Matthias Durrieu

Antoine Wystrach

Patrick Arrufat

Martin Giurfa

Guillaume Isabel

Fruit flies can learn non-elemental olfactory discriminations

Keywords: Training cycles Performance Index (PI) Negative patterning Training cycles Performance Index (PI) Negative Patterning, Feature Negative Discrimination, Associative Learning, Pavlovian Conditioning, Insect, Drosophila melanogaster

0.2 0.0 0.2 0.4 0.6 0.8 1.0 1 cycle 5 cycles

Training cycles

Performance Index (PI)

Feature negative

Introduction

The ability to form a link between meaningful events is the cornerstone of associative learning. One of the most studied forms of associative learning is Pavlovian conditioning, where animals learn a conditional relation between a conditioned stimulus (CS) and an unconditioned stimulus (US) [START_REF] Pavlov | Conditioned reflexes: an investigation of the physiological activity of the cerebral cortex[END_REF]. Several protocols can be used to study Pavlovian conditioning. One of them is differential conditioning, which allows studying how animals learn to associate different CSs with different outcomes (e.g. a reinforced stimulus A + from a non-reinforced stimulus B -) [START_REF] Bower | A contrast effect in differential conditioning[END_REF]. In this case, each CS is associated with a distinct US through a simple, non-ambiguous link so that they are learnt independently of each other. In consequence, this type of learning is also referred to as 'elemental learning' [START_REF] Rescorla | A Theory of Pavlovian Conditioning: Variations in the Effectiveness of Reinforcement and Nonreinforcement[END_REF][START_REF] Giurfa | Cognitive neuroethology: Dissecting non-elemental learning in a honeybee brain[END_REF].

However, stimuli in nature may not appear as isolated, distinct elements. Usually, they are compounds constituted of multiple elements. For the elemental-learning theory, a compound AB is the linear sum of its elements. In other words, an animal presented with AB would learn the independent relation of A and of B with reinforcement. Yet, several compounds with shared elements and different outcomes could co-exist, creating thereby ambiguity at the level of the single elements. This possibility led to the proposal of the configural (non-elemental) theory, which states that a compound is not learned as the linear sum of its components but as a distinct configuration in which elements would not be fully recognized [START_REF] Pearce | Configural Associations in Discrimination Learning[END_REF][START_REF] Rudy | Configural and elemental associations and the memory coherence problem[END_REF][START_REF] Rudy | Configural association theory and the hippocampal formation: An appraisal and reconfiguration[END_REF]. The validity of this hypothesis has been studied using a discrimination task termed 'Negative Patterning' (NP) in which a subject has to learn to respond to two single elements reinforced (A + , B + ) and to inhibit its response to their non-reinforced compound (AB -). For the elemental-learning account, solving this task is impossible as animals reinforced on A and B should respond twice as more to AB. In contrast, for the configural account, NP learning is possible as AB would be perceived as a configuration different from A and B, thus facilitating differentiation [START_REF] Pearce | Configural Associations in Discrimination Learning[END_REF]. Other studies explored alternative explanations for NP solving and argued that the compound AB would be perceived as the sum of A and B plus a unique cue specific to the AB compound (therefore termed 'unique cue theory') [START_REF] Pressing | Configural ' Conditioning in Discrete-Trial[END_REF]. Within this framework, joint presentation to the animal may result in perceptual interferences such as overshadowing, which suggests that in that case, only part of A and B are perceived within the compound [START_REF] Mackintosh | An Analysis of Overshadowing and Blocking[END_REF]. In either Configural and Unique cue theory, animals would learn the NP task by focusing on an unambiguous compound-specific cue.

