Search

Egg storage for fertilization and seed production has great significance in aquaculture. Giant grouper(Epinephelus lanceolatus)is an economically important aquaculture species in China, and its fertilized eggs are much expensive. In this study, we tried to develop an in vitro egg storage system in this species. Storage conditions such as medium, temperature and pH were experimentally investigated. Results showed that eggs stored in L-15 medium(pH=8. 1)at 22 C for 2 h showed a relatively high fertilization rate(66. 4%) and a hatching rate(47. 9%). With supplementation of giant grouper serum and vitamin premix into L-15 medium, the storage time was prolonged and eggs still retained a relatively high viability. The fertilization and hatching rates from eggs stored for 8 h were 64. 4% and 44. 8%, respectively. This optimized short-term egg storage system would facilitate breeding practice and hatchery management in giant grouper. [ABSTRACT FROM AUTHOR]

During their breeding season, estrogen induces vitellogenin (VTG) production in the liver of teleost fish through estrogen receptors (ERs) that support oocyte vitellogenesis. There are at least three ER subtypes in teleost fish, but their roles in mediating E2-induced VTG expression have yet to be ascertained. In this study, we investigated the expression of vtgs and ers in the liver of orange-spotted grouper (Epinephelus coioides). Their expression levels were significantly increased in the breeding season and were upregulated by an estradiol (E2) injection in female fish, except for the expression of erβ1. The upregulation of vtgs, erα and erβ2 by E2 was also observed in primary hepatocytes, but these stimulatory effects could be abolished by ER antagonist ICI182780 treatment. Subsequent studies showed that ERβ antagonist Cyclofenil downregulated the E2-induced expression of vtg, erα, and erβ2, while the ERβ agonist DPN simulated their expression. Knockdown of erβ2 by siRNA further confirmed that ERβ2 mediated the E2-induced expression of vtgs and erα. To reveal the mechanism of ERβ2 in the regulation of erα expression, the erα promoter was cloned, and its activity was examined in cells. E2 treatment simulated the activity of the erα promoter in the presence of ERβ2. Deletions and site-directed mutations showed that the E2 up-regulated transcriptional activity of erα occurs through a classical half-estrogen response element- (ERE) dependent pathway. This study reveals the roles of ER subtypes in VTG expression in orange-spotted grouper and provides a possible explanation for the rapid and efficient VTG production in this species during the breeding season. [ABSTRACT FROM AUTHOR]

Simple Summary: C. sonnerati is an important marine fish species in coral reef ecosystems and has huge commercial value. This species serves as an excellent research model due to its complex social structures and behavior mechanisms. Nevertheless, owing to the lack of genomic resources, molecular genetic studies and genomic breeding remain unexplored in this species. Therefore, it is important to obtain more genome sequences of Cephalopholis grouper species for research on the classification, evolution, genetics, and biology of groupers. In the present study, we first assembled a high-quality, chromosome-level C.sonnerati genome, providing a valuable genome resource for further studies of the genetic conservation, resistance breeding, and evolution of C. sonnerati. The tomato hind, Cephalopholis sonnerati, is a bottom-dwelling coral reef fish, which is widely distributed in the Indo-Pacific and Red Sea. C. sonnerati also features complex social structures and behaviour mechanisms. Here, we present a high-quality, chromosome-level genome assembly for C. sonnerati that was derived using PacBio sequencing and Hi-C technologies. A 1043.66 Mb genome with an N50 length of 2.49 Mb was assembled, produced containing 795 contigs assembled into 24 chromosomes. Overall, 97.2% of the complete BUSCOs were identified in the genome. A total of 26,130 protein-coding genes were predicted, of which 94.26% were functionally annotated. Evolutionary analysis revealed that C. sonnerati diverged from its common ancestor with E. lanceolatus and E. akaara approximately 41.7 million years ago. In addition, comparative genome analyses indicated that the expanded gene families were highly enriched in the sensory system. Finally, we found the tissue-specific expression of 8108 genes. We found that these tissue-specific genes were highly enriched in the brain. In brief, the high-quality, chromosome-level reference genome will provide a valuable genome resource for studies of the genetic conservation, resistance breeding, and evolution of C. sonnerati. [ABSTRACT FROM AUTHOR]

