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ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Although shrimp are a valuable protein source, shrimp aquaculture has numerous challenges from various infectious diseases and understanding molecular mechanisms of disease pathogenesis is crucial for disease management. In this study, a gene-to-gene correlation network generated from a transcriptomic database of the stomach of shrimp infected with acute hepatopancreatic necrosis disease (AHPND) was used to identify a new α-arrestin, termed arrestin domain containing-3 gene (LvARRDC3), with crucial roles in development of both AHPND and white spot disease (WSD). Double stranded RNA-mediated silencing or plasmid-mediated overexpression of LvARRDC3 gene significantly decreased expression of WSSV genes (IE1, VP28, and ICP11) and viral genome copy numbers. Nevertheless, in AHPND, silencing the LvARRDC3 gene increased the AHPND-associated plasmid and Pir toxins copy numbers, whereas overexpression of LvARRDC3 had the opposite effect. An in vitro pathogen binding assay with recombinant LvARRDC3 protein produced robust binding to WSSV virions and AHPND-causing V. parahaemolyticus. Moreover, based on immunofluorescence, LvARRDC3 was localized in the cytoplasm of Spodoptera frugiperda (Sf9) insect cells. Therefore, we inferred that LvARRDC3 has a role in pathogen internalization, making it a valuable target for addressing AHPND and WSD and also a biomarker for marker-associated shrimp breeding.

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Roles of S100A1 and S100A10 from hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in the immune response to Vibrio infection

The S100 proteins are highly conserved EF-hand calcium-binding proteins found only in vertebrates. In the current study, two S100 genes (S100A1 and S100A10) were successfully identified and characterized from hybrid grouper Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀. The deduced S100A10 protein contained two EF-hand domains, and S100A1 only possessed the N-terminal EF-hand. Phylogenetic analysis revealed that S100A1 and S100A10 from hybrid grouper were evolutionarily closely related to their counterparts in other selected vertebrates. Quantitative real-time PCR results revealed that the transcripts of S100A1 and S100A10 mRNA were ubiquitously distributed in all the examined tissues. After Vibrio alginolyticus infection, the expression of S100A1 and S100A10 in the spleen increased significantly. Moreover, overexpression of S100A1 and S100A10 could not only regulate the expression of interleukin 8 (IL-8), IL-10, and IL-16 in the head kidney, liver, and spleen, change the activities of acid phosphatase, catalase, lysozyme, and superoxide dismutase in serum, but also reduce the promoter activities of interferon 3 and nuclear factor kappa-B in vitro. Taken together, this study indicated that S100A1 and S100A10 participate in the immune response of hybrid grouper against bacterial infection.

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Comparative analysis of intestinal microbiota and its function on digestion and immunity of juvenile abalone Haliotis discus hannai fed two different sources of dietary soybean protein

The present study evaluated the replacement of fish meal (FM) with two different soybean protein sources (soybean protein concentrate (SPC) and soybean meal (SBM)) on the intestine microbiota of abalone Haliotis discus hannai and the implications for the host intestinal function and health. The control diet with FM as the main protein source (CON), and the four experimental diets with 50% and 100% SBM replacing FM (SBM50 and SBM100), and 25% and 100% SPC replacing FM (SPC25 and SPC100) were fed to abalone for 110 days. The intestinal microbiota analysis results revealed that there were no significant differences in α-diversity indices (Chao1, Ace, Sobs, and Shannon) among all groups. However, analysis of similarity (ANOSIM) demonstrated dramatic shifts in the intestinal microbiota component at the genus level among the groups. Venn diagram analysis identified 470 overlapping operational taxonomic units (OTUs) across the five groups, with the SBM50 and CON groups exhibiting the highest and lowest number of unique OTUs, respectively. Firmicutes and Proteobacteria were the predominant phyla in abalone, with Mycoplasma being the dominant genus (CON: 42.95%; SBM50: 23.98%; SBM100: 49.32%; SPC25: 27.20%; SPC100: 34.25%). Notably, the pathogens Vibrio abundance in the SPC25 group was significantly lower than in the CON group. The intestinal microbiota networks in the CON, SBM50, SBM100, SPC25, and SPC100 groups consisted of 1757, 2140, 1992, 2281 and 1747 edges, respectively. Furthermore, correlation heatmap results suggested that digestive enzymes and immune indices in abalone were associated with specific intestinal microbiota. Functional prediction via the KEGG pathway analysis revealed that the replacement levels of dietary FM with SBM and SPC significantly affect various biological functions of the intestinal microbiota. In summary, feeding SBM (50%) and SPC (25%) diets to abalone increased the abundance of beneficial bacterium in the intestines, contributing to improved digestion and increased growth rate of abalone.

