TANK-binding kinase 1 (TBK1) plays a central role in the host's defense system. In this research, we identified and analyzed the TBK1 ortholog from large yellow croaker (Larimichthys crocea) and investigated its function in the host innate immunity. The obtained ORF of Lc-TBK1 is 2172 bp and encoding a 723 aa protein. Subcellular localization analysis demonstrated that Lc-TBK1 predominantly resides in the cytoplasm. Lc-TBK1 showed broad expression across multiple tissues, and its transcript abundance was particularly high in the muscle. Its transcription was significantly upregulated after challenge with poly I:C, LPS, PGN, and Pseudomonas plecoglossicida. Overexpression of Lc-TBK1 activated IRF3, IRF7, and IFNd promoters, and such promoter activation were enhanced when Lc-TBK1 co-expressed with Lc-TRAF3 and Lc-TRIF. Co-immunoprecipitation assays confirmed direct interactions between Lc-TBK1 and several key adaptors, including Lc-TRIF, Lc-MAVS_tv1, Lc-MAVS_tv2, Lc-TRAF3, and Lc-TRAF6. Subsequent co-expression analyses revealed adaptor-specific modulation of antiviral gene transcription: Lc-TRIF and Lc-TRAF3 synergistically enhanced RSAD2, IRF3, and IRF7 expression, whereas Lc-MAVS isoforms attenuated these responses. Overall, our findings demonstrate that Lc-TBK1 signaling is differentially regulated by specific adaptor proteins, providing new insights into the evolutionary conservation and diversification of antiviral immune mechanisms in teleost.

Skin coloration is a distinctive phenotype and an economically important trait in ornamental fish. Understanding its regulatory mechanisms is crucial for both aquaculture and selective breeding. In this study, we cloned and analysed the full-length complementary DNA (cDNA) sequences of four key pigmentation-related genes-mitf, mc1r, tyr and sox10-in the black-tailed angelfish (Centropyge vrolikii). We established the fin-derived tissue cell line of C. vrolikii (CVFTCL) and confirmed its transfection efficiency using EGFP, achieving 40%-50% expression 24 h post-transfection, providing an in vitro platform for subsequent gene function verification. Using the primary fin cell culture system, we conducted overexpression and RNA interference (RNAi) experiments to investigate the regulatory relationships among these genes. Overexpression of mitf upregulated mc1r but downregulated sox10 and tyr; overexpression of mc1r enhanced mitf expression while inhibiting sox10 and tyr; overexpression of tyr only increased tyrp1 expression and overexpression of sox10 upregulated both mc1r and mitf, without affecting tyr. RNAi-mediated knockdown confirmed the efficiency of gene silencing and supported the regulatory relationships inferred from overexpression experiments. Collectively, these findings indicate that mitf, mc1r, tyr and sox10 may interact to coregulate melanocyte development and melanin synthesis, thereby influencing skin coloration in C. vrolikii. This study provides a mechanistic framework for pigmentation regulation and a foundation for colour improvement and selective breeding in this species.

ABSTRACT The Japanese eel ( Anguilla japonica ) displays a uniquely complex reproductive strategy shaped by its catadromous life cycle, protracted maturation, and extreme sensitivity to environmental cues. Despite decades of research, key regulatory mechanisms governing its reproduction remain unresolved. This review first establishes the biological and ecological foundations of the Japanese eel reproduction, then examines major biological and technical barriers to successful captive breeding, followed by an integration of current knowledge on neuroendocrine control, pathways of sex differentiation, and the physiological and molecular bases of artificial maturation. Emphasis is placed on both established mechanisms and emerging hypotheses, with particular attention to their implications for aquaculture. By identifying critical knowledge gaps, we highlight priorities for future research essential to both species conservation and the development of sustainable breeding technologies. These insights provide a foundation for optimizing hormonal induction protocols, improving larval performance, and refining nutritional management across life stages, particularly for broodstock conditioning and early larval development, thereby enabling more reliable and scalable closed‐cycle aquaculture for Japanese eel.

