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The study of the coupling coordinated relationship between urbanization and the eco-environment is important to promote regional high-quality development. This paper measured the coupling coordination degree between urbanization and the eco-environment from 2008 to 2020, using the coupling coordination model, depicted its spatiotemporal characteristics with ArcGIS, and explored its driving factors and impacts of the interaction between pairwise factors on it with geo-detection methods. The results show that the coupling coordination level becomes increasingly improved, but there exist widening spatial disparities, and in 2020, its spatial distribution presents characteristics of “decreasing from north to south” and “increasing from west to east”. According to their evolutionary characteristics of coupling coordination categories, cities can be classified into four types: “transition from basic misalignment to primary coordination”, “transition from primary coordination to intermediate coordination”, “transition from primary coordination to senior coordination”, “no hierarchical transition”. Currently, driving factors of industrial structure, opening to the outside world and technological innovation significantly affect the coupling coordination degree, and the interaction between pairwise factors is enhanced. Policies should be implemented in categories to improve the coupling coordination level. Regional cooperation, exchanges, and interconnection of resources and factors should be strengthened to jointly build a coordinated regional development pattern.

In fish and other vertebrates, growth hormone (GH) is an essential polypeptide required for normal growth and development. In an attempt to understand growth regulation in yellowtail kingfish (YTK), the full-length cDNA sequences encoding gh and its receptors (ghr1 and ghr2) were cloned, characterized and the expression profiles of these three genes were investigated during embryonic development. The full-length cDNA sequences of GH and its receptors were obtained by RT-PCR combined with RACE methord. YTK gh cDNA sequence was 852 base pairs (bp) that comprised an open reading frame (ORF) of 615 bp encoding a 204-amino acids (aa) precursor. The preprohormone compassed a signal peptide (17 aa) and the mature peptide (187 aa). YTK GHR1 protein consisted of a signal peptide (28 aa), an extracellular domain (222 aa), a single transmembrane domain (23 aa) and an intracellular domain (361 aa). GHR2 protein included 18 aa, 223 aa, 23 aa, and 321 aa, respectively. Tissue distribution analysis showed that the maximal level of gh expression was observed in the pituitary, and ghr1 mRNA was mainly detected in the liver, while ghr2 transcripts were most abundant in the gonad. Moreover, both ghr1 and ghr2 mRNAs were expressed in all embryonic stages and displayed different gene expression profiles. Overall, these results provide initial evidences for the involvement of the GH/GHR system in the early ontogeny of yellowtail kingfish.

Yellowtail kingfish, Seriola lalandi, is a globally distributing pelagic marine fish species. In recent years, the wild population has suffered drastic decline due to overfishing and environmental deterioration. Thus, the sustainable utilization and conservation management for this species should be implemented urgently. In the present study, we developed SNP markers using 2b-RAD sequencing method from 33 individuals of yellowtail kingfish. Wherein, 100 high-quality SNP markers were obtained based on high throughput sequencing and stringent SNP filter process. The minor allele frequency of these SNPs ranged from 0.0126 to 0.4957. The observed heterozygosity and expected heterozygosity ranged from 0 to 0.9914 and 0.0249 to 0.5000, respectively. The inbreeding coefficient varied from − 0.9828 to 0.7882. Among these SNPs, 10 loci deviated significantly from Hardy–Weinberg equilibrium after Bonferroni correction. These novel SNP markers will serve as valuable tools for population genetics evaluation and natural resources conservation of yellowtail kingfish.

Yellowtail kingfish (Seriola lalandi) is a pelagic marine piscivore with a circumglobal distribution. It is particularly suitable for open ocean aquaculture owing to its large body size, fast swimming, rapid growth, and high economic value. A high-precision genome is of great significance for future genetic breeding research and large-scale aquaculture in the open ocean. PacBio, Illumina, and Hi-C data were combined to assemble chromosome-level reference genome with the size of 648.34 Mb (contig N50: 28.52 Mb). 175 contigs was anchored onto 24 chromosomes with lengths ranging from 12.28 to 34.59 Mb, and 99.79% of the whole genome sequence was covered. The BUSCOs of genome and gene were 94.20 and 95.70%, respectively. Gene families associated with adaptive behaviors, such as olfactory receptors and HSP70 gene families, expanded in the genome of S. lalandi. An analysis of selection pressure revealed 652 fast-evolving genes, among which mkxb, popdc2, dlx6, and ifitm5 may be related to rapid growth traits. The data generated in this study provide a valuable resource for understanding the genetic basis of S. lalandi traits.

