Dean R. Jerry

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Evaluation of visual implant elastomer and alphanumeric internal tags as a method to identify juveniles of the freshwater crayfish, Cherax destructor

In the last few years there has been an increasing interest in using traditional animal breeding techniques to improve important traits such as growth rate in freshwater crayfish. However, a major impediment to such programs is an inability to reliably identify individuals or family groups for long periods because of the non-retention of external tags through ecdysis. Visual implant elastomer (VIE) and visual alphanumeric (VIalpha) internal tags have proven useful in identifying finfish and amphibians. We evaluated the suitability of these two internal tags as a method to identify yabby (Cherax destructor) juveniles. Both tags proved useful in identifying juveniles between 0.1 and 4.6 g, with the VIE having the higher retention rate of the two tags (up to 92%, as opposed to 79% for the VIalpha tag). Mortality, as a direct consequence of tagging juveniles with the VIE and VIalpha tags was 13% and 11%, respectively. The ability to reliably identify C. destructor juveniles will increase the efficiency of selective breeding programs for this species by allowing individuals and family groups to be pooled in growth experiments, thereby decreasing the impact of confounding environmental effects.

Population genetics of a marine bivalve, Pinctada maxima, throughout the Indo‐Australian Archipelago shows differentiation and decreased diversity at range limits

Intraspecific genetic diversity governs the potential of species to prevail in the face of environmental or ecological challenges; therefore, its protection is critical. The Indo‐Australian Archipelago (IAA) is a significant reservoir of the worlds marine biodiversity and a region of high conservation priority. Yet, despite indications that the IAA may harbour greater intraspecific variation, multiple‐locus genetic diversity data are limited. We investigated microsatellite DNA variation in Pinctada maxima populations from the IAA to elucidate potential factors influencing levels of genetic diversity in the region. Results indicate that genetic diversity decreases as the geographical distance away from central Indonesia increases, and that populations located towards the centre of P. maximas range are more genetically diverse than those located peripherally (P < 0.01). Significant partitioning of genetic variation was identified (FST = 0.027; RST = 0.023, P < 0.001) and indicates that historical biogeographical episodes or oceanographic factors have shaped present population genetic structure. We propose that the genetic diversity peak in P. maxima populations may be due to (i) an abundance of suitable habitat within the IAA, meaning larger, more temporally stable populations can be maintained and are less likely to encounter genetic bottlenecks; and/or (ii) the close proximity of biogeographical barriers around central Indonesia results in increased genetic diversity in the region because of admixture of genetically divergent populations. We encourage further genetic diversity studies of IAA marine biota to confirm whether this region has a significant role in maintaining intraspecific diversity, which will greatly assist the planning and efficacy of future conservation efforts.

Transcriptome analysis of biomineralisation-related genes within the pearl sac: host and donor oyster contribution.

Cultured pearl production is a complex biological process involving the implantation of a mantle graft from a donor pearl oyster along with a bead nucleus into the gonad of a second recipient host oyster. Therefore, pearl production potentially involves the genetic co-operation of two oyster genomes. Whilst many genes in the mantle tissue have been identified and linked to shell biomineralisation in pearl oysters, few studies have determined which of these biomineralisation genes are expressed in the pearl sac and potentially linked to pearl biomineralisation processes. It is also uncertain whether the host or donor oyster is primarily responsible for the expression of biomineralisation genes governing pearl formation, with only two shell matrix proteins previously identified as being expressed by the donor oyster in the pearl sac. To further our understanding of pearl formation, the pearl sac transcriptome of Pinctada maxima and Pinctada margaritifera was each sequenced to an equivalent 5× genome coverage with putative molluscan biomineralisation-related genes identified. Furthermore, the host and donor contribution of these expressed genes within the pearl sac were quantified using a novel approach whereby two pearl oyster species harbouring unique genomes, P. maxima or P. margaritifera, were used to produce xenografted pearl sacs. A total of 19 putative mollusc biomineralisation genes were identified and found to be expressed in the pearl sacs of P. maxima and P. margaritifera. From this list of expressed genes, species-diagnostic single nucleotide polymorphisms (SNP) were identified within seven of these genes; Linkine, N66, Perline, N44, MSI60, Calreticulin and PfCHS1. Based on the presence/absence of species diagnostic gene transcripts within xenografted pearl sacs, all seven genes were found to be expressed by the species used as the donor oyster. In one individual we also found that the host was expressing Linkine. These results convincingly show for the first time that the donor mantle tissue is primarily responsible for the expression of biomineralisation genes in the pearl sac.

Effects of chlorpyrifos on cholinesterase activity and stress markers in the tropical reef fish Acanthochromis polyacanthus

Tropical coastal ecosystems, including the Great Barrier Reef (GBR) of Australia are increasingly threatened by pollution; yet few studies have investigated the sensitivity of GBR species to these pollutants. Here we exposed juveniles of the tropical reef fish Acanthochromis polyacanthus (spiny damselfish) to three concentrations of the insecticide chlorpyrifos (CPF) and measured (i) muscle cholinesterase (ChE) activity; (ii) hepatic glutathione-S-transferase (GST) activity; and (iii) coenzyme Q (CoQ) redox balance, after 6h and 96h of exposure. After 96h, muscle ChE activity was significantly inhibited by 26%, 49% and 53% when fish were exposed to 1, 10 or 100μg/L CPF, respectively. Muscle ChE characterization revealed three types of ChEs, including two atypical forms. Hepatic CoQ antioxidant form significantly increased at 10μg/L after 6h of exposure, potentially demonstrating an early response to CPF-induced oxidative stress in liver. Hepatic GST was not affected by CPF exposure.

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

SUMMARY 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.

