Comparative Genomics Reveals Diversification and Chromosomal Organization of Putative Antimicrobial Peptide-Derived Sequences in Amphibious Mudskippers

Antimicrobial peptides (AMPs) and AMP-like fragments are important components of vertebrate innate immunity, but their genome-wide diversification in amphibious fishes remains unclear. Here, we performed integrated bioinformatics and comparative genomic analyses of three representative mudskippers, Boleophthalmus pectinirostris (Bp), Periophthalmus magnuspinnatus (Pma), and Periophthalmus modestus (Pmo), together with zebrafish and humans as the reference vertebrates. Through genomics comparisons, we identified 708 putative AMP-derived genes in the three mudskipper genomes. Compared with zebrafish and humans, mudskippers contained fewer numbers of AMP-derived genes, indicating lineage-associated differences in repertoire size although their evolutionary basis remains unresolved. Several AMP-derived genes showed a pattern of clustered chromosomal distribution, such as histone-associated clusters on the Chr12 and Chr14 of the Bp genome. Histone H2B-derived sequences were highly conserved among various vertebrates, but mudskippers showed a distinct alanine-to-glycine substitution at position 66. In addition, a Misgurin-like fragment was located within TNNT3a rather than as an independent AMP gene. Compared with pond loach Misgurin, the synthetic Bp Misgurin-like fragment showed no detectable antibacterial activity against six tested bacterial strains under the tested assay conditions and concentration range, while this fragment was absent from the human TNNT3 gene. These findings provide new insights into AMP-derived sequence diversity, chromosomal organization, and potential immune adaptation in amphibious mudskippers, and offer candidate resources for future functional validation in medical and aquaculture applications.