Another task that has received wide attention among learning scholars is the 'Feature Negative discrimination' (NF) in which subjects learn to respond to a single element reinforced (A + ) but not to a non-reinforced compound AB - [START_REF] Pearce | A Model for Stimulus Generalization in Pavlovian Conditioning[END_REF]. NF is interesting as it admits both elemental and configural explanations: for the elemental theory, focusing on B (the negative feature) brings an elemental solution to the problem. As stimulus A has an ambiguous valence, being as often reinforced and non-reinforced, B alone suffices as a conditioned inhibitor to respond appropriately. For the non-elemental theory, discrimination is straightforward as the compound AB is a configuration that is unrelated to its elements [START_REF] Pearce | A Model for Stimulus Generalization in Pavlovian Conditioning[END_REF][START_REF] Pearce | Similarity and discrimination: A selective review and a connectionist model[END_REF]. Studying if and how animals solve the NP and NF discriminations is important to access the mechanisms of associative learning in the animal brain. Given the importance of invertebrates for studies on learning and memory [START_REF] Carew | Invertebrate Learning and Memory: From Behavior to Molecules[END_REF][START_REF] Giurfa | Cognition with few neurons: Higher-order learning in insects[END_REF], research on lobsters [START_REF] Livermore | Elemental and Configural Learning and the Perception of Odorant Mixtures by the Spiny Lobster Panulirus argus[END_REF] and honeybees [START_REF] Deisig | Configural Olfactory Learning in Honeybees : Negative and Positive Patterning Discrimination[END_REF][START_REF] Deisig | The effect of similarity between elemental stimuli and compounds in olfactory patterning discriminations[END_REF][START_REF] Deisig | A Modified Version of the Unique Cue Theory Accounts for Olfactory Compound Processing in Honeybees[END_REF] has focused on the capacity of these animals to learn these discrimination problems. In other insect species, other forms of non-elemental learning such as multimodal NF [START_REF] Balkenius | Discrimination training with multimodal stimuli changes activity in the mushroom body of the hawkmoth manduca sexta[END_REF], biconditional discrimination [START_REF] Matsumoto | Context-dependent olfactory learning in an insect[END_REF][START_REF] Sato | Contextual olfactory learning in cockroaches[END_REF] or contextual learning (21) have been described, yet the solving of NP has remained circumscribed to the honeybee. Attempts to study these phenomena in the fruit fly Drosophila melanogaster, an insect model that is used recurrently for studying elemental learning forms [START_REF] Davis | Olfactory Memory Formation In Drosophila : From Molecular to Systems Neuroscience[END_REF][START_REF] Cognigni | Do the right thing : neural network mechanisms of memory formation , expression and update in Drosophila[END_REF] have failed [START_REF] Young | Neurobiology of Learning and Memory Elemental and non-elemental olfactory learning in Drosophila[END_REF]. This lack of success is regrettable as the fruit fly offers a vast spectrum of neurogenetic tools, which would allow comparing circuits and mechanisms underlying elemental and nonelemental forms of learning. This approach has allowed identifying the olfactory pathways and learning circuitry of the fly, both from an anatomical (a full connectome of these circuits has been published recently [START_REF] Takemura S Ya | A connectome of a learning and memory center in the adult Drosophila brain[END_REF]) and functional perspective [START_REF] Cognigni | Do the right thing : neural network mechanisms of memory formation , expression and update in Drosophila[END_REF]. Robust computational models based on said circuits have been developed and predict that non-elemental learning should be achievable by fruit flies, as normalization events at the perceptual level support the emergence of configural representation of compound stimuli [START_REF] Wessnitzer | A model of non-elemental olfactory learning in Drosophila[END_REF]. Thus, studying nonelemental learning from a mechanistic perspective in the fly represents an attractive and accessible goal.

Here we achieved the first demonstration of the fruit fly's capacity to solve NP and NF in the olfactory domain, thus showing that beyond simple discrimination learning, flies can also solve non-elemental discriminations. In showing this capacity, we determined the associative strategies used by the insects and demonstrated that the processing of olfactory compounds moves along a continuum between elemental and configural processing.

Increasing the number of conditioning trials promoted configural processing, enabling flies to solve the NP task.

Material and Methods

Fly rearing: The wild-type line used in this study was a Canton-Special (Canton-S) strain. Flies were raised on standard medium at 25°c, ~60% humidity and a 12h/12h light/dark cycle. The flies were kept in 36x82mm plastic tubes containing ~20mL of medium. Olfactory conditioning: Odours were diluted in a bottle of mineral oil. Odours used were 3-Octanol (termed 'A' for the sake of simplicity, 2.27mM) and 4-Methylcyclohexanol ('B', 2.62mM) (figure 1 and figure 2). Benzaldehyde ('C', 1.89 mM) was used as a novel odour in some conditions (figure 2). IsoamylAcetate ('D') and EthylButyrate ('E') were used as alternative odours (electronic supplementary material, figure S2) to determine if discrimination solving was independent of the pair of odours used. Odours were delivered at the same concentration whether as components (A, B, D or E) or as mixtures (AB or DE). In the case of mixtures, odours were diluted together in the same bottle of mineral oil. All odours and the solvent were from Sigma Aldrich (France). The US consisted of twelve 1.5s pulses of 60V electric shock every 5s delivered through a metallic grid. Each experiment included two groups of ~30 flies (2 -4 days old) and was performed using a semi-automated device based on a previous work [START_REF] Pascual | Localization of long-term memory within the Drosophila mushroom body[END_REF]. In a T-Maze, two main phenomena drive the preference of flies toward a compartment or another: on the one hand, the learnt information about the stimuli acquired during conditioning and on the other hand, the fact that odours are rarely completely neutral; at the concentrations used in our work, they are in fact repulsive to naïve flies. When two odorants are opposed in the absence of punishment in the T-maze, repulsion balances; yet, if one odorant is opposed to a compound, which is more repulsive, a bias towards the less repulsive stimulus is visible. To disentangle learning from non-learning behavioural components, one of the groups experienced an explicit pairing of CS and US ('paired group') while the other group experienced both stimuli unpaired to prevent their association ('unpaired group').

