Mating behaviors in ovoviviparous black rockfish (Sebastes schlegelii): molecular function of prostaglandin E2 as both a hormone and pheromone.

Prostaglandins (PGs) are profound hormones in teleost sexual behavior, especially in mating. PGs act as pheromones that affect the olfactory sensory neurons of males, inducing the initiation of a series of mating behaviors. However, the molecular mechanism by which PGs trigger mating behavior in ovoviviparous teleosts is still unclear. In the present study, we employed the ovoviviparous black rockfish (Sebastes schlegelii), an economically important marine species whose reproductive production is limited by incomplete fertilization, as a model species. The results showed that when the dose of PGE2 was higher than 10 nmol/L, a significant (P < 0.05) increase in mating behaviors was observed. Dual-fluorescence in situ hybridization indicated that PGE2 could fire specific neurons in different brain regions and receptor cells in the olfactory sac. After combining with specific neurons in the central nervous system (CNS), a series of genes related to reproduction are activated. The intracerebroventricular administration of PGE2 significantly increased lhb levels (P < 0.05) in both sexes. Moreover, steroidogenesis in gonads was also affected, inducing an increase (P < 0.05) in E2 levels in males and T levels in females. PGE2 levels were also increased significantly (P < 0.05) in both sexes. The present study revealed that PGE2 can activate mating behavior in black rockfish in both hormone and pheromone pathways, leading to variations in sex steroid levels and activation of reproductive behaviors. Our results provide not only novel insight into the onset of mating behaviors in ovoviviparous teleosts but also solutions for the incomplete fertilization caused by natural mating in cage aquaculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00214-w.

PGE2 functions in ovoviviparous teleost black rockfish (Sebastes schlegelii): evolutionary status between parturition and ovulation†.

Fish have evolved various reproductive strategies including oviparity, viviparity, and ovoviviparity, which undoubtedly affect the survival of the whole species continuity. As the final step in reproduction, parturition in viviparous vertebrate and ovulation in oviparous teleost seem to share a similar mechanism, when prostaglandins (PGs) act as the trigger to launch the whole process. In the present study, ovoviviparous teleost black rockfish (Sebastes schlegelii) is employed as the research object. Intraperitoneal injection showed that PGE2 (500 µg/kg) could activate the delivery reactions in perinatal black rockfish. RNA-seq data of ovary in perinatal period revealed transcriptional change in cell junction, inflammation, and apoptosis, which is related to mammal parturition and teleost ovulation. Further results proved the positive correlation between ptger EP2 and previous mentioned pathways. Subsequent experiment proved that PGE2 was able to induce the ovulation and spawning in unfertilized individuals, which had a bilayer follicular structure compared to monolayer follicular in perinatal period black rockfish. Both unfertilized and perinatal ovary matrix could response to PGE2 stimulation. In conclusion, the function of PGE2 in activating both parturition and ovulation in a relatively different pathways conserved with viviparity or oviparity provided novel evidence of the evolutionary status of ovoviviparous vertebrates.

FGFs function in regulating myoblasts differentiation in spotted sea bass (Lateolabrax maculatus).

Fibroblast growth factors (FGFs) are a family of structurally related peptides that regulate processes such as cell proliferation, differentiation, and damage repair. In our previous study, fibroblast growth factor receptor 4 (fgfr4) was detected in the most significant quantitative trait loci (QTL), when identified of QTLs and genetic markers for growth-related traits in spotted sea bass. However, knowledge of the function of fgfr4 is lacking, even the legends to activate the receptor is unknown in fish. To remedy this problem, in the present study, a total of 33 fgfs were identified from the genomic and transcriptomic databases of spotted sea bass, of which 10 were expressed in the myoblasts. According to the expression pattern during myoblasts proliferation and differentiation, fgf6a, fgf6b and fgf18 were selected for further prokaryotic expression and purification. The recombinant proteins FGF6a, FGF6b and FGF18 were found to inhibit myoblast differentiation. Overall, our results provide a theoretical basis for the molecular mechanisms of growth regulation in economic fish such as spotted sea bass.

Pro-inflammatory cytokine IL1ß1 participates in promoting parturition related pathways in the ovoviviparous teleost black rockfish (Sebastes schlegelii).

