Ontogeny of the Cytochrome P450 Superfamily in the Ornate Spiny Lobster (Panulirus ornatus).

Cytochrome P450s (CYP450s) are a versatile superfamily of enzymes known to undergo rapid evolution. They have important roles across growth and development pathways in crustaceans, although it is difficult to characterise orthologs between species due to their sequence diversity. Conserved CYP450s enzymes in crustaceans are those associated with ecdysteroidogenesis: synthesising and breaking down the active moult hormone, 20-hydroxyecdysone. The complex life cycle of the ornate spiny lobster, Panulirus ornatus, relies on moulting in order to grow and develop. Many of these diverse life stages have been analysed to establish a comprehensive transcriptomic database for this species. The transcripts putatively encoding for CYP450s were mapped using transcriptomic analysis and identified across growth and development stages. With the aid of phylogeny, 28 transcripts of 42 putative P. ornatus CYP450s were annotated, including the well conserved Halloween genes, which are involved in ecdysteroidogenesis. Expression patterns across the life stages determined that only a subset of the CYP450s can be detected in each life stage or tissue. Four Shed transcripts show overlapping expression between metamorphosis and adult tissues, suggesting pleotropic functions of the multiple Shed orthologs within P. ornatus.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

The byssal-producing glands and proteins of the silverlip pearl oyster Pinctada maxima (Jameson, 1901).

Pinctada maxima are most well known for their production of high-quality natural pearls. They also generate another natural material, the byssus, an adhesive thread critical for steadfast attachment underwater. Herein, P. maxima byssal threads were analysed via proteotranscriptomics to reveal 49 proteins. Further characterisation was undertaken on five highly expressed genes: glycine-rich thread protein (GRT; also known as PUF3), apfp1/perlucin-like protein (Pmfp1); peroxidase; thrombospondin 1, and Balbiani ring 3 (BR3), which showed localised tissue expression. The spatial distribution of GRT and Pmfp1 via immunodetection combined with histology helped to identify glandular regions of the foot that contribute to byssal thread production: the byssal gland, the duct gland, and two thread-forming glands of basophilic and acidophilic serous-like cells. This work advanced primary knowledge on the glands involved in the creation of byssal threads and the protein composition of the byssus for P. maxima, providing a platform for the design of marine biopolymers.

Deploying new generation sequencing for the study of flesh color depletion in Atlantic Salmon (Salmo salar).

BACKGROUND: The flesh pigmentation of farmed Atlantic salmon is formed by accumulation of carotenoids derived from commercial diets. In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. In this study, RNA sequencing (RNA-Seq) was performed to investigate the reduction in flesh pigmentation. Library preparation is a key step that significantly impacts the effectiveness of RNA sequencing (RNA-Seq) experiments. Besides the commonly used whole transcript RNA-Seq method, the 3' mRNA-Seq method is being applied widely, owing to its reduced cost, enabling more repeats to be sequenced at the expense of lower resolution. Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3' mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes. RESULTS: In both methods, DEGs between the two color phenotypes were associated with metal ion transport, oxidation-reduction processes, and immune responses. We also found DEGs related to lipid metabolism in the QuantSeq method. In the TruSeq method, a missense mutation was detected in DEGs in different flesh color traits. The number of DEGs found in the TruSeq libraries was much higher than the QuantSeq; however, the trend of DEGs in both library methods was similar and validated by qPCR. CONCLUSIONS: Flesh coloration in Atlantic salmon is related to lipid metabolism in which apolipoproteins, serum albumin and fatty acid-binding protein genes are hypothesized to be linked to the absorption, transport and deposition of carotenoids. Our findings suggest that Grp could inhibit the feeding behavior of low color-banded fish, resulting in the dietary carotenoid shortage. Several SNPs in genes involving in carotenoid-binding cholesterol and oxidative stress were detected in both flesh color phenotypes. Regarding the choice of the library preparation method, the selection criteria depend on the research design and purpose. The 3' mRNA-Seq method is ideal for targeted identification of highly expressed genes, while the whole RNA-Seq method is recommended for identification of unknown genes, enabling the identification of splice variants and trait-associated SNPs, as we have found for duox2 and duoxa1.

