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.

Intensification of Penaeid Shrimp Culture: An Applied Review of Advances in Production Systems, Nutrition and Breeding.

Intensification of the shrimp sector, also referred to as vertical expansion, has been predominately driven by consecutive incidences of global disease outbreaks, which have caused enormous economic loss for the main producer countries. A growing segment of the shrimp farming industry has opted to use production systems with higher density, biosecurity, and operating control to mitigate the risks posed by disease. However, successful super-intensive shrimp production is reliant on an advanced understanding of many important biological and economic parameters in the farming system, coupled with effective monitoring, to maintain optimal production. Compared to traditional extensive or semi-intensive systems, super-intensive systems require higher inputs of feed, energy, labor, and supplements. These systems are highly sensitive to the interactions between these different inputs and require that the biological and economical parameters of farming are carefully balanced to ensure success. Advancing nutritional knowledge and tools to support consistent and efficient production of shrimp in these high-cost super-intensive systems is also necessary. Breeding programs developing breeding-lines selected for these challenging super-intensive environments are critical. Understanding synergies between the key areas of production systems, nutrition, and breeding are crucial for super-intensive farming as all three areas coalesce to influence the health of shrimp and commercial farming success. This article reviews current strategies and innovations being used for Litopenaeus vannamei in production systems, nutrition, and breeding, and discusses the synergies across these areas that can support the production of healthy and high-quality shrimp in super-intensive systems. Finally, we briefly discuss some key issues of social license pertinent to the super-intensive shrimp farming industry.

Considerations for Maintaining Family Diversity in Commercially Mass-Spawned Penaeid Shrimp: A Case Study on Penaeus monodon.

Skewed family distributions are common in aquaculture species that are highly fecund, communally (mass) spawned, and/or communally reared. The magnitude of skews pose challenges for maintaining family-specific genetic diversity, as increased resources are required to detect individuals from underrepresented families, or reliably determine relative survival as a measure of family performance. There is limited understanding of family skews or changes in family proportion of communally reared shrimp under commercial rearing conditions and particularly how this may affect genotyping strategies to recover family performance data in breeding programs. In this study, three separate batches of shrimp, Penaeus monodon, were communally spawned and reared, and then sampled as larvae when ponds were stocked at 30 days of culture (DOC) and as juveniles from commercial ponds during harvest at 150 DOC. A total of 199 broodstock contributed to the 5,734 progeny that were genotyped with a custom multiplex single nucleotide polymorphism (SNP) panel, and family assignments were cross-referenced using two parentage assignment methods, CERVUS and COLONY. A total of 121 families were detected, with some families contributing up to 11% of progeny at 30 DOC and up to 18% of progeny at harvest. Significant changes were detected for 20% of families from 30 to 150 DOC, with up to a 9% change in relative contribution. Family skew data was applied in several models to determine the optimal sample size to detect families, along with the ability to detect changes in relative family contribution over time. Results showed that an order of magnitude increase in sampling was required to capture the lowest represented 25% of families, as well as significantly improve the accuracy to determine changes in family proportion from 30 to 150 DOC. Practical measures may be implemented at the hatchery to reduce family skews; a cost-effective measure may be to address the initial magnitude differences in viable progeny produced among families, by pooling equal quantities of hatched larvae from each family. This study demonstrates the relationships between skews in families under commercial conditions, the ability to accurately detect families, and the balance of sampling effort and genotyping cost in highly fecund species such as shrimp.

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.

Real-time PCR tests to specifically detect IHHNV lineages and an IHHNV EVE integrated in the genome of Penaeus monodon.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can cause mass mortalities in western blue shrimp Penaeus stylirostris, runt deformity syndrome in Pacific white shrimp P. vannamei and scalloped abdominal shell deformities in black tiger shrimp P. monodon. In P. monodon, however, PCR-based diagnosis of IHHNV can be complicated by the presence of a chromosome-integrated, non-replicating endogenous viral element (EVE). To facilitate high-throughput screening of P. monodon for IHHNV infection and/or EVE sequences, here we report real-time PCR tests designed to specifically detect IHHNV Lineage I, II and III but not EVE Type A sequences and vice versa. Using 108 dsDNA copies of plasmid (p)DNA controls containing either IHHNV or EVE-Type A sequences, both tests displayed absolute specificity. The IHHNV-q309 PCR reliably detected down to ≤10 copies of pDNA, at which levels a 309F/R PCR amplicon was just detectable, and the presence of an IHHNV-EVE sequence did not significantly impact its sensitivity. The IHHNV-qEVE PCR was similarly sensitive. Testing of batches of P. monodon clinical samples from Vietnam/Malaysia and Australia identified good diagnostic concordance between the IHHNV-q309 and 309F/R PCR tests. As expected for a sequence integrated into host chromosomal DNA, IHHNV-qEVE PCR Ct values were highly uniform among samples from shrimp in which an EVE was present. The highly specific and sensitive IHHNV-q309 and IHHNV-qEVE real-time PCR tests described here should prove useful for selecting broodstock free of IHHNV infection and in maintaining breeding populations of P. monodon specific pathogen free for IHHNV, and if desired, also free of IHHNV-EVE sequences.

Cytological defects during embryogenesis in heat-induced tetraploid Kuruma shrimp Penaeus japonicus.

Tetraploid shrimp embryos have been induced; however, in all cases no postlarvae were produced. This study determined when tetraploid Penaeus japonicus became non-viable and identified unique abnormalities to aid in elucidating the causes of lethality. Embryonic development was analyzed using flow cytometry to determine ploidy and laser scanning confocal microscopy for cytological examination of embryogenesis. Abnormalities exclusive to tetraploids were identified from the 1-cell stage: an off-centre pronucleus, polypolar spindles, delayed time to first mitosis and polypolar cleavage. Following first mitosis in the tetraploids, 50% of the cells did not contain DNA. This unique abnormality was not resolved later in development and is therefore believed to be a lethal trait. Causes of this phenomenon likely stemmed from abnormal mitotic spindle regeneration following the mitotic heat shock. Consequently, the findings of this study indicate that current methods of tetraploidy induction using heat shock appear unsuitable for viable tetraploid shrimp production.

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.

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.

Problem gambling in Australian PTSD treatment-seeking veterans.

This study explored gambling among Australian veterans entering posttraumatic stress treatment programs (n = 153). Twenty-eight percent reached the South Oaks Gambling Screen (SOGS) criteria for probable problem gambling, as did 17% on the DSM-IV gambling scale (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; American Psychiatric Association, 1994). Almost all problem gamblers reported gambling to escape problems in other areas of their lives. The strongest independent predictor of problem gambling was gambling weekly or more often on electronic gaming machines. There was no significant relationship between problem gambling, posttraumatic stress disorder (PTSD), anxiety, depression, or alcohol use. The study identified an entrenched gambling culture among PTSD treatment-seeking veterans, finding these veterans indulge in many different forms of gambling and that these forms are mediated by situational factors that provide both casual and formal gambling opportunities.