Surface hydrocarbons of queen eggs regulate worker reproduction in a social insect

Abstract

A hitherto largely unresolved problem in behavioral biology is how workers are prevented from reproducing in large insect societies with high relatedness. Signals of the queen are assumed to inform the nestmates about her presence in the colony, which leads to indirect fitness benefits for workers. In the ant Camponotus floridanus, we found such a signal located on queen-laid eggs. In groups of workers that were regularly provided with queen-laid eggs, larvae, and cocoons, with larvae and cocoons alone, or with no brood, only in the groups with queen-laid eggs did workers not lay eggs. Thus, the eggs seem to inform the nestmates about the queen's presence, which induces workers to refrain from reproducing. The signal on queen-laid eggs is presumably the same that enables workers to distinguish between queen- and worker-laid eggs. Despite their viability, the latter are destroyed by workers when given a choice between both types. Queen- and worker-laid eggs differ in their surface hydrocarbons in a way similar to the way fertile queens differ from workers in the composition of their cuticular hydrocarbons. When we transferred hydrocarbons from the queen cuticle to worker-laid eggs, the destruction of those eggs was significantly mitigated. We conclude that queen-derived hydrocarbon labels inform workers about the presence of a fertile queen and thereby regulate worker reproduction.

Fig. 1.

Inhibition of worker egg laying by the presence of queen-laid eggs. After 160 days of separation from the parental colonies, the difference in worker egg laying among the groups was significant. Overall comparison: Cochran Q test, n = 19, Q = 17.64, P < 0.0001. Post hoc comparison: one-tailed Fisher's exact test, group b versus group c, P < 0.0001; group a versus group c, P < 0.002; group a versus group b, not significant.

Fig. 2.

Discrimination of untreated queen- and worker-laid eggs. Only medians are presented. Already after 24 h >62% of the worker-laid eggs disappeared on average whereas the queen-laid eggs remained almost untouched. The differences between the queen-laid eggs and the worker-laid eggs were statistically significant after 120 h [N_groups = 9; Friedman's ANOVA, P < 0.001, Wilcoxon-Wilcox test for multiple comparisons, P < 0.01 (own queen-laid eggs versus worker-laid eggs) and P < 0.05 (foreign queen-laid eggs versus worker-laid eggs); the difference was not significant between queen-laid eggs]. The decrease after 24 h may be partly due to accidental egg destruction during egg counting.

Fig. 3.

Chromatograms of the surface hydrocarbons of eggs and the cuticular hydrocarbons of fertile queens and workers. The compounds have been identified on the basis of retention times (in reference to GC/MS analysis). 1, N-pentacosane; 2, 3-methylpentacosane; 3, 10,14-dimethylhexacosane; 4, N-heptacosane; 5, 9-methyl-, 11-methyl-, and 13-methylheptacosane; 6, 11,15-dimethylheptacosane; 7, 3-methylheptacosane and 7,11-dimethylheptacosane; 8, N-octacosane; 9, 3,7-dimethyl-, 3,9-dimethyl-, 3,11-dimethyl-, and 3,13-dimethylheptacosane; 10, 10-methyl-, 12-methyl-, and 14-methyloctacosane; 11, 12,16-dimethyloctacosane; 12, N-nonacosane; 13, 9-methyl-, 11-methyl-, 13-methyl-, and 15-methylnonacosane; 14, 13,17-dimethyl-, 11,15-dimethyl-, and 9,13-dimethylnonacosane; 15, 3-methylnonacosane; 16, 3,7-dimethyl- and 3,9-dimethylnonacosane; 17, 10-methyl-, 12-methyl-, and 14-methyltriacontane; 18, 4-methyltriacontane and 12,16-dimethyltriacontane; 19, 4,8-dimethyl-, 4,10-dimethyl-, 4,12-dimethyl-, and 4,14-dimethyltriacontane; 20, N-hentriacontane; 21, 4,8,12-trimethyltriacontane; 22, 11-methyl-, 13-methyl-, and 15-methylhentriacontane; 23, 7-methyl- and 9-methylhentriacontane and 13,17-dimethylhentriacontane; 24, 11,15-dimethylhentriacontane; 25, 3-methylhentriacontane; 26, 5,9-dimethyl-, 5,11-dimethyl-, and 5,13-dimethylhentriacontane; 27, 7,11,15-trimethylhentriacontane; 28, N-dotriacontane, 3,7-dimethyl-, and 3,9-dimethylhentriacontane; 29, 3,7,11-trimethylhentriacontane; 30, 3,9,15,21-tetramethyl- and 3,7,11,15-tetramethylhentriacontane; 31, 4,8-dimethyl-, 4,10-dimethyl-, 4,12-dimethyl-, and 4,14-dimethyldotriacontane; 32, N-tritriacontane; 33, 4,8,12,16-tetramethyldotriacontane; 34, 5,9,13,17-tetramethyltritriacontane.

