Intra-colony venom diversity contributes to maintaining eusociality in a cooperatively breeding ant

Abstract

Background: Eusociality is widely considered to evolve through kin selection, where the reproductive success of an individual's close relative is favored at the expense of its own. High genetic relatedness is thus considered a prerequisite for eusociality. While ants are textbook examples of eusocial animals, not all ants form colonies of closely related individuals. One such example is the ectatommine ant Rhytidoponera metallica, which predominantly forms queen-less colonies that have such a low intra-colony relatedness that they have been proposed to represent a transient, unstable form of eusociality. However, R. metallica is among the most abundant and widespread ants on the Australian continent. This apparent contradiction provides an example of how inclusive fitness may not by itself explain the maintenance of eusociality and raises the question of what other selective advantages maintain the eusocial lifestyle of this species.

Results: We provide a comprehensive portrait of the venom of R. metallica and show that the colony-wide venom consists of an exceptionally high diversity of functionally distinct toxins for an ant. These toxins have evolved under strong positive selection, which is normally expected to reduce genetic variance. Yet, R. metallica exhibits remarkable intra-colony variation, with workers sharing only a relatively small proportion of toxins in their venoms. This variation is not due to the presence of chemical castes, but has a genetic foundation that is at least in part explained by toxin allelic diversity.

Conclusions: Taken together, our results suggest that the toxin diversity contained in R. metallica colonies may be maintained by a form of group selection that selects for colonies that can exploit more resources and defend against a wider range of predators. We propose that increased intra-colony genetic variance resulting from low kinship may itself provide a selective advantage in the form of an expanded pharmacological venom repertoire. These findings provide an example of how group selection on adaptive phenotypes may contribute to maintaining eusociality where a prerequisite for kin selection is diminished.

Keywords: Eusocial; Formicidae; Group selection; Kin selection; Peptide; Toxin.

Fig. 1

The polypeptidic venom composition of R. metallica. A R. metallica worker. B Dissected venom apparatus of R. metallica worker. C SDS-PAGE gel of R. metallica venom (pooled) labeled with major venom proteins identified by LC-MS/MS. D Total ion chromatogram from LC-MS/MS analysis of R. metallica venom (pooled from ~100 worker ants) with peaks corresponding to identified venom peptides labeled. Additional peptides were identified only in the reduced and alkylated and/or reduced, alkylated and trypsin-digested venom samples. E Venom component-encoding transcripts (i.e., those encoding peptides detected in the venom itself) comprised 96% of total transcript expression. Of these, transcripts encoding aculeatoxin peptides, EGF-domain peptides, a CNH-domain peptide, and venom proteins comprised 98.81, 0.36, 0.04, and 0.79%, respectively, of venom component expression. F Venom component-encoding transcripts (highlighted in green) constitute almost all the most highly expressed transcripts. TPM, transcripts per million

Fig. 2

Amino acid sequence alignments of the three major aculeatoxin clades of R. metallica venom. Sequence identity plots (sliding window of 2) are shown above each alignment. Methionine, lysine/arginine, aspartate/glutamate, and cysteine residues are highlighted in purple, blue, red, and yellow, respectively. Signal peptides and mature peptides are underlined in purple and gray, respectively. ^ indicates sequences with predicted mature peptide regions.

Fig. 3

Individual R. metallica workers from a single colony have distinct venom compositions. A MALDI mass spectrum, generated in reflectron positive mode, of pooled R. metallica venom. Selected peaks are labeled with observed monoisotopic MH⁺¹. B Gel view of MALDI-MS spectra collected in three technical replicates from pooled venom and venoms of six individual workers illustrates intra-colony variation in R. metallica venom

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

Fig. 5

Venom peptides are pharmacologically diverse. Effect of venom peptides on F11 cells: A Rm1a, B Rm4a, C Rm9a, D Rm20a, E Rm34a, F Rm52d, G Rm53a/b, H Rm53c, I Rm54a, J Rm55a, and K Rm55b. Values are max – min (n = 3), and error bars represent SEM. Curves were fitted using a four-parameter Hill equation (variable slope) in Graphpad Prism v8. Inset are the corresponding fluorescence-time traces; error bars show SEM (n = 3)

Fig. 6

Intra-colony toxin diversity in R. metallica is at least partly due to toxin allelic variation. A Gel view of MALDI MS spectra collected in positive reflectron mode in three technical replicates from venoms of eight individual workers across four different colonies of R. metallica, showing both high intra- and inter-colony variation in venom composition. B Principal component analysis (PCA) and C clustering analysis of MALDI MS spectra of individual workers from different colonies revealed neither colony-specific clusters nor across-colony clusters suggestive of chemically distinct worker castes. Datapoints in the PCA plots and numbered specimens in the clustering analysis are colored according to the colony label in A. D PCR products from genomic DNA using toxin-specific primers show that the intra-colony genetic variation is at least in part due to toxin allelic variants. Genomic was extracted from individual ants belonging to the same colony as those used for our transcriptome and main proteomic data. DNA standards and their corresponding length (bp) are shown in the left- and right-most lanes, individual ants are labeled 1–10, while C represents negative control. Full gel images are shown in Additional file 4: Fig. S4

Fig. 7

Site-specific selection on R. metallica aculeatoxin genes. Log ratio test scores, omega values, and p-values obtained by A fixed effects likelihood (FEL) and B mixed effects model of evolution (MEME) tests, partitioned according to signal-, pro-, and mature peptide domains as indicated by vertical solid lines. Dotted horizontal lines indicate either significance thresholds (FEL and MEME) or positive (> 1) or negative (< 1) selection (FEL). For plotted values see Additional file 6: Table S3. For alignments, see Additional file 3