Mechanisms of sex differentiation and sex reversal in hermaphrodite fish as revealed by the Epinephelus coioides genome.

Most grouper species are functional protogynous hermaphrodites, but the genetic basis and the molecular mechanisms underlying the regulation of this unique reproductive strategy remain enigmatic. In this study, we report a high-quality chromosome-level genome assembly of the representative orange-spotted grouper (Epinephelus coioides). No duplication or deletion of sex differentiation-related genes was found in the genome, suggesting that sex development in this grouper may be related to changes in regulatory sequences or environmental factors. Transcriptomic analyses showed that aromatase and retinoic acid are probably critical to promoting ovarian fate determination, and follicle-stimulating hormone triggers the female-to-male sex change. Socially controlled sex-change studies revealed that, in sex-changing fish, the brain's response to the social environment may be mediated by activation of the phototransduction cascade and the melatonin synthesis pathway. In summary, our genomic and experimental results provide novel insights into the molecular mechanisms of sex differentiation and sex change in the protogynous groupers.

Chromosome-Level Genome Assembly and Transcriptome Comparison Analysis of Cephalopholis sonnerati and Its Related Grouper Species.

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.

Beta-Hydroxysteroid Dehydrogenase Genes in Orange-Spotted Grouper (Epinephelus coioides): Genome-Wide Identification and Expression Analysis During Sex Reversal.

Beta-hydroxysteroid dehydrogenases (ß-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 ß-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 ß-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 ß-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 ß-hsd genes were up- and downregulated, respectively, indicating their important roles in sex reversal. Our results demonstrated that ß-hsd genes may be involved in the sex reversal of grouper by regulating the synthesis and metabolism of sex steroid hormones.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides.

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.

An SNP-Based Genetic Map and QTL Mapping for Growth Traits in the Red-Spotted Grouper (Epinephelus akaara).

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.

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

Socially controlled male-to-female sex reversal in the protogynous orange-spotted grouper, Epinephelus coioides.

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.

Comparative transcriptome analysis of diploid and triploid hybrid groupers (Epinephelus coioides♀ × E. lanceolatus♂) reveals the mechanism of abnormal gonadal development in triploid hybrids.

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-17ß 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.

MT-Feeding-Induced Impermanent Sex Reversal in the Orange-Spotted Grouper during Sex Differentiation.

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.

Molecular regulation of sex change induced by methyltestosterone -feeding and methyltestosterone -feeding withdrawal in the protogynous orange-spotted grouper.

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Transcriptome analysis reveals the molecular mechanisms underlying growth superiority in a novel grouper hybrid (Epinephelus fuscogutatus♀ × E. lanceolatus♂).

BACKGROUND: Groupers (Epinephelus spp.) have been widely cultivated in China and South-East Asian countries. As a novel hybrid offspring crossed between E. fuscogutatus♀ and E. lanceolatus♂, Hulong grouper exhibits significant growth superiority over its female parent, which made it a promising farmed species in grouper aquaculture industry in China. Hulong grouper present a good combination of beneficial traits from both parent species, but the molecular mechanisms of its heterosis still remain poorly understood. RESULTS: Based on RNA sequencing and gene expression profiling, we conducted comparative transcriptome analyses between Hulong grouper and its parents E. fuscoguttatus & E. lanceolatus. Six hundred sixty-two and 5239 differentially expressed genes (DEGs) were identified in the brains and livers, respectively. GO enrichment analysis of these DEGs revealed that metabolic process and catalytic activity were the most enriched GO terms. Further analysis showed the expressions of GnRH1 and GnRH3 in the brain, and GH/IGF axis related genes such as IGF-1, IGF-2b, IGFBP-1, IGFBP-2, IGFBP-4 and IGFBP-5a in the liver of the hybrid F1 were significantly up-regulated, which is in accordance with the growth superiority of hybrid grouper. Meanwhile, expressions of genes related to the protein and glycogen synthesis pathway, such as PI3KC, PI3KR, Raptor, EIF4E3, and PP1 were up-regulated, while PYG expression was down-regulated. These changes might contribute to increased protein and glycogen synthesis in the hybrid grouper. CONCLUSIONS: We identified a number of differentially expressed genes such as GnRH1 and GnRH3, and genes involved in GH/IGF axis and its downstream signaling pathways for protein and glycogen synthesis in Hulong Grouper. These findings provided molecular basis underlying growth superiority of hybrid grouper, and comprehensive insights into better understanding the molecular mechanisms and regulative pathways regulating heterosis in fish.

The complete mitochondrial genome of Epinephelus awoara (Perciformes: Epinephelus) with phylogenetic consideration.

The complete mitochondrial genome of the Epinephelus awoara was presented in this study. The mitochondrial genome is 16 798-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 Epinephelus awoara mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order is 27.35% of A, 16.53% of C, 28.44% of T, and 27.69% of G. 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 Epinephelus awoara was closer to Epinephelus fasciatomaculosus in the phylogenetic relationship.

The complete mitochondrial genome of the Epinephelus lanceolatus (Perciformes: Serranidae).

The complete mitochondrial genome of the Epinephelus lanceolatus was presented in this study. The mitochondrial genome is 16,743 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition of E. lanceolatus mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.55% of A, 15.02% of C, 29.67% of T and 28.76% of G. 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 complete mitochondrial genome of the Platax teira (Osteichthyes: Ephippidae).

We presented the complete mitochondrial genome of the Platax teira in this study. The mitochondrial genome is 16,561 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition of Epinephelus tukula mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 28.80% of A, 31.16% of C, 24.44% of T and 15.60% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and seven tRNA genes, all other mitochondrial genes are encoded on the heavy strand.

The complete mitochondrial genome of the Pampus nozawae (Perciformes: Stromateidae).

The complete mitochondrial genome of the Pampus nozawae was presented in this study. The mitochondrial genome is 16,556 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition of Pampus nozawae mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 30.01% of A, 27.51% of C, 27.29% of T and 15.18% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and seven tRNA genes, all other mitochondrial genes are encoded on the heavy strand.

The complete mitochondrial genome of the Epinephelus fuscoguttatus (Perciformes: Serranidae).

The complete mitochondrial genome of the Epinephelus fuscoguttatus was presented in this study. The mitochondrial genome is 16,648 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region. The gene order and the composition of E. fuscoguttatus mitochondrial genome were similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.91% of A, 15.58% of C, 29.19% of T, and 28.32% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand.