Fabien Démares
email: fabien.demares@cefe.cnrs.fr

Laëtitia Gibert

Pierre Creusot

Benoit Lapeyre

Magali Proffit

Acute ozone exposure impairs detection of floral odor, learning, and memory of honey bees, through olfactory generalization

Keywords: Air pollution, volatile organic compounds, olfaction, generalization, Apis mellifera, plant-pollinator interactions

Highlights (max 85 characters including spaces per bullet point)

• Ozone (O3) at 80 ppb decreases the recall of learned odor association.

• O3 increases the response to new odor suggesting higher generalization.

• Directly after O3 exposure at 80 & 200 ppb, antennal activity decreases.

• But after a two-hour rest, O3 increases antennal activity at 120 & 200 ppb.

• O3 affects olfactory detection and perception of VOCs in honey bees.

Abstract (max 300 words) (word count: 299)

Air pollution stemming from human activities affects the environment in which plant and animal species live and interact. Similar to primary air pollutants which are emitted, secondary air pollutants, such as tropospheric ozone (O3) formed from nitrogen oxides, are also harmful to human health and plant physiology. Yet, few reports studied the effects of O3 on pollinators' physiology, despite that this pollutant, with its high oxidative potential, likely affects pollinators behaviors, especially the perception of signals they rely on to navigate their environment.

Volatile Organic Compounds (VOCs) released by plants are used as signals by different animals. For pollination services, VOCs attract different insects to the flowers and strengthen these interactions. Here, we used the honey bee Apis mellifera as a model to characterize the effects of acute exposure to different realistic mixing ratios of O3 (80-, 120-, and 200-ppb) on two crucial aspects: first, how exposed honey bees detect VOCs; and second, how O3 affects these pollinators' learning and memory processes. With electroantennogram (EAG) recordings, we showed that increasing O3 mixing ratios had a biphasic effect: an initial 25% decrease of the antennal activity when bees were tested directly after exposure (O3 direct effect), followed by a 25% increase in activity and response when bees were allowed a two-hour rest after exposure (O3 delayed effect). In parallel, during olfactory conditioning, increasing O3 mixing ratios in both exposure protocols scarcely affected olfactory learning, followed by a decrease in recall of learned odors and an increase of response to new odors, leading to a higher generalization rate (i.e., discrimination impairment). These results suggest a link between O3related oxidative stress and olfactory coding disturbance in the honey bee brain. If ozone affects the pollinators' olfaction, foraging behaviors may be modified, in addition with a possible longterm harmful effect on pollination services.

Introduction.

Plants release many Volatile Organic Compounds (VOCs) to attract pollinators and seed dispersers, in order to insure their reproduction [START_REF] Raguso | Start making scents: the challenge of integrating chemistry into pollination ecology[END_REF][START_REF] Muhlemann | Floral volatiles: from biosynthesis to function: Floral volatiles[END_REF][START_REF] Burkle | Floral volatiles structure plant-pollinator interactions in a diverse community across the growing season[END_REF]. These compounds spread with a concentration gradient, the higher the concentration the closer to the plant; this enables for instance the pollinators to locate the plant accurately, using floral VOCs as olfactory cues from both long and short distances [START_REF] Cardé | Navigational Strategies Used by Insects to Find Distant, Wind-Borne Sources of Odor[END_REF][START_REF] Riffell | Physical Processes and Real-Time Chemical Measurement of the Insect Olfactory Environment[END_REF][START_REF] Dötterl | The chemical ecology and evolution of bee-flower interactions: a review and perspectivesThe present review is one in the special series of reviews on animal-plant interactions[END_REF]. Thanks to the combination of the VOCs released, insect pollinators are able to extract information such as identifying the plant species, assessing the nectar availability and the quality of the resource [START_REF] Howell | Osmia bees (Hymenoptera: Megachilidae) can detect nectarrewarding flowers using olfactory cues[END_REF][START_REF] Wright | Reward quality influences the development of learned olfactory biases in honeybees[END_REF]). However, since the beginning of the industrial era, this chemical communication between plants and pollinators has been challenged by several factors [START_REF] Yuan | Smelling Global Climate Change: Mitigation of Function for Plant Volatile Organic Compounds[END_REF][START_REF] Jürgens | Changing odour landscapes: the effect of anthropogenic volatile pollutants on plant-pollinator olfactory communication[END_REF], affecting recognition of host plants by the insects, which in turn may affect the crucial pollination services provided by them.

