Sensitization after Brief Pre-Exposure in a Moth Figure 1. Behavioural responses to female pheromone extracts (PHE) of males pre-exposed to olfactory and gustatory stimuli on a locomotion compensator. A) pre-exposure to PHE, test with 0.1FE. B) pre-exposure to LIN and GER, test with 0.1FE. C) pre-exposure to SUC and QUI, test with 0.25FE. D) responses of males to PHE after a non-specific mechanical pre-exposure, test with 0.25FE. 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 different 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). Numbers at the bottom of bars indicate numbers of tested males. 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). Sensitivity to PHE was intra-modally increased by pre- exposure to PHE, LIN and GER, and cross-modally increased by pre-exposure to SUC and QUI. doi:10.1371/journal.pone.0034141.g001 pheromone processing. In both groups, a sigmoidal curve of the cumulative frequency of responding neurons to the main pheromone component was found (Figure 5) with response thresholds ranging between 1025 and 102 ng. Around 20% of very sensitive neurons, a few neurons with intermediate sensitivity and high proportions of neurons with thresholds at 1 ng or higher were found. The distribution of neurons with different thresholds was not statistically different between na¨ıve and sucrose-exposed males for any of the tested doses (G-Test, G(5) = 9.9, p = 0.08). Gustatory responses to SUC using the PER paradigm To test if also gustatory sensitivity could be altered by a brief exposure to tastants, we took advantage of the innate response of males, which extend their proboscis in a dose-dependent manner when their antennae are touched with a sucrose solution. We found this response to be modulated by a gustatory pre-exposure to SUC or QUI 24 h before testing, as more pre-exposed (SUC or QUI) than naive males (C(H2O)) extended their proboscis as response to the contact of 0.03 M SUC (Figure 6A; X2 = 7.87, d.f. = 2 and X2 = 8.98, d.f. = 2, respectively, p,0.05 in both cases). Similar effects of pre-exposure were found along a dose-response curve and were stronger when low doses of SUC were tested (Figure 7). Moreover, if the pre-exposure and test were done on differ- ent antennae of the same individual (contra-lateral), the effect of pre-exposure was even stronger than if pre-exposure and test were done on the same antenna (ipsi-lateral) (Figure 6B; X2 = 4.23, d.f. = 1, p,0.05). When males were pre-exposed to PHE on the locomotion compensator before gustatory tests, a significant increase in the PER response to 0.03 M SUC was observed as compared to na¨ıve males (Figure 6C; X2 = 6.93, d.f. = 1, p,0.01). No effect of a non-specific stress generating pre-exposure (shaking) on the gustatory response of males to 0.1 M SUC was found (Figure 6D; X2 = 1.23, d.f. = 1, p = 0.35). Different concentrations of SUC were tested along experiments because motivation of males to extend their proboscis when their antennae were stimulated with SUC solutions depended on time of the year. However, tested doses were always in the lower part of the dose-response curve, where differences between pre-exposed and control males were obvious (Figure 7). Responses of gustatory receptor neurons to SUC after SUC and QUI pre-exposure Extracellular recordings from 287 antennal Sensilla chaetica in 72 male moths showed dose-dependent responses (Figure 8). The average action potential frequency of gustatory receptor neurons during the first second of stimulation, increased significantly with stimulus intensity (F = 280.63, d.f. = 3, p,0.001). Post-hoc Tukey HSD comparisons showed significant differences between the responses to the 4 doses of SUC tested (p,0.05 in all cases). PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34141