The pluripotency factor Nanog plays a central role in somatic cell reprogramming and stem cell maintenance. In this study, we identified and characterized the Nanog gene in large yellow croaker (Larimichthys crocea), an economically important marine fish. The full length Nanog cDNA of large yellow croaker was cloned, and sequence analysis revealed a conserved homeodomain. Expression profiling showed that Nanog transcripts were abundant during early embryogenesis, but declined sharply after gastrulation, indicating a maternal expression pattern. In adult tissues, Nanog expression was predominantly detected in the gonads, with significantly higher levels in the ovary than that in the testis. Functional assays in a large yellow croaker muscle cell line (LYCMs) demonstrated that RNAi-mediated knockdown of Nanog impaired cell growth and morphology, whereas its overexpression enhanced proliferation and altered cell morphology towards a denser, more rounded phenotype. Furthermore, overexpression of Nanog promoted its own expression as well as the endogenous OSKM (Oct4, Sox2, Klf4, c-Myc) network. Conversely, individual overexpression of each OSKM factor also upregulated Nanog to varying degrees, suggesting a reciprocal regulatory loop. Based on these findings, we propose a preliminary regulatory network linking Nanog and the OSKM factors in modulating cell proliferation. This study provides the first functional insights into Nanog in L. crocea and establishes a foundation for future research on pluripotency regulation and induced pluripotent stem cell (iPSC) induction in fish.

STING serves as a crucial adaptor protein in cytosolic nucleic acid sensing pathways that initiate host antiviral defense mechanisms. Herein, we report the molecular cloning and functional characterization of a STING ortholog namely Lc-STING in large yellow croaker (Larimichthys crocea). The complete coding sequence of Lc-STING comprises 1,227-bp, encoding a 408-aa protein containing a TMEM173 domain and three transmembrane regions. Lc-STING exhibits ubiquitous tissue distribution, with maximal levels in the intestine and minimal in the brain. Upon challenge with poly I:C, LPS, PGN, or Pseudomonas plecoglossicida, Lc-STING expression was markedly elevated in immune-related tissues. As a cytoplasmic distribution protein, Lc-STING overexpression dose-dependently activated NF-κB, IRF3, IRF7, and type I IFN promoters. Notably, the co-expression of Lc-STING with Lc-TRIF, Lc-TRAF3, Lc-IRF3, or Lc-IRF7 substantially influenced antiviral signaling cascade, attenuating NF-κB while amplifying IRF3, IRF7, and type I IFN promoter activation. Such combinatorial expressions also significantly impacted the transcription of antiviral and inflammatory genes, encompassing IL-1β, IRF3, TNF-α, IRF7, ISG15, Mx, and ISG56. These findings collectively establish Lc-STING as a pivotal mediator of antiviral immunity in large yellow croaker, engaging in intricate crosstalk with multiple signaling components to coordinate balanced activation of interferon regulatory pathways while tempering inflammatory NF-κB responses.

The Japanese eel (Anguilla japonica), a commercially important species, has experienced severe population declines in the wild, underscoring the urgent need to improve artificial breeding techniques. However, the complexity of embryonic development and the limited understanding of its molecular regulatory mechanisms have constrained progress in artificial reproduction. To elucidate the dynamics of gene expression during early development, we conducted a comprehensive transcriptomic analysis of eight key embryonic stages using RNA-Seq. A total of 16,728 differentially expressed genes (DEGs) were identified, with the most pronounced pluripotency-related changes observed during the multicellular-blastula and differentiation-related changes in gastrula-embryo body transitions. Functional enrichment revealed distinct stage-specific pathways: early stages (multicellular) dominated by Notch and Wnt signaling, involved early developmental decisions; mid-stages (blastula to gastrula), with enriched pathways like Cell cycle, supporting rapid cell division; mid-late stages (embryo-body-formation to somite appearance) featured extracellular matrix receptor (ECM-receptor) interaction and focal adhesion, contributing to cell connectivity and tissue morphogenesis; and late stages (muscle-effect to newly-hatched-larvae) highlighted calcium signaling and metabolic pathways, providing signaling and energy support for organogenesis and functional maturation. Weighted gene co-expression network analysis (WGCNA) identified four stage-specific modules that correlated with developmental progression. Additionally, key members of the Sox, Hox, and Wnt transcription factor families were screened and found to exhibit dynamic, stage-specific expression patterns. These factors likely form a synergistic regulatory network that coordinates the entire developmental progression. Collectively, these findings delineate the molecular landscape of A. japonica embryogenesis and establish a crucial baseline transcriptomic resource that will facilitate future investigations into the molecular regulation of early development in this species.