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Expression and Antimicrobial Characterization of Rhamnose-Binding Lectin in Pinctada fucata martensii

Rhamnose-binding lectins (RBLs) are key components of pattern recognition molecules involved in pathogen clearance during non-specific immune responses and play an important role in the immune response of Mollusca. Pinctada fucata martensii is an essential species for artificial seawater pearl cultivation in China. With the increasing pollution of seawater, the study of the immune function of P. f. martensii has become increasingly urgent. Therefore, investigating the basic structural characteristics and immunological activity of RBL is of significant interest. This study employed RACE technology to clone and perform bioinformatics analysis on the RBL from P. f. martensii (PmRBL). Real-time quantitative fluorescence (qRT-PCR) technology was used to analyze gene expression following stimulation with pathogen-associated molecular patterns (PAMPs). In addition, a prokaryotic expression vector for PmRBL was constructed, followed by an evaluation of the antibacterial and hemolytic activities of the recombinant protein. The results revealed that the cDNA sequence of the PmRBL gene is 1045 bp in length, with its open reading frame (ORF) encoding 212 amino acids that include two D-galactoside-binding lectin domains. The expression of PmRBL across various tissues and in response to PAMP stimulation showed that PmRBL was most abundantly expressed in the gill tissue of P. f. martensii and exhibited a significant response to stimulation with lipopolysaccharides (LPS). From the perspective of antibacterial activity, PmRBL exhibited significant efficacy against Gram-negative bacteria. Overall, this study enhances our understanding of the functional characteristics of RBLs in Mollusca and provides new insights into the immune molecular polymorphism of P. f. martensii.

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Identification of a novel antimicrobial peptide from amphioxus ribosomal protein L27

Antimicrobial peptides (AMPs), derived from a variety of proteins such as ribosomal proteins, play a pivotal role in the innate immune system. However, information regarding ribosomal protein-derived AMPs is currently limited and their mechanisms of action remain poorly defined. Here we identified and characterized the antibacterial activity of amphioxus RPL27 (BjRPL27) and its core functional region located at residues 51-72 (termed BjRPL2751-72). We found that BjRPL27 expression was upregulated in the hepatic caecum following bacterial infection. Both the recombinant protein rBjRPL27 and the synthetic peptide BjRPL2751-72 effectively killed the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Aeromonas hydrophila via a combined action of disrupting cell membrane integrity, inducing membrane depolarization, and increasing intracellular reactive oxygen species (ROS) production. Additionally, the sequence of BjRPL2751-72 was highly conserved among all eukaryotic RPL27s, implying an ancient origin for the antibacterial activity of the RPL27 family. In vivo assays showed that BjRPL2751-72 not only efficiently protected zebrafish from A. hydrophila infection, but also killed the bacterium S. aureus on the skin wound of rats. Furthermore, neither BjRPL27 nor BjRPL2751-72 exhibited hemolytic activity towards human red blood cells, making them promising lead molecules for designing novel AMPs. These findings highlight the potential of BjRPL2751-72 as a novel AMP with selective bactericidal properties.

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Effects of chronic cold stress and thermal stress on growth performance, hepatic apoptosis, oxidative stress, immune response and gut microbiota of juvenile hybrid sturgeon (Acipenser baerii ♀ × A. schrenkii ♂)

The current study was conducted to investigate the effects of chronic cold stress and thermal stress on the growth performance, hepatic oxidative status, immune response, apoptosis and gut microbiota in juvenile hybrid sturgeon. The fish (initial mean weight: 21.4±0.3g) was reared at three temperatures (14°C, 22°C, and 30°C) for 16d, which were termed as low temperature group (LT), moderate temperature group (MT), and high temperature group (HT), respectively, and the second group was regarded as control group in this study. Each group was assigned randomly to three tanks with 15 fish per replica. The results indicated that cold stress resulted in a significant reduction of growth metrics and a significant increase of feed conversion ratio in fish compared with MT group. Interestingly, cold stress increased hepatocyte apoptosis revealed by TUNEL staining, along with nuclear disappearance in H&E-stained sections and elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Transcriptional levels of apoptosis-related genes and toll-like receptor signaling pathway components were significantly up-regulated in liver under cold stress. Compared with control group, in terms of thermal stress, the growth performance and feed utilization of fish were declined to some extent compared with MT group. Moreover, high temperature significantly elevated hepatic productions of malondialdehyde and hydrogen peroxide, as well as increased activities of some antioxidant enzymes in liver. In addition, low and high temperature induce changes in the composition of gut microbiota. Overall, the results suggested that cold stress decelerated growth performance, induced hepatocyte apoptosis, and enhanced innate immunity in hybrid sturgeon to cope with additional stressors. Whereas, thermal stress resulted in hepatic oxidative stress in liver and the protective responses in the antioxidant enzymes in fish were activated. These results provided insights into the different physiological adaptation strategies in responsive to cold stress and thermal stress in this cold-water fish.

