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Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements (EVEs) have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such EVEs and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of EVEs. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for one generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific EVEs identified an element comprised of a 9,045 bp stretch of repeated, inverted and jumbled genome fragments of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) bounded by a repeated 591/590 bp host sequence. As only near complete linear ~4 kb IHHNV genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear EVE types. The existence of joined inverted IHHNV genome fragments also provides a means by which hairpin dsRNAs could be expressed and processed by the shrimp RNA interference (RNAi) machinery.

Injecting double-stranded (ds)RNA into abdominal muscle of female shrimp may provide a relatively simple means of reducing pre-existing virus infection loads and thus the propensity for infection to be transmitted vertically to seedstock used for aquaculture. However, to obtain regulatory approvals to use such RNA interference (RNAi) technology in hatcheries producing seedstock to be farmed for human consumption, there is a need to demonstrate that injected dsRNA will not transfer from broodstock or be maintained/amplified in their farmed progeny. To investigate this, at the time they were eyestalk ablated, female Black Tiger shrimp (Penaeus monodon) broodstock naturally impregnated with male spermatophores were injected with a 1:1 mix of dsRNAs synthesised to a 954 bp region of the gill-associated virus (GAV) genome and a 464 bp region of the firefly luciferase (Luc) gene. Eggs spawned 6 to 14 days later from each of 3 non-injected control females and 3 females injected at a dosage of 0.5 μg each dsRNA/g shrimp weight were reared separately and sampled at key developmental stages until they reached post-larval (PL) age. To detect residual dsRNA, cDNA synthesised to both native and heat-denatured (HD) total nucleic acid (TNA = RNA + DNA) extracted from the larval samples and pleopod, lymphoid organ and ovary tissues sampled from each female immediately after spawning was amplified using highly-sensitive TaqMan quantitative real-time (q)PCR tests targeting the dsGAV1 and dsLuc dsRNA sequences. Using HD TNA to strand-separate dsRNA and thus improve the detection sensitivity of the RT-qPCR tests, both dsRNAs were detected in all 3 tissues examined from injected females. Direct qPCR testing of the DNA component of each TNA confirmed that the detections were not due to residual template DNA in the purified dsRNA preparations. Similar RT-qPCR testing of both native and HD TNA extracted from pools of 50 embryos, nauplii, protozoea, and mysis and 32 individual PL reared separately for each female generated no unequivocal or consistent detections of dsGAV1 or dsLuc dsRNA. These findings provide confidence that purified dsRNA injected into female broodstock at the time an eyestalk is ablated and at the dosage tested was not effectively transferred to/amplified in progeny, and thus that the human health risks of using this RNAi strategy to reduce virus disease risks during shrimp grow-out will be negligible.

In shrimp aquaculture, the ability to reliably test for the presence and load of pathogens is vital for managing disease. As pathogen infection loads are not distributed homogenously throughout different tissues, detection by PCR can vary based on what tissue type is tested. Moreover, there is potential for infection loads to vary within the same tissue type depending on infection severity and nature of the tissue. This study used reverse transcriptase real-time quantitative PCR (RT-qPCR) to examine the sensitivity and variability in gill-associated virus (GAV) detection levels within and between different tissue types sampled from individual black tiger (Penaeus monodon) shrimp. From the group of juvenile shrimp (Group 1 - 10.9 ± 1.4 g, n = 10) with low-level GAV infection, eight gill filaments and eight pleopods were tested from each individual, and from another group of adult shrimp (Group 2 - 40.4 ± 2.3 g, n = 12) with higher-level GAV infection, 10 gill filaments, 10 pleopods and both lymphoid organ lobes were tested from each individual. For Group 1, 69/80 gill and 67/80 pleopod samples tested PCR-positive, and for Group 2, all samples tested PCR-positive. In some shrimp from each group, GAV detection levels among gill filaments or pleopods varied by up to ~3000-fold, highlighting the variability that is present in infection load among these tissues within individual shrimp. Statistical analyses used to determine the most sensitive and consistent tissue type to test identified no significant differences between mean GAV infection loads detected, either in gill or pleopod tissue, within each group (Group 1, p = 0.852; Group 2, p = 0. 146). Similarly, variability in GAV detection levels among individual gill filaments and pleopods did not differ between either tissue for each group (Group 1, p = 0.922; Group 2, p = 0.151). The mean GAV infection load (GAV RNA copies μg−1 TNA) detected in lymphoid organ lobes tested from Group 2 adult shrimp (5.96 × 107 ± 8.67 × 107 copies), was significantly higher (p

Increased disease tolerance is an important trait commonly incorporated into shrimp breeding programs. Sib-selection using experimental pathogen challenge tests are used to measure family based tolerance. It is critical for challenge tests to be standardised and repeatable for accurate genetic estimation. Accordingly, this study focused on establishing a method for uniformly challenging Black Tiger shrimp ( Penaeus monodon ) to an acute gill-associated virus (GAV) infection. A stock inoculum of GAV (10 5.27 GAV RNA copies μL − 1 inoculum) was prepared from infected shrimp and used as the source of infection for the challenge methodologies investigated. Three challenge approaches compared in groups of juvenile P. monodon (2–10 g) were; intramuscular injection, ingestion of fresh or frozen diced muscle tissue from GAV-infected shrimp and immersion in water containing the GAV inoculum. Intramuscular injection using a weight-standardised dose of the inoculum resulted in cumulative mortality surpassing 50% on Day 7 post-challenge. Immersion of shrimp for 2 h in water comprising a 1:1000 dilution of the inoculum resulted in no mortalities over the 11 day trial period. GAV RT-qPCR analysis also identified no evidence of any shrimp becoming infected by the immersion method. Feeding shrimp either fresh or frozen diced muscle tissue of moribund shrimp with an acute GAV infection generated cumulative mortality that reached/surpassed 50% on day 11 post-challenge. Compared to the survivors of the injection group (mean = 10 7.73 ± 0.49 GAV RNA copies/μg total RNA), RT-qPCR analysis identified GAV infection loads to be less uniform and 100- to 1000-fold lower among survivors of feeding either fresh or frozen muscle tissue (mean = 10 5.71 ± 1.97 or 10 4.77 ± 1.90 GAV RNA copies/μg total RNA, respectively). Coefficient of variation (CV) values for the GAV infection load data supported survivors of injection (CV = 0.06) having a more uniform infection load compared to the survivors of either fresh (CV = 0.41) or frozen (CV = 0.33) tissue ingestion. Of the three challenge approaches assessed for their suitability in measuring GAV tolerance, injection was clearly identified to provide the most uniform and reliable means of infecting shrimp with a defined dose of GAV and therefore is the preferred method of infection to use for GAV challenge tests.

Additional file 1: Supplementary Figure 1.Penaeus monodon distribution across Australia (light grey), approximate locations of current pond based farming operations (dark grey), and location of samples included in DARTcap ‘discovery’ populations (1 – Joseph Bonaparte Bay (n = 34), 2 – Tiwi Islands (n = 56), 3 – Gulf of Carpentaria (n = 43), 4 – Bramston Beach (n = 60), 5 – Etty Bay (n = 50), 6 – Townsville (n = 22) and 7 – Commercial Farm site (n = 394). Supplementary Figure 2. Clustering of samples based upon genetic similarity shown through discriminant analysis of principle components (DAPC). PC1 and PC2 are shown on the x and y axis respectively. (A) Including all samples (n = 418). (B) Including only second generation (G2) individuals (n = 272) obtained from routine commercial spawning.

The ability of domesticated Penaeus monodon, Black Tiger shrimp, to spawn following tail-muscle injection of dsRNA was examined. Ablated domesticated female broodstock infected subclinically with gill-associated virus (GAV) were injected with saline or a cocktail of five-dsRNAs targeting different regions in the GAV ORF1a/1b gene. To track changes in GAV infection loads, TaqMan real-time PCR was used to quantify mean viral RNA amounts in each of three pleopod clips collected at the time of injection (Day 0) and either immediately after a female spawned or on Day 11 when the trial was terminated. Over the trial, 4 of 19 (21%) saline-injected shrimp spawned and 12 of 25 (48%) dsRNA-injected shrimp spawned, with one spawning twice. Egg numbers varied from 25 600 to 459 800 for the saline-injected shrimp and from 4900 to 213 900 for the dsRNA-injected shrimp. Of these, one of the four egg batches hatched from saline-injected shrimp and 9 of the 13 egg batches hatched from dsRNA-injected shrimp. While variable, egg numbers and hatch rates recorded were typical of those obtained from domesticated broodstock at the commercial hatchery and particularly among females previously spawned. Mean GAV RNA amounts detected in pleopod samples increased in five of the eight saline-injected shrimp tested by 1.6–227.4-fold and decreased in 12 of the 15 ds-RNA-injected shrimp tested by −1.1 to −45.1-fold. The study demonstrated that tail-muscle injection of GAV-specific dsRNA does not adversely impact the ability of P. monodon to spawn.

In penaeid shrimp, mesoderm forms from two sources: naupliar mesoderm founder cells, which invaginate during gastrulation, and posterior mesodermal stem cells called mesoteloblasts, which undergo characteristic teloblastic divisions. The primordial mesoteloblast descends from the ventral mesendoblast, which arrests in cell division at the 32-cell stage and ingresses with its sister dorsal mesendoblast prior to naupliar mesoderm invagination. The naupliar mesoderm forms the muscles of the naupliar appendages (first and second antennae and mandibles), while the mesoteloblasts form the mesoderm, including the muscles, of subsequently formed posterior segments. To better understand the mechanism of mesoderm and muscle formation in penaeid shrimp, twist, snail, and mef2 cDNAs were identified from transcriptomes of Penaeus vannamei, P. japonicus, P. chinensis, and P. monodon. A single Twist ortholog was found, with strong inferred amino acid conservation across all three species. Multiple Snail protein variants were detected, which clustered in a phylogenetic tree with other decapod crustacean Snail sequences. Two closely-related mef2 variants were found in P. vannamei. The developmental mRNA expression of these genes was studied by qPCR in P. vannamei embryos, larvae, and postlarvae. Expression of Pv-twist and Pv-snail began during the limb bud stage and continued through larval stages to the postlarva. Surprisingly, Pv-mef2 expression was found in all stages from the zygote to the postlarva, with the highest expression in the limb bud and protozoeal stages. The results add comparative data on the development of anterior and posterior mesoderm in malacostracan crustaceans, and should stimulate further studies on mesoderm and muscle development in penaeid shrimp.

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥108) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1-7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia.

A 40-day reproductive performance trial was conducted to assess the effect of targeted supplementation of Penaeus monodon broodstock grow-out and maturation diets with microbial biomass (MB). Over a seven month grow-out period, shrimp were fed a typical pelleted grow-out diet with or without 10% MB. Broodstock were then transferred to a maturation facility and a subset of animals from each grow-out diet fed on a typical fresh-frozen maturation diet that included a pellet ration with or without 30% MB (5.5% of total diet fed). At nine months of age, female broodstock were unilaterally eyestalk-ablated and reproductive assessments commenced. No significant difference in ovary maturation, hepatosomatic index, spawning and egg and nauplii production parameters was found between diet treatments (p = > 0.05). However, females originating from control ponds displayed a higher gonadosomatic index at first spawn, whilst the percentage of eggs that hatched was lower in females fed a MB-inclusive maturation diet (p =

