Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

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.

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.

Development and validation of a RAD-Seq target-capture based genotyping assay for routine application in advanced black tiger shrimp (Penaeus monodon) breeding programs.

BACKGROUND: The development of genome-wide genotyping resources has provided terrestrial livestock and crop industries with the unique ability to accurately assess genomic relationships between individuals, uncover the genetic architecture of commercial traits, as well as identify superior individuals for selection based on their specific genetic profile. Utilising recent advancements in de-novo genome-wide genotyping technologies, it is now possible to provide aquaculture industries with these same important genotyping resources, even in the absence of existing genome assemblies. Here, we present the development of a genome-wide SNP assay for the Black Tiger shrimp (Penaeus monodon) through utilisation of a reduced-representation whole-genome genotyping approach (DArTseq). RESULTS: Based on a single reduced-representation library, 31,262 polymorphic SNPs were identified across 650 individuals obtained from Australian wild stocks and commercial aquaculture populations. After filtering to remove SNPs with low read depth, low MAF, low call rate, deviation from HWE, and non-Mendelian inheritance, 7542 high-quality SNPs were retained. From these, 4236 high-quality genome-wide loci were selected for baits-probe development and 4194 SNPs were included within a finalized target-capture genotype-by-sequence assay (DArTcap). This assay was designed for routine and cost effective commercial application in large scale breeding programs, and demonstrates higher confidence in genotype calls through increased call rate (from 80.2 ± 14.7 to 93.0% ± 3.5%), increased read depth (from 20.4 ± 15.6 to 80.0 ± 88.7), as well as a 3-fold reduction in cost over traditional genotype-by-sequencing approaches. CONCLUSION: Importantly, this assay equips the P. monodon industry with the ability to simultaneously assign parentage of communally reared animals, undertake genomic relationship analysis, manage mate pairings between cryptic family lines, as well as undertake advance studies of genome and trait architecture. Critically this assay can be cost effectively applied as P. monodon breeding programs transition to undertaking genomic selection.

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 (≥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.

De novo assembly, characterization, functional annotation and expression patterns of the black tiger shrimp (Penaeus monodon) transcriptome.

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 based on nine adult tissues and eight early life-history stages (BUSCO - Complete: 98.2% [Duplicated: 51.3%], Fragmented: 0.8%, Missing: 1.0%). The assembly resulted in 236,388 contigs, which were then further segregated into 99,203 adult tissue specific and 58,678 early life-history stage specific clusters. 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. 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. 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 impacts of modern-use pesticides on shrimp aquaculture: An assessment for north eastern Australia.

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.

Expression of the prospective mesoderm genes twist, snail, and mef2 in penaeid shrimp.

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.

Transcriptome profiles of Penaeus (Marsupenaeus) japonicus animal and vegetal half-embryos: identification of sex determination, germ line, mesoderm, and other developmental genes.

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 ß-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.

Quantitative analysis of low-density SNP data for parentage assignment and estimation of family contributions to pooled samples.

BACKGROUND: While much attention has focused on the development of high-density single nucleotide polymorphism (SNP) assays, the costs of developing and running low-density assays have fallen dramatically. This makes it feasible to develop and apply SNP assays for agricultural species beyond the major livestock species. Although low-cost low-density assays may not have the accuracy of the high-density assays widely used in human and livestock species, we show that when combined with statistical analysis approaches that use quantitative instead of discrete genotypes, their utility may be improved. The data used in this study are from a 63-SNP marker Sequenom® iPLEX Platinum panel for the Black Tiger shrimp, for which high-density SNP assays are not currently available. RESULTS: For quantitative genotypes that could be estimated, in 5% of cases the most likely genotype for an individual at a SNP had a probability of less than 0.99. Matrix formulations of maximum likelihood equations for parentage assignment were developed for the quantitative genotypes and also for discrete genotypes perturbed by an assumed error term. Assignment rates that were based on maximum likelihood with quantitative genotypes were similar to those based on maximum likelihood with perturbed genotypes but, for more than 50% of cases, the two methods resulted in individuals being assigned to different families. Treating genotypes as quantitative values allows the same analysis framework to be used for pooled samples of DNA from multiple individuals. Resulting correlations between allele frequency estimates from pooled DNA and individual samples were consistently greater than 0.90, and as high as 0.97 for some pools. Estimates of family contributions to the pools based on quantitative genotypes in pooled DNA had a correlation of 0.85 with estimates of contributions from DNA-derived pedigree. CONCLUSIONS: Even with low numbers of SNPs of variable quality, parentage testing and family assignment from pooled samples are sufficiently accurate to provide useful information for a breeding program. Treating genotypes as quantitative values is an alternative to perturbing genotypes using an assumed error distribution, but can produce very different results. An understanding of the distribution of the error is required for SNP genotyping platforms.

