Dietary salt concentrations influence growth, nutrient utilization, and fatty acid profiles of turbot (Scophthalmus maximus) reared in brackish water.

Expansion of economically viable turbot (Scophthalmus maximus) aquaculture depends on access to brackish-cold ground water sources in various parts of the world. Since brackish water sources can adversely affect the physiology and zoo technical performance of fish due to the burden of osmoregulation, dietary salt inclusion can alleviate the negative impacts of low-saline waters in several aquaculture species. This study investigated the effects of increasing dietary salt levels on the growth, feed utilization, body composition, and tissue fatty acid composition of juvenile turbot (initial live weight 120.3 ± 0.03 g/fish). Fish were fed five experimental diets supplemented with varying levels of sodium chloride (1.8-6.4%) or a control diet without salt. Each diet was tested in triplicate tanks for 9 weeks. Results showed that increasing dietary salt intake negatively impacted turbot performance, with significant reductions in weight gain, specific growth rate, and feed conversion ratio. Dry matter and ash content in the whole body and filet increased quadratically with increasing salt levels, whereas gill moisture and protein content decreased linearly. Furthermore, the nitrogen, lipid, and energy utilization efficiencies decreased with their respective intake and gain levels. Dietary salt significantly influenced the fatty acid profiles of gill, liver, and filet tissues. In the gill, monounsaturated fatty acids (16:1n-7, ΣMUFA) and n-6 PUFA (20:2n-6) increased, whereas EPA and DHA decreased. Liver ΣSFA (16:0, 18:0) increased, and n-3 PUFA (18:3n-3, 20:5n-3) decreased with increasing dietary salt. Filet saturated fatty acids (14:0, 15:0, 17:0) and n-6 PUFA (20:2n-6, 20:4n-6) increased, while n-3 PUFA (18:3n-3, EPA) decreased with dietary salt. DHA levels in filets showed a quadratic increase. Overall, this study shows that increasing dietary salt negatively impacts turbot growth, feed utilization, and tissue fatty acid composition in brackish water, highlighting the need for further studies on salinity management strategies for turbot aquaculture.

Long-term hypoxia-induced physiological response in turbot Scophthalmus maximus L.

Hypoxia affects fish's survival, growth, and physiological metabolism processes. In this study, turbot plasma glucose and cortisol contents, hepatic glycolysis (hexokinase [HK], phosphofructokinase [PFK], pyruvate kinase [PK]) and lipolysis (fatty acid synthetase [FAS], lipoprotein lipase [LPL]) enzyme activities, anti-oxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px]) activities, malondialdehyde (MDA), lactate and glycogen contents, gill histological parameters (lamellar length [SLL], width [SLW], interlamellar distance [ID]), respiratory frequency (RF), the proportion of the secondary lamellae available for gas exchange (PAGE), and hifs (hif-1α, hif-2α, hif-3α) expression were determined during long-term hypoxia and reoxygenation. Results showed that long-term hypoxia (3.34 ± 0.17 mg L-1) significantly elevated plasma cortisol and glucose contents; increased hepatic HK, PK, PFK, FAS, and LPL activity; decreased hepatic glycogen, lactate contents, and lipid drop numbers; and caused changes of hepatocyte (vacuolation, pyknotic, and lytic nucleus) after treatment for 4 weeks. Hepatic SOD, CAT, GSH-Px activity, and MDA contents; lamellar perimeter, SLL, ID, RF, and PAGE; and hepatic hif-1α, hif-2α, and hif-3α manifested similar results. Meanwhile, hif-1α is significantly higher than hif-2α, and hif-3α. Interestingly, females and males demonstrated no sex dimorphism significantly different from the above parameters (except hepatic FAS, LPL activity, and lipid drop number) under hypoxia. The above parameters recovered to normal levels after reoxygenation treatment for 4 weeks. Thus, long-term hypoxia promotes turbot hepatic glycogenolysis and lipolysis, induces oxidative damage and stimulates hepatic antioxidant capacity, and alters gill morphology to satisfy insufficient energy demand and alleviate potential damage, while hif-1α plays critical roles in the above physiological process.

In-depth histological, lectin-histochemical, immunohistochemical and ultrastructural description of the olfactory rosettes and olfactory bulbs of turbot (Scophthalmus maximus).

