The effect of ionizing irradiation of post-larvae on subsequent survival and reproductive performance in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus (Bate)

Aquaculture 264 (2007) 309 – 322 www.elsevier.com/locate/aqua-online

The effect of ionizing irradiation of post-larvae on subsequent survival and reproductive performance in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus (Bate) Melony J. Sellars a,b,⁎, Greg J. Coman a , Tamera R. Callaghan a , Stuart J. Arnold a , Jan Wakeling a , Bernard M. Degnan b , Nigel P. Preston a a

CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric Research, 233 Middle Street, Cleveland, Qld. 4163, Australia b School of Integrative Biology, The University of Queensland, Brisbane, Qld, 4072, Australia Received 9 October 2006; received in revised form 5 January 2007; accepted 9 January 2007

Abstract Considerable genetic advances in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus, have been achieved in the last decade, creating the demand for a technique to provide genetic copyright and prevent escapees from farm ponds genetically contributing to natural fishery populations. The induction of sexual sterility is one technique that may provide such protection. This study evaluated the potential of sterilizing reproductive age (10 month old) P. japonicus by ionizing irradiation (IR) treatment at an early life-history stage. To achieve this, a lethal dose curve was established for post-larval stage 15 (PL15) shrimp by treating with different doses of IR. From this curve, six irradiation levels — all below the 100% mortality treatment — were trialed, ranging from 0 to 30 Gray (Gy). Another group of PL15 P. japonicus were subsequently exposed to the six IR treatments and then reared until sexual maturity, at which time their survival and reproductive performance were assessed. At 8 months of age, females and males from each of the 0, 10, 15 and 20 Gy treatment groups were reciprocally crossed to give 16 mating combinations, whilst females and males from each of the 25 and 30 Gy treatment groups were directly crossed with 0 Gy shrimp of the opposite gender to give four direct mating crosses. At 10 months of age, all females had their reproductive performance assessed in a standardized 30 day trial. The percentage of females maturing and spawning in the 0 Gy treatment group (80.97 ± 5.5% and 69.9 ± 5.77%) were significantly higher (P b 0.05) than for IR treated females in the 10, 15 and 20 Gy treatment groups (63.93 ± 5.34%, 61.68 ± 5.77% and 43.66 ± 6.03% for percentage maturations and 51.44 ± 5.5%, 49.69 ± 6.04% and 38.94 ± 6.31% for percentage spawnings). Similarly, the number of maturations and spawnings per female in the 0 Gy treatment group (1.50 ± 0.13 and 1.24 ± 0.12) were significantly higher (P b 0.05) than for IR treated females in the 10, 15, and 20 Gy treatment groups (1.13 ± 0.12, 1.04 ± 0.13 and 0.76 ± 0.14 for the number of maturations and 0.88 ± 0.12, 0.84 ± 0.13 and 0.67 ± 0.13 for the number of spawnings). The effects of IR on protozoeal metamorphosis varied. No differences in fecundity, hatch rates, gonadal somatic indices or gonadal histology were found between control and irradiated shrimp. These results indicate that IR of PL15 P. japonicus had some effect on ovarian maturation and spawning, however IR was not able to confer sterility in shrimp. Treatment of PL15s with IR that does not result in death (i.e. between 10 and 30 Gy) is therefore not effective at preventing the production of some viable offspring. © 2007 Elsevier B.V. All rights reserved. Keywords: Shrimp; Selective breeding; Genetic protection; Aquaculture

⁎ Corresponding author. CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric 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 © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.01.009

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1. Introduction World shrimp farming industries are increasingly using elite genotypes generated from genetic improvement programs for commercial culture (Preston and Clifford, 2002; Benzie and Argue, 2006). The elite genotypes generated from these programs are of considerable financial and genetic value to the producer, creating a need for a method to protect these living genetic resources from unlicensed breeding. Techniques to induce sterility provide an avenue to prevent propagation of such elite genotypes and also prevent cultured escapees from breeding with wild stocks and genetically contributing to natural fishery populations. In Australia, the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus (Bate), is one shrimp species that has been the focus of successful genetic improvement programs over the last 15 years (Preston and Clifford, 2002; Preston et al., 2001). It is a high-value species which is sold directly to the live seafood market in Japan. Given the achievements in genetic improvement of stocks and the fact that all produce is sold live, the development of a technique to sexually sterilize P. japonicus is a priority for the industry. Considerable efforts have been invested in P. japonicus polyploidy research as a means to sexually sterilize progeny (Preston et al., 2004). However, while viable polyploids have been induced, 100% sterility has not been conferred in all progeny within a spawning (e.g. Preston et al., 2004; Norris et al., 2005; Sellars et al., 2006). The use of ionizing irradiation (IR) to confer sterility is one technique that has received relatively little research attention despite its success at conferring 100% sterility in other species, such as fruit fly (Dominiak et al., 2003; Suckling, 2003) and male sea lampreys (Petromyzon marinus) (Hanson, 1990). IR has considerable potential for P. japonicus farmers in Australia as it can be easily applied as part of the harvesting process. Additionally, as the purpose of irradiating shrimp under such circumstances is not for food preservation, there are no regulatory or legal restrictions in Australia for such an application (Australia and New Zealand Food Standards Code 1.5.3 Irradiation of Food, 1991). IR works most profoundly on cells which are undergoing constant renewal, such as the gonad cells, and typically has little effect on somatic tissues with low renewal rates. IR causes sterility by damaging cellular DNA through base damage and single and double strand breaks (Coates et al., 2004; Snyder and Morgan, 2004; Lee, 2000). Such aberrations prevent successful replication of affected cells, effectively conferring sterility

by preventing meiotic and mitotic production of viable gonad tissue (Henriksen et al., 1996). In some instances DNA repair processes enable affected cells to continue functioning, however, if the damage is misrepaired or unrepaired, all mitotic descendants of that cell will have radiation-induced genetic changes (Coates et al., 2004; Henriksen et al., 1996), resulting in the production of non-functional gonad cells. Research efforts to determine the effects of IR on shrimp sterility have been limited. A significant reduction in egg production was reported for female Grass shrimp (Palaemonetes pugio) when exposed to 9.75 gray (Gy) (life-history stage at time of exposure not specified) (Rees, 1962), whilst 100% sterility was achieved in male and female Malaysian shrimp (Macrobrachium rosenbergii) when exposed to 10 Gy or more as juveniles (Lee, 2000). Previous research on P. japonicus has shown that treatment with IR at harvest age has a negative impact on the reproductive capacity of females and males at 20 and 10 Gy respectively (Sellars et al., 2005, 2006). However, 100% sterility was not conferred by IR, with some viable offspring being produced (Sellars et al., 2006). The present study assessed the reproductive performance of 10 month old P. japonicus treated with IR at post-larval stage 15 (PL15). Notably, IR of M. rosenbergi at a similar age (PL15) was found to result in 100% sterility (Lee, 2000). Initially, a lethal dose curve of IR for PL15 P. japonicus was established. Based on these results, PL15 shrimp were treated with IR at 0, 10, 15, 20, 25 and 30 Gy to examine the effect of IR dose on their reproductive capacity at 10 months of age. 2. Materials and methods 2.1. Experimental design The aim of this study was to evaluate the potential to sterilize reproductive age (i.e. 10 month old) P. japonicus by IR treatment of an early life-history stage, PL15. To achieve this, it was essential to first establish a lethal dose curve to determine the effect of different IR treatment doses on PL15 survival. From the lethal dose curve an array of treatment doses were selected (that were between the lowest to highest treatment level which did not result in 100% mortality), and a second group of P. japonicus PL15s were treated with IR. The second group of IR treated PL15s were subsequently reared to reproductive age. Survival of the shrimp from the different IR treatments was assessed from 7 to 10 months of age. Shrimp weight was assessed