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange‐spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11‐ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change. [ABSTRACT FROM AUTHOR]

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange‐spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42′ 02.6″ N, 114° 32′ 10.1″ E). Analysis of gonadal restructuring revealed that females with pre‐vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre‐vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17β‐estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11‐ketotestosterone (11‐KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre‐vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female‐to‐male transformation, whereas increased 11‐KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange‐spotted grouper. [ABSTRACT FROM AUTHOR]

Beta-hydroxysteroid dehydrogenases (β-HSDs) are a group of steroidogenic enzymes that are involved in steroid biosynthesis and metabolism, and play a crucial role in mammalian physiology and development, including sex determination and differentiation. In the present study, a genome-wide analysis identified the numbers of β -hsd genes in orange-spotted grouper (Epinephelus coioides) (19), human (Homo sapiens) (22), mouse (Mus musculus) (24), chicken (Gallus gallus) (16), xenopus (Xenopus tropicalis) (24), coelacanth (Latimeria chalumnae) (17), spotted gar (Lepisosteus oculatus) (14), zebrafish (Danio rerio) (19), fugu (Takifugu rubripes) (19), tilapia (Oreochromis niloticus) (19), medaka (Oryzias latipes) (19), stickleback (Gasterosteus aculeatus) (17) and common carp (Cyprinus carpio) (27) samples. A comparative analysis revealed that the number of β -hsd genes in teleost fish was no greater than in tetrapods due to gene loss followed by a teleost-specific whole-genome duplication event. Based on transcriptome data from grouper brain and gonad samples during sex reversal, six β -hsd genes had relatively high expression levels in the brain, indicating that these genes may be required for neurogenesis or the maintenance of specific biological processes in the brain. In the gonad, two and eight β -hsd genes were up- and downregulated, respectively, indicating their important roles in sex reversal. Our results demonstrated that β -hsd genes may be involved in the sex reversal of grouper by regulating the synthesis and metabolism of sex steroid hormones. [ABSTRACT FROM AUTHOR]

In this study, we injected cortisol into the protogynous orange-spotted grouper (Epinephelus coioides) to investigate the role of this hormone in sex change. Following injection, we evaluated gonadal changes, serum levels of steroid hormones, and sex-related gene expression during the processes of cortisol-induced sex change and cortisol withdrawal in the orange-spotted grouper. Cortisol treatment caused the degeneration of oocytes and induced sex change in a dose-dependent manner. Over the long-term, we observed a significant increase in serum 11-ketotestosterone (11-KT) levels in all cortisol-treated groups, although levels of 17β-estradiol did not change significantly. Consistent with the elevation of serum 11-KT levels, the expression of genes related to testicular development was also significantly up-regulated in the cortisol-treated groups. Based on our results, we propose that cortisol may trigger masculinization by inducing the synthesis of 11-KT and by directly activating the expression of sex-related genes. Furthermore, we found that cortisol-induced sex change was not permanent and could be reversed after the withdrawal of cortisol treatment. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. Conclusions: Our study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts. [ABSTRACT FROM AUTHOR]