Air pollution is one of the most impacting environmental hazards arising from human activities (W.H.O. Occupational & Environmental Health 2006): urbanization and industrialization lead to several environmental issues with negative impacts on climate and air quality [START_REF] Baklanov | Megacities, air quality and climate[END_REF]). Among the different pollutants in the atmosphere, the rates of tropospheric ozone (O3) started to increase since the pre-industrial era and will continue to do so for the next decades [START_REF] Vingarzan | A Review of Surface Ozone Background Levels and Trends[END_REF]). Tropospheric O3, through its high oxidative potential and the increased production of reactive oxygen species, can affect the balance between oxidative stress and anti-oxidant defenses; O3 has a direct influence on plant physiology, causing oxidative damages [START_REF] Iriti | Oxidative Stress, the Paradigm of Ozone Toxicity in Plants and Animals[END_REF][START_REF] Pinto | The Role of Ozone-reactive Compounds, Terpenes, and Green Leaf Volatiles (GLVs), in the Orientation of Cotesia plutellae[END_REF]. Further emission of VOCs by plants can be affected by O3 exposure [START_REF] Peñuelas | Effects of ozone concentrations on biogenic volatile organic compounds emission in the Mediterranean region[END_REF], Holopainen & Gerchenzon 2010), including floral compounds [START_REF] Saunier | The Effect of Elevated Ozone on Floral Chemistry of Brassicaceae Species[END_REF]. Additionally, O3 can directly react with VOCs released by these plants in the atmosphere [START_REF] Holopainen | Where do herbivore-induced plant volatiles go?[END_REF][START_REF] Blande | Plant volatiles in polluted atmospheres: stress responses and signal degradation: Plant volatiles in a polluted atmosphere[END_REF][START_REF] Dubuisson | Tropospheric ozone alters the chemical signal emitted by an emblematic plant of the Mediterranean region: the true lavender (Lavandula angustifolia Mill[END_REF]. Incidentally, O3 and other air pollutants will impact plant-pollinator communications [START_REF] Girling | Diesel exhaust rapidly degrades floral odours used by honeybees[END_REF][START_REF] Lusebrink | The Effects of Diesel Exhaust Pollution on Floral Volatiles and the Consequences for Honey Bee Olfaction[END_REF][START_REF] Farré-Armengol | Ozone degrades floral scent and reduces pollinator attraction to flowers[END_REF][START_REF] Fuentes | Air Pollutants Degrade Floral Scents and Increase Insect Foraging Times[END_REF][START_REF] Ryalls | Anthropogenic air pollutants reduce insect-mediated pollination services[END_REF]. While few studies reported the direct effects of air pollution on insect pollinators through carbon oxides, nitrous oxides, and diesel exhausts [START_REF] Leonard | Acute Exposure to Urban Air Pollution Impairs Olfactory Learning and Memory in Honeybees[END_REF][START_REF] Reitmayer | Acute exposure to diesel exhaust induces central nervous system stress and altered learning and memory in honey bees[END_REF], especially on their learning abilities, the effects of O3 are still less known and described.

