Aquaculture Breeding Enhancement: Maturation and Spawning in Sea Cucumbers Using a Recombinant Relaxin-Like Gonad-Stimulating Peptide

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 90–170 min. 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

Development of artificial diets for milkfish (Chanos chanos) larvae

Abstract onlyThis study aims to develop nutritionally balanced and cost-effective artificial diets for milkfish larvae. Two larval diets (Feed A and Feed B) were formulated and prepared to contain 45% protein and 10% lipid. Several larval diet preparation techniques were tried and diets produced were assessed in terms of feed particle size and bouyancy, water stability, and feed acceptability. The larval diet preparation that gave the best particle size and bouyancy as well as good water stability was the one prepared as microbound diet (using K-carrageenan as a binder) and flaked using a drum drier.A series of feeding experiments were then conducted to determine growth and survival of milkfish larvae reared on various feeding schemes involving the use of these artificial diets. The artificial diets were fed either alone or in combination with live foods. Larvae in control treatments were reared on live foods such as Brachionus and Artemia. Larvae were observed to ingest the diets indicating that the feeds had suitable physical characteristics and were attractive to the larvae. Over-all results of the feeding trials showed that the artificial diets could be fed to milkfish larvae in combination with the rotifer Brachionus starting Day 8 or could be fed alone to milkfish larvae starting Day 15 onward. These promising results would reduce dependence of milkfish larvae on live foods and would have significant economic benefits in the form of simplified milkfish hatchery procedures

Molecular studies on the neuroendocrine regulation of pubertal development in grey mullet, Mugil Cephalus

