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Epigenetics is involved in sex differentiation of gonochoristic and hermaphroditic fish species, whereby two genes dmrt1 (pro-male) and cyp19a1 (pro-female) are known to play major roles. Barramundi, Lates calcarifer, is an important tropical aquaculture species that undergo natural and permanent male to female sex change, a process for which the exact underlying molecular mechanisms are still unknown. To elucidate whether DNA methylation is involved in sex control of barramundi, a next-generation bisulfite amplicon sequencing approach was used to target 146 CpG sites within proximal promoters and first exons of seven sex-related genes (dmrt1, cyp19a1, amh, foxl2, nr5a2, sox8 and sox9) of 24 testis and 18 ovaries of captive and wild adult barramundi. Moreover, comparative expression profiles of the key dmrt1 and cyp19a1 genes were further investigated using RT-qPCR and Sanger sequencing approaches, whereas expression levels of remaining targeted genes were based on available literature for the species. Results showed that cyp19a1 and amh were more methylated in males, whereas dmrt1 and nr5a2 were more methylated in females (P < 0.001), with no gender differences found for foxl2, sox8 or sox9 genes (P > 0.05). Sex-biased promoter DNA methylation was inversely related to gene expression only for dmrt1 and nr5a2, and directly related to amh expression, whereas no differences in cyp19a1 expression were found between testes and ovaries. Notably, unique sex-specific alternative splicing of dmrt1 and cyp19a1 were discovered, whereby males lacked the full-length aromatase coding cyp19a1 mRNA due to partial or total exon splicing, and females lacked the dmrt1 exon containing the DM-domain sequence. This study advances the current knowledge aiming to elucidate the genetic mechanisms within male and female gonads of this large protandrous hermaphrodite by providing the first evidence of epigenetics and alternative splicing simultaneously affecting key genes (cyp19a1 and dmrt1) central to sex differentiation pathways.

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.

Barramundi ( Lates calcarifer ) is an important aquaculture species extensively farmed throughout its natural distribution of Australia and Southeast Asia, as well as being increasingly farmed in the Middle East, USA, and Europe. Barramundi has a firm, pink‐white flesh; however, fillets from farmed barramundi often exhibit grey colouration. This grey colouration detracts from its market appeal, leading to challenges in consumer acceptance and competitiveness of the product against other white fillet fish. Selective breeding, environmental manipulation, and dietary adjustments are being investigated to reduce grey flesh colouration. Yet, the absence of a rapid, noninvasive approach to predict greyness in flesh means that large numbers of samples cannot be quickly evaluated, and issues cannot be mitigated preharvest and noninvasively to preserve the fish. To address this issue, rapid analysis of flesh greyness was developed using noninvasive near‐infrared (NIR) spectroscopy through the fish skin. Thirty fish were purchased from a barramundi farm, filleted, and divided into 3 cm sections, yielding a total of 335 samples from both dorsal and ventral fillet regions. NIR spectral data were obtained from the skin side of all samples, and colouration data were collected from the flesh side of the same samples. Data were randomised into a training set (256 spectra) and a validation set (79 spectra). Predictive models were developed using flesh colour as the training input for skin NIR spectra. The refined partial least squares regression model explained 78% of the variation in the medial flesh colour (R 2 pe of 0.776, an RMSEP of 2.820, and an RPD pe of 2.122) demonstrating the ability to adequately predict the flesh quality through skin spectra. This highlights the potential of NIR spectroscopy as a dependable, noninvasive tool, enabling the rapid evaluation of large samples and offering the potential to address flesh colouration issues in barramundi preharvest.

