Genome-wide characterization, phylogenetic and expression analysis of Galectin gene family in Golden pompano Trachinotus ovatus.

Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals (ToGals) were identified in the Golden pompano (Trachinotus ovatus), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae, indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa, and Aeromonas hydrophila. This study lays the foundation for further research on the immune roles of Gals in teleosts.

Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides.

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 µM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Identification of the NOD-like receptor family of golden pompano and expression in response to bacterial and parasitic exposure reveal its key role in innate immunity.

This study presents a genome-wide identification of NOD-like receptors (NLRs) in the golden pompano, key to its innate immunity. We identified 30 ToNLRs, analyzing their chromosomal positions, characteristics, evolutionary relationships, evidence of positive selection, and synteny with the yellowtail kingfish. Our findings categorize these NLRs into three main subgroups: NLRA, NLRC, and the distinct ToNLRX1. Post-exposure to Streptococcus agalactiae, most ToNLRs increased expression in the spleen, whereas NLRC3like13, NLRC3like16, and NLRC3like19 so in the kidneys. Upon Cryptocaryon irritans exposure, we categorized our groups based on the site of infection into the control group (BFS), the trophont-attached skin (TAS), and the nearby region skin (NRS). ToAPAF1 and ToNOD1 expressions rose in the NRS, in contrast to decreased expressions of ToNLRC5, ToNWD1 and ToCIITA. Other ToNLRs showed variable expressions in the TAS. Overall, this research lays the groundwork for further exploration of innate immunity in the golden pompano.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.

Cryopreservation of black seabream (Acanthopagrus schlegelii) sperm.

In recent years, biotechnology has had a significant impact on the aquaculture industry, particularly in the field of breeding. Molecular selection breeding has emerged as a novel approach to breeding. Reducing the cost of genetic information for individuals with desirable traits after breeding has become an important research direction. Cryopreservation technology allows bypassing time and space constraints in genetic breeding, simplifying broodstock management. This study presents a detailed cryopreservation method for black seabream sperm, evaluating extender type, glucose concentration, cryoprotectant type and concentration, sperm-dilution ratio, and cooling protocols. Sperm motility parameters were analyzed using computer-assisted sperm analysis (CASA) before and after two days of freezing. This involved using an RS solution with a glucose concentration of 15 g/L and adding a 5% final concentration of EG as the sperm cryoprotectant. After mixing the sperm and solution at a ratio of 1:2, we subjected it to 5 min fumigation at 5 cm above the liquid nitrogen surface before plunging it into the nitrogen. Sperm motility reached 85.46 ± 7.32% after two days. Various enzymatic activities showed changes over 20 days post-cryopreservation. This improved cryopreservation protocol for black seabream sperm is beneficial for genetic breeding and reproduction and provides reference for studying the cryodamage mechanisms of black seabream sperm.

Characterization of the MMP9 Gene and Its Association with Cryptocaryon irritans Resistance Traits in Trachinotus ovatus (Linnaeus, 1758).

The MMPs are endogenous proteolytic enzymes that require zinc and calcium as cofactors. MMP9 is one of the most complex matrix metalloproteinases in the gelatinase family and has many biological functions. In mammals, mmp9 is thought to be closely associated with cancer. However, studies in fish have rarely been reported. In this study, to understand the expression pattern of the ToMMP9 gene and its association with the resistance of Trachinotus ovatus to Cryptocaryon irritans, the sequence of the MMP9 gene was obtained from the genome database. The expression profiles were measured by qRT-PCR, the SNPs were screened by direct sequencing, and genotyping was performed. The ToMMP9 gene contained a 2058 bp ORF encoding a putative amino acid sequence of 685 residues. The homology of the ToMMP9 in teleosts was more than 85%, and the genome structure of ToMMP9 was conserved in chordates. The ToMMP9 gene was expressed in different tissues of healthy individuals and was highly expressed in the fin, the gill, the liver and the skin tissues. The ToMMP9 expression in the skin of the infected site and its adjacent sites increased significantly after C. irritans infection. Two SNPs were identified in the ToMMP9 gene, and the SNP (+400A/G) located in the first intron was found to be significantly associated with the susceptibility/resistance to C. irritans. These findings suggest that ToMMP9 may play an important role in the immune response of T. ovatus against C. irritans.