Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

The survival, gene expression, and DNA methylation of Paralichthys olivaceus impacted by the decay of green tide and bacterial infection in both laboratory and field simulation experiments.

The recurring appearance of Ulva prolifera green tides has become a pressing environmental issue, especially for marine transportation, tourism, and aquaculture in the stage of decomposition. An abundance of decaying U. prolifera leads to water acidification, hypoxia and pathogenic microorganism proliferation, threatening marine germplasm resources, particularly benthic organisms with weak escape ability. Epigenetic modification is considered to be one of the molecular mechanisms involved in the plastic adaptive response to environmental changes. However, few studies concerning the specific impact of decaying green tide on benthic animals at the epigenetic level. In this study, decomposing algal effluents of U. prolifera, sediments containing uncorrupted U. prolifera, pathogenic microorganism were considered as impact factors, to reveal the effect of decaying U. prolifera on marine economic benthic species, Paralichthys olivaceus, using both field and laboratory simulation experiments. Field simulation experiment showed higher mortality rates and serious histopathological damage than the laboratory simulation experiment. And both the decaying U. prolifera and the sediment containing U. prolifera were harmful to P. olivaceus. Genome-wide DNA methylation and transcription correlation analyses showed that the response of P. olivaceus to green tide stress and bacterial infection was mainly mediated by immune signaling pathways such as PI3K-Akt signaling pathway. DNA methylation regulates the expression of immune-related genes involved in the PI3K-Akt signaling pathway, which enables P. olivaceus to adapt to the adverse environmental stresses by resisting apoptosis. In summary, this research analyzed the potential role of P. olivaceus in decaying U. prolifera, which is of great significance for understanding the impact of decaying green tide on marine commercial fish and also provides some theoretical guidance for the proliferation and release of fish seedlings.

miR-144 affects the immune response and activation of inflammatory responses in Cynoglossus semilaevis by regulating the expression of CsMAPK6.

MicroRNAs are increasingly recognized for their pivotal role in the immune system, yet the specific regulatory functions of fish-derived microRNAs remain largely unexplored. In this research, we discovered a novel miRNA, Cse-miR-144, in the Chinese tongue sole (Cynoglossus semilaevis), characterized by a 73-base pair precursor and a 21-nucleotide mature sequence. Our findings revealed that the expression of Cse-miR-144 was notably inhibited by various Vibrio species. Utilizing bioinformatics and dual-luciferase assay techniques, we established that the pro-inflammatory cytokine gene CsMAPK6 is a direct target of Cse-miR-144. Subsequent in vitro and in vivo western blotting analyses confirmed that Cse-miR-144 can effectively reduce the protein levels of CsMAPK6 post-transcriptionally. Moreover, CsMAPK6 is known to be involved in the activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Additional investigations using qPCR and ELISA demonstrated that suppression of Cse-miR-144 leads to an upsurge in the liver mRNA levels of various immune genes (including MYD88, TRAF6, NF-κB, TRAF2, TRAF3, and TNF), alongside a marked increase in the production and secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) in the bloodstream of C. semilaevis. These findings collectively underscore the potential of Cse-miR-144 as a key inhibitor of CsMAPK and its crucial role in modulating the immune and inflammatory responses in teleost fish. Compared to the siRNA, miRNA is a better tool in controlling the expression of target gene with a lower cost.

Unraveling the transcriptomic landscape of eye migration and visual adaptations during flatfish metamorphosis.

Flatfish undergo a remarkable metamorphosis from symmetrical pelagic larvae to fully asymmetrical benthic juveniles. The most distinctive features of this transformation is the migration of one eye. The molecular role of thyroid hormone in the metamorphosis process in flatfishes is well established. However, the regulatory network that facilitates eye movement remains enigmatic. This paper presents a morphological investigation of the metamorphic process in turbot eyes, using advanced imaging techniques and a global view of gene expression. The study covers migrant and non-migrant eyes and aims to identify the genes that are active during ocular migration. Our transcriptomic analysis shows a significant up-regulation of immune-related genes. The analysis of eye-specific genes reveals distinct patterns during the metamorphic process. Myosin is highlighted in the non-migrant eye, while ependymin is highlighted in the migrant eye, possibly involved in optic nerve regeneration. Furthermore, a potential association between the alx3 gene and cranial restructuring has been identified. Additionally, it confirmed simultaneous adaptation to low light in both eyes, as described by changes in opsins expression during the metamorphic process. The study also revealed that ocular migration activates systems asynchronously in both eyes, providing insight into multifaceted reorganization processes during metamorphosis of flatfish.

Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure.

Black flounder (Paralichthys orbignyanus, Pleuronectiformes) is a commercially significant marine fish with promising aquaculture potential in Argentina. Despite extensive studies on Black flounder aquaculture, its limited genetic information available hampers the crucial role genetics plays in the development of this activity. In this study, we first employed Illumina sequencing technology to sequence the entire genome of Black flounder. Utilizing two independent libraries-one from a female and another from a male-with 150 bp paired-end reads, a mean insert length of 350 bp, and over 35 X-fold coverage, we achieved assemblies resulting in a genome size of ~ 538 Mbp. Analysis of the assemblies revealed that more than 98% of the core genes were present, with more than 78% of them having more than 50% coverage. This indicates a somehow complete and accurate genome at the coding sequence level. This genome contains 25,231 protein-coding genes, 445 tRNAs, 3 rRNAs, and more than 1,500 non-coding RNAs of other types. Black flounder, along with pufferfishes, seahorses, pipefishes, and anabantid fish, displays a smaller genome compared to most other teleost groups. In vertebrates, the number of transposable elements (TEs) is often correlated with genome size. However, it remains unclear whether the sizes of introns and exons also play a role in determining genome size. Hence, to elucidate the potential factors contributing to this reduced genome size, we conducted a comparative genomic analysis between Black flounder and other teleost orders to determine if the small genomic size could be explained by repetitive elements or gene features, including the whole genome genes and introns sizes. We show that the smaller genome size of flounders can be attributed to several factors, including changes in the number of repetitive elements, and decreased gene size, particularly due to lower amount of very large and small introns. Thus, these components appear to be involved in the genome reduction in Black flounder. Despite these insights, the full implications and potential benefits of genome reduction in Black flounder for reproduction and aquaculture remain incompletely understood, necessitating further research.

Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

pparß regulates lipid catabolism by mediating acox and cpt-1 genes in Scophthalmus maximus under heat stress.

Peroxisome proliferator-activated receptor ß (pparß) is a key gene-regulating lipid metabolism pathway, but its function in turbot remains unclear. In this study, the CDS of pparß was cloned from kidney for the first time. The CDS sequence length was 1533 bp encoding 510 amino acids. Structural analysis showed that the pparß protein contained a C4 zinc finger and HOLI domain, suggesting that the pparß gene of turbot has high homology with the PPAR gene of other species. The high expression patterns of pparß, acox, and cpt-1 at high temperatures, as shown through qPCR, indicated that high temperatures activated the transcriptional activity of pparß and increased the activity of the acox and cpt-1 genes. The expression of acox and cpt-1 was significantly inhibited when pparß was downregulated using RNAi technology and inhibitor treatments, suggesting that pparß positively regulated acox and cpt-1 expression at high temperatures and, thus, modulates lipid catabolism activity. These results demonstrate that pparß is involved in the regulation of lipid metabolism at high temperatures and expand a new perspective for studying the regulation of lipid metabolism in stress environments of teleost.

A comprehensive coding and microRNA transcriptome of vertebral bone in postlarvae and juveniles of Senegalese sole (Solea senegalensis).

