Olfactory discrimination conditioning in the moth Spodoptera littoralis

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1 Physiology & Behavior 72 (2001) 159± 165 Olfactory discrimination conditioning in the moth Spodoptera littoralis Ruey-Jane Fan*, Bill S. Hansson Department of Ecology, Lunds University, S Lund, Sweden Received 18 October 1999; received in revised form 15 December 1999; accepted 5 September 2000 Abstract We used a proboscis extension reflex (PER) to study the olfactory discrimination capability in the moth Spodoptera littoralis. Already after a single experience, moths were capable to discriminate a rewarded from an unrewarded odor. In the first experiment, when rewarded and unrewarded odors were substituted for each other, moths were able to undergo reversal conditioning already after two experiences. Both shorter and longer inter-trial intervals (ITIs) supported high degrees of learning. In a second experiment, moths could solve both featurepositive and -negative discrimination tasks. Two hypotheses for the way in which these associations exert their discrimination performance are considered. The moth's olfactory physiology has been extensively studied. This animal thus provides a powerful system in which to study the neurobiology of olfactory discrimination and odor recognition. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Proboscis extension reflex (PER); Odor; Discrimination index; Inter-trial interval; Moths * Corresponding author. Tel.: ; fax: address: ruey-jane.fan@ekol.lu.se (R.-J. Fan). Many insects depend mainly on olfaction for food search and mate location [4,26]. Learning to extract olfactory information and to discriminate among diverse odors are potentially important survival strategies [5,8]. Behavioral analysis of odor discrimination and olfactory recognition in insects has been greatly advanced by the use of a simple preparation such as proboscis extension reflex (PER) conditioning. The PER conditioning paradigm has been extensively used to study olfactory discrimination in honeybees [6,16,20]. In discrimination (differential) conditioning, two odors are presented during training; one odor is paired temporally with positive reinforcement, whereas the other odor is either negatively reinforced or non-reinforced [6,30]. After training, the animal typically extends its proboscis to the rewarded conditioned stimulus (CS+) but does not respond to the CS that has not been rewarded (CSo) [6]. The moth has proven to be a suitable subject for the study of olfactory information processing. The olfactory system of the moth Spodoptera littoralis has been well studied in both behavioral and neurophysiological experiments. Antennal receptor neurons and antenna lobe interneurons have been characterized both anatomically and physiologically [1 ± 3,21]. Recently, it was demonstrated that moths are able to learn to associate an odor (CS) with a sucrose reward (unconditioned stimulus, US) by means of PER conditioning [10,15]. The moth thus provides a useful system for analyzing the neural mechanisms underlying odor processing as well as olfactory conditioned behavior. Several training factors may affect the responses of animals in conditioning. These factors include inter-trial interval (ITI), reversing the roles of the CS+ and CSo, and the effect of a compound stimulus [11,22,27]. The roles and mechanisms of these factors upon olfactory discrimination in moths still remain to be examined. In the present study, we used standard and modified discrimination procedures based on the PER preparation to study olfactory discrimination behavior in the moth [6,23,24,32]. A first step in analyzing olfactory discrimination was to test the ability of moths to reassign the significance of two odors in a reversal discrimination task combined with different ITIs. A second experiment was performed to investigate whether the moths can distinguish a bimodal stimulus, consisting of an odor and a mechanosensory stimulus, from the mechanosensory stimulus alone in feature-positive and -negative discrimination tests /01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S (00)

2 160 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159± General method 1.1. Subjects A total of 120 female and 120 male cotton leaf worm moths S. littoralis were used. Moths were supplied from a culture maintained at the Swedish University of Agricultural Sciences in Alnarp, Sweden. The moths were reared on a potato-based semi-synthetic diet [17]. Pupae were separated according to sex and put in emergence boxes ( cm) in an air-conditioned room (23±25 C, 70% RH, L/D 16:8). Immediately after emergence, the moths were given access to water for 6±8 h. Then, they were placed in other boxes ( cm) without water supply for 42 2 h. 2 ±3-day-old adults were used in all experiments Procedure Conditioned stimuli Two odorants, 100-mg geraniol and 10-mg phenylacetalehyde (PAA), were used as CS (in Experiment 2, only geraniol was used). These odorants were chosen because they are both floral odors related to food sources of S. littoralis [7]. Personal observation showed that the learning performance to 100-mg geraniol and 10-mg PAA appeared in a similar response level. Thus, a higher concentration of geraniol and a lower concentration of PAA were employed in all behavioral experiments. These two stimuli were dissolved in paraffin oil and applied to a piece of filter paper (10 18 mm) in a Pasteur pipette. A purely mechanosensory stimulus (a puff of clean air) was used as a CS in the second experiment