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Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine
Aquaculture 256 (2006) 337 – 345 www.elsevier.com/locate/aqua-online
Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine Melony J. Sellars a,b,⁎, Bernard M. Degnan b , Nigel P. Preston a a
CSIRO Food Futures National Research Flagship, CSIRO Marine Research, 233 Middle Street, Cleveland, Qld 4163, Australia b School of Integrative Biology, The University of Queensland, Brisbane, Qld, 4072, Australia Received 23 November 2005; received in revised form 31 January 2006; accepted 16 February 2006
Abstract This study investigated the chromosome ploidy level of Marsupenaeus (Penaeus) japonicus (Bate) non-viable (unhatched) embryos and nauplii after exposure to 6-dimethylaminopurine (6-DMAP), timed to stop either polar body (PB) I, or PBI and II extrusion. Embryos from eight separate families or spawnings were exposed to 150 or 200 μM 6-DMAP from 1- to 3-min postspawning detection (psd) for a 4- to 5-min duration (timed to stop PBI extrusion). Separate aliquots of embryos from five of the same spawnings were also exposed to 200 μM of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). For one spawning, a third aliquot of embryos was exposed to 400 μM of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). At 18-h psd, non-viable embryo and nauplii samples were taken separately for fluorescent activated cell sorting (FACS). FACS revealed that there were diploids and triploids among all treated non-viable embryos and nauplii. All control non-viable embryos and nauplii were diploid. Percentages of triploid induction for the 4- to 5-min and 16-min durations were not significantly different (P > 0.05). Additionally, no difference was found in the triploidy level of nonviable embryos compared to nauplii in these treatments. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 4- to 5-min duration ranged from 29.57% to 99.23% (average 55.28 ± 5.45%) and from 5.60% to 98.85% (average 46.70 ± 7.20%), respectively. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 ± 11.00%) and from 47.5% to 99.24% (average 79.38 ± 5.24%), respectively. To our knowledge, this is the first documentation of successful PBI or PBI and II inhibition in shrimp. This study conclusively shows that treatment of M. japonicus embryos with 6-DMAP at 1- to 3-min psd for either a 4- to 5-min duration (timed to stop PBI extrusion) or 16-min duration (timed to stop both PBI and II extrusion) results in viable triploid nauplii. © 2006 Elsevier B.V. All rights reserved. Keywords: Genetic protection; Kuruma prawn; Chromosome segregation
1. Introduction ⁎ Corresponding author. CSIRO Food Futures National Research Flagship, CSIRO Marine Research, 233 Middle Street, Cleveland, Qld 4163, Australia. Tel.: +61 7 3826 7359; fax: +61 7 3826 7222. E-mail address: [email protected] (M.J. Sellars). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.052
Shrimp domestication and genetic improvement programs in Australia are most advanced for Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) (Preston et al., 2001). As a result, substantial research
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efforts have focused on developing suitable approaches by which to confer sexual sterilization to prevent downstream loss of genetic resources at the live export market and the introduction of genetically altered strains into natural fisheries (Sellars et al., 2004; Norris et al., 2005). In other invertebrate species, the induction of sexual sterilization has additionally been found to confer desirable traits such as improved growth rates or pathogen resistance. To date, research efforts for M. japonicus have investigated irradiation (Sellars and Preston, 2005; Sellars et al., 2005), triploidy (by prevention of polar body (PB) II extrusion) (Xiang et al., 2001; Sellars et al., 2003, 2004; Norris et al., 2005) and tetraploidy (by prevention of the first mitotic division) (Sellars et al., in press) as potential ways to achieve sexual sterility. While irradiation has been successfully used to impair the reproductive capacity of M. japonicus (Sellars et al., 2005), the most successful sterilization of this species to date has been achieved through induction of triploidy (Sellars et al., 2003, 2004; Norris et al., 2005). Triploid M. japonicus produced through prevention of PBII extrusion using chemical shocks are sterile and all female (Sellars et al., 2003, 2004; Norris et al., 2005). However, prevention of PBII extrusion never results in 100% triploid progeny, due to inherent variation among developing embryos, with induction rates ranging from 0% to 85.9% (Norris et al., 2005). Producing triploids by the mating of tetraploids with diploids may provide a solution to these variable induction rates and result in 100% triploid progeny. This approach has been successful for several marine species including Pacific oysters (Crassostrea gigas) (Guo and Allen, 1994; Guo et al., 1996; Wang et al., 2002), oyster hybrids (C. gigas × C. ariakensis) (Huayong and Allen, 2002), rainbow trout (Oncorhynchus mykiss) (Chourrout et al., 1986) and carp hybrids (Crassius auratus red var. × Cyprinus carpio L.) (Liu et al., 2001). Production of viable tetraploids is a prerequisite for producing triploids in this manner. In a recent study, tetraploid M. japonicus embryos were produced by stopping the first division in mitosis (Sellars et al., in press). However, these tetraploid embryos did not hatch, suggesting that mitotic tetraploid M. japonicus may not be viable. One potential alternative method to mitotic inhibition for tetraploid induction is the prevention of PBI extrusion in meiosis I. The prevention of PBI extrusion has been reported to complicate subsequent chromosome segregation (Guo et al., 1992) and result in many different ploidy combinations including viable tetraploids. Triploid and tetraploid progeny resulted from preventing PBI extrusion in the American oyster (Crassostrea virginica)
(Stanley et al., 1981), the blue mussel (Mytilus edulis) (Yamamoto and Sugawara, 1988) and the Pacific abalone (Haliotis discus hannai) (Arai et al., 1986). In comparison, the prevention of PBI extrusion only produced tetraploids in dwarf surfclams (Mulinia lateralis) (Yang and Guo, 2004) and zhikong scallops (Chlamys farreri) (Yang et al., 2000). In the pacific oyster (C. gigas), the prevention of PBI extrusion using thermal stress resulted in triploids only (Quillet and Panelay, 1986) while using cytochalasin B stress resulted in tetraploids (Stephens and Downing, 1988; Stephens, 1989) or a combination of triploids, tetraploids and aneuploids (Guo et al., 1992). Prevention of both PBI and II extrusion has also been shown to result in tetraploid mussels (Mytilus galloprovincialis) (Scarpa et al., 1993) and dwarf surfclams (M. lateralis) (Peruzzi and Guo, 2002). Recently, however, a contradictory report found that preventing PBI and II in dwarf surfclams produced pentaploids (Yang and Guo, 2004), a more theoretically plausible outcome (Cooper and Guo, 1989). Both PBI triploids and tetraploids have been found to have commercial advantages in several aquatic cultured species. Triploids produced by preventing PBI extrusion have been reported to grow faster than those resulting from preventing PBII extrusion, probably due to increased heterozygosity (e.g. soft-shell clam, Allen et al., 1982; American oyster, Stanley et al., 1984; Pacific oyster, Yamamoto et al., 1988; blue mussel, Beaumonet and Kelly, 1989; pearl oyster, Jiang et al., 1991). As discussed, tetraploids are of commercial value for mating with diploids to produce 100% triploid progeny. Production of PBI triploid and tetraploid M. japonicus may therefore have considerable commercial value to the industry. This study investigated the effect of stopping PBI, or both PBI and PBII extrusion on subsequent chromosome ploidy level of M. japonicus non-viable (unhatched) embryos and nauplii. 2. Materials and methods 2.1. Broodstock, spawning and embryo collection Wild-caught M. japonicus broodstock (25 females:20 males) were acclimated to a reversed light cycle (12h light:12-h dark) for 30 days in 2000-L sand-substrate tanks (Crocos and Coman, 1997). Tanks received 1.6 L min− 1 of 27 ± 2 °C seawater. Shrimp were fed commercial M. japonicus pellets ad libitum once per day and freshly cut squid (2cm3) (Loligo spp.) three times weekly during dark hours.
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Fig. 1. Cross section view of a 100-L spawning tank fitted with an automated spawning detection system, false 2-mm oyster mesh base and glass trays for embryo collection.
light cycle and water source (at a flow rate of 0.2L min− 1) as the acclimation tanks. Females in spawning tanks were fed one piece of freshly cut squid (2cm3) (Loligo spp.) daily during light hours.