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at 7 and 10 months of age for the different IR treatments. Survival and reproductive output of the shrimp were assessed from 10 to 11 months of age in a standardized trial (Sellars et al., 2005) to determine the effect that the different IR doses had on reproductive output. Shrimp were fed a variety of high quality feeds ad libitum, which varied with shrimp age, including freshly hatched Artemia spp. nauplii (brine shrimp), freshly cut squid of various sizes (grated up to 2 cm3) (Loligo spp.) and commercial P. japonicus pellets of various sizes depending on shrimp size (0.2 mm up to 5 mm) (Lucky Star, Taiwan Hung Kuo Industrial Co.) daily at 0900 and 1700 h. 2.2. PL15 survival after treatment with IR For the initial post-treatment survival trial, PL15 P. japonicus were treated at 21 different levels of IR ((0 (control, with handling stress), 0 (control, no handling stress), 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 Gy) to establish a lethal dose curve. PLs obtained from a commercial hatchery (approximately 4000 shrimp) that had been reared according to standard rearing procedures as outlined by Coman (2002), were transported to the Gammacell Facility, University of Queensland, Australia. At the Gammacell Facility 21 aliquots (one for each treatment) of 150 randomly selected PL15s were placed in 4 L of seawater. To expose PL15s to IR, the PLs were first collected by gently pouring each aliquot through a mesh screen (1 mm2 holes). The screen was then gently folded and placed into a metal chamber (10.9 cm inner and 12.2 cm outer ht, 7.5 cm inner and 8.5 cm outer dia.) which fitted within the IR delivery device. IR was delivered at the corresponding treatment level using a cobalt-60 Gammacell-200 irradiator (Atomic Energy of Canada Ltd) calibrated to ± 0.0254 Gy. After treatment, the PLs were washed from the mesh screen into 20 L of seawater within a transport bag and given gentle aeration. Control PLs (0 Gy (control, with handling stress)) received the same handling stress as treatment PLs, whereas control no handling stress PL15s (0 Gy (control, no handling stress)) were placed straight into a transport bag after being randomly selected. Once all aliquots had received their corresponding treatment dose of IR, transport bags were filled with oxygen gas, sealed and transported back to CSIRO Marine and Atmospheric Research (CMAR) laboratories. At CMAR PLs were counted into three replicates of 20 individuals per treatment level (PLs dead on arrival were not used for stocking). At stocking, individuals in a

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random sample of 100 PL15s were weighed to the nearest 0.01 mg. Each replicate of PLs was reared in 5 L of 27 ± 2 °C seawater with gentle aeration for 30 d. Photoperiod was set at 12 h light: 12 h dark. The number of PLs remaining in each replicate was counted daily between 0900 and 1000 h, with dead shrimp being removed during this process. The 5 L seawater volume of each rearing tank received a 100% exchange every third day. Survival of shrimp from the different treatment levels at five day time intervals were analyzed using the repeated measures test (SAS Institute Software, 1999). 2.3. Long-term survival, growth and reproductive performance of PL15 shrimp exposed to IR treatment 2.3.1. PL15 production PLs to be used in the reproductive assessment experiment were spawned from wild-caught P. japonicus broodstock that were maintained at a gender ratio of 20 females: 15 males. Broodstock were maintained in a 2000 L sand-substrate tank which received 1.6 L min− 1 of 27 ± 2 °C seawater, had a 10 mm thin walled polycarbonate (Polygal Inc.) lid to reduce light intensity and a 12 h light: 12 h dark photoperiod (Crocos and Coman, 1997). To produce the experimental PLs, gravid female broodstock (stage IV; Crocos and Kerr, 1983) impregnated with spermatophores were selected from the tanks, unilaterally eyestalk ablated using hot forceps, and spawned in 100 L circular tanks filled to 90 L. Upon spawning, eggs (embryos) were left to hatch in the 100 L tanks. Approximately 3000 nauplii were harvested from each spawning and stocked into four replicate 100 L circular tanks filled to 30 L (100 shrimp L− 1) with 27 °C, 34 ppt salinity seawater. Larvae were reared from nauplius stage I to PL15 in these 100 L tanks according to the methods outlined in Coman (2002). Four families from separate spawnings were produced in total for treatment with IR. 2.3.2. Treatment of PL15 P. japonicus with IR Once shrimp reached PL15, PLs from all replicate rearing tanks of each family were harvested and pooled together (i.e. four pooled tanks, one for each family). Eight 250 mL sub-samples of PL15s were taken from the pooled batches and the number of PL15s counted to estimate the number of PL15 L− 1 seawater. At random, 100 of the PL15s used for counting were weighed to the nearest 0.01 mg. Weights of the PL15s from the four families being used in the reproductive performance trial, and the PL15s from the commercial hatchery used

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to establish lethal dose curves, were analyzed by oneway analysis of variance (SAS Institute Software, 1999). PLs remaining in the pooled batches were evenly divided into eight 5 L aliquots (spawning 1 and 2) or twelve 5 L aliquots (spawning 3 and 4). Two aliquots from each of families 1 and 2 were exposed to 0 (control), 10, 15 and 20 Gy of IR. Two aliquots from each of families 3 and 4 were exposed to 0 (control), 10, 15, 20, 25 and 30 Gy of IR. Treated PL15s were randomly stocked into 2000 L tanks identical to those used for broodstock acclimation with one exception — tanks were divided equally into quadrants by four flyscreen mesh pens (2 mm aperture, 0.75 m2 bottom surface area). The pens were dug into the sand-substrate, providing a 6 to 8 cm layer of sand in each pen. One aliquot of PL15s was stocked per pen. The water source, flow rate, photoperiod and light intensity were identical to the tank used for broodstock acclimation. 2.3.3. Rearing of PL15s through to reproductive maturity Once shrimp reached PL45, they were collected from the pens and each treatment from each family was

stocked into separate 2000 L tanks at densities between 60 and 80 shrimp per tank. In instances where survival or shrimp numbers were low, shrimp from pens of the same family and treatment were stocked into the same tank. Once shrimp reached PL115, they were collected from the tanks and internally tagged with elastomer implants (Northwest Marine Technology Inc.) to allow identification down to the treatment level. Shrimp were then stocked back into the 2000 L tanks (45–50 animals per tank) so that each IR treatment had equal numbers of representatives in each tank. Once shrimp reached 7 months of age they were collected from tanks and sorted into their different treatment groups and genders. Shrimp from each treatment were counted and weighed to the nearest 10 mg. The 0, 10, 15 and 20 Gy treatment groups were reciprocally crossed into their corresponding treatment tank (identical to the 2000 L tanks used previously), whilst females and males from each of the 25 and 30 Gy treatment groups were only crossed with 0 Gy shrimp of

Table 1 Total number of male and female P. japonicus (treated with IR at PL15) that were crossed into tanks at 7 months of age (i.e. stocking densities for the 62 d mating period) Male treatment 0 Gy

10 Gy

15 Gy

20 Gy

25 Gy 30 Gy

# females crossed per treatment Tank

# males

0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

1 2 3 4 5 23⁎ 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24⁎ 25⁎

16 16 16 16 16 18 16 16 16 16 16 16 16 14 14 14 15 15 15 15 15 15 14 18 5

7 7 7 7 7 n.a. 7 7 7 7 7 7 7 6 7 7 7 7 7 7 7 7 7 15 12

5 5 5 5 5 n.a. 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 n.a. n.a.

4 4 4 4 4 n.a. 4 4 4 4 4 4 4 4 4 4 4 5 5 4 4 4 4 n.a. n.a.

3 4 4 3 3 n.a. 3 3 3 3 3 3 3 3 3 4 4 3 4 3 4 3 3 n.a. n.a.

n.a. n.a. n.a. n.a. n.a. 4 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

n.a. n.a. n.a. n.a. n.a. 22 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

Tank numbers with an asterisk (⁎) did not contain a reciprocal cross. n.a. = not applicable. # = number.

Tank density (# shrimp) 35 36 36 35 35 44 35 35 35 35 35 35 35 32 33 34 35 35 36 34 35 34 33 33 17

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the opposite gender as there were too few shrimp to complete the reciprocal cross design for these IR treatments. The resulting mating cross design included 16 reciprocal crosses with five to seven replicates per cross (Table 1) and four direct crosses with one replicate of each (0 Gy female × 25 Gy male, 0 Gy female × 30 Gy male, 25 Gy female × 0 Gy male, 30 Gy female × 0 Gy male) (Table 1, denoted by an asterisk). Once crossed, shrimp were left for 62 d, allowing shrimp to reach sexual maturity and mate within their respective treatments. It should be noted that as all shrimp within a treatment group were pooled, some full-sib matings would have occurred. However, it is known that for first generation P. japonicus progeny (as used in this study) there are no significant effects of inbreeding depression on reproductive performance (Crocos et al., 2000; Keys et al., 2004). At 10 months of age, all shrimp were phenotypically sexed, weighed and counted to establish survival rates for each treatment group over the 62 d mating period. All females were unilaterally eye-stalk ablated using hot

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forceps. From each reciprocal cross, replicates of each treatment group were pooled and then evenly distributed into treatment tanks to ensure that the number of shrimp in each tank were similar for the subsequent reproductive assessment (Table 2). For the four direct crosses, shrimp were returned to their tank of origin (Table 2). Differences in shrimp weight at 7 months of age and at 10 months of age for each treatment group were compared by one-way analysis of variance (SAS Institute Software, 1999) separately for females and males. Differences in weight for the 25 and 30 Gy treatment groups were not included in the analyses due to there being too few shrimp for rigorous statistical comparison. Differences in shrimp survival for the different treatment levels during the 62 d mating period were also analyzed by analysis of variance (SAS Institute Software, 1999). 2.3.4. Reproductive performance assessment After ablation, a 30 d reproductive performance assessment was undertaken. Ovarian development was

Table 2 Total number of male and female P. japonicus (treated with IR at PL15) on which the reproductive performance assessments were completed at 10 months of age (i.e. stocking densities for the 30 d reproductive trial) Male treatment 0 Gy

10 Gy

15 Gy

20 Gy

25 Gy 30 Gy

# females crossed per treatment Tank

# males

0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

1 2 3 4 5 23⁎ 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24⁎ 25⁎

12 12 12 12 12 15 10 10 11 12 11 10 11 11 11 11 11 11 10 9 11 9 9 9 1

5 5 4 5 5 n.a. 5 4 4 5 5 6 4 4 3 3 4 4 8 7 7 7 7 8 8

4 5 4 4 5 n.a. 3 3 4 4 4 4 3 4 4 4 4 4 3 3 5 3 4 n.a. n.a.

2 3 3 3 3 n.a. 2 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 4 n.a. n.a.

3 3 3 4 3 n.a. 2 3 2 3 3 2 3 3 3 4 3 3 2 2 2 1 2 n.a. n.a.

n.a. n.a. n.a. n.a. n.a. 4 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

n.a. n.a. n.a. n.a. n.a. 19 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

Tank numbers with an asterisk (⁎) did not contain a reciprocal cross. n.a. = not applicable. # = number.

Tank density (# shrimp) 26 28 26 28 28 38 22 23 24 27 26 25 24 25 24 24 25 25 26 24 28 23 26 17 9

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assessed by shining a torch beam through the dorsal exoskeleton of the females every second day during dark hours (Crocos and Coman, 1997). Ready to spawn impregnated females (stage IV; Crocos and Kerr, 1983) were transferred to 100 L circular spawning tanks filled to 90 L which were checked daily for the presence of eggs. Spawned females were returned to their corresponding maturation tank and the numbers of eggs and hatch rates of each spawning estimated from the total number of hatched and unhatched eggs in four 250 mL samples taken 2 to 3 h after first hatching (Crocos and Coman, 1997). Mortalities in both maturation and spawning tanks were recorded daily. To determine larval survival, nauplii from three 250 mL samples of each spawning were stocked into separate plastic beakers containing 300 mL of 27 °C seawater and light aeration. The larvae were reared without food at constant temperature for a further 62 h, by which time metamorphosis to protozoeal stage I should have occurred (Hudinaga, 1941). At this time, a number of nauplii and protozoea in each sample were counted. Larval survival was calculated as the percentage of nauplii stocked into each container metamorphosing into protozoea. Reproductive performance was expressed for each mating cross in terms of the percentage of females that matured (reaching ovary stage IV) and spawned; number of maturations and spawnings per female; egg numbers per spawning (fecundity); hatch rate (percentage of eggs hatching into nauplii); and protozoeal metamorphosis rate (percentage of nauplii metamorphosing to protozoeal stage I). Reproductive performance of shrimp from the reciprocally crossed treatments (i.e. 0, 10, 15 and 20 Gy treatment groups) were analyzed by factorial analysis of variance (SAS Institute Software, 1999) with female and male treatment included as main effects. Logarithmic transformation of the data was also completed and the same analyses applied to account for the possibility that stabilization of the variances and normalization the data may be required. The analyses also included a term to determine if there was variability between tank replicates (a tank nested within male treatment term), and a female by tank interaction term. When the female treatment level was fixed at 0 Gy, reproductive performance of the male treatments (0, 10, 15, 20, 25 and 30 Gy) were compared by analysis of variance with male treatment and tank nested within male treatment, included as terms in the model. When male treatment level was fixed at 0 Gy, reproductive performance of the female treatments (0, 10, 15, 20, and 30 Gy; N.B. the 25 Gy female treatment group was not

included as there was no replication for this cross) were compared by analysis of variance with female treatment, tank and the female × tank interaction terms included in the analysis. Differences in shrimp survival from each treatment level during the 30 d reproductive performance period were also analyzed by analysis of variance (SAS Institute Software, 1999). 2.3.5. Histological examination of gonad development and gonad somatic indices Gonad histology at 3 and 11 months of age (i.e. 100 and 300 d after treatment of PL15s with IR) and gonad somatic indices (GSI) taken at 11 months of age (day 30 of the reproductive performance assessment) were also used as measures of reproductive condition. At 3 months of age two females and two males were randomly sampled from the 0, 10, 15 and 20 Gy treatment groups for histological processing. At 11 months of age 5, 7, 7 and 3 females (ovary stage IV) from the 0, 10, 15 and 20 Gy treatment groups respectively, and 5 males from each of the 0, 10, 15, 20 and 25 Gy treatment groups were sampled for GSI calculations and histological processing. All shrimp were weighed to the nearest 0.01 g. Shrimp assessed for GSI were molt staged (Smith and Dall, 1985) and their gonads were dissected out and weighed to the nearest 0.1 mg. GSIs were calculated for each shrimp [GSI = (gonad weight / body weight) × 100] and analyzed separately for females and males from the different treatment groups by one-way analysis of variance (SAS Institute Software, 1999). As done for the reproductive performance data, logarithmic transformation and further analysis of the data was also completed. All samples (cephalothorax region of the head for 3 month samples and dissected gonads for 11 month samples) were prepared for routine histological examination (Bell and Lightner, 1988). Tissue sections (5 μm) were stained with Haematoxylin and Eosin stain (Clinipure, HD scientific). Reproductive tissues were examined using light microscopy and comparisons were made between control and treatment female and male shrimp to determine variations in gonad tissue and cellular structure. 3. Results 3.1. Post-larval survival after treatment with IR Treatment dose and the interaction between IR dose and time interval (5 d intervals) significantly affected shrimp survival (P b 0.001), indicating that the patterns of survival of the treatment groups varied between each

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5 d measure. Consequently, the effect of IR dose on survival was analyzed separately for each 5 d time interval. Overall, treatment of PL15s with N 30 Gy IR resulted in a marked increase in shrimp death when compared to IR treatments of 30 Gy or less (Table 3). In general, shrimp died within a shorter time period when treated with higher levels of IR, with 100% mortality within 10 d for the 95 and 100 Gy treatments, 100% mortality within 15 d for treatments above and including 60 Gy, 100% mortality within 20 d for the 55 Gy treatment, 100% mortality within 25 d for the 45 and 50 Gy treatments and 100% mortality within 30 d for the 40 Gy treatment. At 30 d after irradiation, there was no significant difference between the survivals of shrimp treated with between 35 and 100 Gy of irradiation, with 13 of the 14 treatments in this range having no shrimp alive. Survival of the 30 Gy treatment group was similar to controls between 5 and 10 d. However, survival of the 30 Gy treatment group was significantly lower than the controls by 15 d. Notably, survival of the 30 Gy treatment was significantly higher than for all treatment levels above 30 Gy at 30 d. Generally, survivals of all 0

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to 25 Gy treatment groups were similar throughout the duration of the experiment. Of note, survival in the 5 Gy treatment was statistically higher than the 10 and 25 Gy treatments at a few of the later measures in the trial. At 30 d after irradiation it was evident that treatment with IR up to and including 25 Gy has no significant biological effect on shrimp survival when compared to the controls. Therefore, the subsequent reproductive performance trial assessed the five treatment levels which had no effect on survival (0, 10, 15, 20 and 25 Gy), and the one treatment level which had the smallest effect on survival (30 Gy). 3.2. Treatment with IR and rearing of PL15s through to reproductive maturity The total number of PL15s treated with IR varied between families due to differences in larval survival of the families. For families one, two, three and four there were 85 ± 3.21, 868 ± 7.46, 152 ± 4.21 and 746 ± 5.39 PL15s treated for each treatment group respectively. Despite the difference in larval survival between families, there was no significant difference (P N 0.05) in the weight of PL15s from the four different families

Table 3 Mean (±S.E.) number of P. japonicus shrimp alive in 5 d intervals for the 30 d period after treatment with IR at PL15 Treatment

0 Gy 0 Gy (stressed) 5 Gy 10 Gy 15 Gy 20 Gy 25 Gy 30 Gy 35 Gy 40 Gy 45 Gy 50 Gy 55 Gy 60 Gy 65 Gy 70 Gy 75 Gy 80 Gy 85 Gy 90 Gy 95 Gy 100 Gy

Time since treatment with IR (d) 5

10

15

20

25

30

16.67 ± 1.67 17.67 ± 0.33 17.33 ± 1.45 16.67 ± 0.33 18.00 ± 0.00 17.67 ± 0.33 16.00 ± 0.58 14.33 ± 0.88 15.33 ± 2.18 18.00 ± 1.53 17.67 ± 0.33 17.00 ± 0.00 17.67 ± 0.58 18.33 ± 0.33 17.33 ± 0.33 14.67 ± 0.67 17.33 ± 1.76 16.33 ± 0.67 17.67 ± 1.45 17.67 ± 0.33 14.33 ± 1.76 14.00 ± 1.53 n.s.

