Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

Regulatory roles of miRNA-530 in the post-transcriptional regulation of NF-κB signaling pathway through targeted modulation of IκBα in Sebastesschlegelii.

MicroRNAs (miRNAs) are a crucial type of non-coding RNAs involved in post-transcriptional regulation. The playing essential regulatory roles in the NF-κB signaling pathway and modulate the host immune response to diverse pathogens by targeting IκBα. However, the regulatory mechanism of miRNAs in relation with IκBα in Sebastes schlegelii remains unclear. In our study, we identified two copies of IkBα gene in black rockfish (Sebastes schlegelii), namely IkBα1 and IkBα2. Moreover, we have discovered that miRNA-530 can activate the NF-κB signaling pathway by inhibiting the expression of IκBα, thereby inducing the inflammatory response. This project comprehensively investigated the interactive regulatory roles of miRNA-530 in the NF-κB signaling pathway at both cellular and in vivo levels, while also elucidating the regulatory relationships between miRNA-530 and IκBα. In conclusion, our research confirmed that miRNA-530 can target the 3'UTR region of IκBα, resulting in a decrease in the expression of IκBα at the post-transcriptional level and inhibiting its translation. The findings contribute to the understanding of the regulatory network of non-coding RNA in teleosts and its subsequent regulation of the NF-κB signaling pathway by miRNAs.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Akirin2 enhances antibacterial ability via interacting with 14-3-3ζ in V. splendidus-challenged Apostichopus japonicus.

Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5'-untranslated region (UTR), and a 154 bp 3'-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function.

Identification and characterization of STAT family in silver pomfret (Pampus argenteus) involved in different exogenous stresses.

Members of the Signal Transducer and Activator of Transcription (STAT) family function pivotally as transcriptional activators integral to the modulation of inflammatory responses. The aquaculture of silver pomfret is frequently compromised by the imposition of exogenous stressors, which include thermal fluctuations, notably low-temperatures, diminished oxygen levels, and the onslaught of bacterial pathogens. Notwithstanding the critical impact of these stressors, the scientific literature presents a notable gap in our understanding of the STAT pathway's role in the silver pomfret's adaptive response mechanisms. To address this lacuna, we identified stat genes in the silver pomfret-denominated as Pastat1, Pastat2, Pastat3, Pastat4, and Pastat5-through a thorough and systematic bioinformatics analysis. Further scrutiny of the gene configurations and constituent motifs has elucidated that STAT proteins possess analogous structural frameworks and exhibit significant evolutionary preservation. Subsequently, the expression patterns of five stat genes were verified by RT-qPCR in twelve different tissues and four growth periods in healthy fish, showing that the expression of Pastat genes was temporally and spatially specific, with most of the stat genes expressed at higher levels in the spleen, following muscle, gill, and liver. Transcriptomic analysis of exposure to exogenous stressors, specifically formaldehyde and low-temperature conditions, elucidated that Pastat1 and Pastat2 genes exhibited a heightened sensitivity to these environmental challenges. RT-qPCR assays demonstrated a marked alteration in the expression profiles of jak1 and Pastat gene suites in PaS upon prolonged bacterial infection subsequent to these exogenous insults. Moreover, the gene expression of the downstream effectors involved in innate immunity and apoptosis displayed marked deviations. This study additionally elucidated the Pastat gene family's role in modulating the innate immune response and apoptotic regulation within the silver pomfret during exogenous stressors and subsequent pathogenic incursions.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

STAM2 negatively regulates the MyD88-mediated NF-κB signaling pathway in miiuy croaker, Miichthys miiuy.

Signal transducing adapter molecule 2 (STAM2), a member of the Signal Transducing Adapter Molecule (STAM) family, is a protein with significant implications in diverse signaling pathways and endocytic membrane trafficking. However, the role of the STAM2, especially in fish, remains largely unknown. In this study, we discovered that STAM2 negatively regulates the NF-κB signaling pathway, and its inhibitory effect is enhanced upon LPS induction. Our study confirmed that STAM2 can enhance the degradation of myeloid differentiation primary-response protein 88 (MyD88), an upstream regulator of NF-κB pathway. Furthermore, the UIM domain of STAM2 is important for the inhibition of MyD88. Mechanistically, STAM2 inhibits the NF-κB signaling pathway by targeting the MyD88 autophagy pathway. In addition, we showed that STAM2 promotes the proliferation of Vibrio harveyi. In summary, our study reveals that STAM2 inhibits NF-κB signaling activation and mediates innate immunity in teleost via the autophagy pathway.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Molecular confirmation and functional study of signal transducer and activator of transcription genes in the Pacific oyster Crassostrea gigas.

