Interannual variability in early life phenology is driven by climate and oceanic processes in two NE Atlantic flatfishes.

Early life phenology is a crucial factor for population dynamics in a climate change scenario. As such, understanding how the early life cycle of marine fishes is influenced by key oceanic and climate drivers is of chief importance for sustainable fisheries. This study documents interannual changes in early life phenology of two commercial flatfishes: European flounder (Platichthys flesus) and common sole (Solea solea) from 2010 to 2015 based on otolith microstructure. Using GAMs, we looked for correlations of the North Atlantic Oscillation (NAO), Eastern Atlantic pattern (EA), sea surface temperature (SST), chlorophyl a concentration (Chla) and upwelling (Ui) variation with the onset of hatch, metamorphosis, and benthic settlement day. We concluded that higher SST, more intensive upwelling, and EA were coincident with a later the onset of each stage, while increasing NAO induces an earlier onset of each stage. Although similar to S. solea, P. flesus showed a more complex interaction with the environmental drivers, most possibly because it is at its southern limit of its distribution. Our results highlight the complexity of the relationship between climate conditions and fish early life history, particularly those with complex life cycles that include migrations between coastal areas and estuaries.

Cathepsin H and cathepsin B of Cynoglossus semilaevis are involved in anti-bacterial immunity against Edwardsiella tarda.

Cathepsin H and Cathepsin B are two lysosomal cysteine proteases participating in various physiological processes including immune responses. In fish, the functional roles of Cathepsin H and Cathepsin B during bacterial infection are less understood. In a previous work, we characterized a Cathepsin B homologue (CsCatB) of half-smooth tongue sole (Cynoglossus semilaevis), an economically valuable fish species in China. In this report, we identified a Cathepsin H homologue (CsCatH) from C. semilaevis. In healthy tongue sole, the transcriptional expression of CsCatH was detected in nine different tissues. Laser scanning confocal microscopic analysis showed that ectopically expressed CsCatH and CsCatB were co-localized with the lysosome. Upon infection by Edwardsiella tarda, a significant fish pathogen which caused a severe fish disease termed edwardsiellosis, the expressions of CsCatH and CsCatB were remarkedly upregulated. The knockdown of CsCatH and CsCatB significantly increased the replication of E. tarda and mitigated E. tarda-induced apoptosis in tongue sole tissues. These findings revealed the importance of CsCatH and CsCatB in anti-bacterial immunity of tongue sole.

A novel miRNA, Cse-miR-33, functions as an immune regulator by targeting CsTRAF6 in Chinese tongue sole (Cynoglossus semilaevis).

The tumor necrosis factor receptor-associated factor 6 (TRAF6) can act as a fundamental adaptor protein in a chain reaction of signal transduction and cascade events to finish off immune defenses. However, immunomodulatory research on TRAF6 gene is still limited in fish. In this study, a novel miRNA, Cse-miR-33 was identified from the whole genome of Chinese tongue sole (Cynoglossus semilaevis). After separate infections with three different Vibrio strains (V. harveyi, V. anguillarum, V. parahemolyticus) and one virus (nervous necrosis virus, NNV), the expressions of CsTRAF6 and Cse-miR-33 displayed significant time-dependent changes in immune related tissues and the trends were opposite in general. Through target gene prediction and dual luciferase reporter assay, Cse-miR-33 was proven to regulate CsTRAF6 by combining with 3'-UTR sequence of the gene. The results of qRT-PCR and western blotting (WB) analyses showed that Cse-miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level, rather than degrading the target mRNA. Further experiment indicated that Cse-miR-33 inhibitor largely reduced the death rate of Chinese tongue sole caused by V. harveyi and NNV. The expressions of CsTRAF6-associated immune genes (such as CsIL-1R, CsMYD88, CsIRAK1, CsTNFα, CsIL6 and CsIL8) were also significantly changed in response to Cse-miR-33 agomir and inhibitor. The study suggested that Cse-miR-33 affected the immune response via targeting CsTRAF6 in C. semilaevis, which would provide us deep insights into miRNA-mediated regulatory network and help improve the immunity in fish.

The Role of the Heat Shock Cognate Protein 70 Genes in Sex Determination and Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis).

