Molecular cloning of estrogen receptor and its function on vitellogenesis in pompano (Trachinotus ovatus).

Estrogen receptors (ERs) play a critical role in vitellogenesis (Vtgs). However, the contribution of each ER for the regulation of vtgs expression was not analyzed clearly in teleosts. In the present study, three ers isoforms (erα, erß1, and erß2) were cloned in pompano (Trachinotus ovatus). Real-time PCR and enzyme-linked immunosorbent assay (ELISA) was used to detect the effects of 17ß-estradiol (E2) on ERs and Vtgs in the liver of pompano. In vivo injection experiments showed that E2 significantly increased the expressions of ers and vtgs. ER broad spectrum antagonist Fulvestrant significantly attenuated the E2- induced up-regulation of ers and vtgs in a dose-dependent manner. ERα antagonist Methyl-piperidino pyrazole (MPP) significantly attenuated the up-regulation of erα, erß2, vtg-B and vtg-C, and promoted the expressions of erß1 and vtg-A. ERß antagonist Cyclofenil significantly inhibited the expressions of erß1, erß2, vtg-A and vtg-C, and promoted the expressions of erα and vtg-B. In addition, E2 significantly increased the protein level of Vtg, while Fulvestrant, MPP and Cyclofenil significantly inhibited the protein level of Vtg in a dose-dependent manner. Our results indicate that E2 may regulate the expression of each vtg with different subtypes of ERs, and shows a distinct compensatory expression effect on the regulation for ers and vtgs, which provides a theoretical basis for reproductive endocrinology study in pompano.

Identification of Insulin-like Growth Factor (IGF) Family Genes in the Golden Pompano, Trachinotus ovatus: Molecular Cloning, Characterization and Gene Expression.

Insulin-like growth factors (IGFs) are hormones that primarily stimulate and regulate animal physiological processes. In this study, we cloned and identified the open reading frame (ORF) cDNA sequences of IGF family genes: the insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), and insulin-like growth factor 3 (IGF3). We found that IGF1, IGF2, and IGF3 have a total length of 558, 648, and 585 base pairs (bp), which encoded a predicted protein with 185, 215, and 194 amino acids (aa), respectively. Multiple sequences and phylogenetic tree analysis showed that the mature golden pompano IGFs had been conserved and showed high similarities with other teleosts. The tissue distribution experiment showed that IGF1 and IGF2 mRNA levels were highly expressed in the liver of female and male fish. In contrast, IGF3 was highly expressed in the gonads and livers of male and female fish, suggesting a high influence on fish reproduction. The effect of fasting showed that IGF1 and mRNA expression had no significant difference in the liver but significantly decreased after long-term (7 days) fasting in the muscles and started to recover after refeeding. IGF2 mRNA expression showed no significant difference in the liver but had a significant difference in muscles for short-term (2 days) and long-term fasting, which started to recover after refeeding, suggesting muscles are more susceptible to both short-term and long-term fasting. In vitro incubation of 17ß-estradiol (E2) was observed to decrease the IGF1 and IGF3 mRNA expression level in a dose- (0.1, 1, and 10 µM) and time- (3, 6, and 12 h) dependent manner. In addition, E2 had no effect on IGF2 mRNA expression levels in a time- and dose-dependent manner. The effect of 17α-methyltestosterone (MT) in vitro incubation was observed to significantly increase the IGF3 mRNA expression level in a time- and dose-dependent manner. MT had no effect on IGF2 mRNA but was observed to decrease the IGF1 mRNA expression in the liver. Taken together, these data indicate that E2 and MT may either increase or decrease IGF expression in fish; this study provides basic knowledge and understanding of the expression and regulation of IGF family genes in relation to the nutritional status, somatic growth, and reproductive endocrinology of golden pompano for aquaculture development.

Identification and functional characterization of gonadotropin -releasing hormone in pompano (Trachinotus ovatus).