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

Toll-interacting protein is activated by the transcription factor GATA1 and Sp1 to negatively regulate NF-κB and MAPK pathways in the Japanese eel (Anguilla japonica).

Toll-interacting protein (Tollip) serves as a crucial inhibitory factor in the modulation of Toll-like receptor (TLR)-mediated innate immunological responses. The structure and function of Tollip have been well documented in mammals, yet the information in teleost remained limited. This work employed in vitro overexpression and RNA interference in vivo and in vitro to comprehensively examine the regulatory effects of AjTollip on NF-κB and MAPK signaling pathways. The levels of p65, c-Fos, c-Jun, IL-1, IL-6, and TNF-α were dramatically reduced following overexpression of AjTollip, whereas knocking down AjTollip in vivo and in vitro enhanced those genes' expression. Protein molecular docking simulations showed AjTollip interacts with AjTLR2, AjIRAK4a, and AjIRAK4b. A better understanding of the transcriptional regulation of AjTollip is crucial to elucidating the role of Tollip in fish antibacterial response. Herein, we cloned and characterized a 2.2 kb AjTollip gene promoter sequence. The transcription factors GATA1 and Sp1 were determined to be associated with the activation of AjTollip expression by using promoter truncation and targeted mutagenesis techniques. Collectively, our results indicate that AjTollip suppresses the NF-κB and MAPK signaling pathways, leading to the decreased expression of the downstream inflammatory factors, and GATA1 and Sp1 play a vital role in regulating AjTollip expression.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection.

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

Unraveling the immune functions of large yellow croaker Tmem208 in response to Pseudomonas plecoglossicida: Insights from cloning, expression profiling, and transcriptome analysis.

Pseudomonas plecoglossicida, the causative agent of Visceral White Spot Disease, poses substantial risks to large yellow croaker (Larimichthys crocea) aquaculture. Previous genome-wide association studies (GWAS), directed towards elucidating the resistance mechanisms of large yellow croaker against this affliction, suggested that the transmembrane protein 208 (named Lctmem208) may confer a potential advantage. TMEM proteins, particularly TMEM208 located in the endoplasmic reticulum, plays significant roles in autophagy, ER stress, and dynamics of cancer cell. However, research on TMEM's function in teleost fish immunity remains sparse, highlighting a need for further study. This study embarks on a comprehensive examination of LcTmem208, encompassing cloning, molecular characterization, and its dynamics in immune function in response to Pseudomonas plecoglossicida infection. Our findings reveal that LcTmem208 is highly conserved across teleost species, exhibiting pronounced expression in immune-relevant tissues, which escalates significantly upon pathogenic challenge. Transcriptome analysis subsequent to LcTmem208 overexpression in kidney cells unveiled its pivotal role in modulating immune-responsive processes, notably the p53 signaling pathway and cytokine-mediated interactions. Enhanced phagocytic activity in macrophages overexpressing LcTmem208 underscores its importance in innate immunity. Taken together, this is the first time reported the critical involvement of LcTmem208 in regulating innate immune responses of defensing P. plecoglossicida, thereby offering valuable insights into teleost fish immunity and potential strategies for the selective breeding of disease-resistant strains of large yellow croaker in aquaculture practices.

miR-144 affects the immune response and activation of inflammatory responses in Cynoglossus semilaevis by regulating the expression of CsMAPK6.

MicroRNAs are increasingly recognized for their pivotal role in the immune system, yet the specific regulatory functions of fish-derived microRNAs remain largely unexplored. In this research, we discovered a novel miRNA, Cse-miR-144, in the Chinese tongue sole (Cynoglossus semilaevis), characterized by a 73-base pair precursor and a 21-nucleotide mature sequence. Our findings revealed that the expression of Cse-miR-144 was notably inhibited by various Vibrio species. Utilizing bioinformatics and dual-luciferase assay techniques, we established that the pro-inflammatory cytokine gene CsMAPK6 is a direct target of Cse-miR-144. Subsequent in vitro and in vivo western blotting analyses confirmed that Cse-miR-144 can effectively reduce the protein levels of CsMAPK6 post-transcriptionally. Moreover, CsMAPK6 is known to be involved in the activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Additional investigations using qPCR and ELISA demonstrated that suppression of Cse-miR-144 leads to an upsurge in the liver mRNA levels of various immune genes (including MYD88, TRAF6, NF-κB, TRAF2, TRAF3, and TNF), alongside a marked increase in the production and secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) in the bloodstream of C. semilaevis. These findings collectively underscore the potential of Cse-miR-144 as a key inhibitor of CsMAPK and its crucial role in modulating the immune and inflammatory responses in teleost fish. Compared to the siRNA, miRNA is a better tool in controlling the expression of target gene with a lower cost.