One of the most studied pollinators, the honey bee Apis mellifera, is a polylectic pollinator, meaning the foragers do not have one specialized interaction with one plant, but instead visit different flowers and plant species in order to collect nectar and pollen [START_REF] Robertson | Heterotropic Bees[END_REF]; also, see definitions in the appendix of [START_REF] Müller | Pollen hosts of western palaearctic bees of the genus Colletes (Hymenoptera: Colletidae): the Asteraceae paradox[END_REF]. Honey bees are able to navigate their environment and successfully recognize and remember patches of resource-bearing flowers via visual and olfactory memory associations (Chittka & Raine 2006[START_REF] Dötterl | The chemical ecology and evolution of bee-flower interactions: a review and perspectivesThe present review is one in the special series of reviews on animal-plant interactions[END_REF]. In order to recognize them, bees need first to detect the signals released by plants; for olfactory signals, they can achieve that through VOCs detection and signaling: first, the VOCs are detected by the antennas, through olfactory receptor neurons [START_REF] Kaissling | Insect Olfaction[END_REF][START_REF] Jung | Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana[END_REF], then the olfactory message is mediated towards the honey bee brain; initially processed through the antennal lobes, then carried up to the mushroom bodies via projection neurons [START_REF] Strausfeld | Organization of the honey bee mushroom body: Representation of the calyx within the vertical and gamma lobes[END_REF][START_REF] Paoli | Olfactory coding in honeybees[END_REF]. The mushroom bodies, which receive information from all the sensory modalities, are the higher structures of the honey bee brain, and where the memory processes take place [START_REF] Dujardin | Mémoire sur le Système Nerveux des Insectes[END_REF][START_REF] Menzel | Cognitive architecture of a mini-brain: the honeybee[END_REF]. This whole progression describes the olfactory pathway, which is classically stimulated for the proboscis extension reflex (PER) conditioning through appetitive reward presentation [START_REF] Takeda | Classical conditioned response in the honey bee[END_REF][START_REF] Bitterman | Classical conditioning of proboscis extension in honeybees (Apis mellifera)[END_REF]. The association between olfaction and PER ultimately represents the basis for olfactory learning and memory in honey bees [START_REF] Menzel | Functional Organization of Appetitive Learning and Memory in a Generalist Pollinator, the Honey Bee[END_REF][START_REF] Menzel | Cognitive architecture of a mini-brain: the honeybee[END_REF].

The effect of oxidative stress on pollinators, and honey bees specifically, has been mostly studied under the scope of pesticide poisoning and subsequent detoxification processes (Kodrick et al. 2015[START_REF] Chaitanya | Oxidative Stress in Invertebrate Systems[END_REF]. While the effects of insecticides are largely reported on many aspects of learning and memory, very few looked at the effects of exogenous oxidative stress. [START_REF] Farooqui | Iron-induced oxidative stress modulates olfactory learning and memory in honeybees[END_REF] reported the effects of injections of ferrous ammonium citrate, source of oxidative stress, in the antennal lobes, and looked at the olfactory learning and recall.

Bees subjected to iron-induced oxidative stress had a consistent effect on recall of the learned association, and the acquisition was also affected [START_REF] Farooqui | Iron-induced oxidative stress modulates olfactory learning and memory in honeybees[END_REF]). In the same way, Leonard and colleagues (2019) measured the effects of carbon oxides, nitrous oxides and particle matter altogether, on olfactory learning and memory of honey bees. Pollution treatments were based on peak concentrations measured at city street levels. After a short exposure from low-to highconcentration treatments, bees exerted a lower learning rate than the control group and their olfactory memory was impaired [START_REF] Leonard | Acute Exposure to Urban Air Pollution Impairs Olfactory Learning and Memory in Honeybees[END_REF]. Moreover, Dötterl and colleagues (2016) reported the effect of O3 fumigation on the detection of floral VOCs by cut antenna of honey bee using electroantennography (EAG) recordings: O3 induced a decrease of the antennal activity when stimulated by VOCs [START_REF] Dötterl | Ozone Differentially Affects Perception of Plant Volatiles in Western Honey Bees[END_REF]. But the O3 mixing ratio used in this report (1000 ppb) was highly exceeding naturally-occurring mixing ratios [START_REF] Vautard | Simulation of ozone during the August 2003 heat wave and emission control scenarios[END_REF]).

More recently, [START_REF] Vanderplanck | Ozone Pollution Alters Olfaction and Behavior of Pollinators[END_REF] also reported the direct effects of O3 exposure, using field-relevant mixing ratios (from 80 to 200 ppb), on antennal activity and behavior of two insect pollinators: the fig wasp Blastophaga psenes and the bumble bee Bombus terristris. For the latter, increasing mixing ratios of O3 decreased the antennal activity and suppressed the innate attraction to natural VOCs for both pollinators [START_REF] Vanderplanck | Ozone Pollution Alters Olfaction and Behavior of Pollinators[END_REF]). To date, there are no reports on the effects of O3 exposure on olfactory learning and memory in honeybees, or any other insect.