Leptin (Lep) plays a key role in the regulation of food intake and energy homeostasis in vertebrates. Our previous studies have provided evidence for the existence of two leptin genes (lepa and lepb) and one leptin receptor (lepr) gene in a flatfish, the half-smooth tongue sole (Cynoglossus semilaevis). However, the spatial-temporal expression patterns and possible roles of the leptin system during early development and ovarian maturation are still poorly understood in teleosts. In the current study, we evaluated dynamic expression profiles of lepa, lepb, and lepr mRNAs during various developmental stages in this species. Quantitative RT-PCR analysis indicated that both ligand (lepa and lepb) and receptor (lepr) genes were detected in unfertilized eggs and during embryogenesis but with different expression profiles. In addition, lepa, lepb, and lepr transcripts levels increased significantly during larval development, reaching the peak at 10, 25, and 30 days post-hatching (dph), respectively. On the other hand, changes in mRNA expression of these three genes at the brain-pituitary-gonad (BPG) axis were also investigated during ovarian maturation, and lepa, lepb, and lepr mRNAs varied greatly. Taken together, our results encompass the first study reporting the dynamic expression patterns of leptin and its receptor mRNAs in the order Pleuronectiformes, providing evidence that the leptin system could be functional and play important roles during early development and ovarian maturation in tongue sole.

The structure of intestinal microbiota of black rockfish Sebastes schlegelii in five early development stages were determined in high throughput sequencing with Illumina MiSeq PE300 system. The relationship between intestinal microbial community and the environmental (including culture water and feed) microbiota and the abundance variation trends of core microbiota were investigated, based on which the source of some core microbiota was analyzed in this study. The results show that Proteobacteria and Firmicutes are the most dominant phyla in guts. At the genus level, there are obvious differences between the artificial breeding fish and wild adults in the intestinal microflora structure. The compositions of dominant genera are similar, although the structure of intestinal microbiota gradually changes with the growth of larvae and juveniles. The core microbiota including Bacillus, Acinetobacter, Pseudomonas, Lactobacillus, Lactococcus, Glaciecola, Vibrio, Pseudoalteromonas, Acidovorax, and Aliivibrio were determined in the analysis of dominant and shared species. Compared with the water, the effect of feed microbiota on the structure of the gut microbial community is more obvious. Moreover, the trends of Bacillus, Acinetobacter, Pseudomonas, Lactobacillus, Lactococcus, and Glaciecola were opposite to Vibrio and Pseudoalteromonas in the gut. The correlation analysis suggested that Acidovorax, Glaciecola, Pseudomonas, Lactobacillus, and Acinetobacter might transited from mainly the parents and/or came from the fertilization process. The relative results may provide a theoretical reference for selecting the native probiotics, and supply the basic data for artificially regulating the intestinal microbiota with probiotic during early developmental stage of black rockfish.

Both gonadotropin-inhibitory hormone (GnIH) and kisspeptin play a critical role in the control of reproduction in vertebrates. However, the spatial–temporal expression patterns and possible roles of these two peptides during development are poorly understood. To this end, we evaluated dynamic expression profiles of lpxrfa, the piscine ortholog of gnih, kisspeptin (kiss2) and their receptor mRNAs during embryogenesis and early larval development in a flatfish, Cynoglossus semilaevis. Quantitative RT-PCR analysis indicated that both ligand (lpxrfa and kiss2) and receptor (lpxrfa-r and kiss2r) mRNAs were detected in unfertilized eggs and during embryogenesis, but with different expression profiles. In addition, lpxrfa-r, kiss2 and kiss2r transcripts increased significantly at 6 days post-hatching (dph), while lpxrfa mRNAs did not vary significantly during all larval development. Taken together, these results of the present study suggest that both LPXRFa and kisspeptin systems could be functional and play differential roles during embryogenesis and early larval development in tongue sole.

In this study, the complete mitochondrial genomic sequence of southern flounder Paralichthys lethostigma was determined. The circular mitogenome is 16 843 bp in length and encodes 37 typical animal mitochondrial genes and contains a putative D-loop region. Phylogenetic analysis based on the mitochondrial genomes of southern flounder indicated that P. lethostigma and P. olivaceus are the most closely related species, which strongly supports their close phylogenetic affinity.

