Transcriptomic Analysis across Crayfish (Cherax quadricarinatus) Claw Regeneration Reveals Potential Stem Cell Sources for Cultivated Crustacean Meat.

In the face of rising global demand and unsustainable production methods, cultivated crustacean meat (CCM) is proposed as an alternative means to produce delicious lobster, shrimp, and crab products. Cultivated meat requires starting stem cells that may vary in terms of potency and the propensity to proliferate or differentiate into myogenic (muscle-related) tissues. Recognizing that regenerating limbs are a non-lethal source of tissue and may harbor relevant stem cells, we selected those of the crayfish Cherax quadricarinatus as our model. To investigate stem cell activity, we conducted RNA-Seq analysis across six stages of claw regeneration (four pre-molt and two post-molt stages), along with histology and real-time quantitative PCR (qPCR). Our results showed that while genes related to energy production, muscle hypertrophy, and exoskeletal cuticle synthesis dominated the post-molt stages, growth factor receptors (FGFR, EGFR, TGFR, and BMPR) and those related to stem cell proliferation and potency (Cyclins, CDKs, Wnts, C-Myc, Klf4, Sox2, PCNA, and p53) were upregulated before the molt. Pre-molt upregulation in several genes occurred in two growth peaks; Stages 2 and 4. We therefore propose that pre-molt limb regeneration tissues, particularly those in the larger Stage 4, present a prolific and non-lethal source of stem cells for CCM development.

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.

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.

Induction of Gonadal Development in Protogynous Grouper with Orally Delivered FSH DNA.

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 ß-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 17ß-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.

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.

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.

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.

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.

In-vitro and in-vivo biological activity of recombinant yellowtail kingfish (Seriola lalandi) follicle stimulating hormone.

Biologically active recombinant yellowtail kingfish follicle stimulating hormone (rytkFsh) was produced in yeast Pichia pastoris and its biological activity was demonstrated by both in-vitro and in-vivo bioassays. Incubation of ovarian and testicular fragments with the recombinant hormone stimulated E2 and 11-KT secretion, respectively. In-vivo trial in immature female YTK resulted in a significant increase of plasma E2 levels and development of oocytes. In males at the early stages of puberty, advancement of spermatogenesis was observed, however plasma 11-KT levels were reduced when administered with rytkFsh.

Heritability for body colour and its genetic association with morphometric traits in Banana shrimp (Fenneropenaeus merguiensis).

BACKGROUND: Banana shrimp Fenneropenaeus merguiensis has emerged as an important aquacultured shrimp species in South East Asia and Australia. However, the quantitative genetic basis of economically important traits in this species are currently not available, while for body colour, cooked or uncooked, there are no genetic parameter estimates for any shrimp or indeed any decapod crustacean. In this study, we report for banana shrimp genetic parameters for morphometric traits and, the first time for any shrimp, parameter estimates for body colour. Ten highly polymorphic microsatellite markers were developed from genomic sequences and used to construct a pedigree for 2000 offspring from approximately 60 female and 60 male parents that were sampled from a single routine commercial production pond. RESULTS: Restricted maximum likelihood method applied to a single trait mixed model was used to estimate heritabilities, while correlations were estimated using the multi-trait approach. The estimates of heritability for morphometric traits were moderate to high (h(2) = 0.14 - 0.50). Body colour of uncooked shrimp showed a heritable additive genetic component (h(2) = 0.03 - 0.55), and those estimates obtained for cooked shrimp were significantly different from zero. Genetic correlations among morphometric traits were all positive and very high (close to unity, rg = 0.85 - 0.99). The genetic correlations of body traits (weight, length and width) were positive with both colour after cooking (0.74 - 0.84) and body colour measured on live shrimp (0.59 to 0.70). The positive genetic correlations between the cooked body colour and uncooked body colour (0.64 ± 0.20) suggests these two traits can be simultaneously improved in practical selective breeding programs. This first ever report of genetic parameters for cooked or uncooked colour in crustacean indicates there is potential for genetic improvement of both growth and body colour through selection. CONCLUSIONS: In the present study we demonstrated for banana shrimp that genetic parameters can be estimated from commercial samples (using pedigrees based on DNA markers), that selection for shrimp colour should be successful under such commercial conditions.

Chronic kisspeptin administration stimulated gonadal development in pre-pubertal male yellowtail kingfish (Seriola lalandi; Perciformes) during the breeding and non-breeding season.

The kisspeptin system is now accepted as a key regulator of vertebrate reproductive function, particularly the onset of puberty. In teleosts, the stimulatory effect of exogenous kisspeptins has been demonstrated mainly at the hypothalamic and pituitary levels of the reproductive axis, with very limited information pertaining to gonadal response. We determined the effect of chronic peripheral administration of the conserved kisspeptin decapeptides (YNLNSFGLRY or Kiss1-10; and FNFNPFGLRF or Kiss2-10) on gonadal development of pre-pubertal yellowtail kingfish (Seriola lalandi), a Perciform teleost, during the breeding and non-breeding season. We utilized slow-release implants to chronically deliver the synthesized peptides, which were based on the yellowtail kingfish kiss1 and kiss2 cDNA sequences that we isolated. The expression level of kiss2r and gnrh1 in the brain or hypothalamus did not vary between treated and control groups. Pituitary expression of fshß and lhß was upregulated only with Kiss1-10 treatment regardless of the season. Based on histological evidence, gonadal development was stimulated in male fish with either Kiss1-10 or Kiss2-10, with Kiss2-10 being more effective during the non-breeding period. Overall, our results suggest that kisspeptins modulate the early gonadal development of male yellowtail kingfish, however that may vary with the breeding season.

Neuroendocrine regulation of puberty in fish: insights from the grey mullet (Mugil cephalus) model.

We investigated the molecular regulation of pubertal development in the grey mullet, Mugil cephalus, a relatively late-maturing teleost fish. We have isolated and characterized the cDNAs of key reproductive genes along the brain-pituitary-gonadal (BPG) axis as well as the promoters of genes that modulate the axis at multiple levels. Together with relevant findings from other model species, we propose a conceptual model of the neuroendocrine regulation of puberty in the female grey mullet. Research areas that warrant further investigation are identified in the model.

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.

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 1515bp 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 (650 bp) 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-3 s. 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, alphaT3 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.