In the present study, we tested the effect of exposing honeybees to naturally occurring mixing ratios of O3 [START_REF] Vautard | Simulation of ozone during the August 2003 heat wave and emission control scenarios[END_REF]) on both their ability to detect, learn, and recall floral VOCs.

To do so, in control conditions, honeybees were exposed to four different O3 mixing ratios during one hour; directly after exposure, learning and detection abilities were tested with two different synthetic VOCs mimicking floral scent, using respectively PER conditioning and EAG recordings. Based on the aforementioned literature results, we hypothesize that exposure to increasing mixing ratios of O3 will (1) decrease the learning rate of exposed bees and affect their memory, and (2) decrease the EAG activity of exposed antennas. We also tested the effect of a two-hour rest to assess O3 delayed effect: we investigated how honey bees may cope with the O3 exposure, and determined its effect on perception and detection as well. The effect of a potential recovery after an acute stress on insects has been mostly reported for cold/chill tolerance experiments, with various effects on metabolic rate and neuromuscular functions [START_REF] Lalouette | Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations[END_REF][START_REF] Macmillan | Cold-induced depolarization of insect muscle: differing roles of extracellular K+ during acute and chronic chilling[END_REF][START_REF] Overgaard | The integrative physiology of insect chill tolerance[END_REF]. But we did not find any evidence for a recovery after acute O3 stress. Hence, we can only hypothesize that the two-hour rest in absence of O3 exposure should reduce the decrease in olfactory learning and antennal activity, so the parameters measured should be similar to control groups.

Material and Methods.

Animal samplings

Honey bees (Apis mellifera) were collected from three different hives at the apiary located at the Terrain d'Expérience on the CNRS campus (43°38'19 N, 3°51'49 E), from the LabEx CeMEB platform (Centre Méditérranéen pour l'Environnement et la Biodiversité). We temporarily blocked the hives' entrance, and identified returning foragers thanks to their loaded pollen baskets. Adult bees were collected in small clear plastic tubes and placed shortly under ice (< 5 minutes, no direct contact). While asleep, bees were individually placed and attached to 3-cm high Teflon holders. Once awake, they were given sugar water (50% w/w sucrose) to recover from cold anesthesia, and placed to rest overnight in an incubator at 33.5°C ± 0.2°C / RH>65%. Experiments were conducted from April to September 2021.

Chemical compounds and preparations

We used synthetic versions of two extremely common floral scents [START_REF] Knudsen | Diversity and Distribution of Floral Scent[END_REF], i.e. R-linalool (97 % pure; CAS: 78-70-6), and eucalyptol (99 % pure; CAS: 470-82-6), used as VOCs for electrophysiological recordings (EAG) and associative conditionings (PER), at a unique concentration of 100 µg/µL. Depending on the experiment, different solvents were used: paraffin oil for EAG procedure, and 100% ethanol for PER procedure. For each procedure, a volume of 10 µL of VOC preparation was deposited on a single 20 × 10 mm Whatman no.1 piece of paper, placed in a Pasteur pipette for odor delivery. All compounds were purchased from Sigma Aldrich (St-Louis, MO, USA).

Ozone mixing ratios & exposures

For each procedure, O3 exposure was performed as previously described [START_REF] Vanderplanck | Ozone Pollution Alters Olfaction and Behavior of Pollinators[END_REF]. In short, bees fixed in holders were placed in a 500-mL glass bottle acting as a fumigation chamber, connected to an O3 analyzer-generator (Model 49i, Thermo Fischer, Franklin, MA, USA), with a pump pushing the O3 through Teflon tubes at a 1.5 L/min flow rate. Ozone was produced through oxygen photolysis subjected to UV radiation at 185-nm wavelength. By adjusting the solenoid valves opening level, we can produce fixed mixing ratios of O3: for both procedures, we tested four different mixing ratios: 0 ppb (control group), 80 ppb, 120 ppb, and 200 ppb, similar to those previously tested and reported [START_REF] Vanderplanck | Ozone Pollution Alters Olfaction and Behavior of Pollinators[END_REF]). These mixing ratios correspond to values commonly reported in the South of France: the mean value of O3 measured during a summer day (80 ppb), the peak value reached during this same summer day (120 ppb), and the highest O3 mixing ratio recorded in the South of France during the last 20 years (i.e. the heatwave in 2003) [START_REF] Vautard | Simulation of ozone during the August 2003 heat wave and emission control scenarios[END_REF]. To maintain the O3 at desired ratio and flow rate, air was extracted at the same 1.5 L/min flow rate, from the fumigation chamber toward the O3 analyzer. To keep the honey bees in a humid environment during exposure, a piece of Whatman paper was imbibed with distilled water and placed in the fumigation chamber.

