Deep-sea aquaculture vessels have emerged as promising alternative to conventional nearshore farming, yet their physiological impacts on fish remain poorly understood. This study investigated the effects of deep-sea aquaculture vessel mode on large yellow croaker (Larimichthys crocea) over a 12-month growth trial, with land-based industrial aquaculture as the control. Results showed that no significant differences in specific growth rate (SGR) were observed for fish reared under the two aquaculture modes. While vessel-cultured large yellow croaker exhibited morphological traits more akin to wild populations, evidenced by markedly reduced condition factor (CF) and muscle lipid content. Notably, the long-chain polyunsaturated fatty acid content in muscle was significantly elevated in vessel group. Transcriptomic revealed that hepatic differentially expressed genes were consistently enriched in the process of peroxisome proliferator-activated receptor (PPAR) and unsaturated fatty acid metabolism, where down-regulated genes expression related to lipogenesis, lipid oxidation and lipid transport along with the up-regulated ones related to fatty acid elongation/desaturation were observed in vessel-cultured fish. Histologically, hepatocyte vacuolation and nuclear atrophy were alleviated in vessel-cultured fish. Additionally, aquaculture vessel induced oxidative stress, as indicated by significantly increased malondialdehyde (MDA) level in serum and liver. Concurrently, the nuclear erythroid 2-related factor 2 (Nrf2)-mediated antioxidant system tended to be activated, leading to significantly up-regulated genes expression of superoxide dismutase 1 (sod1), catalase (cat) and glutathione peroxidase (gpx) together with their enhanced enzymes activity. In conclusion, deep-sea aquaculture vessel offers a viable strategy for large yellow croaker farming. While aquaculture vessel mode could trigger lipid metabolism remodeling and oxidative stress of fish, thereby altering morphological index, flesh quality, liver health and oxidative balance.

The yellowtail kingfish(Seriola aureovittata)is a large,fast-swimming fish species characterized by high and aggressive feeding activity,rapid growth rate,and tender flesh.Feed intake is a key factor that influences fish growth and production performance,and appetite is a major determinant of feed intake.To explore the role and potential mechanisms of orexin and its receptor ox2r in the regulation of feeding behavior in SS.aureovittata,we performed homologous cloning to obtain the open reading frame(ORF)sequences of the orexin and ox2r genes in S.aureovittata.Quantitative real-time PCR was used to analyze the expression of these genes across various tissues and developmental stages,as well as their regulatory roles in response to starvation and subsequent refeeding. The results showed that the ORF of orexin in S.aureovittata is 450 bp long,encoding 149 amino acids,including a 46-amino acid signal peptide,a 43-amino acid Orexin A mature peptide,and a 28-amino acid Orexin B mature peptide.The ox2r gene ORF is 1,269 bp,encoding 422 amino acids.Amino acid sequence alignment revealed that orexin and ox2r are highly conserved among teleosts.Phylogenetic analysis indicated that S.aureovittata orexin and ox2r are most closely related to those of Seriola dumerili.The mRNA expression of orexin and ox2r was detected in all 12 examined tissues,with the highest levels observed in the brain,followed by the pituitary gland and stomach.Expression of orexin and ox2r mRNA was also detected in the sperm and eggs,indicating parental inheritance.Both genes are involved in the embryonic and larval development of S.aureovittata,with significant expression changes observed during the transition from endogenous to exogenous nutrition.Starvation stimulated the upregulation of orexin and ox2r mRNA in the brain and pituitary,with expression levels returning to control group levels after 7 d of refeeding. These results suggest that the orexin system may be involved in organ development and feeding regulation during the early growth and feeding of S.aureovittata,thereby providing valuable insights into the mechanisms of feeding regulation in this species.