As a member of the inflammatory caspases, Caspase-1 can increase the host inflammatory response against pathogen invasion and also function dominantly in apoptosis. In this study, we cloned and obtained two transcripts of Caspase-1 in large yellow croaker (Larimichthys crocea), namely Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2. The ORF of Lc-Caspase-1_tv1 is 1239 bp, whereas Lc-Caspase-1_tv2 is 1167 bp in length, encoding a protein of 412 and 388 aa, and both of which contains a CARD and a CASc domain. Subcellular localization analysis showed that Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2 are both cytoplasmically localized, and were widely expressed in the tested tissues/organs, with the highest expression level of Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2 detected in the muscle and intestine, respectively. The expression of Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2 was markedly induced by poly I:C, LPS, PGN stimulation, and Pseudomonas plecoglossicida infection. In addition, overexpression of Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2 significantly induced apoptosis, and the co-expression of Lc-Caspase-1_tv2 but not Lc-Caspase-1_tv1 with Lc-ASC could significantly enhanced the apoptosis, whereas the co-expression of the Caspase-1 transcripts with Lc-NLRC3 could significantly abolished the apoptosis that mediated by Lc-NLRC3. Furthermore, both Lc-Caspase-1_tv1 and Lc-Caspase-1_tv2 could form protein complexes with Lc-ASC and Lc-NLRC3. The co-expression of Lc-Caspase-1_tv1 with Lc-ASC significantly enhanced the expression levels of IRF3, IRF7, TNF-α, and IFNd, whereas the co-expression of Lc-Caspase-1_tv2 with Lc-ASC significantly enhanced IRF3, Mx, and IFNd expression. Nevertheless, co-expression of the Caspase-1 transcripts with Lc-NLRC3 did not significantly impact expression of these immune-related molecules. These results indicate that Caspase-1 transcripts are essential for apoptosis and immune signaling regulation in teleosts.

Vitellogenins (VTGs), being lipoproteins with high molecular weight, are the primary nutrient source for the embryonic development, and they are synthesized and accumulated massively in eggs during vitellogenesis. In this study, a novel vtg gene, Spvtg3, was discovered from our embryo transcriptome data of mud crab (Scylla paramamosain). This gene encodes a 2304-amino-acid protein featuring conserved vitellogenin domains (LPD_N, DUF1943, and VWD) and has a predicted molecular weight of 281.45 kDa. The Spvtg3 was exclusively expressed in the hepatopancreas and ovaries. During embryonic development, Spvtg3 expression dramatically increased in five pairs of appendages period. Liposome-mediated Spvtg3 dsRNA interference resulted in slower embryonic development, reduced hatching rate, and deformities. Transcriptome and metabolome analyses found some important differentially expressed genes (DEGs) in embryonic development signaling pathways (e.g., GAP junction, Wnt, and the MAPK signaling pathway), and some vital differential metabolites in metabolic pathways (e.g., lipid metabolism and pyrimidine metabolism). Furthermore, Spvtg3 and Lysophosphatidylethanolamine (LPE) expression levels were positively correlated with the expression of genes associated with embryonic development (sox21, eg, sox2, sox3, soxb2, foxl2-like, nr2e1, and fshr9). These results indicated that Spvtg3 plays a crucial role in mud crab embryonic development, potentially regulating VTG synthesis, transport, and metabolism.

Vibrio harveyi, a major aquatic pathogen prevalent in tropical and subtropical waters, causes substantial economic losses in global mariculture. This study focused on developing subunit vaccines targeting the outer membrane proteins of a new highly virulent strain of V. harveyi, which are recognized as key protective antigens. We performed whole-genome sequencing of a highly virulent endemic strain, followed by systematic screening and identification of immunogenic OMPs. Two candidate antigens, OmpW and OmpK, were successfully expressed and purified using a prokaryotic expression system. To assess the protective efficacy of the vaccine, pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) were immunized via intraperitoneal injection, followed by a challenge with a virulent strain of V. harveyi. Comprehensive genomic characterization of the pathogen indicated the presence of a wide array of virulence-associated genes, from which 17 antigenic proteins were selected as potential candidates for vaccine development. Compared to the GST control group, grouper immunized with recombinant OmpW or OmpK showed significantly higher serum antibody titers (P < 0.05) and substantial upregulation (2.15-48.6 fold) of key immune-related genes (MHCII and IL1β) in head kidney tissues. Notably, vaccinated fish exhibited a 38.7-50 % increase in serum lysozyme activity, confirming the activation of innate immune mechanisms. These findings collectively demonstrate that OmpW and OmpK provide comprehensive immune protection through synergistic enhancement of both adaptive (specific antibody production) and innate (lysozyme-mediated) immune responses. Histopathological examination confirmed the protective effects, showing significantly reduced inflammatory lesions and cellular necrosis in vital organs (head kidney, spleen, and liver) of immunized fish. Our results demonstrate that recombinant OmpW and OmpK proteins induce comprehensive immune protection through synergistic activation of both humoral and cellular immune pathways, establishing a foundation for developing effective V. harveyi subunit vaccines.

