Complexity of Chemical Emissions Increases Concurrently with Sexual Maturity in Heliconius Butterflies

Abstract

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown.

Keywords: Heliconius; Aging; Androconia; Antiaphrodisiac; GC-MS; Lepidoptera; Pheromones.

Fig. 1

Effect of age on number of compounds detected in H. atthis and H. charithonia (A) wing tissue extracts and (B) genital extracts at all measured time points, also showing the data distribution for each time point in each tissue. Above each set are the linear regression p-values for the effect of age on number of compounds. See Table S2 for the complete linear regression results including all test statistics, as well as results for the effect of age on the total amount of all detected compounds, and Figure S2 for a plot of the effect of age on the total amount of all detected compounds

Fig. 2

Change in chemical blends with age depicted using NMDS plots (k = 2, 1000 permutations) for H. atthis (A, C) and H. charithonia (B, D) male androconia and genitals. Colours represent samples of the same age. See Fig. S1 for NMDS plots for all six tissue types in each species

Fig. 3

Changes in the relative amount with post-eclosion age for each compound detected in extracts of (A) H. atthis wing tissues, (B) H. charithonia wing tissues, (C) H. atthis genitals and (D) H. charithonia genitals. For each compound, the amount was standardized by dividing it by its maximum in that dataset. Compounds that show significant correlations with age are marked with an asterisk in each tissue type. The sixth column of each plot, labelled IV, shows the indicator value (; Table 1) for each compound at 8 days post-eclosion within that tissue. Non-white tiles signify a high indicator value, making that compound an “indicator” within that tissue type. Compounds bearing both an asterisk and a high indicator value are potentially suitable candidates for the role of pheromones. Compounds are arranged by class, and then by retention index