This research project was designed to advance our understanding of the molecular regulation of pubertal development in a late maturing fish, using the grey mullet as model species. The objective was addressed by the molecular cloning of key genes involved in reproductive function followed by the analysis of the regulation of selected genes at the promoter and gene expression level. The cDNAs of the genes encoding for muGPR54, muGnRH1, muGnRH2, muGnRH3, muCyp19a, muCyp19b, muIGF-I, muIGF-II, muERH, and muERI were isolated, and their expression along the BPG axis determined, using RTPCR. This was followed by the isolation of the promoter regions of mudrd2, muCyp19a and muCyp19b genes by genome walking PCR and determination of promoter functionality in vitro by reporter gene assay, using luciferase as reporter gene. The brain, pituitary and ovarian expression profile of muGPR54, muGnRH1, muGnRH2, muGnRH3, mudrd2, muCyp19a and muCyp19b genes was characterised by QPCR in female fish undergoing puberty. The pubertal stages of the experimental fish were defined according to the oocyte developmental stage and the levels of plasma vitellogenin, which was determined by ELISA for mullet vitellogenin developed in the course of the present study. The studies concerning the promoters revealed two putative promoters of the mudrd2 gene. The first putative promoter, located in the region flanking the 5UTR, contained Sp1, progesterone receptor, CREB, GATA and STAT binding sites, GC boxes and CAAT- and TATA- like sequences. The second putative promoter, situated in the region flanking the first coding exon and is therefore an intronic promoter, contained consensus Inr and DPE elements as well as putative AP4, GR, NFkappaB, CREB, AP1, TTF-1, C/EBP, Pit1,GHF-1, Ahr/Arnt, Ptx1, RARH, GATA binding sites, GC boxes and CAAT- and TATA-like sequences. Functionality of the intronic promoter, and the serially deleted constructs derived from it, were demonstrated in COS-7, HT3 and TE671 cell lines. The distinct basal promoter activity of the different constructs indicated the functional relevance of the putative regulatory elements and the tissue-specific regulation of mudrd2 gene expression. The muCyp19a promoter sequence contained consensus TATA box and putative ½ERE, SF-1, AhR/Arnt, PR and GATA sites. The muCyp19b promoter sequence revealed consensus TATA and CCAAT boxes and putative PR, ERE and ½ERE, SP-1, GATA C/EBP, GRE, NFkappaB, STAT, PPAR/RXR, Ahr/Arnt and CRE sites. As in the mudrd2 gene promoter, the functionality of muCyp19a and muCyp19b gene promoters, observed in COS-7, HT3 and TE671 cell lines, pointed to the transcriptional roles of the putative regulatory elements identified on the promoter sequences. Additionally, for the muCyp19b gene, negative This research project was designed to advance our understanding of the molecular regulation of pubertal development in a late maturing fish, using the grey mullet as model species. The objective was addressed by the molecular cloning of key genes involved in reproductive function followed by the analysis of the regulation of selected genes at the promoter and gene expression level. The cDNAs of the genes encoding for muGPR54, muGnRH1, muGnRH2, muGnRH3, muCyp19a, muCyp19b, muIGF-I, muIGF-II, muERH, and muERI were isolated, and their expression along the BPG axis determined, using RTPCR. This was followed by the isolation of the promoter regions of mudrd2, muCyp19a and muCyp19b genes by genome walking PCR and determination of promoter functionality in vitro by reporter gene assay, using luciferase as reporter gene. The brain, pituitary and ovarian expression profile of muGPR54, muGnRH1, muGnRH2, muGnRH3, mudrd2, muCyp19a and muCyp19b genes was characterised by QPCR in female fish undergoing puberty. The pubertal stages of the experimental fish were defined according to the oocyte developmental stage and the levels of plasma vitellogenin, which was determined by ELISA for mullet vitellogenin developed in the course of the present study. The studies concerning the promoters revealed two putative promoters of the mudrd2 gene. The first putative promoter, located in the region flanking the 5UTR, contained Sp1, progesterone receptor, CREB, GATA and STAT binding sites, GC boxes and CAAT- and TATA- like sequences. The second putative promoter, situated in the region flanking the first coding exon and is therefore an intronic promoter, contained consensus Inr and DPE elements as well as putative AP4, GR, NFkappaB, CREB, AP1, TTF-1, C/EBP, Pit1,GHF-1, Ahr/Arnt, Ptx1, RARH, GATA binding sites, GC boxes and CAAT- and TATA-like sequences. Functionality of the intronic promoter, and the serially deleted constructs derived from it, were demonstrated in COS-7, HT3 and TE671 cell lines. The distinct basal promoter activity of the different constructs indicated the functional relevance of the putative regulatory elements and the tissue-specific regulation of mudrd2 gene expression. The muCyp19a promoter sequence contained consensus TATA box and putative ½ERE, SF-1, AhR/Arnt, PR and GATA sites. The muCyp19b promoter sequence revealed consensus TATA and CCAAT boxes and putative PR, ERE and ½ERE, SP-1, GATA C/EBP, GRE, NFkappaB, STAT, PPAR/RXR, Ahr/Arnt and CRE sites. As in the mudrd2 gene promoter, the functionality of muCyp19a and muCyp19b gene promoters, observed in COS-7, HT3 and TE671 cell lines, pointed to the transcriptional roles of the putative regulatory elements identified on the promoter sequences. Additionally, for the muCyp19b gene, negative vi modulation of promoter activity by quinpirole, a DA D2-receptor type agonist, was observed. The gene expression profile of muGPR54, muGnRH1, muGnRH2 and muGnRH3, mudrd2, muCyp19a and muCyp19b genes in pubertal fish revealed their functional relevance during sexual maturation. The significantly high relative expression levels of muGPR54, muGnRH1, muGnRH2 and muGnRH3 genes in the brain at the early stage of puberty (defined by low GSI, no apparent oocyte development and low, but detectable, plasma vitellogenin) pointed to a coordinated stimulatory role of these genes at the beginning of puberty. Data also suggested that muGnRH1 has the gonadotrophic role. In contrast to the expression profiles observed in the brain, expression levels of muGPR54 and muGnRH1 in the ovary were high at the advanced stage of puberty (characterised by high GSI, yolky oocytes and high plasma vitellogenin), suggesting a role of these genes towards sustaining gonadal development. The high expression levels of mudrd2 in the brain at the early stage of puberty and in the pituitary at both the early and intermediate stages, which correspond to a period of pronounced DA inhibition of reproductive function in grey mullet, indicated the role of mudrd2 in mediating the inhibitory effect of DA. Ovarian expression of muCyp19a and brain expression of muCyp19b increased during pubertal development, which implied the functional relevance of E2 biosynthesis to the pubertal process in those tissues. In conclusion, the results provide evidence that the molecular regulation of pubertal development in female grey mullet is contingent upon the functional integration of the regulatory signals coming from a suite of genes, including, at least, the genes characterised in the present study. The molecular tools obtained and the understanding achieved from the present research work provide basis for further investigations dealing with the manipulation of puberty in late maturing species of fish