Training: Each training trial consisted of 90 s of acclimatisation, after which flies were subjected to their respective conditioning protocol. Each of the two odours (CS) was presented once for 1min with an intertrial interval of 1min. For the paired group (figure 1a), one of the olfactory stimulus (CS + ) was paired with the US, while the other stimulus (CS -) remained unpunished. In the unpaired group, flies were exposed to 1min of either shocks or odours, separated by an interval of 1min. This sequence formed one conditioning cycle.

Flies were subjected to one of three training protocols (figure 1b): a differential conditioning (DC) in which they had to learn to discriminate a punished from a non-punished odour (A + vs. B -), a NF discrimination in which they had to learn to discriminate a punished odour from a non-punished odour compound (A + vs. AB -) and a NP discrimination in which they had to learn to discriminate two punished odours from a non-punished odour compound (A + , B + vs. AB -). 3-Octanol was always used as the CS+ for the DC and NF protocols (with 4-Methylcyclohexanol (DC) and 3-Octanol+4-Methylcyclohexanol (NF) as CS-). For the NP protocol, both 3-Octanol and 4-Methylcyclohexanol were used as CS+ when presented alone and as CS-when presented as a compound. Thus, flies subjected to DC training faced a pure elemental discrimination. On the contrary, flies trained in the NP protocol could only solve the problem if they adopted a non-elemental strategy. Finally, flies subjected to NF training could solve the problem using either an elemental or a non-elemental strategy. For each protocol, training consisted of either one or five cycles, which allowed studying if the amount of experience gathered by flies promoted a particular discriminations strategy. Test: After training, flies were transferred to a T-maze [START_REF] Tully | Classical conditioning and retention in normal and mutant Drosophila melanogaster[END_REF] where they could choose between the CS + and the CS -in the absence of shock during 1min. Flies from paired and unpaired protocols were sequentially tested. At the end of the test, flies in each arm of the T-maze were counted. If paired flies learned the discrimination, they should be mostly located in the CS-arm, the arm presenting the odour stimulus that was not associated to the shocks during the training. A Performance Index (PI) was calculated as: (Number of flies in the CS -arm -Number of flies in the CS + arm) / total Number of flies. To control for any experimental bias, each replicate consisted of a 'paired-group' PI (reflecting associative learning + bias) from which an 'unpaired-group' PI (reflecting bias only) was subtracted (electronic supplementary material, figure S1). Statistical analysis: PI data were plotted and analysed using R software (3.5.0 version). Group distributions were tested for normality using the Shapiro-Wilkinson test; homoscedasticity was tested using Bartlett's test. All our experiments met the requirements for parametric statistics. For one or two-sample analyses, we applied one sample or two samples two-tailed Student's tests, respectively. For comparisons involving more than two samples, we used analysis of variance (ANOVA) followed by Tukey's (HSD) post hoc tests. A significance level of p < 0.05 was set for every experiment.

Results

Figure 1c (left panel) shows that flies trained in the DC protocol learned the discrimination and preferred the non-punished odour B-to the punished odour A+. Comparing their PIs against 0 by means of a one-sample t test yielded significant differences after both one training cycle (t = 9.49, df: 17, p = 3.33e-8) and five training cycles (t = 10.83, df = 17, p = 4,76e-9) . Both PIs did not differ from each other (two-sample t-test : N = 18 and 18, t = -0.87, df = 34, p = 0.39), thus providingshowing that the amount of experience of flies did not affect the learning of this elemental discriminationno evidence for an effect of amount of experience.