Deep-sea aquaculture vessels have emerged as promising alternative to conventional nearshore farming, yet their physiological impacts on fish remain poorly understood. This study investigated the effects of deep-sea aquaculture vessel mode on large yellow croaker (Larimichthys crocea) over a 12-month growth trial, with land-based industrial aquaculture as the control. Results showed that no significant differences in specific growth rate (SGR) were observed for fish reared under the two aquaculture modes. While vessel-cultured large yellow croaker exhibited morphological traits more akin to wild populations, evidenced by markedly reduced condition factor (CF) and muscle lipid content. Notably, the long-chain polyunsaturated fatty acid content in muscle was significantly elevated in vessel group. Transcriptomic revealed that hepatic differentially expressed genes were consistently enriched in the process of peroxisome proliferator-activated receptor (PPAR) and unsaturated fatty acid metabolism, where down-regulated genes expression related to lipogenesis, lipid oxidation and lipid transport along with the up-regulated ones related to fatty acid elongation/desaturation were observed in vessel-cultured fish. Histologically, hepatocyte vacuolation and nuclear atrophy were alleviated in vessel-cultured fish. Additionally, aquaculture vessel induced oxidative stress, as indicated by significantly increased malondialdehyde (MDA) level in serum and liver. Concurrently, the nuclear erythroid 2-related factor 2 (Nrf2)-mediated antioxidant system tended to be activated, leading to significantly up-regulated genes expression of superoxide dismutase 1 (sod1), catalase (cat) and glutathione peroxidase (gpx) together with their enhanced enzymes activity. In conclusion, deep-sea aquaculture vessel offers a viable strategy for large yellow croaker farming. While aquaculture vessel mode could trigger lipid metabolism remodeling and oxidative stress of fish, thereby altering morphological index, flesh quality, liver health and oxidative balance.

Sea cucumbers, as typical representatives of deep-sea benthic animals, possess significant scientific and economic importance. This investigation examined the genomic structural characteristics and genetic evolutionary relationships of Holothuria atra through the analysis of its complete mitochondrial genome. The findings indicated that the total length of the mitochondrial genome of the H. atra specimen was 15,788 bp, encompassing seven NADH genes, three cox genes, two ATP genes, and one cob gene. Additionally, two rRNAs and 22 tRNAs were identified. The entire sequence contained a total of 18 non-coding regions and seven overlapping gene regions, with the combined A + T content reaching 59.2%. The lengths of the 22 tRNAs ranged from 62 to 72 base pairs, and their cloverleaf secondary structures were predicted. Regarding codon usage, the PCGs within the mitochondrial genome of H. atra utilized 61 codons, encoding information for 20 amino acids. The most abundantly encoded amino acid in the mitochondrial genome of H. atra is leucine (Leu), representing 16.58%, while cysteine (Cys) is the least represented, accounting for 1.03%. Codons with higher usage frequencies include AGA (Ser 1), CUA (Leu 1), and CCA (Pro), whereas those with comparatively lower frequencies are GCG (Ala), CCG (Pro), and AGG (Ser 1). Evolutionary analysis revealed that H. atra is most closely related to Holothuria polii. By comparing the mitochondrial genomic sequences of 19 species within the class Holothuroidea, it was observed that eight mitochondrial sequences are shared among these species. This study provides valuable data supporting genetic evolutionary research and the development and utilization of H. atra resources within the Kiribati region.

Light colour is an indispensable pathway through which light exerts effects on fish. While gap still exists in research on the mechanisms by which light colour affects fish growth performance and health. To evaluate the effects of different light colour on fish, a 42-day growth trial was conducted on juvenile spotted sea bass (Lateolabrax maculatus) with an initial weight of 36.56 +/- 1.82 g. A total of 225 fish were equally allocated to a full-spectrum (white, WL, the control group) and four monochromatic light colour (yellow, YL; green, GL; red, RL; blue, BL) environments in triplicates. Results showed that the growth performance represented by weight gain rate (WGR) and specific growth rate (SGR) were considerably promoted in fish reared in each light colour as compared with the control (P < 0.05), with fish under blue light displaying highest value in these growth parameters. As to the digestive enzymes, the trypsin (TRY) activity was significantly increased in fish exposed to blue light colour as compared with other groups (P < 0.05). In terms of antioxidant capacity and non-specific immunity, significantly highest activity of SOD, CAT, AKP and ACP accompanied by lowest MDA content were observed in fish cultured under blue light colour (P < 0.05). Compared with the control, the differentially expressed genes in fish subjected to each light colour mainly mapped on the signaling pathways of circadian rhythm and glycine-serine-threonine metabolism, where the gene expression of F-box and leucine-rich repeat protein 3 (fbxl3, degrading cryptochrome) was significantly down-regulated (P < 0.05). As to the glycine-serinethreonine metabolism, the gene expression of glycine C-acetyltransferase (gcat, converting threonine to glycine) was significantly up-regulated in each light colour as compared with the control (P < 0.05). When it comes to the gut flora, the abundance of beneficial bacteria, including Prevotella, Faecalibacterium and Bacteroides, was higher in fish farmed under blue light colour. In conclusion, spotted sea bass reared in blue light environment exhibited superior growth performance, which may be associated with improved protein digestion, antioxidant capacity and non-specific immunity. Furthermore, blue light might promote fish growth by regulating circadian rhythm and glycine-serine-threonine metabolism, as well as increasing abundance of intestinal beneficial flora to enhance nutrient absorption and immune homeostasis.

