Two Foxo1 homologues in the orange-spotted grouper Epinephelus coioides: sequences, expression, and possible involvement in the activation of cyp19a1a expression in the ovary.

Foxo1, a member of Foxo transcription factor family, is involved in a number of physiological processes including metabolism, cell cycle progression, aging, and apoptosis. In the ovarian granulosa cell of mouse, Foxo1 is implicated to inhibit the expression of Cyp19a1, a gene encoding the aromatase that converts androgens into estrogens. Currently, the information about the expression and physiological relevance of Foxo1 homologues in the ovary of teleosts is scarce. In the present study, cDNAs encoding two forms of Foxo1, Foxo1a and Foxo1b, were isolated from the orange-spotted grouper. Phylogenetic analysis indicated that the orange-spotted groupers Foxo1a and Foxo1b were closely related to the counterparts of the ricefield eel. RT-PCR analysis showed that the orange-spotted groupers foxo1a and foxo1b were expressed in a wide range of tissues, with high levels detected in the brain regions, liver, and intestine. Quantitative real-time PCR analysis showed similar expression profiles for cyp19a1a, foxo1a, and foxo1b in the ovary during development from the primary growth to mature stages, with peak values detected at the vitellogenic stage. In situ hybridization detected mRNA of foxo1a, foxo1b, and cyp19a1a in granulosa cells surrounding vitellogenic oocytes. In vitro transfection showed that both Foxo1a and Foxo1b upregulated the orange-spotted grouper cyp19a1a promoter activities, possibly through the conserved Foxo binding site. Collectively, these results suggest that both Foxo1a and Foxo1b may be involved in the regulation of the ovarian functions in the orange-spotted grouper and the physiological roles of Foxo1 homologues in the ovary may be diversified in vertebrates.

Nr5a1b promotes and Nr5a2 inhibits transcription of lhb in the orange-spotted grouper, Epinephelus coioides†.

Nr5a1 (Sf-1) up-regulates lhb expression across vertebrates; however, its regulatory roles on fshb remain to be defined. Moreover, the involvement of Nr5a2 in the regulation of gonadotropin expression is not clear either. In the present study, the involvement of Nr5a1b (a homologue of Nr5a1) and Nr5a2 in the regulation of lhb and fshb expression in the orange-spotted grouper was examined. Dual fluorescent immunohistochemistry using homologous antisera showed that in the pituitary of orange-spotted groupers, Lh cells contain both immunoreactive Nr5a1b and Nr5a2 signals, whereas Fsh cells contain neither of them. In LßT2 cells, Nr5a1b up-regulated basal activities of lhb and fshb promoters possibly via Nr5a sites, and synergistically (on lhb promoter) or additively (on fshb promoter) with forskolin. Surprisingly, Nr5a2 inhibited basal activities of lhb promoter possibly via Nr5a sites and attenuated the stimulatory effects of both forskolin and Nr5a1b. In contrast, Nr5a2 had no effects on fshb promoter. Chromatin immunoprecipitation analysis showed that both Nr5a1b and Nr5a2 bound to lhb promoter, but not fshb promoter in the pituitary of the orange-spotted grouper. The abundance of Nr5a1b bound to lhb promoter was significantly higher at the vitellogenic stage than the pre-vitellogenic stage, whereas that of Nr5a2 exhibited an opposite trend. Taken together, data of the present study demonstrated antagonistic effects of Nr5a1b and Nr5a2 on lhb transcription in the orange-spotted grouper and revealed novel regulatory mechanisms of differential expression of lhb and fshb genes through Nr5a homologues in vertebrates.

Transcriptome analysis of immune response against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides).

Vibrio harveyi is a gram-negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange-spotted grouper (Epinephelus coioides) at 1 and 2 days post-infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P < 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune-related pathway functions, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, NF-κB signaling pathways, and Jak-STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes.

Immunity to nervous necrosis virus infections of orange-spotted grouper (Epinephelus coioides) by vaccination with virus-like particles.

