Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO3, pH, and Na+ and K+ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO3 as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl2·2H2O, 1.1 mM MgSO4·7H2O, 10.0 mM HEPES, 5.6 mM D+ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi

Population structure, genetic connectivity, and signatures of local adaptation of the giant black tiger shrimp (Penaeus monodon) throughout the indo-pacific region

The giant black tiger shrimp (Penaeus monodon) is native to the Indo-Pacific and is the second most farmed penaeid shrimp species globally. Understanding genetic structure, connectivity, and local adaptation among Indo-Pacific black tiger shrimp populations is important for informing sustainable fisheries management and aquaculture breeding programs. Population genetic and outlier detection analyses were undertaken using 10,593 genome-wide single nucleotide polymorphisms (SNPs) from 16 geographically disparate Indo-Pacific P. monodon populations. Levels of genetic diversity were highest for Southeast Asian populations and were lowest for Western Indian Ocean (WIO) populations. Both neutral (n = 9,930) and outlier (n = 663) loci datasets revealed a pattern of strong genetic structure of P. monodon corresponding with broad geographical regions and clear genetic breaks among samples within regions. Neutral loci revealed seven genetic clusters and the separation of Fiji and WIO clusters from all other clusters, whereas outlier loci revealed six genetic clusters and high genetic differentiation among populations. The neutral loci dataset estimated five migration events that indicated migration to Southeast Asia from the WIO, with partial connectivity to populations in both oceans. We also identified 26 putatively adaptive SNPs that exhibited significant Pearson correlation (P < 0.05) between minor allele frequency and maximum or minimum sea surface temperature. Matched transcriptome contig annotations suggest putatively adaptive SNPs involvement in cellular and metabolic processes, pigmentation, immune response, and currently unknown functions. This study provides novel genome-level insights that have direct implications for P. monodon aquaculture and fishery management practices

High-Density Genetic Linkage Map of the Southern Blue-ringed Octopus (Octopodidae: Hapalochlaena maculosa)

Genetic linkage maps provide a useful resource for non-model genomes and can aid in genome reassembly to form more contiguous pseudo-chromosomes. We present the first linkage map of any cephalopod, H. maculosa, composed of 47 linkage groups (LG). A total of 2166 single nucleotide polymorphisms and 2455 presence–absence variant loci were utilised by Lep-Map3 in linkage map construction. The map length spans 2016.62 cM with an average marker distance of 0.85 cM. Integration of the recent H. maculosa genome allowed 1151 scaffolds comprising 34% of the total genomic sequence to be orientated and/or placed using 1278 markers across all 47 LG. The linkage map generated provides a new perspective on HOX gene distribution in octopods. In the H. maculosa linkage map three (SCR, LOX4 and POST1) of six identified HOX genes (HOX1/LAB, SCR, LOX2, LOX4, LOX5, POST1) were located within the same LG (LG 9). The generation of a linkage map for H. maculosa has provided a valuable resource for understanding the evolution of cephalopod genomes and will provide a base for future work

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

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

The State of “Omics” Research for Farmed Penaeids: Advances in Research and Impediments to Industry Utilization

Elucidating the underlying genetic drivers of production traits in agricultural and aquaculture species is critical to efforts to maximize farming efficiency. “Omics” based methods (i.e., transcriptomics, genomics, proteomics, and metabolomics) are increasingly being applied to gain unprecedented insight into the biology of many aquaculture species. While the culture of penaeid shrimp has increased markedly, the industry continues to be impeded in many regards by disease, reproductive dysfunction, and a poor understanding of production traits. Extensive effort has been, and continues to be, applied to develop critical genomic resources for many commercially important penaeids. However, the industry application of these genomic resources, and the translation of the knowledge derived from “omics” studies has not yet been completely realized. Integration between the multiple “omics” resources now available (i.e., genome assemblies, transcriptomes, linkage maps, optical maps, and proteomes) will prove critical to unlocking the full utility of these otherwise independently developed and isolated resources. Furthermore, emerging “omics” based techniques are now available to address longstanding issues with completing keystone genome assemblies (e.g., through long-read sequencing), and can provide cost-effective industrial scale genotyping tools (e.g., through low density SNP chips and genotype-by-sequencing) to undertake advanced selective breeding programs (i.e., genomic selection) and powerful genome-wide association studies. In particular, this review highlights the status, utility and suggested path forward for continued development, and improved use of “omics” resources in penaeid aquaculture

Maturation and spawning performance of hormonally-induced precocious female barramundi (Lates calcarifer) and implications of their use in selective breeding

Breeding programs for barramundi (Lates calcarifer) are impeded by the lack of control over the sexual development of broodstock. As a protandrous hermaphrodite, barramundi initially sexually mature as male at 2–3 years (2–4 kg body weight; BW), before changing sex to female at 4–6 years (>6–8 kg BW). Recently, precocious female barramundi have been generated using a single intramuscular estrogen implant, providing the potential to speed up the sex change process and implement same-age mating of male and female broodfish. However, the functional maturation of hormonally-induced precocious females and their ability to spawn and produce viable progeny remains untested. Male barramundi (~2.2 kg BW, 57.8 cm total length; TL) were hormonally induced to sex change using an estrogen implant. The precocious female barramundi were cannulated at 6 (T1), 8 (T2), 10 (T3), 11.5 (T4) and 13 (T5) months after sex change, with oocytes measured and staged to determine reproductive condition. At T3, four females (~4.2 kg BW, 67.8 cm TL) with oocytes >350 μm in size were transferred to a spawning tank along with four males to assess their spawning performances. Mass spawning events were induced at T3, T4 and T5, and eggs released on the first and second spawning nights were collected to determine rates of fertilization, hatching and larval survival at 24 and 48 h post-hatch. At each cannulation event (T1 - T5), several precocious females were confirmed to have mature oocytes and were considered ready for induced spawning. Conversely, in some individuals, only immature oocytes were during each cannulation event. The four precocious females induced to spawn generated large numbers of eggs, with 7.7 million (T3), 10.9 million (T4) and 9.1 million (T5) eggs obtained across the two nights of each mass spawning event. Fertilization of oocytes was not observed at T3 and was likely due to male inactivity. Spawning performance improved considerably for T4 and T5 spawns, with the highest fertilization rate (64%) and hatching rate (72%) observed during T5 - Night 1. Furthermore, larval survival at 24 and 48 h after hatching was 99% and 94%, respectively. Total larval production across both nights of the T5 spawn exceeded 3 million individuals. Spawning performances of precocious females were comparable to routine commercial stocks of large females (8–15 kg), and confirmed that precociously induced female barramundi can produce the quantity of seedstock required in existing selective breeding programs. Further research examining the factors involved in promoting oocyte growth and maturation is needed to enhance the rate at which mature precocious females are available for spawning