Synaptonemal complex protein 3 (SCP3) is a fundamental structural component of the synaptonemal complex and serves as a well-established molecular marker for germ cell development and meiotic progression in teleost fish. In this study, we focused on the marbled flounder (Pseudopleuronectes yokohamae), a cold-water flatfish species endemic to the Yellow and Bohai Seas with considerable aquaculture potential due to its favorable growth performance and market value. However, despite its economic importance, the molecular mechanisms governing gonadal development in this species, particularly the precise regulation of meiosis initiation during sexual differentiation, remain poorly understood. Therefore, elucidating the expression dynamics of meiosis-associated genes, such as scp3, is crucial for delineating the critical period of sex differentiation and deciphering the regulatory network underlying germ cell development in this commercially valuable flatfish. This study had three main objectives for addressing these knowledge gaps. First, to clone and characterize the complete cDNA sequence of the scp3 gene from P. yokohamae. Second, examine the tissue distribution and stage-dependent expression patterns during gonadal development. Third, to determine the precise cellular localization within the gonadal tissues. These steps will help evaluate scp3 as a molecular marker for meiosis initiation and establish baseline data for understanding sex differentiation in this species. Specimens were collected at various developmental stages. These included juveniles aged 60–150 days post-hatching (dph) and adults aged 8–20 months post-hatching (mph). Total RNA was extracted from the gonadal and somatic tissues. Using integrated RT-PCR and RACE, full-length scp3 cDNA was obtained. Comprehensive bioinformatic analyses were performed to characterize the sequence architecture, predict protein structural features, and reconstruct phylogenetic relationships. The tissue distribution and developmental expression profiles were assessed using semi-quantitative RT-PCR and qRT-PCR. Cellular localization was determined by in situ hybridization (ISH) with digoxigenin-labeled RNA probes. Results showed that the cloned scp3 cDNA was 1022 nucleotides long. It contained a 708-bp open reading frame encoding a 235-amino acid polypeptide. This protein contains characteristic structural motifs, a coiled-coil domain, and a Cor1 conserved region, which are essential for synaptonemal complex assembly and function. Phylogenetic analyses revealed strong amino acid sequence conservation with other flatfish species, especially European flounder (Platichthys flesus) and plaice (Pleuronectes platessa). These species clustered within the Pleuronectiformes clade, confirming their evolutionary conservation. Expression profiling indicated that scp3 transcripts were mainly restricted to the gonads. The testicular expression levels were significantly higher than those in the ovaries. Minimal expression was detected in the brain, spleen, and heart tissues. No expression was observed in any of the other somatic tissues examined. During juvenile development (60–150 dph), scp3 expression levels remained low until 130 dph. A statistically significant increase was observed at 140 dph, suggesting the initiation of meiosis around this time. In adult gonads, testicular scp3 expression progressively increased from 8 to 20 mph and consistently exceeded ovarian expression. The ovarian expression initially decreased from 8 to 16 mph, with a slight recovery at 20 mph. This pattern reflects the fundamental differences in the meiotic progression between spermatogenesis and oogenesis. Cellular localization via ISH confirmed the cell-specific distribution. scp3 transcripts were mainly found in the cytoplasm of Stage II oocytes in ovarian tissue. Testicular sections were strongly associated with primary spermatocytes. The signal intensity was markedly higher in 20-month testis samples than in 8-month ones, consistent with increased meiotic activity during testicular maturation. In conclusion, the study provided a comprehensive molecular characterization of the scp3 gene in P. yokohamae. These distinct spatiotemporal expression patterns support its validity as a reliable marker for meiosis initiation and progression. The key evidence includes upregulation at 140 dph, gonadal-predominant expression, testis-biased accumulation, and localization to meiotic cell types. These findings address important gaps in the knowledge regarding the regulation of germ cell development in this species. They also established a foundation for future research on sexual differentiation pathways. This study has important implications for the development of monosex breeding approaches that utilize female growth advantages, thereby supporting sustainable aquaculture of P. yokohamae.

