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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.

Spermatogenesis is a process of self-renewal and differentiation in spermatogonial stem cells. During this process, germ cells and somatic cells interact intricately to ensure long-term fertility and accurate genome propagation. Spermatogenesis has been intensely investigated in mammals but remains poorly understood with regard to teleosts. Here, we performed single-cell RNA sequencing of ~9500 testicular cells from the male, orange-spotted grouper. In the adult testis, we divided the cells into nine clusters and defined ten cell types, as compared with human testis data, including cell populations with characteristics of male germ cells and somatic cells, each of which expressed specific marker genes. We also identified and profiled the expression patterns of four marker genes (calr, eef1a, s100a1, vasa) in both the ovary and adult testis. Our data provide a blueprint of male germ cells and supporting somatic cells. Moreover, the cell markers are candidates that could be used for further cell identification.

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.

Growth hormone-releasing hormone (GHRH) and the receptor, GHRHR, constitute important components of the hypothalamus-pituitary growth axis and act on the downstream growth hormone (GH). PACAP-related peptide/pituitary adenylate cyclase activating polypeptide (PRP-PACAP) is a paralog of GHRH. These genes all play key roles in development and growth patterns. To improve the quality of cultured fish strains, natural genetic variation must be examined and understood. A mixed linear model has been widely used in association mapping, taking the population structures and pairwise kinship patterns into consideration. In this study, a mass cross population of orange-spotted grouper (Epinephelus coioides) was examined. These candidate genes were found to harbor low nucleotide diversity (θw from 0.00154 to 0.00388) and linkage disequilibrium levels (delay of 50% within 2 kbp). Association mapping was employed, and two single-nucleotide polymorphisms (KR269823.1:g.475A>C and KR269823.1:g.2143T>C) were found to be associated with growth (false discovery rate Q < 0.05), explaining 9.0%–17.0% of the phenotypic variance. The association of KR269823.1:g.2143T>C was also found via haplotype-based association (p < 0.05). The identified associations offer new insights into gene functions, and the associated single-nucleotide polymorphisms (SNPs) may be used for breeding purposes.

In mammals, leptin has been demonstrated to perform important roles in many physiological activities and to influence development, growth, metabolism and reproduction. However, in fish, its function is still unclear. Duplicate leptin genes, leptin-a and leptin-b, have been identified in the orange-spotted grouper. In the present study, the polymorphisms in the leptin-b gene of the orange-spotted grouper were detected, and the relation between these polymorphisms and 12 growth traits were analyzed. Six polymorphisms (including 3 single nucleotide polymorphisms (c.14G>A, c.93A>G, c.149G>A) in exon 1, 2 SNPs (c.181A>G, c.193G>A) in intron 1, and 1 SNP (c.360C>T) in exon 2) were identified and genotyped from 200 different individuals. The results revealed that the SNP c.149G>A was significantly associated with growth traits, that the heterozygous mutation genotype GA having negative effects on growth traits. However, the other five SNPs (c.14G>A, c.93A>G, c.181A>G, c.193G>A, c.360C>T) did not show significant associations with all the growth traits. Compared with our findings in leptin-a gene, the results suggested that the leptin-a hormone has more important physiological effects in fish bodies than the leptin-b type. Moreover, leptin genes were supposed to be one class of major candidate genes of regulating growth traits in the orange-spotted grouper.

Leptin is a multifunctional protein involved in processes such as body weight regulation, energy expenditure, fat metabolism, food intake, and appetite regulation. Duplicate leptin genes, leptin-a and leptin-b, were previously detected in the orange-spotted grouper. In this study, we cloned the full-length open reading frame (ORF) of the leptin-a gene in the orange-spotted grouper, searched for polymorphisms, and performed association analyses between these polymorphisms and seven growth traits. Six polymorphisms, consisting of 2 SNPs in intron 1 (c.182T > G, c.183G > T) and 4 SNPs in exon 2 (c.339C > G, c.345C > T, c.447G > A, c.531C > T), were identified and genotyped in 200 individuals. The c.182T > G and c.183G > T polymorphisms showed complete linkage and were analyzed together. Association analyses revealed that the c.182 + 183TG > GT polymorphism was significantly associated with body weight (BWT) and body width (BWH), with the AB (TG/GT) genotype showing positive effects on growth traits. Additionally, the SNP c.447G > A was significantly associated with BWT, BWH, overall length (OL), trunk width (TW), and head length (HL), with the GA genotype displaying positive effects on growth traits. The c.531C > T SNP showed a close association between the TT genotype and decreased growth. Our results demonstrate that several polymorphisms in the leptin-a gene are associated with growth traits and can be used for marker-assisted selection (MAS) in orange-spotted grouper populations.

The large yellow croaker (Larimichthys crocea) is the most important mariculture fish species in China and the wild stocks of this croaker have collapsed in the past decades due to high fishing pressure and habitat degradation. Due to a lack of wild croaker samples, however, studies concerning the genetic changes of the cultured croaker stocks compared to their wild counterparts were never conducted. Here, we collected three wild populations in the northern and central East China Sea during fisheries survey and investigated the differences in terms of genetic diversity and differentiation between and within cultured stocks and wild populations. Our results demonstrated that the cultured croaker had significantly reduced genetic diversity in contrast to the wild populations, and also presented statistically significant differentiation from the wild, indicating that enhancement of the current wild stock should be conducted with caution. These changes may be caused by founder effects, artificial selection and random genetic drift. With a relatively high level of genetic diversity, the wild populations showed important value for improving the ongoing breeding program of this croaker. Further, we detected no differentiation among the wild populations, suggesting that the wild croaker in the northern and central East China Sea should be considered as one unit for management and conservation.

In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise Fst, structure, molecular variance analysis, as well as multidimensional scaling analysis. Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.

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.

Leptin is a multifunctional protein involved in processes such as body weight regulation, energy expenditure, fat metabolism, food intake, and appetite regulation. Duplicate leptin genes, leptin-a and leptin-b, were previously detected in the orange-spotted grouper. In this study, we cloned the full-length open reading frame (ORF) of the leptin-a gene in the orange-spotted grouper, searched for polymorphisms, and performed association analyses between these polymorphisms and seven growth traits. Six polymorphisms, consisting of 2 SNPs in intron 1 (c.182T > G, c.183G > T) and 4 SNPs in exon 2 (c.339C > G, c.345C > T, c.447G > A, c.531C > T), were identified and genotyped in 200 individuals. The c.182T > G and c.183G > T polymorphisms showed complete linkage and were analyzed together. Association analyses revealed that the c.182 + 183TG > GT polymorphism was significantly associated with body weight (BWT) and body width (BWH), with the AB (TG/GT) genotype showing positive effects on growth traits. Additionally, the SNP c.447G > A was significantly associated with BWT, BWH, overall length (OL), trunk width (TW), and head length (HL), with the GA genotype displaying positive effects on growth traits. The c.531C > T SNP showed a close association between the TT genotype and decreased growth. Our results demonstrate that several polymorphisms in the leptin-a gene are associated with growth traits and can be used for marker-assisted selection (MAS) in orange-spotted grouper populations. [ABSTRACT FROM AUTHOR]