Active Rejection of Non-Host Plants Across Families in Papilio xuthus Revealed by Tarsal Multi-Feature Spike Sorting

Herbivorous insects must identify suitable host plants for egg-laying and reliably reject unsuitable ones, but whether non-host plants are actively rejected through deterrent cues, or simply fail to trigger host-recognition signals, remains unclear. We addressed this question in the swallowtail butterfly Papilio xuthus by combining oviposition behavior trials with electrophysiological recordings from taste sensilla on the female forelegs. Females primed with a Citrus (host plant) extract laid eggs on a plain water control 51.5% of the time, providing an operational post-priming baseline for oviposition. Against this baseline, egg-laying was completely suppressed by extracts of all five non-host species tested from families other than the host family Rutaceae. This supports the interpretation that non-host extracts contain stimulus properties associated with reduced oviposition, rather than simply lacking a stimulatory cue. Because a single recording can contain spikes from several spike-generating cells activated at once, we grouped spikes by six waveform features-amplitude plus five measures of spike shape and timing-rather than amplitude alone, resolving five reproducible spike types (Types 0-4). Type 0 fired consistently more in response to non-host extracts, with this association remaining statistically significant in the female-ID clustered GEE sensitivity analysis; Type 4 fired more in response to host extracts, though this association did not remain significant in the same analysis. Orixa japonica, a non-host species that nonetheless belongs to the host family Rutaceae, elicited a Type 4 firing rate lower than that of host Rutaceae together with elevated Type 0 firing; egg-laying was markedly reduced even though butterflies frequently began, without completing, the egg-laying sequence. This mixed peripheral response pattern supports an ensemble integration model in which oviposition is associated with the combined balance of activating ("go") and inhibitory ("stop") peripheral signals, rather than with a single on/off cue.