Saddleback caterpillar venom proteomics

Limacodidae is a family of lepidopteran insects comprising more than 1,500 species, over half of which produce pain-inducing defensive venom components in the larval stage, but little is known about their venom. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans, but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 confirmed venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A. stimulea venom, indicating strong similarities to D. vulnerans venom, despite their geographic separation. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of these RF-amide toxins displayed potent activation of the human neuropeptide FF1 receptor, insecticidal activity when injected into Drosophila melanogaster, and moderate ability to inhibit the larval development of the parasitic nematode Haemonchus contortus. This study provides insights into venom evolution and toxin activity in Limacodidae, and will allow the synthetic production of A. stimulea peptide toxins towards functional and structural characterisation.