Spexin (SPX) is a newly identified peptide hormone of 14 amino acids (aa) with pleiotropic functions in vertebrates, and recently a second paralog of SPX (named SPX2) has been only found in some non-mammalian species (the previous form of SPX is now designated as SPX1). Results of previous studies have revealed the existence of a functional SPX1 in the half-smooth tongue sole. To clarify the divergence of the spexin family, we further isolated the spx2 gene from the same flatfish species in the current study. The open reading frame of tongue sole spx2 was 378 nucleotides in size that encoded a 125-aa preprohormone, and the tongue sole SPX2 precursor included a 17-aa mature peptide that may be amidated at the C-terminus, rather than a tetradecapeptide reported in most other vertebrates. Tissue distribution analysis showed that spx2 transcripts were found in a wide array of tissues of both sexes with the highest expression in the ovary. In addition, the spx2 mRNA abundance was also ubiquitously detected in different brain regions. Food deprivation for 4 weeks stimulated spx2 mRNA levels at the brain-pituitary-ovary axis, and refeeding of starved fish restored the spx2 mRNA levels back to normal. Taken together, this study provides initial evidence for the existence of a novel spexin gene (spx2) in Pleuronectiformes and suggest its possible involvement in the regulation of energy balance in the female tongue sole.

In fish and other vertebrates, growth hormone (GH) is an essential polypeptide required for normal growth and development. In an attempt to understand growth regulation in yellowtail kingfish (YTK), the full-length cDNA sequences encoding gh and its receptors (ghr1 and ghr2) were cloned, characterized and the expression profiles of these three genes were investigated during embryonic development. The full-length cDNA sequences of GH and its receptors were obtained by RT-PCR combined with RACE methord. YTK gh cDNA sequence was 852 base pairs (bp) that comprised an open reading frame (ORF) of 615 bp encoding a 204-amino acids (aa) precursor. The preprohormone compassed a signal peptide (17 aa) and the mature peptide (187 aa). YTK GHR1 protein consisted of a signal peptide (28 aa), an extracellular domain (222 aa), a single transmembrane domain (23 aa) and an intracellular domain (361 aa). GHR2 protein included 18 aa, 223 aa, 23 aa, and 321 aa, respectively. Tissue distribution analysis showed that the maximal level of gh expression was observed in the pituitary, and ghr1 mRNA was mainly detected in the liver, while ghr2 transcripts were most abundant in the gonad. Moreover, both ghr1 and ghr2 mRNAs were expressed in all embryonic stages and displayed different gene expression profiles. Overall, these results provide initial evidences for the involvement of the GH/GHR system in the early ontogeny of yellowtail kingfish.

Despite its functional significance in mammals and birds, the biological role of gonadotropin-inhibitory hormone (GnIH) in reproduction is still far from being fully understood in teleosts. In the current study, we have identified LPXRFa, the piscine ortholog of GnIH, and its cognate receptor (LPXRFa-R) in yellowtail kingfish (YTK), which is considered as a promising species for aquaculture industry worldwide. The YTK cDNA sequence of lpxrfa was 534 base pair (bp) in length and encoded a 178-amino acids (aa) preprohormone. The LPXRFa precursor comprised three putative peptide sequences that included –MPMRF, –MPQRF, or -LPERL motifs at the C-termini, respectively. The YTK lpxrfa-r cDNA sequence was composed of 1265 bp that gave rise to a LPXRFa-R of 420 aa, encompassing the characteristic seven hydrophobic transmembrane domains. In males, both lpxrfa and lpxrfa-r transcripts could be detected at high levels in the brain and testis. In females, a noteworthy expression of lpxrfa was observed in the brain and ovary, while the expression of lpxrfa-r was especially evident only in the brain. To study the ontogeny of LPXRFa system, transcript levels were also investigated during early life stages. Variable expression of the LPXRFa system was observed during all stages of YTK embryogenesis. The highest expression of lpxrfa and lpxrfa-r were noticed at 7 dph and 15 dph, respectively. Furthermore, LPXRFa peptides stimulated growth hormone (gh), luteinizing hormone (lhβ) and follicle-stimulating hormone (fshβ) gene expression from the pituitary. Taken together, our results provide initial evidence for the existence of the LPXRFa system in yellowtail kingfish and suggest its possible involvement at early development and reproductive functions.

