Independent Evolution of Sex Chromosomes and Male Pregnancy-Related Genes in Two Seahorse Species.

Unlike birds and mammals, many teleosts have homomorphic sex chromosomes, and changes in the chromosome carrying the sex-determining locus, termed "turnovers", are common. Recent turnovers allow studies of several interesting questions. One question is whether the new sex-determining regions evolve to become completely non-recombining, and if so, how and why. Another is whether (as predicted) evolutionary changes that benefit one sex accumulate in the newly sex-linked region. To study these questions, we analyzed the genome sequences of two seahorse species of the Syngnathidae, a fish group in which many species evolved a unique structure, the male brood pouch. We find that both seahorse species have XY sex chromosome systems, but their sex chromosome pairs are not homologs, implying that at least one turnover event has occurred. The Y-linked regions occupy 63.9% and 95.1% of the entire sex chromosome of the two species and do not exhibit extensive sequence divergence with their X-linked homologs. We find evidence for occasional recombination between the extant sex chromosomes that may account for their homomorphism. We argue that these Y-linked regions did not evolve by recombination suppression after the turnover, but by the ancestral nature of the low crossover rates in these chromosome regions. With such an ancestral crossover landscape, a turnover can instantly create an extensive Y-linked region. Finally, we test for adaptive evolution of male pouch-related genes after they became Y-linked in the seahorse.

Isolation and identification of a new strain of nervous necrosis virus from the big-belly seahorse Hippocampus abdominalis.

BACKGROUND: Betanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish, resulting in great economic losses worldwide. METHODS: In this study, we isolated a virus strain named seahorse nervous necrosis virus (SHNNV) from cultured big-belly seahorses Hippocampus abdominalis in Xiamen city, Fujian Province, China. Virus isolation, PCR detection, phylogenetic analysis, qRT-PCR, fluorescence in situ hybridization and histology were used for virus identification and analysis of virus histopathology. Furthermore, an artificial infection experiment was conducted for virulence testing. RESULTS: Brain and eye tissue homogenates of diseased big-belly seahorses were inoculated onto a grouper spleen (GS) cell monolayer at 28 °C. Tissue homogenates induced obvious cytopathic effects in GS cells. PCR and sequencing analyses revealed that the virus belonged to Betanodavirus and shared high sequence identity with red-spotted grouper nervous necrosis virus isolates. qRT-PCR and fluorescence in situ hybridization revealed that SHNNV mainly attacked the brain and eye. Histopathological examination revealed that the virus led to cytoplasmic vacuolation in the brain and retinal tissues. Infection experiments confirmed that SHNNV was highly infectious, causing massive death in big-belly seahorses. CONCLUSION: A novel seahorse betanodavirus from the big-belly seahorse cultured in China was discovered. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

A multi-omic analysis of orange-spotted grouper larvae infected with nervous necrosis virus identifies increased adhesion molecules and collagen synthesis in the persistent state.

Grouper (Epinephelus coioides) is an important commercial maricultural fish, which suffers from nervous necrosis virus (NNV) infection. The molecular mechanisms underlying the pathogenesis of the viral infection are not clear. In this study, we combined deep RNA sequencing and label-free mass spectrum for the first time to analyze the transcriptomic and proteomic profiles in infected/dead, infected/survival (persistent), and infection-free (control)orange-spotted groupers in the larval stage. Further analyses showed that the transcriptome and proteome changed dramatically among the three distinct groups, especially differentially-expressed genes in the infected/dead and infected/survival larvae enriched for pathways related to immune response. Notably, the overlapped genes between transcriptomes and proteomes identified that genes related to collagen synthesis and adhesion molecules were enhanced in the persistent (infected/survival) stage, which might contribute to suppressing the acute and lethal immune responses upon NNV infection. These transcriptomic and proteomic datasets enable the investigation of molecular mechanisms underlying NNV infection, thus may help further development of molecular breeding in marine fishery.

Functional analysis of the CXCR1a gene response to SGIV viral infection in grouper.

