The effectiveness of heat, cold and 6-dimethylaminopurine shocks for inducing tetraploidy in the Kuruma shrimp, Marsupenaeus japonicus (Bate).

ABSTRACT In this study tetraploid Marsupenaeus japonicus (Bate) embryos were produced by preventing the first division in mitosis. The effectiveness of temperature and chemical shocks for producing tetraploid M. japonicus were assessed when applied at different times postspawning and for different durations. Tetraploid M. japonicus embryos (spawned at 27[degrees]C) were produced by heat shocks at 35[degrees]C and 36[degrees]C in three and eight spawning samples respectively, and a cold shock at 5[degrees]C in a single spawning sample. All temperature shocks inducing tetraploidy were applied 18-23 min postspawning for a 5-10 min duration. The percentage of spawnings successfully inducing tetraploid embryos (i.e., frequency of induction) ranged from 33.33% to 66.67% for the 21, 22 and 23 min postspawning heat shock treatment regimes. The percentage of tetraploid embryos within an induction (i.e., induction rate), as determined by flow cytometry, ranged from 8.82% to 98.12% (ave. [+ or -] S.E.) (34.4 [+ or -] 21.4%) for the 35[degrees]C shock treatments, from 13.12% to 61.02% (35.0 [+ or -] 5.0%) for the 36[degrees]C shock treatments and was 15% for the 5[degrees]C cold shock treatment. No tetraploids were produced for spawnings that received heat shocks above 36[degrees]C or below 35[degrees]C, or for cold shocks above 5[degrees]C for any of the tested postspawning treatment and duration times. Chemical shock with 150 [micro]M 6-thylaminopurine did not result in tetraploid M. japonicus embryos at any of the tested postspawning treatment times and durations. Tetraploid M. japonicus embryos were nonviable, with no tetraploid larvae being detected by flow cytometry. Based on our results heat shocking of M. japonicus embryos at 36[degrees]C, 23 min postspawning for a 5-10 min duration is the most effective means to produce tetraploids through inhibition of the first mitotic division (taking into consideration the importance of frequency and induction rate equally).

KEY WORDS: Penaeus japonicus, polyploidy, shrimp, selective breeding, genetic protection

INTRODUCTION

In Australia, shrimp domestication and genetic improvement programs are most advanced for Marsupenaeus japonicus (Bate) (Preston et al. 2001). The development of selectively improved genotypes for over 10 generations and a live export market has prompted industry to seek mechanisms to sterilize stocks to prevent unlicensed breeding and the introduction of genetically improved strains into natural fisheries. Previous efforts to contain selectively bred M. japonicus have focused on inducing sterility via irradiation or triploidy. These techniques have yet to prove effective in providing guaranteed sterility in the target stocks. Irradiation has been reported to significantly impair the reproductive capacity of female M. japonicus when exposed to 20 gray and male M. japonicus when exposed to 10 gray (Sellars et al. 2005b). However, irradiation was not 100% effective at preventing the production of viable offspring. In comparison, successful triploidy induction through prevention of polar body I1 extrusion is 100% effective at preventing reproduction, however, inductions never result in 100% triploid progeny (Sellars et al. 2004, Norris et al. 2005).

Producing triploids by the mating of tetraploids with diploids may provide a solution to these variable induction rates, resulting in 100% triploid progeny. Triploid stocks have been produced through the mating of tetraploid and diploid broodstock in several marine species including Pacific oysters (Crassostrea gigas) (Guo & Allen 1994, Guo et al. 1996, Wang et al. 2002), oyster hybrids (C. gigas x C. ariakensis) (Huayong & Allen 2002), Rainbow trout (Oncorhynchus mykiss) (Chourrout et al. 1986) and carp hybrids (Crassius auratus red vat. x Cyprinus carpio L.) (Liu et al. 2001).

However, production of viable tetraploids and their successful mating with diploids is a prerequisite for producing triploids in this manner.

