Qiaomu Hu

Epigenetic modification and inheritance in sexual reversal of fish

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis).

Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.

Comparative transcriptome reveal the potential adaptive evolutionary genes in Andrias davidianus

To search the evidence of molecular evolution mechanism for aquatic and cave habitat in Andrias davidianus, the evolution analysis was carried out among several species transcriptome data. The transcriptome data of Notophthalmus viridescens, Xenopus tropicalis, Cynops pyrrhogaster, Hynobius chinensis and A. davidianus were obtained from the Genbank and reassembled except Xenopus tropicalis. The BLAST search of transcriptome data obtained 1244 single-copy orthologous genes among five species. A phylogenetic tree showed A. davidianus to have the closest relationship to H. chinensis. Fourteen positively selected genes were detected in A. davidianus and N. vridescens group and fifteen in A. davidianus and H. chinensis group. Five genes were shared in the both groups which involved in the immune system, suggesting that A. davidianus adaptation to an aquatic and cave environment required rapid evolution of the immune system compared to N. viridescens and H. chinensis.

MicroRNA repertoire and comparative analysis of Andrias davidianus infected with ranavirus using deep sequencing

ABSTRACT Andrias davidianus is a large and economically important amphibian in China. Ranavirus infection causes serious losses in A. davidianus farming industry. MicroRNA mediated host‐pathogen interactions are important in antiviral defense. In this study, five small‐RNA libraries from ranavirus infected and non‐infected A. davidianus spleens were sequenced using high throughput sequencing. The miRNA expression pattern, potential functions, and target genes were investigated. In total, 1356 known and 431 novel miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes are associated with apoptotic, signal pathway, and immune response categories. Analysis identified 82 downregulated and 9 upregulated differentially expressed miRNAs, whose putative target genes are involved in pattern‐recognition receptor signaling pathways and immune response. These findings suggested miRNAs play key roles in A. davidianuss response to ranavirus and could provide a reference for further miRNA functional identification, leading to novel approaches to improve A. davidianus ranavirus resistance. HighlightsThe microRNA of Andrias davidianus and Andrias davidianus responding to ranavirus were sequenced.MiRNA repertoire of ranavirus infected and non‐infected A. davidianus was investigated.Differential expression miRNAs immune‐related were analyzed.

Identification and expression of cytochrome P450 genes in the Chinese giant salamander Andrias davidianus

Cytochrome P450 (cyp) genes play vital roles in biological processes, including in metabolism of endogenous and exogenous compounds. Exogenous hormone influences on gene expression that leads to disruption of gonad development have been studied in several species, but whether exogenous hormones affect cyp genes that show sexually dimorphic expression remains to be determined. Here, we identified and characterized cyp genes from the widely-cultured Chinese giant salamander Andrias davidianus. We obtained 20 cyp genes including 11 genes with complete sequences. Phylogenetic analyses supported the classification of cyp genes similar to other vertebrates. Expression profile of female and male salamanders showed multiple cyp genes to exhibit higher expression in ovary than in testis, including cyp26a, cyp19a, cyp1a1, cyp4v2, cyp3a24, cyp2c20, cyp2d14, cyp2d15, and cyp4b; while cyp11a, cyp2b11, cyp11b1, cyp4f22, cyp2j6, cyp2k1, cyp2k4, cyp1a5 was higher in testis than in ovary. Seven sex-biased genes were detected after aromatase inhibitor injection and with exposure to high water temperatures. Cyp2k1, cyp11b1, and cyp2j6 expression were increased, while cyp26a, cyp2c20, cyp4b genes were decreased with aromatase inhibition. In ovary, cyp26a and cyp2c20 expression were significantly reduced; whereas cyp2k1, cyp2j6, and cyp2k4 expression were significantly elevated with no changes in cyp11a and cyp11b1 expression after temperature treatment. These findings provide valuable information for further study of sex differentiation mechanisms and cyp gene evolution.

Identification and analysis of the β-catenin1 gene in half-smooth tongue sole (Cynoglossus semilaevis)

β-catenin is a key signalling molecule in the canonical Wnt pathway, which plays a role in cell adhesion, embryogenesis and sex determination. However, little is known about its function in teleosts. We cloned and characterized the full-length β-catenin1 gene from half-smooth tongue sole (Cynoglossus semilaevis), which was designated CS-β-catenin1. The CS-β-catenin1 cDNA consists of 2,346 nucleotides and encodes a protein with 782 amino acids. Although CS-β-catenin1 was transcribed in the gonads of both sexes, the level was significantly higher in ovaries compared to testes. Furthermore, the mRNA level of CS-β-catenin1 was significantly upregulated at 160 days and constantly increased until 2 years of age. In situ hybridization revealed that CS-β-catenin1 mRNA was mainly localized in oocyte cells, especially in stage I, II and III oocytes. When CS-β-catenin1 expression was inhibited by injection of quercetin in the ovaries, levels of CS-Figla and CS-foxl2 mRNA were significantly down-regulated, and CS-dmrt1 was up-regulated, which suggested that CS-β-catenin1 is a potential upstream gene of CS-Figla and is involved in the development of the ovaries, i.e., folliculogenesis.

Characterization of a low-density lipoprotein receptor, Lrp13, in Chinese tongue sole ( Cynoglossus semilaevis ) and medaka ( Oryzias latipes )

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.