Fish sex determination can be affected by environmental temperature. This process relies on temperature-sensitive proteins such as heat shock proteins (HSPs). Our previous work found that heat shock cognate proteins (HSCs) may participate in high-temperature associated sex reversal of Chinese tongue sole (Cynoglossus semilaevis). However, the role of hsc genes in responding to high temperature and affecting sex determination/differentiation remains unclear. Here, by using C. semilaevis as model, we identified hsc70 and hsc70-like. hsc70 was abundant in the gonads with a testicular-higher expression at all gonadal development stages except for 6 months post fertilization (mpf). Intriguingly, hsc70-like showed higher expression in testes from 6 mpf on. Both long-term heat treatment during the temperature-sensitive sex-determining period and short-term heat stress at the end of this period caused different expression of hsc70/hsc70-like between sexes. The dual-luciferase assay results also suggested that these genes can respond to high temperature rapidly in vitro. Heat treatment of C. semilaevis testis cells overexpressed with hsc70/hsc70-like could affect the expression of sex-related genes sox9a and cyp19a1a. Our results indicated that hsc70 and hsc70-like were key regulators linking external high-temperature signals with sex differentiation in vivo and provide a new idea for understanding the mechanism by which high temperature affects sex determination/differentiation in teleosts.

Genotype by Temperature Interaction for Plasma Physiological Indexes in Turbot (Scophthalmus maximus) under Acute Heat Stress─Exploring a Method for Screening Physiological Biomarkers of Nontoxic Stress in Aquatic Environments.

This paper presents a method for exploring the genetic mechanism underlying the plasma physiological indexes under heat stress in aquatic environments and for screening reliable stress biomarkers based on split-split-plot analysis (SSP), additive main effects and multiplicative interaction (AMMI) analysis, and genotype main effects and genotype × environment interaction (GGE) biplot analysis. The methodology developed was illustrated by applying it to a specific turbot heat stress case study. Five plasma physiological indexes (epinephrine, cortisol, alkaline phosphatase, superoxide dismutase, and blood glucose levels) were measured in turbot (Scophthalmus maximus) under acute heat stress at four temperatures (18, 21, 24, and 27 °C) for various exposure times (3, 6, 9, 12, 24, 48, and 72 h). The SSP analysis showed that exposure time and temperature × gene interactions had significant (P < 0.01) effects on the activity/content of turbot plasma physiological indexes. The AMMI analysis showed the following: (1) that at each exposure time, the genotype effect > the genotype × temperature interaction > the temperature effect; (2) that during the whole experiment, the change trend of the contribution of the genotype × temperature interactions was similar to that of the temperature effect, and the changing trends of the contributions of the genotype × temperature interaction and the genotype effect were clearly completely reversed; and (3) that the 3-24-h period was the key period for the changes in the physiological indexes due to acute heat stress. The GGE biplot analysis showed that blood glucose and cortisol levels were reliable biomarkers and could be used as early warning markers for numerical simulations of physiological behavior.

Genome-wide identification and functional characterization of inhibitor of nuclear factor-κB (IκB) kinase (IKK) in turbot (Scophthalmus maximus).

The inhibitor of nuclear factor-κB (IκB) kinase (IKK) is involved in a variety of intracellular cell signaling pathways and is an important component of the NF-κB signaling pathway. IKK genes have been suggested to play important roles in the innate immune response to pathogen infection in both vertebrates and invertebrates. However, little information is available about IKK genes in turbot (Scophthalmus maximus). In this study, six IKK genes were identified including SmIKKα, SmIKKα2, SmIKKß, SmIKKε, SmIKKγ, and SmTBK1. The IKK genes of turbot showed the highest identity and similarity with Cynoglossus semilaevis. Then, phylogenetic analysis showed that the IKK genes of turbot were most closely related to C. semilaevis. In addition, IKK genes were widely expressed in all the examined tissues. Meanwhile, the expression patterns of IKK genes were investigated by QRT-PCR after Vibrio anguillarum and Aeromonas salmonicida infection. The results showed that IKK genes had varying expression patterns in mucosal tissues after bacteria infection, indicating that they may play key roles in maintaining the integrity of the mucosal barrier. Subsequently, protein and protein interaction (PPI) network analysis showed that most proteins interacting with IKK genes were located in the NF-κB signaling pathway. Finally, the double luciferase report and overexpression experiments showed that SmIKKα/SmIKKα2/SmIKKß involved in the activation of NF-κB in turbot. In summary, our results suggested that IKK genes of turbot played important roles in the innate immune response of teleost, and provide valuable information for further study of the function of IKK genes.

Immunity of turbot Induced by inactivated vaccine of Aeromonas salmonicida from the perspective of DNA methylation.