There are many different methods for inducing tetraploidy in aquatic species, with the inhibition of an early embryonic developmental phase being critical to all. Because triploid M. japonicus do not produce viable gametes (Sellars et al. 2003, Preston et al. 2004, Sellars et al. 2004), there are only two possible ways to induce tetraploidy in this species; inhibition of polar body I extrusion during meiosis and prevention of the first division in mitosis. The prevention of polar body I extrusion has been reported to complicate subsequent chromosome segregation (Guo et al. 1992), and result in many different ploidy combinations including viable tetraploids. Inhibition of polar body I extrusion is difficult in some species because of the very short time frame in which the embryos must be treated and the fragile nature of the newly spawned embryos. In M. japonicus polar body I extrusion occurs at 4 min 10 sec postspawning at 27[degrees]C (Hudinaga 1941). Successful detection, collection, concentration and application of a shock to spawned embryos within this short time frame is a significant challenge. Only one reported study has attempted preventing polar body I extrusion in shrimp, however no tetraploids were successfully induced (Li et al. 2003).

Preventing the first division in mitosis is the alternative strategy that has been the focus of several previous attempts to induce tetraploidy in shrimp (Xiang et al. 1993, Peeters 1996, Li et al. 2003, Deoraj et al. 2005). Viable tetraploid shrimp, produced by inhibition of the first mitotic division, have only been reported in a single study by Xiang et al. (1993) in Ferropenaeus (Penaeus) chinensis using chemical and temperature shocks. Deoraj et al. (2005) reported poor viability of tetraploid and polyploid Litopenaeus vannamei embryos induced through heat shock, whereas Peeters (1996) reported unsuccessful tetraploid inductions in Penaeus indicus using chemical shocks. Li et al. (2003) reported successful induction of tetraploid F. chinensis embryos, however the larvae were not viable. Notably, all reported tetraploid inductions in shrimp have used temperature or chemical shocks (Xiang et al. 1993, Peeters 1996, Li et al. 2003, Deoraj et al. 2005).

This study aimed to assess the effectiveness of different temperature and chemical shock agents to produce tetraploid M. japonicus by preventing the first division in mitosis, which begins at 30 min postspawning at 27[degrees]C (Hudinaga 1941). A number of treatments were tested at various times after spawning and for different durations to determine which treatment regimes could be used to produce tetraploid M. japonicus.

MATERIALS AND METHODS

Source of Broodstock and Maturation 

Wild M. japonicus broodstock were captured from coastal waters near Mackay, Queensland, Australia (21[degrees]09'S, 149[degrees]30'E) by trawling at a depth of approximately 120 m. Broodstock were maintained (25 females: 15 males) in 2,000-L round fiberglass tanks, each fitted with a subsand circulation system (Crocos & Coman 1997). Tanks received 1.6 L [min.sup.-1] of 10-[micro]m filtered, 34 ppt salinity seawater at 27 [+ or -] 2[degrees]C and were maintained on a 12 h light: 12 h dark cycle. Shrimp were fed commercial M. japonicus pellets ad libitum once per day and fresh squid (Loligo spp.) three times a week during dark hours.

Ovarian development was assessed by shining a torch beam through the dorsal exoskeleton of the females during dark hours (Crocos & Coman 1997). Impregnated females that were ready to spawn (stage IV; Crocos & Kerr 1983) were caught, unilaterally eye-stalk ablated using hot forceps, and transferred to 100-L circular spawning tanks filled to 40 L. Spawning tanks received 0.2 L [min.sup.-1] of 10-[micro]m filtered, 34 ppt salinity seawater at 27 [+ or -] 2[degrees]C and were maintained on a 12-h light:12-h dark cycle. Females in spawning tanks were fed one piece (2 [cm.sup.3]) of fresh chopped squid (Loligo spp.) daily during light hours.

Spawning Detection and Embryo Collection

Spawning tanks were fitted with an automated spawning detection system (Coman et al. 2003) that accurately detects spawnings of M. japonicus within 2 min of spawning. The time of alarm from the automated detection system was taken as time zero and referred to from hereon as time postspawning. For ease of explanation spawned eggs, whether fertilized or unfertilized, will be referred to as embryos from hereon. At 10 min postspawning embryos were siphoned onto a 60-[micro]m screen suspended in a 1-L beaker of seawater. Seawater was allowed to flow out of the beaker, resulting in the concentration of embryos on the screen. Once embryos were concentrated (approx. 60,000-100,000 embryos [L.sup.-l]), the screen was lifted out of the beaker and embryos were rinsed into a beaker containing between 200-800 mL of seawater depending on the experimental design for that spawning. All seawater used for embryo collection, hatching and chemical stock solution preparation was 10-[micro]m filtered and maintained at 27 [+ or -] 2[degrees]C and 34 ppt salinity.