Nervous necrosis virus (NNV) is a kind of the betanodaviruses, which can cause viral nervous necrosis (VNN) and massive mortality in larval and juvenile stages of orange-spotted grouper (Epinephelus coioides). Due to the lack of viral genomes, virus-like particles (VLPs) are considered as one of the most promising candidates in vaccine study to control this disease. In this study, a type of VLPs, which was engineered on the basis of orange-spotted grouper nervous necrosis virus (OGNNV), was produced from prokaryotes. They possessed the similar structure and size to the native NNV. In addition, synthetic oligodeoxynucleotide (ODN) containing CpG motif was added in vaccines, and the expression patterns of several genes were analyzed after injecting with VLP and VLP with adjuvant (VA) to assess the regulation effect of vaccine for inducing immune responses. RT-PCR assays showed that six related genes in healthy tissues were ubiquitously expressed in all nine tested tissues. The vaccine alone was able to enhance the expression of genes, including MHCIa, MyD88, TLR3, TLR9 and TLR22 after vaccination, indicating that the vaccine was able to induce immune response in grouper. In liver, spleen and kidney, the gene expressions of VA group were all significantly higher than that of VLP group at 72 h post-stimulation, showing that the fish of VA challenge group obtained the longer-lasting protective immunity and resistance to pathogen challenge than that of VLP group. The data indicated that the efficacy of vaccine could be further enhanced by CpG ODN after vaccination and provided the reference for the development of future viral vaccine in grouper.

A member of the immunoglobulin superfamily, orange-spotted grouper novel immune gene EcVig, is induced by immune stimulants and type I interferon.

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response.

Genome-Wide Mapping of Growth-Related Quantitative Trait Loci in Orange-Spotted Grouper (Epinephelus coioides) Using Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq).

Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish.

Cytoplasmic localization of Lrh-1 down-regulates ovarian follicular cyp19a1a expression in a teleost, the orange-spotted grouper Epinephelus coioides.

Liver receptor homolog-1 (LRH-1) is a conserved member of the NR5A subfamily in vertebrates and a potential regulator of estrogen synthesis in the ovarian granulosa cells. An Lrh-1 homologue was obtained from the orange-spotted grouper Epinephelus coioides that contains the conserved structural features of NR5A and is phylogenetically closely related to NR5A2. The expression of the orange-spotted grouper Lrh-1 is tissue-specific with relatively higher levels in the liver and ovary. The immunoreactive signals for Lrh-1 and Cyp19a1a were present in the ovarian follicular cells and germ cells. In the ovarian follicular cells, Lrh-1 was present both in the nucleus and cytoplasm, and colocalized with Cyp19a1a. The expression levels of both increased during vitellogenesis whereas only Cyp19a1a dramatically decreased toward maturation when Lrh-1 was localized almost exclusively to the cytoplasm of the follicular cells. The orange-spotted grouper Lrh-1 could up-regulate cyp19a1a transcription in vitro via the two conserved Ftz-f1 sites in cyp19a1a promoter. Chromatin immunoprecipitation analysis showed that the orange-spotted grouper Lrh-1 could bind cyp19a1a promoter in vivo with a higher abundance in the vitellogenic ovary, whereas the binding was dramatically decreased in the mature ovary. Taken together, the results of present study demonstrate that Lrh-1 plays an important role in up-regulating cyp19a1a gene in the ovarian follicular cells during vitellogenesis, and the sequestration of Lrh-1 to the cytoplasm may down-regulate cyp19a1a expression in the mature ovary. This mechanism for modifying transcriptional roles of the orange-spotted grouper Lrh-1 may shed new light on the regulation of Cyp19a1 expression in other vertebrates as well.

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.

A homologue of Nr5a1 activates cyp19a1a transcription additively with Nr5a2 in ovarian follicular cells of the orange-spotted grouper.