Differential larval survival of Penaeid shrimp families when reared communally for any of the mainstream species is widely acknowledged as a likely scenario commercially. However, no scientific data exist in the public domain that quantifies the magnitude of this phenomenon. We report on the differential survival of Pacific White shrimp, Litopenaeus vannamei, families when reared communally during commercial larval rearing and provide explanations of the impacts of such occurrences on managing commercial selective breeding programs. In total 76 families are pooled in two groups of 40 and 36 families and reared through to PL15/16, respectively. DNA microsatellite markers were then used to assign PL15/16 to their family of origin, and the abundance of PL in each family was used to calculate survival rates at a family-based level during commercial communal larval rearing. Calculated survival distributions of 76 families during the larval culture phase ranged from 0 to 100 % with three of the 76 families having no survivors present at PL15/16. Despite all families being stocked into larval culture with somewhat equal representation, only 18 of the 76 families made up 50 % of the population at PL15/16 that would then be stocked into ponds. We found no correlation between the number of nauplii produced per spawning (family) versus the calculated number of PL15/16 alive after the larval rearing phase. Together these three factors have significant implications in Penaeid shrimp selective breeding programs. If populations are not managed appropriately, there is a risk of significant reduction in a programs’ effective population size.

The ability of Novacq to improve resilience of black tiger shrimp, Penaeus monodon, to infection and mortality induced by gill-associated virus (GAV) was investigated. Over a 26-d period, shrimp were fed pellets with or without 10% Novacq. Following this, four replicate tanks, each containing 10 shrimp that had been fed either diet, were maintained as-is, injected with saline or injected with GAV inoculum (i.e., 40 shrimp for each of the six groups). For shrimp (n=20) in two of each group of four tanks, survival was monitored daily over 14d and a pleopod was sampled from each shrimp on Days 0 and 14. For the other two tanks, a pleopod was sampled from each shrimp on Days 0, 3, 7, 10, and 14 to track changes in GAV loads over time. Survival was significantly higher (P

Advances in the domestication and selective breeding of Australian Black Tiger shrimp, Penaeus monodon, opens the opportunity for world producers to reconsider the benefits of farming this species. Just over a decade ago this species was the world's most farmed shrimp species, however, difficulty in its domestication, in part, led to the widespread establishment of Penaeus vannamei (Pacific White shrimp) as the most farmed shrimp species in the world. This study empirically evaluates the productivity benefits of commercially domesticating P. monodon against production from wild broodstock of the same species. The evaluation compared the relative production from commercial ponds stocked with the progeny of wild P. monodon broodstock and ponds stocked with the progeny of domesticated stocks. The production data were from 164 ponds of domesticated stocks and 30 ponds of wild stocks, collected over 4 years (2009–2013) from two separate farm sites of the same Australian shrimp farming company. The wild stocks were sourced from the east coast of Australia. The results suggested that the productivity of the selectively bred stocks was 39% greater compared with production from wild stocks given equivalent amounts of feed and other inputs. Furthermore, productivity was additionally enhanced depending on the choice of feeds and whether stocking took place in September rather than later in the year (i.e. in early spring rather than late spring/early summer in the Southern hemisphere). This suggests that there is significant potential to further enhance the productivity of P. monodon farms via integrating advances in domestication, feeds and management practices.

The black tiger shrimp (Penaeus monodon) remains the second most widely cultured shrimp species globally. However, issues with disease and domestication have seen production levels stagnate over the past two decades. To help identify innovative solutions needed to resolve bottlenecks hampering the culture of this species, it is important to generate genetic and genomic resources. Towards this aim, we have produced the most complete publicly available P. monodon transcriptome database to date. The assembly was carried out in multiple assemblers using 2×125 bp HiSeq data from PolyA selected, ribo-depleted RNA extracted from nine adult tissues and eight early life-history stages. In total, approximately 700 million high-quality sequence reads were obtained and assembled into 236,388 clusters. These were then further segregated into 99,203 adult tissue specific clusters, and 58,678 early life-history stage specific clusters. The final transcriptome had a high TransRate score of 0.37, with 88% of all reads successfully mapping back to the transcriptome. BUSCO statistics showed the assembly to be highly complete with low fragmentation, few genes missing, but higher redundancy or transcript duplication (Complete: 98.2% (Duplicated: 51.3%), Fragmented: 0.8%, Missing: 1.0%), and to greatly exceed the completeness of existing P. monodon transcriptomes. While annotation rates were low (approximately 30%), as is typical for a non-model organisms, annotated transcript clusters were successfully mapped to several hundred functional KEGG pathways. To help address the lack of annotation, transcripts were clustered into groups within tissues and early life-history stages, providing initial evidence for their roles in specific tissue functions, or developmental transitions. Additionally, transcripts of shrimp viruses previously not known to occur in Australia were also discovered. We expect the transcriptome to provide an essential resource to investigate the molecular basis of commercially relevant-significant traits in P. monodon and other shrimp species.

The use of pyrethroid and neonicotinoid insecticides has increased in Australia over the last decade, and as a consequence, increased concentrations of the neonicotinoid insecticide imidacloprid have been measured in Australian rivers. Previous studies have shown that non-target crustaceans, including commercially important species, can be extremely sensitive to these pesticides. Most shrimp farms in Australia are predominantly located adjacent to estuaries so they can obtain their required saline water, which support multiple land uses upstream (e.g. sugar-cane farming, banana farming, beef cattle and urbanisation). Larval and post-larval shrimp may be most susceptible to the impacts of these pesticides because of their high surface area to volume ratio and rapid growth requirements. However, given the uncertainties in the levels of insecticides in farm intake water and regarding the impacts of insecticide exposure on shrimp larvae, the risks that the increased use of new classes of pesticide pose towards survival of post-larval phase shrimp cannot be adequately predicted. To assess the potential for risk, toxicity in 20day past hatch post-larval Black Tiger shrimp (Penaeus monodon) to modern use insecticides, imidacloprid, bifenthin, and fipronil was measured as decreased survival and feeding inhibition. Post-larval phase shrimp were sensitive to fipronil, bifenthrin, and imidacloprid, in that order, at concentrations that were comparable to those that cause mortality other crustaceans. Bifenthrin and imidacloprid exposure reduced the ability of post-larval shrimp to capture live prey at environmentally realistic concentrations. Concentrations of a broad suite of pesticides were also measured in shrimp farm intake waters. Some pesticides were detected in every sample. Most of the pesticides detected were measured below concentrations that are toxic to post-larval shrimp as used in this study, although pesticides exceed guideline values, suggesting the possibility of indirect or mixture-related impacts. However, at two study sites, the concentrations of insecticides were sufficient to cause toxicity in shrimp post larvae, based on the risk assessment undertaken in this study.