Introduction: DNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear. Methods: In this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida. Results: A total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene. Discussion: We jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Non-synonymous variation and protein structure of candidate genes associated with selection in farm and wild populations of turbot (Scophthalmus maximus).

Non-synonymous variation (NSV) of protein coding genes represents raw material for selection to improve adaptation to the diverse environmental scenarios in wild and livestock populations. Many aquatic species face variations in temperature, salinity and biological factors throughout their distribution range that is reflected by the presence of allelic clines or local adaptation. The turbot (Scophthalmus maximus) is a flatfish of great commercial value with a flourishing aquaculture which has promoted the development of genomic resources. In this study, we developed the first atlas of NSVs in the turbot genome by resequencing 10 individuals from Northeast Atlantic Ocean. More than 50,000 NSVs where detected in the ~ 21,500 coding genes of the turbot genome, and we selected 18 NSVs to be genotyped using a single Mass ARRAY multiplex on 13 wild populations and three turbot farms. We detected signals of divergent selection on several genes related to growth, circadian rhythms, osmoregulation and oxygen binding in the different scenarios evaluated. Furthermore, we explored the impact of NSVs identified on the 3D structure and functional relationship of the correspondent proteins. In summary, our study provides a strategy to identify NSVs in species with consistently annotated and assembled genomes to ascertain their role in adaptation.

A saposin domain-containing protein of tongue sole Cynoglossus semilaevis: Antimicrobial activity and mechanism.

Prosaposin is a precursor that can be processed into four different saposins, designated as A, B, C, and D, which have multiple functions in mammals, including neuroprotection and immune modulation. The immune function of saposin in teleost remains largely unknown. In the present study, a saposin (SAP) domain-containing protein was identified in half-smooth tongue sole Cynoglossus semilaevis and named CsSDP. CsSDP harbors one SAP A domain and two SAP B domains. When expressed in HEK293T cells, CsSDP was specifically localized in the lysosome. When overexpressed in Escherichia coli, CsSDP markedly inhibited bacterial growth, and the inhibitory effect depended on two specific regions in the SAP A and SAP B domains. Two polypeptides (P32 and P30) derived from the above SAP A and B domains could bind to and inhibit the growth of both Gram-negative and Gram-positive bacteria. The ultrastructural analysis revealed that P32 and P30 killed target bacteria by disrupting the bacterial cell wall and inducing substantial release of cytoplasmic contents. These results shed new lights on the immune function of saposin domain-containing protein in teleost.

A soluble TLR5 is involved in PBLs activation and antibacterial immunity via TLR5M-MyD88-signaling pathway in tongue sole Cynoglossus semilaevis.

In higher vertebrates, there is only a membranal TLR5 (TLR5M), which is crucial for host defense against microbes via MyD88 signaling pathway. In teleost, both TLR5M and soluble TLR5 (TLR5S) are identified, whereas the antibacterial mechanism of TLR5S is largely unknown. In this study, we studied the immune antibacterial mechanism of Cynoglossus semilaevis TLR5S homologue (named CsTLR5S). CsTLR5S, a 71.1 kDa protein, consists of 649 amino acid residues and shares 41.7 %-57.8 % overall sequence identities with teleost TLR5S homologues. CsTLR5S contains a single extracellular domain (ECD) composed of 12 leucine-rich repeats. CsTLR5S expression was constitutively identified and upregulated by bacterial infection in tissues. In vitro recombinant CsTLR5S (rCsTLR5S) could interact with bacteria and tongue sole rTLR5M (rCsTLR5M). Furthermore, rCsTLR5S could bind to the membranal CsTLR5M of peripheral blood leukocytes (PBLs), which led to enhancing the activity and the antibacterial role of PBLs via Myd88-NF-κB pathway. In vivo rCsTLR5S could activate the Myd88-NF-κB pathway, facilitate the release of proinflammatory cytokines, and enhance the host antibacterial response against Vibrio harveyi. Moreover, the knockdown of CsTLR5M or the Myd88 inhibitor could significantly suppress the antibacterial effect of rCsTLR5S. Collectively, our findings added important insights into the TLR5S immune antibacterial property in a TLR5M-MyD88-dependent manner.

Comparative analysis of allergenicity and predicted linear epitopes in α and ß parvalbumin from turbot (Scophthalmus maximus).