Chemokine receptors are a superfamily of seven-transmembrane domain G-coupled receptors and have important roles in immune surveillance, inflammation, and development. In previous studies, a series of CXCRs in grouper (Epinephelus coioides) was identified; however, the function of CXCR in viral infection has not been studied. To better understand the effect of the CXCR family on the fish immune response, full-length CXCR1a was cloned, and its immune response to Singapore grouper iridovirus (SGIV) was investigated. Grouper CXCR1a shared a seven-transmembrane (7-TM) region and a G protein-coupled receptor (GPCR) family 1 that contained a triaa stretch (DRY motif). Phylogenetic analysis indicated that CXCR1a showed the nearest relationship to Takifugu rubripes, followed by other fish, bird and mammal species. Fluorescence microscopy revealed that CXCR1a was expressed predominantly in the cytoplasm. Overexpression of CXCR1a in grouper cells significantly inhibited the replication of SGIV, demonstrating that CXCR1a delayed the occurrence of cytopathic effects (CPE) induced by SGIV infection and inhibited viral gene transcription. Furthermore, our results also showed that CXCR1a overexpression significantly increased the expression of interferon-related cytokines and activated ISRE and IFN promoter activities. Taken together, the results demonstrated that CXCR1a might have an antiviral function against SGIV infection.

Assessment of the Effects of Bisphenol A on Dopamine Synthesis and Blood Vessels in the Goldfish Brain.

Bisphenol A (BPA) is an abundant contaminant found in aquatic environments. While a large number of toxicological studies have investigated the effects of BPA, the potential effects of BPA exposure on fish brain have rarely been studied. To understand how BPA impacts goldfish brains, we performed a transcriptome analysis of goldfish brains that had been exposed to 50 µg L-1 and 0 µg L-1 BPA for 30 days. In the analysis of unigene expression profiles, 327 unigenes were found to be upregulated and 153 unigenes were found to be downregulated in the BPA exposure group compared to the control group. Dopaminergic signaling pathway-related genes were significantly downregulated in the BPA exposure group. Furthermore, we found that serum dopamine concentrations decreased and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) staining was present in dopamine neurons enriched regions in the brain after BPA exposure, suggesting that BPA may disrupt dopaminergic processes. A KEGG analysis revealed that genes involved in the fluid shear stress and atherosclerosis pathway were highly significantly enriched. In addition, the qRT-PCR results for fluid shear stress and atherosclerosis pathway-related genes and the vascular histology of the brain showed that BPA exposure could damage blood vessels and induce brain atherosclerosis. The results of this work provide insights into the biological effects of BPA on dopamine synthesis and blood vessels in goldfish brain and could lay a foundation for future BPA neurotoxicity studies.

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.

Genomic organization, promoter characterization and expression analysis of the leukocyte cell-derived chemotaxin-2 gene in Epinephelus akaraa.

Leucocyte cell-derived chemotaxin 2 (LECT2) was first identified as a chemotactic factor and has been subsequently proven to be a multifunctional protein that mediates the regulation of liver regeneration, carcinogenesis and Natural killer T (NKT) cell homeostasis in mammals. In fish, it has been recently found to be critical for the inflammatory response to stimuli. However, the in vivo function of LECT2 in fish remains obscure. Base on the full-length cDNA of the Epinephelus akaraa LECT2 (EaLECT2) gene we previously isolated, we sought to analyze its genomic structure and context. The genomic DNA of the EaLECT2 gene spans 2866bp from the transcription start site to the termination codon. As in most LECT2 genes in other vertebrates, the EaLECT2 genomic DNA contains four exons and three introns. An analysis of the promoter region revealed the presence of a TATA box and several putative transcription factor-binding sites. And transcriptional activity analysis suggested that most basal DNA regulatory elements required for EaLECT2 transcriptional activity might be contained within the 581bp region upstream of the transcription start codon. A real-time PCR analysis showed that the EaLECT2 expression levels were slightly increased in the head kidney, liver, gill and brain by bacterial challenge with Vibrio harveyi. Furthermore, the transcriptional level of the EaLECT2 gene in the liver was significantly up-regulated within 1h and reached its peak level at 12h post-stimulation. Higher levels of LECT2 expression were also observed in head kidney in challenged individuals.The expression pattern demonstrates the role of EaLECT2 in the immune response and its functions under other conditions. Additionally, we found that the recombinant EaLECT2 could be expressed as a soluble protein using a prokaryotic expression system with the expression vector pET32a(+) and the soluble protein was further proved to be the recombinant EaLECT2 with the rat antiserum against EaLECT2 we obtained. This work provides a unique basis for substantial work in future projects.