Flies trained in the NF protocol (figure 1c, middle panel) also learned the discrimination between the single odour punished A+ and the non-punished odour-compound AB-. Note that AB is innately more repulsive than A but the relative PI shows the expected effect of learning (figure 2b, and electronic supplementary material, figure S1). Performance was significantly different from zero after both one cycle of training (t = 4.13, df: 16, p = 7,78e-4) or after five cycles (t = 6,69, df: 17, p=3.77e-6). In contrast with DC training, performance improved significantly with the amount of training (N=17 and 18, t = -2.11, df = 33, p = 0.042). We also tested the flies' performance after the DC or NF protocols balancing odor contingencies, i.e. using B as CS + and A as CS -(electronic supplementary material, figure S3).

In the case of flies trained in the NP protocol (figure 1c 1c, right panel). These findings show that training repetition is crucial for NP solving as it improved the ability of flies to discriminate the odours with different outcome. To determine if NP solving is independent of the pairs of odours used, we repeated the experiment using different odours (D+, E+ vs. DE-; electronic supplementary material, figure S2). The flies learned the NP discrimination also in this case, showing that the type of odour used during the test phase had no significant effect on performance (i.e. 'D vs. DE' or 'E vs. DE' ; 2-way ANOVA : F(1, 69) = 3.005, p = 0.088). After 1 trial, flies were not attracted by the single odours (t = -0.73, df = 35, p = 0.47). After five trials, they were able to solve the task and showed a significant preference for the compound (t = 5.21, df = 35, p = 8.59e-06). This result demonstrates that the ability to solve NP is not specific to the type of odour used in the protocol.

after a t test(DC and NF) or after a 2-way anova (NP)

Despite their different complexity, the three protocols assayed opposed one or two CS + to a CS -. Under these conditions, an animal may learn to avoid the CS + , to be attracted to the CS-, or both. To determine the nature of the associations inculcated by DC, NF and NP, we studied if the CS -(not reinforced) was learnt as a safe stimulus (inducing attraction), remained neutral (being indifferent to trained animals) or in the case of compounds sharing elements with the reinforced CS + , gained also inhibitory strength, thus eliciting avoidance. To test these options, flies were first trained under the DC, NF or NP protocols (Figure 2a) and then given a choice between the CS -and a novel, neutral odour C. Here again, the preference of trained flies was compared to preference of flies after an unpaired protocol. After being trained in the DC protocol (figure 2b ). Yet, differently from NF training, the flies' aversion for the CS -was reduced after 5 training cycles (N= 30 and 30, t = -2.74, df = 58, p = 8.10e-3), thus showing that increasing the amount of experience diminished the inhibitory strength of the compound. This variation is consistent with a non-linear processing of the compound, which became less similar to the two single odour components, both being inhibitory after having been paired with shocks.

Discussion

Our results provide the first evidence of Negative-Patterning solving in an insect other than the honey bee [START_REF] Deisig | Configural Olfactory Learning in Honeybees : Negative and Positive Patterning Discrimination[END_REF][START_REF] Young | Neurobiology of Learning and Memory Elemental and non-elemental olfactory learning in Drosophila[END_REF]. Our results show that flies solved both a NF and a NP discrimination and preferred, in both cases, the odour compound AB that was unpunished during training. In both cases, the compound acquired some inhibitory strength as shown by its avoidance when confronted with a novel stimulus C, a fact that could reflect an influence of the punished element (A in NF, A and B in NP) in it. Such an influence would be consistent with an elemental processing of the compound (AB = A+B). Yet, in NP, increasing the amount of training diminished this influence, thus promoting a configural processing (AB= new odour). When interpreted in the light of these theories, our results cannot accommodate a strictly elemental or configural account. Even after five training cycles in NP, the flies still exhibited some avoidance of the CS -mixture AB, implying that AB -still carried some of the inhibitory strength associated with its constituents, a result that goes against a purely configural compound representation (figure 2b, right panel). In other words, the compound remained repulsive, but was preferred to the "more repulsive" elements taken alone. This result differs from the response of naïve flies, for which a compound is more repulsive than the single odorants, at the concentrations used (electronic supplementary material, figure S1).