Copper nanoparticles (Cu-NPs) are widely used in aquaculture for their growth-promoting and immunoenhancing properties. However, excessive or over-accumulated Cu-NPs in aquaculture water cause toxicity through waterborne immersion. To explore the toxicity effects of waterborne Cu-NPs on yellowtail kingfish (Seriola aureovittata), a 7-day exposure experiment comprising a control group (no Cu-NPs), a low-concentration group (0.120 mg/L), and a high-concentration group (0.384 mg/L) was conducted, followed by a 7-day recovery period without Cu-NP stress. Exposure to Cu-NPs significantly elevated antioxidant enzyme activities (GSH-Px, Cu/Zn-SOD) and thiobarbituric acid reactive substances (TBARS) level in liver and posterior kidney, indicating significant oxidative stress. In the liver, levels of epinephrine and diacylglycerol were significantly upregulated, the cAMP signaling pathway was disrupted, and the NF-κB signaling pathway was activated, along with the downregulation of ap-1 and erk1-2 and upregulation of gadd45β, reducing cell proliferation and viability. These alterations resulted in hepatocellular vacuolation and blurred outlines of hepatic lobules. In the gill, high-concentration Cu-NPs activated oxidative phosphorylation, phagosome, and cell-adhesion molecules pathways and downregulated genes related to cell proliferation, adhesion, and tight junctions (i.e., l1cam, camd1, cntn1, cdh2, and nectin1), leading to cellular vacuolization and epithelial separation in histology. These changes disrupted osmotic balance and significantly upregulated Na+/K+-ATPase activity. All histological damages were not fully recovered within the tested recovery period. Therefore, under conditions of continuous exposure, the concentration of Cu-NPs used for juvenile yellowtail kingfish in aquaculture should be kept below 0.120 mg/L, with a recovery period exceeding 7 days. These results reveal the toxicity mechanisms of Cu-NPs and provide a scientific basis for establishing safe application guidelines in yellowtail kingfish aquaculture.

Marbled flounder (Pseudopleuronectes yokohamae) is becoming a commercially important flatfish species in Northeast Asia due to its strong environmental adaptability and excellent nutritional value. However, no high-quality marbled flounder reference genome was reported to date, which greatly limits the studies of evolutionary and functional genomics. Here, we reported the first gap-free T2T genome in flatfish (marbled flounder), with length of 582.73 Mb (contig N50: 26.29 Mb) combing short reads, PacBio HiFi long reads, ONT ultra-long reads, and Hi-C data. All of the genome sequences were assembled onto 24 chromosomes, and 48 telomeres were identified on both ends of all chromosomes. 99.29% complete BUSCOs were identified, demonstrating a high level of completeness. The average mapping ratio of short reads, PacBio HiFi reads, and ONT ultra-long reads aligned to the genome was more than 99.89%. 121.02 Mb repeating elements and 22,778 protein-coding genes were identified in the genome assembly. These results provide valuable resources for the evolutionary genomics research and the identification of key candidate genes for economic traits in marbled flounder.

Leptin,a 16 kDa protein hormone encoded by the obesity gene(ob),is secreted by adipose tissue and essential for regulating various physiological processes,such as fat metabolism,feeding,reproduction,and immunity.Seriola aureovittata,prized for its flavor and high nutritional content,often experiences excessive fat accumulation under artificial farming conditions because of spatial constraints in farming facilities and the provision of fresh fish as feed.This fat accumulation can adversely impact their growth and quality.A recombinant leptin protein for S.aureovittata was constructed using a prokaryotic expression vector to investigate the multiple physiological functions of leptin,and its biological activity was verified via intraperitoneal injection.This study provides technical support for further investigation of the physiological regulatory role of leptin in the growth and fat metabolism of S.aureovittata and for developing specialized products for growth and quality control. Total RNA was isolated from the brain tissue of S.aureovittata,and the first strand of cDNA was synthesized.Mature peptide fragments were synthesized based on amino acid sequences encoded by lepa and lepb in S.aureovittata.Using the prokaryotic expression vector pQE30,recombinant plasmids lepa/pQE30 and lepb/pQE30 were constructed and transformed into Escherichia coli M15.Following induction with 0.5 mmol/L IPTG at 37 ℃ for 4 h,the expressed proteins were validated for their expected sizes via western blot analysis,revealing clear bands at approximately 17.9 and 17.3 kDa.This confirmed that the recombinant proteins exhibited antigenic activity and were specifically recognized by 6×His antibodies.Further purification of the target proteins via a Ni2+-NTA affinity chromatography column yielded purified LepA and LepB recombinant proteins,with purity verified at＞90％and endotoxin levels ≤ 1 EU/μg.Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis(SDS-PAGE)analysis of the purified proteins showed distinct bands at approximately 17.9 kDa and 17.3 kDa,aligning with the expected size of the recombinant proteins,thereby confirming the effective purification of the LepA and LepB fusion recombinant proteins.The concentrations of the purified LepA and LepB recombinant proteins were measured at 0.2 and 0.3 mg/mL,respectively,using a protein quantification kit. In the intraperitoneal injection experiment,four groups were established:a control group(0.9％saline solution)and experimental groups with concentrations of 0.05,0.1,and 0.2 μg/kg.Each group consisted of 18 fish reared in a 1 m3 water tank.Each fish received an intraperitoneal injection of recombinant leptin protein at a dose of 1 μL/g body weight.At 6,12,and 24 h after the first injection,brain tissues from six randomly selected fish were used to detect biological activity.After injecting three concentration groups(0.05,0.1,and 0.2 μg/kg)of LepA and LepB proteins at different time points(6,12,and 24 h),the expression of lepa and lepb genes in the brain was either upregulated or downregulated,indicating that the recombinant proteins LepA and LepB possessed biological activity.These results provide technical support for further research into the physiological functions and regulatory mechanisms of leptin in the growth and development of S.aureovittata.

