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High-resolution and high-throughput genotype-to-phenotype studies in fish are rapidly advancing, driven by innovative technologies that aim to address the challenges of modern breeding models. In recent years, machine vision and deep learning techniques, particularly convolutional neural networks (CNNs), have achieved significant success in image recognition and segmentation. Moreover, qualitative and quantitative analysis of disease resistance has always been a crucial field of research in genetics. This motivation has led us to investigate the potential of large yellow croaker visceral white-nodules disease (VWND) in encoding information on disease resistance for the task of accession classification. In this study, we proposed an image segmentation framework for the feature extraction of the spleen after VWND infection based on machine vision. We utilized deep CNNs and threshold segmentation for automatic feature learning and object segmentation. This approach eliminates subjectivity and enhances work efficiency compared to using hand-crafted features. Additionally, we employed spleen-related traits to conduct genome-wide association analysis (GWAS), which led to the identification of 24 significant SNPs and 10 major quantitative trait loci. The results of function enrichment analysis on candidate genes also indicated potential relationships with immune regulation mechanisms. Furthermore, we explored the use of genomic selection (GS) technology for phenotype prediction of extreme individuals, which further supports the predictability of spleen-related phenotypes for VWND resistance in large yellow croakers. Our findings demonstrate that artificial intelligence (AI)-based phenotyping approaches can deliver state-of-the-art performance for genetics research. We hope this work will provide a paradigm for applying deep learning and machine vision to phenotyping in aquaculture species., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Feed terrestrial components can induce intestinal stress in fish, affecting their overall health and growth. Recent studies suggest that seaweed products may improve fish intestinal health. In this experiment, three types of feed were prepared: a basic diet (C group), a diet with 0.2 % fucoidan (F group), and a diet with 3 % kelp powder (K group). These diets were fed to large yellow croaker (Larimichthys crocea) over an 8-week period. Each feed was randomly assigned to three seawater cages (4.0 m × 4.0 m × 5.0 m) containing 700 fish per cage. The study assessed changes in growth and intestinal health, including intestinal tissue morphology, digestive enzyme activities, expression of immune-related genes, and bacterial community structure. Results showed that incorporating seaweed products into the diet improved the growth and quality traits of large yellow croakers and significantly enhanced their intestinal digestive capacity (P < 0.05). Specifically, the 0.2 % fucoidan diet significantly increased the intestinal villus length and the activities of digestive enzymes such as trypsin, lipase, and α-amylase (P < 0.05). The 3 % kelp powder diet significantly enhanced the intestinal crypt depth and the activities of trypsin and lipase (P < 0.05). Both seaweed additives significantly enhanced intestinal health by mitigating inflammatory factors. Notably, the control group's biomarkers indicated a high presence of potential pathogenic bacteria, such as Streptococcus, Pseudomonas, Enterococcus, Herbaspirillum, Neisseria, Haemophilus, and Stenotrophomonas. After the addition of seaweed additives, these bacteria were no longer the indicator bacteria, while the abundance of beneficial bacteria like Ligilactobacillus and Lactobacillus increased. Significant reductions in the expression of inflammatory factors (e.g., il-6, tnf-α, ifn-γ in the fucoidan group and il-8 in the kelp powder group) further supported these findings. Our findings suggested that both seaweed additives helped balance intestinal microbial communities and reduce bacterial antigen load. Considering the effects, costs, manufacturing, and nutrition, adding 3 % kelp powder to the feed of large yellow croaker might be preferable. This study substantiated the beneficial effects of seaweed on the aquaculture of large yellow croaker, particularly in improving intestinal health. These findings advocated for its wider and more scientifically validated use in fish farming practices., Competing Interests: Declaration of competing interest All authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pseudomonas plecoglossicida is one of most important pathogenic bacterial species in large yellow croaker and several other commercially valuable fish species. In our previous study, a GacS deficient mutant (ΔgacS) was constructed and its virulence showed substantially attenuated. In present study, the safety, immunogenicity and protective effect of the ΔgacS were evaluated in large yellow croaker as a live-attenuated vaccine candidate. It was shown that the ΔgacS strain exhibited good safety to large yellow croaker and there was no mortality or clinical symptoms observed in all fish that infected by ΔgacS strain with the doses range from 2 × 10 5 ～10 7  CFU per fish via intraperitoneal injection (IP) or immersion (IM), and almost all bacteria were cleaned up in the spleen of the fish at 14-day post infection. Specific antibodies could be detected at 7-day and 14-day post infection by direct agglutination method, and the valences of antibodies and bactericidal activities of the serum were significant increased with vaccination doses and vaccination time. Moreover, the expressions of some molecules and cytokines involved in specific immune responses were detected in the ΔgacS strain immunization group and control group. After challenged by the wild-type (WT) strain XSDHY-P, the relative percentage survival (RPS) showed highly correlated with the immunized dosage regardless of vaccination methods. It showed that the RPS of the IP groups were 39.47 %, 57.89 %, 71.05 % with the immune dosage in a descending order, respectively, and the RPS of the IM groups were 26.31 %, 36.84 %, 76.31 % with the immune dosage in a descending order, respectively. In summary, the ΔgacS strain exhibited safety and good protective effect to large yellow croaker and was a potential live vaccine candidate., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Galectin-8 (Gal-8) is a versatile carbohydrate-binding protein with pivotal roles in immune regulation and cellular processes. This study introduces a novel galectin-8 protein, LcGal-8, from the large yellow croaker (Larimichthys crocea), showcasing typical characteristics of tandem-repeat-type galectins, including the absence of a signal peptide or transmembrane region and the presence of conserved sugar-binding motifs. Phylogenetic analysis reveals its conservation among fish species. Expression profiling indicates widespread distribution in immune tissues, particularly the spleen, implicating involvement in immune processes. The subcellular localization analysis reveals that LcGal-8 is present in both the cytoplasm and nucleus. Upon bacterial challenge, LcGal-8 is up-regulated in immune tissues, suggesting a role in host defense. Functional assays demonstrate that LcGal-8 can agglutinate gram-negative bacteria. The recombinant LcGal-8 protein agglutinates red blood cells from the large yellow croaker independently of Ca 2 ⁺, however, this activity is inhibited by lipopolysaccharide (LPS) at 2.5 μg/mL. Fluorescence detection kits and scanning electron microscopy (SEM) confirm the agglutination and bactericidal effects of LcGal-8 against various gram-negative bacteria, including Vibrio harveyi, Aeromondaceae hydrophila, Aeromondaceae veronii, Pseudomonas plecoglossicida, Edwardsiella tarda. These findings contribute valuable insights into the genetic basis of disease resistance in the large yellow croaker and could support molecular breeding strategies to enhance disease resistance., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

The non-specific cytotoxic cell receptor protein 1 (NCCRP1) is considered the universal marker for teleost non-specific cytotoxic cells (NCCs). However, the specific distribution characteristics and response patterns of NCCRP1, and the confirmed existence of NCCs in fish species remain debatable. In this study, we investigated the distribution of NCCRP1 in the croaker and observed the most dominant abundance in the head kidney. While most common markers of cytotoxicity were localized in the trunk kidney lymphocytes (TKLs) and spleen lymphocytes (SPLs), NCCRP1-positive cells were predominantly detected in head kidney lymphocytes (HKLs) with a positive rate of approximately 10 %, where present a huge amount of macrophages (Mϕ) as well. Furthermore, the remarkable induction evidence of NCCRP1 in HKLs was determined. Collectively, these findings contribute significantly to comprehending the immunological function of NCCRP1 in fish species and enhancing our understanding of its evolutionary development., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As an adaptor protein functions essentially in the activation of NF-κΒ and MAPK signaling pathways mediated by NOD1 and NOD2, RIP2 plays important roles in the host innate immune responses. In the present study, the RIP2 ortholog termed Lc-RIP2 was identified and characterized in large yellow croaker (Larimichthys crocea). It was revealed that Lc-RIP2 is consisted of an open reading frame (ORF) of 1695 bp, encoding a protein of 564 aa, with an N-terminal kinase domain and a C-terminal caspase activation and recruitment domain (CARD). Subcellular localization assays demonstrated that Lc-RIP2 was a cytosolic protein, which was broadly distributed in the examined tissues/organs, and could be induced in response to poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo according to qRT-PCR analysis. Notably, Lc-RIP2 overexpression in vitro was sufficient to abolish SVCV proliferation in EPC cells, and could significantly induce the activation of NF-κB, IRF3, IRF7, and IFN1 promoters. In addition, luciferase assays found that Lc-RIP2 could cooperate with Lc-MAVS, Lc-TRAF3, Lc-TRAF6, Lc-IRF3, and Lc-IRF7 in NF-κB activation, associate with Lc-TRIF, Lc-MAVS, Lc-TRAF3, Lc-IRF3, and Lc-IRF7 in IRF3 activation, enhance Lc-TRIF, Lc-MAVS, Lc-TRAF3, and Lc-TRAF6 mediated IRF7 activation, and Lc-IRF3 mediated IFN1 activation, whereas suppress NF-κB activation when co-expressed with Lc-TRIF. Co-immunoprecipitation (Co-IP) assays also demonstrated that Lc-RIP2 interacts separately with Lc-TRIF, Lc-MAVS, Lc-TRAF3, Lc-TRAF6, Lc-IRF3, and Lc-IRF7. It is thus collectively indicated that Lc-RIP2 function dominantly in the regulation of the host innate immune signaling., (Copyright © 2024. Published by Elsevier Ltd.)