Overview of the marine fish hatchery industry in Taiwan

Although fish culture itself is an age-old tradition in Taiwan, it was in the 1960s that the first successes on artificial propagation were achieved, with several species of Chinese carps and tilapias. The first marine fish to be bred in captivity was the grey mullet; it was first induced to spawn in 1968. Various other species have since been added to the list of propagated marine fish. The characteristics of the marine fish hatchery industry in Taiwan are outlined, considering both the outdoor pond and indoor tank systems. Future prospects are very good; Taiwan now exports marine fish larvae and fingerlings to many of its Asian neighbours and there are some 60 marine fish species for which commercial larval production is possible

Temporal expression of G-protein-coupled receptor 54 (GPR54) gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2. (c) 2006 Elsevier Inc. All rights reserved

Cytochrome P450 aromatase in grey mullet: cDNA and promoter isolation; brain, pituitary and ovarian expression during puberty

In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515 bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485 bp and encoded 495 predicted amino acid residues. The expression level of muCyp19b significantly increased in the brain as puberty advanced; however, its expression level in the pituitary increased only slightly with pubertal development. In the ovary, the muCyp19a expression level markedly increased as puberty progressed. The promoter regions of the two genes were also isolated and their functionality evaluated in vitro using luciferase as the reporter gene. The muCyp19a promoter sequence (650bp) contained a consensus TATA box and putative transcription factor binding sites, including two half EREs, an SF-1, an AhR/Arnt, a PR and two GATA-3s. The muCyp19b promoter sequence (2500 bp) showed consensus TATA and CCAAT boxes and putative transcription binding sites, namely: a PR, an ERE, a half ERE, a SP-1, two GATA-binding factor, one half GATA-1, two C/EBPs, a GRE, a NFkappaB, three STATs, a PPAR/RXR, an Ahr/Arnt and a CRE. Basal activity of serially deleted promoter constructs transiently transfected into COS-7, alpha T3 and TE671 cells demonstrated the enhancing and silencing roles of the putative transcription factor binding sites. Quinpirole, a dopamine agonist, significantly reduced the promoter activity of muCyp19b in TE671. The results suggest tissue-specific regulation of the muCyp19 genes and a putative alternative promoter for muCyp19b. (c) 2006 Elsevier Ireland Ltd. All rights reserved

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 β 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 FSHβ 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.This research was funded by grants from the Australian Seafood CRC (2008/745) and the Australian Centre for International Agricultural Research (FIS/2012/101)

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 β-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.This project was funded by the Australian Centre for International Agricultural Research (ACIAR) under project FIS 2012 101: Developing technologies for giant grouper (Epinephelus lanceolatus) aquaculture in Vietnam, the Philippines and Australia (SEAFDEC/AQD study code: 6149-T-RD-ACIAR4). LD was a recipient of the University of the Sunshine Coast, Australia, APA Ph.D. scholarship, PP was an ACIAR John Allwright Fellow. Samples were graciously provided by Dr. Kiyoshi Soyano’s lab in Nagasaki, Dr. Evelyn Grace de Jesus-Ayson of SEAFDEC/ AQD, Tigbauan, Philippines, Dr. Stewart Fielder, Port Stephens Fisheries Centre: NSWDPI, Dr. Richard Knuckey from The Company One, Cairns, Australia, and Mr. Josh McNally from Ecomarine, Noosa, Australia

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 (E. 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 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.This study was funded by the Australian Centre for International Agricultural Research (ACIAR) through project no. FIS/2012/101 granted to A. Elizur. P. Palma was a recipient of an ACIAR funded Australia Awards John Allwright Fellowship