Delayed effect of low rearing temperature on gonadal DNA methylation in juvenile barramundi (Lates calcarifer)

Sex differentiation in many gonochoristic fish is highly responsive to temperature treatment. In some species, exposure to high or low temperatures during primary gonadal development induces phenotypic reversal of an otherwise genetically encoded sex. These changes in sexual phenotype occur in the absence of changes in genotype and are often accompanied by epigenetic modifications, such as DNA methylation. It is unknown if sequentially hermaphroditic fish exhibit similar temperature sensitivity to what has been shown in gonochoristic species; however, sex-specific differences in DNA methylation have been observed. The barramundi (Lates calcarifer) is a protandrous (male-first) sequential hermaphrodite that exhibits sex-specific DNA methylation patterns. To examine the effect of temperature on DNA methylation and primary gonadal differentiation in a sequential hermaphrodite, sexually immature barramundi were exposed to high (34 °C), control (29 °C) low (24 °C) and fluctuating (25–33 °C) temperatures from 10 to 90 days post hatch, and then on-reared at 29 °C for 12 months. At 6 months and 12 months, individuals were sampled, and their gonads examined for evidence of changes in DNA methylation and/or gonadal differentiation. Male-specific DNA methylation patterns and advanced testis development were observed at 12 months but were absent at 6 months, suggesting that low temperature treatment had a delayed effect on the gonads of juvenile barramundi. Additionally, there was an initial, but not lasting, negative effect of low temperature treatment on fish length and weight, indicating that compensatory growth may have occurred. This research provides the first evidence for an effect of temperature on gonadal DNA methylation and primary sexual development in a sequential hermaphrodite. A refinement of treatment timing and intensity could lead to the development of a hormone-free method of sex control for this widely cultured species

Validation of advanced tools to evaluate sperm function in barramundi (Lates calcarifer)

Barramundi (Lates calcarifer) is a tropical finfish species rapidly growing in popularity for aquaculture production. However, sperm quality tests have yet to be adapted to enable selection of highly fertile male broodstock in this species. Accordingly, in this study advanced tools were optimized to evaluate barramundi sperm function to facilitate the future study of male fertility and address some of the reproductive constraints currently observed in captive-bred broodstock. Sperm morphology data were used to calibrate and validate automated sperm counting and motility detection by computer-assisted sperm analysis (CASA; AndroVision, Minitube). Several parameters were examined to determine the optimum settings for accurate CASA sperm counting and were compared to manual haemocytometer methods including: sample dilution (1:1000, r = 0.87), minimum number of fields (n = 4, CV = 7.5%), and the effect of motile vs. immotile spermatozoa on automated counting (no effect, r = 0.99, P < .001). Assays for cell viability and DNA damage were also validated for barramundi spermatozoa using 70 °C heat-treated controls and a 5-point intact:damaged dilution curve (r = 0.98, P < .001), and DNase-treated sperm controls, respectively. Data from these optimized assessments indicated high variation between individuals for each parameter assessed and the presence of high rates of DNA and membrane damage in sperm samples tested. Further research building upon this preliminary sperm quality data, is required to identify the cause of DNA and membrane damage in barramundi spermatozoa and understand any potential relationships with paternal performance in commercial spawns

The state of "omics" research for farmed penaeids: advances in research and impediments to industry utilization

Elucidating the underlying genetic drivers of production traits in agricultural and aquaculture species is critical to efforts to maximize farming efficiency. "Omics" based methods (i.e., transcriptomics, genomics, proteomics, and metabolomics) are increasingly being applied to gain unprecedented insight into the biology of many aquaculture species. While the culture of penaeid shrimp has increased markedly, the industry continues to be impeded in many regards by disease, reproductive dysfunction, and a poor understanding of production traits. Extensive effort has been, and continues to be, applied to develop critical genomic resources for many commercially important penaeids. However, the industry application of these genomic resources, and the translation of the knowledge derived from "omics" studies has not yet been completely realized. Integration between the multiple "omics" resources now available (i.e., genome assemblies, transcriptomes, linkage maps, optical maps, and proteomes) will prove critical to unlocking the full utility of these otherwise independently developed and isolated resources. Furthermore, emerging "omics" based techniques are now available to address longstanding issues with completing keystone genome assemblies (e.g., through long-read sequencing), and can provide cost-effective industrial scale genotyping tools (e.g., through low density SNP chips and genotype-by-sequencing) to undertake advanced selective breeding programs (i.e., genomic selection) and powerful genome-wide association studies. In particular, this review highlights the status, utility and suggested path forward for continued development, and improved use of “omics” resources in penaeid aquaculture