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Glycerol monolaurate (GML) is a potential candidate for regulating metabolic syndrome and inflammatory response. However, the role of GML in modulating intestinal health in fish has not been well determined. In this study, a 70-d feeding trial was conducted to evaluate the effect of GML on intestinal barrier, antioxidant capacity, inflammatory response and microbiota community of large yellow croaker (13.05 ± 0.09 g) fed with high level soybean oil (SO) diets. Two basic diets with fish oil (FO) or SO were formulated. Based on the SO group diet, three different levels of GML 0.02% (SO0.02), 0.04% (SO0.04) and 0.08% (SO0.08) were supplemented respectively. Results showed that intestinal villus height and perimeter ratio were increased in SO0.04 treatment compared with the SO group. The mRNA expressions of intestinal physical barrier-related gene odc and claudin-11 were significantly up-regulated in different addition of GML treatments compared with the SO group. Fish fed SO diet with 0.04% GML addition showed higher activities of acid phosphatase and lysozyme compared with the SO group. The content of malonaldehyde was significantly decreased and activities of catalase and superoxide dismutase were significantly increased in 0.02% and 0.04% GML groups compared with those in the SO group. The mRNA transcriptional levels of inflammatory response-related genes (il-1β, il-6, tnf-α and cox-2) in 0.04% GML treatment were notably lower than those in the SO group. Meanwhile, sequencing analysis of bacterial 16S rRNA V4-V5 region showed that GML addition changed gut microbiota structure and increased alpha diversity of large yellow croaker fed diets with a high level of SO. The correlation analysis results indicated that the change of intestinal microbiota relative abundance strongly correlated with intestinal health indexes. In conclusion, these results demonstrated that 0.02%-0.04% GML addition could improve intestinal morphology, physical barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis of large yellow croaker fed diets with a high percentage of SO., Competing Interests: Declaration of competing interest The authors announce no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

The large yellow croaker (Larimichthys crocea) is the most productive mariculture fish in China, and its aquaculture scale is expanding along the southeastern coast of China, but that development is causing environmental damage by increasing the use of antibiotics and other chemicals. How to improve fish immunity through non-antibiotic substances is still a problem facing aquaculture industry. At present, the experiments have shown that Isaria cicadae spent substrate (IC) can improve the growth performance and immunity of Oreochromis niloticus. Therefore, I. cicadae may be a natural alternative to antibiotic for aquaculture. In order to study the effects of IC on growth performance, serum biochemical indices, intestinal microbiota, and intestinal metabolism of large yellow croakers, the fish were divided into three groups with three replicates in each group. Basal diet, basal diet with 2% and 6% IC supplementation (IC2 and IC6 groups), respectively. The results showed that weight gain rate (WG) and specific growth rate (SGR) of large yellow croaker significantly increased (P < 0.05) in IC6 group. The content of triglyceride (TG), low density lipoprotein cholesterol (LDL-C), total protein (TP) and albumin (ALB) increased significantly (P < 0.05), and total cholesterol (T-CHO) decreased significantly (P < 0.05) in IC2 group. Compared to IC0 group, the activity of malondialdehyde (MDA) , superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased significantly (P < 0.05) in IC2 group, the activity of total antioxidant capacity (T-AOC) and GSH-Px increased significantly (P < 0.05) in IC6 group, and the activity of lysozyme (LZM) increased significantly in IC2 and IC6 groups. The addition of IC in the diets significantly increased the diversity of the microbial community in the intestine of large yellow croaker (P < 0.05), significantly improved the relative abundance of Acidobacteriota (P < 0.05) at the phylum level, and reduced the relative abundance of Bacteroidota, Desulfobacterota, and Synergistota (P < 0.05). At the genus level, the relative abundance of Bacteroides, Cetobacterium and Mycoplasma, which are dominant bacteria in fish gut, significantly increased (P < 0.05). The relative abundance of Ruminofilibacter, Desulfomicrobium, DMER64, Syntrophomonas, Hydrogenophaga, and Aminobacterium reduced significantly (P < 0.05). Among them, Ruminofilibacter, DMER64, Syntrophomonas and Hydrogenophaga are bacteria that can participate in the hydrolysis and acidification of organic matter, while DMER64 is the hydrogen carrier. The intestinal metabolome analysis showed that IC could improve metabolic composition and function, which was related to host immunity and metabolism. In conclusion, I. cicadae can improve the growth performance, regulate the lipid metabolism and immune and antioxidant capacity of large yellow croakers by regulating intestinal microbiota and intestinal metabolism. This study provides a reference for the application of IC in aquaculture., Competing Interests: Declaration of competing interest All the authors declare there are no conflict of interest in this work., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ladderlectin is unique C-type lectin because it has been so far found only in teleost fish. In this study, large yellow croaker (Larimichthys crocea) Ladderlecin (LcLL) sequence was identified and characterized. LcLL encodes a polypeptide of 186 amino acids that includes a signal peptide and a C-type lectin-like domains (CTLD) with two sugar-binding motifs of WSD and EPN. Tissues distribution analysis revealed that LcLL is a ubiquitous gene, with the highest expression in head kidney and gill. Subcellular localization showed that LcLL was in cytoplasm and nucleus of HEK 293T cells. Transcripts of LcLL were significantly up regulated after immune challenge with P. plecoglossicida. In contrast to this, a sharp down-regulation occurred after Scuticociliatida infection. Moreover, recombinant LcLL (rLcLL) was prepared and exhibited hemagglutination on L. crocea and N. albiflora erythrocytes in a Ca 2+ -dependent manner, which can be only inhibited by LPS. rLcLL showed a strong ability of binding to Gram + bacteria (M. lysodeikticus, S. aureus, B. subtilis) and Gram-bacteria (P. plecoglossicida, E. coli, V. Vulnificus, V. harveyi, V. alginolyticus, V. parahaemolyticus. A. hydrophila, and E. tarda), and could agglutinate all tested bacteria except for P. plecoglossicida. Further study showed that rLcLL promoted the gathered bacteria death through damaging cell membrane based on PI staining and SEM observation. However, rLcLL does neither kill bacteria directly nor have complement-activating activities. Altogether, these results demonstrated that LcLL played a vital role in L. crocea innate immune towards bacterial and parasitic challenge., (Copyright © 2023. Published by Elsevier Ltd.)