Chemical communication through olfaction is crucial for fish behaviours, mediating in socio-sexual behaviours as reproduction. Turbot, a flatfish with significant aquaculture production, possesses a well-developed olfactory system from early developmental stages. After metamorphosis, flatfish acquire their characteristic bilateral asymmetry with an ocular side facing the open water column, housing the dorsal olfactory rosette, and a blind side in contact with the sea bottom where the ventral rosette is located. This study aimed to address the existing gap in specific histological, ultrastructural, lectin-histochemical and immunohistochemical studies of the turbot olfactory rosettes and olfactory bulbs. We examined microdissected olfactory organs of adult turbots and premetamorphic larvae by using routine histological staining techniques, and a wide array of lectins and primary antibodies against G-proteins and calcium-binding proteins. We observed no discernible structural variations in the olfactory epithelium between rosettes, except for the dorsal rosette being larger in size compared to the ventral rosette. Additionally, the use of transmission electron microscopy significantly improved the characterization of the adult olfactory epithelium, exhibiting high cell density, small cell size, and a wide diversity of cell types. Moreover, specific immunopositivity in sensory and non-sensory cells provided us of essential information regarding their olfactory roles. The results obtained significantly enriched the scarce morphological and neurochemical information available on the turbot olfactory system, revealing a highly complex olfactory epithelium with distinct features compared to other teleost species, especially with regard to olfactory cell distribution and immunolabelling patterns.

Dietary fishmeal replacement by Clostridium autoethanogenum protein meal influences the nutritional and sensory quality of turbot (Scophthalmus maximus) via the TOR/AAR/AMPK pathways.

Clostridium autoethanogenum protein (CAP) is a promising protein source for aquaculture; however, how CAP influences fish quality is worth extensive research. We randomly allocated 630 turbot with initial body weights of about 180 g into 6 groups, with fishmeal-based control diet or diet with CAP replacing 15% (CAP15), 30% (CAP30), 45% (CAP45), 60% (CAP60), or 75% (CAP75) of fishmeal protein. After a 70-d feeding trial, the fillet yield (P = 0.015) and content of protein (P = 0.017), collagen (P < 0.001), hydroxyproline (P < 0.001), C20:5n-3 (P = 0.007), and ∑n-3/∑n-6 polyunsaturated fatty acids ratio (P < 0.001) in turbot muscle was found to decrease linearly with increasing CAP. However, turbot fed CAP15 diet maintained these parameters (P > 0.05). By contrast, the muscle hardness increased linearly with increasing CAP (P = 0.004), accompanied by linear reduction of muscle fiber area (P = 0.003) and expression of myogenesis-related genes, including cathepsin D (ctsd P < 0.001) and muscle ring finger protein 1 (murf 1, P < 0.001). Phosphorylation of protein kinase B (Akt, P < 0.001), target of rapamycin (TOR, P = 0.001), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1, P < 0.001), and ribosomal protein S6 (S6, P < 0.001) decreased linearly; however, phosphorylation of AMP-activated protein kinase (AMPK, P < 0.001), eukaryotic initiation factor 2α (eIF2α, P < 0.001), and the abundance of activating transcription factor 4 (ATF4, P < 0.001) increased with increasing CAP, suggesting that the TOR signaling pathway was inhibited, and the amino acid response (AAR) and AMPK pathways were activated. Additionally, expression of genes related to protein degradation, including myogenic factor 5 (myf 5, P < 0.001), myogenic differentiation (myod, P < 0.001), paired box 7 (pax 7, P < 0.001), and ctsd (P < 0.001), decreased linearly with increasing CAP. In conclusion, CAP could be used to replace up to 15% of fishmeal without negatively impacting turbot quality. However, higher levels of CAP decreased fillet yield, muscle protein content, and muscle fiber diameter while increasing muscle hardness, which could be attributed to the inhibition of the TOR pathway and activation of the AAR and AMPK pathways.

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.

Identification and genomic analysis of a pathogenic circovirus associated with maricultured Scophthalmus maximus L. in China.

In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.

EPA and DHA promote cell proliferation and enhance activity of the Akt-TOR-S6K anabolic signaling pathway in primary muscle cells of turbot (Scophthalmus maximus L.).

Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish.

Effect of tea polyphenol-trehalose complex coating solutions on physiological stress and flesh quality of marine-cultured Turbot Scophthalmus maximus during waterless transport.

OBJECTIVE: The waterless transport of live fish has changed the present situation of live-fish transport. However, the waterless transport environment may cause stress in fish. This research evaluated the effect of tea polyphenol-trehalose (TPT) coating solutions on Turbot Scophthalmus maximus during waterless transport. METHODS: After cold acclimation, Turbot were coated and subsequently transported in a waterless environment for 18 h. Physiological and biochemical parameters were measured, including lysozyme (LZM) and immunoglobulin M (IgM) activities, serum creatinine (Cr) and uric acid (UA) concentrations, and nutritional flavor. RESULT: The results showed that the nonspecific immunity of Turbot was inhibited during the waterless transport; the LZM activity first increased and then decreased, and the serum Cr and UA concentrations significantly increased. In addition, the waterless transport promoted the breakdown of Turbot flesh proteins, leading to changes in nucleotides and free amino acids (FAAs). After waterless transport, the LZM and IgM activities in the TPT-treated Turbot were higher than those in the control group (CK), and the changes in FAA content and nucleotides were smaller than those observed in the CK group. CONCLUSION: This study shows that the use of TPT coating solution can reduce the impact of waterless transportation stress on the immune and metabolic functions of Turbot and can maintain the meat quality and flavor of Turbot.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Characterization of ccl20a.3 and ccl20l as gene markers for Th17 cell in turbot.