The ability of RNA interference (RNAi) based on injected dsRNA was investigated here for its ability to reduce the severity of pre-existing subclinical Gill-associated virus (GAV) infections in farm stocks of juvenile Black Tiger shrimp (Penaeus monodon). Following tail muscle injection of single or multiple long dsRNAs targeted to sequences positioned across the GAV ORF1a/1b replicase genes, pleopods were sampled sequentially from individuals at regular intervals over a 2. week period to track changes in GAV RNA loads by quantitative real-time RT-PCR. Mean GAV RNA amounts showed statistically significant (P

Knowledge about the specification of the germ line in penaeid shrimp would allow development of techniques to control germ cell formation and/or fate to produce reproductively sterile shrimp for genetic copyright purposes. Recent studies have traced the localization of an RNA-enriched intracellular body (ICB) in the putative germ line of four penaeid shrimp species. It is hypothesized that the ICB may serve as a putative germ granule and marker of germ line fate. In this study semi-thin and ultra-thin sections of Marsupenaeus japonicus embryos were prepared, and the dimensions and ultrastructure of the ICB was examined at different stages of embryogenesis. The ICB was an aggregation of electron dense granules, small vesicles and multi-vesicular bodies (MVBs), similar to germ granules from other species. Lamellar membranes and mitochondria were localized at the periphery of the ICB. Using fluorescence microscopy, microtubules were also observed between the centrosome and the ICB. The localization of the ICB in the D lineage and putative germ cell line, the enrichment of RNA in the ICB, and the ultrastructural similarities to other germ granules characterized in this study support the hypothesis that the ICB contains germ granules.

Custom-built single-nucleotide-polymorphism (SNP) marker systems that are compatible with Sequenom((R)) chemistry are compared with a general purpose microsatellite marker system in their ability to accurately assign Black Tiger shrimp parentage. The microsatellite system consisted of 13 markers, while the SNP systems comprised of 63 (SNPa), 59 (SNPb) or 122 markers (SNPab). Comparisons were made using animals from commercial breeding lines with thresholds for assignment derived using simulated genotypes. Pedigree assignment for commercial lines was highest when panel SNPab was used. Panel SNPa, panel SNPb and Msat functioned with an overall similar level of power for pedigree assignment, however, for some families, the Msat panel was not as powerful. Pedigree assignment for the simulated diploid genotypes was higher for all SNP panels compared with Msat. Overall the three SNP panels provided parentage assignment rates suitable for commercial shrimp breeding programs with assignment rates in the simulated genotypes greater than 96.8% and correct assignments greater than 99.3%. Compared with the microsatellite panel, custom-built SNP panels, whether they operate as single panels, or as a combined panel, have improved power to perform dam and sire assignment to progeny and provide faster turnaround time as they are compatible with Sequenom((R)) chemistry.

Genetic protection of improved lines of Giant Tiger shrimp, Penaeus monodon, may be achieved through the reliable production of all triploid families, as triploid shrimp are reproductively sterile. The problem with current triploid induction methods is in producing 100 % triploid progeny reliably and doing so on a commercial scale. All triploid offspring may be achievable by mating tetraploids with diploids, overcoming current limitations. However, tetraploidy in shrimp to date has been unsuccessful, with all tetraploids dying very early in development. As the induction method of heat was found to be lethal, this study investigated an alternative method using cold water to assess induction rates and lethality. Cold-water treatments between −2 and 14 °C were applied at 18, 20, and 22 min post-spawning detection for various durations with the aim of inhibiting first mitosis. Tetraploidy was induced in treatments at or less than 1 °C; however, no nauplii hatched from these treatments. As no diploid or tetraploid nauplii hatched from any of the treatments below 3 °C, it was determined that this cold-water treatment method is not suitable for tetraploid production in P. monodon.

Sibling harvest age Black Tiger shrimp triploids and diploids of both sexes were reared to reproductive maturity, crossed with wild caught females and males, conditioned for spawning and a comprehensive reproductive performance trial was undertaken. Ovarian development, spawning frequency, fecundity, hatch rate, gonad morphology, male reproductive tracts and thelycum impregnation rates of the wild female × triploid male cross were assessed. After ablation, ovarian development and cycling between wild G0 diploid and G1 diploids was not significantly different, whereas G1 triploids failed to show any signs of ovarian development and cycling, thus resulting in no G1 triploid female spawnings. There were 10 G0 diploid female × G0 diploid male first-spawnings and 9 G0 diploid female × G1 diploid male first-spawnings, all of which produced viable nauplii. In comparison, there were 7 G0 diploid female × G1 triploid male first-spawnings, none of which produced viable nauplii. The 26 wild G0 diploid female spawnings had more eggs than the 1 G1 diploid female spawning. Gonad morphology and male reproductive tract assessments showed impaired reproductive development in triploid gonadal tissues of both sexes (compared with sibling diploids and wild shrimp) to a point where complete maturation had not occurred. The thelycum of 16 wild G0 diploid females crossed with G1 triploid males had no visible spermatophore present, suggesting that G1 triploid males are incapable of developing viable spermatophores and mating with females. This study demonstrates that the triploid females and males are incapable of producing viable gametes and are thus reproductively sterile.