Understanding vertebral bone development is essential to prevent skeletal malformations in farmed fish related to genetic and environmental factors. This is an important issue in Solea senegalensis, with special impact of spinal anomalies in postlarval and juvenile stages. Vertebral bone transcriptomics in farmed fish mainly comes from coding genes, and barely on miRNA expression. Here, we used RNA-seq of spinal samples to obtain the first comprehensive coding and miRNA transcriptomic repertoire for postlarval and juvenile vertebral bone, covering different vertebral phenotypes and egg-incubation temperatures related to skeleton health in S. senegalensis. Coding genes, miRNA and pathways regulating bone development and growth were identified. Differential transcriptomic profiles and suggestive mRNA-miRNA interactions were found between postlarvae and juveniles. Bone-related genes and functions were associated with the extracellular matrix, development and regulatory processes, calcium binding, retinol and lipid metabolism or response to stimulus, including those revealed by the miRNA targets related to signaling, cellular and metabolic processes, growth, cell proliferation and biological adhesion. Pathway enrichment associated with fish skeleton were identified when comparing postlarvae and juveniles: growth and bone development functions in postlarvae, while actin cytoskeleton, focal adhesion and proteasome related to bone remodeling in juveniles. The transcriptome data disclosed candidate coding and miRNA gene markers related to bone cell processes, references for functional studies of the anosteocytic bone of S. senegalensis. This study establishes a broad transcriptomic foundation to study healthy and anomalous spines under early thermal conditions across life-stages in S. senegalensis, and for comparative analysis of skeleton homeostasis and pathology in fish and vertebrates.

Effects of acute hypoxia on nutrient metabolism and physiological function in turbot, Scophthalmus maximus.

Acute hypoxia is a common stress in aquaculture, and causes energy deficiency, oxidative damage and death in fish. Many studies have confirmed that acute hypoxia activated hif1α expression, anaerobic glycolysis and antioxidant system in fish, but the effects of acute hypoxia on lipid and protein metabolism, organelle damage, and the functions of hif2α and hif3α in economic fishes have not been well evaluated. In the present study, turbot was exposed to acute hypoxia (2.0 ± 0.5 mg/L) for 6 h, 12 h, and 24 h, respectively. Then, the contents of hemoglobin (HB), metabolite, gene expressions of hifα isoforms, energy homeostasis, endoplasmic reticulum (ER) stress, and apoptosis were measured. The results suggested that turbot is intolerant to acute hypoxia and the asphyxiation point is about 1.5 mg/L. Acute hypoxia induced perk-mediated ER stress, and increased lipid peroxidation and liver injury in turbot. The blood HB level and liver vegfab expression were increased under hypoxia, which enhances oxygen transport. At hypoxia stress, hif3α, anaerobic glycolysis-related genes expression, and lactate content were increased in the liver, and glycogen was broken down to ensure ATP supply. Meanwhile, hif2α, lipid synthesis-related genes expression, and TG content were increased in the liver, but the lipid catabolism and protein synthesis were suppressed during hypoxia, which reduced the oxygen consumption and ROS generation. Our results systematically illustrate the metabolic and physiological changes under acute hypoxia in turbot, and provide important guidance to improve hypoxia tolerance in fish.

Functional relation of agouti signaling proteins (ASIPs) to pigmentation and color change in the starry flounder, Platichthys stellatus.

We investigated the involvement of agouti-signaling proteins (ASIPs) in morphological pigmentation and physiological color change in flatfishes. We isolated ASIP1 and 2 mRNAs from the skin of starry flounder (Platichthys stellatus), and compared their amino acid (aa) structures to those of other animals. Then, we examined the mRNA expression levels of two ASIPs (Sf-ASIPs) in the pigmented ocular body and in the unpigmented blind body, as well as in the ordinary skin and in albino skin, in flatfishes. To investigate the role of Sf-ASIPs in physiological color change (color camouflage), we compared the expression of the two genes in two background colors (dark-green and white). Sf-ASIP1 cDNA had a 375-bp open reading frame (ORF) that encoded a protein consisting of 125 aa residues, and Sf-ASIP2 cDNA had a 402-bp ORF that encoded a protein consisting of 132 aa residues. RT-PCR revealed that the strongest Sf-ASIP1 and Sf-ASIP2 expression levels were observed in the eye and blind-skin, respectively. In Sf-ASIP1, the gene expression did not differ between the ocular-side skin and blind-side skin, nor between ordinary skin and abnormal skin of the fish. However, in Sf-ASIP2, the expression level was significantly higher in blind-side skin, compared to ocular-side skin, suggesting that the ASIP2 gene is related to the countershading body pigment pattern of the fish. In addition, the Sf-ASIP2 gene expression level was lower in the pigmented spot regions than in the unpigmented spot regions of the malpigmented pseudo-albino skins on the ocular side, implying that ASIP2 is responsible for the ocular-side pseudo-albino. Additionally, ASIP2 gene expression in the blind-side skin of ordinary fish was enhanced by a white tank, implying that a bright background color could inhibit hypermelanosis in the blind-side skin of cultured flounder by increasing the activity of the Sf-ASIP2 gene. However, we did not find any relationship of ASIPs with camouflage color changes. In conclusion, the ASIP2 gene is related to the morphological pigmentation (countershading and malpigmentation) of the skin in starry flounder, but not with physiological color changes (color camouflage) in the ocular-side skin.