To explore the regulatory effect of exogenous recombinant leptin protein (rlepA/rlepB) on fatty acid metabolism and reproductive function in vivo, normal saline (control group), 50 mu g/kg (rlepA, rlepB), 150 mu g/kg (rlepA, rlepB) were injected intraperitoneally into 1-year-old female tongue sole (weighted about 169 g) for four consecutive weeks. Each group had three replicates, with six fish in each replicate. As a result, the low concentrate rlepB group significantly reduced the plumpness, liver acetyl-CoA and muscle crude fat content of fish in the experimental group, while other groups had no significant effect compared with the control group. The transcriptome analysis showed that compared with the control group, KEGG results of each experimental group were enriched in steroid biosynthesis pathway and endocrine resistance. The results of metabolome showed that leptin in all experimental groups significantly decreased the content of saturated fatty acids, and significantly increased the content of unsaturated fatty acids such as docosahexaenoic acid (DHA, C22:6n3), docosapentaenoic acid (DPA, C22:5n3) and arachidonic acid (ARA, C20:4n6). Based on the joint analysis, high concentration rlepA upregulated the expression of pla2 and acot gene sets, which enriched in linoleic acid metabolome and promoted the synthesis of unsaturated fatty acids. Finally, the expression of gnrh3 mRNA in the brain and fsh beta mRNA in the pituitary were upregulated in the rlepB-lc group, as well as the expression of gnrh3 gene and kiss1 gene in the low concentrate rlepA group. Through the joint analysis of transcriptome and metabolome, as well as RT-qPCR, we proposed the possible mechanism of rlepA and rlepB in regulating liver fatty acid metabolism and the possible role in reproduction-related, and analyzed the functional transduction pathway, providing a new idea for the study of leptin on lipid metabolism and reproductive function regulation mechanism of tongue sole. In the future, our research will focus on the regulation of fatty acid metabolism by leptin to achieve the control of reproductive function, which is also an innovative hint for the control of precocious puberty in fish.

We evaluated the impact of stocking density on the growth performance and physiological parameters of 1-year-old yellowtail kingfish (Seriola aureovittata) farmed in offshore net cages. A 90-day experiment was conducted with high (HSD, 7.41 ind/m3), medium (MSD, 5.93 ind/m3), and low (LSD, 4.44 ind/m3) stocking densities. Results showed that weight gain rate (WGR) and specific growth rate (SGR) were decreased with increase in density. Serum adrenaline, cortisol, and glucose levels and SOD and LZM activities were all increased with increase in density, and there were significant differences between HSD and MSD. HSD upregulated immune-related gene mRNA levels and activated the cytokine-cytokine receptor interaction pathway. These findings suggest that the fish in HSD suffered from crowding stress and exhibited immune stress, and the fish accelerated protein catabolism and phosphoenolpyruvate metabolism through significantly increasing AST, ALT, and PK activities to obtain more energy and cope with this stress. Meantime, the fish in HSD and MSD significantly downregulated gck expression and upregulated g6pc2 expression in glycolysis/gluconeogenesis to support the elevation of serum glucose levels. The fish in MSD experienced endoplasmic reticulum (ER) stress, which ultimately triggered the ER-associated degradation to restore the normal physiological function of ER. Considering the non-significant difference in AST, ALT, LZM, and PK activities between MSD and LSD, it is speculated that the stress level in MSD is lower than that in HSD. Interestingly, growth-related gene mRNA levels and K value in MSD were higher than those in HSD and LSD, indicating that the fish in MSD have greater growth potential. In this study, an MSD (5.93 ind/m3) was appropriate for 1-year-old yellowtail kingfish farmed in offshore net cages based on welfare and growth performance. These findings provide a technical foundation for the stocking regulation and healthy culture of yellowtail kingfish farmed in net cages.

