information appears in a known environment. Although the establishment of cognitive maps in vertebrates have been shown in paradigms using reinforcement, mere exposure to sensory cues might be sufficient to acquire information about the environment. Once a map of spatial relationships between cues within the environment has been established, an animal might use short-cuts even through unexplored areas [47–49]. In our model, S. littoralis, the brain might acquire a sort of cognitive map of its sensory environment through pre-exposure to different behaviourally relevant stimuli (i.e. odours of sex and food, sound of a predator, appetitive or repulsive tastants), which improves behavioural responses to the same or different signals in order to optimize survival and reproduction. Materials and Methods Insects S. littoralis males and females were kept in separate climatic chambers (16L:8D, 22uC and 70–90% relative humidity) from the pupal stage. Emerged adults were kept without feeding. For behavioural olfactory tests, wings of adult males were cut at the day of emergence (day-0). Olfactory or gustatory pre-exposure was performed 2–5 h into the scotophase at day-1. Behavioural and electrophysiological tests were carried out 2–5 h into the scotophase at day-2, i.e. at least 24 h after pre-exposure. Olfactory stimuli Pheromone extracts (PHE) were prepared from 2/3-day-old females as described previously [33]. Dilutions of 0.1FE (female equivalent), 0.25FE or 1FE were used for stimulation. For electrophysiological recordings, the main sex pheromone compo- nent ((Z,E)-9,11-tetradecadienyl acetate; .97% purity, synthe- sized in Versailles by Martine Lettere) diluted in hexane (HEX) was used at doses from 0.01 pg to 100 ng. The synthetic plant-related odours linalool (LIN; Sigma, 97% purity, Saint-Quentin Fallavier, France) and geraniol (GER; Sigma, 96% purity, Saint-Quentin Fallavier, France) were used at the dose of 0.1% v/v diluted in mineral oil (MO) (Sigma, Saint- Quentin Fallavier, France). This dose was chosen because preliminary data using different doses of both compounds showed no differences in eliciting behavioural responses of males, and because it is biologically active in males of S. littoralis [50]. Gustatory stimuli Aqueous solutions of 1022, 1021.5, 1021, 1020.5 and 1 M sucrose (SUC; .99.5% purity, Sigma, Saint-Quentin Fallavier, France) and 0.1 M quinine (QUI; = 98.0% purity, Sigma, Saint- Quentin Fallavier, France) were prepared every day. These concentrations are well detected by gustatory receptor neurons on the antennae of S. littoralis [51]. Pre-exposure procedures Olfactory pre-exposure was carried out on the locomotion compensator (see below). Males, placed 2 h prior to pre-exposure in the experimental room, were individually pre-exposed during 1 min either to 1FE of PHE, to 0.1% LIN or to 0.1% GER, under red light. Two control groups were exposed to HEX or to MO. Behaviour in response to the stimulus was monitored, and males were carefully returned to the rearing chamber until the behavioural tests or electrophysiological recordings were carried out. Gustatory pre-exposure was done by using the proboscis extension reflex (PER). Antennal contact with a sugar solution elicits extension of the proboscis in S. littoralis [27], which can be Sensitization after Brief Pre-Exposure in a Moth used to determine the detection threshold for a sugar solution. Males were inserted in cut Pipette tips with the head protruding. Individual pre-exposure was carried out by moving a tooth pick soaked with either distilled water (H2O, control), 1 M SUC or 0.1 M QUI evenly over one antenna for 10 s to make sure that a maximum number of gustatory sensilla got in contact with the solution. PER responses were monitored, and males were returned to the rearing chamber until the behavioural tests or electrophys- iological recordings were carried out. In an additional series of experiments, we provided a non- specific mechanical stimulation by shaking 20 males together in a plastic box on a laboratory vortex mixer (2400 rpm) during 1 min, without application of chemical stimuli. A control group was handled without shaking in parallel. Males were then returned to the rearing chamber until the olfactory or gustatory tests were carried out. Behavioural olfactory tests A locomotion compensator (LC-300, Syntech, Kirchzarten, Germany) was used to analyze the olfactory behaviour of males in presence of a PHE source as described previously [52]. Briefly, wingless males were released at the top of the plastic sphere (30 cm Ø), which is rotated opposite to the insect displacement by motors controlled by a camera located above the insect, while the insect is maintained in a constant position. Rotational movements are transferred to a computer, and incremented as X and Y coordinates in 0.1 s intervals from which the trajectory of each insect was reconstructed. Males were exposed to a constant charcoal filtered humidified airflow (17 ml/s). An additional airflow (7 ml/s) was switched to a parallel tube with the pipette or vial containing the odorant, using a stimulus controller (CS 55, Syntech, Kirchzarten, Germany). A pipette, containing HEX on a filter paper or a vial containing 1 ml of MO was used as control. Na¨ıve or pre-exposed males were placed in the experimental room before the onset of scotophase. At the time of the experiment, males were individually placed on the locomotion compensator, left for 1 min for acclimatization, and their behaviour was recorded in response to PHE during 2 min. The following experimental series were performed: – pre-exposure to PHE, test with PHE – pre-exposure to LIN or GER, test with PHE – pre-exposure to SUC or QUI, test with PHE – pre-exposure to shaking, test with PHE Different pheromone doses were used in different experiments because ‘‘absolute’’ response thresholds to PHE varied between seasons. A sub-optimal pheromone dose depending on the ‘‘absolute’’ threshold during a given test-period was chosen to reveal possible differences between pre-exposed and naive males for each experimental series. The existence of a preferred direction of males in relation to the olfactory stimulus was analyzed using circular statistics and graphs (Oriana software, Kovach Computing Services, Anglesey, Wales). The position of the air stream outlet was designated at 0u. The statistical evidence of a uniform distribution around a circle was tested using the Rayleigh test [53]. The locomotor behaviour of males was analyzed using the TrackSphere 3.1 software (Syntech, Kirchzarten, Germany) and the following parameters were calculated for individual trajectories: Upward length is the net displacement towards the stimulus in mm. Positive or negative values denote an overall preference to walk towards or against the stimulus, respectively. PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e34141