After acclimation, gravid females (average weight 33 ± 3.41g) were selected by shining a torch beam through their dorsal exoskeleton during dark hours. Gravid females impregnated with spermatophores were unilaterally eyestalk ablated using hot forceps and spawned in 100-L circular spawning tanks filled to 40L and fitted with an automated spawning detection system (Coman et al., 2003). Tanks were fitted with a false 2-mm oyster mesh base suspended 10cm above the bottom of the tank (Fig. 1). Two glass trays (21 × 15 × 6 cm, length × width × depth) were placed beneath the false oyster mesh base to collect embryos as they were released from the female (three glass trays were used in the case of spawning 4). For ease of explanation, spawned eggs, whether fertilized or unfertilized, will be referred to as embryos from herein. Spawning tanks received the same
2.2. Experimental design Spawnings from eight different females were exposed to various concentrations of 6-DMAP at 1to 3-min post-spawning detection (psd) for either a 4to 5-min duration or both a 4- to 5-min duration and a 16-min duration. All 4- to 5-min duration treatments will be referred to from herein as a 5-min duration for ease of explanation. The experimental design for each spawning was dependant upon the availability of freshly prepared 6-DMAP solution, efficiency of
Table 1 Experimental design for treatment aliquots or glass trays for the eight separate spawnings of Marsupenaeus japonicus Spawning
Aliquot 1 Aliquot 2 Aliquot 3
6-DMAP (μM) Duration (min) 6-DMAP (μM) Duration (min) 6-DMAP (μM) Duration (min)
n.a. = not applicable.
1
2
3
4
5
6
7
8
150 4 to 5 150 4 to 5 n.a.
200 4 to 5 200 4 to 5 n.a.
200 4 to 5 200 4 to 5 n.a.
200 4 to 5 200 16 400 16
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
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glass tray removal from spawning tanks and rate of embryo concentration. As a result, experimental designs had slight variations between spawnings (Table 1). Spawning 1 had two aliquots of collected embryos (each glass tray of embryos making up an aliquot) that were both exposed for a 5-min duration to 150 μM 6-DMAP. Spawnings 2 and 3 also had two aliquots of embryos each that were exposed for a 5min duration; however, the 6-DMAP concentration was 200 μM. Spawning 4 had three aliquots of embryos, with two aliquots being exposed to 200μM 6-DMAP for either a 5- or 16-min duration, and the third aliquot being exposed to 400 μM 6-DMAP for a 16-min duration. Spawnings 5 to 8 all had two aliquots of embryos, which were exposed to 200μM 6-DMAP, with one aliquot of each spawning being exposed for a 5-min duration and the other aliquot being exposed for a 16-min duration. A control (untreated) aliquot of embryos was collected from spawning tanks for each spawning at 1- to 4-min psd.
trays that were present in the tank during spawning, and from glass trays added to the tank 1-min post-spawning (data not presented). At 1min prior to 6-DMAP treatment being due to cease, excess seawater was pumped off leaving embryos in a 2- to 3-mm layer of seawater. Once 6-DMAP treatment time had elapsed, glass trays containing embryos and remaining seawater were then placed in 9L of 60 μM filtered, UV sterilized, 27 °C seawater. After embryos had settled (approximately 2 min), 8 L of seawater was decanted off and replaced by a fresh 8L of seawater. Each aliquot of embryos received three of these 8-L water exchanges to remove all remaining 6DMAP. Exchanges were completed by 20-min psd for all 5-min duration treatments and by 30-min psd for all 16-min duration treatments. Aliquots of control embryos were exposed to the same handling stress as treatment embryos. After treatments and water exchanges were complete, embryos were incubated in 9 L of seawater at 27 ± 2 °C with light aeration until hatching approximately 12 h later (Hudinaga, 1941).
2.3. Ploidy induction using 6-dimethylaminopurine The initiation of spawning, as detected by the automated detection system (Coman et al., 2003), was taken as time 0. Glass trays were removed immediately upon spawning detection (i.e. plastic lid was fastened to the glass tray which was subsequently lifted out of the tank) and the water level was reduced to 200 mL by siphoning (except for spawning 1 which was 280 mL and the 400μM 6-DMAP treatment in spawning 4 which was 150 mL), leaving approximately 80% of the embryos which were originally in the tray when lifted out of the tank. The resulting number of embryos in each glass tray varied with spawning size (average 72.5 ± 14.11 embryos/glass tray). For the 200μM 6-DMAP treatments, a freshly prepared 50-mL aliquot of 1 mM 6DMAP (made up in 60 μM filtered, UV sterilized 27 °C seawater) was added to each glass tray containing the 200 mL of seawater and embryos. For the 400 μM 6DMAP treatments, two 50-mL aliquots of 1mM 6DMAP were added to the glass trays. For the 150μM 6DMAP treatments, the water level was at 280mL and a 50-mL aliquot of 1mM 6-DMAP was added. Time of treatment varied from 1-min to 3-min psd and was dependent on human proximity to the spawning tanks at time of alarm. Immediately after 6-DMAP treatments were applied, another glass tray was placed in the spawning tank to collect embryos for a control (untreated) aliquot which was removed after 1 min. Preliminary studies showed that there was no difference between hatch rates of embryos when taken from glass
2.4. Monitoring of embryonic development and determination of hatch rate Using light microscopy the development rate of 10 to 20 embryos from each treatment and control were monitored until they reached the four-cell stage. Brief notes were made on the psd time when embryos were one-cell, elongating, kinked and about to divide, twocell and four-cell (Hudinaga, 1941). Where the number of non-viable embryos and nauplii present in a treatment were estimated to be more than required for fluorescent activated cell sorting (FACS) analysis, samples were collected to establish hatch rate data. Within each treatment and control, three 50-mL samples of non-viable embryos and nauplii were taken from the 9-L incubation buckets at approximately 4 h after the first signs of hatching in controls. Prior to samples being taken, the contents of each bucket were mixed so that samples were homogeneous. The number of non-viable embryos and nauplii within each replicate sample were counted within 24 h of being sampled. Percentage hatch rates of embryos in the control and treatment aliquots were analysed separately for each spawning using one-way ANOVA (SAS Institute, 1999). 2.5. FACS analysis and ModFit LT analysis Two replicate samples of between 25 to 50 nonviable embryos and 10 to 30 nauplii were separately
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Fig. 2. Example of a FACS data output after ModFit LT analysis for spawning three nauplii from a (A) control and (B) 200 μM 6-DMAP (4- to 5-min duration) treatment. Gated cells were analyzed using a FL2 histogram for the DNA content to determine the percentage ploidy and respective cell cycle distribution (i). Cells were gated to remove doublets and eliminate debris from the analysis (ii).
sampled at approximately 16-h psd for each spawning from all controls and treatments, with each sample containing less than 100 μL of sample and seawater. Samples were snap frozen in liquid nitrogen and stored at − 65 °C for up to 7 days before being analysed. The limitation of only having 1 to 3min post-spawning to collect embryos for treatment meant that in some instances there were only enough non-viable embryos or nauplii for one replicate sample for FACS analysis. In three instances, there were too few non-viable embryos for FACS analysis.
For FACS analyses, 500μL of marine phosphate buffered solution (MPBS) (11.0g L− 1 NaCl, 0.2g L− 1 KCl, 1.15g L− 1 Na2HPO4·2H2O) propidium iodide (PI) stain (MPBS containing 0.1% Triton X-100, 0.02mg mL− 1 RNase A, 0.02mg mL− 1 PI) was added to each sample. Samples were then homogenized individually by aspiration eight times through a 25-G needle pushed firmly against the side of the sample tube. After homogenation 8μL of a 1:100 dilution of the internal standard, glutaraldehyde fixed, chicken red blood cells (CRBC, Handbook of Flow Cytometry Methods, 1993) was added to each sample. Cell
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Table 2 Means (±S.E.) of the percentage hatch rate (nauplii/total embryos) in controls and treatments for spawnings five, six, seven and eight of Marsuepnaeus japonicus Treatment
Spawning 5
Spawning 6
a
Spawning 7 Spawning 8
a
Control 79.15 ± 2.59 67.88 ± 2.12 66.67 ± 3.21 56.03 ± 1.28a 5-min 26.93 ± 2.39b 48.89 ± 1.11b 71.03 ± 2.41 27.03 ± 1.21b duration 16-min 26.93 ± 2.39b 72.34 ± 2.73a 58.33 ± 4.81 24.95 ± 4.60b duration Treatment concentrations for these four spawnings were 200μM 6DMAP. Different superscripts indicate significant differences between control and treatment hatch rates within spawning.