Genome and gene evolution of seahorse species revealed by the chromosome-level genome of Hippocampus abdominalis.

Seahorses belong to the teleost family Syngnathidae that evolved a distinct body plan and unique male pregnancy compared to other teleosts. As a classic model for studying evolution of viviparity and sexual selection of teleosts, seahorse species still lack a publicly available high-quality reference genome. Here, we generated the genome assembly of the big-belly seahorse, Hippocampus abdominalis with long-read and Hi-C technologies. We managed to place over 99% of the total length of 444.7 Mb of assembled genome into 21 linkage groups with almost no gaps. We reconstructed a phylogenomic tree with the big-belly seahorse genome and other representative Syngnathidae and teleost species. We also reconstructed the historical population dynamics of four representative Syngnathidae species. We found the gene families that underwent expansion or contraction in the Syngnathidae ancestor were enriched for immune-related or ion transporter gene ontology terms. Many of these genes were also reported to show a dynamic expression pattern during the pregnancy stages of H. abdominalis. We also identified putative positively selected genes in the Syngnathidae ancestor or in H. abdominalis, whose mouse mutants are enriched for abnormal craniofacial and limb morphological phenotypes. Overall, our study provides an important genome resource for evolutionary and developmental studies of seahorse species, and candidate genes for future experimental works.

The ERß-CXCL19/CXCR4-NFκB pathway is critical in mediating the E2-induced inflammation response in the orange-spotted grouper (Epinephelus coioides).

The main physiological function of 17ß-estradiol (E2) in vertebrates is to regulate sexual development and reproduction. In fish, especially hermaphroditic fish, estrogen is often used to aid reproduction, but it also can trigger an inflammatory response. However, the molecular mechanism for this E2-induced inflammatory reaction is not clear. In this study, we found that the ERß-CXCL19/CXCR4-NFκB cascade regulated the E2-induced inflammatory response in the orange-spotted grouper (Epinephelus coioides). Strikingly, E2 treatment resulted in significantly high expression of inflammatory cytokines and induced phosphorylation and degradation of IκBα and translocation of NFκB subunit p65 to the nucleus in grouper spleen cells. However, the E2-induced inflammatory response could be prevented by the broad estrogen receptor (ER) ligand ICI 182,780. Moreover, the luciferase assay showed that E2 induced the inflammatory response by activating the promotor of chemokine CXCL19 through ERß1 and ERß2. Knockdown of CXCL19 blocked the E2-induced inflammatory response and NFκB nucleus translocation. Additionally, knockdown of chemokines CXCR4a and CXCR4b together, but not alone, blocked the E2-induced inflammatory response. The immunofluorescence assay and co-immunoprecipitation analysis showed that CXCL19 mediated the E2-induced inflammatory response by activating CXCR4a or CXCR4b. Taken together, these results showed that the ERß-CXCL19/CXCR4-NFκB pathway mediated the E2-induced inflammatory response in grouper. These findings are valuable for future comparative immunological studies and provide a theoretical basis for mitigating the adverse reactions that occur when using E2 to help fish reproduce.

The complete mitochondrial genome of the Picasso clownfish: genomic comparisons and phylogenetic inference among Amphiprioninae.