T-helper 17 lymphocytes (Th17) are the most common inflammatory cells identified in mammals. However, the identification of Th17 cells and the clarification of their function in teleost fish remain largely unknown. In this study, we took advantage of the single-cell RNA sequencing-based immune cell atlas that was identified in turbot (Scophthalmus maximus), and revealed two chemokine-related genes, ccl20a.3 and ccl20l, that were specifically expressed in Th17 cells. Moreover, through immuno-fluorescence analysis, we found that CCL20a.3 or CCL20l was co-expressed with the classical makers in Th17 cells, including IL17a/f1 and IL22. Furthermore, through a Th17 lineage-specific transcription factor RORc inhibitor GSK805 treatment, we found that the expression of ccl20a.3 and ccl20l was significantly impaired, compared with other T cell markers. Besides, we also found that ccl20a.3 and ccl20l exhibited the same dynamic response with the classical markers that were identified in Th17 cells during bacterial infection. Taken together, these results provide potential gene markers for better understanding of the dynamic immune responses of Th17 cells in teleost fish.

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.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1ß, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

Genome-wide identification, evolution and expression analysis of tight junction gene family and the immune roles of claudin5 gene in turbot (Scophthalmus maximus L.).

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost.

Effect of ovarian fluid on sperm performance in teleost with internal and external fertilization strategies.

Ovarian fluid is essential for successful fertilization by maintaining the viability, motility, and velocity of sperm. The organic compounds and inorganic ions in ovarian fluid significantly influence spermatozoa's motility, velocity, and longevity. However, the effect of ovarian fluid on sperm performance is limited in teleost fish. In this study, the effect of ovarian fluid on sperm performance and its components in external fertilization species (Scophthalmus maximus, turbot) and internal fertilization species (Sebastes schlegelii, black rockfish) was investigated using computer-assisted sperm analysis, high-performance liquid chromatography, and metabolome analysis. The ovarian fluid had a distinct and species-specific effect on both species. In the black rockfish, the ovarian fluid from turbot significantly increased sperm motility (74.07% ± 4.09%), as well as VCL (45 ± 1.67 µm/s), VAP (40.17 ± 1.6 µm/s), and VSL (36.67 ± 1.86 µm/s), and longevity (352 ± 11.31 min) (P < 0.05). In the turbot, only the longevity (71.33 ± 5.69 min) and fertilization rate (65.27% ± 11.59%) showed significantly improvement (P < 0.05). The ovarian fluid was rich in organic compounds, suggesting enrichment in the glycolysis/gluconeogenesis pathways. The results suggest that glycometabolism plays a crucial role in improving sperm performance in teleost with internal fertilization. Thus, incorporating ovarian fluid into the sperm activation medium can enhance artificial fertilization in fish breeding.

Identification and functional characterization of caspases in turbot (Scophthalmus maximus) in response to bacterial infection.

Apoptosis is the autonomous and orderly death of cells under genetic control to maintain the stability of the internal environment, and is a programmed cell death process with unique morphological and biochemical properties that is regulated by a variety of factors. Caspase gene family has a significant function in the process of apoptosis. However, the knowledge of caspases in turbot remains largely unknown. In present study, a total of nine turbot caspase genes were identified. The mRNA length of these caspase genes was ranged from 1225 bp (caspase-7) to 3216 bp (caspase-2), and the protein length was ranged from 281 aa (caspase-3a) to 507 aa (caspase-10). Phylogenetic analysis showed these caspase genes were divided into three subfamilies. The qRT-PCR results showed that turbot caspase genes were expressed in all the examined organs, especially the intestine, kidney, blood and gills. Meanwhile, we explored the expression patterns of caspase genes in the intestine, skin and gills after Vibrio anguillarum and Aeromonas salmonids infections. The results showed that caspase genes showed different expression patterns in mucosal tissues after bacterial infection, demonstrating the critical role of caspase genes in mucosal immune responses. In addition, protein-protein interaction analysis showed that caspase proteins interacted with immune molecules such as NLR, IL-1ß, and birc. The results of interference and overexpression experiments showed that caspase-1 might play key roles in the regulation of the IL-1ß production, but the detailed mechanism needs to be further studied. The results of this study provide valuable information for further study the roles of caspase genes in turbot, which could help us to further understand the inflammatory pathways in teleost.

Derivation and characterization of new cell line from intestine of turbot (Scophthalmus maximus).