Gonadotropin-releasing hormone (GnRH) is an important neuropeptide in the reproductive system. Although GnRH analogues have been used to artificially spawn pompano (Trachinotus sp.), the native forms of GnRH have not been described in this species. In this study three GnRH subtypes [sea bream GnRH (sbGnRH), chicken GnRH-Ⅱ (cGnRH-Ⅱ) and salmon GnRH (sGnRH)] were identified in pompano (Trachinotus ovatus). cgnrh-Ⅱ and sgnrh were mainly expressed in the brain of male and female fish, showing a tissue-specific expression pattern, while sbgnrh was expressed at different transcriptional levels in all tested tissues. In vivo injection experiment showed that sbGnRH significantly increased fsh and lh genes expression in a dose-dependent manner, but a high concentration of sbGnRH could desensitize the expression of lh. High concentrations of cGnRH-Ⅱ and sGnRH could induce the expression of fsh and lh. In addition, the results of in vitro incubation experiments showed that the high concentration of sbGnRH peptide could induce the expression of fsh and lh, while cGnRH-Ⅱ and sGnRH peptides could only induce the expression of fsh. 17ß-estradiol (E2) and 17α-methyltestosterone (MT) significantly inhibited sbgnrh mRNA expression in a dose-dependent manner, but did not affect the expression of cgnrh-Ⅱ and sgnrh mRNA. sbGnRH is the main GnRH subtype in pompano. E2 and MT can play a negative role in the regulation of sbgnrh. This study provides a theoretical basis for the reproductive endocrinology of pompano.

ddRADseq-assisted construction of a high-density SNP genetic map and QTL fine mapping for growth-related traits in the spotted scat (Scatophagus argus).

BACKGROUND: Scatophagus argus is a popular farmed fish in several countries of Southeast Asia, including China. Although S. argus has a highly promising economic value, a significant lag of breeding research severely obstructs the sustainable development of aquaculture industry. As one of the most important economic traits, growth traits are controlled by multiple gene loci called quantitative trait loci (QTLs). It is urgently needed to launch a marker assisted selection (MAS) breeding program to improve growth and other pivotal traits. Thus a high-density genetic linkage map is necessary for the fine mapping of QTLs associated with target traits. RESULTS: Using restriction site-associated DNA sequencing, 6196 single nucleotide polymorphism (SNP) markers were developed from a full-sib mapping population for genetic map construction. A total of 6193 SNPs were grouped into 24 linkage groups (LGs), and the total length reached 2191.65 cM with an average marker interval of 0.35 cM. Comparative genome mapping revealed 23 one-to-one and 1 one-to-two syntenic relationships between S. argus LGs and Larimichthys crocea chromosomes. Based on the high-quality linkage map, a total of 44 QTLs associated with growth-related traits were identified on 11 LGs. Of which, 19 significant QTLs for body weight were detected on 9 LGs, explaining 8.8-19.6% of phenotypic variances. Within genomic regions flanking the SNP markers in QTL intervals, we predicted 15 candidate genes showing potential relationships with growth, such as Hbp1, Vgll4 and Pim3, which merit further functional exploration. CONCLUSIONS: The first SNP genetic map with a fine resolution of 0.35 cM for S. argus has been developed, which shows a high level of syntenic relationship with L. crocea genomes. This map can provide valuable information for future genetic, genomic and evolutionary studies. The QTLs and SNP markers significantly associated with growth-related traits will act as useful tools in gene mapping, map-based cloning and MAS breeding to speed up the genetic improvement in important traits of S. argus. The interesting candidate genes are promising for further investigations and have the potential to provide deeper insights into growth regulation in the future.

Genome Sequencing and Analysis of Thraustochytriidae sp. SZU445 Provides Novel Insights into the Polyunsaturated Fatty Acid Biosynthesis Pathway.

Thraustochytriidae sp. have broadly gained attention as a prospective resource for the production of omega-3 fatty acids production in significant quantities. In this study, the whole genome of Thraustochytriidae sp. SZU445, which produces high levels of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), was sequenced and subjected to protein annotation. The obtained clean reads (63.55 Mb in total) were assembled into 54 contigs and 25 scaffolds, with maximum and minimum lengths of 400 and 0.0054 Mb, respectively. A total of 3513 genes (24.84%) were identified, which could be classified into six pathways and 44 pathway groups, of which 68 genes (1.93%) were involved in lipid metabolism. In the Gene Ontology database, 22,436 genes were annotated as cellular component (8579 genes, 38.24%), molecular function (5236 genes, 23.34%), and biological process (8621 genes, 38.42%). Four enzymes corresponding to the classic fatty acid synthase (FAS) pathway and three enzymes corresponding to the classic polyketide synthase (PKS) pathway were identified in Thraustochytriidae sp. SZU445. Although PKS pathway-associated dehydratase and isomerase enzymes were not detected in Thraustochytriidae sp. SZU445, a putative DHA- and DPA-specific fatty acid pathway was identified.