Along with the evolution process, the reproductive strategies evolved including oviparity, viviparity and ovoviviparity, to fit the residential environment maximize the survival rate of the off spring. In mammals, the key to the initiation of parturition is the inflammatory response at the maternal-fetal interface. As a pro-inflammatory cytokine, interleukin 1 beta (IL1ß) plays an important role in the process of human parturition. While less is known about IL1ß1 in teleost parturition, identification of the functions of IL1ß1 in inducing the parturition, black rockfish, an ovoviviparity teleost, which provides over 60% nutrition supply for over 50 000 embryos though a placenta like structure during pregnant, was employed as the research model. In the present study, based on the gene cloning, we detected the expression pattern of both Il1b1 and its receptor perinatal period, as well as the localization to the ovary by in situ hybridization. The different expression genes in transcriptomic data of perinatal primary ovarian cells treated with the recombinant IL1ß1 (rIL1ß1) obtained by prokaryotic expression system were analyzed. Differentially expressed genes, functional enrichment and pathway analysis mainly included immune response, signal transduction and cell death. In summary, our research provides novel insights into the potential role of IL1ß1 in the parturition of ovoviviparity teleost.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors.

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Comparative transcriptomic analysis and genome-wide characterization of the Semaphorin family reveal the potential mechanism of angiogenesis around embryo in ovoviviparous black rockfish (Sebastes schlegelii).

To guarantee the quality and survival rate of their offspring, ovoviviparous teleost evolved special characteristics of in vivo fertilization and embryo development. Maternal black rockfish, having over 50 thousand embryos developing within the ovary simultaneously, provided around 40% nutrition throughout oocyte development, while the capillaries around each embryo contributed the rest 60% during pregnancy. Since fertilization, capillaries started to proliferate and developed into a placenta-like structure that covered over half of each embryo. Aimed to characterize the potential mechanism behind, comparative transcriptome analysis of samples collected according to the process of pregnancy. Three important time point in the process, including mature oocyte stage, fertilization and sarcomere period, were chosen for the transcriptome sequencing. Our study identified key pathways and genes involved in the cell cycle as well as DNA replication and repair, cell migration and adhesion, immune, and metabolic functions. Notably, several of the semaphoring gene family members were differently expressed. To confirm the accuracy of these genes, total of 32 sema genes were identified from the whole genome and distinct expression pattern of sema genes was observed in different pregnant stages. Our results revealed a novel insight for further investigating the functions of sema genes in reproduction physiology and embryo processes in ovoviviparous teleost.

TAC3/TACR3 System Function in the Catadromous Migration Teleost, Anguilla japonica.

Neurokinin B (NKB), a member of the tachykinin (TAC) family, plays important roles in mammalian neuropeptide secretion in related to reproduction. However, its potential role in spawning migration teleost is less clear. In the present study, Japanese eel (Anguilla japonica) was employed to study the performance of NKB in regulating reproduction. Results showed that two tac3 and one tacr3 genes were identified in Japanese eel. Sequence analysis showed that two tac3 transcripts, tac3a and tac3b, encode four NKBs: NKBa-13, NKBa-10, NKBb-13, and NKBb-10. However, compared with other species, a mutation caused early termination of TACR3 protein was confirmed, leading to the loss of the 35 amino acid (aa) C-terminal of the receptor. Expression analysis in different tissues showed that both tac3a and tac3b mRNAs were highly expressed in the brain. In situ hybridization localized both tac3a and tac3b mRNAs to several brain regions, mainly in the telencephalon and hypothalamus. Because of the mutation in TACR3 of Japanese eel, we further analyzed whether it could activate the downstream signaling pathway. Luciferase assay results showed the negative regulation of cAMP Response Element (CRE) and Sterol Response Element (SRE) signal pathways by Japanese eel NKBs. Intraperitoneal injection of four different NKB mature peptides at 100 ng/g had negative effect on either gnrh or gth gene expression. However, the high concentration of NKBa-10 and NKBb-13 (1,000 ng/g) upregulated mgnrh and fshb or lhb expression level significantly, which may be mediated by other receptors. In general, the NKBs/NK3Rs system has important functions in regulating eel puberty onset.

Neurosecretory protein GL in GIFT tilapia (Oreochromis niloticus): cDNA cloning, tissue distribution and effects of feeding on its expression.

