Zining Meng

Structural diversity of the GnIH/GnIH receptor system in teleost: its involvement in early development and the negative control of LH release.

Gonadotropin inhibitory hormone (GnIH), via binding to GnIH receptor (GnIHR), plays a negative role on the avian and mammalian reproductive axis by inhibiting luteinizing hormone (LH) release. However, the biological significance of the GnIH/GnIHR system in other vertebrates is controversial. To demonstrate the presence of such a system in teleost, we have identified the orthologous gnih genes in zebrafish, stickleback, medaka and Takifugu. Three orthologous genes (gnihr1, gnihr2 and gnihr3) for the gnihr were also identified in zebrafish. The zebrafish gnih precursor contains three putative LPXRFamide peptides. The three zebrafish gnihrs are typical seven transmembrane G protein-coupled receptors sharing high sequence homology with the mammalian and avian GnIHRs (GPR147). Tissue expression studies revealed that zebrafish gnih is mainly expressed in the brain, eye, testis, ovary and spleen, corroborating largely with the tissue expression patterns of the gnihrs in zebrafish. The expression patterns of gnih and its receptors at different developmental stages of zebrafish were also studied. Gnih expression first appeared in the prim-5 stage, and thereafter maintained at a relatively constant level. The three gnihrs could be detected at all embryonic stages of zebrafish and also during early development after hatching. The biological action of the teleost gnih on LH release was further investigated in goldfish in vivo. Intraperitoneal administration of the mature zebrafish gnih peptide (LPXRFa peptide-3) could significantly reduce the basal serum LH level in goldfish. These results provided the first evidence that gnih plays an important role in the negative regulation of LH release in teleost.

Molecular Identification of the Kiss2/Kiss1ra System and Its Potential Function During 17Alpha-Methyltestosterone-Induced Sex Reversal in the Orange-Spotted Grouper, Epinephelus coioides

The Kiss1/Kiss1r system is a component of the hypothalamus-pituitary-gonadal (HPG) axis, which plays a crucial role in regulating gonadotropins and gonadotropin-releasing hormone. The sex reversal process is a special reproductive phenomenon regulated by the HPG axis. To better understand the neuroendocrine mechanisms of sex reversal, cDNAs encoding kiss2 and kiss1ra have been cloned and functionally characterized from the orange-spotted grouper Epinephelus coioides, a protogynous hermaphroditic teleost. The core mature peptide (Kiss2-10) of grouper Kiss2 shared high similarity to other KISS orthologs. In phylogenetic analyses, the grouper Kiss was clustered with the teleost Kiss2 clade and termed grouper kiss2. The predicted amino acid sequence of grouper kiss1ra contained three putative glycosylation sites at its N-terminus, showing high similarity to that of other teleosts. Synthesized Kiss2-10 was able to functionally interact with Kiss1ra in cultured COS-7 cells to trigger downstream signaling. Both kiss2 and kiss1ra mRNAs were expressed in all tissues examined, with highest levels in the olfactory bulb and moderate levels in the hypothalamus among brain areas and highest levels in ovary among peripheral tissues. Intraperitoneal injection of Kiss2-10 significantly increased gnrh1 mRNA levels in hypothalamus and follicle-stimulating hormone beta (fshb) mRNA levels in the pituitary at 6 and 12 h postinjection. During the process of sex reversal induced by 17 alpha-methyltestosterone (MT), kiss2 and kiss1ra mRNA expression were significantly decreased in the first week, but kiss2 increased in the fourth week, in accordance with the expression pattern of gnrh1 mRNA in the grouper hypothalamus. This is the first description of the Kiss2/Kiss1ra system during MT-induced sex reversal in orange-spotted grouper.

The complete mitochondrial genome of the Trachinotus ovatus (Teleostei, Carangidae)

Abstract We present the complete mitochondrial genome of the Trachinotus ovatus in this study. The mitochondrial genome is 16,563 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 T. ovatus mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 29.03% of A, 28.86% of C, 26.23% of T and 15.88% of G. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Two copies of tandem repeat sequence (56 bp) was observed in the 5′ end of the control region.

Molecular cloning and functional characterization of the first non-mammalian 26RFa/QRFP orthologue in Goldfish, Carassius auratus.