In this study we induced triploids in a single family of Black Tiger shrimp ( Penaeus monodon ) using chemical shock induction to stop the second meiotic division. The family was reared from egg to harvest age (post-larval stage 184) in a combination of commercial hatchery and experimental controlled environment tank rearing systems. During the rearing phase ploidy, survival, weight, sex ratio and gonad morphology of sibling diploids and induced triploids were periodically assessed. This is the first published report of the culture performance of triploid P. monodon . The induction rate of triploids at the nauplii stage was 83.4 ± 0.04% but this proportion had dropped to 62.45% by the time shrimp metamorphosed to the protozeal stage. Proportions of triploids remained fairly constant throughout the remaining larval and early post-larval stages (ranging from 60.47% to 72.38%), but dropped again to 56.32% by the time shrimp reached harvest age. The reduction in triploid rate from egg to harvest age is thought to be due to a combination of factors including heightened sensitivity of triploids to continual handling and general lower survival of triploids compared to diploids. Once shrimp reached harvest age diploid females were significantly heavier (42.95 ± 2.43 g, P P > 0.05) between triploid females, diploid males and triploid males (35.14 ± 2.26 g, 34.02 ± 0.89 g and 31.79 ± 1.56 g respectively). The final sex ratio of harvest age triploid P. monodon was 1 female:1.625 males. Histological examination of harvest age female and male gonads revealed a drastic reduction in the development of both ova and sperm in triploid shrimp compared to diploids. The limited numbers of oocytes and sperm produced in triploids failed to fully mature by harvest age. No sperm were observed within spermatophores of triploids at harvest age when a gross morphological examination was performed and no sperm bundles were seen in the vas deferens. Using the same examination technique, diploid harvest age males had sperm within the spermatophores and vas deferens, and sperm bundles. Results indicate that reproductive sterility is likely for both female and male triploid P. monodon .

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.

In those shrimp species comprehensively studied, polyploidy is currently the only known technique that can achieve the dual outcomes of reproductive sterility for genetic protection and skewing sex ratios towards a high proportion of females, which are larger than males in all of the penaeid shrimp. This review article aims to collate the scientific knowledge and global shrimp industries experience, thoughts, perceptions and publications from numerous formats and languages on shrimp polyploidy to provide an overview of its global status and future direction. At present there is a significant amount of literature on polyploidy in penaeid shrimp: however, some is difficult to access and several observations by Dr. Xiang's research group at the Institute of Oceanology, China, and Dr. Preston's CSIRO Food Futures Flagship research group at CSIRO Marine and Atmospheric Research, Australia have not yet been published. For any researcher or shrimp industry member who has attempted to, or whom would like to thoroughly search the literature to gain a complete understanding of the current state of shrimp polyploidy, this publication will be an invaluable source of reference material, some of which is published here for the first time. (C) 2010 Elsevier BM. All rights reserved.

Recent studies in penaeid shrimp and various crustaceans showing specific antiviral responses to the DNA based White spot syndrome virus (WSSV) induced by pre-exposure to viral proteins are of particular research interest as invertebrates lack the classical adaptive immune response characterised in vertebrate animals. However, this protein-induced defence response has not been reported for any other invertebrate virus, and the mechanisms remain unknown. Here we investigated the generality of this defence response by challenging Penaeus monodon (Black Tiger shrimp) with the RNA based Gill-associated virus (GAV) following pre-exposure to fragments of the GAV gp116 envelope glycoprotein or to the p20 nucleocapsid protein. Shrimp were injected twice with recombinant GAV protein fragments expressed and purified from bacteria before challenge with GAV. Protein vaccination did not protect the shrimp, with mortality rates in shrimp injected with any of the GAV proteins not differing from shrimp either not injected with protein or injected with a non-specific protein. In addition, there was no significant difference in GAV RNA loads amongst the virus challenged shrimp groups. The data suggest that the efficacy of protection following protein-based vaccination may vary amongst different shrimp viruses.

A previous study suggested that mesendoderm (ME) cell arrest occurred at the 64-cell stage and a ring of eight presumptive naupliar mesoderm cells or crown cells surrounded the blastopore in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus. Since this varied from the pattern observed in other penaeoidean shrimp, cleavage and gastrulation was re-examined in P. japonicus using the nucleic acid stain Sytox Green and confocal microscopy. In contrast to the earlier study, cleavage and gastrulation followed the pattern observed in other penaeoidean shrimp. The ME cells arrested at the 32-cell stage, ingressed into the blastocoel, and resumed division after a three cell cycle delay. Nine naupliar mesoderm or crown cells surrounded the blastopore and their descendants invaginated during gastrulation. An intracellular body (ICB) was detected by Sytox Green and SYTO RNASelect staining to be segregated to one ME cell in P. japonicus, as described previously in Penaeus monodon. Staining of the ICB was eliminated by pre-treatment with RNase but not DNase. The ICB was also found in two other penaeoidean shrimp, Penaeus vannamei (Family Penaeidae) and Sicyonia ingentis (Family Sicyoniidae). The results support the hypothesis that the ICB is a germ granule found in the Dendrobranchiata.

