Multiple mechanisms of action of an extremely painful venom.

Evolutionary arms races between predator and prey can lead to extremely specific and effective defense mechanisms. Such defenses include venoms that deter predators by targeting nociceptive (pain-sensing) pathways. Through co-evolution, venom toxins can become extremely efficient modulators of their molecular targets. The venom of velvet ants (Hymenoptera: Mutillidae) is notoriously painful. The intensity of a velvet ant sting has been described as "Explosive and long lasting, you sound insane as you scream. Hot oil from the deep fryer spilling over your entire hand." [1] The effectiveness of the velvet ant sting as a deterrent against potential predators has been shown across vertebrate orders, including mammals, amphibians, reptiles, and birds [2-4]. The venom's low toxicity suggests it has a targeted effect on nociceptive sensory mechanisms [5]. This leads to the hypothesis that velvet ant venom targets a conserved nociception mechanism, which we sought to uncover using as a model system. larvae have peripheral sensory neurons that sense potentially damaging (noxious) stimuli such as high temperature, harsh mechanical touch, and noxious chemicals [6-9]. These polymodal nociceptors are called class IV multidendritic dendritic arborizing (cIV da) neurons, and they share many features with vertebrate nociceptors, including conserved sensory receptor channels [10,11]. We found that velvet ant venom strongly activated nociceptors through heteromeric Pickpocket/Balboa (Ppk/Bba) ion channels. Furthermore, we found a single venom peptide (Do6a) that activated larval nociceptors at nanomolar concentrations through Ppk/Bba. Ppk/Bba is homologous to mammalian Acid Sensing Ion Channels (ASICs) [12]. However, the Do6a peptide did not produce behavioral signs of nociception in mice, which was instead triggered by other non-specific, less potent, peptides within the venom. This suggests that Do6a is an insect-specific venom component that potently activates insect nociceptors. Consistent with this, we showed that the velvet ant's defensive sting produced aversive behavior in a predatory praying mantis. Together, our results indicate that velvet ant venom evolved to target nociceptive systems of both vertebrates and invertebrates, but through different molecular mechanisms.