Neurosecretory protein GL (NPGL), a novel neuropeptide, has been identified in the hypothalamus of chicks and rodents. NPGL plays a crucial role in monitoring energetic status via the regulation of feeding and metabolism. However, no study on NPGL has been reported in fish thus far. In the present study, the full-length cDNA of NPGL was identified from the hypothalamus of GIFT tilapia (Oreochromis niloticus). The ORF of tilapia NPGL is 471 bp and encodes a precursor peptide with a size of 156 a.a, consisting of a 26 a.a signal peptide and an 82 a.a mature peptide. Tissue distribution profiles of npgl in tilapia were acquired using semiquantitative PCR and in situ hybridization (ISH). The results showed that the highest npgl mRNA is expressed in the telencephalic-preoptic complex, which comprises both the telencephalon and the anterior preoptic area (POA) of male tilapia, and in the ovary of female tilapia. In addition, in male tilapia, the ISH results showed that the cells containing npgl mRNA were distributed exclusively in the anterior periventricular pretectal nucleus (Ppa) of the POA. FISH results demonstrated that npgl mRNA is also expressed in the lateral tuberal nucleus of the hypothalamus (NLT). Real-time PCR showed that npgl mRNA significantly increased in the telencephalic-preoptic complex of male tilapia that were fasted for 24 h and then fed a full diet for 20 min compared with the unfed group. Results of the FISH study showed that parvocellular cells containing npgl mRNA in the Ppa of fed fish were apparently more abundant than those of the unfed group. Few npgl positive signals also appeared in the NLT after full feeding, where pomc mRNA is highly expressed. These results indicate that NPGL may be a short-term satiety factor in fish and that the coexpression of NPGL and POMC may be present in the hypothalamus of male tilapia.

Molecular characterization and expression patterns of glucocorticoid receptors in the viviparous black rockfish Sebastes schlegelii.

Glucocorticoid receptors (GRs) are ligand-activated transcription factors associated with anti-inflammation, stress, metabolism and gonadal development. In this study, two gr genes (gr1 and gr2) were cloned and analyzed from a viviparous teleost, black rockfish (Sebastes schlegelii). The phylogenetic analysis of GRs showed that GR1 and GR2 clustered into teleost GR1 and GR2 separately and differed from the GRs of tetrapods or basal ray-finned fishes. Black rockfish GRs possess four modular domains of the nuclear receptor superfamily: an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR) and a ligand-binding domain (LBD). Nine conserved amino acid inserts were found in the GR1 DBD, and the ligand cavity-related amino acids of GR1 and GR2 LBD were slightly different. Tissue distribution analysis revealed that grs was widely expressed in various tissues, while cyp11b was mainly expressed in the testis and head kidney. The cyp11b transcripts were localized in the interrenal glands of the head kidney, the main source of cortisol; grs transcripts were detected in oocytes, the follicle layer and the ovarian wall. Histologically, significant blood vessel dilation was observed in the fetal membrane during or after parturition of black rockfish. The highest levels of serum cortisol and ovarian cyp11b mRNA were detected in parturition. In addition, the relative expression level of gr1 was upregulated significantly after delivery, while the levels of gr2 showed no significant change. In addition, in vitro GC treatment inhibited the expression of il1b but significantly upregulated the transcription of il1r1. These data provide evidence that GRs are likely to work as anti-inflammatory factors by inhibiting the functions of pro-inflammatory factors in the parturition of black rockfish.

Cyclooxygenases of ovoviviparous black rockfish (Sebastes schlegelii): Cloning, tissue distribution and potential role in mating and parturition.

Prostaglandins are a series of unsaturated fatty acids that play critical roles in regulating reproductive events. The prostaglandins endoperoxide H synthases-1/2 (PGHS-1/2; also named cyclooxygenases-1/2, COX-1/2) catalyse the commitment step in prostaglandin synthesis. However, the of the cox genes in teleosts, especially ovoviviparous teleosts, is still unclear. The aim of the present study was to determine the potential role of cox genes in mating and parturition behaviour using black rockfish (Sebastes schlegelii) as a model species. Two transcripts, cox1 and cox2, were cloned. The phylogenetic analysis results revealed that both cox genes were closely related to mammalian coxs. qPCR analyses of their tissue distribution showed that cox1 was mainly expressed in the heart in both sexes, while cox2 was mainly expressed in the testis and ovary. Detection of cox expression in samples from reproductive-related stages further showed that both cox genes may play important roles in mating and parturition processes. In situ hybridization further detected positive cox mRNA signals in the testis and ovary, where they are known to be involved in mating and parturition behaviour. These data suggest that cox1 and cox2 are crucial in inducing mating, gonad regeneration and parturition behaviour.