Socially controlled sex change in teleosts is a dramatic example of adaptive reproductive plasticity. In many cases, the occurrence of sex change is triggered by a change in the social context, such as the disappearance of the dominant individual. The orange‐spotted grouper Epinephelus coioides is a typical protogynous hermaphrodite fish that changes sex from female to male and remains male throughout its life span. In this study, male‐to‐female sex reversal in male Epinephelus coioides was successfully induced by social isolation. The body length and mass, gonadal change, serum sex steroid hormone levels and sex‐related gene expression patterns during the process of socially controlled male‐to‐female sex reversal in E. coioides were systematically examined. This report investigates the physiological mechanisms of the socially controlled male‐to‐female sex reversal process in a protogynous hermaphrodite grouper species. The results enable us to study the physiological control of sex change, not only from female to male, but also from male to female. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the gonadal development of 10‐ to 43‐mo‐old diploid and triploid orange‐giant grouper hybrids, Epinephelus coioides♀ × Epinephelus lanceolatus♂, was investigated. The diploid hybrid groupers exhibited normal gonadal development and experienced vitellogenesis at 43 mo of age. In contrast, the triploid gonad was masculinized and gradually filled with spermatogenic cysts. Radioimmunoassays revealed that the diploid hybrids showed high serum E2 levels at 10 and 43 mo and high T levels at 43 mo, while the triploid hybrids showed high E2 levels at 18 and 30 mo and high T levels at 18 mo. Furthermore, the messenger RNA (mRNA) expression of genes related to reproductive regulation was examined in these hybrids. The mRNA levels of fshb, fshr, lhb, lhr, dmrt1, and sox9 varied significantly during gonadal development. Interestingly, the expression levels of fshr, lhr, dmrt1, and sox9 were higher in triploid hybrids than in diploid hybrids. The hyperactive expression of these genes may be associated with the gonadal masculinization of triploid hybrids. [ABSTRACT FROM AUTHOR]

Fish-related antibiotic resistance genes (ARGs) have attracted attention for their potentially harmful effects on food safety and human health through the food chain transfer. However, the potential factors affecting these ARGs have not been fully explored. In this study, ARGs and bacterial communities in the fish gut, mucosal skin, and gill filaments in fish were comprehensively evaluated in four different mariculture systems formed by hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), Gracilaria bailinae , and Litopenaeus vannamei using different combinations. The results showed that 9 ARGs were detected in the gut and mucosal skin and 6 ARGs in the gill filaments. The detection rate of aphA1 was the highest, and the abundance was 1.91 × 10−3 - 6.30 × 10−2 copies per 16 S rRNA gene. Transposase gene (tnpA-04) was detected in all samples with the abundance of 3.57 × 10−3 - 3.59 × 10−2 copies per 16 S rRNA gene, and was strongly correlated with multiple ARGs (e.g., aphA1 , tet(34) , mphA-02). Proteobacteria, Deinococcus-Thermus, Firmicutes, and Bacteroidetes were the dominant phyla in the four mariculture systems, accounting for 65.1%–96.2% of the total bacterial community. Notably, the high relative abundance of Stenotrophomonas , a potential human pathogen, was elevated by 20.5% in the hybrid grouper gut in the monoculture system. In addition, variation partitioning analysis (VPA) showed that the difference in bacterial communities between mariculture systems was the main driving factor of ARGs distribution differences in hybrid groupers. This study provides a new comprehensive understanding of the characterization of fish-related ARGs contamination in different mariculture systems and facilitates the assessment of potential risks of ARGs and pathogen taxa to human health. [Display omitted] • The mucosal skin and gill in IMTA systems had higher ARGs abundance. • High abundance of the pathogen Stenotrophomonas in grouper monoculture system. • Bacterial differences between aquaculture systems drove ARGs differences. • AphA1 / tnpA-04 result suggests aminoglycoside resistance/transmission risk. • IMTA of fish-algae is a recommended production method for groupers. [ABSTRACT FROM AUTHOR]

Chromatin structure plays a pivotal role in maintaining the precise regulation of gene expression. Accessible chromatin regions act as the binding sites of transcription factors (TFs) and cis-elements. Therefore, information from these open regions will enhance our understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. We employed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq analyses in the gonads of protogynous hermaphroditic orange-spotted groupers during sex reversal to profile open chromatin regions and TF binding sites. We focused on several crucial TFs, including ZNF263, SPIB, and KLF9, and analyzed the networks of TF-target genes. We identified numerous transcripts exhibiting sex-preferred expression among their target genes, along with their associated open chromatin regions. We then investigated the expression patterns of sex-related genes as well as the mRNA localization of certain genes during sex reversal. We found a set of sex-related genes that—upon further study—might be identified as the sex-specific or cell-specific marker genes that trigger sex reversal. Moreover, we discovered the core genes (gnas, ccnb2, and cyp21a) of several pathways related to sex reversal that provide the guideposts for future study. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the hybrid grouperEpinehelus moara ♀×Epinephelus lanceolatus ♂is presented in this study. The mitochondrial genome is 16,418 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition of the hybrid grouper mitochondrial genome were similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order was 28.86% of G, 28.61% of T, 26.46% of A and 16.08% of C. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method shows that the hybrid grouper has closer relationship toEpinehelus moaraandEpinehelus bruneus. [ABSTRACT FROM PUBLISHER]