Octopus sinensis is an important economic species in China that relies mainly on the innate immune system to resist pathogenic invasion. Scavenger receptors (SRs), as key members of pattern recognition receptors (PRRs), initiate immune responses by recognizing pathogen-associated molecular patterns (PAMPs). However, the composition and immune functions of SR family members in O. sinensis remain unclear. In this study, six OsSR genes were identified from the O. sinensis genome, all of which contain conserved SR family domains. These SRs were classified into five subclasses (A, B, E, H, and I), with one member in each subclass except subclass B, which contains two genes. Subcellular localization prediction revealed that MARCO, SRCRB4D, and MRC1 localize to the extracellular matrix, STAB2 and DMBT1 to both the extracellular matrix and plasma membrane, and SCARB1 to both the endoplasmic reticulum and plasma membrane-a distribution pattern that may support their differential immune recognition functions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the six OsSR genes were expressed in eight normal tissues (including gill, hepatopancreas, and caecum) with varying expression levels. After stimulation with peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly I:C), and Vibrio parahaemolyticus, the expression levels of OsSR genes in the hepatopancreas and gill showed significant upregulation at different time points (6 h, 12 h, 24 h, 48 h) (p < 0.05), while a few genes remained unchanged or were downregulated. Specifically, OsMARCO was significantly upregulated in both the gill and hepatopancreas after stimulation with PGN, poly I:C, and V. parahaemolyticus (p < 0.05); OsSCARB1 was significantly upregulated in both gill and hepatopancreas after V. parahaemolyticus stimulation (p < 0.05) but showed no significant change in gill tissues under PGN and poly I:C stimulation. These qRT-PCR results demonstrate that OsSRs are differentially regulated by pathogenic and PAMP challenges, thus not only defining their role in the transcriptional immune response but also laying a foundation for research on innate immune signaling pathways in cephalopods.

As a member of the NLRs family, NLRC3 has been determined to function in the NF-κB, MAPK, and type I IFN signaling, which are crucial for the host innate immunity and inflammatory response. In this study, an NLRC3 ortholog, named as Lc-NLRC3, was cloned and identified in large yellow croaker (Larimichthys crocea). The gene characteristics analysis revealed that Lc-NLRC3 consists of 18 exons and 17 introns, with a full-length open reading frame (ORF) of 3405 bp, encoding a protein of 1134 amino acids (aa), that containing a N-terminal CARD domain, a central NACHT domain, and a C-terminal LRRs domain. It was shown that Lc-NLRC3 is predominantly found in the cytosol, and was widely distributed across various tissues/organs, with the highest expression detected in the intestine, and could be induced by poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Importantly, Lc-NLRC3 overexpression significantly activate NF-κB, TNFα, IL-1β, IRF3, IRF7, and IFN1 promoters, whereas when co-expressed with RIP2, STING, or TBK1, it down-regulated those promoter activation compared to their individual overexpression alone, thereby suppressing downstream antiviral and inflammatory gene expression. Interestingly, Lc-NLRC3 associated with TRAF6 in IRF3/IRF7 promoter activation, and enhanced the expression of IRF7, Mx, ISG15, and TNF-α. Co-immunoprecipitation assays also confirmed interactions of Lc-NLRC3 with RIP2, STING, TBK1, and TRAF6. The above results imply that Lc-NLRC3 is an important regulator in RIP2, STING, TBK1, and TRAF6 mediated type I IFN signaling and inflammatory response.