With the antibiotics prohibition in feedstuffs worldwide, antimicrobial peptides (AMPs) are considered a more promising substitute for antibiotics to be used as feed additives, and positive results have been reported in livestock feeding studies. However, whether dietary supplementation of AMPs could promote the growth of mariculture animals such as fish and the underlying mechanism has not been elucidated yet. In the study, a recombinant AMP product of Scy-hepc was used as a dietary supplement (10 mg/kg) to feed mariculture juvenile large yellow croaker (Larimichthys crocea) with an average initial body weight (BW) of 52.9 g for 150 days. During the feeding trial, the fish fed with Scy-hepc showed a significant growth-promoting performance. Especially at 60 days after feeding, fish fed with Scy-hepc weighed approximately 23% more than the control group. It was further confirmed that the growth-related signaling pathways such as the GH-Jak2-STAT5-IGF1 growth axis, the PI3K-Akt and Erk/MAPK pathways were all activated in the liver after Scy-hepc feeding. Furthermore, a second repeated feeding trial was scheduled for 30 days using much smaller juvenile L. crocea with an average initial BW of 6.3 g, and similar positive results were observed. Further investigation revealed that the downstream effectors of the PI3K-Akt pathway, such as p70S6K and 4EBP1, were significantly phosphorylated, suggesting that Scy-hepc feeding might promote translation initiation and protein synthesis processes in the liver. Taken together, as an effector of innate immunity, AMP Scy-hepc played a role in promoting the growth of L. crocea and the underlying mechanism was associated with the activation of the GH-Jak2-STAT5-IGF1 axis, as well as the PI3K-Akt and Erk/MAPK signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography-tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection., Competing Interests: Declaration of competing interest The authors declare that they have no commercial interests or personal relationships in the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

NEMO (nuclear factor-κB essential modulator) plays an important role in activating NF-κB signaling pathway, p65 is a pivotal positive-regulator of NF-κB family. However, the role of NEMO in p65-triggered immune activation in teleost is largely unknown. In the present study, the cDNA sequence of LcNEMO was identified from the large yellow croaker (Larimichthys crocea). The predicated LcNEMO protein encoded 565 amino acids, consisting of a N-terminal NEMO domain, followed by two coiled coil (CC) motifs, a CC2-leucine zipper (CC2-LZ) domain, and a C-terminal zinc finger (ZnF) domain. Quantitative PCR showed that the strongest constitutive expression of LcNEMO was detected in blood and the inductive expression of it significantly enhanced after LPS and poly I:C challenge. The effect of LcNEMO on p65, RelB and cRel associated-immune activation detected by dual-luciferase reporter system assay indicated that Lcp65-triggered NF-κB, TNF-α and IL-1β activation could be significantly enhanced by LcNEMO. Furthermore, Co-IP revealed that the protein-protein interaction was existed between LcNEMO and Lcp65. Western blot and confocal microscope observation displayed that Lcp65 nuclear translocation could be promoted by LcNEMO with a dose- and time-dependent manner, which was further verified by RNA interference of LcNEMO expression. Our findings suggest that LcNEMO may be crucial in immune response by promoting p65-mediated immune activation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The terminal differentiation of B cells into plasma cells is central to the generation of protective, long-lived humoral immune responses. In mammals, interleukin-2 (IL-2) has been shown to play a role in B cell proliferation and differentiation. However, it remains unclear whether fish IL-2 is involved in B cell proliferation and differentiation. To this end, we investigated the regulatory role of IL-2 in B cell proliferation and differentiation in large yellow croaker (Larimichthys crocea). We found that L. crocea IL-2 (LcIL-2) significantly increased IgM +  B cells proliferation both in vivo and in vitro and facilitated IgM +  B cells differentiation into plasma cells. Furthermore, LcIL-2 increased the production of specific antibodies after immunization with the Vibrio alginolyticus subunit vaccine, recombinant dihydrolipoamide dehydrogenase (rDLD); simultaneous administration of LcIL-2 and rDLD prior to challenge with Vibrio parahaemolyticus or V. alginolyticus significantly increased relative percent survival. Mechanistically, LcIL-2 promoted B cell proliferation and regulated B cell differentiation by triggering the JAK-STAT5 signaling pathway. Collectively, our results demonstrated that LcIL-2 improved B cell proliferation and specific antibody production via the conserved JAK-STAT5 signaling pathway in large yellow croaker, providing valuable insights into the mechanisms underlying the IL-2-mediated regulation of the humoral immune response in fish., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As a member of the tumor necrosis factor receptor-associated factor (TRAF) family, TRAF5 acts as a crucial adaptor molecule and plays important roles in the host innate immune responses. In the present study, the typical form and a splicing variant of TRAF5, termed Lc-TRAF5&#95;tv1 and Lc-TRAF5&#95;tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-TRAF5&#95;tv1 protein is constituted of 577 aa, contains a RING finger domain, two zinc finger domains, a coiled-coil domain, and a MATH domain, whereas Lc-TRAF5&#95;tv2 protein is constituted of 236 aa and only contains a RING finger domain due to a premature stop resulted from the intron retention. Subcellular localization analysis revealed that both of Lc-TRAF5&#95;tv1 and Lc-TRAF5&#95;tv2 were localized in the cytoplasm, with Lc-TRAF5&#95;tv2 found to aggregate around the nucleus. It was revealed that Lc-TRAF5&#95;tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-TRAF5&#95;tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo. Interestingly, overexpression of Lc-TRAF5&#95;tv1 and Lc-TRAF5&#95;tv2 could significantly induce NF-κB but not IFN1 activation, whereas co-expression of them remarkably induced IFN1 activation but impaired NF-κB activation. In addition, both Lc-TRAF5&#95;tv1 and Lc-TRAF5&#95;tv2 were associated with TRAF3 and RIP1 in IFN1 activation, whereas only Lc-TRAF5&#95;tv1 cooperated with TRAF3 and RIP1 in NF-κB activation. These results collectively indicated that the splicing variant together with the typical form of TRAF5 function importantly in the regulation of host immune signaling in teleosts., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this study, we conducted functional analyses for two ISG15 homologues of Larimichthys crocea (LcISG15-1 and LcISG15-2). Our results of qRT-PCR showed that both LcISG15-1 and LcISG15-2 were significantly changed in head kidney and peripheral blood, after poly (I:C) stimulation. Western blot analyses with prepared polyclonal antibodies suggested that LcISG15-1 and LcISG15-2 both could be secreted by primary head kidney lymphocytes into the extracellular milieu. The purified recombinant LcISG15-1 (rLcISG15-1) and LcISG15-2 (rLcISG15-2) could both activate primary macrophages as extracellular cytokines and significantly enhance macrophage respiratory burst, NO production and bactericidal activity and induce the expression of proinflammatory cytokine genes of the cells. Moreover, rLcISG15-2 exhibited much stronger cytokine-like activities than those of