The red-spotted grouper (Epinephelus akaara) is one of the most commercially important aquatic species in China. However, its seedstock has low larval survival rates, and its stability is confronted with the danger of overexploitation. In this study, a high-density genetic map was constructed using 3435 single nucleotide polymorphisms (SNPs) from 142 first generation (F1) full-sib offspring and two parents of a red-spotted grouper population. The total genetic length of the map was 2300.12 cM with an average intermarker distance of 0.67 cM. Seventeen genome-wide significant quantitative trait loci (QTLs) for growth-related traits were detected on 24 linkage groups, including 5 QTLs for full length, 7 QTLs for body length, and 5 QTLs for body weight. The contribution values of explained phenotypic variance ranged from 10.7% to 12.9%. Moreover, 13 potential candidate genes for growth-related traits were identified. Collectively, these findings will be useful for conducting marker-assisted selection of the red-spotted grouper in future studies. [ABSTRACT FROM AUTHOR]

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper. [ABSTRACT FROM AUTHOR]

The relaxin family, a group of peptides, is now considered as a major player in the reproductive processes in teleosts fishes. Insulin-like peptide 3 (INSL3) is a core relaxin, plays a key role in testis development and male fertility. However, its regulatory mechanism remains unclear. In the present research, insl3 gene of the hermaphroditic orange-spotted grouper was isolated. The entire coding sequence of insl3 was found to be 384 bp encoding a protein of 127 amino acids. Insl3 expression was mainly detected in gonad, kidney, heart, and head kidney. Compared to ovary, testis showed a significantly higher expression level of insl3. In the gonad, INSL3 could induce a significant increase of Akt and ERK protein phosphorylation. Overexpression of insl3 promoted the proliferation of gonidium-like cells and the expression of male-related factors. These results contribute to the better understanding of INSL3 functions and its involvement in the regulation mechanisms behind gonad development and sex reversal in orange-spotted grouper. • The insl3 gene of orange-spotted grouper was cloned and analyzed. • The expression patterns of the insl3 gene were investigated. • INSL3 induced a significant increase in the phosphorylation of Akt and ERK. • Insl3 overexpression promoted gonidium-like cell proliferation and male-related factors expression. [ABSTRACT FROM AUTHOR]

Using Ringer solution as extender, the present study examined the protective effect of dimethyl sulphoxide (Me 2 SO; 8-12%, v/v) on the cryopreservation of giant grouper (Epinephelus lanceolatus) sperm. The cryopreserved sperm was then successfully applied in interspecific hybridization with tiger grouper (E. fuscoguttatus). Higher motility (90.56 ± 6.58%) and fertilization rate (69.61 ± 4.83%) was achieved in 10% Me 2 SO with Ringer solution as extender (dilution ratio 1:1), which should no significant difference in comparison with fresh sperm (95.88 ± 1.64% and 73.10 ± 1.28%). There were no statistical differences in both fertilization and hatching rates between hybrid and non-hybrid tiger grouper by using cryopreserved sperm for fertilization, but malformation rate of the hybrid was higher than non-hybrid (17%) (P < 0.05). Survival rate of the hybrid was lower than that of the controls at 15 days post hatching (23% vs 48%). However, hybrids showed survival rate equal to the controls at the end of the 60-day study period. Hybridization of E. fuscoguttatus x E. lanceolatus was successfully achieved using cryopreserved sperm from giant grouper. The cryopreservation of giant grouper sperm and its application in hybridization provided a technical support for further grouper breeding work. [ABSTRACT FROM AUTHOR]