suspensions were screened through 62-μm mesh prior to fluorescent activated cell sorting (FACS) on a Calibur Flow Cytometer (Beckton Dickinson Immunocytometry Systems San Jose, California, USA). A total of 5000 to 20,000 shrimp cells were analysed by FACS for each sample. The level of ploidy in each treatment and control non-viable embryo and nauplii sample were analysed using ModFit LT software (Verity Software House, Topsham, Maine, USA). The manual model option was used for all analyses, allowing the use of the diploid and aneuploid function, while also allowing the inclusion of an internal standard control (CRBC). Prior to analysis, cells were gated to remove doublets and eliminate debris
from statistical calculations (Fig. 2ii). ModFit analysis resulted in a diagrammatic output, statistical calculation of the different cell sizes and calculation of the quantity of cells within each sample (Fig. 2i). Ploidy levels of non-viable embryos compared to nauplii were analysed using the paired t-test separately for the 5- and 16-min duration treatment groups (SAS Institute, 1999). Comparisons of the ploidy levels between the 5-min and 16-min duration treatments were analysed by t-test separately for the non-viable embryos and the nauplii (SAS Institute, 1999). 3. Results 3.1. Embryo development and hatch rate A 1- to 2-min delay in embryonic development was observed in treated embryos compared to controls for each mitotic division up to the four-cell stage for all eight spawnings. However, beyond these initial delays in development up to the four-cell stage, the rate of embryo development was similar in controls and treatments for all spawnings, with the first three mitotic divisions occurring between 32- to 38-min psd, 36–42min psd and 64- to 74-min psd, respectively. Hatch rates of treated and control embryos were highly variable within each spawning. Control hatch
Table 3 Percentage triploidy of treatment non-viable embryo and nauplii samples when analyzed using ModFit LT software Spawning
Percentage triploidy 6-DMAP treatment
1 1 2 2 3 3 4 5 6 7 8 4 4 5 6 7 8
Non-viable embryos
Nauplii
Concentration
psd time on
psd time off
Duration
Replicate 1
Replicate 2
Average ± S.E.
Replicate 1
Replicate 2
Average ± S.E.
150μM 150μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 400μM 200μM 200μM 200μM 200μM 200μM
3min 3min 2min 2min 40s 1min 30s 2min 20s 1min 20s 1min 10s 2min 1min 10s 1min 2min 3min 2min 2min 2min 2min
8min 8min 6min 7min 6min 7min 6min 6min 6min 6min 6min 18min 19min 18min 18min 18min 18min
5min 5min 4min 4min 20s 4min 30s 4min 40s 4min 40s 4min 40s 4min 4min 50s 5min 16min 16min 16min 16min 16min 16min
37.81 51.80 81.80 52.62 n.a. 29.57 99.23 58.11 55.00 43.24 43.15 n.a. n.a. 11.71 98.96 56.43 49.03
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 57.01 n.a. 54.04 n.a. n.a. 24.92 66.63 24.90 87.36
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 56.01 ± 1.01 n.a. 48.60 ± 5.45 n.a. n.a. 18.32 ± 6.61 82.80 ± 16.17 40.67 ± 15.77 68.20 ± 19.17
48.95 20.21 25.72 5.60 68.95 17.63 49.24 49.33 98.28 17.78 84.27 56.85 47.50 66.23 88.22 93.30 75.76
50.61 15.70 22.21 7.64 n.a. 17.86 83.11 54.59 97.46 n.a. 98.85 n.a. n.a. 81.46 99.24 95.47 89.75
49.78 ± 0.83 17.96 ± 2.26 23.97 ± 1.75 6.62 ± 1.02 n.a. 17.75 ± 0.12 66.18 ± 16.94 51.96 ± 2.63 97.87 ± 0.41 n.a. 91.56 ± 7.29 n.a. n.a. 73.86 ± 7.61 93.73 ± 5.51 94.39 ± 1.09 82.76 ± 7.00
No significant difference was found (P > 0.05) between triploid levels of non-viable Marsupenaeus japonicus embryos and nauplii, and between the 4- to 5-min and 16-min treatment durations. n.a. = not applicable.
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rates were either greater than (P < 0.05) or similar to (P > 0.05) treatment hatch rates in all spawnings (Table 2). 3.2. Level of ploidy ModFit LT analysis of FACS outputs revealed that there were diploids and triploids among treated nonviable embryos and nauplii for all spawnings and induction durations, while all control non-viable embryos and nauplii were diploid. There was no significant difference (P > 0.05) in the triploidy level of non-viable embryos compared to nauplii for either 5min or 16-min treatment durations. The percentage of triploid non-viable embryos and nauplii when exposed to 6-DMAP for a 5-min duration ranged from 29.57% to 99.23% (average 55.28 ± 5.45%) and from 5.60% to 98.85% (average 46.70 ± 7.20%), respectively (Table 3). The percentage of triploid non-viable embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 ± 11.00%) and from 47.5% to 99.24% (average 79.38 ± 5.24%), respectively (Table 3). Triploid induction rates for the 5-min compared to the 16-min durations were not significantly different (P > 0.05) for the non-viable embryos and nauplii. Both the 5- and 16-min durations resulted in successful triploid induction for all spawning aliquots that were treated with 6DMAP. 3.3. Noteworthy observations As PBI begins to appear at 4-min 10-s post-spawning in M. japonicus embryos (Hudinaga, 1941), the immediate collection and sensitive detection of spawned embryos is critical to prevent PBI extrusion. From the present study, one observation of the spawning behavior of M. japonicus was essential for development of the final spawning tank design and method of embryo collection used. It was critical that the embryo collection trays were in place and ready to collect embryos prior to spawning so that by the time the spawnings were detected by the automated alarm system the trays had already collected suitable numbers of embryos for induction and could be removed immediately. The reduced water volume of 100-L spawning tanks to 40 L also increased the number of collected embryos in such a short time frame. Initially, tanks were not fitted with a false 2-mm oyster mesh base and shrimp were allowed to come into contact with embryo collection trays. Eight out of eight stage IV gravid, impregnated female M. japonicus that were ablated and placed in spawning
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tanks without a false oyster mesh base regressed and molted without spawning. It was thought that the embryo collection trays were interfering with the spawning behavior of the females, preventing them from swimming around the tank. Without the false base, the females were observed sitting in the trays bumping into its side for hours at a time. By using the false base (Fig. 1) all eight ablated stage IV impregnated females, which were from the same batch of females used previously, spawned viable embryos. 4. Discussion In this study, treatment of M. japonicus embryos with 6-DMAP applied during meiosis I was effective at preventing polar body release at 1- to 8-min psd, a time corresponding with the release of PBI (Hudinaga, 1941; Norris et al., 2005). Similarly, 6-DMAP treatment applied during meiosis I and II was effective at stopping PB release at 2- to 19-min psd, a time corresponding to the release of both PBI and II (Hudinaga, 1941; Norris et al., 2005). All treatment durations resulted in the induction of triploids with no other alternative ploidy levels to the normal diploids being observed. The production of triploids from preventing PBI extrusion in this study is consistent with results from a study on Pacific oyster (C. gigas) in which only triploid embryos were produced by preventing PBI extrusion (Quillet and Panelay, 1986). In M. japonicus embryos which have retained PBI, it is likely that the chromosomes underwent united bipolar segregation (perfect alignment and subsequent segregation; Guo et al., 1992). If the chromosomes of PBI inhibited embryos underwent separated bipolar segregation, which is thought to be the only other chromosome segregation pattern than can result in PBI triploids (Guo et al., 1992), resulting ploidy levels would have included tetraploids as well as the observed triploids and diploids. Other successful reports of triploid induction in marine species by preventing PBI extrusion also resulted in progeny of other ploidy levels. For example, prevention of PBI extrusion resulted in triploid, tetraploid and aneuploid Pacific oysters (Guo et al., 1992) and triploid and tetraploid sea cucumbers (Apostichopus japonicus Liao) (Chang and Xiang, 2002). It appears that the prevention of PBI extrusion complicates subsequent chromosome segregation as suggested by Guo et al. (1992), and can result in many different ploidy levels. Resulting ploidy levels from preventing PBI extrusion are clearly species dependant but are also likely to be influenced by the type, amount and duration of shock applied (e.g. chemical, thermal, pressure).