BACKGROUND: Parvalbumin (PV) can be subdivided into two phylogenetic lineages, αPV and ßPV. The bony fish ßPV is considered a major fish allergen. However, there is no available report on the immunological property and epitope mapping of bony fish αPV. RESULTS: To characterize the allergenic property of bony fish αPV and investigate the difference in allergenic property of bony fish αPV and ßPV, turbot (Scophthalmus maximus) αPV and ßPV were identified by mass spectrometry and were expressed in Escherichia coli system in this study. Spectra analysis and three-dimensional (3D) modeling showed the similar structure between αPV and ßPV. However, αPV exhibited lower immunoglobulin E/immunoglobulin G (IgE/IgG) binding capacity than ßPV. Three identified ßPV epitopes possessed higher IgE reactivity and more hydrophobic residues than three identified αPV epitopes. In addition, less similarity in sequence homology of αPV epitopes was observed with allergen sequences in database. CONCLUSION: These finding expanded information on fish PV epitopes and substantiated the difference in allergenicity and epitope mapping between fish αPV and ßPV, which will improve the epitope-based detection tools of PV and diagnostic of PV induced fish allergy. © 2023 Society of Chemical Industry.

Phylogenetic relationships and the origin of New World soles (Teleostei: Pleuronectiformes: Achiridae): The role of estuarine habitats.

Even though the monophyletic status of Achiridae has been supported by morphological and molecular data, the interrelationships within the representatives of this family are poorly resolved. In the present study, we carried out the most complete molecular phylogenetic analysis of this group, encompassing all genera and employing both nuclear (Rhodopsin, Recombination activator [Rag 1], Mixed - lineage Leukemia [MLL] and Early Growth Response Protein 3 [EGR3]) and mitochondrial (Cytochrome C Oxidase Subunit I [COI], Cytochrome B [CytB], ATPase 6.8, 16S and 12S RNAr) genes. All topologies based on Maximum Likelihood, Bayesian inferences and Bayesian Inference of the Multispecies Coalescent confirmed the monophyletism of Achiridae, in spite of some incongruences in relation to Achirus mucuri, A. lineatus, Apionichthys finis and Trinectes microphthalmus. In fact, Achirus and Trinectes proved to be non-monophyletic genera while Hypoclinemus mentalis was closely related to A. achirus, suggesting this species should be reevaluated. We provided evidence that Achiridae has first arisen in estuaries (about 23.5 million years ago) and some lineages have evolved independently to either marine or freshwater habitats. Furthermore, we propose a diversification scenario of New World soles involving at least two events of marine incursions during Miocene and Pliocene - Pleistocene associated with natural geographic barriers (Victoria-Trindade chain), the width and exposure of continental shelf and headwater capture along the Amazon basin. Finally, the evolutionary dependence of Achirid soles on estuaries, characterized as highly dynamic environments, has probably driven the recent divergence of many species of Achiridae.

The immune-related circRNA-miRNA-mRNA ceRNA regulatory network in the liver of turbot (Scophthalmus maximus L.) induced by Vibrio anguillarum.

Recently, Vibrio anguillarum, a Gram-negative pathogenic bacterium, has been becoming a major constraint on the development of the turbot aquaculture industry because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. Although the functions of protein-coding mRNAs in the immune response against bacterial infection have been reported, as well as several non-coding RNAs (ncRNAs), such as circular RNAs (circRNAs) and microRNAs (miRNAs), the relationships between mRNAs and ncRNAs in the immune system of turbot liver are still limited during bacterial infection. In present study, the comprehensive analyses of whole-transcriptome sequencing were conducted in turbot liver infected by V. anguillarum. The differential expression was analyzed in the data of circRNAs, miRNAs, and mRNAs. The interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted basing on the negative regulatory relationships between miRNAs and their target circRNAs\mRNAs. The circRNA-related ceRNA regulatory networks were constructed for the analyses of regulated mechanism in turbot immune system. Subsequently, the RT-qPCR was carried out to verify the results of sequencing. Finally, we identified 31 circRNAs, 53 miRNAs and 948 mRNAs with differential expression. Gene set enrichment analyses using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that innate immunity was principally activated at the early stages of infection, while adaptive immunity was activated after 24 h. Finally, 65 circRNA-miRNA-mRNA pathways were constructed, based on the hypothesis of ceRNA regulatory networks. In conclusion, our findings provide new insights on the underlying immune response to bacterial infection and identify novel target genes for the prevention and control of disease in turbot.

Vitamin D3 enhances the antibacterial ability in head-kidney macrophages of turbot (Scophthalmus maximus L.) through C-type lectin receptors.