and is described below. The duration of the CS was 2 s Unconditioned stimulus A wooden toothpick moistened with 40% sucrose solution was used as an US. The US duration was 2 s. The interval between the CS and the US was 1 s, measured from the offset of the CS to the onset of the US. This interstimulus interval has previously been demonstrated to elicit the highest response level in S. littoralis [10] Training The moths were brought to the laboratory 1±2 h before the start of the dark period and were restrained. Each moth was introduced into the wide end of an Eppendorf tube, from which the narrow end had been cut to allow the head and antennae to protrude. The animals were then kept in the laboratory to adapt to the environment. The experiments started 2 h into the dark period under red light (25 lx). During discrimination conditioning, moths were grouped into squads of 10. The members in each squad were treated sequentially, such that the first animal was returned for its second trial only after the last one in the squad had been given its first trial. During training, the head of the moth was placed 1 cm away from the outlet of a glass tube (id, 8 mm), so that the antennae were continuously flushed with a charcoal-filtered and humidified air stream (0.5 ml/s.) At the beginning of each conditioning trial, each moth was left in the airflow for 10 s to adapt to the mechanical stimulation of the airflow. Then, the animals were trained with two types of CS; one stimulus was followed by the sucrose solution (CS+), but the other was not (CSo). Odor pulses (2 s, 5 ml/s) were injected by a stimulus controller (Syntech) from the Pasteur pipette containing the CS into the continuously flowing air stream through an opening (id, 3 mm) in the glass tube. The CS without odors (air puff) was also injected into the continuous air stream. Due to different flow rates between the permanent air stream and the injected stimulus, all stimulation with odors also involve a mechanosensory component. Following the CS+, the moth antennae were touched briefly with the sucrosemoistened toothpick to elicit the PER. The moth was subsequently rewarded with sucrose solution from the toothpick for 2 s while it extended its proboscis. For the CSo, the moths received only a 2-s CS without the sucrose solution. This basic discrimination procedure was manipulated as described below for each experiment Response measurement and statistical analysis A conditioned response (CR) to the rewarded odor (CS) was scored if the moth displayed a PER during training and/or testing periods. The PER was defined as when the moth extended its proboscis to the rewarded odor during the observation period, 3 s between onset of the CS and US [10]. A spontaneous PER was noted when the moth extended its proboscis before the training. The CR was recorded as 1 (responded to the CS) or 0 (failed to respond). The degree of discrimination was calculated as a discrimination index (DI) of subject group. The measurement of DI was based on the percentage of CR in each training trial. At the ith trial with each stimulus (CS+ and CSo), i =1,...,6: DI% i ˆ %CR to CS i %CR to CSo i Percentages of CR probability (% DI) were obtained for each subject group of 10 moths each in each CS+/CSo pair trial. The mean percentage of CR probability of each CS+/ CSo pair trial was calculated for statistical analysis. An analysis of variance (ANOVA) was used to determine the effects of variables in the experiments. Polynomial contrasts were tested with SYSTAT [31] to analyze the trends of training curves and their interactions [19]. The Tukey honest significant difference (HSD) test was also employed for multiple comparisons of between-subject factors. In no groups were there significant differences between the responses of males and females or between the different odors as the CS+. Therefore, the effects of sex and of different odors as the CS+ were not considered for further analysis.

3 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159± Fig. 1. The 5- and 10-min ITI groups received Odor 1 with the sucrose reward (CS 1 +) and Odor 2 without the reward (CS 2 o) in the first training phase. Then in the reversal phase, the moths were trained with the reversal relationship of CS 1 (CS 1 +! CS 1 o) and CS 2 (CS 2 o! CS 2 +). CR indicates a conditioned response (conditioned PER) and CS a conditioned stimulus. `` + '' and ``o'': with and without the sucrose reward. Geraniol (100-mg) or PAA (10-mg) was used as either CS 1 or CS 2. ITI indicates inter-trial intervals. The arrow shows the level of the spontaneous PER. Numbers of subject groups are eight each for 5- and 10-min ITI groups, respectively (n = 8) Experiment 1: reversal discrimination In reversal odor discrimination, animals are conditioned to respond to a rewarded odor (CS 1 +) but show no response to an unrewarded one (CS 2 o). Once the discrimination has been mastered, the animals are presented with a reversal of this task in which they should respond to CS 2 but not CS 1. In such a task, honeybees were able to express or withhold a conditioned PER depending upon the properties of the CS [16,30]. Like bees, moths are capable of forming CS ±US associations and performing CS+ CSo discrimination [9]. However, whether moths are capable of reversal discrimination is not known. Fig. 2. Mean percentages of group DI (%) of the two ITI groups. Same letter symbols as above indicate that the values within the same CS+ CSo acquisition trial are not significantly different when tested by Tukey HSD test (n = 8).

