Leptins regulate the migration, proliferation, apoptosis, and synthesis of sexual steroid in tongue sole (Cynoglossus semilaevis) ovarian cells.

Leptin is an important hormone in mammals, which plays a key role in regulating reproduction and energy metabolism. However, there are few studies on the function of leptin in reproductive regulation in fish, especially on tongue sole (Cynoglossus semilaevis). Thus, in this study, we firstly exploited the basic function of tongue sole leptins, the migration and growth rate of ovarian cells were reduced after knocking down lepA and lepB in ovarian cells, while increasing the apoptosis rate. Then both rlepA and rlepB were proved to be combined with lepR to further exert functions by dual luciferase assay. Transcriptome sequencing showed that differentially expressed genes (DEGs) were mainly enriched in KEGG pathways related to membrane receptors, fatty acid synthesis, growth, etc. when lepA and lepB were knocked down or additionally added in vitro. Additionally, the estradiol (E2) hormone was increased significantly after knocking down lepB. Finally, based on DEGs and the signaling pathways they participated in, we proposed a hypothesis about the signaling pathways in which leptin may be involved in ovarian cells. Taken together, these results provide new insights into the role of leptin in the regulation of physiological functions such as ovarian growth and development.

Mitochondrial Genome Characteristics Reveal Evolution of Acanthopsetta nadeshnyi (Jordan and Starks, 1904) and Phylogenetic Relationships.

In the present study, the mitochondrial genomic characteristics of Acanthopsetta nadeshnyi have been reported and have depicted the phylogenetic relationship among Pleuronectidae. Combined with a comparative analysis of 13 PCGs, the TN93 model was used to review the neutral evolution and habitat evolution catalysis of the mitogenome to verify the distancing and purification selectivity of the mitogenome in Pleuronectidae. At the same time, a species differentiation and classification model based on mitogenome analysis data was established. This study is expected to provide a new perspective on the phylogenetic relationship and taxonomic status of A. nadeshnyi and lay a foundation for further exploration of environmental and biological evolutionary mechanisms.

Multi-organ transcriptomic profiles and gene-regulation network underlying vibriosis resistance in tongue sole.

Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs.

Immune and regulative characterization of complement-related gene cfhl5 in response to Vibrio harveyi challenge in Cynoglossus semilaevis.

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1ß (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole.

Genome-Wide Identification and Interaction Analysis of Turbot Heat Shock Protein 40 and 70 Families Suggest the Mechanism of Chaperone Proteins Involved in Immune Response after Bacterial Infection.

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

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.

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.

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.

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.

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.

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.

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.