Induction and Hatching of Collected Embryos

Initial experiments using heat and cold shocks were conducted to assess a range of temperatures for 3 durations (5, 10 and 15 min). Once a suitable temperature shock was established for inducing tetraploidy (based on consistent inductions at the different durations), we further assessed different postspawning treatment times using the 5- and 10-min treatment durations. For the chemical shock experiments a predetermined treatment concentration was chosen based on results of M. japonicus triploid induction studies (Norris et al. 2005), which inhibited early embryonic processes. Initial experiments using chemical shock were therefore focused at assessing different postspawning treatment times for variable treatment durations (5-15 min).

Tetraploid inductions were timed to prevent the first division in mitosis. Various shock agents were trialed including heat, cold and chemical shocks. Shocks were applied between 16-28 min postspawning, with durations varying from 5-15 min. Control embryos were included in each experiment or spawning sample and received the same handling stress as their treated siblings. Control and treatment embryos from all spawnings were incubated at 27 [+ or -] 2[degrees]C with light aeration until hatching. Notably, only spawnings with hatching rates of >40% in the controls were used in this study. Depending on the quantity of eggs spawned and number of available people to complete inductions, in some instances spawnings were divided into two or three spawning samples.

Heat Shock Inductions

Heat shock inductions were completed on 26 spawning samples in total. Heat shocks varied from 32[degrees]C to 46[degrees]C and were applied from 18-24 min postspawning for a 5, 10 or 15 min duration (Table 1). A similar technique as described by Sellars et al. (2005a) to expose M. japonicus embryos to ozone was used to expose embryos to heat shocks. Briefly, concentrated embryos were divided into enough 100-mL aliquots so that there was one for each postspawning treatment time. These aliquots were further divided into enough subaliquots so that there was one for each treatment duration. At the correct postspawning treatment time each subaliquot of embryos was poured through a 60-[micro]m screen, which was placed directly into a seawater bath at the specified heat shock temperature. Each 60-[micro]m screen was removed from the seawater bath after the treatment duration time had lapsed and embryos were rinsed into 200 mL of 27[degrees]C seawater. Before, during and after treatment, seawater bath temperature was monitored using a mercury thermometer.

Cold Shock Inductions

Cold shock inductions were completed on 31 samples from different spawnings in total. Cold shocks ranged from 5[degrees]C to 23[degrees]C and were applied from 16-28 min postspawning for a duration of 5-15 min (Table 2). For samples of spawnings where there was only one postspawning and duration time, treatment was applied by the addition of cold seawater to concentrated embryos. For these spawnings cold shock was stopped by decanting off the cold water once embryos had settled during the shock treatment, followed by the addition of 27[degrees]C seawater at the correct time to give the different treatment durations. When a spawning sample was exposed to a cold shock treatment that had different postspawning or duration times, the same procedures as outlined earlier for heat shock inductions were used.

Chemical Shock Inductions

Chemical inductions using a final concentration of 150-[micro]M 6-dimethylaminopurine (6-DMAP) (Sigma) were completed on 14 spawning samples in total. Chemical shock was applied between 18-28 min postspawning for a duration of 5-15 min (Table 3). 6-DMAP was applied to embryos from spawning samples 58-63 using the same procedures outlined by Norris et al. (2005). In brief, 28 mL of a l-mM stock solution of 6-DMAP was added to 160 mL of seawater containing the embryos. For spawning samples 64-71 a similar technique as described for temperature treatment earlier and by Sellars et al. (2005a) to expose M. japonicus embryos to ozone was used to expose embryos to 6-DMAP. Briefly, concentrated embryos were divided into 4 x 100-mL aliquots (one for each of the tour postspawning treatment times) to which 50 mL of a fresh 450 [micro]M 6-DMAP stock solution (made up in seawater) was added at the correct postspawning treatment time. Each aliquot was then divided into 3 x 50-mL subaliquots. Subaliquots were poured through a 60-[micro]m screen after the appropriate treatment duration to collect the embryos, which were then transferred to 200 mL of seawater to cease chemical exposure.