Gonadal transcriptomes associated with sex phenotypes provide potential male and female candidate genes of sex determination or early differentiation in Crassostrea gigas, a sequential hermaphrodite mollusc.

BACKGROUND: In the animal kingdom, mollusca is an important phylum of the Lophotrochozoa. However, few studies have investigated the molecular cascade of sex determination/early gonadal differentiation within this phylum. The oyster Crassostrea gigas is a sequential irregular hermaphrodite mollusc of economic, physiological and phylogenetic importance. Although some studies identified genes of its sex-determining/-differentiating pathway, this particular topic remains to be further deepened, in particular with regard to the expression patterns. Indeed, these patterns need to cover the entire period of sex lability and have to be associated to future sex phenotypes, usually impossible to establish in this sequential hermaphrodite. This is why we performed a gonadal RNA-Seq analysis of diploid male and female oysters that have not changed sex for 4 years, sampled during the entire time-window of sex determination/early sex differentiation (stages 0 and 3 of the gametogenetic cycle). This individual long-term monitoring gave us the opportunity to explain the molecular expression patterns in the light of the most statistically likely future sex of each oyster. RESULTS: The differential gene expression analysis of gonadal transcriptomes revealed that 9723 genes were differentially expressed between gametogenetic stages, and 141 between sexes (98 and 43 genes highly expressed in females and males, respectively). Eighty-four genes were both stage- and sex-specific, 57 of them being highly expressed at the time of sex determination/early sex differentiation. These 4 novel genes including Trophoblast glycoprotein-like, Protein PML-like, Protein singed-like and PREDICTED: paramyosin, while being supported by RT-qPCR, displayed sexually dimorphic gene expression patterns. CONCLUSIONS: This gonadal transcriptome analysis, the first one associated with sex phenotypes in C. gigas, revealed 57 genes highly expressed in stage 0 or 3 of gametogenesis and which could be linked to the future sex of the individuals. While further study will be needed to suggest a role for these factors, some could certainly be original potential actors involved in sex determination/early sex differentiation, like paramyosin and could be used to predict the future sex of oysters.

CrustyBase: an interactive online database for crustacean transcriptomes.

Transcriptome sequencing has opened the field of genomics to a wide variety of researchers, owing to its efficiency, applicability across species and ability to quantify gene expression. The resulting datasets are a rich source of information that can be mined for many years into the future, with each dataset providing a unique angle on a specific context in biology. Maintaining accessibility to this accumulation of data presents quite a challenge for researchers.The primary focus of conventional genomics databases is the storage, navigation and interpretation of sequence data, which is typically classified down to the level of a species or individual. The addition of expression data adds a new dimension to this paradigm - the sampling context. Does gene expression describe different tissues, a temporal distribution or an experimental treatment? These data not only describe an individual, but the biological context surrounding that individual. The structure and utility of a transcriptome database must therefore reflect these attributes. We present an online database which has been designed to maximise the accessibility of crustacean transcriptome data by providing intuitive navigation within and between datasets and instant visualization of gene expression and protein structure.The site is accessible at https://crustybase.org and currently holds 10 datasets from a range of crustacean species. It also allows for upload of novel transcriptome datasets through a simple web interface, allowing the research community to contribute their own data to a pool of shared knowledge.

Ecdysis triggering hormone modulates molt behaviour in the redclaw crayfish Cherax quadricarinatus, providing a mechanistic evidence for conserved function in molt regulation across Pancrustacea.

