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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.

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.

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.

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.

The complete mitochondrial genomic sequence of Yellowtail Kingfish Seriola aureovittata was determined in this study. The circular mitogenome contains 16,609 bp, encoding 37 typical animal mitochon...

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.

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.

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHβ, LHβ and CGα) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHβ, LHβ and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHβ transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHβ expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system 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.

We determined the complete nucleotide sequence of the mitochondrial genome for the summer flounder, Paralichthys dentatus (Pleuronectiformes, Paralichthyidae). This mitochondrial genome, consisting of 17,033 base pairs (bp), encoded genes for 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a putative D-loop region like those found in other vertebrates, with the gene order identical to that of typical vertebrates. Phylogenetic analysis showed that P. dentatus clustered into one clade with its Paralichthyidae counterparts, Paralichtchys lethostigma and Paralichtchys olivaceus, which showed the close relationship among these species.

We sequenced the complete mitochondrial genome of the marbled flounder Pseudopleuronectes yokohamae collected from the Yellow Sea off China. The mitogenome comprised a 16 864-bp circular DNA molecule containing 37 genes and an AT-rich control region known as the D-loop. Phylogenetic analysis based on the mitochondrial genomes of marbled flounder indicated that P. yokohamae and Verasper variegatus are the most closely related species, which strongly supports their close phylogenetic affinity.