Differential tissue-regulation of myostatin genes in the teleost fish Lates calcarifer in response to fasting. Evidence for functional differentiation

Gene or genome duplication is a fundamental evolutionary mechanism leading towards the origin of new genes, or gene functions. Myostatin (MSTN) is a negative regulator of muscle growth that in teleost fish, as a result of genome duplication, is present in double copy. This study provides evidence of differentiation of MSTN paralogs in fish by comparatively exploring their tissue-regulation in the Asian sea bass (Lates calcarifer) when subjected to fasting stress. Results showed differential regulation as well as specific tissue-responses in the muscle, liver, gill and brain of L. calcarifer after nutritional deprivation. In particular, the LcMstn-1 expression increased in liver (∼4 fold) and muscle (∼3 fold) and diminished in brain (∼0.5 fold) and gill (∼0.5 fold) while that of LcMstn-2 remained stable in brain and muscle and was up regulated in gill (∼2.5 fold) and liver (∼2 fold). Differential regulation of Mstn paralogs was supported by in silico analyses of regulatory motifs that revealed, at least in the immediate region upstream the genes, a differentiation between Mstn-1 and Mstn-2. The Mstn-1 in particular showed a significantly higher conservation of regulatory sites among teleost species compared to its paralog indicating that this gene might have a highly conserved function in the taxon.

DNA barcoding to support conservation: species identification, genetic structure and biogeography of fishes in the Murray)Darling River Basin, Australia

Freshwater fish stocks worldwide are under increasing threat of overfishing, disease, pollution and competition from introduced species. In the Murray—Darling Basin (MDB), the largest river system of Australia, more than half the native species are listed as rare or endangered. Active management is required to counteract reduction in population sizes, prevent local extinctions and to maintain genetic diversity. We describe the first comprehensive set of DNA barcodes able to discriminate between all 58 native and introduced species of freshwater fish recorded in the MDB. These barcodes also distinguish populations from those in adjacent basins, with estimated separation times as short as 0.1 million years ago. We demonstrate the feasibility of using DNA fingerprinting of ribosomal RNA (12S and 18S rRNA) genes and mitochondrial DNA control region (mtDNA CR) sequences to identify species from eggs, larvae, tissues and predator gut contents as well as differentiate populations, morphologically cryptic species and hybrids. The DNA barcode resource will enhance capacity in many areas of fish conservation biology that can benefit from improved knowledge of genetic provenance. These include captive breeding and restocking programs, life history studies and ecological research into the interactions between populations of native and exotic species.

CRYOPRESERVATION OF BLACK-LIP PEARL OYSTER (PINCTADA MARGARITIFERA, L.) SPERMATOZOA: EFFECTS OF CRYOPROTECTANTS ON SPERMATOZOA MOTILITY

Abstract Cryopreservation of sperm is seen as an important step in developing effective hatchery culture techniques for the black-lip pearl oyster, Pinctada margaritifera. As a preliminary investigation into cryopreservation of the gametes of this species we tested 5 cryoprotectant agent combinations for their ability to retain sperm motility: (1) 1 M trehalose and 5, 10 and 15% dimethyl sulphoxide (DMSO); (2) Hanks calcium-free balanced salt solution (C-F HBSS) and 5%, 10% and 15% DMSO; (3) C-F HBSS and 5, 10 and 15% propylene glycol (PG); (4) 1 M trehalose and an equal combination of DMSO and PG making up 5, 10, 15% total volume; and (5) C-F HBSS and an equal combination of DMSO and PG making up 5%, 10% and 15% total volume. Total, rapid and progressive sperm motilities were estimated through computer assisted sperm analysis (CASA). Sperm cryopreserved in 1 M trehalose and 5% DMSO retained the highest total (86.0 ± 1.2% SE), progressive (46.0 ± 1.2% SE) and rapid (25.1 ± 0.6% SE) motilities of all cryoprotectant solutions, whereas those cryopreserved with PG generally retained poor motility. Although the 1 M trehalose and 5% DMSO treatment compared favorably with that of fresh sperm for total motility (P < 0.01), all cryoprotectant treatments were poor at retaining the proportion of original rapid and progressively moving sperm. This study highlights the potential for cryopreservation of gametes from P. margaritifera, which will benefit selective breeding and conservation programs with this important commercial species.

Fine-tuning for the tropics: application of eDNA technology for invasive fish detection in tropical freshwater ecosystems

Invasive species pose a major threat to aquatic ecosystems. Their impact can be particularly severe in tropical regions, like those in northern Australia, where >20 invasive fish species are recorded. In temperate regions, environmental DNA (eDNA) technology is gaining momentum as a tool to detect aquatic pests, but the technologys effectiveness has not been fully explored in tropical systems with their unique climatic challenges (i.e. high turbidity, temperatures and ultraviolet light). In this study, we modified conventional eDNA protocols for use in tropical environments using the invasive fish, Mozambique tilapia (Oreochromis mossambicus) as a detection model. We evaluated the effects of high water temperatures and fish density on the detection of tilapia eDNA, using filters with larger pores to facilitate filtration. Large‐pore filters (20 μm) were effective in filtering turbid waters and retaining sufficient eDNA, whilst achieving filtration times of 2–3 min per 2‐L sample. High water temperatures, often experienced in the tropics (23, 29, 35 °C), did not affect eDNA degradation rates, although high temperatures (35 °C) did significantly increase fish eDNA shedding rates. We established a minimum detection limit for tilapia (1 fish/0.4 megalitres/after 4 days) and found that low water flow (3.17 L/s) into ponds with high fish density (>16 fish/0.4 megalitres) did not affect eDNA detection. These results demonstrate that eDNA technology can be effectively used in tropical ecosystems to detect invasive fish species.