Adult workers of M. gulosa were collected from a single colony near Kooringal, Moreton Island, Queensland, Australia. Venom was collected from three individuals through a combination of defensive stinging (movie S1) followed by electrostimulation of the abdomen (in an effort to deplete the venom reservoir and maximize the expression of toxin genes before RNA extraction for transcriptome sequencing). Defensive stings were collected by holding the ant with a pair of forceps while allowing it to sting through a layer of parafilm covering the opening of a 1.5-ml tube. Each ant aggressively and repeatedly stung the parafilm layer presented to it, depositing, on most occasions, a small drop of clear colorless venom. When no more defensive venom was secreted, the ant was allowed to rest for approximately 10 min. The procedure was then repeated while applying a mild electric current (15 V, ~1 mA) to the abdomen as soon as the stinger was inserted into the parafilm membrane, resulting in additional venom being expelled. Venom was collected from the bottom of the parafilm layer and the sides of the tube by rinsing with 10 μl of ultrapure water. Venom was then stored at −80°C until further analysis. We found no observable differences by LC-MS/MS of native venom (see below) between voluntary secreted venom and that obtained with electrostimulation, and pooled venom drops were used for subsequent analyses.

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