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The induction of triploid M. japonicus by treatment with 6-DMAP for a duration of 16 min, corresponding to a time period over which both PBI and II would be normally extruded, was unexpected. Theoretically the prevention of PBI and II extrusion should result in pentaploids (Cooper and Guo, 1989), as has been proven true for the dwarf surfclam (M. lateralis) (Yang and Guo, 2004). As developmental observations in the present study showed that treatment embryos were only 1 to 2 min behind that of controls up until the four-cell stage, and that treatment embryos underwent their first nuclear division in mitosis at a time similar to that reported for normal M. japonicus development (Hudinaga, 1941; Norris et al., 2005), it is possible to conclude that PBI and II inhibition in M. japonicus results in triploid progeny. However, this is difficult to explain if the PBI triploids were produced through united bipolar segregation. The fact that triploid M. japonicus are produced by 6DMAP treatments, which are timed to stop PBI or PBI and II extrusion, may prove interesting from a commercial viewpoint if they are more heterozygous than PBII triploid M. japonicus. Improved growth rates have been reported for PBI triploids of numerous aquatic species when compared to PBII triploids and normal diploids (i.e. soft-shell clam (Mya arenaria), Allen et al., 1982; American oyster (C. viginica), Stanley et al., 1984; Pacific oyster (C. gigas), Yamamoto et al., 1988; blue mussel (M. edulis), Beaumonet and Kelly, 1989; pearl oyster (Pinctada martensii), Jiang et al., 1991). This improved performance is attributed to increased heterozygosity of PBI triploids, resulting either from the maternal parent being more heterozygous due to the retention of both maternal chromosomes, or because the spermatozoa carries a heterologous allele. In the present study, 6-DMAP had varying effects on embryo hatch rate for the different spawnings. However, control embryos always hatched as well as if not better than treated embryos. In an earlier ploidy study on M. japonicus, final 6-DMAP concentrations of 200 and 400 μM significantly reduced hatch rates (Norris et al., 2005). Differences in handling stresses and variation in water temperatures during the treatment process are possible causes of variation in hatch rates between the two studies and between spawnings in the present study. In conclusion, this study clearly shows that treatment of M. japonicus embryos with 6-DMAP timed to stop PBI or PBI and II extrusion results in the production of viable triploid nauplii. To our knowledge, this is the first report of successful PBI, and PBI and II inhibition in shrimp, and production of triploids using these inhibi-
tion regimes. Future studies aim to use M. japonicus microsatellite markers as developed by Moore et al. (1999) to conclusively determine if PBI triploids are more heterozygous than PBII triploids. Acknowledgements The authors would like to thank Frank Coman, Tamera Callaghan, Greg Coman and Lucy Hurrey for their experimental support, and Paula Hall and Grace Chojnowsky from the Queensland Institute of Medical Research for their assistance with flow cytometry. References Allen Jr., S.K., Gagnon, P.S., Hidu, H., 1982. Induced triploidy in the soft-shell clam. Journal of Heredity 73, 421–428. Arai, K.F., Naito, F., Fujino, K., 1986. Triploidization of the Pacific abalone with temperature and pressure treatments. Bulletin of the Japanese Society of Scientific Fisheries 52 (3), 417–422. Beaumonet, A.R., Kelly, K.S., 1989. Production and growth of triploid Mytilus edulis larvae. Journal of Experimental Marine Biology and Ecology 132, 69–84. Chang, Y., Xiang, J., 2002. Inducement of polyploidy sea cucumber (Apostichopus japonicus Liao). Journal of Dalian Fisheries 17 (1), 1–7. Chourrout, D., Chevassus, B., Krieg, F., Happe, A., Burger, G., Renard, P., 1986. Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females—potential of tetraploid fish. Theoretical and Applied Genetics 72 (2), 193–206. Coman, F.E., Norris, B.J., Pendrey, R.C., Preston, N.P., 2003. A simple spawning detection and alarm system for penaeid shrimp. Aquaculture Research 34, 1359–1360. Cooper, K., Guo, X., 1989. Polyploid Pacific oyster produced by blocking polar body I and II with cytochalasin B. Journal of Shellfish Research 8, 412. Crocos, P.J., Coman, G.J., 1997. Seasonal and age variability in the reproductive performance of Penaeus semisulcatus broodstock: optimising broodstock selection. Aquaculture 155, 55–67. Guo, X., Allen Jr., S.K., 1994. Viable tetraploids in the Pacific oyster (Crassostrea gigas Thunberg) produced by inhibiting polar body I in eggs from triploids. Molecular Marine Biology and Biotechnology 3, 42–50. Guo, X., Cooper, K., Hershberger, W.K., Chew, K.K., 1992. Genetic consequences of blocking polar body I with cytochalasin B in fertilised eggs of the Pacific oyster, Crassostrea gigas: I. Ploidy of resultant embryos. Biological Bulletin 183, 381–386. Guo, X., DeBrosse, G., Allen Jr., S.K., 1996. All triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids. Aquaculture 142, 149–161. Huayong, A., Allen Jr., S.K., 2002. Hybridisation of tetraploid and diploid Crassostrea gigas (Thunberg) with diploid C. ariakensis (Fujita). Journal of Shellfish Research 21 (1), 137–143. Hudinaga, K., 1941. Reproduction development and rearing of Penaeus japonicus Bate. Japanese Journal of Zoology 10, 305–390. Jiang, W., Xu, G., Lin, Y., Li, G., 1991. Comparison of growth between triploid and diploid of Pinctada martensi (D). Journal of Tropical Oceanography 10 (3), 1–7.
M.J. Sellars et al. / Aquaculture 256 (2006) 337–345 Liu, S., Liu, Y., Zhou, G., Zhang, X., Luo, C., Feng, H., He, X., Zhu, G., Yang, H., 2001. The formation of tetraploid stocks of red crucian carp × common carp hybrids as an effect of interspecific hybridisation. Aquaculture 192, 171–186. Moore, S.S., Whan, V., Davis, G.P., Byrne, K., Hetzel, D.J.S., Preston, N., 1999. The development and application of markers for the Kuruma prawn Penaeus japonicus. Aquaculture 173, 19–32. Norris, B.N., Coman, F.E., Sellars, M.J., Preston, N.P., 2005. Triploid induction in Penaeus japonicus (Bate) with 6-dimethylaminopurine. Aquaculture Research 36, 202–206. Peruzzi, S., Guo, X., 2002. Tetraploid induction by meiosis inhibition with cytochalasin B in the dwarf surfclam, Mulinia lateralis Say: effects of temperature. Journal of Shellfish Research 21, 677–684. Preston, N.P., Crocos, P., Jackson, C., Duncan, P., Zipf, M., Koenig, R., 2001. Farming of the Kuruma shrimp Penaeus japonicus in Australia —a case history. Aquaculture 2001 Book of Abstracts, p. 540. Quillet, E., Panelay, P.J., 1986. Triploidy induction by thermal shocks in the Pacific oyster, Crassostrea gigas. Aquaculture 57, 271–279. Robinson, J.P. (Ed.), 1993. Handbook of Flow Cytometry Methods. Wiley-Liss, New York. SAS Institute Software, 1999. SAS/STAT software, version 7. SAS Institute Inc., Cary, NC, USA. Scarpa, J., Wada, K.T., Komaru, A., 1993. Induction of tetraploid mussel by suppression of polar body formation. Bulletin of the Japanese Society of Fisheries Science 59, 2017–2023. Sellars, M.J., Preston, N.P., 2005. The effects of ionizing radiation on the reproductive capacity of adult Penaeus (Maruspenaeus) japonicus (Bate). Aquaculture Research 36 (11), 1144–1147. Sellars, M., Coman, F., Norris, B., Preston, N., 2003. Relative survival and growth rates of triploid and diploid female Penaeus japonicus. World Aquaculture Society Book of Abstracts, p. 713. Sellars, M., Coman, F., Preston, N., 2004. Protecting genetically improved shrimp via induced sterility. Australasian Aquaculture Book of Abstracts, p. 265. Sellars, M.J., Degnan, B.M., Carrington, L.E., Preston, N.P., 2005. The effects of ionizing radiation on the reproductive capacity of adult Penaues (Marsupenaeus) japonicus (Bate). Aquaculture 250, 194–200.