Current penaeid shrimp ploidy induction techniques allow the production of two different classes of triploid: Polar Body (PB) I triploids and PB II triploids. In the present study we quantified the heterozygosity at eight microsatellite loci of PB I and PB II full-sibling triploid Penaeus (Marsupenaeus) japonicus Bate (Kuruma shrimp) compared with full-sibling diploid controls in six separate families. Differences in harvest weight, survival from PL120 to PL210, sex at harvest age and gonad development in females at PL310 of the different triploid classes compared with diploid controls were also assessed for three families, two of which were also included in the heterozygosity assessments. We found no consistent trends in heterozygosity between the PB I and PB II triploids and diploid controls. Furthermore, no consistent trends in the harvest weight and no significant differences ( P > 0.05) in survival from PL120 to PL210 of shrimp in the different triploidy classes and diploid controls within each of the three families were observed when reared in controlled environment tanks. However, we demonstrate for the first time that PB I triploid P. japonicus are predominantly female with only two of the 34 individuals being male, both of which were from the same family. All 115 PB II triploids from the three families were female. We also demonstrate that PB I and PB II triploid females do not produce gonad tissue that is visible through the cuticle by the time they reach PL360, at which life-stage gonad is visible in sibling diploid controls. Provided commercial induction techniques can be developed, this study indicates that triploid induction has the potential to improve harvest yields through the production of predominantly female populations that are potentially reproductively sterile. Findings from this study indicate that there is no benefit in using the harder to perform PB I triploidy induction methods over PB II triploidy induction methods for commercial applications.

Differences in genetic diversity between wild and domesticated Black Tiger shrimp (Penaeus monodon) were assessed in five stocks using molecular markers. These stocks represent an Australian wild population from a single location, and four populations of domesticated animals originating from the same location. Two established high-throughput genotyping systems with a total of 12 microsatellite markers were used to assess diversity of farmed stocks in comparison to wild-caught animals. Genetic diversity analyses showed a large decline in diversity from the wild-caught stocks to the domesticated stocks. Diversity shifts were noted in both allele numbers per locus corrected for sample size (allelic richness) and heterozygosity, declining from 14.2 to 4.6, and 0.77 to 0.61, respectively. The effective population size (Ne) of a commercial fourth generation domesticated strain, following multiple introductions of wild stocks was estimated at 10, highlighting the bottleneck effects possible within a few generations. Significant differences were noted between all pairs of stocks using FST analysis (0.042 to 0.160, P < 0.05), with the greatest difference observed between wild animals and a fourth generation domesticated stock sampled from a commercial farm. This study illustrates the rapid loss of genetic diversity and reduction in effective population size that can occur during domestication when rigid genetic management of the stocks is not possible. This study also demonstrates the value of using molecular (microsatellite) marker systems in shrimp breeding programs to monitor genetic diversity and the effective population size.

In this study the effect of triploidy on survival, growth and gender of the Kuruma shrimp, Penaeus ( Marsupenaeus ) japonicus (Bate), was assessed by making comparisons to diploid sibling performance. Triploidy was induced by preventing polar body II extrusion using 6-dimethylaminopurine (6-DMAP) chemical shock. Four families with a triploid induction rate greater than 70% were reared for approximately 300 days (10 months), during which time their survival, growth, gender ratios and individual ploidy level were assessed. All triploids were found to be female resulting in significantly skewed gender ratios. Males treated with 6-DMAP were either diploid or mosaic (having both diploid and triploid cell populations). 6-DMAP treated shrimp in families 1 and 2 had significantly poorer ( P P > 0.05) over the same period. Similarly, for all four families, survival of 6-DMAP treated and control shrimp was not significantly different ( P > 0.05) from tagging through to assessment age (PL251 for families 1 and 2, and PL173 for families 3 and 4). Treatment with 6-DMAP had a significantly negative effect on weight of females at assessment age when compared to controls of families 1, 2 and 3, and males of families 1 and 4. Diploid females that had received treatment with 6-DMAP were significantly heavier ( P P. japonicus are female could be of significant commercial interest in the future, as shrimp growth rates are sexually dimorphic with females growing faster and larger than males. However, triploidy induced by 6-DMAP chemical shock appears to have detrimental effects on growth and survival performance from sub-adult to reproductive size when compared to diploid siblings. The production of only female triploids supports the hypothesis that the female is the heterogametic gender for P . japonicus .

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 Pjaponicus 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 < 0.05) than for I R 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 < 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. (c) 2007 Elsevier B.V. All rights reserved.

The tolerance of Penaeus monodon embryos from five spawnings (families) to four different ozone doses in seawater [0.0, 0.5, 1.0 and 2.0 mg L−1, measured by the residual oxidant concentration (ROC)] was examined when applied for three exposure times (1, 2 and 4 min) at three post-spawning treatment times (25, 120 and 480 min post-spawning). Ozone dose typically had a larger affect on embryo hatching than exposure time and the ozone dose × exposure time interaction for most combinations of family and post-spawning treatment time. At ozone doses of 2.0 mg L−1, embryos had lower hatchings than controls for all families at 25 and 120 min post-spawning, and for several combinations of family and exposure time at 480 min post-spawning. At ozone doses of 1.0 mg L−1, the effect on embryo hatching was more varied between families and exposure times for the three post-spawning treatment times, but typically embryos were less affected when exposed at later post-spawning treatment times. Ozone doses of 0.5 mg L−1 typically had minimal effects on hatching for all exposure times and post-spawning treatment times. In summary, later-stage P. monodon embryos typically tolerated ozone doses of up to 1.0 mg L−1 in seawater for durations of up to 4 min.

In this study tetraploid Marsupenaeus japonicus (Bate) embryos were produced by preventing the first division in mitosis. The effectiveness of temperature and chemical shocks for producing tetraploid M. japonicus were assessed when applied at different times postspawning and for different durations. Tetraploid M. japonicus embryos (spawned at 27°C) were produced by heat shocks at 35°C and 36°C in three and eight spawning samples respectively, and a cold shock at 5°C in a single spawning sample. All temperature shocks inducing tetraploidy were applied 18–23 min postspawning for a 5–10 min duration. The percentage of spawnings successfully inducing tetraploid embryos (i.e., frequency of induction) ranged from 33.33% to 66.67% for the 21, 22 and 23 min postspawning heat shock treatment regimes. The percentage of tetraploid embryos within an induction (i.e., induction rate), as determined by flow cytometry, ranged from 8.82% to 98.12% (ave. ± S.E.) (34.4 ± 21.4%) for the 35°C shock treatments, from 1...

