The venom system of bees has been primarily studied with a strong emphasis on the honeybee (Apis mellifera), yet this represents only a small part of the diversity in the Anthophila (20,000 bee species). The short amphipathic pore-forming peptide melittin, is the main pain-causing component in honeybee venom, but it remains uncertain how conserved this peptide is across other bee lineages. Here, we investigated the evolution and functional diversification of venom peptides across bees. Using genomic, transcriptomic and proteomic data from a total of 75 species, spanning all major bee families, we identified a diverse suite of melittin orthologues as well as novel cysteine rich ‘anthophilin’ peptides. We synthesized 35 representative melittin-family peptides and tested their activity at mammalian sensory neurons, as a proxy for pain-causing activity.
We show that the melittin family of peptides share common ancestry yet exhibit variation in primary structure and pain-causing activity. The most potent pain-causing peptides have evolved within eusocial Apis species, suggesting lineage-specific adaptation associated with colony-level defence against vertebrate predators. We also demonstrate that some solitary species of bee utilise venom dominated by anthophilin peptides, suggesting a possible role for these peptides in defence.This study expands upon the putative simplicity of bee venom and redefines melittin not as a taxonomically restricted molecule, but as one member of a larger, evolutionarily dynamic toxin family. Our findings provide new insights into the origin and diversity of pain-causing toxins in bees and highlight a wealth of unexplored peptide diversity.