16.33 ± 1.86A 17.33 ± 0.33A 17.33 ± 1.45A 15.57 ± 0.33AB 16.67 ± 0.67A 17.33 ± 0.67A 15.67 ± 0.33AB 14.33 ± 0.88AB 12.67 ± 2.91B 16.67 ± 1.45A 15.67 ± 0.88AB 15.33 ± 1.20AB 6.33 ± 2.40C 0.67 ± 0.33E 1.67 ± 0.88DE 1.00 ± 0.58DE 1.00 ± 0.00DE 1.67 ± 0.33DE 1.00 ± 1.58DE 4.00 ± 0.58CD 0.00 ± 0.00E 0.00 ± 0.00E P b 0.0001

16.33 ± 1.86AB 16.67 ± 0.33AB 17.33 ± 1.45A 14.67 ± 0.33BC 16.67 ± 0.67AB 16.67 ± 0.88AB 15.33 ± 0.33ABC 13.00 ± 0.58CD 6.67 ± 1.20F 9.67 ± 2.40E 11.00 ± 0.00DE 7.00 ± 1.53F 1.00 ± 1.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G 0.00 ± 0.00G P b 0.0001

16.33 ± 1.86ABC 16.67 ± 0.33AB 17.33 ± 1.45A 14.67 ± 0.33CD 16.67 ± 0.67AB 16.00 ± 1.00ABC 15.33 ± 0.33BC 13.00 ± 0.58D 2.33 ± 0.88FG 4.00 ± 0.58EF 5.00 ± 1.15EF 2.00 ± 0.58G 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H 0.00 ± 0.00H P b 0.0001

16.33 ± 1.86AB 16.67 ± 0.33AB 17.33 ± 1.45A 14.67 ± 0.33BC 16.33 ± 0.88AB 16.00 ± 1.00AB 15.33 ± 0.33BC 13.00 ± 0.58C 1.67 ± 0.88D 0.33 ± 0.33D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D 0.00 ± 0.00D P b 0.0001

16.00 ± 2.08ABC 15.67 ± 0.67ABC 17.33 ± 1.45A 14.33 ± 0.33C 16.33 ± 0.88AB 16.00 ± 1.00ABC 15.33 ± 0.33BC 11.67 ± 0.33D 1.67 ± 0.88E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E 0.00 ± 0.00E P b 0.001

Means with different superscripts are significantly different (P b 0.05) for that measure day (within column). n.s. = no significant difference for that measure day (P N 0.05). d = day.

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Table 4 Mean (±S.E.) weights of female and male P. japonicus at 7 and 10 months of age for the different treatment groups Age

Gender

7 mo

Female Male

10 mo

Female Male

Treatment 0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

25.04 ± 0.40A n = 154 18.05 ± 0.27B n = 161 32.79 ± 0.53A n = 140 22.66 ± 0.29B n = 119

22.40 ± 0.46B n = 111 17.58 ± 0.28B n = 126 30.16 ± 0.75B n = 80 21.44 ± 0.41B n = 71

21.68 ± 0.58B n = 66 19.30 ± 0.29A n = 72 30.26 ± 0.59B n = 69 26.63 ± 0.42A n = 56

21.10 ± 0.76B n = 105 15.82 ± 0.36C n = 43 31.0 ± 1.38B n = 58 20.41 ± 0.55B n = 49

25.25 ± 1.00 n = 22 18.68 ± 0.73 n = 22 33.80 ± 0.94 n = 19 23.36 ± 0.80 n=9

26.84 ± 3.89 n=4 20.87 ± 1.69 n=5 34.41 ± 3.70 n=5 29.46 n=1

Statistical analyses were only completed on the reciprocally crossed treatments which are the boldface values. Means with different superscripts are significantly different (P b 0.05) within rows. mo = month. Italicized values are the number of shrimp or values used to calculate the mean value.

treated with IR. Furthermore, weights of PL15s from the four families used in the reproductive assessment trial were not significantly different from the mean weight of the commercially produced PL15s used to establish the lethal dose curves. Mean family weights ranged from 2.13 ± 0.31 mg for family one to 2.65 ± 0.07 mg for family three.

Females in the 0 Gy treatment group were significantly larger (P b 0.05) than females in the 10, 15 and 20 Gy treatment groups at both 7 and 10 months of age (Table 4). No differences were found between the weights of females in the 10, 15 and 20 Gy treatment groups at 7 and 10 months of age. Differences in male weights were also found between treatments

Table 5 Mean (±S.E.) number of maturations per female and percentage of maturing P. japonicus for the different crosses during the 30 d reproductive assessment Female treatment

Male treatment

0 Gy

Mat. % Mat.

10 Gy

Mat. % Mat.

15 Gy

Mat. % Mat.

20 Gy

Mat. % Mat.

25 Gy

Mat. % Mat.

30 Gy

Mat. % Mat.

0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

Ave.1

1.33 ± 0.19 79.17 ± 8.47 n = 24 1.50 ± 0.23 68.18 ± 10.16 n = 22 1.85 ± 0.22 100.00 ± 0.00 n = 13 1.35 ± 0.30 76.47 ± 10.60 n = 17 1.06 ± 0.24 66.67 ± 11.43 n = 18 2.25 ± 0.25 100.00 ± 0.00 n=4

1.40 ± 0.22 73.33 ± 8.21 n = 30 1.32 ± 0.24 72.73 ± 9.72 n = 22 0.95 ± 0.23 55.00 ± 11.41 n = 20 0.85 ± 0.27 53.85 ± 14.39 n = 13 n.a. n.a.

1.22 ± 0.27 66.67 ± 11.43 n = 18 0.75 ± 0.24 50.00 ± 11.47 n = 20 1.00 ± 0.30 61.54 ± 14.04 n = 13 1.19 ± 0.23 68.75 ± 11.97 n = 16 n.a. n.a.

1.06 ± 0.19 60.60 ± 8.64 n = 33 0.71 ± 0.19 52.94 ± 12.48 n = 17 0.73 ± 0.27 40.00 ± 13.09 n = 15 0.56 ± 0.38 22.22 ± 14.70 n=9 n.a. n.a.

0.67 ± 0.33 50.00 ± 22.36 n=6 n.a. n.a.

1.14 ± 0.34 71.43 ± 18.44 n=7 n.a. n.a.

1.50 ± 0.13A 80.97 ± 5.50A 1.13 ± 0.12B 63.93 ± 5.34B

n.a. n.a.

n.a. n.a.

1.04 ± 0.13BC 61.68 ± 5.77B

n.a. n.a.

n.a. n.a.

0.76 ± 0.14C 43.66 ± 6.03C

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

Boldface values are the reciprocally crossed treatments. Average of reciprocally crossed mean values only. Means with different superscripts are significantly different (P b 0.05). Mat. = number of maturations to ovary stage IV (Crocos and Kerr, 1983) per female during the 30 d reproductive period. % Mat. = the percentage of females maturing during the 30 d reproductive period. n.a. = not applicable. Italicized values are the number of maturation values used to calculate the mean value.