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Sellars, M.J., Coman, F.E., Degnan, B.M., Preston, N.P., in press. The effectiveness of heat, cold and 6-dimethylaminopurine shocks for inducing tetraploidy in the Kuruma shrimp, Marsupenaeus japonicus (Bate). Journal of Shellfish Research. Stanley, J.G., Allen Jr., S.K., Hidu, H., 1981. Ploidy induced in the American oyster, Crassostrea virginica, with cytochalasin B. Aquaculture 23, 1–10. Stanley, J.G., Hidu, H., Allen Jr., S.K., 1984. Growth of American oysters increased by polyploidy induced by blocking meiosis I but not meiosis II. Aquaculture 37, 147–155. Stephens, L.B., 1989. Inhibition of the first polar body formation in Crassostrea gigas produces tetraploids, not meiotic I triploids. MS thesis, University of Washinton, Seattle. Stephens, L.B., Downing, S.L., 1988. Inhibition of the first polar body formation in Crassostra gigas produces tetraploids, not meiotic I triploids. Journal of Shellfish Research 7 (3), 550–551. Wang, Z., Guo, X., Allen Jr., S.K., Wang, R., 2002. Heterozygosity and body size in triploid Pacific oysters, Crassostrea gigas Thunberg, produced from meiosis II inhibition and tetraploids. Aquaculture 204 (3–4), 337–348. Xiang, J., Zhou, L., Zhang, F., 2001. Triploid induction in penaeidae shrimps with special reference to a new chemical inducing reagent 6-DMAP (6-dimethylaminopurine). Aquaculture 2001: Book of Abstracts, p. 705. Yamamoto, S., Sugawara, Y., 1988. Induced triploidy in the mussel, Mytilus edulis, by temperature shock. Aquaculture 72, 21–29. Yamamoto, S., Sugaware, Y., Nomaru, T., Oshino, A., 1988. Induced triploid in the Pacific oyster, Crassostrea gigas, and performance of triploid larvae. Tohoku Journal of Agricultural Research 39 (1), 47–59. Yang, H., Guo, X., 2004. Tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis (Say 1822): effects of cytochalasin B duration. Aquaculture Research 35, 1187–1194. Yang, H., Zhang, F., Guo, X., 2000. Triploid and tetraploid zhikong scallop, Chlamys farreri Jones et Preston, produced by inhibiting polar body I. Marine Biotechnology 2, 466–475.
Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine Melony J. Sellars a,b,⁎, Bernard M. Degnan b , Nigel P. Preston a a
CSIRO Food Futures National Research Flagship, CSIRO Marine Research, 233 Middle Street, Cleveland, Qld 4163, Australia b School of Integrative Biology, The University of Queensland, Brisbane, Qld, 4072, Australia Received 23 November 2005; received in revised form 31 January 2006; accepted 16 February 2006
Abstract This study investigated the chromosome ploidy level of Marsupenaeus (Penaeus) japonicus (Bate) non-viable (unhatched) embryos and nauplii after exposure to 6-dimethylaminopurine (6-DMAP), timed to stop either polar body (PB) I, or PBI and II extrusion. Embryos from eight separate families or spawnings were exposed to 150 or 200 μM 6-DMAP from 1- to 3-min postspawning detection (psd) for a 4- to 5-min duration (timed to stop PBI extrusion). Separate aliquots of embryos from five of the same spawnings were also exposed to 200 μM of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). For one spawning, a third aliquot of embryos was exposed to 400 μM of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). At 18-h psd, non-viable embryo and nauplii samples were taken separately for fluorescent activated cell sorting (FACS). FACS revealed that there were diploids and triploids among all treated non-viable embryos and nauplii. All control non-viable embryos and nauplii were diploid. Percentages of triploid induction for the 4- to 5-min and 16-min durations were not significantly different (P > 0.05). Additionally, no difference was found in the triploidy level of nonviable embryos compared to nauplii in these treatments. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 4- to 5-min duration ranged from 29.57% to 99.23% (average 55.28 ± 5.45%) and from 5.60% to 98.85% (average 46.70 ± 7.20%), respectively. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 ± 11.00%) and from 47.5% to 99.24% (average 79.38 ± 5.24%), respectively. To our knowledge, this is the first documentation of successful PBI or PBI and II inhibition in shrimp. This study conclusively shows that treatment of M. japonicus embryos with 6-DMAP at 1- to 3-min psd for either a 4- to 5-min duration (timed to stop PBI extrusion) or 16-min duration (timed to stop both PBI and II extrusion) results in viable triploid nauplii. © 2006 Elsevier B.V. All rights reserved. Keywords: Genetic protection; Kuruma prawn; Chromosome segregation
1. Introduction ⁎ Corresponding author. CSIRO Food Futures National Research Flagship, CSIRO Marine Research, 233 Middle Street, Cleveland, Qld 4163, Australia. Tel.: +61 7 3826 7359; fax: +61 7 3826 7222. E-mail address: [email protected] (M.J. Sellars). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.052
Shrimp domestication and genetic improvement programs in Australia are most advanced for Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) (Preston et al., 2001). As a result, substantial research
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efforts have focused on developing suitable approaches by which to confer sexual sterilization to prevent downstream loss of genetic resources at the live export market and the introduction of genetically altered strains into natural fisheries (Sellars et al., 2004; Norris et al., 2005). In other invertebrate species, the induction of sexual sterilization has additionally been found to confer desirable traits such as improved growth rates or pathogen resistance. To date, research efforts for M. japonicus have investigated irradiation (Sellars and Preston, 2005; Sellars et al., 2005), triploidy (by prevention of polar body (PB) II extrusion) (Xiang et al., 2001; Sellars et al., 2003, 2004; Norris et al., 2005) and tetraploidy (by prevention of the first mitotic division) (Sellars et al., in press) as potential ways to achieve sexual sterility. While irradiation has been successfully used to impair the reproductive capacity of M. japonicus (Sellars et al., 2005), the most successful sterilization of this species to date has been achieved through induction of triploidy (Sellars et al., 2003, 2004; Norris et al., 2005). Triploid M. japonicus produced through prevention of PBII extrusion using chemical shocks are sterile and all female (Sellars et al., 2003, 2004; Norris et al., 2005). However, prevention of PBII extrusion never results in 100% triploid progeny, due to inherent variation among developing embryos, with induction rates ranging from 0% to 85.9% (Norris et al., 2005). Producing triploids by the mating of tetraploids with diploids may provide a solution to these variable induction rates and result in 100% triploid progeny. This approach has been successful for several marine species including Pacific oysters (Crassostrea gigas) (Guo and Allen, 1994; Guo et al., 1996; Wang et al., 2002), oyster hybrids (C. gigas × C. ariakensis) (Huayong and Allen, 2002), rainbow trout (Oncorhynchus mykiss) (Chourrout et al., 1986) and carp hybrids (Crassius auratus red var. × Cyprinus carpio L.) (Liu et al., 2001). Production of viable tetraploids is a prerequisite for producing triploids in this manner. In a recent study, tetraploid M. japonicus embryos were produced by stopping the first division in mitosis (Sellars et al., in press). However, these tetraploid embryos did not hatch, suggesting that mitotic tetraploid M. japonicus may not be viable. One potential alternative method to mitotic inhibition for tetraploid induction is the prevention of PBI extrusion in meiosis I. The prevention of PBI extrusion has been reported to complicate subsequent chromosome segregation (Guo et al., 1992) and result in many different ploidy combinations including viable tetraploids. Triploid and tetraploid progeny resulted from preventing PBI extrusion in the American oyster (Crassostrea virginica)
(Stanley et al., 1981), the blue mussel (Mytilus edulis) (Yamamoto and Sugawara, 1988) and the Pacific abalone (Haliotis discus hannai) (Arai et al., 1986). In comparison, the prevention of PBI extrusion only produced tetraploids in dwarf surfclams (Mulinia lateralis) (Yang and Guo, 2004) and zhikong scallops (Chlamys farreri) (Yang et al., 2000). In the pacific oyster (C. gigas), the prevention of PBI extrusion using thermal stress resulted in triploids only (Quillet and Panelay, 1986) while using cytochalasin B stress resulted in tetraploids (Stephens and Downing, 1988; Stephens, 1989) or a combination of triploids, tetraploids and aneuploids (Guo et al., 1992). Prevention of both PBI and II extrusion has also been shown to result in tetraploid mussels (Mytilus galloprovincialis) (Scarpa et al., 1993) and dwarf surfclams (M. lateralis) (Peruzzi and Guo, 2002). Recently, however, a contradictory report found that preventing PBI and II in dwarf surfclams produced pentaploids (Yang and Guo, 2004), a more theoretically plausible outcome (Cooper and Guo, 1989). Both PBI triploids and tetraploids have been found to have commercial advantages in several aquatic cultured species. Triploids produced by preventing PBI extrusion have been reported to grow faster than those resulting from preventing PBII extrusion, probably due to increased heterozygosity (e.g. soft-shell clam, Allen et al., 1982; American oyster, Stanley et al., 1984; Pacific oyster, Yamamoto et al., 1988; blue mussel, Beaumonet and Kelly, 1989; pearl oyster, Jiang et al., 1991). As discussed, tetraploids are of commercial value for mating with diploids to produce 100% triploid progeny. Production of PBI triploid and tetraploid M. japonicus may therefore have considerable commercial value to the industry. This study investigated the effect of stopping PBI, or both PBI and PBII extrusion on subsequent chromosome ploidy level of M. japonicus non-viable (unhatched) embryos and nauplii. 2. Materials and methods 2.1. Broodstock, spawning and embryo collection Wild-caught M. japonicus broodstock (25 females:20 males) were acclimated to a reversed light cycle (12h light:12-h dark) for 30 days in 2000-L sand-substrate tanks (Crocos and Coman, 1997). Tanks received 1.6 L min− 1 of 27 ± 2 °C seawater. Shrimp were fed commercial M. japonicus pellets ad libitum once per day and freshly cut squid (2cm3) (Loligo spp.) three times weekly during dark hours.