Brown tiger shrimp (Penaeus esculentus) were intensively grown from PL17 to approximately 1.0 g in a tank system with artificial substrates (AquaMat®). Shrimp growth and survival performance were compared between shrimp stocked at a density of 5720 and 11,430 m− 3. Mean shrimp weight was significantly higher (P 0.05) in survival and biomass between the two stocking densities. Increasing the stocking density from 5720 to 11,430 shrimp m− 3 did not significantly increase shrimp biomass production and resulted in greater production costs and greater risk of losing stock.

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 mu M 6-DMAP from 1- to 3-min post-spawning 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 mu M 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 p M 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 pscl for either a 4- to 5-min duration (timed to stop PBl extrusion) or 16-min duration (timed to stop both PBI and II extrusion) results in viable triploid nauplii. (c) 2006 Elsevier B.V. All rights reserved.

A previous study of captive breeding of Penaeus ( Marsupenaeus ) japonicus demonstrated that broodstock reared from egg to maturity in controlled environment tanks had significantly higher survival rates than sibling broodstock sourced from farm ponds. The pronounced difference in survival of these siblings prompted the current study of Mourilyan virus (MoV) loads in the tank-reared and pond-reared stocks. We examined variation in MoV prevalence in each of five batches of sibling P. japonicus that had been reared for 6 months in either controlled environment tanks or earthen farm ponds before being transferred to maturation tanks. Screening using RT-nested PCR showed no detectible levels of MoV in any of the batches of 20-day-old postlarvae (PL 20 ) at the time that the tanks and ponds were stocked. Six months later, at the start of the 56-day maturation phase, MoV remained undetectable in any of the five batches of tank-reared shrimp. However, among the pond-reared shrimp, MoV was variably detected in all five sibling batches. After maturation for 56 days, MoV remained undetectable in all of 15 shrimp sampled from three of the tank-reared batches but was detectable by RT-PCR in 2 of the 10 shrimp sampled in the other two batches. In contrast, MoV was detected by RT-PCR in most (28/30) shrimp from the five sibling batches of P. japonicus that had previously been reared in ponds. The low prevalence of MoV in the tank-reared stocks was associated with high mean survival (± S.E.) (75.81 ± 7.12%). Conversely, the high prevalence of MoV in the pond-reared stocks was associated with low mean survival (11.29 ± 1.42%). The results indicate that P. japonicus broodstock reared in controlled environment systems are less at risk from MoV, due to lower infection rates or a greater capacity to tolerate infection, compared to putative broodstock sourced from farm ponds.

The reproductive capacity of adult Penaeus (Marsupenaeus) japonicus (Bate) was assessed after exposure to ionizing gamma radiation from a cobalt-60 source. Males and females were each exposed to 0, 10 and 20 Gray (Gy) of ionizing radiation (IR) and reciprocally crossed to give nine mating combinations. Fecundity and hatch rate of resulting spawnings were used as measures of reproductive capacity. IR significantly (P

Brown tiger shrimp ( Penaeus esculentus ) were grown from PL 16 (16-day-old postlarvae) at low density (400 shrimp m −3 ) for 61 days or at high density (1430 shrimp m −3 ) for 75 days. The shrimp were cultured in a raceway (10,500 L) subdivided into six independent, flow through tanks (1750 L). Artificial substrates (buoyant and non-buoyant) placed within the tanks provided a 3-dimensional habitat. Shrimp growth and survival performance was compared at the two stocking densities for three replicate tanks. Growth and survival were significantly higher ( P −3 and a mean biomass of 0.62 ± 0.05 kg m −3 . When harvested after 75 days, shrimp at high density reached a mean weight of 1.48 ± 0.21 g with a survival of 59.8 ± 6.0% and, although the survival was significantly less than the low density, a significantly higher ( P −3 ) and mean biomass (1.23 ± 0.06 kg m −3 ) was produced. The results of this study indicate that P. esculentus has potential for intensive production in tank systems with artificial substrates.

This study investigated the tolerance of Penaeus (Marsupenaeus) japonicus embryos to ozone disinfection to establish a suitable treatment level that has no detrimental effect on embryo hatch rate. In a factorially design experiment, three families were exposed to four different ozone concentrations (C) (0, 0.5, 1 and 2 ppm), for five different exposure times (T) (0.5, 1, 2, 4 and 8 min), to give a total of eight ozone concentration×exposure time (CT) combinations, with an exception for family 1 which had no 8 min exposure time. This was conducted at 16, 120 and 480 min post-spawning for all three families. Embryos were least tolerant to ozonation 16 min post-spawning, with a CT value as low as 0.5 for family 1, and a CT value of 1 for families 2 and 3, significantly (P 0.05) on hatch rate relative to controls in any of the three families. Families 1 and 2 mean hatch rate was not significantly (P

The toxicities of several CT values generated from different combinations of ozone concentration (C) and exposure time (T) were evaluated using hatch rates of Penaeus (Marsupenaeus) japonicus embryos as a measure of ozone toxicity. Embryo hatch rates were investigated at five different CT values (0.5, 1, 2, 4 and 8), produced from different combinations of C (0.5, 1 and 2 ppm) and T (0.5, 1, 2, 4 and 8 min). The experiment was conducted at 16, 120 and 480 min post-spawning for two P. japonicus families. The effect of C on embryo hatch rate when treated at different CT values varied between post-spawning treatment times and families. Where differences in hatch rates were found between C treatments at a single CT value, hatch rates decreased with increasing C. These results indicate that CT values generated by higher C and shorter T values are more toxic to P. japonicus embryos than CT values generated by lower C and longer T values. Our findings indicate that ozone dose cannot be prescribed solely by the CT value, but must also be qualified by describing the C and T values used to generate the CT value. These results have significant implications for designing treatment protocols that aim to use ozonation as a disinfection agent for cultured organisms.

