Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.
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