Sensitization after Brief Pre-Exposure in a Moth Figure 2. Behavioural responses to different concentrations of female pheromone extracts (PHE) of males pre-exposed to PHE on a locomotion compensator. Grey and black columns show the percentage of males walking towards and against the source, respectively. The length of the whole column (grey+black) shows their activity level. Within each frame (A, B, C, D), locomotor activity was significantly differ- ent between columns with different lower case letters (Chi-Square, p,0.05), and orientation levels differed significantly between columns with different numbers (Chi-Square, p,0.05). The number of tested males is n = 30 for each column. Circular diagrams show the mean angle of individual males (the stimulus is situated at 0u). Asterisks in circular diagrams show groups of insects that showed non-uniform distributions (Rayleigh test, p,0.05). Pheromone responses increased with the tested dose, but more pre-exposed than na¨ıve males responded independently of the tested concentration. doi:10.1371/journal.pone.0034141.g002 Contrarily, no effect of pre-exposure was observed (F = 0.38, d.f. = 2, p = 0.68); responses in SUC-exposed and QUI-exposed males did not differ significantly in their spike frequency nor when compared with responses in na¨ıve males. The analysis of 100 ms bins of action potentials to evaluate the initial phasic and the following tonic part of the responses separately did not reveal any differences in firing rates between pre-exposed and na¨ıve males either. Discussion In the present study we show that a brief pre-exposure to different olfactory and gustatory stimuli elicits behavioural intra- and reciprocal cross-modal long-term sensitization in a male moth. Interestingly, although the sensitivity of AL neurons to pheromone was recently shown to change after pheromone and bat sound pre- exposure [19,33], our new data allow us to exclude early processing stages (the antennal and AL level) to be at the origin of gustatory-evoked pheromone sensitization. Effects of brief odour exposure on locomotion responses to odours Although moths generally orient to pheromone sources mainly by flying, S. littoralis males showed clear walking orientation on the locomotion compensator, as observed before in other moth species such as Bombyx mori [34,35]. Our observations confirm the increase of attraction to the sex pheromone exhibited by S. littoralis males in both wind tunnel and olfactometer bioassays, 24 h after a brief pre-exposure to the pheromone [13,19,33]. In addition to a higher percentage of males orienting towards the pheromone source after pre-exposure, we have now evidence that pre-exposed males are not only more prone to respond to the pheromone but also walked more rapidly towards the stimulus than na¨ıve males, whereas their response latency did not change. Although pre-exposure with linalool or geraniol increased subsequent responses to the sex pheromone, no changes in the responses to the plant compounds themselves were found. Taking into consideration that pre-exposure to suboptimal pheromone stimuli had a much lower effect on subsequent pheromone res- ponses than pre-exposure to a natural stimulus [13], it is possible that the doses used here for pre-exposure with plant odours were sufficient to show an effect on pheromone responses in most cases, but too low to have an effect on subsequent responses to plant odours. Another explanation could be that only single plant compounds were used in this study. As for pheromone, it is well known that insects show better responses to blends of plant odours than to single compounds [36]. Here pre-exposure to single plant odours had a positive effect on the response to the main component of the pheromone, but no effect on responses to these plant odours themselves. Possibly, pre-exposure is not sufficient to change a non- attractive stimulus into an attractive one, but can modulate the sensitivity to an attractive stimulus (the pheromone blend). PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34141