Each treated group was exposed for one hour; from here, we either tested the exposed bees right away, to assess ozone direct effects (a), or placed them back in the incubator for a 2-hr rest before testing them, to assess ozone delayed effects (b). This paradigm serves a proxy to foraging behaviors where bees can be exposed to O3 pollution outside (a) and return to the colony to recover from possible oxidative stress (b).

Olfactory conditioning (PER)

Effects of O3 exposure on honey bees' learning ability were assessed through a classical olfactory conditioning using the proboscis extension reflex (PER; [START_REF] Bitterman | Classical conditioning of proboscis extension in honeybees (Apis mellifera)[END_REF]. Honey bees underwent an acquisition phase where they learned to associate a neutral odor (later acting as the conditioned stimulus, CS) with an appetitive reward eliciting the PER (acting as the unconditioned stimulus, US). One trial of training session consists of the CS presentation for 7 seconds, accompanied by the US presentation (a small drop of sucrose solution) for 5 seconds; both stimuli overlapped for 3 seconds. This trial was repeated five times, with a 1-minute interval (minimum) between trials. This acquisition phase results in long-term memory formation [START_REF] Gerber | Temporal Determinants of Olfactory Long-Term Retention in Honeybee Classical Conditioning: Nonmonotonous Effects of the Training Trial Interval[END_REF][START_REF] Démares | Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera[END_REF]. This memory can be tested afterwards with recall tests: it consists of the presentation of CS alone, to check if the bee remembers and recalls the reinforced association by exerting the PER. In parallel, we presented a new odor (NO) to check for generalization, and to test the specificity of the CS response. Similar to the training sessions, stimuli were randomly presented and separated by 1-minute interval, one hour after the last trial of the acquisition phase.

Ozone exposures, training sessions and memory tests were performed between late morning and mid-afternoon (from 09.00AM to 04.00PM). Bees were fed briefly in the early morning and late-afternoon (08.00AM and 06.00PM): this allowed to check for normal PER to sucrose feeding. Bees not responding to sucrose were discarded from the experiments. Bees responding to the CS odor at the first acquisition trial were also discarded from analysis. The odors used as CS were the aforementioned VOCs, R-linalool and eucalyptol, equally randomized within treated groups. When one VOC was used as CS, the other was used as NO. In total, between all protocols, O3 mixing ratios, and VOC tested as CS (N=16 groups), we used 308 adult honey bees for this section (ranging from 18 to 22 bees per group).

Electroantennography (EAG)

EAG recordings allow assessment of VOCs detection from individualized antenna. Specifically, it measures the amplitude of depolarization of all active olfactory sensory neurons in response to an olfactory stimulus [START_REF] Roelofs | Electroantennogram Assays: Rapid and Convenient Screening Procedures for Pheromones[END_REF]. Here, we measured how O3 exposure might affect such amplitude in response to either R-linalool or eucalyptol stimulation. After O3 exposures, the right antenna of individual exposed honey bees was cut and mounted between glass capillaries, filled with Ringer's solution (composition, in mM: NaCl, 131; KCl, 5; CaCl2, 2; NaHCO3, 29; pH 7). Those were connected to silver electrodes of an EAG probe. A continuous humidified air flow (450 mL/min), purified by activated carbon, was blown at the antenna through a circulating tube to keep it from drying. Single VOCs were applied to a Whatman no.1 paper placed in a Pasteur pipette; the tip of this pipette was inserted in a hole in the circulating airflow tube. A short pulse of purified air (0.5 second, 900 mL/min) through the Pasteur pipette released the VOC in the airflow leading to the antenna. The continuous air flow and the pulsed air stimulation are both set and delivered by the stimulus controller (CS-55), recorded by an acquisition controller (IDAC-2), and analyzed with the GcEad 1.2.5 software (all EAG equipment purchased from Ockenfels SYNTECH Buchenbach, Germany). The maximum amplitude of depolarization was recorded for each stimulus. Intervals between two stimulations were at least 45 seconds (up to one minute), in order for the antennal activity to return to a stable baseline. Every antenna went through a balanced randomized sequence of VOC stimulations, with paraffin controls always coming first and last of this sequence. The response amplitude of each VOC was adjusted with subtracted mean paraffin-response from their own sequence. In total, with all the exposure protocols and O3 mixing ratios (N= 8 groups), we tested 208 antennae, each tested for both VOCs (n=26 for each group).

