Venom Composition in a Phenotypically Variable Pit Viper (Trimeresurus insularis) across the Lesser Sunda Archipelago

The genus Trimeresuruscomprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularissampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresuruscomplex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis.Despite the geographical isolation, T. insularisvenoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P–III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2(PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2inhibitor, phospholipase B, cobra venom factor, 5′-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularisvenoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularisvenoms exhibited overall similarity to the other Trimeresuruscomplex species examined, with the exception of P. flavoviridisvenom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularisvenom proteins were recognized by Green Pitviper (T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurusspecies, but incomplete antivenom–venom recognition was observed against P. flavoviridisvenom proteins. These results demonstrate significant conservation in the venom composition of T. insularisacross the Lesser Sunda archipelago relative to the other Trimeresurusspecies examined.