Toll-like receptors (TLRs) are central to pathogen recognition in teleost innate immunity. In this study, we surveyed 41 genomes from four representative teleost orders (i.e., Cypriniformes, Siluriformes, Perciformes, and Pleuronectiformes) for 15 TLR genes (TLR1–9, 12, 13, 18, 20–22) revealed a conserved core (TLR2/3/7 in nearly all examined species) alongside lineage-specific losses (TLR4/9/18/20/21/22), indicating both strong conservation and dynamic diversification of the TLR repertoire. We further identified and characterized 12 TLR genes in economically important darkbarbel catfish (Pelteobagrus vachellii). Corresponding cDNAs span 2089–4456 bp and encode proteins of 789–1,087 aa, with canonical extracellular LRR arrays and C-terminal TIR domains but notable “non-classical” features (such as absence of signal peptides in TLR1/13; no transmembrane segment in TLR7; multiple transmembranes in TLR3/8/13/18/22), suggesting subcellular and functional heterogeneity of various TLR genes. Subsequent gene-structure comparisons uncovered gene-specific exon–intron organizations and variable UTR lengths, implicating differential post-transcriptional regulation. Predicted 3D structures retain the traditional hallmark LRR horseshoe fold with subtle variations potentially tuning ligand specificity. Genomic synteny with Pseudobagrus ussuriensi and Pangasianodon hypophthalmus reveals conserved chromosomal organization, and phylogeny construction resolves each TLR subtype into well-supported monophyletic clades, which underscore evolutionary stability. Functionally, exogenous Aeromonas hydrophila challenge triggered rapid, tissue-dependent TLR up-regulation in the kidney, liver, and especially gill (with some transcripts > 1000-fold), highlighting coordinated mucosal and systemic surveillance in darkbarbel catfish. Taken together, these valuable data provide a comprehensive framework for the structural, evolutionary, and inducible expression landscape of catfish TLRs and establish a foundation for in-depth studies on antibacterial immunity in diverse teleost species.

DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in multiple important biological processes in fish, such as gonadal development. The cellular DNA methylation level is tightly regulated by DNA methyltransferases (Dnmt). However, detailed investigations of this family in fish are very scarce. In this study, our results confirmed that teleost genomes contain 4 to 16 Dnmt genes, with diversity likely resulting from a combination of whole-genome duplication (WGD), tandem duplication, and gene loss. Differences were observed in tissue distribution, transcription abundance, and protein structure of Dnmt duplicates, supporting their subfunctionalization or neofunctionalization after duplication. Interestingly, we found that fish Dnmt3b duplicates likely have acquired the functions of mammalian Dnmt3l, which may compensate for the absence of fish Dnmt3l. Furthermore, transcriptome analysis and qPCR results indicated that DNA methyltransferase genes (Dnmt1, Dnmt3aa, Dnmt3ab, Dnmt3ba, and Dnmt3bb.1) possibly play important roles in the natural sex change of protandrous hermaphrodite blackhead seabream (Acanthopagrus schlegelii) and inferred that global remodeling of gonadal DNA methylation, regulated by DNA methyltransferase genes, was closely associated with sex change in sequentially hermaphroditic fishes. Overall, our results may help provide a better understanding of the evolution and function of DNA methyltransferases in fish.

Extrachromosomal circular DNAs (eccDNAs) play a significant role in regulating various biological processes, including abnormal muscle development. The molecular functions and impact of eccDNAs in the muscle development of fish are poorly understood. To investigate the potential roles of eccDNAs in the muscle development of fish, we analyzed and compared the expression profile of muscle eccDNAs of crisp grass carp, fed a faba bean meal‐based diet, and ordinary grass carp, fed a practical diet. Using the Circle‐seq strategy, we found the eccDNA abundance in crisp grass carp (211,920 eccDNAs) was significantly higher than that in ordinary grass carp (25,857 eccDNAs), suggesting that the faba bean diet likely independently influences eccDNA production. Compared to ordinary grass carp, crisp grass carp exhibited 10,565 upregulated and 129 downregulated eccDNAs, indicating eccDNAs were possibly associated with the muscle development of grass carp. GO and KEGG enrichment analyses indicated that the upregulated eccDNAs were related to muscle fiber development, cellular structure, and cell junctions. Based on our results, we speculated that the overexpression of genes involved in muscle fiber, calcium metabolism, and collagen driven by eccDNAs likely contributes to the observed increase in muscle fiber density, calcium levels, and collagen content in crisp grass carp, thereby enhancing muscle hardness. Notably, eccDNAs were identified as potential innate immunostimulants capable of eliciting immune responses in fish. In summary, our findings demonstrate that eccDNAs are aberrantly expressed in the muscles of fish fed a faba bean diet, offering novel insights into the molecular mechanisms underlying muscle hardening in fish.

This study investigates the genomic basis of immune adaptation in the transparent glass catfish (Kv: Kryptopterus vitreolus), focusing on the loss of the Toll-like receptor 21 (TLR21) gene. Comparative genomic analysis with closely related non-transparent North African catfish (Cg: Clarias gariepinus) revealed 11 TLR genes in the latter, while only 8 TLR genes (KvTLR1, 2, 3, 5, 7, 9, 13, and 20) were retained in the glass catfish, with TLR21 specifically absent. Collinearity analysis confirmed that the genomic region containing TLR21 is conserved across eight siluriform species, with loss exclusively in the glass catfish, supporting its lineage-specific absence. Structural expansion was notable in KvTLR5, KvTLR7, and KvTLR20. Molecular docking indicated that binding stability between CpG oligonucleotides and TLR21 varies significantly, with CpG-B 1681 showing the strongest interaction, which highlights sequence-dependent ligand recognition. Interestingly, absence of the TLR1 gene in another transparent teleost, the X-ray tetra (Pristella maxillaris), suggests that transparent fishes may share an evolutionary trend of lineage-specific TLR gene loss. Together, these findings reveal a distinctive evolutionary trajectory in the innate immune receptor family of transparent fishes and provide new molecular insights into their adaptive immune strategies. These insights will benefit the academic community by improving comparative frameworks for fish innate immunity, and they may inform disease prevention and health management strategies in aquaculture and the ornamental fish trade.