rLcISG15-1, indicating the ISG15-2 gene copy evolved enhanced activity after gene duplication of ISG15 in sciaenid fishes. These results indicated important roles of LcISG15-1 and especially LcISG15-2 in immune regulation and host immune defense of large yellow croaker against viral and bacterial infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Membrane-associated RING-CH (MARCH) family, as Ring-type E3 ligases, have attracted extensive attention to their immune functions. MARCH4 plays an essential role in regulating immune response in mammal. In the present study, it is the first to report on MARCH4 characteristics and signal pathway in fish. MARCH4 in large yellow croaker Larimichthys crocea (named as LcMARCH4) encodes a RING-CH domain and two TM domains, as well as other function domains, including an N-terminal proline rich domain, an AxxxG-motif in TM1, a tyrosine-based YXXØ motif, and a C-terminal PDZ-binding domain. LcMARCH4 is a tissue-specific protein with highly significant expression in brain. The mRNA transcripts of LcMARCH4 were significantly induced in the main organs (skin, gill, spleen, and head-kidney) by C. irritans infection. Consistently, significant increase was observed in spleen and head-kidney after LPS, Poly I:C stimulation and V. parahaemolyticus infection. Subcellular localization analysis showed that LcMARCH4 was localized in the cytoplasm and membrane. Moreover, we found 46 DEGs in a comparative transcriptome analysis between the LcMARCH4 overexpression group and control vector group. The analysis showed that HSPA6, HSPA1B and DNAJB1 might play important regulatory roles to MARCH4 in fish. Notably, two noncoding RNA, both RN7SL1 and RN7SL2, the expression levels went up in MARCH4 overexpression cells. Taken together, this study will provide new insights into finfish MARCH4 and its potential regulatory signaling pathway as well., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced-NF-κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

High mobility group box 1 (HMGB1) and HMGB2 have been demonstrated to be key regulators not only in DNA recombination, replication, gene transcription, but also in host inflammation and immune responses. In the present study, orthologs of HMGB1 and HMGB2 named Lc-HMGB1 and Lc-HMGB2 were characterized in large yellow croaker (Larimichthys crocea). The ORFs of Lc-HMGB1 and Lc-HMGB2 are 621 bp and 648 bp, encoding proteins of 206 aa and 215 aa, with the putative Lc-HMGB1 and Lc-HMGB2 proteins both contain two HMG domains, respectively. The genome organizations of Lc-HMGB1 and Lc-HMGB2 are both composed of four exons and three introns, which are conserved in vertebrates. Lc-HMGB1 and Lc-HMGB2 were identified as cell nucleus localized proteins, and were ubiquitously distributed in the examined organs/tissues. Additionally, Lc-HMGB1 was significantly up-regulated under LPS and PGN stimulation, whereas the stimulation of poly I:C, LPS, PGN, and Pseudomonas plecoglossicida infection could significantly induce Lc-HMGB2 expression in vivo. Notably, both Lc-HMGB1 and Lc-HMGB2 overexpression could significantly up-regulated the expression of diverse immune-related genes, including IFN1, IRF3, ISG15, ISG56, RSAD2, g-type lysozyme, and TNF-α. Moreover, overexpression of Lc-HMGB1 could also induce the expression of IRF7 and Mx. These results collectively indicate that Lc-HMGB1 and Lc-HMGB2 play important roles in host immune responses against pathogen infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Receptor interacting protein 1 (RIP1) plays important roles not only in cell-death pathways but also in host innate immune responses. In the present study, a RIP1 ortholog named Lc-RIP1 was cloned and characterized in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-RIP1 is 2,046 bp, encoding a protein of 681 amino acids (aa), with an N-terminal kinase domain, an RHIM domain, and a C-terminal death domain. Subcellular localization analysis revealed that Lc-RIP1 was a cytosolic protein, which was broadly expressed in examined tissues/organs, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo based on qRT-PCR analysis. Notably, Lc-RIP1 could induce NF-κB, but not IRF3, IRF7 or type I IFN promoter activation. In addition, Lc-RIP1 overexpression could enhance Lc-MAVS, Lc-TRAF3, and Lc-TRAF6 mediated NF-κB promoter activation, and also Lc-TRIF and Lc-MAVS mediated IRF3 promoter activation, whereas suppress Lc-TRIF mediated NF-κB and type I IFN promoter activation, as well as Lc-TRAF3 and Lc-IRF3 mediated IRF3 promoter activation, Lc-IRF3 mediated type I IFN promoter activation and Lc-IRF7 mediated IRF7 promoter activation. These results collectively indicated that Lc-RIP1 function importantly in regulation of host innate immune signaling., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fish tend to rely more on their innate immunity to executing defense against viral infection by inducing antiviral gene production. However, the expression pattern and underlying mechanism of fish antiviral responses have yet to be fully defined. In the present study, an in vitro viral infection model was established by exposing head kidney-derived macrophages of large yellow croaker to virus analog, poly(I:C). Transcriptome analysis indicated that poly(I:C) appeared to induce potent antiviral activity featuring dominant interferon a3 (IFNa3) expression through activation of toll-like receptors (TLRs)/TIR-domain-containing adapter-inducing interferon-β (TRIF) and retinoic acid-inducible gene I-like receptors (RLRs)/mitochondrial antiviral signaling protein (MAVS) pathways. Inhibition of nuclear factor κB (NF-κB) and stimulator of interferon genes (STING)/interferon regulatory factor 3 (IRF3) pathways diminished the expression of IFNa3. Mechanistically, transcription factors including p65 and IRF3 could promote expression of IRF3, and activated IRF3 alone further increased the transcriptional activity of IFNa3. We also characterized the promoter of IFNa3 with direct IRF3 binding site which was sufficient to render the transcription of IFNa3. This effect was attenuated after deletion or mutation of the IRF3 binding sites. Taken together, our findings illustrate the distinct transcriptional profiling of fish macrophages triggered by poly(I:C). Also, this work provides new insights into the molecular mechanism underpinning coordinated activation of pathogen recognition and signaling transduction in the antiviral responses of non-model fish species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Interleukin (IL)-11 is a multifunctional cytokine that exerts a series of important immunomodulatory effects and exists in many tissues and cells. A 1106-bp nucleotide sequence representing the complete cDNA of IL-11 was obtained from large yellow croaker (Larimichthys crocea), containing an open reading frame (ORF) of 603 bp encoding for 200 amino acids (aa). The predicted LcIL-11 protein included a 12aa signal peptide and a conserved IL-11 domain. The polypeptide sequence identities between LcIL-11 and its counterparts in mammals and other fish are from 84% to 92% with known fish IL-11a and 22%-27% with fish IL-11b. LcIL-11 mRNA existed in most tissues with the most predominant expression in the gill. After immune challenge, the expression levels of LcIL-11 were induced largely in vivo and in vitro, with the peak-value of 32 times as much as the control in the liver at 24 h after Vibrio parahaemolyticus injection (p < 0.05) and the greatest value of 13.9 times as much as the control in LCK cells at 12 h after poly I:C stimulation (p < 0.05). Furthermore, the overexpression