K., Downing, S.L., Performance of triploid Pacific oyster, Crassostrea gigas (Thunberg), part I: survival, growth, glycogen content and sexual maturation in yearlings. Journal of Experimental Marine Biology and Ecology 102, Arai, K., Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture 197, Avnemelech, Y., Using the pond as a biofilter: review of theory and practice. International Journal of Recirculating Aquaculture 6, Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., Nutrients and microbial dynamics in high-density, zero-exchange shrimp ponds in Belize. Aquaculture 219, Coman, F.E., Sellars, M.J., Norris, B.J., Coman, G.J., Preston, N.P., The effects of triploidy on Penaeus (Marsupenaeus) japonicus (Bate) survival, growth and gender when compared to diploid siblings. Aquaculture 276, Dumas, S., Campos-Ramos, R., Triploidy induction in the Pacific white shrimp Litopenaeus vannamei (Boone). Aquaculture Research 30, Garnica-Rivera, C., Arredondo-Figueroa, J.L., Barriga-Sosa, I.L.A., Optimization of triploidy induction in the pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture 16, Guo, X., Allen Jr., S.K., Reproductive potential and genetics of triploid Pacific oysters, Crassostrea gigas (Thunberg). The Biological Bulletin 187, Han, Y., Liu, M., Zhang, L.L., Simpson, B., Zhang, G.X., Comparison of reproductive development in triploid and diploid female rainbow trout Oncorhynchus mykiss. Journal of Fish Biology 76, Hulata, G., Genetic manipulations in aquaculture: a review of stock improvement by classical and modern technologies. Genetica 111, Kiyomoto, M., Komaru, A., Scarpa, J., Wada, K.T., Danton, E., Awaji, M., Abnormal gametogenesis, male dominant sex ratio, and sertoli cell morphology in induced triploid mussels, Mytilus galloprovincialis. Zoological Science 13, Lakra, W.S., Kumar, P., Das, M., Goswami, U., Improved techniques of chromosome preparation from shrimps and prawn. Asian Fisheries Science 10, Li, F., Xiang, J., Zhou, L., Wu, C., Zhang, X., 2003a. Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis. Aquaculture 219, Li, F., Xiang, J., Zhang, X., Zhou, L., Zhang, C., Wu, C., 2003b. Gonad development characteristics and sex ratio in triploid Chinese shrimp (Fenneropenaeus chinensis). Marine Biotechnology 5, Li, F., Zhang, C., Yu, K., Liu, X., Zhang, X., Zhou, L., Xiang, J., Larval metamorphosis and morphological characteristic analysis of triploid shrimp Fenneropenaeus chinensis (Osbeck, 1765). Aquaculture Research 37, Norris, B.J., Coman, F.E., Sellars, M.J., Preston, N.P., Triploid induction in Penaeus japonicus (Bate) with 6-dimethylaminopurine. Aquaculture Research 36, Pongtippatee, P., Luppanakane, R., Thaweethamsewee, P., Kirirat, P., Weerachatyanukul, W., Withyachumnarnkul, B., Delay of the egg activation process in the black tiger shrimp Penaeus monodon by manipulation of magnesium levels in spawning water. Aquaculture Research 41, Pongtippatee-Taweepreda, P., Chavadej, J., Plodpai, P., Pratoomchart, B., Sobhon, P., Weerachatyanukul, W., Withyachumnarnkul, B., Egg activation in the black tiger shrimp Penaeus monodon. Aquaculture 234, Preston, N.P., Sellars, M.J., Coman, F.E., Norris, B.J., Lyons, R., Ploidy manipulation induces sterility in Kuruma prawns. Global Aquaculture Advocate 7, Sellars, M.J., Wood, A., Dixon, T.J., Dierens, L.M., Coman, G.J., A comparison of heterozygosity, sex ratio and production traits in two classes of triploid Penaeus (Marsupenaeus) japonicus (Kuruma shrimp): Polar Body I vs II triploids. Aquaculture 296, Sellars, M.J., Li, F., Preston, N.P., Xiang, J., Penaeid shrimp polyploidy: global status and future direction. Aquaculture 310, 1 7. Sellars, M.J., Wood, A., Murphy, B., McCulloch, R.M., Preston, N.P., Triploid black tiger shrimp (Penaeus monodon) performance from egg to harvest age. Aquaculture ( ). Thorgaard, G.H., Chromosome set manipulation and sex control in fish. In: Hoar, W.S., Randall, D.J., Donalson, E.M. (Eds.), Fish Physiology. Academic Press, London, pp Wood, A.T., Coman, G.J., Foote, A.R., Sellars, M.J., Triploid induction of black tiger shrimp, Penaeus monodon (Fabricius) using cold shock. Aquaculture Research 42 (11),