Leptin (Lep) is a key factor for the regulation of food intake and energy homeostasis in mammals. To date, a number of studies have provided evidence for the existence of multiple leptin genes in teleosts, but not much information is available in fish regarding the regulation of leptin genes by sex steriods. As a first step, two leptin genes (lepa and lepb) and a leptin receptor (lepr) gene were cloned from the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNAs of tongue sole lepa and lepb were 1265 bp and 1157 bp in length, encoding for proteins of 160 aa and 158 aa, respectively. The three-dimensional structures modeling of tongue sole LepA and LepB showed strong conservation of tertiary structure with other vertebrates. The full-length cDNA of tongue sole lepr was 4576 bp, encoding a protein of 1133 aa which contained all functionally important domains conserved among vertebrate LepRs. Tissue distribution analysis showed that tongue sole lepa mRNA was highly detectable in the ovary and brain, while lepb mRNA was ubiquitously expressed in various tissues. Notably, the tongue sole lepr mRNA was most abundant in the ovary. Using a primary hepatocyte culture system, we evaluated the effects of sex steroids on lep/lepr gene expression. Both 17β-estradiol (E2) and testosterone (T) inhibited hepatic lepa and lepr mRNAs without affecting lepb mRNA levels. In addition, T also suppressed growth hormone receptor 1 (ghr1), ghr2, and insulin-like growth factor 2 (igf-2) mRNA levels, and stimulated expression of igf-1 gene. On the other hand, none of these four genes were altered by E2. To the best of our knowledge, this is the first description of a direct and differential regulation of lep/lepr gene expression by sex steroids at the hepatocyte level of a flatfish, supporting that individual leptin peptide may possess different biological roles in teleosts.

In this study, to understand the role of the insulin-like growth factor (IGF) system in the regulation of early development in yellowtail kingfish (YTK, Seriola lalandi), an economically important marine fish species with a high potential for aquaculture, we first cloned the full-length cDNAs for igf1 and igf2 from the liver. YTK igf1 cDNA was 1946 base pairs (bp) in length with an open reading frame (ORF) of 558 bp encoding preproIGF1 of 185 amino acids (aa). The preproIGF1 consisted of 44 aa for the signal peptide, 68 aa for the mature peptide comprising B, C, A, and D domains, and 73 aa for the E domain. YTK igf2 cDNA had an ORF of 648 bp that encoded a total of 215 aa spanning the signal peptide (47 aa), the mature peptide (70 aa), and the E domain (98 aa). At the protein level, both YTK IGF1 and IGF2 exhibited high sequence identities with their corresponding fish counterparts, respectively. Subsequently, quantitative RT-PCR analysis indicated that the highest level of igf1 mRNA expression was recorded in the gonad and liver, while the igf2 mRNA expression was most abundant in the gill and liver. In addition, both igf1 and igf2 were detected in all stages of embryonic development and exhibited different gene expression patterns, supporting that IGF1 and IGF2 could be functional and play important roles during YTK embryogenesis. Overall, this initial study of IGF1 and IGF2 provides an insight into the endocrine mechanism involved in the early development of yellowtail kingfish.