vector pcDNA3.1-LcIL-11 was constructed and transfected to LCK cells. Our results showed that the transcriptional expression levels of tumor necrosis factor (TNF)-α and myxovirus resistant protein (Mx) significantly up-regulated in LCK cells after LcIL-11 overexpression (p < 0.05). However, no significant changes of IL-1β, janus kinase (JAK)2 and signal transducers and activators of transcription (STAT)5 was detected. Our finding indicated that LcIL-11 might enhance TNF-α and antiviral protein Mx expression in large yellow croaker., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Large yellow croaker (Larimichthys crocea, LYC) aquaculture is being threatened by intensive infectious diseases. Relevant studies have focused on LYC immune responses to infection. By contrast, little is known how and to what extent the gut microbiota responds to infection. Here, we explored the interactions between LYC immune responses and gut bacterial communities during Pseudomonas plecoglossicida infection. P. plecoglossicida successfully colonized into LYC gut microbiota, resulting in an increasing mortality rate. Relative gene expressions of pro-inflammatory cytokines (TNF-α1, TNF-α2 and IL-1β) and anti-inflammatory cytokine (IL-10) were consistently and significantly induced by P. plecoglossicida infection, whereas non-specific immune enzymes activities were only enhanced at the early infection stages. P. plecoglossicida infection caused an irreversible disruption in the gut microbiota, of which infection and hours post infection constrained 16.2% and 5.6% variations, respectively. In addition, top 18 discriminatory taxa that were responsible for the difference between treatments were identified, whose abundances were significantly associated with the immune activities of LYC. Using a structural equation modeling (SEM), we found that gut bacterial communities were primarily governed by the conjointly direct (-0.33) and indirect (0) effects of infection, which subsequently affect host immune responses. Our results suggest that an irreversible dysbiosis in gut microbiota could be the causality of increasing mortality. To our knowledge, this is the first study to provide an integrated overview among pathogen infection, immune response and gut microbiota of LYC., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Apart from mitigating endoplasmic reticulum (ER) stress, vast studies have demonstrated the crucial role of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) - spliced X-box binding protein 1 (XBP1s) signaling pathway in inflammatory response in mammals. In addition, palmitic acid (PA)-induced inflammation has been verified in large yellow croaker (Larimichthys crocea). However, whether the IRE1α-XBP1s signaling pathway is involved in inflammatory response caused by PA remains poorly studied in fish. The present study was aimed at elucidating the role of the IRE1α-XBP1s signaling pathway in inflammatory response induced by PA in primary hepatocytes from large yellow croaker. In the present study, the full-length cDNA of ire1α and xbp1s were cloned and comprised 3793 bp and 1789 bp with an open reading frame of 3279 bp and 1170 bp, encoding 1093 and 390 amino acids, respectively. IRE1α protein possessed a protein kinase and endoribonuclease domain and XBP1s protein possessed a basic-leucine zipper domain. The IRE1α protein and XBP1s protein located to the ER membrane and nucleus respectively. The ire1α and xbp1s were widely transcribed in various tissues with the higher level in intestine, liver, adipose and head kidney. The ER stress-inducing agent tunicamycin (Tm) and PA treatment significantly activated the IRE1α-XBP1s signaling pathway and increased the pro-inflammatory genes expression including tumor necrosis factor α (tnfα), interleukin 6 (il-6) and interleukin 1β (il-1β) (P < 0.05). When KIRA6, the IRE1α kinase inhibitor, was used to block the IRE1α-XBP1s signaling pathway, the Tm and PA-induced pro-inflammatory genes expression was significantly suppressed (P < 0.05). These data indicated that the IRE1α-XBP1s signaling pathway was involved in the PA-induced inflammatory response in large yellow croaker., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As a member of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family, TRAF3 is an important regulator of NF-κB and type I interferon (IFN) activation, especially in Toll-like receptors (TLRs)- and retinoic acid inducible gene I (RIG-I)-like receptors (RLRs)-mediated signaling pathway. In the present study, a TRAF3 homologue named Lc-TRAF3 was characterized in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-TRAF3 contains 1788 bp encoding a protein of 595 amino acids (aa). Sequence analysis indicated that Lc-TRAF3 is conserved in vertebrates, constituted with a N-terminal RING finger, two TRAF-type zinc fingers, and a C-terminal TRAF-MATH domain. The genome organization of Lc-TRAF3 is conserved in fish, with 13 exons and 12 introns, but different from that in birds or mammals, which contains 10 exons and 9 introns. Lc-TRAF3 was identified as cytosolic protein base on fluorescence microscopy analysis. Expression analysis revealed that Lc-TRAF3 was broadly distributed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression Lc-TRAF3 could induce the activation of NF-κB, and Lc-TRAF3 co-transfected with Lc-TRIF induced a significantly higher level of NF-κB and IRF3 promoter activity, implying that Lc-TRAF3 may function as an enhancer in Lc-TRIF-mediated signaling pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-β (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A 70-day feeding trial was conducted to investigate the effects of dietary fish oil (FO) replaced by palm oil (PO) on growth, biochemical and antioxidant response as well as inflammatory response in the liver of large yellow croaker (initial weight 15.87 ± 0.14 g). Four iso-proteic and iso-lipidic experimental diets were formulated with 0% (the control group), 33.3%, 66.7% and 100% FO replaced by PO. Fish fed the diet with 100% PO showed significantly lower growth performance than the control group. As expected, the contents of C16:0, C18:1n-9 and C18:2n-6 were increased with increasing dietary PO levels. There were remarkable increases in total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels in fish fed the diet with 100% PO compared to the control group. Moreover, dietary PO significantly increased activities of plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) in fish fed the diet with 100% PO compared to the control group. The total antioxidant capacity (T-AOC) and the activity of catalase (CAT) in plasma were significantly decreased in fish fed the diet with 100% PO compared to the control group, and meanwhile no significant differences were found in T-AOC and CAT activity in fish fed diets with no more than 66.7% PO. Fish fed the diet with 100% PO exerted significantly higher toll like receptors (TLRs) and myeloid differentiation factor (MyD88) mRNA expression levels than the control group. The IFNγ, IL-1β and TNFα mRNA expressions were increased with increasing dietary PO levels. The increase of pro-inflammatory gene expression may be due to the activation of NF-κB signaling as the ratio of nucleus p65 to total p65 protein was elevated with the increase of dietary PO levels. These results showed that relatively higher PO levels in diets suppressed the growth and antioxidant capacity as well as induced the inflammatory response by activating TLR-NF-κB signaling pathway in juvenile large yellow croaker., (Copyright © 2019. Published by Elsevier Ltd.)