Integrative taxonomy of Serrasentis gibsoni n. sp. (Acanthocephala: Isthmosacanthidae) from flatfishes in the Gulf of Mexico.

The Isthmosacanthidae acanthocephalan species of the genus Serrasentis are parasites of marine teleosts and an elasmobranch. In this study, Serrasentis gibsoni n. sp. is described from the intestines of four flatfish species (Paralichthyidae), namely Ancyclopsetta quadrocellata, Cyclopsetta chittendeni, Syacium gunteri, and S. papillosum from 10 oceanic sites in the Gulf of Mexico (GoM). Twenty sequences of the 'barcoding' region of cytochrome C oxidase subunit I gene were obtained from 20 adults of Serrasentis gibsoni n. sp. Additionally, five sequences of the barcoding region were obtained from five adults of rhadinorhynchid Gorgorhynchus lepidus from C. chittendeni, S. papillosum and one species of Haemulidae, Haemulom aurolineatum, from five oceanic sites from the GoM. Two phylogenetic approaches were followed: Bayesian inference and maximum likelihood. In both phylogenetic reconstructions, the sequences of Serrasentis gibsoni n. sp. were recovered as a monophyletic group within the genus Serrasentis and placed as a sister group to G. lepidus. However, due to the lack of molecular data for species of the Isthmosacanthidae and Rhadinorhynchidea, these phylogenetic inferences must be taken with caution. Serrasentis gibsoni n. sp. is the first species of Serrasentis described from Paralichthyidae flatfish species from marine waters of the Americas and from the GoM. Based on the barcoding data set analyzed, Serrasentis gibsoni n. sp. appears to have high intraspecific genetic variation; thus, it is necessary to continue exploring the genetic diversity of this species to infer its intraspecific evolutionary patterns.

Assembly of the poorly differentiated Verasper variegatus W chromosome by different sequencing technologies.

The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes.

Genome-Wide Identification and Involvement in Response to Biotic and Abiotic Stresses of lncRNAs in Turbot (Scophthalmus maximus).

Long non-coding RNAs (lncRNAs) play crucial roles in a variety of biological processes, including stress response. However, the number, characteristics and stress-related expression of lncRNAs in turbot are still largely unknown. In this study, a total of 12,999 lncRNAs were identified at the genome-wide level of turbot for the first time using 24 RNA-seq datasets. Sequence characteristic analyses of transcripts showed that lncRNA transcripts were shorter in average length, lower in average GC content and in average expression level as compared to the coding genes. Expression pattern analyses of lncRNAs in 12 distinct tissues showed that lncRNAs, especially lincRNA, exhibited stronger tissue-specific expression than coding genes. Moreover, 612, 1351, 1060, 875, 420 and 1689 differentially expressed (DE) lncRNAs under Vibrio anguillarum, Enteromyxum scophthalmi, and Megalocytivirus infection and heat, oxygen, and salinity stress conditions were identified, respectively. Among them, 151 and 62 lncRNAs showed differential expression under various abiotic and biotic stresses, respectively, and 11 lncRNAs differentially expressed under both abiotic and biotic stresses were selected as comprehensive stress-responsive lncRNA candidates. Furthermore, expression pattern analysis and qPCR validation both verified the comprehensive stress-responsive functions of these 11 lncRNAs. In addition, 497 significantly co-expressed target genes (correlation coefficient (R) > 0.7 and q-value < 0.05) for these 11 comprehensive stress-responsive lncRNA candidates were identified. Finally, GO and KEGG enrichment analyses indicated that these target genes were enriched mainly in molecular function, such as cytokine activity and active transmembrane transporter activity, in biological processes, such as response to stimulus and immune response, and in pathways, such as protein families: signaling and cellular processes, transporters and metabolism. These findings not only provide valuable reference resources for further research on the molecular basis and function of lncRNAs in turbot but also help to accelerate the progress of molecularly selective breeding of stress-resistant turbot strains or varieties.