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at 7 and 10 month of age (P b 0.05) (Table 4). Importantly, shrimp from all treatments were of a size and age considered suitable for reproduction in this species (Hudinaga, 1941; Coman et al., 2004). During the 62 d mating period mean survivals for the different treatment groups and genders were not significantly different (P N 0.05) (mean ± S.E. 70.7 ± 6.7%). 3.3. Reproductive performance assessment When analyzed for the reciprocal crossed treatments, female IR treatment significantly (P b 0.05) affected the percentage of females maturing, and the number of maturations per female (Table 5). The average percentage of maturations for 0 Gy females was significantly greater (80.97 ± 5.50%) than for 10 and 15 Gy females (63.93 ± 5.34% and 61.68 ± 5.77% respectively), which was significantly greater than for 20 Gy females (43.66 ± 6.03). The average number of maturations per female was significantly greater in the 0 Gy treatment (1.50 ± 0.13) compared to the 10, 15 and 20 Gy treatments (1.13 ± 0.12, 1.04 ± 0.13 and 0.76 ± 0.14 respectively). Male IR treatment did not affect (P N 0.05) the percentage of females

317

maturing or the number of maturations per female (Table 5). When analyzed for the 0 Gy male crosses, no differences were found (P N 0.05) in the percentage of maturations per female or the number of maturations per female between the six female IR treatments (0, 10, 15, 20, 25 and 30 Gy) (Table 5). Likewise, when analyzed for the 0 Gy female crosses, no differences (P N 0.05) in the percentage of maturations per female or the number of maturations per female were found between male IR treatments (Table 5). When analyzed for the reciprocal crossed treatments, female IR treatment significantly (P b 0.05) affected the percentage of females spawning, and the number of spawnings per female (Table 6). The percentage of spawnings for 0 Gy females was significantly greater (69.90 ± 5.77%) than for 10, 15 and 20 Gy females (51.44 ± 5.50%, 49.69 ± 6.04% and 38.94 ± 6.31% respectively). The average number of spawnings per female was significantly greater in the 0 Gy treatment (1.24 ± 0.12) compared to the 10, 15 and 20 Gy treatments (0.88 ± 0.12, 0.84 ± 0.13 and 0.67 ± 0.13 respectively). Male IR treatment did not affect

Table 6 Mean (±S.E.) number of spawnings per female and percentage of spawnings in P. japonicus for the different crosses during the 30 d reproductive assessment Female treatment

Male treatment

0 Gy

Spawn. % Spawn.

10 Gy

Spawn. % Spawn.

15 Gy

Spawn. % Spawn.

20 Gy

Spawn. % Spawn.

25 Gy

Spawn. % Spawn.

30 Gy

Spawn. % Spawn.

0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

Ave.1

1.08 ± 0.19 70.83 ± 9.48 n = 24 1.23 ± 0.27 59.09 ± 10.73 n = 22 1.54 ± 0.27 84.62 ± 10.42 n = 13 1.12 ± 0.31 64.71 ± 11.95 n = 17 1.00 ± 0.24 61.11 ± 11.82 n = 18 1.75 ± 0.25 100.00 ± 0.00 n=4

1.67 ± 0.21 63.33 ± 8.95 n = 30 1.05 ± 0.23 59.09 ± 10.73 n = 22 0.75 ± 0.20 45.00 ± 11.41 n = 20 0.54 ± 0.24 38.46 ± 14.04 n = 13 n.a. n.a.

1.11 ± 0.23 66.67 ± 11.43 n = 18 0.50 ± 0.22 30.00 ± 10.51 n = 20 0.85 ± 0.32 46.15 ± 14.39 n = 13 0.88 ± 0.22 56.25 ± 12.81 n = 16 n.a. n.a.

0.94 ± 0.18 54.45 ± 8.80 n = 33 0.59 ± 0.17 47.06 ± 12.48 n = 17 0.60 ± 0.25 33.33 ± 12.60 n = 15 0.56 ± 0.58 22.22 ± 14.70 n=9 n.a. n.a.

0.33 ± 0.21 33.33 ± 21.08 n=6 n.a. n.a.

0.86 ± 0.26 71.43 ± 18.44 n=7 n.a. n.a.

1.24 ± 0.12A 69.90 ± 5.77A 0.88 ± 0.12B 51.44 ± 5.50B

n.a. n.a.

n.a. n.a.

0.84 ± 0.13B 49.68 ± 6.04B

n.a. n.a.

n.a. n.a.

0.67 ± 0.13B 38.94 ± 6.31B

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

Boldface values are the reciprocally crossed treatments. Average of reciprocally crossed mean values only. Means with different superscripts are significantly different (P b 0.05). Spawn. = number of spawnings per female during the 30 d reproductive period. % Spawn. = the percentage of females spawning during the 30 d reproductive period. n.a. = not applicable. Italicized values are the number of spawning values used to calculate the mean value.

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Table 7 Mean (±S.E.) fecundity and hatch rate of spawnings for all female and male P. japonicus treatment groups Female treatment

Male treatment

0 Gy

Fecun. Hatch

10 Gy

Fecun. Hatch

15 Gy

Fecun. Hatch

20 Gy

Fecun. Hatch

25 Gy

Fecun. Hatch

30 Gy

Fecun. Hatch

0 Gy

10 Gy

15 Gy

20 Gy

25 Gy

30 Gy

58,999 ± 6048 55.6 ± 6.4 n = 26 61,313 ± 7873 50.5 ± 7.1 n = 26 39,706 ± 7663 48.3 ± 7.2 n = 21 49,808 ± 8681 49.8 ± 6.7 n = 19 38,244 ± 4937 58.1 ± 8.0 n = 18 60,552 ± 7928 52.2 ± 11.2 n=7

55,901 ± 6489 54.0 ± 5.6 n = 35 51,553 ± 7790 47.4 ± 7.4 n = 20 53,031 ± 8003 59.6 ± 7.6 n = 18 52,401 ± 13,303 56.4 ± 11.6 n=7 n.a. n.a.

49,912 ± 6284 43.8 ± 8.0 n = 21 43,848 ± 7990 54.4 ± 8.9 n=9 36,828 ± 11,082 47.4 ± 10.6 n = 11 53,887 ± 10,123 54.1 ± 9.4 n = 13 n.a. n.a.

62,396 ± 6886 50.1 ± 5.9 n = 30 46,748 ± 10,225 46.9 ± 12.4 n=9 52,004 ± 11,231 57.1 ± 10.3 n = 10 85,084 ± 11,625 49.8 ± 17.3 n=4 n.a. n.a.

32,618 ± 19,633 24.8 ± 24.8 n=2 n.a. n.a.

80,170 ± 12,817 35.9 ± 11.7 n=6 n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

n.a. n.a.

Boldface values are the reciprocally crossed treatments. Fecun. = fecundity. Hatch = the number of eggs (embryos) hatching to nauplii (hatch rate). n.a. = not applicable. Italicized values are the number of values used to calculate the mean value.

(P N 0.05) the percentage of females spawning or the number of spawnings per female (Table 6). When analyzed for the 0 Gy male crosses, no differences were found (P N 0.05) in the percentage of spawnings per female or the number of spawnings per female were found between the six female IR treatments (0, 10, 15, 20, 25 and 30 Gy) (Table 6). Likewise, when analyzed for the 0 Gy female crosses, no differences (P N 0.05) in the percentage of spawnings per female or the number of spawnings per female were found between male IR treatments (Table 6).

No significant differences (P N 0.05) in fecundity or hatch rate were found between IR treatment groups for either the reciprocal or the direct crosses (Table 7). Compared to other reproductive performance comparisons for P. japonicus and other penaeid species, the fecundity and hatch rate results for the different treatment categories in the present study were extremely consistent (CSIRO Unpublished Information, Coman et al., 2005, 2006). Mean fecundity ranged from 32,618 eggs (0 Gy female × 25 Gy male) to 85,084 eggs (20 Gy female × 20 Gy male), whilst the mean hatch rate ranged

Table 8 Mean (±S.E.) protozoeal metamorphosis rates per P. japonicus spawning that are hatched for all female and male reciprocal crosses Female treatment

Male treatment 0 Gy

10 Gy

15 Gy

20 Gy

Ave.