Comparative transcriptomic analysis of gonadal development and renewal in the ovoviviparous black rockfish (Sebastes schlegelii).

BACKGROUND: The black rockfish (Sebastes schlegelii) has an ovoviviparous reproductive pattern and long-term sperm storage, resulting in asynchronous gonadal development between the sexes. However, the comprehensive understanding of gonadal development in black rockfish has not yet been achieved. Here, we studied gonadal development and germ cell renewal using histology and RNA-seq. RESULTS: In this study, RNA-seq was performed on testes and ovaries to characterize key pathways and genes that are active during development and gamete maturation in black rockfish. Differentially expressed genes (DEGs) were identified and annotated in 4 comparisons (F_III vs. F_IV, F_IV vs. F_V, M_III vs. M_IV and M_IV vs. M_V). Based on analysis of DEGs enriched in the testis, 11 and 14 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mapped to the M_III vs. M_IV group and the M_IV vs. M_V group, respectively. DEGs in ovarian development were also classified into 10 groups according to their biological functions. The expression patterns of the selected genes determined by qPCR were significantly correlated with the RNA-Seq results, supporting the reliability and accuracy of the RNA-Seq analysis. E2 levels showed down regulation from previtellogenesis to mature stage in female and T level showed down regulation from spermatogenesis to regressed stage in the male. CONCLUSIONS: The categories "intercellular interaction and cytoskeleton", "molecule amplification" and "repair in the cell cycle" were revealed to be crucial in testis development and spermatogenesis, as was the biosynthesis of a series of metabolites. Our results provide comprehensive insight into black rockfish gonadal development and provide a basis for further study of reproductive physiology and molecular biology in ovoviviparity teleosts.

Brain and Pituitary Transcriptome Analyses Reveal the Differential Regulation of Reproduction-Related LncRNAs and mRNAs in Cynoglossus semilaevis.

Long noncoding RNAs (lncRNAs) have been identified to be involved in half-smooth tongue sole (Cynoglossus semilaevis) reproduction. However, studies of their roles in reproduction have focused mainly on the ovary, and their expression patterns and potential roles in the brain and pituitary are unclear. Thus, to explore the mRNAs and lncRNAs that are closely associated with reproduction in the brain and pituitary, we collected tongue sole brain and pituitary tissues at three stages for RNA sequencing (RNA-seq), the 5,135 and 5,630 differentially expressed (DE) mRNAs and 378 and 532 DE lncRNAs were identified in the brain and pituitary, respectively. The RNA-seq results were verified by RT-qPCR. Moreover, enrichment analyses were performed to analyze the functions of DE mRNAs and lncRNAs. Interestingly, their involvement in pathways related to metabolism, signal transduction and endocrine signaling was revealed. LncRNA-target gene interaction networks were constructed based on antisense, cis and trans regulatory mechanisms. Moreover, we constructed competing endogenous RNA (ceRNA) networks. In summary, this study provides mRNA and lncRNA expression profiles in the brain and pituitary to understand the molecular mechanisms regulating tongue sole reproduction.

Integrated lncRNA and mRNA Transcriptome Analyses in the Ovary of Cynoglossus semilaevis Reveal Genes and Pathways Potentially Involved in Reproduction.