Mechanisms of colour adaptation in the prawn Penaeus monodon.

Exposure of prawns to dark- or light-coloured substrates is known to trigger a strong colour adaptation response through expansion or contraction of the colouration structures in the prawn hypodermis. Despite the difference in colour triggered by this adaptive response, total levels of the predominant carotenoid pigment, astaxanthin, are not modified, suggesting that another mechanism is regulating this phenomenon. Astaxanthin binds to a specific protein called crustacyanin (CRCN), and it is the interaction between the quantities of each of these compounds that produces the diverse range of colours seen in crustacean shells. In this study, we investigated the protein changes and genetic regulatory processes that occur in prawn hypodermal tissues during adaptation to black or white substrates. The amount of free astaxanthin was higher in animals adapted to dark substrate compared with those adapted to light substrate, and this difference was matched by a strong elevation of CRCN protein. However, there was no difference in the expression of CRCN genes either across the moult cycle or in response to background substrate colour. These results indicate that exposure to a dark-coloured substrate causes an accumulation of CRCN protein, bound with free astaxanthin, in the prawn hypodermis without modification of CRCN gene expression. On light-coloured substrates, levels of CRCN protein in the hypodermis are reduced, but the carotenoid is retained, undispersed in the hypodermal tissue, in an esterified form. Therefore, the abundance of CRCN protein affects the distribution of pigment in prawn hypodermal tissues, and is a crucial regulator of the colour adaptation response in prawns.

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 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.

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.

Cleavage and gastrulation in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus (Bate): a revised cell lineage and identification of a presumptive germ cell marker.

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.

Expression of sex and reproduction-related genes in Marsupenaeus japonicus.

Expressed sequence tags (ESTs) were identified from reciprocal suppression subtractive hybridization cDNA libraries from Marsupenaeus japonicus (Kuruma shrimp) female and male gonads. The expression profiles of 24 of these ESTs were determined in female and male gonads and developing postlarvae by real-time quantitative reverse transcription-polymerase chain reaction. When expression was determined in gonads, six of the ESTs were expressed in ovaries only, and five of the ESTs were expressed in testes only. When expression was determined in whole individuals during postlarval development, expression of the ESTs was low and inconsistent until stage PL110 (110 days since metamorphosis from mysis stage to the first postlarval stage). At PL110, seven of the ESTs were detected in females only, and seven ESTs were detected in males only. Sex-specific expression at this developmental stage indicates that these ESTs act as important gonadal development markers and may have a role in gametogenesis.

A PL10 vasa-like gene in the kuruma shrimp, Marsupenaeus japonicus, expressed during development and in adult gonad.

A PL10 vasa-like gene was isolated from the Kuruma shrimp Marsupenaeus japonicus and therefore called Mjpl10. It is differentially expressed during embryonic, larval, and postlarval development, and in female and male gonads. Using absolute real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrate that Mjpl10 transcripts are present in the two-cell embryo, suggesting it is maternally expressed, and continually at low levels throughout embryogenesis. Mjpl10 expression increases significantly in the first 25 h after hatching (nauplii IV) and then decreases in a linear fashion by 316-fold over the next 52-day period. Its continued expression throughout embryonic and larval development is compatible with a conserved role in early germ cell specification. Transcript levels of Mjpl10 are also detected in the ovary and testes of mature adults.

Real-time RT-PCR quantification of Kuruma shrimp transcripts: a comparison of relative and absolute quantification procedures.

Housekeeping genes are often used as references when quantifying the relative abundance of transcripts of interest, because it is assumed that they are stably expressed across tissues and developmental stages. Standard housekeeping genes are targeted particularly in organisms where there is no detailed information on gene expression profiles. Here, the validity of using the two widely accepted housekeeping genes, 18S rRNA and beta-actin, as reference genes to normalize real-time RT-PCR gene expression data from the Kuruma shrimp, Marsupenaeus japonicus, was tested. Expression patterns of two target genes in a diverse sample set of embryonic, larval, post-larval and gonad mRNAs were quantified using relative and absolute real-time RT-PCR procedures. Comparison of these approaches revealed significant differences (P<0.0001) in transcript level profiles between the relative and absolute procedures for both target genes. When 18S rRNA was used as a reference, target gene expression was more similar to that of the absolute method than when beta-actin was used as a reference. Variability between the relative and absolute procedures occurred for a greater percentage of the embryonic stages compared to later developmental stages. This study indicates that the use of 18S rRNA and beta-actin for studying relative gene expression patterns in Kuruma shrimp embryonic, larval, post-larval and gonad samples will give significantly variable results, and illustrates the proposition that housekeeping genes are not necessarily appropriate references for real-time RT-PCR data normalization. Until suitable reference genes are characterized, gene expression experiments using the studied Kuruma shrimp tissues of different morphological developmental stages should use absolute quantification procedures.