Gonadotropin-inhibitory hormone (GnIH) has been characterized by its ability to inhibit either basal or gonadotropin-releasing hormone (GnRH)-induced gonadotropin synthesis and release in birds and mammals. However, the physiological role of GnIH on the reproductive axis in fish remains inconclusive, with most studies focusing on the orders Cypriniformes and Perciformes. To gain insight into the role of GnIH in the regulation of reproduction in the order Pleuronectiformes, we first cloned the LPXRFa gene, the piscine ortholog of GnIH, in the half-smooth tongue sole. The full-length cDNA of LPXRFa was 918bp in size with an open reading frame (ORF) of 585bp that encoded a 194 amino acids preprohormone with a calculated molecular mass and isoelectric point of 21.73kDa and 6.52, respectively. The LPXRFa precursor encoded two putative peptide sequences that included -MPMRF or -MPQRF motifs at the C-terminal. Tissue distribution analysis showed that LPXRFa transcripts could be detected at high levels in the brains of both sexes and to a lesser extent in the ovary, heart and stomach of females, while a noteworthy expression was observed in the kidney and muscle of males. Furthermore, the expression patterns of LPXRFa mRNA during ovarian maturation were also investigated. In the brain, the mRNA expression of LPXRFa increased significantly at stage III, declined at stage V and reached a maximum at stage VI. In the pituitary, the levels of LPXRFa mRNA remained stable during ovarian maturation and increased significantly to the top level at stage V and then declined back to basal levels. In contrast, the ovarian LPXRFa mRNA levels declined sharply at stage III and remained depressed over the course of ovarian maturation. Taken together, our results provide further evidence for the existence of LPXRFa in the order Pleuronectiformes and suggest its possible involvement in the regulation of reproduction in the female tongue sole.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide that inhibits the reproductive axis of mammals and birds. Little information is available in teleosts regarding its actions on the brain neuroendocrine systems controlling reproduction. Here, we analyzed the effects of intraperitoneal injection of the piscine GnIH ortholog LPXRFa peptides (LPXRFa-1, LPXRFa-2, and LPXRFa-3) on the expression of reproduction-related genes in the brain of yellowtail kingfish. Our results revealed an inhibitory effect of the three LPXRFa peptides on gnrh1, lpxrfa, kiss1r, kiss2r, and lpxrfa-r mRNA levels. Both LPXRFa-1 and LPXRFa-2 were found to suppress kiss2 expression, and LPXRFa-1 reduced gnrh2 expression. Moreover, LPXRFa-3 induced a decrease in kiss1 and gnrh2 expression. Overall, these data suggest the inhibitory actions of each LPXRFa peptide encoded by the same precursor on the brain reproductive genes in yellowtail kingfish.

Simple Summary CircRNAs: as molecules involved in gene regulation, have become a new research hotspot in the non-coding RNA field. CircRNAs show tissue- or developmental stage-specific patterns of expression and can influence the expression levels of their parental genes. Recent studies have documented the potential biological roles of circRNAs in the growth, development, reproduction and health of humans and animals. Tongue sole (Cynoglossus semilaevis) is a marine flatfish that is an economically important farmed species in China. The commercial aquaculture of tongue sole has developed in the last few years because wild resources have decreased. Reproduction is regulated by brain-pituitary-gonad-liver axis which limits the development of artificial tongue sole culture. However, the roles of circRNAs in the ovarian development and maturation of tongue sole has never been reported. The identification of the potential functions of circRNAs provides a foundation for understanding the genetic mechanisms that regulate oocyte growth and maturation, which will allow the efficiency of tongue sole reproduction to be improved. Moreover, our findings extend the knowledge about a new type of endogenous RNA involved in regulating the ovarian development and maturation of tongue sole. Abstract CircRNAs are novel endogenous non-coding small RNAs involved in the regulation of multiple biological processes. However, little is known regarding circRNAs in ovarian development and maturation of fish. Our study, for the first time, provides the genome-wide overview of the types and relative abundances of circRNAs in tongue sole tissues during three ovarian developmental stages. We detected 6790 circRNAs in the brain, 5712 in the pituitary gland, 4937 in the ovary and 4160 in the liver. Some circRNAs exhibit tissue-specific expression, and qRT-PCR largely confirmed 6 differentially expressed (DE) circRNAs. Gene Ontology and KEGG pathway analyses of DE mRNAs were performed. Some DE circRNA parental genes were closely associated with biological processes in key signalling pathways and may play essential roles in ovarian development and maturation. We found that the selected circRNAs were involved in 10 pathways. RNase R digestion experiment and Sanger sequencing verified that the circRNA had a ring structure and was RNase R resistant. qRT-PCR results largely confirmed differential circRNA expression patterns from the RNA-seq data. These findings indicate that circRNAs are widespread in terms of present in production-related tissues of tongue sole with potentially important regulatory roles in ovarian development and maturation.

Although recent studies have revealed its involvement in fish reproduction, the physiological role of neurokinin B (NKB), which is encoded by the tachykinin 3 (tac3) gene, is still largely unclear in teleosts, with most researches on the orders Perciformes and Cypriniformes. In the current study we identified the tac3 gene in a representative species of Pleuronectiformes, the half-smooth tongue sole. The full-length cDNA sequence of tongue sole tac3 was 742 base pairs (bp) in length with an open reading frame of 372 bp that encoded a 123-amino acids preprohormone. The TAC3 precursor contained NKB and an additional peptide NKB-related peptide (NKBRP), both of which had an –FVGLM motif at the C-termini. Tissue distribution analysis indicated that tac3 transcripts were highly expressed in the brain, intestine and gonads of both sexes, and to a lesser extent in other tissues. Among various brain regions, tac3 transcripts were most abundant in the hypothalamus. In addition, the expression profiles of tac3 mRNA were also evaluated along the brain-pituitary-gonadal axis during tongue sole ovarian maturation. The brain tac3 mRNA levels remained unchanged, however, in both pituitary and ovary, the mRNA expression of tac3 increased significantly to the maximal levels at stage III and then declined back to basal levels. Taken together, our results encompass the first study reporting the existence of tac3 gene in a flatfish species and suggest its possible involvement in the control of reproduction in the half-smooth tongue sole.