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as Ec IKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKβ-NEMO-binding domain, was identified in Ec IKKα. Phylogenetic analysis suggested that Ec IKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with Ec IKKα plasmid. Moreover, overexpression of Ec IKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that Ec IKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens. • Epinephelus coioides IKKα was identified with a highly conserved structure. • NF-κB activity was up-regulated when Ec IKKα was expressed in HEK 293T cells. • Ec IKKα regulated cytokines expression induced by LPS and flagellin in GS cells. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) as essential pattern recognition receptors in innate immunity, can recognize pathogens and trigger immune response to eliminate invading pathogens. MicroRNAs regulates multiple biological processes by suppressing mRNA translation or resulting in mRNA degradation. MiR-182 has previously been implicated in DNA repair, disease and cancer aspects. The potential role of miR-182-3p in TLR signaling pathway against pathogens is unclear. In this study, we found that the expression of miR-182-3p was up-regulated after Vibrio parahaemolyticus flagellin stimulation in grouper spleen (GS) cells, and negatively correlated with the expression of orange-spotted grouper (Epinephelus coioides) TLR5M (Ec TLR5M). Then we found that miR-182-3p could directly target Ec TLR5M by using bioinformatic analysis and dual-luciferase reporter assay. Dual-luciferase reporter assay also showed that miR-182-3p down-regulated the wild-type Ec TLR5M 3′UTR in luciferase activity rather than the mutant group in HEK 293T cells. We further verified the effect of miR-182-3p on the activation of Nuclear factor-κB (NF-κB) signaling pathway, and found that miR-182-3p inhibitors significantly augmented flagellin-induced NF-κB phosphorylation. Additionally, we also demonstrated that the increased expression of miR-182-3p significantly suppressed the flagellin-induced Ec TLR5M, pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expression. And the endogenous miR-182-3p knockdown experiments reversely verified the regulatory effect of miR-182-3p. These results suggested that miR-182-3p post-transcriptionally controls Ec TLR5M expression and thereby suppresses the expression of pro-inflammatory cytokines. This study is the first to demonstrate that miR-182-3p suppresses pro-inflammatory cytokines expression by regulating the TLR signaling pathway. • Ec TLR5M is a direct target gene of miR-182-3p in orange-spotted grouper. • MiR-182-3p suppressed V. parahaemolyticus flagellin-induced Ec TLR5M expression. • MiR-182-3p suppressed V. parahaemolyticus flagellin-induced cytokines expression. [ABSTRACT FROM AUTHOR]

In our previous studies, diploid and triploid hybrids have been detected from the hybridization of Epinephelus coioides ♀ × E. lanceolatus ♂. The triploid groupers have been found to be delayed in gonadal development, but the mechanism remains poorly understood. In this study, we examined the gonadal development, assayed the serum steroid hormone levels, and compared the BP (brain and pituitary) and G (gonad) transcriptomes of 18-month-old diploid and triploid hybrids. The results showed that levels of serum estradiol-17β and testosterone were significantly higher in triploid groupers. The RNA-seq data revealed that 1518 and 14,963 differentially expressed genes were identified in the BP and G transcriptome, respectively. Further analysis revealed that the expression levels of genes involved in the sexual differentiation pathway and sex steroid synthesis pathway are significantly higher in triploid hybrids. Our findings provided a comprehensive insight into a better understanding of the regulatory mechanisms of sterility in triploid hybrid fish. • The gonadal development of triploid hybrids seems to be disrupted. • RNA-seq analysis of 18-month-old diploid and triploid hybrids was performed. • Sexual differentiation pathway and sex steroid synthesis pathway are significantly activated in triploid hybrids. [ABSTRACT FROM AUTHOR]

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhβ mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhβ expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro . Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17β-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species. [ABSTRACT FROM AUTHOR]

