Sensitization after Brief Pre-Exposure in a Moth Figure 8. Responses of single gustatory sensilla to sucrose (SUC) in males pre-exposed to gustatory (SUC and QUI) stimuli. Each point represents the mean action potential frequency during the first second of stimulation and its standard error. Whereas responses of gustatory sensilla were dose-dependent, they did not differ statistically between pre-exposed and na¨ıve males as revealed by a two-way ANOVA with Repeated Measures on one factor (stimulus concentration, see text). doi:10.1371/journal.pone.0034141.g008 to sucrose improved not only after pre-exposure to stimuli of the same modality, but also after pre-exposure to the respective other modality. The idea of a general multimodal sensitization presented here was already suggested in a previous study [33], where it was shown that a behaviourally meaningful auditory stimulus can modify the sensitivity of S. littoralis males to the sex pheromone. Twenty-four hours after hearing an artificial bat sound (i.e. a natural predator sound) the behavioural response threshold of males to the female-emitted pheromone was lower than in na¨ıve males. In our study, we extend this paradigm and show that the olfactory responses of males to pheromone were also cross-modally improved after an experience with both an attractive and an aversive gustatory stimulus (sucrose and quinine). Furthermore, we show that also gustatory sensitivity to sucrose improved after pre-exposure to tastants and to pheromone. These results support the hypothesis that meaningful sensory stimuli, i.e. aversive or attractive signals, might contribute to a general sensitization of different sensory modalities. Whereas previous studies have focused on intra-and cross-modal effects of brief pre-exposure on pheromone responses [19,33], we show here for the first time that brief pre-exposure with an olfactory signal also modifies responses to gustatory stimuli. On the other hand mechanical shaking, a stimulus without a precise biological significance to the moth, did not elicit increased sensitivity to olfactory and gustatory stimuli. Biologically meaningful stimuli of the natural environment thus seem to prepare the nervous system for future encounters with the same or other environmental signals to improve behavioural responses. Although olfactory and gustatory input reaches the brain via the same antennal nerve, their neurons target different zones. Modulatory neurons are interconnecting different brain areas, including the antennal lobe, the antennal mechanosensory and motor centre, the tritocerebrum and the subesophageal ganglion, representing the different target zones of olfactory and gustatory receptor neurons [43,44]. An interaction of olfactory and gustatory stimuli could thus occur within any of these neural structures accounting for the cross-modal effects of pre-exposure. However, in our work, no changes of sensitivity to the pheromone were found in MGC neurons when pre-exposing males to sucrose. These results suggest that the neuronal basis of the behavioural ‘‘cross-modal’’ effect of gustatory pre-exposure on olfactory responses is not located at the AL level but most likely at higher brain levels (e.g. mushroom bodies, etc), as opposed to previously described strong effects of acoustic pre-exposure on the sensitivity of AL neurons to both sex pheromone and plant odours [33]. Conclusions Our results on cross-modal effects of brief pre-exposure, together with another study [33] show clearly that the observed pre-exposure effects are not a case of selective attention, but rather support the hypothesis of general sensitization by different meaningful sensory inputs. We propose that behaviourally relevant stimuli occurring in the environment might contribute to a maturation process increasing the sensitivity of olfactory, gustatory and possibly other sensory systems in moths. Sensory inputs elicit developmental processes in the nervous system of insects, including the modulation of sensory systems. Most likely, any behaviourally relevant sensory stimulus might contribute to physiological or anatomical changes, leading ultimately to increasing sensitivity inducing changes in behaviour. Since increasing the sensitivity of sensory neurons is energetically costly [45], the existence of a low/ high sensitivity switch that may allow insects to economize energy by being less sensitive when there is no need to be (i.e. when no cues are present) and reactive when biologically relevant cues are present in the environment, might be biologically meaningful. Pre-exposure effects obtained in the present study could be compared with pre-exposure effects during spatial learning [46]. In vertebrates, a cognitive map is thought to be a highly flexible representation of space that automatically updates whenever novel PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34141