pheromone within the primary olfactory centre, whereas no change was observed at the detection level as measured by electroantennogram (EAG) recordings. We now asked if this brief exposure to the pheromone could also affect the behavioural responses and the perception of other rele- vant odours: e.g. plant odours, which are processed along different neural pathways within the olfactory system than pheromones. Moths use plant odour cues to find host plants and food sources, even if oriented responses towards plant odours are in general far less striking than towards sex pheromones. Male moths have shown attraction to flower odours in both laboratory and field experiments [20–22]. Although the input channels for sex pheromones and plant odours are well separated in male moths [23], at least up to the first olfactory integration centre, synergistic interactions between plant volatiles and the female pheromone have been shown, improving in some cases their orientation efficiency to find females [24]. Plant compounds thus might play a role not only for food search, but also in the context of mating. Whereas olfaction is the main sense involved in detection of food sources and a mating partner, taste represents a key sense for the final assessment of plants and food sources. As a general pattern in phytophagous insects, the presence of sucrose is a sign of high food quality, whereas the presence of secondary compounds, such as quinine, may indicate toxicity or non-palatability [25]. The taste system of moths is quite accessible, as taste sensilla sensitive to sucrose or quinine are distributed over the mouthparts (the proboscis) and antennae, as it is the case in Heliothis virescens [26] and most other moths including S. littoralis. Upon contacting the antennae with a sucrose solution, these insects respond by extending their proboscis [27,28] in search for food. This proboscis extension reflex (PER) stimulated by sugars, inhibited by alkaloids and modulated by internal factors such as hunger or age, has been widely studied in many insect species [29–32], and provides thus a simple way to estimate the sensitivity of their taste system. Investigating the hypothesis of general sensitization, we explored in the present study if pre-exposure effects occur also intra-modally with non-pheromonal odours, intra-modally in the gustatory system and if cross-modal sensitization effects occur reciprocally between olfactory and gustatory stimuli relevant in different behavioural contexts. We pre-exposed male S. littoralis with different behaviourally relevant attractive and repulsive chemosensory cues and tested their behavioural response to olfactory stimuli on a locomotion compensator and to gustatory stimuli using the PER paradigm. Electrophysiological recordings from pheromone sensitive AL neurons and gustatory receptor neurons were carried out to provide an indication on the neural level from which behavioural modifications might originate. Results Olfactory responses to pheromone on the locomotion compensator When tested on a locomotion compensator with a pheromone stimulus (PHE), both na¨ıve and pre-exposed males responded to 0.1 or 0.25 female equivalents (FE) of PHE orienting towards the source (Figure 1, Rayleigh test p,0.05 in all cases). However, olfactory (Figure 1A, B) and gustatory (Figure 1C) pre-exposure 24 h before caused a significant increase in the percentage of active males (letters in Figure 1) and males that walked mainly towards PHE (numbers in Figure 1) as compared to na¨ıve ones. Results of a detailed statistical analysis are given below. As expected from previous results [13,19], significantly more males pre-exposed to PHE (1FE) were activated (Figure 1A, Sensitization after Brief Pre-Exposure in a Moth X2 = 7.63, d.f. = 1, p,0.01) and walked mainly towards 0.1FE PHE (X2 = 7.21, d.f. = 1, p,0.01) as compared to the control group, C(hex). Moreover, both activity and orientation levels increased with increasing PHE concentrations in control and pre- exposed males (Figure 2, positive dose-response curves) and significant differences between pre-exposed and control-exposed males were observed for all three doses of PHE (0.1, 0.25 and 0.5 FE): in all cases, responses of pre-exposed males were higher than in na¨ıve males. In addition to the differences in the percentage of males activated and oriented to the source, we analyzed if active males changed their locomotion behaviour while approaching the PHE after PHE pre-exposure. Our detailed analysis of walking tracks (Figure 3) showed that neither the latency of response (X2 = 0.42, d.f. = 1, p = 0.51) nor the total walked time (X2 = 0.014, d.f. = 1, p = 0.90) changed with pre-exposure, but changed significantly with the tested PHE dose (X2 = 10.23, d.f. = 2, p,0.01 and X2 = 15.67, d.f. = 2, p,0.001, respectively). On the other hand, the walked distance (X2 = 13.71, d.f. = 1, p,0.001) and the mean walking speed (X2 = 10.99, d.f. = 1, p,0.001) were significantly higher in PHE pre- exposed males than in control males and did not change with doses (X2 = 3.11, d.f. = 2, p = 0.21 and X2 = 1.87, d.f. = 2, p = 0.39, respectively). This result shows that pre-exposed males not only respond more frequently to the pheromone, but that responding males also improve their oriented locomotion behaviour (i.e. they walk faster towards the source) as compared to control males. Males did not respond clearly to stimulation with 0.1% linalool (LIN) or geraniol (GER) (Figure 4A, B). Pre-exposure to LIN and GER (0.1%) modified, however, the response level of males to 0.1FE PHE as compared to na¨ıve males, C(oil) (Figure 1B; Figure 4C, D). Pre-exposure to LIN significantly increased the activity and orientation of males to PHE (X2 = 7.67, d.f. = 2 and X2 = 6.12, d.f. = 2, respectively, p,0.05 in both cases). Pre- exposure to GER did not modify the activity level of males (X2 = 4.82, d.f. = 2, p = 0.09) but increased their oriented response (X2 = 6.98, d.f. = 2, p,0.05). Gustatory pre-exposure by touching the antennae with 1 M sucrose (SUC) or 0.1 M quinine (QUI) (Figure 1C) significantly increased the activity (X2 = 7.20, d.f. = 2 and X2 = 6.83, d.f. = 2, respectively, p,0.05 in both cases) and orientation (X2 = 7.12, d.f. = 2 and X2 = 6.98, d.f. = 2, respectively, p,0.05 in both cases) of males towards the pheromone source on the locomotion compensator as compared to control-exposed males, C(H2O). However, these dif- ferences were relatively small possibly due to the much lower absolute response rates to PHE males exhibited during this experimental series as compared to other series (e.g. Figure 1A, B; Figure 2). To test if only pre-exposure to stimuli relevant to resource location causes changes in pheromone responses, or if contrarily, experience of a new context was sufficient to elicit a similar increased sensitivity, a non-specific mechanical stimulus was generated by shaking males for 1 minute on a laboratory shaker. No changes in the behaviour of shaken males caused by this non- specific pre-exposure was found (Figure 1D, for activity X2 = 1.72, d.f. = 1, p,0.05; for orientation X2 = 1.18, d.f. = 1, p,0.05) when comparing with non-shaken males. During shaking males were highly active inside the box. Preliminary assays revealed that this activation lasted for a few minutes and that males were not physically damaged by this treatment. Responses of AL neurons to PHE after SUC pre-exposure Intracellular recordings were done from 76 neurons in 35 control males (exposed to distilled water) and from 80 neurons in 40 sucrose-exposed males, penetrated within the male-specific macroglomerular complex (MGC) of the AL, dedicated to PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34141