In this study, we used data mining approach to predict 26RFa/QRFP precursors from fish, amphibian, reptile and avian species and subsequently cloned a 26RFa/QRFP precursor cDNA from goldfish brain based on the predicted sequences information. The goldfish 26RFa/QRFP precursor cDNA encoded a propeptide of 168 amino acids (aa) with predicted signal peptide of 30 aa at N-terminal and putative mature peptides, including 26RFa (26 aa) and 7RFa (7 aa) located at the C-terminal. Multiple sequence alignment showed almost all of the 26RFa/QRFP mature peptides possessed KGGFXFRF-amide motifs (X=G, S, A or N) at their C-terminus, and the last three residues FRF were fully conserved across vertebrates, indicating that the evolutionary pressure has exerted to conserve several C-terminal amino acid residues among the known and predicted 26RFa/QRFP precursors. Real-time PCR revealed that 26RFa/QRFP gene was expressed abundantly in goldfish hypothalamus, optic tectum-thalamus and testis. The regulation of goldfish hypothalamic 26RFa/QRFP gene expression by negative energy balance and putative role of goldfish 26RFa/QRFP in the control of luteinizing hormone (LH) release were studied. Hypothalamic 26RFa/QRFP gene expression was pronouncedly increased at 4 days after food deprivation. Furthermore, intraperitoneal (IP) injection of synthesized goldfish 26RFa/QRFP at a dose of 1 microg/g bodyweight significantly increased serum LH levels at 1h. However, LH levels were not significantly changed by IP injection of goldfish 26RFa/QRFP at lower dosage or at other time points (3 and 6 h), or by incubation of goldfish primary cell cultures. These results suggested that goldfish 26RFa/QRFP shared some similar features with its mammalian counterparts and partly exerted the regulatory function in energy homeostasis and hypothalamic-pituitary-gonadal (HPG) axis as observed in mammalian species.

Population genetic studies revealed local adaptation in a high gene-flow marine fish, the small yellow croaker (Larimichthys polyactis).

The genetic differentiation of many marine fish species is low. Yet local adaptation may be common in marine fish species as the vast and changing marine environment provides more chances for natural selection. Here, we used anonymous as well as known protein gene linked microsatellites and mitochondrial DNA to detect the population structure of the small yellow croaker (Larimichthys polyactis) in the Northwest Pacific marginal seas. Among these loci, we detected at least two microsatellites, anonymous H16 and HSP27 to be clearly under diversifying selection in outlier tests. Sequence cloning and analysis revealed that H16 was located in the intron of BAHCC1 gene. Landscape genetic analysis showed that H16 mutations were significantly associated with temperature, which further supported the diversifying selection at this locus. These marker types presented different patterns of population structure: (i) mitochondrial DNA phylogeny showed no evidence of genetic divergence and demonstrated only one glacial linage; (ii) population differentiation using putatively neutral microsatellites presented a pattern of high gene flow in the L. polyactis. In addition, several genetic barriers were identified; (iii) the population differentiation pattern revealed by loci under diversifying selection was rather different from that revealed by putatively neutral loci. The results above suggest local adaptation in the small yellow croaker. In summary, population genetic studies based on different marker types disentangle the effects of demographic history, migration, genetic drift and local adaptation on population structure and also provide valuable new insights for the design of management strategies in L. polyactis.

Loss of Genetic Diversity in the Cultured Stocks of the Large Yellow Croaker, Larimichthys crocea , Revealed by Microsatellites

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.

Genetic Diversity and Differentiation of the Orange-Spotted Grouper (Epinephelus coioides) Between and Within Cultured Stocks and Wild Populations Inferred from Microsatellite DNA Analysis

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.

Signatures of selection in tilapia revealed by whole genome resequencing

Natural selection and selective breeding for genetic improvement have left detectable signatures within the genome of a species. Identification of selection signatures is important in evolutionary biology and for detecting genes that facilitate to accelerate genetic improvement. However, selection signatures, including artificial selection and natural selection, have only been identified at the whole genome level in several genetically improved fish species. Tilapia is one of the most important genetically improved fish species in the world. Using next-generation sequencing, we sequenced the genomes of 47 tilapia individuals. We identified a total of 1.43 million high-quality SNPs and found that the LD block sizes ranged from 10–100 kb in tilapia. We detected over a hundred putative selective sweep regions in each line of tilapia. Most selection signatures were located in non-coding regions of the tilapia genome. The Wnt signaling, gonadotropin-releasing hormone receptor and integrin signaling pathways were under positive selection in all improved tilapia lines. Our study provides a genome-wide map of genetic variation and selection footprints in tilapia, which could be important for genetic studies and accelerating genetic improvement of tilapia.

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235bp for gERα, 1967bp for gERβ1 and 2158bp for gERβ2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERβ1 mainly expressed in immature ovaries and gERβ2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERβ1 increased gradually, and gERβ2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.

Ion Torrent next-generation sequencing reveals the complete mitochondrial genome of black and reddish morphs of the Coral Trout Plectropomus leopardus.

Abstract Using Ion Torrent next-generation sequencing (NGS) technology, we sequenced the complete mitochondrial genome (mitogenome) of black and reddish morphs of the coral trout Plectropomus leopardus. High-throughput sequencing generated a total of 958,614 sequence reads covering 164.80 Mb of two mitogenomes with a coverage of 4800X. Thirty-seven mitochondrial genes and gene order of P. leopardus was quite similar to that of other teleostean fishes. Most genes were either abutted or overlapped, and all the protein-coding genes began with an ATG start codon except for COX1 and ATP6. The number of stop codon was different for the black and reddish P. leopardus. Comparisons between the mitochondrial sequences of the two morphs revealed a total of 74 variable sites and one indel. Nucleotide diversity across protein-coding gene varied from 0.0006 (16s rRNA) to 0.0070 (Cytochrome b). As expected, the highest level of nucleotide diversity (0.0291) was detected in the control region. Our results demonstrate the NGS technology based on Ion torrent platform can be used to assemble the mitogenome of fish species.