Marbled flounder (Pseudopleuronectes yokohamae) exhibits a distinct female growth advantage and an XX/XY sex determination system. To exploit these traits, we investigated 17α-methyltestosterone (MT)-induced transcriptomic changes in gonadal tissue with the goal of generating pseudomale XX broodstock for all-female fry production. Full-sibling diploid juveniles (60 days post-hatching, dph) were fed diets containing 0 (control), 0.5, or 2 mg/kg MT for 120 days, followed by a 60-day recovery period on a commercial diet prior to sampling. Testicular transcriptomes were profiled via high-throughput sequencing, and key differentially expressed genes were validated using qPCR. Both MT treatments resulted in 100% masculinization. Testicular transcriptome analysis revealed 972 differentially expressed genes (DEGs) (180 up, 792 down) in the 0.5 mg/kg MT-treated males (MT05M) compared to the control males, and 1245 DEGs (842 up, 403 down) in the 2 mg/kg MT group (MT20M). Gene Ontology terms were enriched for extracellular space and signaling receptor regulator activity. KEGG pathway analysis indicated significant enrichment in neuroactive ligand–receptor interaction, ovarian steroidogenesis, and TGF-β signaling. qPCR confirmed significant downregulation (p < 0.05) of sox17, bmp4, and smad6, while dmrt1 was downregulated only in the MT20M group. These findings demonstrate that MT effectively masculinizes P. yokohamae by modulating key sex-related genes and signaling pathways, providing a transcriptomic foundation and potential mechanistic insights for optimizing pseudomale induction to enable all-female aquaculture production.

Spexin (SPX1) is a novel neuropeptide composed of 14 amino acids and well conserved across vertebrates, and it has been implicated in various physiological functions via galanin receptor 2 (GALR2) and GALR3. However, the detailed signaling pathways mediating its actions in target cells are still largely unknown. Accordingly, we addressed this issue in the present study using yellowtail kingfish as a model. SPX1 significantly increased CRE-luc activity in COS-7 cells expressing its cognate receptors GALR2a and GALR2b, and this stimulatory effect was attenuated by two inhibitors of the PKA pathway. Similarly, an evident induction of SRE-luc activity was observed when COS-7 cells transfected with GALR1b, GALR2a, GALR2b, GALR type 1, or GALR type 2 were challenged with SPX1, and two blockers of the PKC pathway suppressed this stimulatory action. Moreover, SPX1 markedly elevated NFAT-RE-luc activity in COS-7 cells expressing GALR1a, GALR2a, or GALR2b, and this promotion was inhibited by two antagonists of the Ca2+ route. Overall, our results have revealed that activation of six yellowtail kingfish galanin receptors by the SPX1 peptide may occur with different downstream signaling events, which could account for its pleotropic functions.

Leptin is a peptide hormone primarily produced by adipose tissue, which plays a crucial role in regulating energy balance by controlling appetite and energy expenditure. To better understand the intricate physiological functions of leptin in hepatocytes in tongue sole (Cynoglossus semilaevis), this study presents an integrated transcriptomic and metabolomic analysis of tongue sole hepatocytes following stimulation with lepA and lepB. Comparative analysis identified 2971 and 2900 upregulated differentially expressed genes (DEGs) and 1895 and 1821 downregulated DEGs in response to lepA and lepB stimulation, respectively. Notably, 5706 genes were commonly regulated by both stimulations, suggesting overlapping functional roles of these two leptin proteins. GO and KEGG enrichment analysis indicated significant enrichment in immune response (JAK-STAT signaling pathway, NF-κB signaling pathway, etc.) and lipid metabolism pathways, such as fatty acid synthesis, with similar findings for lepB. Metabolomic analysis demonstrated clear separation between treatment and control groups, with 34 medium- to long-chain fatty acids and numerous differentially expressed metabolites (DEMs) identified. KEGG analysis of the metabolome paralleled the transcriptomic findings, with shared pathways in lipid metabolism and immune function. Finally, joint analysis highlighted co-enrichment in linoleic acid metabolism and leishmaniasis pathways. Detailed mechanistic insights revealed the activation of JAK-STAT and NF-κB signaling pathways, modulation of arachidonic acid metabolism, and the influence on the MAPK signaling pathway. Additionally, lepA uniquely co-enriched in the fatty acid synthesis pathway, with specific gene regulation affecting the synthesis of various fatty acids. The study concluded that lepA and lepB exerted significant regulatory effects on lipid metabolism and immune responses in tongue sole hepatocytes through complex molecular networks. To our current knowledge, this represents the first direct evidence of leptin's role in immune function within teleost fish and offers valuable insights into the molecular mechanisms of lipid metabolism underlying the actions of lepA and lepB.