Polyploidy Detection and Analysis

Pooled samples of between 5-30 nauplii (less than 30 nauplii were sampled only in instances when less than this hatched) and 50-100 embryos were separately sampled for each spawning from all controls and treatments. In treatments where there were no nauplii hatched, it was only possible to take an unhatched embryo sample. In some instances samples were snap frozen in liquid nitrogen and stored at -20[degrees]C for up to 30 days. If polyploidy analysis could be completed on the day of hatching, nauplii were sampled and taken live to the laboratory and embryos were transported on ice. Snap frozen samples were transported to the laboratory and defrosted on ice. Live nauplii were chilled on ice at the laboratory until they died. Once dead nauplii or embryos had settled, excess seawater was removed, <200 [micro]L of seawater and sample remained in each tube.

At the laboratory 500 [micro]L of marine phosphate buffered solution (MPBS) (11.0 g [L.sup.-1] NaCl, 0.2 g [L.sup.-1] KCl, 1.15 g [L.sup.-1] [Na.sub.2]HP[O.sub.4].2[H.sub.2]O) propidium iodide (PI) stain (MPBS containing 0.1% Triton X-100, 0.2 mg [mL.sup.-1]Rnase A, 0.02 mg [mL.sup.-1] PI) was added to each sample. Samples were then homogenated individually by aspiration eight times through a 25-G needle pushed firmly against the side of the sample tube. After homogenation 10 [micro]L of a 1:100 dilution of the internal standard, glutaraldehyde fixed, chicken red blood cells (CRBC, Handbook of Flow Cytometry Methods) was added to each sample. Cell suspensions were screened through 62-[micro]m mesh prior to fluorescent activated cell sorting (FACS) on a Calibur Flow Cytometer (Beckton Dickinson Immunocytometry Systems San Jose, California, USA). A total of 30,000 shrimp cells were analyzed for each sample, however, in some instances only 15,000 shrimp cells were analyzed because there were too few cells in a sample.

Induction rates were calculated for each treatment sample. Initially the proportion of diploid cells in the G2 phase relative to the GI phase was calculated from a control spawning sample of the same life-history stage. This proportion was used to calculate the number of diploid G2 cells in treatment samples relative to the number if diploid G1 cells in the treatment sample. Once the total number of diploid G2 cells in treatment samples was estimated, the tetraploid G1 peak was calculated by subtracting the diploid G2 estimation from the total number of cells where the tetraploid G1 peak falls (Fig. 1) (within the cell cycle G1 cells are in the growth phase, whereas G2 cells have double the normal chromosomal content and are preparing for division). Where the number of cells counted with a DNA content the same as a diploid G2 cell was greater than expected (by 5% or more), the level of polyploidy in that sample was analyzed using ModFit software (Verity Software House, Topsham, Maine, USA). A five percent threshold was chosen based on observed variability among multiple samples taken from the same control groups.

The frequency of producing tetraploid embryos was calculated for the trialed heat shock treatment regimes by dividing the number of spawning samples within a treatment regimen, which had tetraploid embryos by the total number of induction attempts for that treatment regimen.

The effect of heat shock treatment (control, 35[degrees]C, 36[degrees]C) and duration (5 min, 10 min) on induction rates at 22 min postspawning were analyzed by 2-way ANOVA (PROC GLM; SAS Institute Software, 1999) for spawning samples in which one or more of the treatment combinations resulted in tetraploid embryos. Pairwise comparisons between shock and duration treatments were performed using the least significant difference test.

RESULTS

Heat Shock Inductions

From the initial scoping experiments it was evident that 35[degrees]C and 36[degrees]C heat shocks were most suitable for inducing tetraploidy. Tetraploid embryos were produced most frequently by a 35[degrees]C heat shock applied 22 min postspawning for a 10 min duration (66.67% of the time) (Table 4). Tetraploid induction rates for this 35[degrees]C treatment regimen were 8.82, 20.9, 9.9 and 98.1% (ave. [+ or -] S.E.) (34.4 [+ or -] 21.4%) for spawning samples 21, 22 and spawning sample 23 replicate 1 and 2 respectively. The frequency of tetraploid embryo induction was lower for the 36[degrees]C heat shock treatments ranging from 33.33% to 45.45% for the 21, 22 and 23 min postspawning treatments at the 5 and 10 min duration times trialed (Table 4). Tetraploid induction rates for the 36[degrees]C shock treatments ranged from 13.12% to 61.01%. (ave. 35.0 [+ or -] 5.0%). Only one 18 min postspawning induction was applied at 36[degrees]C for a l0 min duration which did produce tetraploid embryos, resulting in a 100% tetraploid induction frequency (Table 4).