Kisspeptin (Kiss) plays a critical role in mediating gonadal steroid feedback to the gonadotropin-releasing hormone (GnRH) neurons in mammals. However, little information regarding the regulation of kisspeptin gene by sex steroids is available in teleosts. In this study, we examined the direct actions of estradiol (E2) and testosterone (T) on hypothalamic expression of kisspeptin and other key factors involved in reproductive function of half-smooth tongue sole. As a first step, a partial-length cDNA of kiss2 was identified from the brain of tongue sole and kiss2 transcript levels were shown to be widely expressed in various tissues, notably in the ovary. Then, the actions of sex steroids on kiss2 and other reproduction-related genes were evaluated using a primary hypothalamus culture system. Our results showed that neither kiss2 nor its receptor kiss2r mRNA levels were significantly altered by sex steroids. Moreover, sex steroids did not modify hypothalamic expression of gonadotropin-inhibitory hormone (gnih) and its receptor gnihr mRNAs, either. However, E2 markedly stimulated both gnrh2 and gnrh3 mRNAs levels. Overall, this study provides insights into the role of sex steroids in the reproductive function of Pleuronectiform teleosts.

Kisspeptin (Kiss) acts as a positive regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the intricate web of intracellular signal transduction pathways in response to Kiss is still far from being fully understood in teleosts. Accordingly, we investigated the molecular mechanism of Kiss action and its possible interaction with LPXRFa signaling in this study. In vitro functional analysis revealed that synthetic tongue sole Kiss2 decapeptide increased the cAMP responsive element-dependent luciferase (CRE-luc) activity in COS-7 cells transfected with its cognate receptor, while this stimulatory effect was markedly reduced by two inhibitors of the adenylate cyclase (AC)/protein kinase A (PKA) pathway. Similarly, Kiss2 also significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity, whereas this stimulatory effect was evidently attenuated by two inhibitors of the phospholipase C (PLC)/protein kinase C (PKC) pathway. In addition, LPXRFa-2 suppressed Kiss2-elicited CRE-luc activity in a dose-dependent manner. Taken together, Kiss2 utilizes both AC/PKA and PLC/PKC pathways to exert its functions via its cognate receptor and LPXRFa may antagonize the action of Kiss2 by inhibiting kisspeptin signaling. As far as we know, this study is the first to characterize the half-smooth tongue sole kisspeptin and LPXRFa signaling pathway in COS-7 cells transfected with their cognate receptors and provides novel information on the interaction between LPXRFa system and kisspeptin system in teleosts.

Kisspeptin (Kiss) and its receptor, KissR (previously known as GPR54), play a critical role in the control of reproduction and puberty onset in mammals. Additionally, a number of studies have provided evidence of the existence of multiple Kiss/KissR systems in teleosts, but the physiological relevance and functions of these kisspeptin forms (Kiss1 and Kiss2) still remain to be investigated. To this end, we examined the direct actions of Kiss2 on hypothalamic functions in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. As a first step, the full-length cDNA for kiss2r was identified and kiss2r transcripts were shown to be widely expressed in various tissues, notably in the brain of tongue sole. Then, the effects of Kiss2 decapeptide on reproduction-related gene expression were evaluated using a primary hypothalamus culture system. Our results showed that neither gnrh2 nor gnrh3 mRNA levels were altered by Kiss2. However, Kiss2 significantly increased the amounts of gnih and kiss2 mRNAs. In contrast, Kiss2 elicited an evident inhibitory effect on both gnihr and kiss2r mRNA levels. To the best of our knowledge, this is the first description of a direct and differential regulation of reproduction-related gene expression by Kiss2 at the hypothalamus level of a teleost fish. Overall, this study provides novel information on the role of Kiss2/Kiss2R system in the reproductive function of teleosts.