Our result could thus be interpreted as supporting the 'unique cue theory', which is consistent with observations both in honeybee and Ddrosophila. In vivo imaging of the Aantennal Llobes, the primary olfactory centreer in the insect brain, suggests that binary mixtures are not coded as the linear sum of their constituents but rather show both similarities and features that are unique to the compound considered [START_REF] Joerges | Representations of odours and odour mixtures visualized in the honeybee brain[END_REF][START_REF] Silbering | Processing of odor mixtures in the Drosophila antennal lobe reveals both global inhibition and glomerulus-specific interactions[END_REF][START_REF] Deisig | Neural representation of olfactory mixtures in the honeybee antennal lobe[END_REF][START_REF] Deisig | Antennal lobe processing increases separability of odor mixture representations in the honeybee[END_REF]. However, the fact that the flies' initial choice, which was closer to an elemental representation, was reversed (although not entirely) after NP training shows that increasing training gradually changed the odour representation.

Our results show that the experience of an individual modulates the kind of processing adopted. This modulation is consistent with previous work on honeybees showing, in both the visual and the olfactory modality, that increasing the number of training trials promotes a configural stimulus representation [START_REF] Giurfa | The effect of cumulative experience on the use of elemental and configural visual discrimination strategies in honeybees[END_REF][START_REF] Stach | The influence of training length on generalization of visual feature assemblies in honeybees[END_REF]. Such a plastic stimulus representation has also been described in other learning paradigms (in crustaceans [START_REF] Livermore | Elemental and Configural Learning and the Perception of Odorant Mixtures by the Spiny Lobster Panulirus argus[END_REF]; larvae [START_REF] Chen Y Chun | Behavioral evidence for enhanced processing of the minor component of binary odor mixtures in larval Drosophila[END_REF] and adults Drosophila [START_REF] Brembs | Context and occasion setting in Drosophila visual learning[END_REF][START_REF] Barth | in Drosophila melanogaster[END_REF]; rabbits [START_REF] Sinding | Experience influences elemental and configural perception of certain binary odour mixtures in newborn rabbits[END_REF]; humans [START_REF] Howard | Configural and elemental coding of natural odor mixture components in the human brain[END_REF]). How such a change of odour representation could be implemented in the fly's brain? Firstly, associative learning has been shown to modulate neural representation of odours in the honeybee Antennal Lobes, a structure involved in olfactory processing, also present in Ddrosophila. Over the course of CS/US associations, odours representation was modified, amplifying the CS+ representation and reducing CS+/CS-correlation. This could be a first explanatory mechanism for enhanced discrimination abilities necessary for NP resolution [START_REF] Faber | Associative learning modifies neural representations of odors in the insect brain[END_REF]. Moreover, pioneer work in the field identified the mushroom bodies as critical structures for the encoding of learnt behaviour in insect brain [START_REF] Erber | Localization of short-term memory in the brain of the bee, Apis mellifera[END_REF][START_REF] Heisenberg | Drosophila Mushroom Body Mutants are Deficient in Olfactory Learning[END_REF][START_REF] De Belle | Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies[END_REF]. Interestingly, bees without functional mushroom bodies learn simple olfactory discriminations but are unable to solve NP tasks [START_REF] Devaud | Neural substrate for higherorder learning in an insect : Mushroom bodies are necessary for configural discriminations[END_REF]. Such a failure in NP tasks (as well as in other tasks such as reversal learning) was related to the pharmacological blockade of a specific subset of feedback neurons providing GABAergic signaling to the mushroom bodies [START_REF] Devaud | Neural substrate for higherorder learning in an insect : Mushroom bodies are necessary for configural discriminations[END_REF][START_REF] Boitard | GABAergic feedback signaling into the calyces of the mushroom bodies enables olfactory reversal learning in honey bees[END_REF]. The functional equivalent of these neurons in Drosophila, the Anterior Paired Lateral neurons (APL), are equally necessary for reversal learning [START_REF] Wu | The GABAergic anterior paired lateral neurons facilitate olfactory reversal learning in Drosophila The GABAergic anterior paired lateral neurons facilitate olfactory reversal learning in Drosophila[END_REF]. Together, this suggests that APL neurons play a crucial role for the modulation of odour representation, and thus for NP solving in flies. The inhibitory feedback mediated by APL neurons enables a sparse encoding of sensory information by maintaining a low level of activity in the MB, which is needed to discriminate between similar olfactory stimuli [START_REF] Lin | Sparse , decorrelated odor coding in the mushroom body enhances learned odor discrimination[END_REF]. Previous modeling work suggested that normalization at the Antennal Lobes level is crucial for non-elemental forms of learning as it leads to elements-specific features inhibition, thus enhancing compound-specific (unique cue) salience [START_REF] Wessnitzer | A model of non-elemental olfactory learning in Drosophila[END_REF]. APL neurons activity also acts as a gain control mechanism and thus further enhances discrimination at the MB level, which would confirm a hypothesis already proposed in a previous work on NP in honeybee [START_REF] Devaud | Neural substrate for higherorder learning in an insect : Mushroom bodies are necessary for configural discriminations[END_REF]. Moreover, by modulating its inhibitory activity depending on the task at hand, the APL neurons could in theory mediate a shift along the generalization / discrimination balance as observed here. Indeed, after 1 cycle of NP conditioning, flies respond to AB as if it were A or B, consistent with odour generalization. Yet, after 5 cycles, flies change their behaviour and choose AB even though the second experiment clearly shows that they still perceive the A and B constituents as aversive. This result, in contrast can be attributed to better discrimination abilities. Interestingly, dopamine signals originating from PPL1 neurons (responsible for aversive US transmission) inhibit APL activity through D2-like receptors, thus modulating olfactory learning [START_REF] Zhou | Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning[END_REF]. Moreover, APL neurons are activated differently by the CS+ and CS- [START_REF] Liu | The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning[END_REF]. More specifically, the association between the CS and the US results in a diminished APL activity (leading to a reduction of inhibition towards the CS+, facilitating -in principle-its reinforcement). Thus, it is possible that during trials, a differential activation of APL in response to the CS+ vs CS-produces a form of pruning, leading to the unequivocal reinforcement of the KC associated only with A or B while inhibiting the KC associated both to A/B (the CS+) and AB (the CS-). If this process is slow compared to the acquisition of the original CS-US association, APL activity would gradually modulate stimulus representation to facilitate the task resolution. In the case of NP, it would help segregating the representation of AB from those of its elements. APL neurons could thus continually adjust and update representation along a generalization/discrimination continuum based on learning events, besides their role as regular gain control system. Understanding how APL activity might modulate non elemental olfactory learning constitutes an interesting future research agenda. So far, olfactory learning in fruit flies was limited to DC protocols, thus reducing the study of associative learning and its neural and molecular mechanisms to its most simple form. Our results thus open new perspectives to explore these mechanisms for higher-order forms of learning and determine if the same or different circuits intervene when different learning strategies are employed.