When treated at 22 min postspawning, tetraploid induction rates of embryos were significantly higher (P < 0.05) for the 36[degrees]C shock applied for a 5 min duration (ave. 12.81 [+ or -] 5.80%) compared with the control (27[degrees]C) (ave. 0.00 [+ or -] 0.00%) and 35[degrees]C (ave. 0.00 [+ or -] 0.00%) shock treatments (Table 5). However, when the duration of treatment was increased to 10 min, there was no significant difference between tetraploid induction rates for the 35[degrees]C and 36[degrees]C shock treatments (ave. 34.43 [+ or -] 21.39 and 18.52 [+ or -] 8.53% respectively) and both were significantly higher than controls (27[degrees]C) (ave. 0.00 [+ or -] 0.00%) (Table 5). Because tetraploid embryos were produced at two of the three duration times trialed for the 36[degrees]C shock, compared with only one for the 35[degrees]C shock, further experimentation to optimize postspawning treatment times used a 36[degrees]C temperature shock.

Although no statistical comparisons were possible as too few spawning samples had tetraploid embryos in the different 36[degrees]C shock postspawning treatment times trialed, tetraploid induction rates of embryos were higher on average for the 21, 22 and 23 min postspawning times when the treatment duration was 10 min compared with 5 min (i.e., 61.00%, 18.52% and 53.40% respectively compared with 12.5%, 12.81% and 45.00%) (Table 6). These results suggest that duration had a stronger effect than postspawning time on tetraploid induction.

There were no tetraploid nauplii produced in any of the heat shock treatments trialed. It is also worth noting that all treatment groups for the 39[degrees]C and 46[degrees]C spawning samples had zero hatch, and FACS outputs of embryos from these treatments were difficult to analyze because the different cell phases could not be discriminated between because of tissue degradation.

Cold Shock Inductions

Only one of the trialed cold shock treatment regimes produced tetraploid embryos. In this treatment, spawning embryos treated at 5[degrees]C for 8 min at 20 min postspawning had a tetraploid induction rate of 15.0%. Nauplii sampled from this same treatment were found to be diploid. All other cold shock inductions failed to induce tetraploidy in any of the embryo and nauplii samples. All spawnings on which cold shocks were applied hatched resulting in all control and treatment nauplii FACS samples containing ~30 nauplii.

6-DMAP Shock Inductions

6-DMAP inductions using a 150-[micro]M final concentration failed to produce tetraploid M. japonicus when applied 18-28 min postspawning, for 5-15 min duration. All embryo and nauplii samples in the control and treatment groups were found to be diploid. All spawnings on which 6-DMAP shocks were applied hatched resulting in all control and treatment nauplii FACS samples containing ~30 nauplii.

DISCUSSION

The results of this study demonstrate that heat and cold shock can prevent the first division in mitosis to produce tetraploid Marsupenaeus japonicus embryos. These findings are consistent with those of Xiang et al. (1993) and Li et al. (2003) who successfully inhibited the first division in mitosis in Ferropenaeus (Penaeus) chinensis using temperature shocks to produce tetraploid embryos. To our knowledge there are no other reports of successful tetraploid induction in Penaeid shrimp.

Tetraploid induction rates and frequency of induction of M. japonicus embryos were highly variable (0% to 98% and 33.33% to 100% respectively). This finding is similar to other polyploidy induction studies that have attempted to prevent an early embryonic development phase (Xiang et al. 1993, Li et al. 2003, Sellars et al. 2004, Norris et al. 2005). In general, polyploidy induction rates are dependant on three main variables; magnitude of shock (e.g., change in temperature), timing of shock and shock duration. This study showed that increasing temperature by 9[degrees]C (from 27[degrees]C) between 21-23 min postspawning most consistently produced tetraploid embryos. Shocking the embryos at this time immediately precedes the disappearance of egg-jelly and the first division in mitosis, which begins at 30 min postspawning (Hudinaga 1941).