Molting enables growth and development across ecdysozoa. The molting process is strictly controlled by hormones - ecdysteroids. Ecdysteroidogenesis occurs in theprothoracic glands and stimulated by prothoracicotropic hormone in insects, while it ensues in the Y-organ and regulated by the molt inhibiting hormone in crustaceans. A peak in ecdysteroids in the hemolymph induces a cascade of multiple neuropeptides including Ecdysis Triggering Hormone (ETH) and Corazonin. The role of ETH is well defined in controlling the molt process in insects, but it is yet to be defined in crustaceans. In this study, we investigated the behavioral response of intermolt crayfish to ETH and Corazonin injections as well as the impact of ETH on the molt period using in vivo assays. Injection of Corazonin and ETH resulted in a clear and immediate eye twitching response to these two neuropeptides. The Corazonin injection induced eye twitching in slow and asynchronous manner, while ETH injection caused eye twitching in a relatively fast and synchronous way. A single injection of ETH to crayfish resulted in a remarkable prolong molt period, at twice the normal molting cycle, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. Bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species. ETH can be classified into specific groups based on the associated motif in each insect order and shows an insect motif -KxxPRx to be conserved in crustaceans.

Neural remodelling in spiny lobster larvae is characterized by broad neuropeptide suppression.

Neuropeptides are ancient endocrine components which have evolved to regulate many aspects of biology across the animal kingdom including behaviour, development and metabolism. To supplement current knowledge, we have utilized a transcriptome series describing larval development in the ornate spiny lobster, Panulirus ornatus. The biology of this animal has been leveraged to provide insights into the roles of molting, metamorphosis and metabolism across the neuropeptide family. We report an extensive list of neuropeptides across three distinct life phases of the animal. We show distinct groups of neuropeptides with differential expression between larval phases, indicating phase-specific roles for these peptides. For selected neuropeptides, we describe and discuss expression profiles throughout larval development and report predicted peptide cleavage sites and mature peptide sequences. We also report the neuropeptide nesfatin for the first time in a crustacean, and report secondary peptide products with a level of evolutionary conservation similar to the conventional mature peptide nesfatin-1, indicating a conserved role in these secondary products which are widely regarded as biologically inactive. In addition, we report a trend of downregulation in the neuropeptides as the animal undergoes extensive neural remodelling in fulfillment of metamorphosis. We suggest that this downregulation in neuropeptides relates to the brief, yet dramatic changes in morphology experienced by the central nervous system in the process of metamorphosis.

Development of a giant grouper Luteinizing Hormone (LH) Enzyme-Linked Immunosorbent Assay (ELISA) and its use towards understanding sexual development in grouper.

A recombinant giant grouper Luteinizing Hormone (LH) consisting of tethered beta and alpha subunits was produced in a yeast expression system. The giant grouper LH ß-subunit was also produced and administered to rabbits for antibody development. The recombinant LH and its antibody were used to develop an Enzyme Linked Immunosorbent Assay (ELISA). This ELISA enabled detection of plasma LH levels in groupers at a sensitivity between 391 pg/ml and 200 ng/ml. Different species of grouper were assayed with this ELISA in conjunction with gonadal histology and body condition data to identify links between circulating LH levels and sexual development. We found that circulating levels of LH decreased when oocytes began to degenerate, and sex-transition gonadal characteristics were apparent when LH levels decreased further. When circulating LH levels were related to body condition (body weight/ body length), transitioning-stage fish had relatively high body condition but low plasma LH levels. This observation was similar across multiple grouper species and indicates that plasma LH levels combined with body condition may be a marker for early male identification in the protogynous hermaphrodite groupers.

Transcriptomic changes across vitellogenesis in the black tiger prawn (Penaeus monodon), neuropeptides and G protein-coupled receptors repertoire curation.

The black tiger prawn (Penaeus monodon) is one of the most commercially important prawn species world-wide, yet there are currently key issues that hinder aquaculture of this species, such as low spawning capacity of captive-reared broodstock females and lack of globally available fully domesticated strains. In this study, we analysed the molecular changes that occur from vitellogenesis to spawning of a fully domesticated population of P.monodon (Madagascar) using four tissues [brain and thoracic ganglia (central nervous system - CNS), eyestalks, antennal gland, and ovary] highlighting differentially expressed genes that could be involved in the sexual maturation. In addition, due to their key role in regulating multiple physiological processes including reproduction, transcripts encoding P.monodon neuropeptides and G protein-coupled receptors (GPCRs) were identified and their expression pattern was assessed. A few neuropeptides and their putative GPCRs which were previously implicated in reproduction are discussed. We identified 573 differentially expressed transcripts between previtellogenic and vitellogenic stages, across the four analysed tissues. Multiple transcripts that have been linked to ovarian maturation were highlighted throughout the study, these include vitellogenin, Wnt, heat shock protein 21, heat shock protein 90, teneurin, Fs(1)M3, hemolymph clottable proteins and some other candidates. Seventy neuropeptide transcripts were also characterized from our de novo assembly. In addition, a hybrid approach that involved clustering and phylogenetics analysis was used to annotate all P. monodon GPCRs, revealing 223 Rhodopsin, 100 Secretin and 27 Metabotropic glutamate GPCRs. Given the key commercial significance of P.monodon and the industry requirements for developing better genomic tools to control reproduction in this species, our findings provide a foundation for future gene-based studies, setting the scene for developing innovative tools for reproduction and/or sexual maturation control in P. monodon.

