How do male danaine butterflies locate pyrrolizidine alkaloids? The foreleg as an olfactory alkaloid-sensing organ

Most male danaine butterflies are well known for their curious behaviour of visiting some particular plants to acquire pyrrolizidine alkaloids (PAs), which they sequester and utilize for sex pheromone production and chemical defence. However, a robust understanding of the mechanism by which they locate PA sources and their preferences for different structural types of PAs is lacking. An experiment testing the attractiveness or repellency of four PAs to adult male Parantica sita (Danainae) revealed that a mixture of intermedine and lycopsamine (I/L) significantly attracted them, whereas the other PAs, viz. monocrotaline, heliotrine and retronecine, showed potent repellency in decreasing order of intensity. By contrast, these PAs were neither significantly attractive nor repellent to male Ideopsis similis, which is also a PA-phile danaine species. Organ ablation tests in male P. sita on the antenna, foreleg (nonwalking leg) and labial palp as potential PA-receptive organs, carried out in combination with a proboscis extension reflex test in response to I/L odour, indicated that only these organs can be used to detect PAs. Of these organs, the forelegs, especially the foretarsi, appeared to be mostly responsible for the olfactory sensing of PAs. Scanning electron micrographs of the male foretarsi of the abovementioned two species and another danaine butterfly, Euploea mulciber, which is also known to be attracted specifically to I/L, revealed ampullaceous sensilla on their foretarsal surface. In electrophysiological examinations (tip recordings), I/L elicited distinct responses (spikes) from the sensilla of the three species, indicating that the ampullaceous sensilla have both olfactory and gustatory functions. These findings indicate that male nonwalking legs play a key role in PA-seeking danaine butterflies.