Identification, functional characterization, and estrogen regulation on gonadotropin-releasing hormone in the spotted scat, Scatophagus argus.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of the reproductive system. However, little is known about the role of GnRH in the spotted scat (Scatophagus argus). Here, three GnRH subtypes (cGnRH-II, sGnRH, and sbGnRH) were identified in the spotted scat. cGnRH-II and sGnRH were only expressed in the brains and gonads of both male and female fish, exhibiting a tissue-specific expression pattern, while sbGnRH was expressed at different transcription levels in all examined tissues. During ovarian maturation, hypothalamus-associated sbGnRH was upregulated, while the expression of sGnRH was variable and cGnRH-II first increased and then decreased. In vivo experiments showed that sbGnRH significantly promoted the expression of fsh and lh genes in a dose-dependent manner and exhibited a desensitization effect on lh expression at high concentrations. For sGnRH and cGnRH-II, only high concentrations could induce fsh and lh expression. Furthermore, treatment with highly concentrated sbGnRH peptide also induced fsh and lh expression, whereas the sGnRH and cGnRH-II peptides only induced fsh expression in vitro. 17ß-Estradiol (E2) significantly inhibited the expression of sbGnRH mRNA in a dose-dependent manner and did not impact sGnRH and cGnRH-II mRNA levels in vivo or in vitro. The inhibitory effect of E2 on sbGnRH expression was attenuated by the estrogen receptor (ER) broad-spectrum antagonist (fulvestrant) and the ERα-specific antagonist (methyl-piperidinopyrazole), respectively, implying that the feedback regulation on sbGnRH is mediated via ERα. This study provides a theoretical basis for the reproductive endocrinology of the spotted scat by studying GnRH.

Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus).

Scatophagus argus is a new emerging aquaculture fish in East and Southeast Asia. To date, research on reproductive development and regulation in S. argus is lacking. Additionally, genetic and genomic information about reproduction, such as gonadal transcriptome data, is also lacking. Herein, we report the first gonadal transcriptomes of S. argus and identify genes potentially involved in reproduction and gonadal development. A total of 136,561 unigenes were obtained by sequencing of testes (n = 3) and ovaries (n = 3) at stage III. Genes upregulated in males and females known to be involved in gonadal development and gametogenesis were identified, including male-biased dmrt1, amh, gsdf, wt1a, sox9b, and nanos2, and female-biased foxl2, gdf9, bmp15, sox3, zar1, and figla. Serum estradiol-17ß and 11-ketotestosterone levels were biased in female and male fish, respectively. Sexual dimorphism of serum steroid hormone levels were interpreted after expression analysis of 20 steroidogenesis-related genes, including cyp19a1a and cyp11b2. This gonadal transcript dataset will help investigate functional genes related to reproduction in S. argus.

The role of PKG in oocyte maturation of zebrafish.

Recently the importance of cyclic guanosine monophosphate (cGMP) signaling pathway in oocyte maturation has been well demonstrated in several species. However, as the primary downstream effector of the cGMP signaling pathway, little is known on the role of cGMP-dependent protein kinase (PKG) in oocyte maturation. In the present study, the expression, regulation and function of PKG in oocyte maturation was investigated in zebrafish. We identified four distinct PKG coding genes (named Prkg1a, Prkg1b, Prkg2, and Prkg3) in zebrafish. All prkgs are expressed in the ovary, and both prkg1a and prkg1b could be regulated by human chronic gonadotropin in follicular cells during oocyte maturation. We found that a cGMP analogue, 8-Br-cGMP, could stimulate oocyte maturation in a dose- and time-dependent manner. Such stimulatory effects of cGMP could be totally blocked by a PKG specific inhibitor, KT-5823. Intriguingly, we further found KT5823 could significantly attenuate spontaneous oocyte maturation in intact follicles but not in the denuded oocytes, suggesting that the activity of PKG in follicular cells is important for oocyte maturation. All of these results clearly demonstrate that PKG is involved in oocyte maturation in zebrafish.