Transcription factors of nuclear receptor 5A (Nr5a) subfamily play pivotal roles in regulation of steroidogenic enzymes in vertebrates including teleosts. In the orange-spotted grouper, the expression of Nr5a1a was only detectable in the ovary, spleen, and head kidney in the female. The immunoreactive Nr5a1a was present in ovarian follicular and germ cells. In the ovarian follicular cells surrounding vitellogenic oocytes, Nr5a1a was detected both in the nucleus and cytoplasm, and co-localized with Cyp19a1a and Nr5a2. In the ovarian follicular cells surrounding fully grown oocytes, Nr5a1a was localized almost exclusively to the cytoplasm together with Nr5a2. Nr5a1a could up-regulate cyp19a1a promoter activities through Nr5a sites, and further increase the responses elicited by Nr5a2 at sub-maximal doses. Chromatin immunoprecipitation analysis showed that Nr5a1a bound to cyp19a1a promoter in the vitellogenic but not fully grown ovary. Taken together, Nr5a1a up-regulates cyp19a1a additively with Nr5a2 during vitellogenesis, and its cytoplasmic sequestration may also contribute to the down-regulation of cyp19a1a in the fully grown ovary.

Foxo3b but not Foxo3a activates cyp19a1a in Epinephelus coioides.

FOXO3 has been shown to be a critical transcription factor for folliculogenesis in mammals, while the information on its roles in reproduction of nonmammalian vertebrates remains scarce. In this study, two foxo3 homologs, namely foxo3a and foxo3b, were identified in a teleost, the orange-spotted grouper Epinephelus coioides. foxo3a was mainly expressed in the central nervous system, ovary, and gut whereas foxo3b was expressed ubiquitously in tissues examined. In contrast to the dominant expression of mammalian FOXO3 in germ cells but barely detectable in ovarian follicular cells, immunoreactive Foxo3a and Foxo3b were identified both in the ovarian germ cells and follicular cells. The immunointensities of both Foxo3a and Foxo3b in ovarian follicular cells during vitellogenesis were significantly increased stage-dependently, and co-localized with Cyp19a1a. In the nucleus of ovarian follicular cells, both Foxo3a and Foxo3b immunostaining could be detected at the vitellogenic stages. Transient transfection and EMSA showed that Foxo3a and Foxo3b upregulated cyp19a1a promoter activities in vitro through a conserved Foxo-binding site, with the latter being a more potent activator. However, ChIP analysis showed that only Foxo3b binds to cyp19a1a proximal promoter region containing the conserved Foxo-binding site in the vitellogenic ovary. Taken together, these results suggested that Foxo3a and Foxo3b are involved in the ovarian development possibly through regulating the ovarian germ cells as well as follicular cells, and Foxo3b but not Foxo3a may activate cyp19a1a in the ovarian follicular cells during vitellogenesis in the orange-spotted grouper.

EcVig, a novel grouper immune-gene associated with antiviral activity against NNV infection.

VHSV-induced genes (VIGs) were first identified in rainbow trout (Oncorhynchus mykiss) and subsequently isolated in a variety of fish. Recent studies have shown that most VIGs have immunological functions against pathogenic infections. However, most research has focused on Vig1, such that our present understanding of these genes in other fish species remains limited. This study isolated a homologue of the uncharacterized O. mykiss Vig-B319 (EcVig) from orange-spotted grouper (Epinephelus coioides). Genomic organization suggests that four EcVig isoforms (EcVig A-D), are generated through alternative splicing. Due to the encoding of 2 immunoglobulin (Ig) domains, the EcVig protein can be considered a member of the immunoglobulin superfamily. The expression of EcVig increased 3 days after hatching (dph) and peaked at 9 dph. This pattern is similar to that displayed by EcMx, an important grouper antiviral gene. Additionally, a tissue tropism assay revealed that EcVig A is the major EcVig isoform present in the tissues considered by this study, with the expression of EcVig A exceeding that of EcVig B. We subsequently investigated whether EcVig expression was induced by the viral pathogen nervous necrosis virus (NNV) or the bacterial pathogen Vibrio anguillarum. Following injection with NNV, the expression levels of EcVig showed significant up-regulation. Conversely, a significant reduction was observed in EcVig expression in brain samples collected from V. anguillarum injected grouper. The overexpression of EcVig A suppressed the replication of NNV in grouper GF-1 cell lines, suggesting that EcVig is an important antiviral factor in the grouper immune responses.