Social regulation of insulin signaling and the evolution of eusociality in ants

Abstract

Queens and workers of eusocial Hymenoptera are considered homologous to the reproductive and brood care phases of an ancestral subsocial life cycle. However, the molecular mechanisms underlying the evolution of reproductive division of labor remain obscure. Using a brain transcriptomics screen, we identified a single gene, insulin-like peptide 2 (ilp2), which is always up-regulated in ant reproductives, likely because they are better nourished than their nonreproductive nestmates. In clonal raider ants (Ooceraea biroi), larval signals inhibit adult reproduction by suppressing ilp2, thus producing a colony reproductive cycle reminiscent of ancestral subsociality. However, increasing ILP2 peptide levels overrides larval suppression, thereby breaking the colony cycle and inducing a stable division of labor. These findings suggest a simple model for the origin of ant eusociality via nutritionally determined reproductive asymmetries potentially amplified by larval signals.

Figure 1:. Brain gene expression in seven ant species identifies one conserved differentially expressed gene.

The figure shows the summary cladogram of the seven ant species used in this study in the context of the entire ant phylogeny with all subfamilies labeled. Five of the focal species have queens, while two (D. quadriceps and O. biroi) are queenless. Although H. saltator is not queenless, the data compared reproductive and non-reproductive workers (Table S1). The dot plots show variance-stabilized transformed read counts for ilp2. Blue and orange dots indicate reproductive and non-reproductive ants, respectively. Horizontal bars indicate means, and asterisks indicate statisically significant differences between groups (Wald test: * p<0.05; *** p<0.001). All images except for A. echinatior are from A. Nobile, S. Hartman, and E. Prado (www.antweb.org). Scale bars represent 2mm. The phylogeny is based on (30). Species numbers are from (6).

Figure 2:. Larvae regulate ilp2 in adults.

(A-C) Immunohistochemistry with anti-ILP2 antibody on an O. biroi brain localizes ILP2 peptide to a single cluster of insulin-producing cells (IPCs) in the pars intercerebralis (body-axis dorsal view). Green: anti-ILP2; blue: DAPI; magenta: phalloidin. MB: mushroom body; AL: antennal lobe. (D) Total intensity of ILP2 in the insulin-producing cells is higher in the brood care phase than in the reproductive phase (n≥14, t-test; p=0.046). (E) RNA-Seq time course shows that the addition of larvae downregulates ilp2, whereas the removal of larvae upregulates ilp2 (n≥4, time:transition interaction, Likelihood Ratio Test with 5% FDR correction; p<10⁻¹⁵). The black arrow indicates when ants with larvae were fed, i.e. changes in expression beyond that time point are confounded by differences in nutrition. Error bars depict SEM. Data from (28). (F) RNA-Seq on ant brains shows that under nutritionally controlled conditions, ilp2 is upregulated eight days after larvae are removed from O. biroi workers in the brood care phase (n=4, Wald test with 5% FDR correction; p<10⁻⁶). Data are variance-stabilized transformed read counts. Horizontal bars indicate means.

Figure 3:. ILP2 supplementation overrides larval suppression of adult reproduction.

(A) Workers injected with 100 µM ILP2 in the brood care phase activate their ovaries relative to controls injected with 100 µM ILP2 B chain despite being in contact with larvae (n≥10, Welch’s t-test with Bonferroni correction (related data in Fig. S8); p=0.0005). (B and C) Confocal images of ovaries from ants injected with either 100 µM ILP2 (B) or 100 µM ILP2 B chain (C). Shown are the pairs of ovaries closest to the mean value from each treatment; the largest oocyte in each pair is circled in blue.

Figure 4:. Intercastes respond less to larvae and have more ILP2 than regular workers.

(A) Intercastes have more active ovaries than age-matched regular workers in the brood care phase, despite both being in contact with larvae (n≥16, Welch’s t-test; p<0.0001). (B) In the brood care phase (n=19, Mann-Whitney U test; p<0.0001) and (C) in the reproductive phase (n≥12, Mann-Whitney U test; p=0.0043), intercastes have more ILP2 in their insulin-producing cells than age-matched regular workers. Horizontal bars indicate means on all dot plots.