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Fig. 1. Cross section view of a 100-L spawning tank fitted with an automated spawning detection system, false 2-mm oyster mesh base and glass trays for embryo collection.
light cycle and water source (at a flow rate of 0.2L min− 1) as the acclimation tanks. Females in spawning tanks were fed one piece of freshly cut squid (2cm3) (Loligo spp.) daily during light hours.
After acclimation, gravid females (average weight 33 ± 3.41g) were selected by shining a torch beam through their dorsal exoskeleton during dark hours. Gravid females impregnated with spermatophores were unilaterally eyestalk ablated using hot forceps and spawned in 100-L circular spawning tanks filled to 40L and fitted with an automated spawning detection system (Coman et al., 2003). Tanks were fitted with a false 2-mm oyster mesh base suspended 10cm above the bottom of the tank (Fig. 1). Two glass trays (21 × 15 × 6 cm, length × width × depth) were placed beneath the false oyster mesh base to collect embryos as they were released from the female (three glass trays were used in the case of spawning 4). For ease of explanation, spawned eggs, whether fertilized or unfertilized, will be referred to as embryos from herein. Spawning tanks received the same
2.2. Experimental design Spawnings from eight different females were exposed to various concentrations of 6-DMAP at 1to 3-min post-spawning detection (psd) for either a 4to 5-min duration or both a 4- to 5-min duration and a 16-min duration. All 4- to 5-min duration treatments will be referred to from herein as a 5-min duration for ease of explanation. The experimental design for each spawning was dependant upon the availability of freshly prepared 6-DMAP solution, efficiency of
Table 1 Experimental design for treatment aliquots or glass trays for the eight separate spawnings of Marsupenaeus japonicus Spawning
Aliquot 1 Aliquot 2 Aliquot 3
6-DMAP (μM) Duration (min) 6-DMAP (μM) Duration (min) 6-DMAP (μM) Duration (min)
n.a. = not applicable.
1
2
3
4
5
6
7
8
150 4 to 5 150 4 to 5 n.a.
200 4 to 5 200 4 to 5 n.a.
200 4 to 5 200 4 to 5 n.a.
200 4 to 5 200 16 400 16
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
200 4 to 5 200 16 n.a.
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glass tray removal from spawning tanks and rate of embryo concentration. As a result, experimental designs had slight variations between spawnings (Table 1). Spawning 1 had two aliquots of collected embryos (each glass tray of embryos making up an aliquot) that were both exposed for a 5-min duration to 150 μM 6-DMAP. Spawnings 2 and 3 also had two aliquots of embryos each that were exposed for a 5min duration; however, the 6-DMAP concentration was 200 μM. Spawning 4 had three aliquots of embryos, with two aliquots being exposed to 200μM 6-DMAP for either a 5- or 16-min duration, and the third aliquot being exposed to 400 μM 6-DMAP for a 16-min duration. Spawnings 5 to 8 all had two aliquots of embryos, which were exposed to 200μM 6-DMAP, with one aliquot of each spawning being exposed for a 5-min duration and the other aliquot being exposed for a 16-min duration. A control (untreated) aliquot of embryos was collected from spawning tanks for each spawning at 1- to 4-min psd.
trays that were present in the tank during spawning, and from glass trays added to the tank 1-min post-spawning (data not presented). At 1min prior to 6-DMAP treatment being due to cease, excess seawater was pumped off leaving embryos in a 2- to 3-mm layer of seawater. Once 6-DMAP treatment time had elapsed, glass trays containing embryos and remaining seawater were then placed in 9L of 60 μM filtered, UV sterilized, 27 °C seawater. After embryos had settled (approximately 2 min), 8 L of seawater was decanted off and replaced by a fresh 8L of seawater. Each aliquot of embryos received three of these 8-L water exchanges to remove all remaining 6DMAP. Exchanges were completed by 20-min psd for all 5-min duration treatments and by 30-min psd for all 16-min duration treatments. Aliquots of control embryos were exposed to the same handling stress as treatment embryos. After treatments and water exchanges were complete, embryos were incubated in 9 L of seawater at 27 ± 2 °C with light aeration until hatching approximately 12 h later (Hudinaga, 1941).
2.3. Ploidy induction using 6-dimethylaminopurine The initiation of spawning, as detected by the automated detection system (Coman et al., 2003), was taken as time 0. Glass trays were removed immediately upon spawning detection (i.e. plastic lid was fastened to the glass tray which was subsequently lifted out of the tank) and the water level was reduced to 200 mL by siphoning (except for spawning 1 which was 280 mL and the 400μM 6-DMAP treatment in spawning 4 which was 150 mL), leaving approximately 80% of the embryos which were originally in the tray when lifted out of the tank. The resulting number of embryos in each glass tray varied with spawning size (average 72.5 ± 14.11 embryos/glass tray). For the 200μM 6-DMAP treatments, a freshly prepared 50-mL aliquot of 1 mM 6DMAP (made up in 60 μM filtered, UV sterilized 27 °C seawater) was added to each glass tray containing the 200 mL of seawater and embryos. For the 400 μM 6DMAP treatments, two 50-mL aliquots of 1mM 6DMAP were added to the glass trays. For the 150μM 6DMAP treatments, the water level was at 280mL and a 50-mL aliquot of 1mM 6-DMAP was added. Time of treatment varied from 1-min to 3-min psd and was dependent on human proximity to the spawning tanks at time of alarm. Immediately after 6-DMAP treatments were applied, another glass tray was placed in the spawning tank to collect embryos for a control (untreated) aliquot which was removed after 1 min. Preliminary studies showed that there was no difference between hatch rates of embryos when taken from glass
2.4. Monitoring of embryonic development and determination of hatch rate Using light microscopy the development rate of 10 to 20 embryos from each treatment and control were monitored until they reached the four-cell stage. Brief notes were made on the psd time when embryos were one-cell, elongating, kinked and about to divide, twocell and four-cell (Hudinaga, 1941). Where the number of non-viable embryos and nauplii present in a treatment were estimated to be more than required for fluorescent activated cell sorting (FACS) analysis, samples were collected to establish hatch rate data. Within each treatment and control, three 50-mL samples of non-viable embryos and nauplii were taken from the 9-L incubation buckets at approximately 4 h after the first signs of hatching in controls. Prior to samples being taken, the contents of each bucket were mixed so that samples were homogeneous. The number of non-viable embryos and nauplii within each replicate sample were counted within 24 h of being sampled. Percentage hatch rates of embryos in the control and treatment aliquots were analysed separately for each spawning using one-way ANOVA (SAS Institute, 1999). 2.5. FACS analysis and ModFit LT analysis Two replicate samples of between 25 to 50 nonviable embryos and 10 to 30 nauplii were separately
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Fig. 2. Example of a FACS data output after ModFit LT analysis for spawning three nauplii from a (A) control and (B) 200 μM 6-DMAP (4- to 5-min duration) treatment. Gated cells were analyzed using a FL2 histogram for the DNA content to determine the percentage ploidy and respective cell cycle distribution (i). Cells were gated to remove doublets and eliminate debris from the analysis (ii).