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Comprehensive genome-wide identification and functional characterization of mapk gene family in northern snakeheads (Channa argus)

The mitogen-activated protein kinase (MAPK) signaling cascade, integral to cellular regulation, orchestrates cell growth, differentiation, stress response, and inflammatory reactions to adapt to challenging environments. The northern snakeheads (Channa argus), a valuable freshwater species known for its hypoxia tolerance, rapid growth, and high nutritional value, lacks comprehensive research on its mapk gene family. In this study, we identified 16 mapk genes in northern snakeheads, among which mapk8, mapk12 and mapk14 have duplicate copies. Phylogenetic analysis confirmed the evolutionary conservation of this gene family. Structural and motif analyses further underscored the conserved nature of these genes. Expression pattern analysis under abiotic and biotic stress conditions showed significant differences expression of mapks in the gills and suprabranchial organ (SBO) after air exposure, as well as in the brain following cold stress, highlighting the extensive role of mapks in stress regulation. It was worth noting that the significant expression differences of mapks were also observed in the spleen after N. seriolae infection, implicating that these genes may be involved in the regulation of innate immune responses. Additionally, analysis of protein-protein interaction (PPI) networks suggested that the co-activation of multiple MAPK signaling pathways may play a key role in regulating an organism's response to biotic and abiotic stresses. This study provides a detailed description of the mapk gene family in the northern snakeheads and elucidates its biological functions under various stress conditions, offering valuable insights into the regulatory mechanisms of the mapk gene family.

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Characterization of tripartite motif containing 59 (TRIM59) in Epinephelus akaara: Insights into its immune involvement and functional properties in viral pathogenesis, macrophage polarization, and apoptosis regulation

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H2O2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara.

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Supplementation of recombinant human lysozyme into diets affects the growth performance, muscle quality, immunity and intestinal microbiota in large yellow croaker Larimichthys crocea

The aim of the present study was to investigate the influence of supplementation of lysozyme (LZM) into diet on the growth performance, muscle quality, immunity, intestinal microbiota in large yellow croaker (Larimichthys crocea) with initial body weight of 194.5±0.27g. After a 70-day feeding trial, 6 experimental diets with LZM supplementation at 0 (LZM0), 10 (LZM10), 30 (LZM30), 50 (LZM50), 70 (LZM70) and 90mg/kg (LZM90) were tested. Results showed that the fish in the LZM70 group exhibited the lowest feed conversion ratio and the highest weight gain (WG), along with the highest trypsin and Na+/K+ ATPase activities in intestine (P<0.05). The LZM activity in serum and intestine was significantly reduced in all dietary LZM supplemented groups compared to the LZM0 group (P<0.05). Compared with that in the LZM0 group, the gene expressions of claudin 11, bcl-2, nlrp 3, tnf α, il-10 and tgf β in intestine in the LZM90 group were significantly elevated, while bax and caspase3 were significantly downregulated (P<0.05). Meanwhile, the group supplemented with 90mg/kg of dietary LZM also increased muscle crude lipid content, springiness and drip loss, along with decreased crude protein content, shear force and hardness compared with other groups (P<0.05). Furthermore, the results of intestinal microbiota showed that compared to those in the LZM0 group, relative abundances of Fusobacterium in the LZM30 and LZM90 groups were decreased, and the relative abundances of Achromobacter, Mycoplasma and Cetobacterium were increased. In conclusion, appropriate supplementation of LZM in diet promoted the growth performance, improved immunity, adjusted intestinal microbiota and muscle quality of large yellow croaker. Furthermore, the optimal level of dietary LZM supplementation for large yellow croaker was estimated to be 67.14mg/kg based on the quadratic regression for WG.

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