Genome-wide analysis of BMP/GDF family and DAP-seq of YY1 suggest their roles in Cynoglossus semilaevis sexual size dimorphism.

Sexual size dimorphism (SSD) characterized by different body size between females and males have been reported in various animals. Gonadectomy experiments have implied important regulatory roles of the gonad in SSD. Among multiple factors from the gonad, TGF-ß superfamily (especially BMP/GDF family) attracted our interest due to its pleiotropy in growth and reproduction regulations. Thus, whether BMP/GDF family members serve as crucial regulators for SSD was studied in a typically female-biased SSD flatfish named Chinese tongue sole (Cynoglossus semilaevis). Firstly, a total of 26 BMP/GDF family members were identified. The PPI network analysis showed that they may interact with ACVR2a, ACVR2b, ACVR1, BMPR2, SMAD3, BMPR1a, and other proteins. Subsequently, DAP-seq was employed to reveal the binding sites for yin yang 1 (yy1), a transcription factor involved in gonad function and cell growth partly by regulating TGF-ß superfamily. The results revealed that two yy1 homologues yy1a and yy1b in C. semilaevis could regulate Hippo signaling pathway, mTOR signaling pathway, and AMPK signaling pathway. Moreover, BMP/GDF family genes including bmp2, bmp4, bmp5, gdf6a, and gdf6b were important components of Hippo pathway. In future, the crosstalk among yy1a, yy1b, and TGF-ß family would provide more insight into sexual size dimorphism in C. semilaevis.

Regulatory mechanism of miR-722 on C5aR1 and its functions against bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse-miR-722) was significantly differentially expressed after infection with Vibrio anguillarum, reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse-miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse-miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse-miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse-miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse-miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse-miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse-miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse-miR-722 regulated the expression of CsC5aR1, CsC5a, and the inflammatory factors CsIL1-ß, CsIL6, CsIL8, and CsTNF-α both in vitro and in vivo. In addition, Cse-miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse-miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse-miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost.

Transcriptome-based nutrigenomics analysis reveals the roles of dietary taurine in the muscle growth of juvenile turbot (Scophthalmus maximus).

The present study explored transcriptomics and gene regulation variations in the muscle of turbot fed with dietary taurine. A 70-day feeding trial was conducted using turbot (initial body weight: 3.66 ± 0.02 g) fed with different levels of dietary taurine: 0 % (C), 0.4 % (T2), 1.2 % (T4) and 2.0 % (T6). Two methods were used to analyze and verify the taurine effects on muscle growth: (1) real-time quantitative PCR (qRT-PCR) for the key muscle growth-related genes and (2) transcriptomic analysis by next-generation sequencing (NGS). The results showed that 1.2 % of dietary taurine supplementation significantly increased the expression of muscle growth stimulatory genes, including TauT, myoD, Myf5, myogenin and follistatin. And also, the 1.2 % level significantly decreased the expression of the muscle growth-restricting gene (myostatin). Meanwhile, transcriptomics analysis found that 1.2 % dietary taurine supplementation significantly increased the number of up-regulated genes linked to metabolic pathways. In contrast, taurine significantly enriched the actin cytoskeleton and metabolic pathways in the T4 and T2 groups, respectively. These findings align with the gene ontology (GO) analysis, which indicated a higher number of cellular component (CC) gene expressions at a 1.2 % of dietary taurine compared to a 0.4 % of dietary taurine supplementation. In conclusion, dietary taurine had positive impacts on the growth-stimulatory genes. Moreover, 1.2 % of dietary taurine supplementation is important to the metabolic pathway enrichment.