Long non-coding RNAs (lncRNAs) have been reported to be involved in multiple biological processes. However, the roles of lncRNAs in the reproduction of half-smooth tongue sole (Cynoglossus semilaevis) are unclear, especially in the molecular regulatory mechanism driving ovarian development and ovulation. Thus, to explore the mRNA and lncRNA mechanisms regulating reproduction, we collected tongue sole ovaries in three stages for RNA sequencing. In stage IV vs. V, we identified 312 differentially expressed (DE) mRNAs and 58 DE lncRNAs. In stage V vs. VI, we identified 1,059 DE mRNAs and 187 DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DE mRNAs were enriched in ECM-receptor interaction, oocyte meiosis and steroid hormone biosynthesis pathways. Furthermore, we carried out gene set enrichment analysis (GSEA) to identify potential reproduction related-pathways additionally, such as fatty metabolism and retinol metabolism. Based on enrichment analysis, DE mRNAs with a potential role in reproduction were selected and classified into six categories, including signal transduction, cell growth and death, immune response, metabolism, transport and catabolism, and cell junction. The interactions of DE lncRNAs and mRNAs were predicted according to antisense, cis-, and trans-regulatory mechanisms. We constructed a competing endogenous RNA (ceRNA) network. Several lncRNAs were predicted to regulate genes related to reproduction including cyp17a1, cyp19a1, mmp14, pgr, and hsd17b1. The functional enrichment analysis of these target genes of lncRNAs revealed that they were involved in several signaling pathways, such as the TGF-beta, Wnt signaling, and MAPK signaling pathways and reproduction related-pathways such as the progesterone-mediated oocyte maturation, oocyte meiosis, and GnRH signaling pathway. RT-qPCR analysis showed that two lncRNAs (XR_522278.2 and XR_522171.2) were mainly expressed in the ovary. Dual-fluorescence in situ hybridization experiments showed that both XR_522278.2 and XR_522171.2 colocalized with their target genes cyp17a1 and cyp19a1, respectively, in the follicular cell layer. The results further demonstrated that lncRNAs might be involved in the biological processes by modulating gene expression. Taken together, this study provides lncRNA profiles in the ovary of tongue sole and further insight into the role of lncRNA involvement in regulating reproduction in tongue sole.

Gonadal lipidomics profile of an ovoviviparity teleost, black rockfish, during gonadal development.

In order to study the variation of gonad lipidomics during reproductive cycle, black rockfish was employed as the research model in the present study. Using histology, lipidomics, and qPCR, the profile of gonad lipidomics and the expression levels of related genes during different developmental stages were detected and analyzed to show the potential regulatory network of lipid metabolism. Based on Ultra High-Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometry (UHPLC-QTOFMS), four significant differential glycerophospholipid metabolic pathways including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA) were enriched by KEGG. Pathway-related enzyme-coding genes, including phosphatidylserine decarboxylase (pisd), phosphatidylserine synthase (ptdss1, ptdss2), and phospholipase D (pld1, pld2) were identified from the whole genome data and confirmed by cloning. The expression profiles of these genes were tested by qPCR in the tissues and gonads in developmental stages, and we found that pisd, pld, and ptdss genes were all downregulated through the developmental process in the brain of male, and the latter two genes were upregulated in the liver and testis at stage IV, which were the opposite trend observed in the female. Thus, our findings would be helpful in further understanding the substance metabolism and regulation during gonad development in ovoviviparity teleosts.

Characterization of CYP11A1 and its potential role in sex asynchronous gonadal development of viviparous black rockfish Sebastes schlegelii (Sebastidae).

Mitochondrial cytochrome P450 side-chain cleavage (P450scc), encoded by the cyp11a1 gene, initiates the first step of steroid biosynthesis. In this study, a 1554-bp open reading frame (ORF) of black rockfish (Sebastes schlegelii) cyp11a1 was cloned. The cyp11a1 gene is located on chromosome 5 and has 9 exons. The ORF encodes a putative precursor protein of 517 amino acids, and the predicted cleavable mitochondrial targeting peptide is located at amino acids 1-39. P450scc shares homology with other teleosts and tetrapods, which have relatively conserved binding regions with heme, cholesterol and adrenodoxin. Tissue distribution analysis revealed that the highest expression levels of cyp11a1 were detected in mature gonads and head kidney but that low levels were detected in gestational/regressed ovaries, regressed testes and other tissues. Immunostaining of P450scc was observed in testicular Leydig cells, ovarian theca cells, interrenal glands of head kidney, pituitary and multiple regions of brain. Particularly, two kinds of fish-specific P450scc-positive cells, including coronet cells of brain saccus vasculosus and hypophyseal somatolactin cells, were identified in black rockfish. Our results provide novel evidence for the potential role played by P450scc in reproduction behavior by mediating steroidogenesis in viviparous teleost.