Neurokinin B signaling in hermaphroditic species, a study of the orange-spotted grouper (Epinephelus coioides).

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhß mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhß expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro. Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17ß-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species.

Molecular cloning, characterization and expression analysis of spexin in spotted scat (Scatophagus argus).

Spexin (Spx), a novel neuropeptide, composed of 14 amino acid residues, is evolutionally conserved from fish to mammals. It has been suggested that Spx has pleiotropic functions in mammals. However, reports about Spx are very limited. To clarify the roles of Spx in the regulation of reproduction and food-intake in the spotted scat, the spx (ssspx) gene was cloned and analyzed. Analysis of the tissue distribution by RT-PCR showed that ssspx expression was widespread. During ovary development, expression of ssspx was found to be highest in phase II, moderate in phase III, and at its lowest level in phase IV. Ssspx expression was significantly down-regulated in the hypothalamus after treatment with E2 both in vitro and in vivo. A significant increase of ssspx was observed after 2 and 7 days of food deprivation. However, the ssspx transcript levels in the 7 day fasting group decreased significantly after refeeding 3 h after the scheduled feeding time. This suggests that ssSpx may be involved in the regulation of reproduction and food-intake in the spotted scat.

Cloning, expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus.

Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erß1 and erß2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erß1 and erß2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erß1 and erß2 were not, with three vtgs in female liver. The effects of 17ß-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erß1, vtgs mRNA and Vtg protein increased significantly after 24h incubation with E2 (0.1, 1 and 10µM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erß1 and vtg-A. Erß selective antagonist Cyclofenil (0.01, 0.1 and 1µM) attenuated the up-regulation effects of E2 on erß1, erß2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.

Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

Phenotypic sorting of individual male and female intersex Cherax quadricarinatus and analysis of molecular differences in the gonadal transcriptome.

Cherax quadricarinatus exhibit sexual dimorphism, with males outpacing females in size specification and growth rate. However, there is limited understanding of the molecular mechanisms underlying sex determination and sex differentiation in crustaceans. To study the differences between intersex individuals and normal individuals, this study counted the proportion of intersex individuals in the natural population, collected the proportion of 7 different phenotypes in 200 intersex individuals, and observed the differences in tissue sections. RNA-seq was used to study the different changes in the transcriptome of normal and intersex gonads. The results showed that: the percentage of intersex in the natural population was 1.5 %, and the percentage of different types of intersex ranged from 0.5 % to 22.5 %; the sections revealed that the development of normal ovaries was stagnant at the primary oocyte stage when intersex individuals with ovaries were present; We screened for pathways and genes that may be associated with gonadal development and sex, including ovarian steroid synthesis, estrogen signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, etc. Relevant genes including tra2a, dmrta2, ccnb2, foxl2, and smad4. This study provides an important molecular basis for sex determination, sex-controlled breeding, and unisex breeding in red crayfish.

Molecular cloning, characterization and expression profiles of multiple leptin genes and a leptin receptor gene in orange-spotted grouper (Epinephelus coioides).

Leptin plays key roles in body weight regulation, energy metabolism, food intake, reproduction and immunity in mammals. However, its function in teleosts is still unclear. In the present study, two leptin genes (gLepA and gLepB) and one leptin receptor gene (gLepR) were cloned and characterized in orange-spotted grouper (Epinephelus coioides). The cDNAs of gLepA and gLepB were 671 bp and 684 bp in length, encoding for proteins of 161 amino acid (aa) and 158 aa, respectively. The three-dimensional (3D) structures modeling of gLepA and gLepB showed strong conservation of tertiary structure with that of other vertebrates. The total length of gLepR cDNA was 4242 bp, encoding a protein of 1169 aa which contained all functionally important domains conserved among vertebrate LEPR. Tissue distribution analysis showed that gLepA was highly expressed in cerebellum, liver and ovary, while gLepB mRNA abundantly in the brain regions, as well as in the ovary with some extend. The gLepR was mainly expressed in kidney, head kidney and most of brain regions. Analysis of expression profiles of gLep and gLepR genes during the embryonic stages showed that high expression of gLepR was observed in the brain vesicle stage, while neither gLepA nor gLepB mRNA was detected during different embryonic stages. Finally, fasting and refeeding experiments were carried out to investigate the possible function of leptin genes in food intake and energy metabolism, and the results showed that a significant increase of gLepA expression in the liver was induced by food deprivation in both short-term (7 days) and long-term (3 weeks) fasting and gLepA mRNA upregulation was eliminated after refeeding, while gLepB wasn't detected in the liver of grouper during fasting. No significant differences in hypothalamic leptin and leptin receptor expression were found during short-term fasting and refeeding. Hepatic expression of gLepA mRNA increased significantly 9h after a single meal. These results suggested gLepA, other than gLepB, functioned in the regulation of energy metabolism and food intake in this Perciform fish.

