Fig. 4

Functional radiation of R. metallica aculeatoxins. A Phylogenetic analysis showed R. metallica aculeatoxins form functionally distinct clades and that individual workers contained representatives from several of these clades. Phylogeny was calculated by maximum likelihood under the JTT+G4 model and is displayed as a midpoint rooted tree. The names of each peptide synthesized and screened for activity are highlighted and shown in bold, with the prevalence of each indicated on the right and the highlight color corresponding to the type of activity detected in b–d; insecticidal (red), defensive (purple), cytolytic (blue), unknown (cyan). B Intra-abdominal injection of Rm1a (but not other venom peptides) in crickets (Acheta domesticus) caused dose-dependent, irreversible, and lethal incapacitation (n = 3 per group). Curves were fitted using a four-parameter Hill equation (variable slope) in Graphpad Prism8 (top). At a dose of 40 nmol/g, only Rm1a resulted in incapacitation of crickets even after 30 min (bottom). C Upon intraplantar injection of 200 pmol peptide (n = 3 per group), only Rm4a caused spontaneous nocifensive behavior in mice as measured by pain behavior counts in 5-min bins (top) or cumulative counts (bottom) across 30 min. D 10 μM Rm20a, Rm34a, Rm54a and Rm55b significantly reduced HAP cell viability; n = 3 per group. 100 μM (bottom) Rm55a significantly reduced HAP cell viability to 24.6 ± 8.1% (n = 3) and has therefore also been labeled as cytotoxic in panel A. All data are expressed as mean ± SEM. Sequence alignments can be found in Additional file 3, individual datapoints for time courses in B and C are shown in Additional file 4: Fig. S1