Juvenile brown tiger shrimp, Penaeus esculentus, have been observed to sustain external damage to their appendages and exoskeleton when reared at high densities above 2000 shrimp m−3 for the purpose of restocking into natural populations. This damage principally includes missing appendage segments, broken rostrums and necrotic lesions on the external body surface. Such damage could reduce post-release survival when the juveniles are stocked into wild populations. This study examined both the physical condition of juveniles reared to approximately 1.0 g in a high-density production system and their post-production recovery. Physical condition, measured as the level of damage sustained to a shrimps appendages and exoskeleton, was compared among four treatment groups relative to a control. Two of the treatments were reared at a single density (2860 shrimp m−3) on two different physical substrate types (A and B), and the other two treatments were reared on a single physical substrate (C) but at two different stocking densities, 5720 shrimp and 11,430 shrimp m−3. Juvenile P. esculentus reared in sandy-bottom tanks at 75 shrimp m−3 were maintained as a control. Shrimp in the treatment groups were reared until they reached an average weight of 1 g. The rate of recovery of damage sustained during high-density rearing was determined for all density treatments and the control, when released into sandy-bottom tanks at 100 shrimp m−3 over a period of 22 days. Shrimp reared at all treatment densities (2860, 5720 and 11,430 m−3) became significantly more damaged over time (P 0.05). Similarly, physical substrate type (A and B) had no significant effect on the level of damage sustained by shrimp stocked at 2860 m−3 (P>0.05). These results indicate that shrimp become damaged when reared at densities of 2860 m−3 or greater under these production conditions, and that damage increases over time. The physical signs of appendage recovery of juvenile P. esculentus reared under high-density conditions were studied by tagging and releasing shrimp from each of the stocking densities (2860, 5720 and 11,430 m−3) and the low-density (75 m−3) sandy-bottom tank control treatment, into sandy-bottom tanks at 100 shrimp m−3. Damage decreased significantly (P 0.05) between the rates of appendage recovery by shrimp reared at different densities. These results indicate that appendage recovery for shrimp reared in the three high-density treatments was rapid. Despite shrimp sustaining damage during the production phase, high-density rearing is still a suitable method of high throughput seedstock production, provided that shrimp can survive the 7 day recovery period once released.

The contribution of epiphytes associated with physical substrates to the nutritional requirements of post-larval shrimp, Penaeus esculentus Haswell, was determined in high-density rearing systems (3000, 6000 and 11 000 m−3). Stable isotope signatures of epiphytes on polyethylene mesh substrate, AquaMats™ and tank walls were compared with shrimp signatures. Two methods were used: the determination of carbon and nitrogen natural abundance ratios; and 15N-nitrogen enrichment ratios after the addition of 15N-ammonium to tanks. Using the natural abundance technique and a simple mixing model, epiphytes were found to contribute substantially to the carbon requirements of post-larval shrimp (39–53%). This was despite the addition of formulated feed at satiation levels. There was no indication of a reduced contribution of carbon from epiphytes to shrimp nutrition at higher shrimp densities. The lack of a difference in the 15N/14N ratios of the two food sources meant that mixing models could not be used to calculate the contribution of nitrogen from epiphytes vs. artificial feed to shrimp nutrition. Using the 15N-nitrogenenrichment method, the amount of nitrogen contributed by epiphytes to shrimp nutrition over 24 h could be determined. This method showed that nitrogen from epiphytes was assimilated by shrimp. 15N-enrichment methods provided a more accurate alternative to natural abundance techniques, particularly when the stable isotope signals ofthe food sources are similar. This experiment hasshown the benefits in providing substrates for P.esculentus in high-density rearing systems to provide an additional food source for shrimp.

There is virtually no knowledge of the molecular events controlling early embryogenesis in Penaeid shrimp. A combination of controlled spawning environment, shrimp embryo micro-dissection techniques, and next-generation sequencing was used to produce transcriptome EST datasets of Penaeus japonicus animal and vegetal half-embryos. Embryos were collected immediately after spawning, and then blastomeres were separated at the two-cell stage and allowed to develop to late gastrulation, then pooled for RNA isolation and cDNA synthesis. Ion Torrent sequencing of cDNA from approximately 500 pooled animal and vegetal half-embryos from multiple spawnings resulted in 560,516 and 493,703 reads, respectively. Reads from each library were assembled and Gene Ontogeny analysis produced 3479 annotated animal contigs and 4173 annotated vegetal contigs, with 159/139 hits for developmental processes in the animal/vegetal contigs, respectively. Contigs were subject to BLAST for selected developmental toolbox genes. Some of the genes found included the sex determination genes sex-lethal and transformer; the germ line genes argonaute 1, boule, germ cell-less, gustavus, maelstrom, mex-3, par-1, pumilio, SmB, staufen, and tudor; the mesoderm genes brachyury, mef2, snail, and twist; the axis determination/segmentation genes β-catenin, deformed, distal-less, engrailed, giant, hairy, hunchback, kruppel, orthodenticle, patched, tailless, and wingless/wnt-8c; and a number of cell-cycle regulators. Animal and vegetal contigs were computationally subtracted from each other to produce sets unique to either half-embryo library. Genes expressed only in the animal half included bmp1, kruppel, maelstrom, and orthodenticle. Genes expressed only in the vegetal half included boule, brachyury, deformed, dorsal, engrailed, hunchback, spalt, twist, and wingless/wnt-8c.