Hormonal pleiotropy helps maintain queen signal honesty in a highly eusocial wasp

Abstract

In insect societies, both queens and workers produce chemicals that reliably signal caste membership and reproductive status. The mechanisms that help to maintain the honesty of such queen and fertility signals, however, remain poorly studied. Here we test if queen signal honesty could be based on the shared endocrine control of queen fertility and the production of specific signals. In support of this "hormonal pleiotropy" hypothesis, we find that in the common wasp, application of methoprene (a juveline hormone analogue) caused workers to acquire a queen-like cuticular hydrocarbon profile, resulting in the overproduction of known queen pheromones as well as some compounds typically linked to worker fertility. By contrast, administration of precocene-I (a JH inhibitor) had a tendency to have the opposite effect. Furthermore, a clear gonadotropic effect of JH in queens was suggested by the fact that circulating levels of JH were ca. 2 orders of magnitude higher in queens than those in workers and virgin, non-egg-laying queens, even if methoprene or precocene treatment did not affect the ovary development of workers. Overall, these results suggest that queen signal honesty in this system is maintained by queen fertility and queen signal production being under shared endocrine control.

Figure 1

Heatmap showing JH-induced changes in cuticular hydrocarbon profiles. Effect of treating workers with the JH-analog methoprene and the JH inhibitor precocene-I on cuticular hydrocarbon profiles and known queen and fertility signals. Colors indicate the fold difference in relative abundance of each CHC peak compared to control workers with undeveloped ovaries. Hence, a value of two implies that the relative abundance of a given compound would be twice as high as in the reference acetone solvent control group. Columns of the heatmap correspond to the different treatments, which were split up by observed ovary development. The compound cluster analysis was based on a UPGMA hierarchical clustering using one minus the Pearson correlation as the distance metric. Values indicate mean absolute quantities per worker in ng and asterisks indicate significance levels for the following contrasts: control developed vs control undeveloped, precocene developed vs control developed, precocene undeveloped vs control undeveloped, methoprene developed vs control developed, methoprene undeveloped vs control undeveloped and queens vs methoprene developed, derived from a LMM with Benjamini & Yekutieli FDR p value correction: *p < 0.05, **p < 0.01, ***p < 0.001. For detailed statistical results see Supplemental Table S2.

Figure 2

Principal component analysis of JH-induced changes in cuticular hydrocarbon profiles. (a) PC factor scores (calculated from a PCA on Aitchison transformed relative compound abundances) show that treatment with the JH-analog methoprene (red) induced worker profiles to become more queen-like (orange), whereas treatment with the JH inhibitor precocene-I had a slight reverse effect (n = 40 per group for workers and n = 14 for egg-laying queens). Workers with developed ovaries (circles) can also be observed to be more queen-like along PC axis 1 than workers with undeveloped ovaries (triangles). (b) Corresponding factor loadings for the different hydrocarbon compounds. The four known queen pheromones appear in the lower right quadrant (n-C₂₇, n-C₂₈, n-C₂₉ and 3-MeC₂₉).

Figure 3

Juvenile hormone regulates known queen signals in the common wasp. Treatment of workers with the JH analog methoprene or the JH inhibitor precocene-I enhances or suppresses the production of four known sterility-inducing hydrocarbon queen pheromones (bars and whiskers = mean relative abundances and 95% confidence intervals calculated on log transformed relative abundance and then backtransformed to the original scale, asterisks above bars indicate the significance of contrasts with their respective control, whereas the asterisks above each pair of bars indicate differences in abundance between workers with or without developed ovaries of a given group; significance levels are based on linear mixed models on Aitchison transformed abundances, using Benjamini & Yekutieli FDR p value correction: *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

Juvenile hormone titers support a gonadotropic effect in common wasp queens. Circulating JH levels in pg/μl are 88 times higher in mature, egg-laying queens than in non-reproductive virgin queens, and 325 times higher than in nonreproductive workers (black circles and whiskers = averages and 95% confidence intervals calculated on a log scale and then backtransformed to the original scale, boxes = quartiles, horizontal bars = medians, n = 5 per group, but using pools of 5 individuals per sample for the workers; significance levels based on Anova and Tukey posthoc tests performed on log transformed data: **p < 0.01, ***p < 0.001).

Figure 5

Juvenile hormone does not have a gonadotropic effect in workers. Treatment of workers with the JH analog methoprene or the JH inhibitor precocene-I did not affect rates of worker ovary development compared to their respective control when scored on a five point scale (I = undeveloped, V = fully developed) after a period of 13 days (panel a = stacked bar charts, panel b = mean ovary development and 95% confidence intervals shown on an ordinal scale, n.s. = non-significant differences based on an ordered logistic model, n = 40 workers per treatment group).

Figure 6

Scheme of the experimental setup. The combs of each of four natural colonies were divided into equal halves and put into separate boxes together with ca. 50 untreated workers. The abdomen of ca. 80 newly emerged workers per box and treatment was then topically treated once with 5 µl of either 20 µg/µl methoprene in acetone solution (green), 6 µg/µl precocene-I in acetone solution (blue), or an acetone solvent control (red). After two weeks the workers were frozen at −20 °C for later analysis of the CHCs.