Characterization of G-protein coupled receptors from the blackback land crab Gecarcinus lateralis Y organ transcriptome over the molt cycle.

BACKGROUND: G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands (Y-organs; YOs) through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process. RESULTS: Ninety-nine G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation. CONCLUSIONS: Molting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.

Correction to: Transcriptional profiling of spiny lobster metamorphosis reveals three new additions to the nuclear receptor superfamily.

Following publication of the original article.

Transcriptional profiling of spiny lobster metamorphosis reveals three new additions to the nuclear receptor superfamily.

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.

Gonadal response of juvenile protogynous grouper (Epinephelus fuscoguttatus) to long-term recombinant follicle-stimulating hormone administration†.

The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 µg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 µg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.

Development of specific enzyme-linked immunosorbent assay for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone using recombinant gonadotropins.

We developed a specific competitive enzyme-linked immunosorbent assay (ELISA) for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone (FSH). We previously produced a full-length single chain recombinant yellowtail kingfish FSH using the Pichia pastoris expression system. We used the same method to produce the ß subunit of the hormone, against which polyclonal antibodies were raised in rabbits. We first confirmed immunoreactivity of the polyclonal antibodies with the recombinant full length FSH and FSHß as well as plasma and pituitary FSH of sexually immature and mature yellowtail kingfish by Western blot analysis. We then developed a precise and reproducible ELISA for yellowtail kingfish FSH and validated the assay in plasma and pituitary extracts. The intra- and inter-assay coefficients of variation was <2.2% and 10.2%, respectively. The sensitivity of the assay was 78 pg/ml. For further validation of the assay, we measured the plasma FSH in immature yellowtail kingfish treated with increasing doses (blank, 50, 100 and 150 µg/kg) of kisseptin2-10 peptide from a previous study. The dose response observed in treated females was not significant, however the increased plasma FSH levels coincided with the significantly higher estradiol levels we previously reported in the treated groups. We assessed the applicability of the assay in measuring circulating FSH in other species. We observed parallelism between the linearized FSH standard curve and displacement curves of serially diluted plasma from Atlantic bluefin tuna (Thunnus thynnus) and tilapia (Oreochromis niloticus). We also observed similar parallelism with full length recombinant giant grouper (Epinephelus lanceolatus) FSH. The ELISA we developed for yellowtail kingfish FSH will be useful in understanding the reproductive biology of the species as well as enhancing its aquaculture.

Identification of neuropeptides in the sea cucumber Holothuria leucospilota.

Neuropeptides play important roles in the regulation of physiological processes such as growth, metabolism and reproduction. In sea cucumbers (Phylum Echinodermata), numerous neuropeptides have been identified and some are attributed to reproductive processes. In this study, our goal was to gain a better understanding of the neuropeptide repertoire for the black sea cucumber Holothuria leucospilota, a species that has been severely overfished from the wild due to human consumption. We applied in silico transcriptome analysis of the adult H. leucospilota radial nerve cord, gonad and body wall to elucidate 35 neuropeptides that are conserved throughout the Bilateria. Then, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of radial nerve cord was employed and showed an additional 8 putative novel neuropeptide precursors, whose predicative cleaved peptides do not share sequence similarity with any reported neuropeptides. These data provide an important basis for experimental approaches to manipulate H. leucospilota broodstock reproduction and growth in culture, which will hopefully re-establish population numbers.