4 162 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159±165 The ITI has been shown to be important in determining excitatory conditioning in vertebrate [14]. Gerber et al. [13] showed that the length of ITIs have an effect on training and memory consolidation in honeybees. However, the effect of the ITI has not been studied in moth's discrimination tasks. We therefore conducted the experiment to investigate the capacity of moths in reversal discrimination, and how different ITI affect their behavior in an odor discrimination task Experimental design A total of 16 subject groups (10 moth each) of female and male moths were randomly assigned to 5- and 10-min ITI groups. The ITI was defined as the interval between two consecutive trials. Training procedures consisted of a differential conditioning phase and a subsequent reversal phase. In the first session, the animals received either geraniol (100 mg) or PAA (10 mg) as the CS 1 + and PAA or geraniol as the CS 2 o. Then in the second session, PAA and geraniol were reversed as the CS 2 + and CS 1 o, respectively. The break between two training sessions was 1 h. The presentation sequences of the two CS were pseudorandomized (either or ) [12]. A four-factor repeated-measures ANOVA was used to analyze the effects of ITI (ITI: 5- and 10-min), the reversed training procedure (reversal: first and reversal training phases), the CS followed by or without reward (reward), and the number of acquisition trials (training). The ITI was designed as a between-subjects factor and the reversal, reward and training were planned as within-subjects factors Result No differences were found between performances of male and female moths and between geraniol or PAA as the CS+; therefore, the data were pooled together for further analysis. Moths were able to learn the association to the CS+ both in the first and reversal training phases [ F(1,14) = 61.6, P <.001]. The differential response of the 10-min ITI group was significantly higher than that of the 5-min group in both training phases [ F(1,14) = 14.0, P <.01; Fig. 1]. In the first and the reversal training phases, the CS+ responses of the two ITI groups occurred at an identical level. However, the CSo response level of the 5-min ITI group was higher than that of the 10-min ITI (Fig. 1). During training, the acquisition trials gradually separated the CS+ responses from the CSo response for both ITI groups [ F(5,70) = 29.9, P <.001]. Significant interaction effects of reward±iti [ F(1,14) = 16.6, P <.01], training±iti [ F(5,70) = 3.2, P <.05], reward±training [ F(5,70) = 76.7, P <.001], and reversal training±iti [ F(5,70) = 2.8, P <.05] were observed in each case. These interaction effects show that differential performance were better with longer ITI. The Tukey HSD test showed that the CS 1 + responses of the 5-min ITI group were significantly different from those of the 10-min group only in the fifth trial of the first phase and second trial of the reversal phase (Fig. 1). When the first and reversal training phases were compared, the 5-min group responded to the CS 1 o at a higher level compared to the CS 2 o. However, the CS 1 o and CS 2 o of the 10-min group responses appeared at an identical level (Fig. 1). The comparison of the group DI reveals that longer ITIs led to more stable discrimination performance over time (Fig. 2). The DI of the 5-min ITI group was significantly lower compared with that of the 10-min group [ F(1,14) = 16.4, P <.01]. The DI of both ITI groups was linearly related to the number of acquisition trials [ F(5,70) = 74.5, P <.001], indicating that the number of training trials affected the discrimination performance (Fig. 2). Fig. 3. Mean percentages of the CR probability of CS+ CSo response to the GA+ Ao, A+ GAo, and A+ Ao treatments. CR indicates a conditioned response (conditioned PER) and CS a conditioned stimulus. `` + '' and ``o'': with and without the sucrose reward. G: an odor, 100-mg geraniol. A: mechanosensory stimulus, air puff. The arrow indicates the level of the spontaneous PER. Numbers of subject groups are four each for the GA+ Ao, A+ GAo, and A+ Ao treatments, respectively (n = 4).

