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Galectins are proteins that play a crucial role in the innate immune response against pathogenic microorganisms. Previous studies have suggested that Galectin-3 could be a candidate gene for antibacterial immunity in the large yellow croaker Larimichthys crocea. In this study, we cloned the Galectin-3 gene in the large yellow croaker, and named it LcGal-3. The deduced amino acid sequence of LcGal-3 contains a carbohydrate recognition domain with two conserved β-galactoside binding motifs. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that LcGal-3 was expressed in all the organs/tissues that were tested, with the highest expression level in the gill. In Larimichthys crocea kidney cell lines, LcGal-3 protein was distributed in both the cytoplasm and nucleus. Moreover, we found that the expression of LcGal-3 was significantly upregulated upon infection with Pseudomonas plecoglossicida, as demonstrated by qRT-PCR analyses. We also purified the LcGal-3 protein that was expressed in prokaryotes, and found that it has the ability to agglutinate large yellow croaker red blood cells in a Ca2+-independent manner. The agglutination activity of LcGal-3 was inhibited by lipopolysaccharides (LPS) in a concentration-dependent manner, as shown in the sugar inhibition test. Additionally, LcGal-3 exhibited agglutination and antibacterial activities against three Gram-negative bacteria, including P. plecoglossicida, Vibrio parahaemolyticus, and Vibrio harveyi. Furthermore, we studied the agglutination mechanism of the LcGal-3 protein using blood coagulation tests with LcGal-3 deletion and point mutation proteins. Our results indicate that LcGal-3 protein plays a critical role in the innate immunity of the large yellow croaker, providing a basis for further studies on the immune mechanism and disease-resistant breeding in L. crocea and other marine fish. [ABSTRACT FROM AUTHOR]

Galectins are proteins that are involved in the innate immune response against pathogenic microorganisms. In the present study, the gene expression pattern of galectin-1 (named as NaGal-1) and its function in mediating the defense response to bacterial attack were investigated. The tertiary structure of NaGal-1 protein consists of homodimers and each subunit has one carbohydrate recognition domain. Quantitative RT-PCR analysis indicated that NaGal-1 was ubiquitously distributed in all the detected tissues and highly expressed in the swim-bladder of Nibea albiflora, and its expression could be upregulated by the pathogenic Vibrio harveyi attack in the brain. Expression of NaGal-1 protein in HEK 293T cells was distributed in the cytoplasm as well as in the nucleus. The recombinant NaGal-1 protein by prokaryotic expression could agglutinate red blood cells from rabbit, Larimichthys crocea, and N. albiflora. The agglutination of N. albiflora red blood cells by the recombinant NaGal-1 protein was inhibited by peptidoglycan, lactose, D-galactose, and lipopolysaccharide in certain concentrations. In addition, the recombinant NaGal-1 protein agglutinated and killed some gram-negative bacteria including Edwardsiella tarda, Escherichia coli, Photobacterium phosphoreum, Aeromonas hydrophila, Pseudomonas aeruginosa, and Aeromonas veronii. These results set the stage for further studies of NaGal-1 protein in the innate immunity of N. albiflora. [ABSTRACT FROM AUTHOR]

Phosphoribosyl pyrophosphate synthetases (EC 2.7.6.1) are key enzymes in the biological synthesis of phosphoribosyl pyrophosphate and are involved in diverse developmental processes. In our previous study, the PRPS1 gene was discovered as a key disease-resistance candidate gene in yellow drum, Nibea albiflora, in response to the infection of Vibrio harveyi, through genome-wide association analysis. This study mainly focused on the characteristics and its roles in immune responses of the PRPS1 gene in yellow drum. In the present study, the NaPRPS1 gene was cloned from yellow drum, encoding a protein of 320 amino acids. Bioinformatic analysis showed that NaPRPS1 was highly conserved during evolution. Quantitative RT-PCR demonstrated that NaPRPS1 was highly expressed in the head-kidney and brain, and its transcription and translation were significantly activated by V. harveyi infection examined by RT-qPCR and immunohistochemistry analysis, respectively. Subcellular localization revealed that NaPRPS1 was localized in cytoplasm. In addition, semi-in vivo pull-down assay coupled with mass spectrometry identified myeloid differentiation factor 88 (MyD88) as an NaPRPS1-interacting patterner, and their interaction was further supported by reciprocal pull-down assay and co-immunoprecipitation. The inducible expression of MyD88 by V. harveyi suggested that the linker molecule MyD88 in innate immune response may play together with NaPRPS1 to coordinate the immune signaling in yellow drum in response to the pathogenic infection. We provide new insights into important functions of PRPS1, especially PRPS1 in the innate immunity of teleost fishes, which will benefit the development of marine fish aquaculture. [ABSTRACT FROM AUTHOR]