It has been known that vitamin D3 (VD3) not only plays an important role in regulating calcium and phosphorus metabolism in animals, but also has extensive effects on immune functions. In this study, the mechanism how VD3 influences bactericidal ability in turbot was explored. The transcriptomic analysis identified that dietary VD3 significantly upregulated the gene expression of C-type lectin receptors (CLRs), including mannose receptors (mrc1, mrc2, pla2r1) and collectins (collectin 11 and collectin 12) in turbot intestine. Further results obtained from in vitro experiments confirmed that the gene expression of mannose receptors and collectins in head-kidney macrophages (HKMs) of turbot was induced after the cells were incubated with different concentrations of VD3 (0, 1, 10 nM) or 1,25(OH)2D3 (0, 10, 100 pM). Meanwhile, both phagocytosis and bactericidal functions of HKMs were significantly improved in VD3 or 1,25(OH)2D3-incubated HKMs. Furthermore, phagocytosis and bacterial killing of HKMs decreased after collectin 11 was knocked down. Moreover, VD3-enhanced antibacterial activities diminished in collectin 11-interfered cells. Interestingly, the evidence was provided in the present study that inactive VD3 could be metabolized into active 1,25(OH)2D3 via hydroxylases encoded by cyp27a1 and cyp27b1 in fish macrophages. In conclusion, VD3 could be metabolized to 1,25(OH)2D3 in HKMs, which promoted the expression of CLRs in macrophages, leading to enhanced bacterial clearance.

Subtle ecosystem effects of microplastic exposure in marine mesocosms including fish.

For two months, communities in 5.8 m3 outdoor marine mesocosms were exposed to 700 µm sphere-shaped polystyrene (PS) beads in dosages between 0.08 and 80 g/m2. Barnacle (Semibalanus balanoides) densities were reduced at dosages of 0.8 g/m2 onwards without following a standard dose response curve. Lugworms and fish (Solea solea) ingested PS-beads without accumulating them. Lugworms (Arenicola marina) ingested the beads nonselective with the sediment without negative effects. The fish seemed to ingest the plastics only occasionally and at the final sampling day even in the highest dosed mesocosms (>30 beads/cm2) only 20% contained plastic. The condition index of the fish was slightly reduced in mesocosms with dosages of 0.8 g/m2 onwards. No difference in condition was found between fish with and without ingested plastic across mesocosms, illustrating the difficulty to relate plastic ingestion with condition from field data. The fish also ingested mollusks with shells exceeding the size of the PS-beads. Bivalves rejected the PS-beads as pseudofeces, without obvious impact on their condition. Mussel's (Mytilus edulis) pseudofeces present an effective matrix to monitor microplastic presence in the water column. Species richness and diversity of the pelagic and benthic community were not affected although, a trend was found that the lower microplastic dosages had a positive effect on the total abundance of benthic invertebrates. In general, the observed effects at even the highest exposure concentrations were that subtle that they will be obscured by natural variation in the field. This underlines the importance of experiments under semi-field conditions for meaningful assessment of the ecological impact of microplastics. This study was performed with the real life, non-toxic, sphere-shaped polystyrene beads as were lost during an actual spill near the Dutch Wadden sea in January 2019. We recommend future mesocosm studies with other types of microplastics, including microfibers, weathered microplastics from sea, and smaller sized particles down to nanoplastics.

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&nbsp;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.

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.

Additional supplementation of sulfur-containing amino acids in the diets improves the intestinal health of turbot fed high-lipid diets.

An eight-week feeding trial was conducted to investigate the effects of diets supplemented with three sulfur-containing amino acids (SAA), namely, methionine, cysteine, and taurine, on the intestinal health status of juvenile turbot (Scophthalmus maximus) fed high-lipid diets. Four diets were formulated, namely, a high-lipid control diet (16% lipid, HL) and three SAA-supplemented diets, which were formulated by supplementing 1.5% methionine (HLM), 1.5% cysteine (HLC), and 1.5% taurine (HLT) into the HL control diet, respectively. Each diet was assigned to triplicate tanks, and each tank was stocked with 30 juvenile fish (appr. initial weight, 8 g). The histological and morphometric results showed that dietary SAA supplementation obviously improved the intestinal morphology and integrity, in particular as reflected by higher height of microvilli and mucosal folds. Dietary SAA supplementation, in particular cysteine, up-regulated the gene expression of mucin-2 and tight junction proteins (ZO-1, Tricellilun and JAM). Dietary SAA supplementation remarkably down-regulated the gene expression of apoptosis-related factors such as p38, JNK, and Bax, expression of pro-inflammatory factors (e.g., NF-κB, AP-1 IL-1ß, IL-8, and TNF-α). SAA supplementation resulted in higher antioxidative abilities in the intestine. Additionally, dietary SAA supplementation largely altered the communities of intestinal microbiota. Compared with the HL group, higher relative abundance of potential beneficial bacteria, and lower relative abundance of opportunistic pathogens were observed in SAA-supplemented groups. Dietary taurine supplementation significantly increased the relative abundance of Ligilactobacillus (in particular Lactobacillus murinus) and Limosilactobacillus (especially Lactobacillus reuteri). In conclusion, dietary sulfur-containing amino acids supplementation have promising potential in ameliorating the intestinal inflammation of turbot fed high-lipid diets. Especially dietary cysteine and taurine supplementation have more positive effects on the communities of the intestinal microbiota of turbot.