Picasso clownfish belong to the subfamily Amphiprioninae and are considered a variant of the genus Amphiprion. In this study, we first sequenced the complete mitochondrial genome of the Picasso clownfish by Illumina next-generation sequencing technology. The length of the whole mitogenome is 16,727 bp long, with a gene arrangement and composition similar to those of two other Amphiprion species (Amphiprion ocellaris and Amphiprion percula). The topological structure of the phylogenetic tree shows that the Picasso clownfish is more closelyrelated to A. percula than it is to A. ocellaris, suggesting that the Picasso clownfish may be a variant of A. percula.

The CXC chemokines and CXC chemokine receptors in orange-spotted grouper (Epinephelus coioides) and their expression after Singapore grouper iridovirus infection.

Chemokines comprise a group of small molecular weight (6-14 kDa) cytokines; chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors. Both chemokines and their receptors have important roles in immune surveillance, inflammation, and development. Recently, 9 CXC chemokine ligands (CXCLs) and 8 CXC chemokine receptors (CXCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CXCLs and CXCRs in healthy tissues of E. coioides. Our data show that CXCL genes are highly expressed in the spleen, kidney and liver and that CXCR genes are ubiquitously expressed, rather than being expressed only in immune organs. Analysis of gene expression after Singapore grouper iridovirus infection indicated that CXCL and CXCR genes are regulated in a gene-specific manner. CXCL8 and CXCL12a were significantly upregulated in the spleen, kidney and liver of resistant fish, indicating potential roles in immunity against the pathogen. Additionally, CXCR4a was upregulated in all three organs in resistant fish, suggesting that CXCL8 or CXCL12a may participate in the immune response via interaction with CXCR4a. In addition, the new orange-spotted grouper receptor CXCR1b was found to be upregulated in the spleen and kidney of resistant fish, indicating that this receptor plays an important role in immune responses to viral infection. These results are valuable for comparative immunological studies and provide insight into the roles of these genes in viral infection.

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.

Toxic effects of bisphenol A on goldfish gonad development and the possible pathway of BPA disturbance in female and male fish reproduction.

Bisphenol A (BPA) is an abundant endocrine-disrupting compound that is found in the aquatic environment and has adverse effects on fish reproduction; however, the exact pathway of these impacts is unclear. In this study, the different effects of BPA on ovarian and testis development in goldfish (Carassius auratus) and the different mechanisms underlying these effects were investigated. The gonadosomatic index (GSI) and gonadal histology demonstrated that BPA diminished ovarian maturation in goldfish, which recovered after BPA treatment withdrawal. In males, BPA disrupted testis maturation, but this disruption could not be recovered after BPA treatment withdrawal. The hypothalamic-pituitary-gonad (HPG) axis-related genes sgnrh, fshß and lhß were significantly decreased in BPA-treated female fish, while no changes in sex steroid hormone levels and no TUNEL and PCNA staining were found in the ovary, suggesting that BPA may reduce ovarian maturation through the HPG axis. In male fish, TUNEL staining was found in 1 µg L-1 BPA-exposed germ cells and 50 and 500 µg L-1 BPA-exposed Leydig cells. Decreases in 11-KT levels were also found in 50 and 500 µg L-1 BPA-exposed fish, but BPA did not affect genes associated with the HPG axes. This result shows that BPA disrupts testis maturation through apoptosis of germ cells and Leydig cells, thus inducing decreases in 11-KT levels that disrupt spermatogenesis. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying BPA disturbance of goldfish reproduction.

De novo assembly of a chromosome-level reference genome of red-spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi-C.

The red-spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South-East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome-level reference genome of E. akaara by taking advantage of long-read single-molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi-C. A red-spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96-fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi-C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA-seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein-coding sequences. The high-quality chromosome-level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red-spotted grouper in the future.

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

We presented the complete mitochondrial genome of the Epinephelus corallicola in this study. The mitochondrial genome is 16,647 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 corallicola mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.60% of A, 15.89% of C, 28.66% of T and 28.85% 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.