Figure 1 (

 1 a) schematic representation of a typical training cycle. Blue and orange boxes show CS presentation while red bars show US delivery. (b) schematic representation of the conditioning protocols. Clouds represent the CS odorants while light bolts indicate the delivery of electric shock during training.A = 3-Octanol, B = 4-Methylcyclohexanol (c) CorrectedRelative performance indices (PI) computed as the difference between paired and unpaired scores. Performances were compared within the same protocol (i.e. 1 cycle vs. 5 cycles) but not between protocols. Data are plotted as boxplots. The middle linerepresents the median while the upper and lower limits of the box are the 25 and 75 percent quantiles. The whiskers are the maximum and minimum values of the data that are respectively within 1.5 times the interquartile range over the 75 th percentile and under the 25th percentile. Raw data are superimposed as jittered dots. "n.s" stands for "non-significant", * indicates p < 0.05, ** indicates p < 0.

  , left panel), flies tested with the nonpunished odour B vs. the neutral odour C did not show any preference either after one training cycle (t = 0.48, df = 26, p = 0.64) or after five training cycles (t = 0.73, df = 26, p = 0.47). There was no significant difference between PIs corresponding to these different training amounts (N = 27 and 27, t = -0.31, df = 52, p = 0.75). This result thus shows suggests that the non-punished odour was not perceived as attractive after DC training and that it conserved this status irrespective of the amount of training, and that the number of training cycles has little impact if at all. . After NF conditioning (figure2b, middle panel), flies preferred significantly the novel odour C to the non-punished compound AB both after one (t = -3.78, df = 26, p = 8.41e-4) and five training cycles (t = -4.15, df = 26, p = 3.16e-4). This preference was maintained despite the increase in the amount of training (N = 27 and 27, t = -0.03, df = 52, p = 0.97), thus showing that in learning this discrimination, flies assigned an aversive valence to the CS -despite the fact that it was not paired with shocks. This inhibitory strength must have been acquired via the presence of the punished element A in the compound AB. Finally, after NP training (figure 2b, left panel), flies also preferred significantly preferred the new odour C to the non-punished compound AB both after one (t = -7.36, df = 29, p = 4.12e-8) and five training cycles (t=-4.46, df = 29, p = 1.12e-4