Vitamin D3 deficiency induced intestinal inflammatory response of turbot through nuclear factor-κB/inflammasome pathway, accompanied by the mutually exclusive apoptosis and autophagy.

Vitamin D3 (VD3) participated widely in the nuclear factor-κB (NF-κB)-mediated inflammation, apoptosis, and autophagy through the vitamin D receptor (VDR). However, the molecular mechanisms remain not understood in teleost. The present study investigated the functions of VD3/VDR on intestinal inflammation, autophagy, and apoptosis of turbot in vivo and in vitro. Triple replicates of 30 fish were fed with each of three diets with graded levels of 32.0 (D0), 1012.6 (D1), and 3978.2 (D2) IU/kg VD3. Obvious intestinal enteritis was observed in the D0 group and followed with dysfunction of intestinal mucosal barriers. The intestinal inflammatory response induced by VD3 deficiency was regulated by the NF-κB/inflammasome signalling. The promotion of intestinal apoptosis and suppression of intestinal autophagy were also observed in the D0 group. Similarly, VD3 deficiency in vitro induced more intense inflammation regulated by NF-κB/inflammasome signalling. The mutually exclusive apoptosis and autophagy were also observed in the group without 1,25(OH)2D3 in vitro, accompanied by similar changes in apoptosis and autophagy increased apoptosis. The gene expression of VDRs was significantly increased with the increasing VD3 supplementation both in vivo and in vitro. Moreover, VDR knockdown in turbot resulted in intestinal inflammation, and this process relied on the activation of inflammasome mediated by NF-κB signalling. Simultaneously, intestinal apoptosis was promoted, whereas intestinal autophagy was inhibited. In conclusion, VD3 deficiency could induce intestinal inflammation via activation of the NF-κB/inflammasome pathway, intestinal apoptosis, and autophagy formed a mutually exclusive relation in teleost. And VDR is the critical molecule in those processes.

Effects of long-term sex steroid hormones (estradiol and testosterone)-supplemented feeds on the growth performance of Chinese tongue sole (Cynoglossus semilaevis).

The phenomenon of sexual size dimorphism (SSD), existing in mammals, birds, reptiles, spiders, amphibians, insects, and fishes, is generally related to feeding efficiency, energy allocation, sex steroids, and somatotropic and reproductive endocrine axes. Recently, positive and negative regulations of sex steroids have been reported on SSD in various species. Chinese tongue soles (Cynoglossus semilaevis) at 4 months were fed with 17ß-estradiol (E2) and testosterone (T) supplemented feeds for 8 months to assess the effect of sex steroids on growth traits in different sexes. The potential genetic regulation was examined using several growth-related genes. The results showed that two sex steroid hormones had inhibitory effects on the growth performance of different sexes of C. semilaevis. At the age of 8 months, the expression of insulin-like growth factor 2 gene (igf2), 24-dehydrocholesterol reductase (dhcr24), leptin, and estrogen receptor 2 (esr2) in the liver showed an overall downward trend. The expression of insulin-like growth factor 1 (igf1) was reduced, while thyroid hormone receptor-associated protein 3 (thrap3) expression tended to increase in the gonad after T and E2 treatments. In the brain, somatostatin 1, tandem duplicate 2 (sst1.2) expression increased with the treatment of T and E2 (P < 0.05), while growth hormone-releasing hormone (ghrh) expression decreased. E2 and T had different effects on growth differentiation factor 8 (gdf8) and insulin-like growth factor-binding protein 7 (igfbp7) expression in the muscle. Expression of gdf8 increased in the treated fishes in contrast to the reduction expression of igfbp7. This study provided important clues for understanding the role of sex steroids in flatfish SSD.