0 Gy

62.8 ± 2.1 n = 17 62.4 ± 2.6 n = 20 62.2 ± 1.8 n = 15 66.6 ± 2.0 n = 15 63.5 ± 2.13

64.9 ± 0.9 n = 31 63.1 ± 3.2 n = 15 60.6 ± 3.6 n = 13 71.5 ± 2.6 n=5 65.0 ± 2.6

67.8 ± 1.0 n = 12 65.6 ± 1.8 n=8 61.6 ± 6.5 n=7 66.0 ± 4.7 n = 11 65.3 ± 3.5

67.7 ± 1.6 n = 25 62.4 ± 2.9 n=7 58.11 ± 5.1 n=7 66.8 ± 1.4 n = 25 63.8 ± 2.8

65.8 ± 1.4AB

10 Gy 15 Gy 20 Gy Ave.

Ave. = average for that column or row. Ave. values with different superscripts are significantly different (P b 0.05). Italicized values are the number of samples used to calculate the mean value.

63.4 ± 2.6B 60.6 ± 4.3B 67.7 ± 2.7A

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from 24.8% (0 Gy female× 25 Gy male) to 58.1% (25 Gy female× 0 Gy male) (Table 7). When analyzed for the reciprocal crossed treatments, significant differences (P b 0.05) in protozoeal metamorphosis rate were found between female IR treatments, with the 20 Gy female treatment group having a significantly higher protozoeal metamorphosis rate (67.7 ± 2.7%) than the 10 and 15 Gy treatments (63.4 ± 2.6% and 60.6 ± 4.3% respectively) (Table 8). The protozoeal metamorphosis rate was not found to be different between the 0, 10 and 15 Gy treatment groups. Male IR treatment had no affect (P N 0.05) on protozoeal metamorphosis rate (Table 8). The protozoeal metamorphosis rates for the different female treatment groups were not statistically different when analyzed for the 0 Gy male crosses separately (Table 9). When the 0 Gy female crosses were analyzed separately, the protozoeal metamorphosis rate of the 30 Gy male treatment was significantly lower (P b 0.05) than the 0, 10, 15, 20 and 25 Gy treatment groups (Table 9). Survival of females was significantly lower (P b 0.05) than the males throughout the 30 d reproductive period (39.61 ± 2.91% for females and 64.99 ± 5.01% for males). No differences in mean survival between treatment groups was found for either gender during the 30 d reproductive period. 3.4. Histological examination of gonad development and GSI At 3 months of age there was no significant difference (P N 0.05) between the average weight of Table 9 Mean (±S.E.) protozoeal metamorphosis rates per P. japonicus spawning that hatched for all 0 Gy female crosses and all 0 Gy male crosses Cross (female × male)

Mean ± S.E.

Cross (female × male)

Mean ± S.E.

0 × 0 Gy

62.8 ± 2.1A n = 17 62.4 ± 2.6A n = 20 62.2 ± 1.8A n = 15 66.6 ± 2.0A n = 15 65.3 ± 1.5A n = 14 50.2 ± 10.1B n=5

0 × 0 Gy

62.8 ± 2.1 n = 17 64.9 ± 0.9 n = 31 67.8 ± 1.0 n = 12 67.7 ± 1.6 n = 25 32.7 n=1 59.6 ± 7.3 n=4

0 × 10 Gy 0 × 15 Gy 0 × 20 Gy 0 × 25 Gy 0 × 30 Gy 1

10 × 0 Gy 15 × 0 Gy 20 × 0 Gy 25 × 0 Gy1 30 × 0 Gy

25 Gy treatment group was not included in ANOVA when male treatment was fixed at 0 Gy. Mean values with different superscripts are significantly different (P b 0.05). Italicized values are the number of shrimp or values used to calculate the mean value.

319

females (10.39 ± 0.54 g) and males (9.77 ± 0.50 g) processed for histology and no differences (P N 0.05) between the different treatment groups when each gender was analyzed separately (i.e. 11.5 ± 0.50 g, 10.76 ± 0.40 g, 8.69 ± 1.79 g and 9.89 ± 0.99 g for 0, 10, 15 and 20 Gy females, and 10.36 ± 0.76 g, 10.37 ± 0.69 g, 8.47 ± 1.57 g and 10.62 ± 0.49 g for 0, 10, 15 and 20 Gy males). Histological examination of the gonads of control and IR treated 3 month old shrimp revealed that there were no visual differences between the tissue and cellular structure within each gender. At 11 months of age there was no significant difference (P N 0.05) between the average weight of shrimp in the different treatment groups when each gender was analyzed separately (i.e. 33.47 ± 0.71 g, 31.33 ± 2.36 g, 29.22 ± 0.79 g and 32.19 ± 3.16 g for 0, 10, 15 and 20 Gy females, and 26.33 ± 2.09 g, 23.91 ± 2.18 g, 25.36 ± 0.81 g, 24.74 ± 1.50 g and 23.71 ± 0.83 g for 0, 10, 15, 20 and 25 Gy males). At 11 months of age no significant differences (P N 0.05) were found between GSI values of the different treatment groups for females and males when analyzed separately. The average GSI values (±S.E.) for females from the 0, 10, 15 and 20 Gy treatment groups were 3.772 ± 0.381, 3.159 ± 0.351, 3.670 ± 0.361 and 2.814 ± 0.606 respectively. The average GSI values (±S.E.) for males from the 0, 10, 15, 20 and 25 Gy treatment groups were 0.824 ± 0.460, 0.382 ± 0.017, 0.444 ± 0.029, 0.501 ± 0.034 and 0.393 ± 0.032 respectively. Histological examination of the gonads used for GSI calculations revealed no visual differences between the tissue and cellular structure within each gender. 4. Discussion In this study, ionizing irradiation (IR) doses higher than 35 Gy resulted in 100% mortality (i.e. lethal dose rate) of PL15 P. japonicus within 30 days after treatment, whilst doses of 25 and 30 Gy significantly reduced PL survival compared to controls. Females treated with 0 Gy of IR matured and spawned more frequently than females treated with 10, 15 and 20 Gy of IR. There were, however, no other significant effects of IR observed on P. japonicus reproductive performance. These findings indicate that IR doses that do not result in 100% mortality can reduce the reproductive capacity if P. japonicus in terms of maturation and spawning ability, however, IR does not confer complete sterility. Whilst IR doses higher than 35 Gy were lethal to P. japonicus within 30 days after treatment and doses of 25 and 30 Gy significantly reduced PL survival compared to controls, IR doses of 20 Gy and lower typically had no significant effect on survival. The lethal