Background Genetic structure in many widely-distributed broadcast spawning marine invertebrates remains poorly understood, posing substantial challenges for their fishery management, conservation and aquaculture. Under the Core-Periphery Hypothesis (CPH), genetic diversity is expected to be highest at the centre of a species’ distribution, progressively decreasing with increased differentiation towards outer range limits, as populations become increasingly isolated, fragmented and locally adapted. The unique life history characteristics of many marine invertebrates such as high dispersal rates, stochastic survival and variable recruitment are also likely to influence how populations are organised. To examine the microevolutionary forces influencing population structure, connectivity and adaptive variation in a highly-dispersive bivalve, populations of the black-lip pearl oyster Pinctada margaritifera were examined across its ~18,000 km Indo-Pacific distribution. Results Analyses utilising 9,624 genome-wide SNPs and 580 oysters, discovered differing patterns of significant and substantial broad-scale genetic structure between the Indian and Pacific Ocean basins. Indian Ocean populations were markedly divergent ( F st  = 0.2534–0.4177, p  < 0.001), compared to Pacific Ocean oysters, where basin-wide gene flow was much higher ( F st  = 0.0007–0.1090, p  < 0.001). Partitioning of genetic diversity (hierarchical AMOVA) attributed 18.1% of variance between ocean basins, whereas greater proportions were resolved within samples and populations (45.8% and 35.7% respectively). Visualisation of population structure at selectively neutral loci resolved three and five discrete genetic clusters for the Indian and Pacific Oceans respectively. Evaluation of genetic structure at adaptive loci for Pacific populations (89 SNPs under directional selection; F st  = 0.1012–0.4371, FDR = 0.05), revealed five clusters identical to those detected at neutral SNPs, suggesting environmental heterogeneity within the Pacific. Patterns of structure and connectivity were supported by Mantel tests of isolation by distance (IBD) and independent hydrodynamic particle dispersal simulations. Conclusions It is evident that genetic structure and connectivity across the natural range of P. margaritifera is highly complex, and produced by the interaction of ocean currents, IBD and seascape features at a broad scale, together with habitat geomorphology and local adaptation at regional levels. Overall population organisation is far more elaborate than generalised CPH predictions, however valuable insights for regional fishery management, and a greater understanding of range-wide genetic structure in a highly-dispersive marine invertebrate have been gained.

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 F ST  = 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.

Calanoid copepods are an important food source for most fish larvae. Their role as a natural prey item means that it is important to develop culture technology for copepods to meet the requirements of larvae culture in aquaculture hatcheries. Copepods have been cultured successfully for some time; however, the implications of long-term cultivation or domestication on copepod life history traits have not yet been assessed. Therefore, the aim of this study was to determine if the domesticated and wild populations of Parvocalanus crassirostris are phenotypically or physiologically different. Wild-caught P. crassirostris were compared to a long-held domesticated strain (>2 years) for size of developmental stage, fecundity and lifespan of adult females, culture density, ingestion rates, faecal pellet production and fatty acid profiles. The domesticated strain was significantly different from the wild strain in size (eggs, nauplii, copepodites and adults were larger in the domesticated strain), egg production (112.3 ± 1.8 eggs female(-1) vs. 64.6 ± 3.3 eggs female(-1)) and adult female lifespan (8.8 ± 0.1 days vs., 7.5 ± 0.1 days). At 1, 3 and 5 adults mL(-1), the domesticated strain performed significantly better than the wild strain in egg production (4189.8 ± 61.2, 11224.0 ± 71.7 and 21860.6 ± 103.6 eggs vs. 1319.5 ± 54.3, 2374.5 ± 80.9 and 4933.8 ± 269.5 eggs, respectively) and mean daily mortality rate (5.6% across all densities vs. 22.9 ± 1.6, 29.8 ± 1.2 and 31.3 ± 1.3%, respectively). The domesticated strain had significantly higher ingestion rates than the wild stain (888.4 ± 9.9 ng C l(-1) and 775.3 ± 11.2 ng C l(-1), respectively), while faecal pellet production was not significantly different between strains. Fatty acid profiles indicated higher levels (as % of total fatty acid) of long-chain polyunsaturated fatty acids in the domesticated strain (36.2±0.4%) than the wild strain (16.1±0.1%). Overall, this study found that the reproductive capacity and tolerance to the culture environment of the calanoid P. crassirostris have improved significantly due to domestication.

Background: Fish skin mucus contains proteins involved in diverse biological pathways, representing a valuable non-invasive diagnostic of fish health. Methods: Skin mucus from three male and three female barramundi was analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following protein extraction and S-Trap digestion. Results and Discussion: A total of 1801 protein groups were matched to the L. calcarifer reference proteome and functionally annotated using Gene Ontology (GO) terms via UniProt ID mapping, with representation across Biological Process, Cellular Component, and Molecular Function categories. Functional classification using eggNOG-mapper further associated leading protein group sequences with Clusters of Orthologous Groups (COGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. GO-based screening prioritised 352 putatively immune-relevant protein groups and 24 protein groups associated with sex- and reproduction-related processes, highlighting the functional complexity of the skin mucus proteome. Comparative analysis revealed sex-associated patterns in protein group detection and relative abundance, with differential abundance analysis identifying 244 protein groups exhibiting statistically significant differences between male and female samples. Conclusions: This study provides the first comprehensive discovery-based characterisation of the barramundi skin mucus proteome and establishes a baseline reference dataset for this aquaculture-relevant species. The findings support the utility of skin mucus proteomics for exploring immune and sex-associated molecular patterns and provide a baseline dataset for future validation studies investigating non-invasive health and reproductive monitoring.