sampled at approximately 16-h psd for each spawning from all controls and treatments, with each sample containing less than 100 μL of sample and seawater. Samples were snap frozen in liquid nitrogen and stored at − 65 °C for up to 7 days before being analysed. The limitation of only having 1 to 3min post-spawning to collect embryos for treatment meant that in some instances there were only enough non-viable embryos or nauplii for one replicate sample for FACS analysis. In three instances, there were too few non-viable embryos for FACS analysis.
For FACS analyses, 500μL of marine phosphate buffered solution (MPBS) (11.0g L− 1 NaCl, 0.2g L− 1 KCl, 1.15g L− 1 Na2HPO4·2H2O) propidium iodide (PI) stain (MPBS containing 0.1% Triton X-100, 0.02mg mL− 1 RNase A, 0.02mg mL− 1 PI) was added to each sample. Samples were then homogenized individually by aspiration eight times through a 25-G needle pushed firmly against the side of the sample tube. After homogenation 8μL of a 1:100 dilution of the internal standard, glutaraldehyde fixed, chicken red blood cells (CRBC, Handbook of Flow Cytometry Methods, 1993) was added to each sample. Cell
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Table 2 Means (±S.E.) of the percentage hatch rate (nauplii/total embryos) in controls and treatments for spawnings five, six, seven and eight of Marsuepnaeus japonicus Treatment
Spawning 5
Spawning 6
a
Spawning 7 Spawning 8
a
Control 79.15 ± 2.59 67.88 ± 2.12 66.67 ± 3.21 56.03 ± 1.28a 5-min 26.93 ± 2.39b 48.89 ± 1.11b 71.03 ± 2.41 27.03 ± 1.21b duration 16-min 26.93 ± 2.39b 72.34 ± 2.73a 58.33 ± 4.81 24.95 ± 4.60b duration Treatment concentrations for these four spawnings were 200μM 6DMAP. Different superscripts indicate significant differences between control and treatment hatch rates within spawning.
suspensions were screened through 62-μm mesh prior to fluorescent activated cell sorting (FACS) on a Calibur Flow Cytometer (Beckton Dickinson Immunocytometry Systems San Jose, California, USA). A total of 5000 to 20,000 shrimp cells were analysed by FACS for each sample. The level of ploidy in each treatment and control non-viable embryo and nauplii sample were analysed using ModFit LT software (Verity Software House, Topsham, Maine, USA). The manual model option was used for all analyses, allowing the use of the diploid and aneuploid function, while also allowing the inclusion of an internal standard control (CRBC). Prior to analysis, cells were gated to remove doublets and eliminate debris
from statistical calculations (Fig. 2ii). ModFit analysis resulted in a diagrammatic output, statistical calculation of the different cell sizes and calculation of the quantity of cells within each sample (Fig. 2i). Ploidy levels of non-viable embryos compared to nauplii were analysed using the paired t-test separately for the 5- and 16-min duration treatment groups (SAS Institute, 1999). Comparisons of the ploidy levels between the 5-min and 16-min duration treatments were analysed by t-test separately for the non-viable embryos and the nauplii (SAS Institute, 1999). 3. Results 3.1. Embryo development and hatch rate A 1- to 2-min delay in embryonic development was observed in treated embryos compared to controls for each mitotic division up to the four-cell stage for all eight spawnings. However, beyond these initial delays in development up to the four-cell stage, the rate of embryo development was similar in controls and treatments for all spawnings, with the first three mitotic divisions occurring between 32- to 38-min psd, 36–42min psd and 64- to 74-min psd, respectively. Hatch rates of treated and control embryos were highly variable within each spawning. Control hatch
Table 3 Percentage triploidy of treatment non-viable embryo and nauplii samples when analyzed using ModFit LT software Spawning
Percentage triploidy 6-DMAP treatment
1 1 2 2 3 3 4 5 6 7 8 4 4 5 6 7 8
Non-viable embryos
Nauplii
Concentration
psd time on
psd time off
Duration
Replicate 1
Replicate 2
Average ± S.E.
Replicate 1
Replicate 2
Average ± S.E.
150μM 150μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 200μM 400μM 200μM 200μM 200μM 200μM 200μM
3min 3min 2min 2min 40s 1min 30s 2min 20s 1min 20s 1min 10s 2min 1min 10s 1min 2min 3min 2min 2min 2min 2min
8min 8min 6min 7min 6min 7min 6min 6min 6min 6min 6min 18min 19min 18min 18min 18min 18min
5min 5min 4min 4min 20s 4min 30s 4min 40s 4min 40s 4min 40s 4min 4min 50s 5min 16min 16min 16min 16min 16min 16min
37.81 51.80 81.80 52.62 n.a. 29.57 99.23 58.11 55.00 43.24 43.15 n.a. n.a. 11.71 98.96 56.43 49.03
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 57.01 n.a. 54.04 n.a. n.a. 24.92 66.63 24.90 87.36
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 56.01 ± 1.01 n.a. 48.60 ± 5.45 n.a. n.a. 18.32 ± 6.61 82.80 ± 16.17 40.67 ± 15.77 68.20 ± 19.17
48.95 20.21 25.72 5.60 68.95 17.63 49.24 49.33 98.28 17.78 84.27 56.85 47.50 66.23 88.22 93.30 75.76
50.61 15.70 22.21 7.64 n.a. 17.86 83.11 54.59 97.46 n.a. 98.85 n.a. n.a. 81.46 99.24 95.47 89.75
49.78 ± 0.83 17.96 ± 2.26 23.97 ± 1.75 6.62 ± 1.02 n.a. 17.75 ± 0.12 66.18 ± 16.94 51.96 ± 2.63 97.87 ± 0.41 n.a. 91.56 ± 7.29 n.a. n.a. 73.86 ± 7.61 93.73 ± 5.51 94.39 ± 1.09 82.76 ± 7.00
No significant difference was found (P > 0.05) between triploid levels of non-viable Marsupenaeus japonicus embryos and nauplii, and between the 4- to 5-min and 16-min treatment durations. n.a. = not applicable.