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

Results of previous studies indicated the existence of LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone (GnIH), and kisspeptin (Kiss2) in tongue sole (Cynoglossus semilaevis), and that LPXRFa exerts an inhibitory effect on Kiss2 activation in the protein kinase A (PKA) pathway. The functions in the control of reproduction and whether LPXRFa antagonizes the action of Kiss2 by inhibiting the protein kinase C (PKC) pathway, however, are still unknown. In the present study, there was an initial investigation of the direct effects of LPXRFa and Kiss2 on relative abundance of pituitary hormone mRNA transcripts using a whole pituitary culture system. Results indicated that LPXRFa-1 specifically functioned to increase relative abundance of lhβ mRNA when there were comparisons with the control, without any effect on relative abundance of gh, gthα and fshβ mRNA. Treatment with LPXRFa-2 resulted in a reduction in relative abundance of gthα and lhβ mRNA, and did not alter relative abundance of fshβ mRNA. Treatment of LPXRFa-2 resulted in a greater relative abundance of gh mRNA. Treatment with Kiss2, however, resulted in an increase in relative abundance of gthα and fshβ mRNA transcripts, without altering relative abundances of gh and lhβ mRNA. Subsequently, there was valuation of the potential interaction between LPXRFa and kisspeptin in COS-7 cells transfected with the cognate receptors. Both LPXRFa-1 and LPXRFa-2 suppressed serum responsive element-dependent luciferase (SRE-luc) activity when compared to stimulation with Kiss2 alone, indicating an inhibitory effect of LPXRFa on kisspeptin activation on the PKC pathway. Overall, data from the present study provide novel evidence for differential actions of LPXRFa and kisspeptin on pituitary hormone synthesis as well as for the interaction between LPXRFa and kisspeptin systems in teleosts.

The hypothalamo–pituitary–gonadal (HPG) axis plays a major role in coordinating the reproduction of fish and other vertebrates. Gonadotropin-releasing hormone (GnRH) is the primary stimulatory factor responsible for the hypothalamic control of gonadotropin secretion. In 2000, a previously unidentified hypothalamic neuropeptide was isolated from the brain of Japanese quail and termed gonadotropin-inhibitory hormone (GnIH) based on its ability to directly inhibit gonadotropin release from the cultured quail anterior pituitary gland. One year later, the cDNA sequence that encodes the quail GnIH precursor polypeptide was cloned and was found to encompass two further peptides (GnIH-related peptide (RP)-1 and GnIH-RP-2) besides GnIH. To date, GnIH orthologous have been detected in a variety of vertebrates from fish to humans. These peptides possess a characteristic-LPXRFa (X = L or Q) motif at the C-terminus and are designated as LPXRFa peptides. It is generally accepted that LPXRFa peptides act on GnRH neurons in the hypothalamus to inhibit gonadotropin synthesis and release in addition to affecting the pituitary function in birds and mammals. However, the exact physiological role of LPXRFa is still uncertain in fish and dual actions of LPXRFa on the HPG axis have been observed. Research aiming to elucidate the detailed signaling pathways mediating the actions of LPXRFa on target cells may contribute to understanding the functional divergence of the LPXRFa system in teleosts. Accordingly, this review will discuss the recent advances in LPXRFa receptor signaling, as well as the potential interactions on cell signaling induced by other factors, such as GnRH and kisspeptin.

Gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide, serves as a key player in the regulation of reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. However, little information is available in teleosts regarding the intracellular signal transduction pathway in response to GnIH. To this end, we first cloned the gene of LPXRFa (the piscine ortholog of GnIH) receptor in the half-smooth tongue sole ( Cynoglossus semilaevis ), a representative species of the order Pleuronectiformes. The full-length cDNA of LPXRFa receptor was 2201 bp in size with an open reading frame (ORF) of 1365 bp that encoded 454 amino acids. Tissue distribution showed that LPXRFa receptor transcripts could be detected at high levels in the brain, to a lesser extent in the pituitary, and at low levels in the ovary and other peripheral tissues. In vitro functional analysis revealed that putative tongue sole LPXRFa-1 and LPXRFa-2 peptides significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity in COS-7 cells transfected with the novel receptor, and these stimulatory effects were evidently reduced by two inhibitors of the PLC/PKC pathway. In addition, neither LPXRFa-1 nor LPXRFa-2 altered the cAMP-responsive element (CRE)-luc activity, but only LPXRFa-2 could markedly decrease forskolin-induced CRE-luc activity in COS-7 cells expressing its cognate receptor. Taken together, our results encompass the first study reporting the existence of LPXRFa receptor in the order Pleuronectiformes and provide novel evidence of differential activation of signaling pathways by LPXRFa peptides in fish.