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 Ca2⁺, 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. • A novel tandem-repeat-type galectin-8 was identified from the large yellow croaker (Larimichthys crocea). • LcGal-8 expression was up-regulated in immune tissues following Pseudomonas plecoglossicida infection. • LcGal-8 exhibited hemagglutinating, bacterial agglutinating activities, specifically targeting and killing gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora , which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi , the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida , Vibrio parahemolyticus , V. harveyi , and Aeromonas hydrophila , and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora. [ABSTRACT FROM AUTHOR]

Cytoglobin (Cygb) is a 21-kDa heme-protein that belongs to the globin superfamily and is expressed in vertebrate tissues. It can participate in the oxidative stress response in organisms through the porphyrin ring. Previous studies have shown that this protein, also known as YdCygb, has potential immune abilities in the infection of Vibrio harveyi in yellow drum (Nibea albiflora). In this study, we report the role of Cygb in the immune response of teleost fish for the first time. Quantitative RT-PCR analysis indicated that YdCygb was highly expressed in the liver and intestine of yellow drum, and its expression can be upregulated by pathogenic attack. The cellular distribution of YdCygb-EGFP proteins was observed in cell membrane, cytoplasm, and nucleus in the kidney cells of N. albiflora. Furthermore, a comparative transcriptome analysis between the YdCygb overexpression group and control vector group identified 28 differentially expressed genes (DEGs). The analysis showed that ANPEP, CLDN5, ORM1/2, SERPINC1 and HPN and ITGAM might play important regulatory roles to Cygb in fish. Notably, using GST-pull down technology, we identified 3-phosphoglyceraldehyde dehydrogenase and intermediate filament protein as direct interactors with YdCygb, playing a role against V. harveyi. The molecular and functional characterization of YdCygb provides better understanding of the genetic basis of disease resistance traits in yellow drum and sheds new light on the functioning of Cygb and its potential regulatory signaling pathway as well. • YdCygb was reported on the role in the immune response of teleost fish for the first time. • YdCygb was in the cell membrane, cytoplasm, and nucleus in Nibea albiflora kidney cells. • The expression of YdCygb mRNA was significantly induced after Vibrio harveyi stimulation. • ANPEP, CLDN5, ORM1/2, SERPINC1 and HPN and might play important regulatory roles to Cygb in fish. • YdCygb resists Vibrio harveyi by interacting with 3-phosphoglyceraldehyde dehydrogenase and intermediate filament protein. [ABSTRACT FROM AUTHOR]