5 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159± Experiment 2: solving feature-positive and -negative discrimination tasks Animals that rely upon olfactory information must be able to extract critical elements from a mixture or a background stimulus context in order to obtain information about the identity and location of the resources indicated by the stimulus [18,29]. In this experiment, we analyzed whether moths can distinguish a bimodal stimulus (an olfactory and a mechanosensory component) from a mechanosensory stimulus and if they can learn to identify the mechanosensory stimulus as the CS Experimental design Feature-positive and -negative discrimination tasks were used in the experiment (see Refs. [24,25,32] for detailed method). Three treatment groups were designed to test whether the moths can discriminate between a compound stimulus (odor and air puff) and the air puff alone (indicated as A). In the first treatment group, the animals received geraniol and air puff (GA) followed by the presentation of a sucrose solution (US) as the CS+ (GA+) and an air puff without the US as the CSo (Ao). In the second group, the moths received a rewarded air puff as the CS+ (A+) and an unrewarded compound stimulus as the CSo (GAo). The third group was trained with two air puffs, one was a rewarded (A+) and the other was unrewarded (Ao). Differences between the response levels of the CS+ and the CSo could potentially be caused by subjective handling bias, e.g. handling the sucrose reward in front of the moth before the odor was delivered. Thus, the third group was included as a control group to exclude these potential subjective biases. A total of 120 animals were randomly divided into three treatment groups; each group consisted of 20 female and 20 male moths, hence, there were four subject groups in each treatment group. The moths were trained according to a sequential discrimination paradigm in order to ensure that the moths learned the properties of the CS but not the order in which the two CS were presented. Six trials of CS+ and CSo were presented to the moths according to a pseudorandom schedule (either or ; + indicates CS+; indicates the CSo). The interval between CS+ and CSo (ITI) was 5 min. An ANOVA was applied to analyze the effect of three treatment groups (treatment: GA+ Ao, A+ GAo, and A+ Ao), the CS with and without the reward (reward), and acquisition trials (training). The first variable was designed as a between-subjects factor, and the last two factors were assigned as within-subject factors Results The moths were able to associate either the compound stimulus (odor and air puff) or the air puff with the sucrose reward (Fig. 3). The main effects of reward [CS+ vs. CSo: F(1,9) = 45.7, P <.001] and training [numbers of CS+ and CSo pairing: F(5,45) = 17.1, P <.001] were significant. Interaction effects of treatment±reward [ F(2,9) = 32.9, P <.001] and reward±training [ F(5,45) = 11.7, P <.001] were also significant. The results show that the GA+ Ao and A+ GAo groups reveal a higher response level of GA+ and A+ than that of Ao and GAo, respectively. The GA+ and A+ responses increase linearly with the number of training trials, showing that increasing the number of trials can improve discrimination performance of the moths. Fig. 4. Mean percentages of group DI (%) of the three treatment groups. Means with the same letters within a CS+ CSo acquisition trial are not significant. The difference was tested by Tukey HSD test (n = 4).

6 164 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159±165 When the DI of each group was compared, the GA+ Ao group displayed a greater discrimination performance than other groups [treatment groups, GA+ Ao, A+ GAo, and A+ Ao: F(2,9) = 35.2, P <.001]. The degree of discrimination increased linearly with the number of acquisition trials [ F(5,45) = 11.2, P <.001], showing that increasing the number of training trials allows the animals to learn the properties of the CS. Multiple comparisons using Tukey HSD tests showed that the GA+ Ao and A+ GAo groups had a greater degree of discrimination than the A+ Ao group in the two to six trials (Fig. 4). 2. General discussion The moth S. littoralis is capable of performing both feature-positive (AGt, Ao) and -negative (A+, AGo) discrimination tasks. It is also able to quickly learn the significance of the CS 1 and CS 2 in a reversal paradigm. Further, the ITI appears to be a significant variable affecting discriminative performance in moths. In the first experiment, we aimed to investigate whether the moths could learn the CS 1 and CS 2 as rewarded stimuli (CS+) in the first and second phases, respectively. The result showed that the moths could associate the CS 1 and CS 2 with the sucrose reward in both phases, implying that they learn the properties of the CS 1 and CS 2 in the first and reversal phases, correspondingly (Figs. 1 and 2). However, if they relearn the significance of the CS 2 in the reversal training phases, one may expect the response level to the CS 1 on the first trial of the reversal phase to be identical to that on the last trial of the first phase [27]. However, it was not the case in this experiment. One reason could be that the pause between both phases was too long (1 h), hence, the outcome of the first phase did not