Spexin (SPX1) is a neuropeptide of 14 amino acids (aa), originally identified by bioinformatics, which has been implicated in various physiological functions in vertebrates via galanin receptors 2 and 3 (GALR2/3). To clarify the biological role of SPX1 in the control of reproduction in yellowtail kingfish, which is regarded as a promising species for offshore aquaculture worldwide, cDNA sequences of spx1 and six potential receptors were identified in the current study. The open reading frame of yellowtail kingfish spx1 was 363 nucleotides in size that encoded a 120-aa preprohormone, and its mature peptide was highly conserved among other species. The cDNA sequences of six GALRs (galr1a, galr1b, galr2a, galr2b, galr type 1, and galr type 2) were 1053 base pairs (bp), 1068 bp, 981 bp, 1137 bp, 1038 bp, 924 bp, which encoded G protein-coupled receptors of 350 aa, 355 aa, 326 aa, 378 aa, 345 aa, 307 aa, respectively. Tissue distribution analysis showed that spx1, galr1b, and galr2b transcripts were mainly detected in the brain. The highest mRNA levels of galr1a and galr2a were observed in the pituitary, followed by the brain and ovary. Both galr type 1 and galr type 2 were widely expressed in various tissues, with a peak level in the kidney. Moreover, all spx1 and galr genes significantly fluctuated during early ontogeny, exhibiting different expression patterns. Intraperitoneal injection of SPX1 significantly increased brain gnrh1, gnih, spx1, gal, and tac3 expression, while it inhibited gnrh2, kiss1r, and kiss2r mRNA levels. In the pituitary, SPX1 injection reduced transcript levels of gh, lhβ, and fshβ. Overall, our results have revealed the involvement of SPX1 in the reproductive functions in yellowtail kingfish.

Leptins and other related genes have been proven to play vital roles in food intake, weight control, and other life activities. While the function of leptins in yellowtail kingfish (Seriola lalandi) has not yet been explored, in the present study, we investigated the structure and preliminary function of four leptin-related genes in S. lalandi. In detail, the sequence of two leptin genes (lepa and lepb), one leptin receptor gene (lepr), and one leptin receptor overlapping transcript (leprot) gene were obtained by homology cloning and RACE methods, in which lepa and lepb have similar structure. Moreover, homologous sequence alignment and evolutionary analysis of all four genes were clustered with Seriola dumerili. The tissue distribution of these four genes in thirteen tissues of yellowtail kingfish was detected by RT-qPCR. Both lepa and leprot were highly expressed in the brain and ovary, while lepb was highly expressed in the pituitary, gill, muscle, and ovary; lepr was highly expressed in the gill, kidney, and ovary. Additionally, these four genes also played roles in embryo development and early growth and development of larvae and juveniles of yellowtail kingfish. Finally, the function of leptin and leptin-related genes was investigated during fasting and re-feeding adaption of yellowtail kingfish. The results showed that these four genes have different regulation functions in five tissues; for example, the mRNA levels of lepa, lepr, and leprot in the brain decreased during fasting and immediately increased after re-feeding, while the mRNA level of lepb did not show significant fluctuation during starvation but significantly lowered after re-feeding. However, lepa and lepb mRNA levels were significantly elevated during fasting and returned to control levels after re-feeding, and there were no significant changes in the expression of lepr and leprot in the liver during fasting and after re-feeding. Moreover, the body mass of fish in the experimental group was measured, and compensatory growth was found after the resumption of feeding. These results suggested that leptin and receptor genes play different functions in different tissues to regulate the physiological state of fish in food deficiency and gain processes.