Comparative Transcriptome Analysis Reveals the Effect of Aurantiochytrium sp. on Gonadal Development in Zebrafish.

Aurantiochytrium sp. has received much attention as a potential resource for mass production of omega-3 fatty acids, which contribute to improved growth and reproduction in aquatic animals. In this study, we evaluated the gonadal index changes in zebrafish supplemented with 1-3% Aurantiochytrium sp. crude extract (TE) and the effects of ex vivo environmental Aurantiochytrium sp. on oocytes. 1% TE group showed significant improvement in the gonadal index, and both in vitro incubation and intraperitoneal injection promoted the maturation of zebrafish oocytes. In contrast, the transcriptome revealed 576 genes that were differentially expressed between the 1% TE group and the control group, including 456 up-regulated genes and 120 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analysis of differentially expressed genes indicated that Aurantiochytrium sp. potentially affects pathways such as lipid metabolism, immune regulation, and oocyte development in zebrafish. The results of this study enriched the knowledge of Aurantiochytrium sp. in regulating gonadal development in zebrafish and provided a theoretical basis for its application in aquaculture.

Structural and functional characterization of neuropeptide Y in a primitive teleost, the Japanese eel (Anguilla japonica).

In the present study, the first full-length cDNA encoding Neuropeptide Y (NPY) was cloned from the brain of Japanese eel (Anguilla japonica). The open reading frame of Japanese eel NPY gene is 294 bp in length, encoding a precursor protein of 97 amino acids, which contains a 36-amino-acid mature peptide. Sequence analysis showed that the Japanese eel NPY peptide is similar to that of other species. Real-time PCR revealed that NPY in Japanese eel is mainly expressed in the brain, especially in the hypothalamus and the optic tectum thalamus. The effect of a negative energy balance on NPY gene expression was examined subsequently. The mRNA level of NPY in the hypothalamus and the optic tectum thalamus showed a pronounced increase after 4 days of food deprivation. The biological activities of Japanese eel NPY were further investigated in vivo and in vitro. Intraperitoneal injection of the NPY peptide into Japanese eel could potently elevate the expression of the mammalian gonadotropin-releasing hormone (mGnRH) in hypothalamus and the follicle-stimulating hormone beta (FSHß), the luteinizing hormone beta (LHß) and growth hormone (GH) in pituitary. In static incubation studies, the stimulatory effects of NPY on mGnRH expression in hypothalamic fragments and on FSHß, LHß and GH expression in pituitary cells were also observed. However, in vivo and in vitro studies showed that NPY exhibits an inhibitory action on the expression of thyroid-stimulating hormone beta (TSHß) in pituitary. The results indicate that NPY is involved in the regulation of multiple physiological processes in Japanese eel.

Physiological Effects and Transcriptomic Analysis of sbGnRH on the Liver in Pompano (Trachinotus ovatus).

