Abigail Elizur

Background: The Crustacea are an evolutionarily diverse taxon which underpins marine food webs and contributes significantly to the global economy. However, our knowledge of crustacean endocrinology and development is far behind that of terrestrial arthropods. Here we present a unique insight into the molecular pathways coordinating crustacean metamorphosis, by reconciling nuclear receptor (NR) gene activity from a 12-stage, 3-replicate transcriptome in the ornate spiny lobster (Panulirus ornatus) during larval development. Results: We annotated 18 distinct nuclear receptor genes, including three novel NRs which are upregulated prior to metamorphosis and have hence been named the "molt-associated receptors" (MARs). We also demonstrate the ecdysone-responsive expression of several known molt-related NRs including ecdysone receptor, fushi-tarazu-F1 and E75. Phylogenetic analysis of the curated NR family confirmed gene annotations and suggested that the MARs are a recent addition to the crustacean superfamily, occurring across the Malacostraca from the Stomatopoda to the Decapoda. The ligand-binding domain of these receptors appears to be less conserved than that of typical group-1 NRs. Expression data from two other crustacean species was utilized to examine MAR expression. The Y-organ of the tropical land crab showed a decline in expression of all MARs from intermolt to post-molt. Tissue distributions showed gonad-enriched expression in the Eastern rock lobster and antennal gland-enriched expression in the tropical land crab, although expression was evident across most tissues. Conclusion: By mining transcriptome data, we have curated an extensive list of NR genes expressed during the metamorphic molts of P. ornatus, including three novel crustacean NRs which appear to play a role in the molting process. Divergence of the E-region of these new receptors indicates that they may have adopted a function that is unconventional for NRs. Based on expression patterns, we can confirm that a number of NRs play a role in the ecdysone cassette which regulates molting in crustaceans. This study describes in detail the molecular events surrounding crustacean molting and metamorphosis by taking advantage of the distinctive life history unique to achelatan crustaceans.

Wild sea cucumber resources have been rapidly exhausted and therefore there is an urgent need to develop approaches that will help restocking. Currently, there is a lack of information regarding the genes involved in sea cucumber reproductive processes. The neurohormone relaxin-like gonad-stimulating peptide (RGP) has been identified as the active gonad-stimulating peptide in sea stars (Asteroidea), which could also be present in other echinoderm groups. In this study, a sea cucumber RGP was identified and confirmed by phylogenetic analysis. A recombinant Holothuria scabra RGP was produced in the yeast Pichia pastoris and confirmed by mass spectrometry. To assess bioactivity, four levels of purification were tested in an in vitro germinal vesicle breakdown (GVBD) bioassay. The most pure form induced 98.56±1.19% GVBD in H. scabra and 89.57±1.19% GVBD in Holothuria leucospilota. Cruder levels of purification still resulted in some GVBD. Upon single injection into female H. scabra, the recombinant RGP induced head waving behavior followed by spawning within 90-170min. Spawned oocytes were fertilized successfully, larvae settled and developed into juveniles. Our results provide a key finding for the development of a break-through new artificial breeding approach in sea cucumber aquaculture.

The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle-stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH β-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.

In salmonids, exposure to elevatedtemperature impairsoogenesis. As such, there is a need to understand the molecular mechanisms that underpin this process, and develop mitigation strategies that maintain or rescue reproductive development inbroodstock.In thisstudy, follicle stimulating hormone (Fsh)and/or insulin-like growth factor1(Igf1) treatment were assessed for their ability to promotereproductive functionat 14 and 22 °C in ovarian follicles from coho salmon in vitro. Maintenance at 22°C resulted in the downregulation of fsh receptor, 17α-hydroxylase/C17,20-lyaseand p450 aromatase a(cyp19a1a), and connexin 34.3(cx34.3).While combined treatment with Fsh and Igf1stimulated the expression of cyp19a1aat 14 °C, thistreatment was not effective at 22 °C. Upregulation of cx34.3 occurred inresponse to treatments that contained Igf1regardless of temperature, and there is evidence to suggest that apoptosis was inhibited to some extent at 22 °C following combined treatment with Fsh and Igf1. This studydemonstratesthe thermal impairmentof key reproductive genes, and highlights the potential for novel hormone treatments to rescue oogenesis in salmonids.

The giant grouper is presumed to follow the reproductive pattern of most Epinephelus species, characterized by protogynous hermaphroditism wherein male maturation is attained through sex reversal of a functional female. This hypothesis, however, has not been verified due to lack of biological data. The present study addresses this gap by investigating the reproductive development of giant groupers from juvenile stage through sexual maturity. Gonad histological analysis of hatchery-bred juvenile giant grouper from Queensland, Australia (0.8-5.2 kg, n = 43) have shown earliest occurrence of primary oocytes (i.e. ovarian differentiation) in 47.8 cm and 2.5 kg fish. Monitoring of sexual maturity by gonadal biopsy was performed in a stock of wild-caught giant groupers (2-20 kg) held in sea cages in the Philippines and Vietnam from 2015 to 2017. Onset of female sexual maturity was at 96.9 ± 1.6 cm and 23.5 ± 1.5 kg in the Philippines, and 103.0 ± 4.1 cm and 33.5 ± 2.5 kg in Vietnam. In both locations, development of primary males was observed wherein fish produced milt (or spermiated) without passing through a functional female phase. The ratio of primary males to females in both locations was about 1:2. Size at maturity of primary males is 86.5 ± 4.8 cm and 17.1 ± 2.1 kg in the Philippines, and 97.3 ± 1.3 cm and 34.3 ± 0.9 kg in Vietnam. To aid in the monitoring of female maturation, we developed a non-invasive method based on immunoassay of vitellogenin in skin mucus and this was shown to be effective in detecting female maturation 9 ± 2 months prior to first observation of oocytes through gonadal biopsy. Our findings suggest that giant grouper is a diandric protogynous hermaphrodite. This study provides novel information on the reproductive biology of giant grouper, an economically important and vulnerable species.