Antimicrobial peptides (AMPs) are small molecular peptides that widely exist in organisms to resist external microbial invasion and play a crucial role in the host's immune defense system. Owing to their functions of efficient broad-spectrum killing of pathogenic microorganisms, immune enhancement, and intestinal health improvement, they have emerged as a focal point in research on the immune defense of aquatic animals in recent years. In this study, a total of 105 putative AMP-derived genes from the genome were screened, and seven candidate AMPs were finally identified by analyzing the differential expression results of the hepatopancreas and the white body transcriptomes combined with machine learning algorithms. Furthermore, the seven synthesized antimicrobial peptides were demonstrated to have good antimicrobial activity. Among them, GAP1 and Big Defensin showed the strongest antibacterial activity. GAP1 and Big Defensin exhibited antibacterial activity against four bacteria (Escherichia coli, Vibro parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis) at low concentrations of 5-10 mu M and 3.2-12.9 mu M respectively. These data will contribute to the development of AMP-based aquatic drugs.

Antimicrobial peptides (AMPs) are small molecular peptides that widely exist in organisms to resist external microbial invasion and play a crucial role in the host's immune defense system. Owing to their functions of efficient broad-spectrum killing of pathogenic microorganisms, immune enhancement, and intestinal health improvement, they have emerged as a focal point in research on the immune defense of aquatic animals in recent years. In this study, a total of 105 putative AMP-derived genes from the genome were screened, and seven candidate AMPs were finally identified by analyzing the differential expression results of the hepatopancreas and the white body transcriptomes combined with machine learning algorithms. Furthermore, the seven synthesized antimicrobial peptides were demonstrated to have good antimicrobial activity. Among them, GAP1 and Big Defensin showed the strongest antibacterial activity. GAP1 and Big Defensin exhibited antibacterial activity against four bacteria (Escherichia coli, Vibro parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis) at low concentrations of 5-10 μM and 3.2-12.9 μM respectively. These data will contribute to the development of AMP-based aquatic drugs.

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.

The feedback regulatory effects of estrogen (E2) and androgen (T) on the gonadotropin-releasing hormone (GnRH) and gonadotropin (GtH) within the brain–pituitary–gonad (BPG) axis in eels with undeveloped ovaries were investigated through in vivo studies. However, the regulatory role of the BPG axis only became apparent during ovary development in the migratory stage. To further elucidate the direct feedback regulation of the BPG axis, female Anguilla japonica underwent artificial induction of vitellogenesis, and the regulation of BPG axis tissues by GtH (human chorionic gonadotropin, hCG), E2, and T was explored through in vitro exposure. The mRNA expression levels of GnRH (mGnRH), GtH (fshb and lhb), and steroid biosynthesis enzymes (cyp11a1, hsd3b, cyp17a1, and cyp17a2) in the diencephalon, pituitary, and ovary, respectively, were determined. The results showed that the expression level of mGnRH in the diencephalon was significantly downregulated by 0.1 IU/mL hCG but upregulated by both 1 nM E2 and higher concentrations of T, suggesting a direct positive feedback regulation of E2 on mGnRH. In the pituitary, the expression levels of fshb and lhb were upregulated by E2, while fshb was suppressed by T. In the ovaries, the expression of cyp11a1 and hsd3b was upregulated by 1 nM E2, whereas T exposure resulted in an opposite effect. Cyp17a1 mRNA levels did not differ significantly with E2 treatment but were upregulated by 1 nM T. These findings suggest that low concentrations of E2 exhibited positive feedback regulation on all three levels (diencephalon, pituitary, and ovary) of the BPG axis, while T showed weaker and differential feedback regulation in BPG axis tissues. Overall, this study’s results revealed the direct feedback regulation of hCG, E2, and T on the BPG axis in eels, a phylogenetic base of teleosts.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Takifugu bimaculatus is a marine fish with high nutritional value. Its ovary contains tetrodotoxin (TTX) which is a severe neurotoxin that limits its edible value of it. To understand the mechanism of oogenesis and production of TTX in T. bimaculatus, an ovarian cell line named TBO from an adolescent ovary was established. TBO was composed of fibroblast-like cells that expressed the ovarian follicle cells marker gene Foxl2 and highly expressed TTX binding protein 2 (PSTBP2) but did not express the germ cells marker gene Vasa. Therefore, TBO seems to be mainly composed of follicle cells and possibly a small percentage of oocytes. Electroporation was used to successfully transfect the pEGFP-N1 and pNanog-N1 vectors into the TBO cell line with a high transfection efficiency. The morphological changes and survival rates of the exposed cells proved that this cell line was effective for exposure to conotoxins (CTXs), another group of toxins related to food safety. Furthermore, PSTBP2 was knocked out in TBO using CRISPR/Cas9 technology, showing that sgRNA2 could mutate PSTBP2. The results suggested that TBO will be more convenient, efficient, and rapid for reproduction and toxicology investigation, and gene editing. This study laid the groundwork for future research into the fish gonadal cell culture and food-related marine toxins. In conclusion, a cell line has been generated from T. bimaculatus, which might represent a valuable model for fish studies in the fields of toxicology and gene editing.