Cubozoan jellyfish pose a risk of envenomation to humans and a threat to many businesses, yet crucial gaps exist in determining threats to stakeholders and understanding their ecology. Environmental DNA (eDNA) provides a cost-effective method for detection that is less labour intensive and provides a higher probability of detection. The objective of this study was to develop, optimise and trial the use of eDNA to detect the Australian box jellyfish, Chironex fleckeri. This species was the focus of this study as it is known to have the strongest venom of any cubozoan; it is responsible for more than 200 recorded deaths in the Indo-Pacific region. Further, its ecology is poorly known. Herein, a specific and sensitive probe-based assay, multiplexed with an endogenous control assay, was developed, and successfully utilised to detect the deadly jellyfish species and differentiate them from closely related taxa. A rapid eDNA decay rate of greater than 99% within 27 h was found with no detectable influence from temperature. The robustness of the technique indicates that it will be of high utility for detection and to address knowledge gaps in the ecology of C. fleckeri; further, it has broad applicability to other types of zooplankton.

Here we utilize a combination of genetic data, oceanographic data, and local ecological knowledge to assess connectivity patterns of the ornate spiny lobster Panulirus ornatus (Fabricius, 1798) in the South-East Asian archipelago from Vietnam to Australia. Partial mitochondrial DNA control region and 10 polymorphic microsatellites did not detect genetic structure of 216 wild P. ornatus samples from Australia, Indonesia and Vietnam. Analyses show no evidence for genetic differentiation among populations (mtDNA control region sequences ΦST = -0.008; microsatellite loci FST = 0.003). A lack of evidence for regional or localized mtDNA haplotype clusters, or geographic clusters of microsatellite genotypes, reveals a pattern of high gene flow in P. ornatus throughout the South-East Asian Archipelago. This lack of genetic structure may be due to the oceanography-driven connectivity of the pelagic lobster larvae between spawning grounds in Papua New Guinea, the Philippines and, possibly, Indonesia. The connectivity cycle necessitates three generations. The lack of genetic structure of P. ornatus population in the South-East Asian archipelago has important implications for the sustainable management of this lobster in that the species within the region needs to be managed as one genetic stock.

Geological evolution of the African continent has been subject to complex processes including uplift, volcanism, desert formation and tectonic rifting. This complex geology has created substantial biogeographical barriers, and coupled with anthropogenic introductions of freshwater fishes, has influenced the genetic diversity, connectivity and sub-structuring of the teleost fauna. Nile perch, Lates niloticus, is an iconic fish in Africa and is of high commercial importance, both in the species' native range and where it has been translocated. However, the species is in decline and there is a need to understand its population genetic structure to facilitate sustainable management of the fishery and aquaculture development. Nile perch tissue samples were acquired from two West and four East (Lakes; Albert, Kyoga, Victoria and Turkana) African locations. Nineteen polymorphic microsatellite loci were used to study the genetic variation among populations across regions (West and East Africa), as well as between native and introduced environments within East Africa. Results revealed strong and significant genetic structuring among populations across the sampled distribution (divergence across regions, FCT = 0.26, P = 0.000). STRUCTURE analysis at a broad scale revealed K = 2 clusters, the West African individuals were assigned to one cluster, while all individuals from the East African region, regardless of whether native or introduced, were assigned to another cluster. The distinct genetic clusters identified in the current study between the West and East African Nile perch, appear to have been maintained by presence of biogeographic barriers and restricted gene flow between the two regions. Therefore, any translocations of Nile perch should be carefully considered across the regions of West and East Africa. Further analysis at a regional scale revealed further structuring of up to K = 3 genetic clusters in East African Nile perch. Significantly (P < 0.05) lower genetic diversity based on analysis of allelic richness (AR) was obtained for the two translocated populations of Lake Kyoga (AR = 3.61) and Lake Victoria (AR = 3.52), compared to Nile perch populations from their putative origins of Lakes Albert (AR = 4.12) and Turkana (AR = 4.43). The lower genetic diversity in the translocated populations may be an indication of previous bottlenecks and may also indicate a difficulty for these populations to persist and adapt to climatic changes and anthropogenic pressures that are currently present in the East African region.