Wild-caught hāpuku (Polyprion oxygeneios) spawn readily in captivity, but although first filial (F1) hāpuku complete vitellogenesis, females fail to undergo oocyte maturation and spawn or produce poor quality eggs. This study investigated whether administration of a synthetic agonist of gonadotropin-releasing hormone (GnRHa) could improve F1 hāpuku spawning and complete the life-cycle in captivity. Spawning trials were conducted over 2 years in 2013 and 2014, when F1 were aged five and six years. In 2013, females previously conditioned under a variable or constant temperature regime were implanted with GnRHa (100 μg/kg−1) or blank implants constructed of powdered cellulose and cholesterol. Spawning was erratic and egg quality very poor in all tanks. No F2 offspring were produced by communal spawning. In contrast, viable F2 larvae were produced by strip-spawning and in vitro fertilization after a series of GnRHa injections. In 2014, two additional trials were conducted: females received ethylene-vinyl acetate copolymer (EVAc) matrix implants containing GnRHa (100 μg/kg−1) or blank implants and in the second trial, two GnRHa doses (100 μg/kg−1 and 50 μg/kg−1) were tested. Eggs were first detected in all tanks 12–17 days post-implantation when females received 100 µg/kg−1 GnRHa implants, but not in the lower dose or control tanks. In summary, this study achieved induction of female spawning with GnRHa implants (target dose 100 μg/kg−1) and the successful production of F2 hāpuku in captivity by strip-spawning.

The apparent digestibility of 14 raw materials by yellowtail kingfish (Seriola lalandi) was examined using the diet substitution method and yttrium oxide as the inert marker. Each raw material was examined in triplicate and faecal material was collected from fish using manual stripping techniques. All raw materials were tested at 30% inclusion, except for blood meal (BLM), which was tested at 15% inclusion. The reference diet was primarily composed of fishmeal (FM). The raw materials examined included: two sources of FM (FM-1; prime quality & FM-2; recycled tuna trimmings); poultry by-product meal (PBM-1 & PBM-2); lupin kernel meal (LKM-1 & LKM-2) and soy protein concentrate (SPC-1 & SPC-2) and a single source of krill meal (KRM), meat meal (MM), BLM, faba beans (FBM), corn gluten meal (CGM) and wheat flour (WH). With the exception of FM-2 and BLM, marine and land animal proteins were well digested, recording protein ADCs between 66.5 and 79.2%. The energy from marine and land animal proteins was also well digested, ranging from 67.0-83.5%, with the exception of BLM, which recorded a very low energy ADC of 43.0%. Digestibility of protein from plant sources was highest in WH (97.7%), LKM-2 (95.0%), FBM (94.7%) and LKM-1 (86.3%). The energy from LKM-1, LKM-2 and FBM was also well digested (67.4-76.9%); however, energy digestibility was poor in SPC-1 (35.5%), SPC-2 (31.7%), WH (34.0%) and CGM (19.4%). Generally, the ADCs recorded from plant proteins were greater variability than the ADCs recorded from marine and land animal proteins. Apparent digestibility of amino acids from marine and land animal proteins was fairly consistent and reflected the crude protein ADCs of these raw materials. The recorded ADCs of amino acids from plant proteins was more erratic and the error variance among replicates was higher than observed among replicated marine and land animal proteins. Mean ADCs of many amino acids were > 100% in FBM, LKM-2 and WH whereas the mean ADCs of amino acids recorded from YTK fed CGM were close to zero and in some cases negative. The results from this study indicate that yellowtail kingfish are generally efficient at digesting nutrients and energy from marine and land animal protein sources. Plant proteins such FBM, LKM-1 and LKM-2 appear to have relatively high protein and energy digestibility in yellowtail kingfish and may prove useful as alternative protein and energy sources in aquafeeds. The poor digestibility of the BLM and CGM used in this study suggests these products interfere with digestibility in yellowtail kingfish or there was some form of interaction between these raw materials and other raw materials in the reference diet. The ADCs derived for the raw materials examined in this study will assist in the formulation of research and commercial aquafeeds for this developing aquaculture species.

Oysters are keystone species in estuarine ecosystems and are of substantial economic value to fisheries and aquaculture worldwide. Contending with disease and environmental stress are considerable challenges to oyster culture. Here we report a draft genome of the Sydney Rock Oyster, Saccostrea glomerata, an iconic and commercially important species of edible oyster in Australia known for its enhanced resilience to harsh environmental conditions. This is the second reference genome to be reported from the family Ostreidae enabling a genus-level study of lophotrochozoan genome evolution. Our analysis of the 784-megabase S. glomerata genome shows extensive expansions of gene families associated with immunological non-self-recognition. Transcriptomic analysis revealed highly tissue-specific patterns of expression among these genes, suggesting a complex assortment of immune receptors provide this oyster with a unique capacity to recognize invading microbes. Several gene families involved in stress response are notably expanded in Saccostrea compared with other oysters, and likely key to this species' adaptations for improved survival higher in the intertidal zone. The Sydney Rock Oyster genome provides a valuable resource for future research in molluscan biology, evolution and environmental resilience. Its close relatedness to Crassostrea will further comparative studies, advancing the means for improved oyster agriculture and conservation.