More to legs than meets the eye: Presence and function of pheromone compounds on heliothine moth legs.

Chemical communication is ubiquitous in nature and chemical signals convey species-specific messages. Despite their specificity, chemical signals may not be limited to only one function. Identifying alternative functions of chemical signals is key to understanding how chemical communication systems evolve. Here, we explored alternative functions of moth sex pheromone compounds. These chemicals are generally produced in, and emitted from, dedicated sex pheromone glands, but some have recently also been found on the insects' legs. We identified and quantified the chemicals in leg extracts of the three heliothine moth species Chloridea (Heliothis) virescens, Chloridea (Heliothis) subflexa and Helicoverpa armigera, compared their chemical profiles and explored the biological function of pheromone compounds on moth legs. Identical pheromone compounds were present on the legs in both sexes of all three species, with no striking interspecies or intersex differences. Surprisingly, we also found pheromone-related acetate esters in leg extracts of species that lack acetate esters in their female sex pheromone. When we assessed gene expression levels in the leg tissue, we found known and putative pheromone-biosynthesis genes expressed, which suggests that moth legs may be additional sites of pheromone production. To determine possible additional roles of the pheromone compounds on legs, we explored whether these may act as oviposition-deterring signals, which does not seem to be the case. However, when we tested whether these chemicals have antimicrobial properties, we found that two pheromone compounds (16:Ald and 16:OH) reduce bacterial growth. Such an additional function of previously identified pheromone compounds likely coincides with additional selection pressures and, thus, should be considered in scenarios on the evolution of these signals.