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Neurokinin B (NKB) is a hypothalamic neuropeptide containing 10 amino acids and is an essential member of the tachykinin family. Based on studies in mammals, Kiss, NKB, and dynorphin A are located in the common neurons, named KNDy neurons, and NKB and Kiss can stimulate the GnRH pulses. In humans, the mutations of NKB and its receptor NK3R can lead to hypogonadotropic hypogonadism and infertility. NKB is also involved in many other physiological activities in mammals and has received increasing attention. NKB was first purified from porcine spinal cord extracts and named neurokinin B or neuromedin K. Subsequently, NKB has been identified in various species, and its encoding gene was named tac2 in ruminants and rodents, and tac3 in other mammals, birds, reptiles, amphibians, and fish. In teleosts, the NKB system was first identified in zebrafish by three laboratories in 2012; it was also found in grass carp, goldfish, Nile tilapia, European eel, orange-spotted grouper, and half-smooth tongue.As teleosts have experienced the third round of genome duplication (3R), the bony fish have two forms of tac3 genes, namely tac3a and tac3b. However, only tac3a is present in highly evolved fish species, such as Nile tilapia and orange-spotted grouper, whereas the tac3b gene has been lost. tac3 in fish and amphibians can encode two mature peptides, NKB and an NKB-related peptide (NKBRP), whereas there is only NKB in mammals, birds, and reptiles. NKB and NKBRP sequences are highly conserved, and sequence analysis showed that NKB and NKBRP share an identical -FXGLM motif at the C-terminus, and X is a hydrophobic or aromatic amino acid residue, which plays an important role in binding to homologous receptors.NKB exerts biological effects by activating the endogenous receptor NK3R. The NK3R receptor belongs to the G protein-coupled receptor family and has a typical seven-transmembrane structure. NK3R is encoded by the tacr3 gene. There are two tacr3 subtypes in teleosts: tacr3a and tacr3b. There was an additional gene, called tacr3a2, in the zebrafish and grass carp, and it was produced by local genome duplication of tacr3a1. The tissue distribution shows that tac3 and tacr3 are widely expressed in the central nervous system and peripheral tissues, indicating that they have essential physiological functions. The specific expression patterns vary depending on the species. They are mainly expressed in the brain, with high expression levels in the pituitary, intestine, and gonads. NKB may participate in regulating reproduction and feeding in teleosts.Currently, studies in teleosts mainly focus on reproductive and feeding regulation. Due to the existence of multiple forms of tac3 and tacr3 genes in teleosts, the action of the NKB system on reproduction control is more complex. Using different experiment methods, such as intraperitoneal injection, intramuscular injection, and incubation of pituitary cells or pituitaries, NKB affected the expression of gnrh, kiss, lhβ, and fshβ and the secretion of LH, FSH, and E2 in fish. The physiological effects varied depending on the gonadal development stages, species, sex, treatment methods, treatment time, and dose. In addition, NKB can act as an anorectic peptide to inhibit food intake and promote gastrointestinal motility. It could also affect the expression of growth-related genes. In summary, as a neurotransmitter or neuromodulator in the central nervous system and a major member of the brain-gut peptides, the NKB system plays an important role in teleosts.Tachykinins activate receptors by coupling to Gαs and Gαq proteins, transducing its signals via PKA and PKC pathways. NKB and NKBRP could activate the PKA/PKC pathway via cognate receptors in some teleosts. This could be verified because different pathway inhibitors could block the effects of NKB and NKBRP. Upon binding to NK3R, NKB induces the secretion of related neuropeptides and the expression of related genes through the AC/cAMP/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-Ⅱ cascades. The C-terminal motif of NKB is crucial to binding receptors; once it is changed, NKB cannot activate the downstream signaling of NK3R, indicating that the integrity of the NKB/NK3R system is essential for the normal functioning of NKB. There are many deficiencies in the research regarding the function of NKB in teleosts. The specific effects and mechanisms of regulating reproductive endocrinology remain unclear, and the functional and signaling interactions between NKB and other neuroendocrine factors such as Kiss, GnIH, and GnRH require further study. In addition, the physiological functions of NKB in fish are mainly focused on reproductive regulation, with less attention given to feeding regulation and other physiological effects.In conclusion, this review provides a summary of the research progress on the NKB system in teleosts, including the identification, tissue distribution, physiological functions, and signaling mechanisms of NKB and its receptors, to enhance the understanding of the NKB system in fish and provide a reference for future research.