As a TIR domain-containing molecular, sterile α-and armadillo motif-containing protein (SARM) acts as an adaptor in Toll-like receptor (TLR) signaling, and also plays important roles in mediating apoptosis and neuronal injury. In the present study, the ortholog of SARM, named as Lc-SARM, was cloned and identified in large yellow croaker (Larimichthys crocea). The full-length ORF of Lc-SARM consists of 2,154 bp, encoding a protein of 717 amino acids (aa), which is comprised of an N-terminal ARM domain, two SAM domains, and a C-terminal TIR domain. Confocal microscopy revealed that Lc-SARM was mainly distributed in the cytoplasm, and the mRNA expression level of Lc-SARM was broadly distributed in all the detected organs/tissues, with the highest expression level found in the brain. The expression patterns of Lc-SARM could be induced in response to poly I:C, LPS, PGN stimulations, and Pseudomonas plecoglossicida infection. Notably, although the overexpression of Lc-SARM could significantly induce NF-κB, IRF3, IRF7, and type I IFN promoter activation, whereas the co-expression of Lc-SARM with Lc-TRIF, Lc-TRAF3, Lc-IRF3, or Lc-IRF7 significantly down-regulated the induction of NF-κB, IRF3, IRF7, or type I IFN promoter activation, and suppressed the antiviral effects as well as the downstream antiviral-related genes expression compared to the only overexpression of Lc-TRIF, Lc-TRAF3, Lc-IRF3, or Lc-IRF7. Co-immunoprecipitation (Co-IP) assays also demonstrated that Lc-SARM interacts separately with Lc-TRIF, Lc-TRAF3, Lc-IRF3, and Lc-IRF7. It is thus collectively suggested that Lc-SARM functions as a negative regulator in Lc-TRIF, Lc-TRAF3, and Lc-IRF3/7 involved antiviral signaling.

The skin coloration is one of the key characteristics of fish morphological diversity; understanding the molecular regulation mechanism of fish skin coloration has attracted a mounting number of attention and is also an important prerequisite for the future research on fish skin coloration genetics and skin coloration improvement breeding of fish. Pearlscale angelfish (Centropyge vrolikii) is a unique marine ornamental fish with tremendous market potential and is well favored by consumers in China, which has a wide variety of colorations and shapes. In this study, the distribution of pigment cells in the fins and scales was observed, and the pigment cells were mainly composed of melanocytes, xanthophore, iridophore, and erythrophore in C. vrolikii. The distribution of pigment cells in the tail scales is more concentrated than that in other parts. There are mainly melanocytes and yellow pigment cells in the anal fin, tail fin, and dorsal fin, and mainly xanthophore with a few melanocytes in the pectoral fin and abdominal fin. The tissues of the pectoral fin (PF), anal fin (AF), tail fin (TF), and eyeballs (EB) were sequenced by SOAPdenovo-Trans technology, the differentially expressed genes (DEGs) of skin coloration were screened, and the expression pattern of these genes in different tissues was analyzed. The results represented that a total of 159,213 all-unigene was assembled and gene annotation was carried out using COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG, and NR functional database. The Tyrp1, Slc45a2, Xdh, Mitf, Tyr, Hps5, Pomc, Pmel, and other genes related to the skin coloration were annotated. Through KEGG pathway enrichment analysis, it was found that tyrosine metabolism, MAPK, Wnt, and melanin production were involved in the regulation of skin coloration. At the same time, the DEGs related to melanin production (Mitf, Mc1r, Sox10, Tyr, Tyrp1, and Pomc) were screened to verify their expression in various tissues. The results also showed that Mitf and Tyr were highly expressed in eyeballs, Tyrp1 was highly expressed in skin, Sox10 and Pomc were highly expressed in the tail fin, and Mc1r was highly expressed in the heart and tail fin. The results suggested that these genes may play an important role in the process of skin coloration in C. vrolikii. This study will help to further understand the molecular regulatory mechanism of skin coloration and provide essential clues for accelerating skin coloration improvement in the future in C. vrolikii.

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.