Shock duration was also shown to significantly affect induction rates, with a 10 min duration resulting in more tetraploid embryos compared with the 5 min duration. Li et al. (2003) also suggested that extended treatment durations may increase tetraploid induction rate. This likely results from shrimp spawning over a period of time, allowing more embryos to reach the embryonic developmental phase at which mitotic division can be prevented when a longer duration of shock is applied. Notably, no tetraploid M. japonicus embryos were produced at the longer 15 min duration. This is likely caused by the prolonged duration negatively interfering with the next stages of embryonic development.

Although tetraploid M. japonicus embryos were produced, they were nonviable, as determined by FACS analysis of the nauplii. Problems with tetraploid viability have been reported across a range of aquatic species. Li et al. (2003) reported similar problems for F. chinensis and were unable to produce viable tetraploid postlarvae by preventing the first division in mitosis. Quiet al. (1997) documented low viability among tetraploid freshwater prawns (Macrobrachium nipponense). Poor viability of tetraploids is commonly reported in numerous fish and shellfish species (e.g., Grass carp, Ctenopharyngdon idella, Cassani et al. 1990; Black carp, Mylopharyngodon piceus, Rothbard et al. 1997; Sydney rock oysters, Saccostrea commerciali, Nell et al. 1998; Manila clams, Rditapes philippinarum, Direr & Dufy 1990; Pacific oysters, Crassostrea gigas, Guo 1991, cited by Guo & Allen 1994, Guo et al. 1994 and scallops, Chlamys azumapecten, Yang et al. 1997).

The genetic, biochemical or biological mechanisms that prevent tetraploid embryos from developing and hatching into viable nauplii remains unknown. In this study, it is unlikely that temperature shock alone was the cause of embryo deaths. This was evidenced by the presence of diploid nauplii hatching within the same temperature shock treatments as the nonviable tetraploids. One explanation proposed by Guo (1992) is that the diploid eggs have insufficient cytoplasmic reserves to develop as a tetraploid. Li et al. (2003) gives no explanation for their nonviable F. chinensis tetraploids, however, concluded that tetraploid embryos may have limited viability or ability to hatch.

In this study 6-dimethylaminopurine (6-DMAP) was also assessed as a potential shock agent to prevent the first division in mitosis. However, this treatment was not effective in inducing tetraploidy. Temperature and cytochalasin-B are the only shock agents that have been reported elsewhere to successfully inhibit the first division in mitosis in penaeid shrimp (Xiang et al. 1993, Li et al. 2003).

CONCLUSION

This study shows that the first division in mitosis can be inhibited in Marsupenaeus japonicus to produce tetraploid embryos. The most suitable treatment regimen for inducing tetraploidy in M. japonicus, giving frequency and induction rate equal importance, was a 36[degrees]C shock administered 23 min postspawning for a 5 or 10 min duration. Notably, none of the tetraploid embryos produced in this study were viable. If sterilization of penaeid shrimp is to be achieved through tetraploidy, future studies will first need to determine why tetraploid embryos are not viable.

ACKNOWLEDGMENTS

The authors thank Greg Coman, Stuart Arnold and Michelle Jones for their experimental support, and Paula Hall and Grace Chojnowsky from the Queensland Institute of Medical Research for their assistance with flow cytometry.

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Yang, H.. R. Wang, X. Guo & Y. Yu. 1997. Tetraploid induction with cytochalasin B treatment in scallop Chlamys azumapecten F. In 11th International Pectinid Workshop, La Paz, BCS, Mexico, Book of abstracts, pp. 187-188.

M. J. SELLARS, (1,2,3) * F. E. COMAN, (1,2) B. M. DEGNAN (3) AND N. P. PRESTON (1,2) (1) CSIRO Food Futures National Research Flagship; (2) CSIRO Marine Research, 233 Middle Street, Cleveland, Qld, 4163, Australia; (3) School of Integrative Biology, The University of Queensland, Brisbane, Qld, 4072, Australia

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Heat shock treatments applied to different spawning samples to
attempt the prevention of the first division in mitosis in
Marsupenaeus japonicus to induce tetraploidy. Durations with an
asterisk (*) had three replicate treatments.