Pompano (Trachinotus ovatus) is one of the important economic marine fishes in the south coast of China. At present, the research on the basic biology of pompano is relatively weak, which has seriously affected the development of this economic important fish. The liver is an important digestive and metabolic organ of fish which plays an important regulatory role in its growth and development. It is necessary to clarify the effects of sea bream gonadotropin releasing hormone (sbGnRH) on liver physiology and metabolic enzyme activity. The effects of sbGnRH peptides (10 ng/gbw) on the physiological and biochemical indices and metabolic enzyme activities of pompano liver were studied. It was found that after injection of 10 ng/gbw sbGnRH peptides, the contents of albumin, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, creatine kinase, iron, magnesium, aspartate aminotransferase, alanine aminotransferase and creatinine increased, while of cholesterol and calcium contents decreased. The activities of amylase, lipase, pyruvate kinase, acyl CoA oxidase, superoxide dismutase, phospholipid hydroperoxide glutathione peroxidase, catalase, glucose-6-phosphate dehydrogenase, fatty acid synthase and lipoprotein lipase increased, while the activities of malic enzyme, carnitine acyl, carnitine translocation, acetyl CoA carboxylase and malondialdehyde decreased. Three hours after the injection of different concentrations of sbGnRH peptides (0 and 10 ng/gbw), the transcriptome sequences of the two groups of livers were sequenced. After quality control and removal of some low-quality data, clean reads of 21,283,647、19,427,359、21,873,990、21,732,174、23,660,062 and 21,592,338 were obtained respectively. In this study, 99 genes were screened and identified as differentially expressed genes, including 77 up-regulated genes and 22 down-regulated genes. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses, these pathways and the typical genes involved can be divided into cellular processes, environmental information processing, genetic information processing, diseases, metabolism and organismal systems. The results from this study provide a the oretical basis for studying the effects of sbGnRH on the physiology, biochemistry and metabolic enzyme activities of liver in pompano.

Effects of Dietary Supplementation with Aurantiochytrium sp. on Zebrafish Growth as Determined by Transcriptomics.

The marine protist Aurantiochytrium produces several bioactive chemicals, including EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid), and other critical fish fatty acids. It has the potential to improve growth and fatty acid profiles in aquatic taxa. This study evaluated zebrafish growth performance in response to diets containing 1% to 3% Aurantiochytrium sp. crude extract (TE) and single extract for 56 days. Growth performance was best in the 1% TE group, and therefore, this concentration was used for further analyses of the influence of Aurantiochytrium sp. Levels of hepatic lipase, glucose-6-phosphate dehydrogenase, acetyl-CoA oxidase, glutathione peroxidase, and superoxide dismutase increased significantly in response to 1% TE, while malic enzyme activity, carnitine lipid acylase, acetyl-CoA carboxylase, fatty acid synthase, and malondialdehyde levels decreased. These findings suggest that Aurantiochytrium sp. extract can modulate lipase activity, improve lipid synthesis, and decrease oxidative damage caused by lipid peroxidation. Transcriptome analysis revealed 310 genes that were differentially expressed between the 1% TE group and the control group, including 185 up-regulated genes and 125 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses of the differentially expressed genes revealed that Aurantiochytrium sp. extracts may influence liver metabolism, cell proliferation, motility, and signal transduction in zebrafish.

Analysis of circRNA and miRNA expression profiles in IGF3-induced ovarian maturation in spotted scat (Scatophagus argus).

Insulin-like growth factor 3 (IGF3) induces ovarian maturation in teleosts; however, research on its molecular regulatory mechanism remains deficient. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in various biological processes, including reproduction. In this study, circRNAs and miRNAs involved in IGF3-induced ovarian maturation were evaluated in spotted scat (Scatophagus argus). In ovarian tissues, we identified 176 differentially expressed (DE) circRNAs and 52 DE miRNAs between IGF3 treatment and control groups. Gene Ontology (GO) enrichment analyses showed that host genes of DE circRNAs and target genes of DE miRNAs were enriched for various processes with a high degree of overlap, including cellular process, reproduction, reproductive process, biological adhesion, growth, extracellular region, cell junction, catalytic activity, and transcription factor activity. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included cell adhesion molecules, ECM-receptor interaction, regulation of actin cytoskeleton, focal adhesion, cell cycle, Hedgehog signaling pathway, phosphatidylinositol signaling system, PI3K-Akt signaling pathway, Apelin signaling pathway, Notch signaling pathway, insulin signaling pathway, and Rap1 signaling pathway. A circRNA-miRNA-mRNA regulatory network was constructed, including DE genes involved in reproduction (e.g., oocyte maturation, oocyte meiosis, and ECM remodeling), such as ccnd2, hecw2, dnm2, irs1, adam12, and cdh13. According to the regulatory network and tissue distribution, we identified one circRNA (Lachesis_group5:6245955|6270787) and three miRNAs (novel_miR_622, novel_miR_980, and novel_miR_64) that may exert regulatory effects in IGF3-induced ovarian maturation in S. argus. Taken together, this study provides a novel insight into the molecular mechanisms by which IGF3 functions in ovaries and highlights the effects of circRNAs and miRNAs in reproduction in S. argus.