A continuous intestine cell line from turbot (Scophthalmus maximus) designated as SMI was established utilizing the tissue explant technique. Primary SMI cell was cultured at 24 °C in a medium with 20% fetal bovine serum (FBS), then subcultured in 10% FBS after 10 passages. Impacts of medium or temperature on the growth of SMI were examined and the results indicated it grew well in DMEM supplemented with 10% FBS at 24 °C. The SMI cell line was subcultured more than 60 times. Karyotyping, chromosome number, and ribosomal RNA genotyping analysis revealed that SMI had a modal diploid chromosome number of 44 and originated from turbot. After being transfected with pEGFP-N1 and FAM-siRNA, a large number of green fluorescence signals were observed in SMI, indicating that SMI could be used as an ideal platform to explore gene function in vitro. In addition, the expression of epithelium-associated genes such as itga6, itgb4, gja1, claudin1, zo-1, and E-cadherin in SMI suggested the SMI had some characteristics of epidermal cells. The upregulation of immune-associated genes such as TNF-ß, NF-κB, and IL-1ß in SMI after stimulation with pathogen-associated molecular patterns suggested the SMI might exhibit immune functions similar to the intestinal epithelium in vivo.

Effects of Lysophosphatidylcholine on Intestinal Health of Turbot Fed High-Lipid Diets.

An 8-week feeding trial was conducted, where turbot were fed four experimental diets, containing different LPC levels (0%, 0.1%, 0.25%, and 0.5%, named LPC0, LPC0.1, LPC0.25, and LPC0.5, respectively). The intestinal morphology results showed that there were no widened lamina propria and mixed inflammatory cells in the LPC-supplemented groups. Dietary LPC remarkably decreased the expression of TLRs (TLR3, TLR8, TLR9, and TLR22), MyD88, and signaling molecules (NF-κB, JNK, and AP-1). Similarly, diets with LPC supplementation markedly depressed the gene expression of NF-κB and JNK signaling pathway downstream genes (TNF-α, IL-1ß, Bax, Caspase9, and Caspase-3). Furthermore, dietary LPC modified the intestinal microbial profiles, increasing the relative abundance of short-chain fatty acids-producers, lactic acid bacteria, and digestive enzyme-producing bacteria. Predictive functions of intestinal microbiota showed that turbot fed LPC diets had a relatively higher abundance of functions, such as lipid metabolism and immune system, but a lower abundance of functions, such as metabolic diseases and immune system diseases. The activities of intestinal acid phosphatase and alkaline phosphatase were also increased by dietary LPC. In conclusion, LPC supplementation could regulate the intestinal mucosal barrier via the TLR signaling pathway and alter the intestinal microbiota profile of turbot fed high-lipid diets.

Genetic Mechanism of Tissue-Specific Expression of PPAR Genes in Turbot (Scophthalmus maximus) at Different Temperatures.

In this study, we used PCR to measure the levels of the peroxisome proliferator activated receptor genes PPARα1, PPARα2, PPARß, and PPARγ in the intestine, liver, gill, heart, kidney, brain, muscle, spleen, skin, and stomach of turbot (Scophthalmus maximus) cultured under different temperature conditions (14, 20, 23, 25, and 28 °C). We used split-split-plot (SSP) analysis of variance, additive main effects and multiplicative interaction (AMMI) analysis, and genotype main effects and genotype × environment interaction (GGE) biplot analysis to evaluate the genotype × tissue interaction effects on gene expression. The results of the SSP analysis of variance showed that temperature and tissue × gene have highly significant (p < 0.01) effect on the expression of S. maximus PPAR genes. The AMMI analysis results revealed that the expression of PPAR genes at the appropriate temperature (14 °C) mainly depended on genotype × tissue interaction and tissue effects. Under stress temperatures, genotype effects, tissue effects, and genotype × tissue interaction, all had significant effects on the expression of PPAR genes. The contribution of the genotype effect slowly increased with increasing temperature; it increased faster at 20 °C and then slowly declined at 25 °C. The contribution of the tissue effect slowly increased from 14 to 20 °C, where it sharply decreased, and then it stabilized after a slight fluctuation. The contribution of the genotype × tissue interaction effect showed a fluctuating upward trend throughout the experiment, and it had a significant impact on PPAR gene expression. The key temperature at which the three effects changed was 20 °C, indicating that it is the limit temperature for active lipid metabolism under high-temperature stress. The GGE biplot analysis results showed that under suitable water temperature, the expression difference of PPAR genes in the liver was the largest; at 20 and 23 °C, the expression difference in the gill was the largest; and at 25 and 28 °C, the expression difference in the brain was the largest. Overall, our results suggest that the mechanism responsible for PPAR gene expression under the three high temperatures (23, 25, and 28 °C) was relatively consistent, but it differed from that at 20 °C.