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dose rates reported for P. japonicus when treated with IR at harvest age are slightly lower in comparison, where 25 Gy or more resulted in 100% mortality of females within 30 days after treatment (Sellars et al., 2005) and 20 Gy significantly reduced male survival (Sellars et al., 2006). Similar lethal IR dose rates for M. rosenbergii have been reported when treated with 30 Gy or more, resulting in 100% mortality of 1 d old larvae within 9 days, 55 d old post-larvae within 12 days and 90 d old juveniles within 14 days (Lee, 2000). It appears that P. japonicus PL15s are slightly more resilient to treatment with IR than harvest age P. japonicus and 1, 55 and 90 d old M. rosenbergii. The duration from treatment to 100% mortality was notably longer for PL15 M. japonicus compared to 1, 55 and 90 d old M. rosenbergii. Female P. japonicus that had been treated with IR were found to be smaller at 7 and 10 months of age compared to females that were not treated with IR. Lee (2000) reported a similar result when juvenile M. rosenbergii were treated with 10 and 15 Gy of IR, with controls being significantly larger at harvest age (110 d after treatment with IR). In contrast, male P. japonicus in the present study had variable size across the treatment groups at 7 and 10 months of age. As it is difficult to separate the contributions of IR versus random variation on shrimp weight, it is possible that the observed differences in weights across the treatments are due to biological variation as opposed to a true treatment effect. In the present study control females (0 Gy) matured and spawned more frequently than females treated with IR doses of 10, 15 and 20 Gy. Notably, no significant difference was found between the number of maturations and spawnings for control females and females treated at 25 and 30 Gy, however this was likely due to low numbers of animals within these highest IR treatments. The reduced number of maturations and spawnings per female, and percentage of maturations and spawnings for the 10, 15 and 20 Gy treatment groups in the present study indicate that IR impairs the ability of P. japonicus to develop mature ovaries subsequently reducing the number of spawnings per female. This is supported by the fact that IR of female P. japonicus resulted in reduced body weight compared to controls. The fact that the size of female penaeids is directly linked to their ability to mature and spawn is well documented (Cavalli et al., 1997; Crocos and Coman, 1997; Hoang et al., 2002; Menasveta et al., 1994; Peixoto et al., 2004). IR was not found to affect the fecundity and hatch rate of spawnings in the present study, indicating that, of the

shrimp that did contribute spawnings, IR did not impair their reproductive performance. This is further supported by the histological assessments of the testes and ovarian tissue where no abnormal cell structure was observed from treatment shrimp at 3 and 11 months of age (100 and 300 d after treatment with IR). It should be noted that histology at 11 months of age was only completed on females with mature stage IV ovaries and that those shrimp that were not maturing throughout the trial would not have been represented in this sample. Previous reports on the reproductive capacity of P. japonicus when treated with IR at harvest age showed a reduction in fecundity from a female effect and a reduction in hatch rate from a male effect (Sellars et al., 2006). Results from the present study are undoubtedly different from these due to the life-history stage when IR was administered. However, in M. rosenbergii Lee (2000) found a significant female effect on fecundity, where 15 Gy of IR (administered to juveniles) reduced the number of eggs spawned once at a reproductively mature age. Rates of metamorphosis to protozoeal stage I for the different female and male treatment groups were statistically different. However, patterns were inconsistent with some of the higher treatment levels having greater rates of metamorphosis than the controls. This is likely an artifact of natural biological variation as opposed to it being a true treatment effect, as the range of mean values and replication was low (less than 8% difference). Gonad weights relative to body weights (GSI) for each gender were similar across all six treatment levels of IR. Lee (2000) reported the same result for M. rosenbergii females and males when treated with 0, 5, 10, 15 and 20 Gy of IR, however, in this study the same shrimp that had been treated with 10 Gy or more were also reported to be 100% sterile. It should be noted that the female GSI values from the present study were taken in a bias manner and did not represent those females that were not maturing or developing gravid stage IV ovaries. Despite IR not being suitable for the indented application of the present study, this is the first report or IR being used on any penaeid shrimp species. As IR can be used in research for a wide range of applications (e.g. gynogenesis), the lethal dose rates for post-larval M. japonicus established in the present study will provide a solid starting point for future IR research experiments. 5. Conclusion This study clearly shows that treatment of PL15 P. japonicus with more than 35 Gy of IR results in 100% mortality within 30 d of treatment and that 25 Gy of IR

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or more significantly reduces PL15 survival compared to controls. Treatment of female P. japonicus with 10 Gy or more significantly reduces the number of maturations per female, the percentage of maturations, the number spawnings per female and the percentage of spawnings. Despite this, some PL15 P. japonicus females treated with 10 Gy or more were still capable of developing mature ovaries and spawning viable offspring. Treatment of PL15s with IR that does not result in death is not 100% effective at preventing the production of viable offspring and will therefore not provide a suitable means by which to sterilize genetically improved P. japonicus shrimp. Acknowledgements The authors would like to thank their colleagues Frank Coman, Maggie Barclay, Lucy Hurrey, and Louise Coles for their experimental support. We would also like to thank Lyle Carrington and acknowledge the Gamma Cell Facility at the University of Queensland for kindly allowing us to use their equipment. Many thanks also to Russell McCulloch from CSIRO Livestock Industries who helped with histological interpretations. References Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. Baton Rouge, Louisiana, USA. Benzie, J.A.H., Argue, B., 2006. Genomics in Penaeid Shrimp Breeding. Genetics in Aquaculture IX Book of Abstracts, p. 9. Cavalli, R.O., Scardua, M.P., Wasielesky, W.J., 1997. Reproductive performance of different-sized wild and pond-reared Penaeus paulensis females. Journal of the World Aquaculture Society 28, 260–267. Coates, P.J., Lorimore, A.A., Wright, E.G., 2004. Damaging and protective cell signalling in the untargeted effects of ionizing radiation. Mutation Research 568, 5–20. Coman, G.J., 2002. Factors affecting the efficiency of genetic selection of the Kuruma prawn Penaeus japonicus. PhD Thesis, The University of Queensland, Queensland, Australia. Coman, G.J., Crocos, P.J., Preston, N.P., Fielder, D., 2004. The effects of density on the growth and survival of different families of juvenile Penaeus japonicus Bate. Aquaculture 229, 215–223. Coman, G.J., Crocos, P.J., Arnold, S.J., Keys, S.J., Preston, N.P., Murphy, B., 2005. Growth, survival and reproductive performance of domesticated Australian stocks of the giant tiger prawn, Penaeus monodon, reared in tanks and raceways. Journal of the World Aquaculture Society 36 (4), 464–479. Coman, G.J., Arnold, S.J., Peixoto, S., Crocos, P.J., Coman, F.E., Preston, N.P., 2006. Reproductive performance of reciprocally crossed wild-caught and tank-reared Penaeus monodon broodstock. Aquaculture 252 (2–4), 372–384. 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.

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