In this study, we sequenced and characterized two homologues of interferon-stimulated gene ISG15, termed as LcISG15-1 and LcISG15-2, from the large yellow croaker (Larimichthys crocea). The LcISG15-1 encodes 159 amino acids and the LcISG15-2 encodes 155 amino acids, both of which contain two tandem ubiquitin-like domains and the conserved C-terminal LRGG conjugation motif. The sequence analyses showed that both the LcISG15-1 and LcISG15-2 exhibit high similarity with ISG15 from other fishes. A putative IFN-stimulatory response element (ISRE) was detected in promoter regions of both the LcISG15-1 and LcISG15-2. Phylogenetic analyses revealed a close evolutionary relationship of both the LcISG15-1 and LcISG15-2 with other teleostean ISG15. Molecular evolutionary analyses suggested a gene duplication event of ISG15 in the ancestor of the Sciaenidae, with a signature of positive selection was found in the ISG15-2 gene copy of sciaenid fishes. The Real-time PCR analyses showed that the LcISG15-1 and LcISG15-2 were both found to be ubiquitously expressed in ten examined organs in large yellow croaker, with predominant expressions both in peripheral blood. Expression analyses showed that both the LcISG15-1 and LcISG15-2 were rapidly and significantly upregulated in vivo after poly (I:C) challenge in liver and spleen organs. However, the LcISG15-1 and LcISG15-2 were both significantly induced after pathogen Vibrio parahemolyticus infection only in the liver but not in the spleen. These results indicated that there are two ISG15 homologues in the large yellow croaker, both of which are likely to be involved in host immune defense against viral and bacterial infection., (Copyright © 2018. Published by Elsevier Ltd.)

The shortage of fish oil (FO) leads to the extensive use of vegetable oil (VO) in marine fish diets. High replacement percentage of dietary FO by VO induced pro-inflammatory response of adipose tissue (AT) and liver tissue (LT) in large yellow croaker (Larimichthys crocea). Mammalian studies showed that the secretion of cytokines by AT affected the immune response of LT. To investigate whether or not the inflammation response of LT is related to AT in large yellow croaker, LT and AT cells from fish fed FO diet (FOL and FOA) and VO diet (VOL and VOA) were co-cultured in a trans-well system, which resulted in an assembly of the two cells types sharing the culture medium but being separated by the membrane of the insert. Co-culture of FOL and FOA was selected as the control group (FOL-FOA). Results indicated that, when compared with the control group, the expression of pro-inflammatory genes (toll like receptors [TLRs], tumour necrosis factor α [TNFα], interleukin 1β [IL1β], suppressor of cytokine signalling 3 [SOCS3] and cyclooxygenase 2 [COX2]) in FOL was significantly increased in the co-culture group of FOL and VOA (FOL-VOA), while the expression of anti-inflammatory genes (arginase I [ArgI] and transforming growth factor β1 [TGFβ1]) in FOL was significantly depressed. On the contrary, a significantly depressed expression of pro-inflammatory genes (TLRs, TNFα, IL1β and COX2) and increased expression of anti-inflammatory genes (interleukin 10 [IL10]) in VOL was observed in the co-culture group of VOL and FOA (VOL-FOA) when compared with the co-culture group of VOL and VOA (VOL-VOA). The change of immune-related gene expressions in LT cells was attributed to nuclear factor κB (NF-κB) signalling since the expression of the p65 protein was observed to show a similar trend to the expression of pro-inflammatory genes. It is speculated that dietary VO increased the secretion of cytokines, which induced pro-inflammatory response in LT cells. These ex vivo results indicate that AT plays a vital role in LT pro-inflammatory response in fish fed VO diet., (Copyright © 2018. Published by Elsevier Ltd.)

Mammalian interleukin-4 (IL-4) and -13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1β and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-β and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Increasing demand, uncertain availability and high price of fish oil with the expansion of aquaculture made it essential to search alternative lipid sources. Vegetable oil has been proved to be the best candidate for the replacement of fish oil in aquafeeds. However, this replacement especially in high level potentially has some negative effects on fish. The present study was conducted to investigate the growth performance, anti-oxidative and inflammatory responses of large yellow croaker to replacement of dietary fish oil by soybean oil. Three isonitrogenous (46% crude protein) and isolipidic (13% crude lipids) diets were formulated to feed fish (initial body weight: 36.80 ± 0.39 g) for 12 weeks. The control diet was designed to contain 6.5% of fish oil, and named as FO. On the basis of the control diet, the fish oil was 50% and 100% replaced by soybean oil, and these two diets were named as FS and SO, respectively. Results showed that the specific growth rate significantly decreased in the SO group. Crude lipid contents in muscle and liver of fish fed SO diet were significantly higher than those in the FO group. The ratio of n-3 poly-unsaturated fatty acids (PUFAs) to identified fatty acids in liver decreased significantly, while n-6 PUFAs increased significantly with increasing dietary soybean oil inclusion. The levels of triacylglycerol, non-esterified fatty acid and tumour necrosis factor α, and the activity of aspartate aminotransferase in serum significantly increased in SO group. The total anti-oxidative capacity and expressions of the anti-oxidation-related genes (superoxide dismutase 1 and 2, catalase, glutathion peroxidase and nuclear factor erythroid 2-related factor 2) were significantly decreased by dietary soybean inclusion. Dietary soybean oil significantly decreased the gene expressions of the anti-inflammatory cytokines (arginase I and interleukin 10), and increased the pro-inflammatory cytokines (tumour necrosis factor α and interleukin 1β). The replacement of dietary fish oil by soybean oil also induced an over-expression of toll-like receptor 22 and myeloid differentiation factor 88 in liver. In conclusion, dietary soybean oil could suppress growth performance and liver anti-oxidative capacity, and induce inflammatory responses of large yellow croaker., (Copyright © 2018. Published by Elsevier Ltd.)