Fig. 4.

Differences in surface hydrocarbons between adult queens and workers, between queen- and worker-laid eggs, and between worker-laid eggs treated with either queen or worker extracts of cuticular hydrocarbons. n = 28 (queens), 33 (workers), 16 (queen-laid eggs), 13 (worker-laid eggs), 9 (worker-laid eggs treated with queen extracts), and 5 (worker-laid eggs treated with worker extracts). Within a group all samples originated from different colonies to obtain independent data points. (A) Major differences exist between queens and workers in 15 compounds. The proportions were calculated in relation to the total amount of the other 19 compounds, leading sometimes to proportions >100%. The differences in the medians of each compound between queens and workers and between queen-laid eggs and worker-laid eggs are significant (Wilcoxon test for paired samples, P < 0.001). The manipulation of the worker-laid eggs simulated either queen origin or worker origin as before, because the direction of the differences in the medians between untreated eggs and between treated eggs were not different (sign test, P > 0.6). Single variations are described by abbreviation of compound names. The numbers in the upper right corners of the panels correspond to the compound names in Fig. 3. (B) Differences in the profile of the remaining compounds of adult queens and workers were determined by a stepwise discriminant analysis. The resulting discriminant function was used to determine the similarity of the profiles of the untreated and treated eggs. Four compounds were excluded from the analysis. Compounds 20 and 34 were not normally distributed, and compounds 16 and 17 did not show variance homogeneity according to Levene's test. In both cases the significance levels were corrected for multiple comparison according to Bonferroni. The stepwise procedure selected the compounds 31, 24, 23, and 27 (see compound names in Fig. 3). Only one discriminant function was extracted. The differences between the queens and workers are statistically significant (Wilk's lambda = 0.181 and P < 0.001). The discriminant function correctly assigned queens and workers, with the exception of three misclassifications of queens (leave-one-out criterion used). The plot of the egg hydrocarbon profiles employing this discriminant function shows that the profiles of the selected compounds of the treated eggs are within the range of the natural profiles.

Fig. 5.

The survival of eggs treated with cuticular hydrocarbons in comparison to untreated eggs. The difference between the untreated eggs from queens and workers and the eggs treated with queen profile is statistically significant after 24 h (Friedman's ANOVA; N_groups = 9; P < 0.0005). The important difference is between the worker-laid eggs treated with queen cuticular hydrocarbons and the untreated worker-laid eggs (Wilcoxon test for paired samples, P < 0.02). The sample size of the last group is smaller due to an insufficient number of eggs available at that time. However, the difference among all groups remains statistically significant with only the five samples including the control with manipulated worker-laid eggs (Friedman's ANOVA, P < 0.005). The important differences between worker-laid eggs treated with either queen extracts versus worker extracts are each significant (Wilcoxon test for paired samples, P < 0.05). The medians of workers and worker profiles overlap.