Sexual repurposing of juvenile aposematism in locusts

Abstract

Adaptive plasticity requires an integrated suite of functional responses to environmental variation, which can include social communication across life stages. Desert locusts (Schistocerca gregaria) exhibit an extreme example of phenotypic plasticity called phase polyphenism, in which a suite of behavioral and morphological traits differ according to local population density. Male and female juveniles developing at low population densities exhibit green- or sand-colored background-matching camouflage, while at high densities they show contrasting yellow and black aposematic patterning that deters predators. The predominant background colors of these phenotypes (green/sand/yellow) all depend on expression of the carotenoid-binding "Yellow Protein" (YP). Gregarious (high-density) adults of both sexes are initially pinkish, before a YP-mediated yellowing reoccurs upon sexual maturation. Yellow color is especially prominent in gregarious males, but the reason for this difference has been unknown since phase polyphenism was first described in 1921. Here, we use RNA interference to show that gregarious male yellowing acts as an intrasexual warning signal, which forms a multimodal signal with the antiaphrodisiac pheromone phenylacetonitrile (PAN) to prevent mistaken sexual harassment from other males during scramble mating in a swarm. Socially mediated reexpression of YP thus adaptively repurposes a juvenile signal that deters predators into an adult signal that deters undesirable mates. These findings reveal a previously underappreciated sexual dimension to locust phase polyphenism, and promote locusts as a model for investigating the relative contributions of natural versus sexual selection in the evolution of phenotypic plasticity.

Keywords: locust swarming; male–male mounting; phenotypic plasticity; sexual dichromatism; sexual selection.

Fig. 1.

Color phenotypes and YP expression in the desert locust. (A) Color phenotypes of the desert locust, with those expressing YP highlighted in yellow. (i) Solitarious juveniles of both sexes (male shown) exhibit cryptic coloration: green at high humidity (=among vegetation) or sand at low humidity (not shown; figure 2 in ref. 6). (ii) Upon reaching adulthood, solitarious males (shown) and females retain a dull cryptic coloration. (iii) Gregarious juveniles of both sexes (male shown) exhibit black and yellow aposematic warning coloration. (iv) Upon reaching adulthood, gregarious males (shown) and females are light pink while sexually immature. (v) Gregarious females become a beige-brown color upon sexual maturation (∼10 d). (vi) Gregarious males (including sham-injected controls in this paper) become bright yellow upon sexual maturation. (vii) RNAi of YP, via dsRNA injection into sexually immature males, leads to a nonyellow, beige coloration upon maturation. (B) Reflectance spectra for 10 each of mature gregarious females, mature gregarious males, and dsYP mature gregarious males (v, vi, and vii in A, respectively). Central line and surrounding color per strip represent the mean reflectance at that wavelength ±1 SE. Approximate spectral sensitivity curves of the three desert locust photoreceptors are also shown (73, 74) (SI Appendix, Results). (C) qRT-PCR of YP expression in the abdominal cuticle of adult male locusts. Boxplots show the relative expression (median ± IQR [interquartile range] and range) in control and dsYP males. RNAi led to a significant reduction in YP mRNA in dsYP males (Mann–Whitney U test, n = 6 per group, U = 36, P = 0.0022; **P < 0.01). Image credits: Tom Fayle and Steve Rogers, University of Cambridge, Cambridge, UK, and Timon Smeets, KU Leuven, Leuven, Belgium.

Fig. 2.

Male yellowing is an intrasexual signal in S. gregaria. (A) Observation chamber and assay setup for MMM assays. Twelve dsYP males and 12 controls were placed in the main chamber and observed for 3 h (n = 8). Twenty-four mature virgin females in the smaller chamber served as an olfactory releaser of male sexual behavior, and ensured that the assay environment more closely achieved the normal balance of male and female pheromones that exists in natural high-density groups. (B) Outcome for all 192 males assayed, plotted as the number of mounts given against the number of mounts received. Treatment is indicated by color (yellow, control; blue, dsYP), trial number 1 to 8 is indicated by decreasing transparency, and count at each made/received combination is indicated by point size (see key, Inset). (C) Mounter choice. Irrespective of their own phenotype, what color conspecifics did male locusts mount throughout each 3-h trial? Males were categorized according to their behavior; boxplots show the number of males per category across eight trials (median ± IQR and range). Significantly more males mounted only a dsYP conspecific(s) than mounted either a control conspecific(s) only or mounted males of both phenotypes (Kruskal–Wallis χ² = 24.829, n = 8 per group, degrees of freedom [d.f.] = 3, P < 0.001. Dunn’s test of multiple comparisons using rank sums with Bonferroni correction: significant differences [*P < 0.05, **P < 0.01, and ***P < 0.001]). Overall, 51.0% of the 192 males assayed mounted only a dsYP conspecific(s), compared with 2.1% that only mounted a control(s). (D) Signaler outcome. Boxplots show the number of males that were mounted by any other conspecific(s) throughout each of the eight trials (median ± IQR and range). Significantly more dsYP males were mounted per trial than were control males (Mann–Whitney U test, n = 8 per group, U = 0.5, P = 0.0011: significant difference **P < 0.01). Overall, 16.7% of the 96 control males assayed were mounted by another male, compared with 63.5% of the 96 dsYP males. Full breakdowns of all phenotype-by-phenotype interactions are given in SI Appendix, Figs. S2 and S4 and Tables S1–S4.

Fig. 3.

Mate-choice assay. (A) Observation chamber and assay setup for female mate-choice assays. One mature virgin female and one each of dsYP and control virgin males were observed for 2 h (n = 45). (B) Female mate choice. Out of 41 trials that resulted in mating, control males mounted and copulated with the females on 21 occasions while dsYP males mounted and copulated with the females on 20 occasions. This difference was not significant (χ² = 0.02439, d.f. = 1, P = 0.8759; n.s., not significant, P > 0.05). Boxplots show the latency to copulate with the female in minutes (median ± IQR and range), which did not significantly differ between the two groups (Mann–Whitney U test, n = 21 for controls, 20 for dsYP, U = 248, P = 0.3278; raw data are in SI Appendix, Table S5).

Fig. 4.

Effect of YP-RNAi (or its resulting color phenotype) on release of male volatiles, as measured by GC-MS. Boxplots (yellow [Left] for controls; beige [Right] for dsYP) show the absolute abundance (log scale) per treatment (median ± IQR and range, with values farther than 1.5× IQR from the median presented as outliers). Chemical structure, Mann–Whitney U, and P are given for each volatile (n.s., P > 0.05; **P < 0.01). Previous studies showed that these six volatiles form the bulk of the mature male-specific pheromone in S. gregaria, with PAN accounting for ∼80% of the total emission (39). This is supported by our analysis. YP-RNAi led to a significant increase in PAN emission (Mann–Whitney U test, n = 12 of each group, U = 21.5, P = 0.0039), and did not significantly affect any of the other volatiles measured (raw data are in SI Appendix, Table S6).