Melanocortin-4 receptor regulation of reproductive function in black rockfish (Sebastes schlegelii).

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor with multiple functions in mammals. However, the functions of MC4R in fish have not been investigated extensively. The purpose of this study was to determine potential regulation of reproduction by the MC4R. We cloned the black rockfish MC4R and analyzed its tissue distribution and function. The results showed that black rockfish mc4r cDNA consisted of 981 nucleotides encoding a protein of 326 amino acids. The quantitative PCR data showed that mc4r mRNA was primarily expressed in the brain, gonad, stomach and intestine. In the brain, mc4r was found to be primarily located in the hypothalamus. Both α-MSH and ß-MSH increased gnih expression and decreased sgnrh and cgnrh expression (P < 0.05). α-MSH and ß-MSH had opposite effects on kisspeptin expression. In contrast, α-MSH and ß-MSH increased the expression of cyp11, cyp19, 3ß-hsd and star. In summary, our study shows that MC4R in black rockfish might regulate reproductive function and that the effects of α-MSH and ß-MSH might differ.

TAC3 Gene Products Regulate Brain and Digestive System Gene Expression in the Spotted Sea Bass (Lateolabrax maculatus).

Neurokinin B (NKB) is a member of the tachykinin (tac) family that plays important roles in mammalian growth by modulating prolactin (PRL) synthesis and secretion and causing contraction of the stomach and intestine. However, its potential role in regulating growth of teleosts is less clear. We aimed to explore the role that NKB plays in regulating fish growth using the spotted sea bass (Lateolabrax maculatus) as a model. In the present study, two tac3 and two tacr3 genes were identified in the spotted sea bass. Sequence analysis showed that two tac3 transcripts, tac3a and tac3b, encode four NKBs: NKBa-13, NKBa-10, NKBb-13, and NKBb-10. Expression analysis in different tissues showed that both genes are highly expressed in the brain, stomach and intestine of the spotted sea bass. In situ hybridization indicated that the tac3a and tac3b mRNAs are both localized in several brain regions, such as the telencephalon and hypothalamus, and that tacr3a and tacr3b are localized in the intestinal villus and gastric gland. To investigate the potential role of NKBs in regulating growth, in vitro experiments were performed to detect the effect of NKBs on growth-related gene expression in the brain and brain-gut peptide (BGP)-related genes in the stomach and intestine. NKBb-13 was the most critical ligand in regulating the expression of growth-related genes in the brain and brain-gut peptide (BGP)-related genes in the stomach. The expression of cholecystokinin (cck) was enhanced by NKBa-13, NKBa-10, and NKBb-10 but not NKBb-13 in the intestine. In general, our results showed that NKBs participate in regulating the growth of spotted sea bass.

Deep Transcriptomic Analysis of Black Rockfish (Sebastes schlegelii) Provides New Insights on Responses to Acute Temperature Stress.

In the present study, we conducted an RNA-Seq analysis to characterize the genes and pathways involved in acute thermal and cold stress responses in the liver of black rockfish, a viviparous teleost that has the ability to cope with a wide range of temperature changes. A total of 584 annotated differentially expressed genes (DEGs) were identified in all three comparisons (HT vs NT, HT vs LT and LT vs NT). Based on an enrichment analysis, DEGs with a potential role in stress accommodation were classified into several categories, including protein folding, metabolism, immune response, signal transduction, molecule transport, membrane, and cell proliferation/apoptosis. Considering that thermal stress has a greater effect than cold stress in black rockfish, 24 shared DEGs in the intersection of the HT vs LT and HT vs NT groups were enriched in 2 oxidation-related gene ontology (GO) terms. Nine important heat-stress-reducing pathways were significantly identified and classified into 3 classes: immune and infectious diseases, organismal immune system and endocrine system. Eight DEGs (early growth response protein 1, bile salt export pump, abcb11, hsp70a, rtp3, 1,25-dihydroxyvitamin d(3) 24-hydroxylase, apoa4, transcription factor jun-b-like and an uncharacterized gene) were observed among all three comparisons, strongly implying their potentially important roles in temperature stress responses.