Genome-Wide Identification of Aqp Family Related to Spermatogenesis in Turbot (Scophthalmus maximus).

The development and maturation of sperm entails intricate metabolic processes involving water molecules, amino acids, hormones, and various substances. Among these processes, the role of aquaporins (aqps) in the testis is crucial. Turbot (Scophthalmus maximus) is a significant marine flatfish species in China; however, natural egg laying in females is not feasible under cultured conditions. Consequently, artificial insemination becomes necessary, requiring the retrieval of sperm and eggs through artificial methods. In this study, we combined genomic, transcriptomics, RT-qPCR, computer-assisted sperm analysis (CASA), and immunohistochemistry to investigate the involvement of the aqp family in spermatogenesis in turbot. Through genomic data analysis, we identified 16 aqps genes dispersed across 13 chromosomes, each exhibiting the characteristic major intrinsic protein (MIP) domain associated with AQPs. The results from RNA-seq and RT-qPCR analysis revealed prominent expression of aqp4, 10, and 12 during the proliferative stage, whereas aqp1 showed primary expression during the mature stage. aqp11 displayed high expression levels during both MSII and MSV stages, potentially contributing significantly to the proliferation and maturation of male germ cells. Conversely, aqp8 showed elevated expression levels during the MSIII, MSIII-IV, and MSIV stages, suggesting its direct involvement in spermiogenesis. Immunohistochemical analysis unveiled the predominant localization of AQP1 protein in male germ cells rather than Sertoli cells, specifically concentrated in the head of sperm within cysts. Furthermore, a noteworthy decline in sperm motility was observed when sperm were subjected to treatment with either the AQP1-specific inhibitor (HgCl2) or the AQP1 antibody. However, no direct correlation was found between the expression of Smaqp1 and sperm quality. Overall, these findings provide new insights into the involvement of aqps in teleost spermatogenesis. Moreover, they hold potential for improving techniques related to sperm activation and cryopreservation, offering valuable knowledge for future advancements in this field.

Genome-wide characterization of integrin (ITG) gene family and their expression profiling in half-smooth tongue sole (Cynoglossus semilaevis) upon Vibrio anguillarum infection.

Integrins (ITGs) are transmembrane heterodimer receptors with ITGα subunit and ITGß subunit, participating in various physiological processes, including immunity. At present, systematic research on ITGs in teleost is scarce, especially in half-smooth tongue sole (Cynoglossus semilaevis). In this study, a set of 28 ITG genes in half-smooth tongue sole have been identified and characterized. The phylogenetic analysis showed that ITGα and ITGß subunits were respectively classified into five and two clusters, consistent with previous studies. The selection pressure analysis indicated that most of ITG genes were under purifying selection, except for ITGα11b and ITGαL with positive selection. The expression profiles of eight selected ITG genes, including ITGα1, ITGα5, ITGα8, ITGα11, ITGß1, ITGß2, ITGß3, and ITGß8, were analyzed in healthy tissues and after infection with Vibrio anguillarum, revealed their implications in immune response. The study provided a comprehensive characterization and expression analysis of ITG genes in half-smooth tongue sole, setting a solid foundation for further functional studies and promising potential in disease control.

Identification and Functional Analysis of foxo Genes in Chinese Tongue Sole (Cynoglossus semilaevis).

The Chinese tongue sole (Cynoglossus semilaevis) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation mechanisms receives a great deal of attention. Forkhead Box O (FoxO) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that foxo genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six Csfoxo members (Csfoxo1a, Csfoxo3a, Csfoxo3b, Csfoxo4, Csfoxo6-like, and Csfoxo1a-like) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4. The siRNA-mediated knockdown of the Csfoxo1a, Csfoxo3a, and Csfoxo3b genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of foxo's function and provide valuable data for studying the male differentiation of tongue sole.

Genome-wide chromatin accessibility and gene expression profiling during flatfish metamorphosis.

Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.