The morphological and ultrastructural features of the spermatozoon in Brachionus calyciflorus are described using light, fluorescence and transmission electron microscopy (TEM). The mature spermatozoon, which appears to be thread-like, is composed of a slightly expanded anterior of cell body region and a flagellum region without acrosome. The cell body region and flagellum region are respectively 16–27 μm and 20–33 μm in length (n = 60). The spermatozoon is characterized by a mass of dense tubular materials, which occupy most of the cell. Some mitochondria are distributed around the nuclear region in the anterior of the cell body region, while in the posterior portion of cell body, the chromatin often contains a single lobated nucleus arranged at the center of cell. The flagellum contains the classic axoneme (9 × 2 + 2) and possesses lateral undulating membrane. Mature B. calyciflorus males have no germ cell stages earlier than the spermatids in the testis. TEM examination reveals rigid rods as well as predominant typical spermatozoon in the testis. Observations, based on successive photographs and videos, enabled a first-time recording of the unique inverted movement of the spermatozoon, which indicated that the movement of the spermatozoon is driven by the flagellum. Our study also provides further supplementary insights into the phylogenetic systematics of the Rotifera.

The membrane progestin receptor α (mPRα) plays an important role in the regulation of oocyte meiotic maturation in fish. However, the endocrine regulation of multiple spawning in fish with asynchronous ovarian development remains to be determined. We identified four genes (FSHβ, LHβ, CGα and mPRα) that are widely expressed in both the reproductive and non-reproductive tissues of rock bream Oplegnathus fasciatus and examined their expression during ovarian maturation. The level of FSHβ transcripts in the pituitary remained high during vitellogenic stages, when the plasma levels of estradiol-17β reached maximum levels and oocyte development was stimulated. LHβ expression in the pituitary and ovary reached maximum levels during oocyte maturation when the plasma levels of testosterone also reached a maximum. A significant correlation between mPRα transcript levels and the gonadosomatic index was observed during vitellogenesis and final maturation. Brain gonadotropin (FSHβ and LHβ) and mPRα were continuously expressed at different stages of ovarian development. The dynamic and diverse nature of gonadotropins and mPRα gene expression patterns suggests that these genes might jointly regulate ovarian development and maturation through the brain–pituitary–ovary axis in the rock bream endocrine system. J. Exp. Zool. 323A: 430–444, 2015. © 2015 Wiley Periodicals, Inc.

In order to elucidate the possible roles of insulin-like growth factors I and II (IGF-I and IGF-II) in the embryonic development of Platichthys stellatus, their cDNAs were isolated and their spatial expression pattern in adult organs and temporal expression pattern throughout embryonic development were examined by quantitative real-time PCR assay. The IGF-I cDNA sequence was 1,268 bp in length and contained an open reading frame (ORF) of 558 bp, which encoded 185 amino acid residues. With respect to IGF-II, the full-length cDNA was 899 bp in length and contained a 648-bp ORF, which encoded 215 amino acid residues. The amino acid sequences of IGF-I and IGF-II exhibited high identities with their fish counterparts. The highest IGF-I mRNA level was found in the liver for both sexes, whereas the IGF-II gene was most abundantly expressed in female liver and male liver, gill, and brain. The sex-specific and spatial expression patterns of IGF-I and IGF-II mRNAs are thought to be related to the sexually dimorphic growth and development of starry flounder. Both IGF-I and IGF-II mRNAs were detected in unfertilized eggs, which indicated that IGF-I and IGF-II were parentally transmitted. Nineteen embryonic development stages were tested. IGF-I mRNA level remained high from unfertilized eggs to low blastula followed by a significant decrease at early gastrula and then maintained a lower level. In contrast, IGF-II mRNA level was low from unfertilized eggs to high blastula and peaked at low blastula followed by a gradual decrease. Moreover, higher levels of IGF-I mRNA than that of IGF-II were found from unfertilized eggs to high blastula, vice versa from low blastula to newly hatched larva, and the different expression pattern verified the differential roles of IGF-I and IGF-II in starry flounder embryonic development. These results could help in understanding the endocrine mechanism involved in the early development and growth of starry flounder.