NLRX1, an intracellular immune receptor, negatively modulates the proinflammatory signals and also regulates immune cell metabolic function in human. However, in teleost, there is limited understanding of molecular mechanisms that how NLRX1 interacts with the host immune system. This research cloned and identified NLRX1 from orange-spotted grouper (Epinephelus coioides). The cDNA contains a 2889 bp open reading frame (ORF) which encodes 962 amino acids, including three typical (a N-terminal, a NACHT and five leucine-rich repeat (LRR)) domains. The Ec NLRX1 protein presents a mitochondrial localization and shows highly constitutive expression in mitochondria-rich tissues. Reactive oxygen species (ROS) are mainly generated in mitochondria to induce cellular oxidative stress. EcNLRX1 could potently induce ROS generation. After Vibrio alginolyticus infection, Ec NLRX1 mRNA expression in spleen is greatly increased in the early stage (24 h and 48 h, p < 0.001) of immune response. Under Poly I:C stimulation, Ec NLRX1 synergistically triggers ROS release. And the Ec NLRX1-LRR domain is a key structural domain for ROS activation. In summary, this result indicated that teleost NLRX1 is a critical component in controlling innate immune responses and oxidative stress at mitochondria, which provides a novel insight for understanding of fish immunometabolism. • The Ec NLRX1 protein was localized in mitochondria and highly expressed in mitochondria-rich tissues. • After Vibrio alginolyticus infection, Ec NLRX1 mRNA expression was greatly increased in early phase of immune response. • Ec NLRX1 mediated ROS production through its LRR domain, but not need MTS domain. • Under Poly I:C stimulation, Ec NLRX1 synergistically triggers ROS release. [ABSTRACT FROM AUTHOR]

In aquaculture, oocyte maturation, ovulation and spawning are important for successful breeding and seed production. Most marine teleost fish can be induced to spawn regularly using intramuscular injections of human chorionic gonadotropin (HCG). In this study, an intra-ovarian injection method was developed in orange-spotted grouper to deal with the broodstocks unresponsive to HCG. Successful oocyte maturation and ovulation in the unilateral injected ovary was induced within 35 h of treatment. An in vitro method was then established to induce HCG-primed oocyte maturation. HCG or maturation-inducing hormones (MIHs) induced the maturation of oocytes with a diameter between 490 and 560 μm. Biochemical, physiological and molecular changes during oocyte maturation and hydration were also characterized. The concentration of free amino acids except the cysteine increased during oocyte maturation, whereas Na+ decreased steadily and K+ increased. The expression of genes encoding cathepsin B, D, L, and the subunits of Na+, K+-ATPase were upregulated during hydration. Specific inhibitors for cathepsin and Na+, K+-ATPase were used to further confirm their crucial roles in oocyte hydration both in vivo and in vitro. These results provide valuable data for inducing oocyte maturation and evaluating oocyte quality in marine teleost fish. • A novel intra-ovarian injection method is established to deal with the broodstocks unresponsive to HCG. • An effective method is established to induce oocyte maturation in vitro. • Characterization of histological changes during oocyte maturation and hydration. • Biochemical, physiological and molecular changes during oocyte maturation and hydration. [ABSTRACT FROM AUTHOR]

• Gonadal meiosis initiated at approximately 180 dah in the orange-spotted grouper. • RA synthesis inhibition resulted a delayed meiotic initiation. • Meiotic initiation was delayed during female-to-male sex change induced by androgen. Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa , dazl , sycp3 and rec8 , which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen. [ABSTRACT FROM AUTHOR]

Vasa and dazl genes have been reported to play pivotal roles in germ cell development and differentiation both in vertebrates and invertebrates; however, little is known about their functions in germ cell differentiation during gametogenesis and sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl (Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). The full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and encoded 646 and 214 amino acid residues, respectively. Reversal transcription PCR showed that Ecvasa and Ecdazl mRNA were highly expressed in the gonads. Further, in situ hybridization revealed that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper. Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of ovo-testes gonads, indicating that the expression of germ cell genes could be suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is the first time to describe the expression profiles of vasa and dazl mRNA in germ cells during gametogenesis and sex reversal in the orange-spotted grouper. These findings will provide new insights into understanding the mechanisms through which vasa and dazl regulate germ cell differentiation in hermaphrodite fish species. • Ecvasa and Ecdazl mRNA were highly expressed in gonads. • Expression of germ cell genes could be suppressed in oocytes during sex reversal. • Vasa and dazl can be used as marker genes to trace the germ cells development during sex reversal in orange-spotted grouper. [ABSTRACT FROM AUTHOR]