                      Treatment Time

Heat             Postspawning      Duration      Spawning
Shock               (min)           (min)         Sample

32[degrees]C          18          5, 10 & 15     1 to 4
                      20          5, 10 & 15     1 to 4
                      22          5, 10 & 15     1 to 4
                      24          5, 10 & 15     1 to 4
35[degrees]C          21          5 & 10         10 to 12
                      22          5 & 10         10 to 12
                      22          5 * & 10 *     21 to 23
                      23          5 & 10         10 to 12
36[degrees]C          18          5, 10 & 15     5
                      20          5, 10 & 15     5
                      21          5 & 10         13 to 15
                      22          5, 10 & 15     5
                      22          5 & 10         13 to 15
                      22          5 & 10         16 to 19
                      22          5 * & 10 *     24 to 26
                      23          5 & 10         13 to 15
                      24          5, 10 & 15     5
38[degrees]C          22          5 & 10         20
39[degrees]C          18          5, 10 & 15     6 to 8
                      20          5, 10 & 15     6 to 8
                      22          5, 10 & 15     6 to 8
                      24          5, 10 & 15     6 to 8
46[degrees]C          18          5, 10 & 15     9
                      20          5, 10 & 15     9
                      22          5, 10 & 15     9
                      24          5, 10 & 15     9

TABLE 2.
Cold shock treatments applied to different spawning samples to
attempt the prevention of the first division in mitosis in
Marsupenaeus japonicus to induce tetraploidy.

                      Treatment Time

Cold             Postspawning      Duration      Spawning
Shock               (min)           (min)         Sample

5[degrees]C           20          8              40
6.5[degrees]C         22          5 & 10         54 to 57
7[degrees]C           16          5 & 10         42
                      25          5              29
                      28          7              34 & 35
8[degrees]C           16          10             45
                      20          8              39
9[degrees]C           18          5, 10 & 15     46 to 49
                      20          5, 10 & 15     46 to 49
                      21          5 & 10         50 to 53
                      22          5, 10 & 15     46 to 49
                      22          5 & 10         50 to 53
                      23          5 & 10         50 to 53
                      24          5, 10 & 15     46 to 49
9.5[degrees]C         16          5 & 10         41
                      16          10             44
10[degrees]C          16          10             43
11[degrees]C          20          8              38
12[degrees]C          28          7              32 & 33
14 [degrees]C         20          8              37
17[degrees]C          25          5              28
                      28          7              30 & 31
22[degrees]C          25          5              27
23[degrees]C          20          8              36

TABLE 3.
150 [micro]M 6-dimethylaminopurine shock treatments applied to
different spawning samples to attempt the prevention of the first
division in mitosis in Marsupenaeus japonicus to induce tetraploidy.
Durations with an asterisk (*) had three replicate treatments.

     Treatment Time

 Postspawning      Duration        Spawning
    (min)           (min)           Sample

      18         5, 10 & 15       64 to 71
      20         10               59
      20         8                60 & 61
      20         6                62 & 63
      20         5, 10 & 15       64 to 71
      22         10               59
      22         8                60 & 61
      22         6                62 & 63
      22         5, 10 & 15       64 to 71
      24         5, 10 & 15       64 to 71
      25         10               59
      25         8                60 & 61
      25         6                62 & 63
      26         10               59
      26         8                60 & 61
      26         6                62 & 63
      28         10 *             58

TABLE 4. Frequency of producing tetraploid Marsupenaeus japonicus
embryos at the different heat shock treatment combinations trialled.