affect the training during the reversal phase. Alternatively, this result might reflect that the moths, at least partially, forgot the CS 1 ±US association and learned the CS 2 +CS 1 o as a new task in the reversal phase. The ITI could be an important factor that affects moth's PER discrimination performance. When the effect of ITI was examined, the DI of the 5-min ITI group was significantly lower than that of the 10-min ITI group in both training phases (Fig. 2). The result thus suggested that the longer the ITI, the better discrimination performance [11]. However, the length of the ITI did not monotonically influence acquisition and memory consolidation in honeybees' appetitive conditioning [13]. The bees showed higher response levels during training with longer ITI (20 min) than shorter ones (30 s, 1 and 3 min). In the memory test, long-term retention after 4 days was reduced for 30-s and 3- min ITIs compared to 1- and 20-min ITIs. The role of ITI in honeybees is, however, debated as both Bitterman et al. [6] and Sandoz et al. [28] demonstrated that ITI had no effect on training and memory. It is important to note that in differential conditioning, as in our experiment, the relevant interval is between two trials with the same stimulus. This interval varied due to the pseudorandomized sequences, and the ITIs refer to the interval between two consecutive stimuli, either rewarded or unrewarded. Furthermore, not only the ITI but also the total duration time may influence acquisition and memory [13]. As the duration of the first phase of the long ITI group was twice as long as of the short ITI group, the impact of experimental duration can not be ruled out. In the second experiment, the moths had no difficulties recognizing the positive feature of the GA+, i.e. geraniol, and could perform the discrimination of the compound signal from pure air after they had received one pair of GA+ Ao. They could also identify geraniol as a negative feature in the A+ GAo group, i.e. they could learn to associate pure air with a reward and did not respond to the compound stimulus. However, three pairs of A+ GAo (air puff +, odor, air puff o) were necessary for the A+ GAo group to achieve the same response level as the GA+ Ao group (Fig. 4). If the relative salience of stimuli is not equal, some stimuli may enter into associations more readily than other [27], which might explain this result. The A+ GAo group seemed to have more difficulty in the beginning of conditioning (Figs. 3 and 4), which might depend on the fact that odors but not plain air normally carry biologically significant information [22,27]. The animals may therefore respond more readily to the odor than to the air puff at the start of the training. However, a further experiment could be conducted to train the moths with GA ±sucrose association and then compare response levels to G and A after training. Then, we may be able to rule out the existence of different levels of salience in these two stimuli. The fact that the moths could solve the feature-positive and -negative tasks might be alternatively explained by the configural theory [22,23]. This theory assumes that when two or more stimuli are presented together for conditioning or discrimination, then a configural representation will be formed of the entire pattern of stimulation. This representation will then enter into a single association with the outcome of the trial. The association with the US is assumed to develop gradually over trials, and its strength is reflected in the power of the CR that will occur to that particular pattern of stimulation. If the pattern of stimulation should change in any way, then a weaker CR will be performed with a vigor that is related to the similarity of the training and test patterns [22,23]. In the GA+ Ao group, we might assume that a GA±sucrose (GA+) and A±absence of sucrose (Ao) associations were gradually developed over trials. The inhibition associated with Ao was sufficient to completely counter the excitation that was transferred to it from GA, therefore, the GA+ Ao group was able to perform the feature-positive discrimination [24]. However, when we look at the A+ GAo group, the stimulus A+ could be assumed to enter individually into excitatory association, and the compound GAo entered into an inhibitory association. Since the effect of excitatory association could be

7 R.-J. Fan, B.S. Hansson / Physiology & Behavior 72 (2001) 159± generalized to GA, the discrimination was developed more slowly than the GA+ Ao group [32]. The moth S. littoralis provides a powerful system for investigating learning processes and odor discrimination on both behavioral and cellular levels. The well-studied olfactory system of the moth and the many identified pheromones and behaviorally active host-plant-associated odors allow differences in discrimination processing for odors with different meaning to the moth to be investigated. In parallel, an investigation of the neural elements responsible for the cellular chain of events occurring from the initial stimulation with the CS to the actual discrimination performances can be achieved. Acknowledgments We wish to thank Drs. B. Gerber and M. Stopfer for valuable comments on the manuscript; M. Carlsson and D. 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