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rates were either greater than (P < 0.05) or similar to (P > 0.05) treatment hatch rates in all spawnings (Table 2). 3.2. Level of ploidy ModFit LT analysis of FACS outputs revealed that there were diploids and triploids among treated nonviable embryos and nauplii for all spawnings and induction durations, while all control non-viable embryos and nauplii were diploid. There was no significant difference (P > 0.05) in the triploidy level of non-viable embryos compared to nauplii for either 5min or 16-min treatment durations. The percentage of triploid non-viable embryos and nauplii when exposed to 6-DMAP for a 5-min duration ranged from 29.57% to 99.23% (average 55.28 ± 5.45%) and from 5.60% to 98.85% (average 46.70 ± 7.20%), respectively (Table 3). The percentage of triploid non-viable embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 ± 11.00%) and from 47.5% to 99.24% (average 79.38 ± 5.24%), respectively (Table 3). Triploid induction rates for the 5-min compared to the 16-min durations were not significantly different (P > 0.05) for the non-viable embryos and nauplii. Both the 5- and 16-min durations resulted in successful triploid induction for all spawning aliquots that were treated with 6DMAP. 3.3. Noteworthy observations As PBI begins to appear at 4-min 10-s post-spawning in M. japonicus embryos (Hudinaga, 1941), the immediate collection and sensitive detection of spawned embryos is critical to prevent PBI extrusion. From the present study, one observation of the spawning behavior of M. japonicus was essential for development of the final spawning tank design and method of embryo collection used. It was critical that the embryo collection trays were in place and ready to collect embryos prior to spawning so that by the time the spawnings were detected by the automated alarm system the trays had already collected suitable numbers of embryos for induction and could be removed immediately. The reduced water volume of 100-L spawning tanks to 40 L also increased the number of collected embryos in such a short time frame. Initially, tanks were not fitted with a false 2-mm oyster mesh base and shrimp were allowed to come into contact with embryo collection trays. Eight out of eight stage IV gravid, impregnated female M. japonicus that were ablated and placed in spawning
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tanks without a false oyster mesh base regressed and molted without spawning. It was thought that the embryo collection trays were interfering with the spawning behavior of the females, preventing them from swimming around the tank. Without the false base, the females were observed sitting in the trays bumping into its side for hours at a time. By using the false base (Fig. 1) all eight ablated stage IV impregnated females, which were from the same batch of females used previously, spawned viable embryos. 4. Discussion In this study, treatment of M. japonicus embryos with 6-DMAP applied during meiosis I was effective at preventing polar body release at 1- to 8-min psd, a time corresponding with the release of PBI (Hudinaga, 1941; Norris et al., 2005). Similarly, 6-DMAP treatment applied during meiosis I and II was effective at stopping PB release at 2- to 19-min psd, a time corresponding to the release of both PBI and II (Hudinaga, 1941; Norris et al., 2005). All treatment durations resulted in the induction of triploids with no other alternative ploidy levels to the normal diploids being observed. The production of triploids from preventing PBI extrusion in this study is consistent with results from a study on Pacific oyster (C. gigas) in which only triploid embryos were produced by preventing PBI extrusion (Quillet and Panelay, 1986). In M. japonicus embryos which have retained PBI, it is likely that the chromosomes underwent united bipolar segregation (perfect alignment and subsequent segregation; Guo et al., 1992). If the chromosomes of PBI inhibited embryos underwent separated bipolar segregation, which is thought to be the only other chromosome segregation pattern than can result in PBI triploids (Guo et al., 1992), resulting ploidy levels would have included tetraploids as well as the observed triploids and diploids. Other successful reports of triploid induction in marine species by preventing PBI extrusion also resulted in progeny of other ploidy levels. For example, prevention of PBI extrusion resulted in triploid, tetraploid and aneuploid Pacific oysters (Guo et al., 1992) and triploid and tetraploid sea cucumbers (Apostichopus japonicus Liao) (Chang and Xiang, 2002). It appears that the prevention of PBI extrusion complicates subsequent chromosome segregation as suggested by Guo et al. (1992), and can result in many different ploidy levels. Resulting ploidy levels from preventing PBI extrusion are clearly species dependant but are also likely to be influenced by the type, amount and duration of shock applied (e.g. chemical, thermal, pressure).
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The induction of triploid M. japonicus by treatment with 6-DMAP for a duration of 16 min, corresponding to a time period over which both PBI and II would be normally extruded, was unexpected. Theoretically the prevention of PBI and II extrusion should result in pentaploids (Cooper and Guo, 1989), as has been proven true for the dwarf surfclam (M. lateralis) (Yang and Guo, 2004). As developmental observations in the present study showed that treatment embryos were only 1 to 2 min behind that of controls up until the four-cell stage, and that treatment embryos underwent their first nuclear division in mitosis at a time similar to that reported for normal M. japonicus development (Hudinaga, 1941; Norris et al., 2005), it is possible to conclude that PBI and II inhibition in M. japonicus results in triploid progeny. However, this is difficult to explain if the PBI triploids were produced through united bipolar segregation. The fact that triploid M. japonicus are produced by 6DMAP treatments, which are timed to stop PBI or PBI and II extrusion, may prove interesting from a commercial viewpoint if they are more heterozygous than PBII triploid M. japonicus. Improved growth rates have been reported for PBI triploids of numerous aquatic species when compared to PBII triploids and normal diploids (i.e. soft-shell clam (Mya arenaria), Allen et al., 1982; American oyster (C. viginica), Stanley et al., 1984; Pacific oyster (C. gigas), Yamamoto et al., 1988; blue mussel (M. edulis), Beaumonet and Kelly, 1989; pearl oyster (Pinctada martensii), Jiang et al., 1991). This improved performance is attributed to increased heterozygosity of PBI triploids, resulting either from the maternal parent being more heterozygous due to the retention of both maternal chromosomes, or because the spermatozoa carries a heterologous allele. In the present study, 6-DMAP had varying effects on embryo hatch rate for the different spawnings. However, control embryos always hatched as well as if not better than treated embryos. In an earlier ploidy study on M. japonicus, final 6-DMAP concentrations of 200 and 400 μM significantly reduced hatch rates (Norris et al., 2005). Differences in handling stresses and variation in water temperatures during the treatment process are possible causes of variation in hatch rates between the two studies and between spawnings in the present study. In conclusion, this study clearly shows that treatment of M. japonicus embryos with 6-DMAP timed to stop PBI or PBI and II extrusion results in the production of viable triploid nauplii. To our knowledge, this is the first report of successful PBI, and PBI and II inhibition in shrimp, and production of triploids using these inhibi-
tion regimes. Future studies aim to use M. japonicus microsatellite markers as developed by Moore et al. (1999) to conclusively determine if PBI triploids are more heterozygous than PBII triploids. Acknowledgements The authors would like to thank Frank Coman, Tamera Callaghan, Greg Coman and Lucy Hurrey for their experimental support, and Paula Hall and Grace Chojnowsky from the Queensland Institute of Medical Research for their assistance with flow cytometry. References Allen Jr., S.K., Gagnon, P.S., Hidu, H., 1982. Induced triploidy in the soft-shell clam. Journal of Heredity 73, 421–428. Arai, K.F., Naito, F., Fujino, K., 1986. Triploidization of the Pacific abalone with temperature and pressure treatments. Bulletin of the Japanese Society of Scientific Fisheries 52 (3), 417–422. Beaumonet, A.R., Kelly, K.S., 1989. Production and growth of triploid Mytilus edulis larvae. Journal of Experimental Marine Biology and Ecology 132, 69–84. Chang, Y., Xiang, J., 2002. Inducement of polyploidy sea cucumber (Apostichopus japonicus Liao). Journal of Dalian Fisheries 17 (1), 1–7. Chourrout, D., Chevassus, B., Krieg, F., Happe, A., Burger, G., Renard, P., 1986. Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females—potential of tetraploid fish. Theoretical and Applied Genetics 72 (2), 193–206. Coman, F.E., Norris, B.J., Pendrey, R.C., Preston, N.P., 2003. A simple spawning detection and alarm system for penaeid shrimp. Aquaculture Research 34, 1359–1360. Cooper, K., Guo, X., 1989. Polyploid Pacific oyster produced by blocking polar body I and II with cytochalasin B. Journal of Shellfish Research 8, 412. Crocos, P.J., Coman, G.J., 1997. Seasonal and age variability in the reproductive performance of Penaeus semisulcatus broodstock: optimising broodstock selection. Aquaculture 155, 55–67. Guo, X., Allen Jr., S.K., 1994. Viable tetraploids in the Pacific oyster (Crassostrea gigas Thunberg) produced by inhibiting polar body I in eggs from triploids. Molecular Marine Biology and Biotechnology 3, 42–50. Guo, X., Cooper, K., Hershberger, W.K., Chew, K.K., 1992. Genetic consequences of blocking polar body I with cytochalasin B in fertilised eggs of the Pacific oyster, Crassostrea gigas: I. Ploidy of resultant embryos. Biological Bulletin 183, 381–386. Guo, X., DeBrosse, G., Allen Jr., S.K., 1996. All triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids. Aquaculture 142, 149–161. Huayong, A., Allen Jr., S.K., 2002. Hybridisation of tetraploid and diploid Crassostrea gigas (Thunberg) with diploid C. ariakensis (Fujita). Journal of Shellfish Research 21 (1), 137–143. Hudinaga, K., 1941. Reproduction development and rearing of Penaeus japonicus Bate. Japanese Journal of Zoology 10, 305–390. Jiang, W., Xu, G., Lin, Y., Li, G., 1991. Comparison of growth between triploid and diploid of Pinctada martensi (D). Journal of Tropical Oceanography 10 (3), 1–7.
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