     Treatment Time                      Heat Shock

Postspawning  Duration
   (min)       (min)    32[degrees]C    35[degrees]C    36[degrees]C

     18           5      0% (n = 4)                        0% (n = 1)
     18          10      0% (n = 4)                      100% (n = 1)
     18          15      0% (n = 4)                        0% (n = 1)
     20           5      0% (n = 4)                        0% (n = 1)
     20          10      0% (n = 4)                        0% (n = 1)
     20          15      0% (n = 4)                        0% (n = 1)
     21           5                       0% (n = 3)   33.33% (n = 3)
     21          10                       0% (n = 3)   33.33% (n = 3)
     22           5      0% (n = 4)       0% (n = 6)   45.45% (n = 11)
     22          10      0% (n = 4)   66.67% (n = 6)   45.45% (n = 11)
     22          15      0% (n = 4)                        0% (n = 1)
     23           5                       0% (n = 3)   33.33% (n = 3)
     23          10                       0% (n = 3)   33.33% (n = 3)
     24           5      0% (n = 4)                        0% (n = 1)
     24          10      0% (n = 4)                        0% (n = 1)
     24          15      0% (n = 4)                        0% (n = 1)

     Treatment Time             Heat Shock

Postspawning  Duration
   (min)       (min)    38[degrees]C   39[degrees]C

     18           5                      0% (n = 3)
     18          10                      0% (n = 3)
     18          15                      0% (n = 3)
     20           5                      0% (n = 3)
     20          10                      0% (n = 3)
     20          15                      0% (n = 3)
     21           5
     21          10
     22           5      0% (n = 1)      0% (n = 3)
     22          10      0% (n = 1)      0% (n = 3)
     22          15                      0% (n = 3)
     23           5
     23          10
     24           5                      0% (n = 3)
     24          10                      0% (n = 3)
     24          15                      0% (n = 3)

TABLE 5.
Average tetraploid induction rate ([+ or -] SE) of Marsupenaeus
japonicas embryos from spawning samples in which tetraploid embryos
were produced in one or more treatment category. Means with different
superscripts are significantly different (P < 0.05) within duration.

                     Temperature Shock Applied 22 min Postspawning

Duration              27[degrees]C                 35[degrees]C

 5 min           0.00 [+ or -] 0.00% (b)      0.00 [+ or -] 0.00% (b)
                         (n = 8)                      (n = 4)
10 min           0.00 [+ or -] 0.00% (b)     34.43 [+ or -] 21.39% (a)
                         (n = 8)                      (n = 4)

                     Temperature Shock Applied 22 min Postspawning

Duration                                  36

 5 min                         12.81 [+ or -] 5.80% (a)
                                        (n = 8)
10 min                         18.52 [+ or -] 8.53% (a)
                                        (n = 8)

TABLE 6.
Tetraploid induction rates of Marsupenaeus japonicas embryos when
exposed to a 36[degrees]C shock at different postspawning times for
a 5 or 10 min duration.

    Time                              Duration

Postspawning              5 min                       10 min

   18 min                                         32.60% (n = 1)
   20 min             0.00 (n = 1)                 0.00% (n = 1)
   21 min            12.50% (n = 1)               61.00% (n = 1)
   22 min      12.81 % [+ or -] S.E. 5.80%   18.52% [+ or -] SE 8.53%
                         (n = 8)                      (n = 8)
   23 min            45.00% (n = 1)              53.40% (n = 1)

Figure 1. Example of a (i) control and (ii) treatment fluorescent
activated cell sorting output from Marsupenaeus japonicus embryos. The
expected size of the diploid G2 peak in (ii) is calculated from
dividing the events in the diploid G2 peak in (i) by the diploid G1
peak in (i), and then multiplying this factor by the events in the
diploid G1 peak in (ii). When the total number of events of the G2 peak
in (ii) was greater than expected (by 5% or more), ModFit analysis was
performed to determine the different levels of polyploidy. In this
instance the expected diploid G2 peak in (ii) would be
([1470/13531]*3226) = 350.47 events. Because the actual number of
events with a DNA content the same as a diploid G2 cell in (ii) is
9911, and we would only expect -350 diploid G2 cells, this output
would be analyzed by ModFit to determine the tetraploidy level. CRBC
is an internal standard control (chicken red blood cells).

                                %
M    Low    High    Events    Total    GMean      CV

1     15      72      9203     29.8     44.27    26.8
2    105     153     13531    43.81    130.34    8.59
3    234     272      1470     4.76    251.98    4.23
                                %
M    Low    High    Events    Total    GMean      CV

1     20      70      7344    23.93     45.27    25.8
2    110     153      3226    10.51     131.6    8.47
3    231     298      9911     32.3     263.9    5.77
4    514     547       330     1.08     526.7    1.78