Galectins are a family of evolutionarily conserved lectins that contain carbohydrate recognition domains (CRDs) specifically recognizing β-galactoside. Galectin-9 plays a crucial role in various biological processes during pathogenic infections. In a previous study, galectin-9 was identified as a candidate gene for resistance to Vibrio harveyi disease in yellow drum using a genome-wide association study (GWAS) analysis. In this study, a galectin-9 gene was identified from Nibea albiflora and named YdGal-9. The mRNA transcripts of YdGal-9 were distributed in all the detected tissues and the highest level was found in the kidney. The subcellular localization of YdGal-9-EGFP proteins was observed in both nucleus and cytoplasm in the kidney cells of N. albiflora. The expression of YdGal-9 in the brain increased significantly after infection with Vibrio harveyi. The red blood cells from rabbits, Larimichthys crocea , and N. albiflora were agglutinated by the purified recombinant YdGal-9 proteins. The results of the agglutination activity of deletion mutants of YdGal-9 proved that the conserved sugar binding motifs (H-NPR and WG-EE-) were critical for YdGal-9's agglutination activity. In addition, YdGal-9 killed some gram-negative bacteria by inducing cell wall destruction including Pseudomonas plecoglossicida , Aeromonas hydrophila , Escherichia coli , V. parahemolyticus , V. harveyi , and V. alginolyticus. Taken together, these results suggested that the YdGal-9 protein of N. albiflora played a vital role in fighting bacterial infections. • A galectin-9 was identified from Nibea albiflora and named YdGal-9. • YdGal-9 was ubiquitously distributed in all the detected tissues. • YdGal-9 expression in the brain significantly increased after Vibrio harveyi infection. • YdGal-9 had agglutination activity towards red blood cells. • YdGal-9 killed some gram-negative bacteria and resisted bacterial infections. [ABSTRACT FROM AUTHOR]

Vibrio harveyi is the primary pathogenic bacteria affecting Nibea albiflora aquaculture. In a previous phase, our laboratory intentionally exposed N. albiflora to V. harveyi and analyzed the outcomes using a combination of genome-wide association study (GWAS) and RNA-seq. The results revealed that the antimicrobial peptide NK-lysin (YdNkl-1) was a candidate gene for resistance to V. harveyi disease in N. albiflora. To investigate the role of the antimicrobial peptide NK-lysin in N. albiflora 's antimicrobial immunity, we screened the YdNkl-1 gene from the transcriptome database. The full-length cDNA of YdNkl-1 gene is 508 bp, with an open reading frame (ORF) of 477 bp, encoding 158 amino acids. The deduced amino acid sequence of YdNkl-1 contains a signal peptide (1st-22nd amino acids) and a Saposin B domain (50th-124th amino acids), akin to mammalian NK-lysin. Phylogenetic tree analysis confirmed that the NK-lysin of teleost fish clustered into a single species, and YdNkl-1 was most closely related to Larimichthys crocea. Subcellular localization showed that YdNkl-1 was distributed in cytoplasm and nucleus of yellow drum kidney cells. Furthermore, YdNkl- 1 mRNA transcripts were significantly up-regulated in the skin, gill, intestine, head-kidney, liver, and spleen after V. harveyi infection, suggesting a critical role in N. albiflora's defense against V. harveyi infection. Additionally, we purified and observed the YdNkl-1 protein, which exhibited a potent membrane-disrupting effect on V. harveyi, Pseudomonas plecoglossicida, Vibrio parahaemolyticus, Escherichia coli and Bacillus subtilis. These findings underscore the significance of NK-lysin in N. albiflora 's resistance to V. harveyi infection and provide new insights into the crucial role of NK-lysin in the innate immunity of teleost fishes. • YdNkl-1 was constitutively expressed in tissues with the highest expression in skin. • YdNkl-1 possessed a signal peptide and a Saposin B domain. • YdNkl-1 was in cytoplasm and nucleus of yellow drum cells. • The expression of YdNkl-1 mRNA was significantly induced after Vibrio harveyi stimulation. • YdNkl-1 has potent antibacterial activity and broad-spectrum antibacterial properties. [ABSTRACT FROM AUTHOR]

Molecular dissection of disease resistance against Vibrio harveyi infection in yellow drum at the genome-wide level uncovered a C-type lectin-like receptor cluster of differentiation CD302 (named as YdCD302) in our previous study. Here, the gene expression pattern of YdCD302 and its function in mediating the defense response to V. harveyi attack were investigated. Gene expression analysis demonstrated that YdCD302 was ubiquitously distributed in various tissues with the highest transcript abundance in liver. The YdCD302 protein exhibited agglutination and antibacterial activity against V. harveyi cells. Binding assay indicated that YdCD302 can physically interact with V. harveyi cells in a Ca2+-independent manner, and the interaction can activate reactive oxygen species (ROS) production in the bacterial cells to induce RecA/LexA-mediated cell death. After infection with V. harveyi , the expression of YdCD302 can be up-regulated significantly in the main immune organs of yellow drum and potentially further trigger the cytokines involved innate immunity. These findings provide insight into the genetic basis of the disease resistance trait in yellow drum and shed light on the functioning of the CD302 C-type lectin-like receptor in host-pathogen interactions. The molecular and functional characterization of YdCD302 is a significant step towards a better understanding of disease resistance mechanisms and the development of new strategies for disease control. [ABSTRACT FROM AUTHOR]