Transforming growth factor-β-activating kinase 1 (TAK1) is triggered by foreign pathogenic infection and involves in proinflammatory response through the activation of nuclear factor-κB (NF-κB), which is specifically regulated by TAK1-binding protein 1 (TAB1). However, the expression and regulatory characterizations of TAK1 and TAB1 in fish immune response remain largely unknown. In the present study, the cDNA sequences of TAK1 (LcTAK1) and TAB1 (LcTAB1) were identified from large yellow croaker, Larimichthys crocea. The open reading frame (ORF) of LcTAK1 was 1725 bp in length, encoding 574 amino acids. The putative LcTAK1 protein contained a protein kinase domain and a C-terminal coiled-coil region. The ORF of LcTAB1 was 1518 bp encoding 505 amino acids. And a typical PP2Cc domain and a conserved sequence motif (PYVDFSQFYLLWGSDH) at C-terminal were identified in the predicted LcTAB1 protein. Multiple alignments showed that LcTAK1 shared 74.0-97.9% and LcTAB1 shared 37.4-95.8% sequence identities with TAK1 and TAB1 proteins from other species, respectively. Quantitative PCR analysis indicated that both LcTAK1 and LcTAB1 were broadly expressed in all examined tissues, with the most predominant expression in brain and the weakest expression in muscle, respectively. Subcellular localization revealed that both LcTAK1 and LcTAB1 expressed in the cytoplasm. In addition, LcTAK1 transcripts increased significantly in LCK cells after flagellin, LPS and poly I:C stimulation while LcTAB1 enhanced greatly after LPS and poly I:C challenge. Furthermore, the roles of them in NF-κB activation were investigated by overexpression of LcTAK1 and LcTAB1 in HEK293T cells. Our results revealed that NF-κB luciferase promoter expression could not be induced by overexpression of LcTAK1 or LcTAB1 alone, however, it could be induced by co-expression of LcTAK1 and LcTAB1 together. Moreover, the roles of LcTAK1 and LcTAB1 in immune response analysis showed that NF-κB activation enhanced significantly in co-overexpressed HEK293T cells following LPS and poly I:C stimulation. However, the expression levels of tumor necrosis factor (TNF)-α, Interleukin-6 (IL-6) and IL-8 were induced only after LPS challenge (p < .05). These findings suggested that the TAK1-TAB1 complex of large yellow croaker might play an important role in pro-inflammatory cytokines and chemokine release after LPS stimulation via inducing NF-κB activation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hepatic steatosis induced inflammation is becoming increasingly prevalent in farmed fish. This study was conducted to investigate the protective effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) against hepatic steatosis-induced inflammation and its potential molecular mechanisms in hepatocyte of large yellow croaker (Larmichthys crocea). We found that the hepatic steatosis-induced inflammation was relieved by ω-3 PUFAs, meanwhile, the Sirt1 activity and transcript expression was increased by ω-3 PUFAs. The increased Sirt1 activity can decrease the hepatic steatosis-induced inflammation. The protective effects of ω-3 PUFAs against hepatic steatosis-induced inflammation was reversed by the treatment with Sirt1 inhibitor EX-527. The nuclear translocation of nuclear transcription factor kappa-B (NF-κB) p65 was significantly decreased after ω-3 PUFAs treatments compared to the palmitic acid stimulation group. The ω-3 PUFAs induced cytoplasm translocation of NF-κB p65 was reversed by EX-527. Together, ω-3 PUFAs alleviate hepatic steatosis-induced inflammation through Sirt1-mediated nuclear translocation of NF-κB p65 subunit in hepatocytes of large yellow croaker. The present study provides important insight into the mechanisms of the protective effects of ω-3 PUFAs, providing theory bases for alleviating the hepatic steatosis induced inflammation of farmed fish, thereby offering great benefits to the aquaculture industry and fish consumers., (Copyright © 2017. Published by Elsevier Ltd.)

The large yellow croaker (Larimichthys crocea) has a well-developed innate immune system. We studied a component of this system, chemokine receptor CXCR family. In this study, we report the full-length open reading frames, as well as the identification and characterization of the chemokine receptor genes CXCR2 (LycCXCR2), CXCR3 (LycCXCR3), and CXCR4 (LycCXCR4) of large yellow croaker. We report that LycCXCR3 and LycCXCR4 are evolving neutrally according to PAML analyses. Quantitative real-time PCR analysis revealed that CXCR transcripts were expressed in all examined tissues. The expression of chemokine receptors LycCXCR2, LycCXCR3, and LycCXCR4 was elevated in the kidney, spleen, and particularly the liver of the large yellow croaker after challenge with Vibrio anguillarum and polyinosinic:polycytidylic acid (poly I:C). These results suggest that LycCXCR2, LycCXCR3, and LycCXCR4 may be important immune-related genes, playing crucial roles in immune defence against bacterial infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As crucial signaling transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling pathway, IL-1R-associated kinase 4 (IRAK4) mediates downstream signaling cascades and plays important roles in innate and adaptive immune responses. In the present study, an IRAK4 orthologue was characterized from large yellow croaker (Larimichthys crocea), named Lc-IRAK4, with a conservative N-terminal death domain and a C-terminal protein kinase domain. The genome of Lc-IRAK4 is structured into eleven exons and ten introns. Expression analysis indicated that Lc-IRAK4 was widely expressed in tested tissues, with the highest level in liver and weakest in muscle. Additionally, in the spleen, liver tissues and blood, it could be induced by poly I:C and LPS stimulation, but not be induced by Vibrio parahemolyticus infection. Fluorescence microscopy assays revealed that Lc-IRAK4 localized in the cytoplasm in HEK 293T cells. It was also determined that Lc-IRAK4 could interact with MyD88, whereas MyD88-mediated NF-κB activation was significantly impaired when co-transfected the two in HEK 293T cells. These findings collectively indicated that although Lc-IRAK4 was evolutionarily conserved in vertebrates, the exact function especially the signaling transduction mediated by IRAK4 in fish immune response was different from that in mammals, which impaired MyD88-mediated NF-κB activation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

High level of vegetable oil (VO) in diets could induce strong inflammatory response, and thus decrease nonspecific immunity and disease resistance in most marine fish species. The present study was conducted to investigate whether dietary VO could exert these anti-immunological effects by altering TLR-NF-κB signalling, macrophages infiltration and polarization in adipose tissue of large yellow croaker (Larimichthys crocea). Three iso-nitrogenous and iso-lipid diets with 0% (FO, fish oil, the control), 50% (FV, fish oil and vegetable oil mixed) and 100% (VO, vegetable oil) vegetable oil were fed to fish with three replicates for ten weeks. The results showed that activities of respiratory burst (RB) and alternative complement pathway (ACP), as well as disease resistance after immune challenge were significantly decreased in large yellow croaker fed VO diets compared to FO diets. Inflammatory response of experimental fish was markedly elevated by VO reflected by increase of pro-inflammatory cytokines (IL1β and TNFα) and decrease of anti-inflammatory cytokine (arginase I and IL10) genes expression. TLR-related genes expression, nucleus p65 protein, IKKα/β and IκBα phosphorylation were all significantly increased in the AT of large yellow croaker fed VO diets. Moreover, the expression of macrophage infiltration marker proteins (cluster of differentiation 68 [CD68] and colony-stimulating factor 1 receptor [CSF1R]) was significantly increased while the expression of anti-inflammatory M2 macrophage polarization marker proteins (macrophage mannose receptor 1 [MRC1] and cluster of differentiation 209 [CD209]) was significantly decreased in the AT of large yellow croaker fed VO diets. In conclusion, VO could induce inflammatory responses by activating TLR-NF-κB signalling, increasing macrophage infiltration into adipose tissue and polarization of macrophage in large yellow croaker., (Copyright © 2016. Published by Elsevier Ltd.)

