Characterization of a Specialized Venomous Spine System and Identification of a Cytolytic Toxin, Cytolytic Toxin-Alpha-like (CTα-L/BGT-1), in the Bagrid Catfish Leiocassis longirostris

Venomous spines are widespread defensive structures in fishes, yet the anatomical organization, molecular composition, and cellular basis of venom systems in many catfishes remain poorly characterized. Here, we combined morphology, proteomics, FISH, scRNA-seq, sequence comparison, and in vivo puncture to study L. longirostris spines. Histology revealed a spine architecture with longitudinal grooves, epidermal glands, a basement layer, and internal canals. DIA-MS identified a spine-enriched toxin repertoire in which CTα-L accounted for 95.34% of toxin abundance and was among the top proteins. Based on its sequence characteristics and close relationship to CTα-L homologs from bagrid catfishes, we designated this protein Bagridae toxin-1 (BGT-1). Spatial and single-cell analyses localized CTα-L-family transcripts to glandular and basement-layer compartments, with enrichment in several specialized cellular populations, including proliferating endothelial cells, immature epithelial progenitor cells, and junctional vascular smooth muscle cells. Sequence and structural comparisons revealed that BGT-1 retains conserved features of fish cytolytic toxins while exhibiting a distinctive multidomain architecture containing SNTX-like, Stonustoxin-associated, thioredoxin-like, FNIII, and tandem galectin domains. Structural modeling further identified a predicted pore-compatible architecture within the conserved toxin-associated core and provided a basis for a putative working model of BGT-1-mediated cytolysis. Finally, puncture with active spines caused rapid hemorrhage, edema, myofiber injury, inflammatory infiltration, and increased TUNEL-positive cells in vivo, whereas heat-inactivated spines produced substantially milder tissue damage. Together, these findings link specialized anatomy, a predominant toxin-associated protein, its cellular localization, molecular architecture, and in vivo activity in this venom system, providing a framework for comparative venom studies across catfishes.