Long-read transcriptomics highlights venom gland specialization and Inhibitor Cystine Knot (ICK) rich toxin diversity in Philippine tarantulas

Animal venoms are a rich source of bioactive molecules, yet their diversity remains incompletely characterized in many species. Here we present the first long-read transcriptomic analysis of venom glands from Philippine tarantulas (Theraphosidae), a highly endemic but understudied group. Using Oxford Nanopore sequencing, we reconstructed near full-length venom gland transcriptomes across multiple species and identified extensive repertoires of toxin-encoding peptides. Venom glands were enriched in cysteine-rich inhibitor cystine knot (ICK) peptides, which dominated the toxin landscape and are known modulators of ion channels. Cross-species comparative analyses revealed a distinct transcriptional signature separating venom from non-venom tissues, driven by coordinated expression of toxin-associated and regulatory gene families. Phylogenomic reconstruction based on orthologous peptides recovered expected taxonomic relationships while revealing potential lineage-specific diversification and potential cryptic taxa. Despite a conserved core set of toxin families, substantial variation in toxin composition was observed among species, consistent with rapid evolution driven by gene duplication and functional divergence. Analysis of highly expressed ICK peptides showed a conserved cysteine framework alongside marked sequence variability in inter-cysteine regions, supporting a model in which structural stability is maintained while functional diversification proceeds. Together, these findings establish the first long-read transcriptomic resource for Philippine theraphosid spiders, reveal a conserved molecular signature underlying venom gland specialization, and provide new insights into the diversification of ICK toxin repertoires that may facilitate future evolutionary and functional studies, including the discovery and characterization of bioactive venom peptides.