Yellow drum (Nibea albiflora), a commercially important fish species in the coastal regions of southeast China, is highly susceptible to red-head disease caused by Vibrio harveyi B0003. Probiotics have been shown to enhance disease resistance in fish, but whether commensal probiotics could improve of the resistance to red-head disease in yellow drum and possible mechanisms has yet not been reported. A six-week feeding trial was conducted to investigate the red-head disease resistance potentials of five probiotic candidates (Bacillus megaterium B1M2, B. subtilis B0E9, Enterococcus faecalis AT5, B. velezensis DM5 and B. siamensis B0E14), and the liver health, serum and skin immunities, gut and skin mucosal microbiota of yellow drum were determined to illustrate the possible mechanisms. The results showed that autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5, P < 0.05) effectively improved red-head disease resistance in yellow drum. Furthermore, B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) efficiently improve liver health by improving liver morphology and decreasing serum glutamic oxaloacetic transaminase and glutamic propylic transaminase activities pre and post challenged with V. harveyi B0003 (P < 0.05). B. subtilis B0E9 and E. faecalis AT5 led to significant improvement (P < 0.05) in the serum complement 3 content (un-detected after challenged with V. harveyi B0003), lysozyme activity and skin mucosal immunity (such as IL-6, IL-10 and lysozyme expression) pre and post challenged with V. harveyi B0003, which was generally consistent with the cumulative mortality after challenged with V. harveyi B0003. This induced activations of serum and skin mucosal immunities were consistent with the microbiota data showing that B. subtilis B0E9 and E. faecalis AT5 modulated the overall structure of intestinal and skin mucosal microbiota, and in particular, the relative abundance of potentially pathogenic Achromobacter decreased while beneficial Streptococcus , Rothia , and Lactobacillus increased in fish fed with B. subtilis B0E9 and E. faecalis AT5. Overall, autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) can improve liver health, serum and skin immunities (especially up-regulated lysozyme activity and inflammation-related genes expression), positively shape gut and skin mucosal microbiota, and enhance red-head disease resistance of yellow drum. • Autochthonous probiotic E. faecalis AT5 and B. subtilis B0E9 improved red-head disease resistance. • Commensals improve liver health, mucosal immunity and microbiota of yellow drum. • Lysozyme activity may serve as a biomarker for assessing red-head disease resistance. [ABSTRACT FROM AUTHOR]

Yellow drum (Nibea albiflora), a commercially important fish species in the coastal regions of southeast China, suffers greatly from attacks from bacteria, viruses, and parasites. To uncover their immune responses, the yellow drums were artificially challenged with the pathogenic Vibrio harveyi , and genome-wide association study (GWAS) was performed to identify the associated SNPs and genetic loci for disease resistance trait. Sequence analysis in 197 susceptible and 148 resistant fish identified 23 significantly associated SNPs and 8 candidate genes distributed in chromosome 14. The estimated proportion of phenotypic variance explained (PVE) by SNPs ranged from 10.64% to 13.68%. The discovery of sphingosine kinase (SPHK), CACNA1H , and MGAT5B harboring or within the flanking region of trait-associated SNPs may account for the underlying antibacterial mechanism in yellow drum. These results provide molecular basis for prevention of disease spread and a reference for genetic breeding in fish aquaculture. • The genomic loci conferring resistance against Vibrio harveyi infection, explained 13.68% phenotypic variances, was mapped in the yellow drum. • A total of 54 significantly associated SNPs and 8 candidate genes located on chromosome 14 were identified. • SPHK1 , CACNA1H and MGAT5B within the flanking region of top associated SNPs may play important roles in antibacterial mechanism in the yellow drum. [ABSTRACT FROM AUTHOR]