In this study, we sequenced and characterized melanoma differentiation-associated antigen 5 (LcMDA5), laboratory of genetics and physiology 2 (LcLGP2) and mitochondrial antiviral signaling protein (LcMAVS) from large yellow croaker (Larimichthys crocea). The LcMDA5 encodes 969 amino acids and contains two caspase-associated and recruitment domains (CARDs), a DExDc (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal domain) and a C-terminal regulatory domain (RD). The LcLGP2 encodes 679 amino acids and contains a DExDc, a HELICc and a RD. The LcMAVS encodes 512 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif ((269)PVQDT(273)). Phylogenetic analyses showed that all the three genes of large yellow croaker are clustered together with their counterparts from other teleost fishes. The Real-time PCR analyses showed that all the three genes were found to be constitutively expressed in all examined tissues in large yellow croaker, but all with relatively low expression levels. Expression analyses showed that the three genes were all rapidly and significantly upregulated in vivo after poly (I:C) challenge in peripheral blood, liver, spleen and head kidney tissues. The results indicate that the LcMDA5, LcLGP2 and LcMAVS might play important roles in antiviral immune responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rab GTPases, members of the Ras superfamily, encode monomeric G-proteins. Rab proteins regulate key steps in membrane traffic transport and endocytic pathway of host immune responses. Rab5A is involved in immune regulation, particularly in T cell migration and macrophage endocytosis in higher vertebrates. However, little is known of the molecular structure of Rab5A gene in marine teleost fish species and its expression profile during the parasite infection. In this study, the full-length cDNA sequence and genomic structure of Rab5A gene of the large yellow croaker (Larimichthys crocea) (LycRab5A), one of the most economical marine fishes, were identified and characterized. The LycRab5A protein, containing the ATPase/GTPase binding motifs and the effector molecules binding motifs, was highly homologous to that of other animals. The expression plasmid containing LycRab5A cDNA fused with GST was engineered and transformed into Escherichia coli to produce recombinant protein GST-LycRab5A, which was purified to prepare a polyclonal antibody specifically against LycRab5A. Subcellular localization revealed that LycRab5A expressed in the membrane and cytoplasm. Based on real-time PCR and Western blot analysis, we found that both mRNA and protein of LycRab5A were expressed in all tissues we examined; especially it was highly expressed in blood and gill. Interestingly, both mRNA and protein of LycRab5A were substantially up-regulated when parasitic ciliate protozoan (Cryptocaryon irritans) was infected. The expression of LycRab5A was reached to the maximal level at 24 h after infection. The line of evidence suggested that LycRab5A might play an important role in large yellow croaker defense against parasite infection. Moreover, on the basis of protein interaction, it was found that the LycRab5A interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. This discovery might contribute better understanding to the molecular events involved in fish immune responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We evaluated the effects of acute Zn exposure (4 and 8 mg L(-1) Zn) on lipid peroxidation, and activities and mRNA levels of antioxidant enzyme genes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecule at different exposure times (0, 6, 12, 24, 48, and 96 h) in the spleen of large yellow croaker. Lipid peroxidation remained relatively constant during 6-48 h and 6-24 h and sharply increased at 96 h and during 48-96 h in fish exposed to 4 and 8 mg L(-1) Zn, respectively. Activities of all tested enzymes increased during the early stage of exposure and decreased towards the end of the exposure in both groups. However, mRNA levels of antioxidant enzyme genes were dramatically up-regulated by 4 and 8 mg L(-1) Zn during the late stage of exposure. During the early stage of exposure for 6 h, the 8 mg L(-1) Zn exposure sharply increased mRNA levels of Cu/Zn-SOD, CAT, GPx1b, Nrf2, and Keap1, whereas, the 4 mg L(-1) Zn exposure did not significantly affect the expression of these genes. Our data also showed positive relationships between Nrf2 expression and mRNA levels of its target genes, suggesting that Nrf2 was required for the protracted induction of these genes. Furthermore, a sharp increase in Keap1 expression levels was observed in fish exposed to 4 mg L(-1) at 96 h, and 8 mg L(-1) at 6, 48, and 96 h. In conclusion, the present study demonstrated that Zn-induced antioxidant defenses were involved in modifications at enzymatic and transcriptional levels and the transcriptional regulation of the Nrf2-Keap1 signaling molecule; these results may contribute to the understanding of mechanisms that maintain the correct redox balance in the immune organ of the large yellow croaker., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5' untranslated region (UTR) of 71 bp and a 3' UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-α transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-α significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1β, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-α expression., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fish is highly affected by many environmental stresses such as temperature and invasive infection. The extracellular signal-regulated kinase (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is found to act as crucial mediators for cell differentiation, proliferation and cell response to various stresses. In the present study, ERK2 (LcERK2) and ERK5 (LcERK2) were cloned and characterized from large yellow croaker, Larimichthys crocea. The full length cDNA sequence of LcERK2 was of 1910 bp, including an ORF of 1110bp encoding a polypeptide of 369 amino acids. The full length cDNA sequence of LcERK5 was of 3720bp, including an ORF of 3375bp encoding a polypeptide of 1124 amino acids. Multiple alignments showed that both LcERK2 and LcERK5 contained highly conserved TEY motif and S&#95;TKc domain in MAPK family and the unique catalytic and active structures of ERK2 and ERK5. Subcellular localization revealed that both LcERK2 and LcERK5 expressed in the cytoplasm and cell nucleus. The expression of LcERK2 and LcERK5 were detected in most tissues of large yellow croaker, with the most predominant expression of LcERK2 in brain and LcERK5 in gill, and the weakest expression of LcERK2 in liver and LcERK5 in intestine, respectively. The expression levels of LcERK2 and LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK (L. crocea kidney) cells. After temperature stress, significant down-regulations of LcERK2 transcripts were detected after 10 °C stress (p < 0.05) whereas LcERK2 transcripts increased significantly after 35 °C stress (p < 0.05). However, significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress (p < 0.05). However, significant up-regulations of LcERK2 and LcERK5 transcripts were found after immune challenge (p < 0.05). Our results showed that LcERK2 transcripts enhanced after heat stress and both LcERK2 and LcERK5 transcripts could be induced by immune challenge. These findings indicated that LcERK2 might be more important in fish response to high temperature stress and both LcERK2 and LcERK5 might play an important role in fish immune response., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Since it was proposed to be a tumor suppressor in 1991, QM protein has attracted intensive and wide attention in plants, animals and fungi research fields. Up to date, however, the function of QM protein in fish immunity remains unknown. In this investigation, a QM gene (named as LycQM gene) was cloned from large yellow croaker (Larimichthys crocea), and LycQM protein was expressed in Escherichia coli and purified. The LycQM gene was ubiquitously transcribed in multi-tissues, including spleen, muscle, heart, liver, intestine, blood and head kidney. By quantitative real-time RT-PCR analysis, we found the highest and the lowest expression level of LycQM gene in head kidney and in heart, respectively. Time course analysis showed that LycQM expression was obviously up-regulated in blood and head kidney after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus and bacterial lipopolysaccharides (LPS). Moreover, as demonstrated by RNAi assays, LycQM protein could regulate the activity of phenoloxidase, a key enzyme in the proPO activation system of immunity. These results suggested that LycQM protein might play an important role in the immune response against microorganisms in large yellow croaker., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in the host innate immune system. In the present study, two LEAP-2 genes (LcLEAP-2A and LcLEAP-2C) from large yellow croaker (Larimichthys crocea) were cloned, both of which consist of 3 exons and 2 introns. The LcLEAP-2A transcripts were expressed in a wide range of tissues, with the highest mRNA levels found in the liver and intestine, while LcLEAP-2C transcripts showed obvious lower mRNA levels in all tested tissues compared to LcLEAP-2A. Upon infection by Vibrio alginolyticus, LcLEAP-2A transcripts were significantly up-regulated in liver, trunk kidney, spleen, head kidney, and gill, but down-regulated in intestine. In addition, significant up-regulation of LcLEAP-2C transcripts were also detected in all tissues tested, including intestine. The LcLEAP-2A and LcLEAP-2C mature peptides were chemically synthesized and found to exhibit selective antimicrobial activity in vitro against various species of bacteria. LcLEAP-2C, but not LcLEAP-2A, had antimicrobial activity against V. alginolyticus. Moreover, LcLEAP-2C treatment at low concentrations was evaluated and found to improve survival rate in V. alginolyticus-infected large yellow croaker, resulting in a decrease in bacterial load and expression of inflammatory cytokines. These results suggest that LcLEAP-2 isoforms play an important role in innate immunity by killing bacteria and inhibiting early inflammatory response in large yellow croaker., (Copyright © 2014 Elsevier Ltd. All rights reserved.)