Feed Containing Novacq Improves Resilience of Black Tiger Shrimp, Penaeus Monodon, to Gill-associated Virus-induced Mortality

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<0.05) from Day 7 onward among the group fed Novacq. GAV infection loads appeared to vary more between individuals in the Novacq diet cohort, but overall were not reduced significantly at any time points post-challenge compared to shrimp tested from the Control diet cohort.&nbsp

Reduced loads of pre-existing Gill-associated virus (GAV) infection in juvenile Penaeus monodon injected with single or multiple GAV-specific dsRNAs

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 (

Fine-scale population structure and evidence for local adaptation in Australian giant black tiger shrimp (Penaeus monodon) using SNP analysis

Background: Restrictions to gene flow, genetic drift, and divergent selection associated with different environments are significant drivers of genetic differentiation. The black tiger shrimp (Penaeus monodon), is widely distributed throughout the Indian and Pacific Oceans including along the western, northern and eastern coastline of Australia, where it is an important aquaculture and fishery species. Understanding the genetic structure and the influence of environmental factors leading to adaptive differences among populations of this species is important for farm genetic improvement programs and sustainable fisheries management. Results: Based on 278 individuals obtained from seven geographically disparate Australian locations, 10,624 high-quality SNP loci were used to characterize genetic diversity, population structure, genetic connectivity, and adaptive divergence. Significant population structure and differentiation were revealed among wild populations (average FST = 0.001–0.107; p <  0.05). Eighty-nine putatively outlier SNPs were identified to be potentially associated with environmental variables by using both population differentiation (BayeScan and PCAdapt) and environmental association (redundancy analysis and latent factor mixed model) analysis methods. Clear population structure with similar spatial patterns were observed in both neutral and outlier markers with three genetically distinct groups identified (north Queensland, Northern Territory, and Western Australia). Redundancy, partial redundancy, and multiple regression on distance matrices analyses revealed that both geographical distance and environmental factors interact to generate the structure observed across Australian P. monodon populations. Conclusion: This study provides new insights on genetic population structure of Australian P. monodon in the face of environmental changes, which can be used to advance sustainable fisheries management and aquaculture breeding programs

Spawning of female Black tiger shrimp (Penaeus monodon) is not impacted by muscle injection of dsRNA targeted to gill-associated virus

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\ua0600 to 459\ua0800 for the saline-injected shrimp and from 4900 to 213\ua0900 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.\ua0monodon to spawn

Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

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 doublestranded (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 ß-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

TaqMan real-time and conventional nested PCR tests specific to yellow head virus genotype 7 (YHV7) identified in giant tiger shrimp in Australia

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 (≥10) 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

The effects of adding microbial biomass to grow-out and maturation feeds on the reproductive performance of black tiger shrimp, Penaeus monodon

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= < 0.05). These results indicate that the inclusion of MB within broodstock grow-out and maturation diets (at the rates presented in this study) did not enhance reproductive performance of domesticated broodstock

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

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

Growing backwards: an inverted role for the shrimp ortholog of vertebrate myostatin and GDF11

Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins involved in muscle cell growth and differentiation as well as neurogenesis of vertebrates. Both MSTN and GDF11 negatively regulate their functions. Invertebrates possess a single ortholog of the MSTN/GDF11 family. In order to understand the role of MSTN/GDF11 in crustaceans, the gene ortholog was identified and characterized in the penaeid shrimp Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, pmMstn/Gdf11 gene expression was downregulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp, but suggest that, compared with higher vertebrates, this gene has an opposite role in invertebrates such as shrimp, where levels of gene expression may positively regulate growth