Figure 2 (

 2 a) Schematic representation of the tests performed after the three conditioning protocols to determine the nature of the CS-representation. A = 3-Octanol, B = 4-Methylcyclohexanol, C = Benzaldehyde (b) CorrectedRelative performance indices (PI) computed as the difference between paired and unpaired scores. Performances were compared within a given protocol (i.e. 1 cycle vs. 5 cycles) but not between protocols. Data are plotted as boxplots. the middle line represents the median. The upper and lower limits of the box are the 25 and 75 percent quantiles. The whiskers are the maximum and minimum values of the data that are respectively within 1.5 times the interquartile range over the 75 th percentile and under the 25th percentile. Raw data are superimposed as jittered dots. "n.s" stands for "non-significant", * indicates p < 0.05. after a t test. Gray shading indicates performances that were not significantly different from chance level while white filling indicates a significant difference from chance level (t test against zero).

  , right panel), the type of odour used during tests had no significant effect on performance (i.e. 'A vs. AB' or 'B vs. AB'; 2-way ANOVA: F(1,69) = 2.72, p = 0.10). Yet, the PI varied significantly depending on the number of training cycles ( F(1,69) = 34.92, p = 1.18e-7). After 1 training cycle, the PI was negative, i.e. biased towards the single odours associated with the shocks during training (A + or B

+ ) (t = -2.47, df = 35, p = 0.018). However, after 5 training cycles, preference was reversed and flies preferred the non-punished compound AB-over the single punished odorants A+,B+ (t = 5.37, df = 35, p = 5.16e-6; figure

http://mc.manuscriptcentral.com/prsb Submitted to Proceedings of the Royal Society B: For Review Only

Acknowledgements

We thank Gérard Latil and Audrey Dussutour for providing the necessary material and making the flies food medium; Stéphane Ferrere for the conditioning setups.

Funding This work was supported by the French Ministry of Higher Education and Research to M.D. and by the Agence Nationale pour la Recherche (ANR) in the form of the projects entitled funded Toulouse Initiative of Excellence IDEX UNITI (ANR11-IDEX-0002-02) transversality grant and the MoleCulture (ANR-18-CE37-0015) to G.I. M.G. thanks the Institut Universitaire de France for support. G.I. benefited from a CNRS Excellence Chair.

No data available. Data It is a condition of publication that data, code and materials supporting your paper are made publicly available. Does your paper present new data?: Yes Data available from the Dryad Digital Repository (temporary link, doi:10.5061/dryad.v9s4mw6t1): https://datadryad.org/stash/share/gXEA6n_fJLubzjdmB3VkAkm1WBAqJohvqvjTOeTTg5U. http://mc.manuscriptcentral.com/prsb

Data accessibility

Data available from the Dryad Digital Repository (temporary link, doi:10.5061/dryad.v9s4mw6t1): https://datadryad.org/stash/share/gXEA6n_fJLubzjdmB3VkAkm1WBAqJohvqvjTOeTTg5U.

Conflict of interest I/We declare we have no competing interests

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CUST_STATE_CONFLICT :No data available.

Authors' contributions

This paper has multiple authors and our individual contributions were as below

Statement (if applicable):

MD carried out all the experiments, analysed data, participated in the design of the study and drafted the manuscript; AW participated in the design of the study and critically revised the manuscript; PA performed the semi-automated device; MG participated in the design and conceptualization of the study and critically revised the manuscript; GI conceived, designed and coordinated the study, and helped draft the manuscript. All authors gave final approval for publication and agree to be held accountable for the work performed therein.

Authors' contibutions

MD carried out all the experiments, analysed data, participated in the design of the study and drafted the manuscript; AW participated in the design of the study and critically revised the manuscript; PA performed the semi-automated device; MG participated in the design and conceptualization of the study and critically revised the manuscript; GI conceived, designed and coordinated the study, and helped draft the manuscript. All authors gave final approval for publication and agree to be held accountable for the work performed therein. M.D. carried out all experimental work and analysed the data. P.A. devised an electric generator and its programming. M.D., A.W., M.G. and G.I. wrote the manuscript. G.I supervised the project and designed all experiments with M.D., A.W. and M.G.

Competing interests

The authors declare no competing interests.