A Crown-of-Thorns Seastar recombinant relaxin-like gonad-stimulating peptide triggers oocyte maturation and ovulation.

The Acanthaster planci species-complex [Crown-of-Thorns Seastar (COTS)] are highly fecund echinoderms that exhibit population outbreaks on coral reef ecosystems worldwide, including the Australian Great Barrier Reef. A better understanding of the COTS molecular biology is critical towards efforts in controlling outbreaks and assisting reef recovery. In seastars, the heterodimeric relaxin-like gonad stimulating peptide (RGP) is responsible for triggering a neuroendocrine cascade that regulates resumption of oocyte meiosis prior to spawning. Our comparative RNA-seq analysis indicates a general increase in RGP gene expression in the female radial nerve cord during the reproductive season. Also, the sensory tentacles demonstrate a significantly higher expression level than radial nerve cord. A recombinant COTS RGP, generated in a yeast expression system, is highly effective in inducing oocyte germinal vesicle breakdown (GVBD), followed by ovulation from ovarian fragments. The findings of this study provide a foundation for more in-depth molecular analysis of the reproductive neuroendocrine physiology of the COTS and the RGP.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Y-linked iDmrt1 paralogue (iDMY) in the Eastern spiny lobster, Sagmariasus verreauxi: The first invertebrate sex-linked Dmrt.

Sex determination pathways are extensively diverse across species, with the master sex-determinants being the most variable element. Despite this, there is a family of DM-domain transcription factors (Dmrts), which hold a highly conserved function in sexual development. This work is the first to describe a heterogametic sex-linked Dmrt in an invertebrate species, the Eastern spiny lobster, Sagmariasus verreauxi. We have termed the Y-linked, truncated paralogue of the autosomal iDmrt1, Sv-iDMY. Considering the master sex-determining function of both DMY in medaka and DM-W in frog, we hypothesised a similar function of Sv-iDMY. By conducting temporal expression analyses during embryogenesis we have identified a putative male sex-determining period during which iDMY>iDmrt1. Employing a GAL4-transactivation assay we then demonstrate the dominant negative suppression of iDMY over its autosomal iDmrt1 paralogue, suggesting the mechanism with which iDMY determines sex. Comparative analyses of Sv-iDMY, DM-W and medaka DMY, highlight the C'-mediated features of oligomerisation and transactivation as central to the mechanism that each exerts. Indeed, these features may underpin the plasticity facilitating the convergent emergence of these three sporadic sex-linked master-Dmrts.

Effects of neuropeptides and sex steroids on the pituitary-gonadal axis of pre-pubertal F1 wreckfish (hapuku) Polyprion oxygeneios in vivo: Evidence of inhibitory effects of androgens.

The ability to advance puberty in broodstock that have a long generation interval and mature at large size is a highly valuable tool in contemporary aquaculture enterprise. Juvenile male and female wreckfish 'hapuku' (Polyprion oxygeneios), a candidate for commercialization in aquaculture, were subjected to treatment for 8weeks with two implants, one containing steroid (blank; estradiol-17ß, E2; 11-ketotestosterone, KT; 17 α-methyltestosterone, MT), the other peptide (blank; gonadotropin-releasing hormone analog, GnRHa; kisspeptin, Kiss2-12). The expression of target genes (glycoprotein homone α-subunit, gpa; follicle stimulating-hormone ß-subunit, fshb; luteinizing hormone ß-subunit, lhb; GnRH receptor, gnrhr) in the pituitary was assayed by quantitative PCR. KT and MT decreased mRNA levels of all target genes in both male and female hapuku, suggestive of a strong inhibitory tone by these steroid hormones. E2, GnRHa and Kiss2-12 were largely ineffective, regardless of whether they were administered alone or in combination with steroid implants. Clear differences in release and/or clearance rates between E2 and KT from implants were evident, in part explaining our observations. Advancement of puberty was not achieved, and we pose that different hormone doses and/or administration during more advanced stages of gonadogenesis need to be considered to move this field forward.