Statistical analyses

To assess honey bees' performance in learning, we performed generalized linear mixed models (GLMM) with a binomial distribution on the PER response of honey bees (binary data) with 'O3 mixing ratio' and 'acquisition trials' as fixed factors, along with their interactions. Bee individual was implemented as a random factor in the model. As reported in Tables 1&2,each GLMM resulted in F-values as statistical indicators related to degrees of freedom, which enabled us to calculate a p-value for each factor and interaction. Between the two VOCs used as CS and the two exposure protocols, we performed four GLMMs for each combination of CS & exposure protocol. Memory recall tests were also analyzed using GLMM with 'O3 mixing ratio' and 'odor tested' (CS vs NO) as fixed factors, and 'bee individual' as random factor. In GLMM pairwise comparisons between all groups were conducted using contrasts, and p-values were adjusted with False Discovery Rate (FDR) corrections. In addition to memory recall test analyses, we assessed the distribution of response categories at one-hour time point. Responses to memory tests fell into four distinct categories: PER to CS only ('CS + '), response to new odor only ('CS -/NO + '), response to both CS and NO ('All Odors'), and response to none ('no PER').

For each condition, all bees were categorized this way and the overall frequency distribution of the four categories was compared with the response distribution of the control group using Pearson's chi-square tests (as described in [START_REF] Urlacher | C-type allatostatins mimic stress-related effects of alarm pheromone on honey bee learning and memory recall[END_REF]. For post-hoc comparisons against control group, subsequent chi-square tests were done and adjusted with the FDR method.

For EAG recordings, we focused on the effect of O3 mixing ratio on EAG response amplitude, depending on the VOC tested for stimulation (eucalyptol & R-linalol) and on the exposure protocol (i.e. direct & delayed effect): we performed one-way ANOVAs with 'O3 mixing ratio' as factors, followed by post-hoc contrasts corrected with the FDR method. All statistical analyses were conducted using IBM SPSS 23 and GraphPad Prism 7. All data used for each figure are reported in Supplementary Table 1. All statistical analysis results with complete numbers are reported in Supplementary Table 2.

Results.

Ozone alters VOC perception

3.1.1. Effect on the responses to reinforced VOC

Effect on learning

For each O3 exposure protocol and each VOC used as conditioned stimulus (CS) for the association with sugar reward, acquisition is achieved (Figs 1 &2): the olfactory learning rate is significantly increasing with each trial increment, for both eucalyptol and R-linalool (GLMM 'acquisition trial' factor; Table 1, O3 direct effect, p < 0.001, see also Fig. 1A; O3 delayed effect, p < 0.001, see also Fig. 1D; Table 2, O3 direct effect, p < 0.001, see also Fig. 2A; O3 delayed effect, p < 0.001, see also Fig. 2D). Also, O3 mixing ratios affect the level of PER response rate in each condition (Table 1, GLMM 'O3 mixing ratio' factor; Eucalyptol, O3 direct effect, p = 0.001; O3 delayed effect, p = 0.038; Table 2, R-linalool, O3 delayed effect; p = 0.023). In all these conditions, bees exposed to 80 ppb of O3 have a significantly lower acquisition rate than control bees, especially when eucalyptol is used as CS (see Figs 1A&D and Fig. 2D; for complete data, see Supplementary Table 1). There is only one condition where the O3 does not significantly affect the acquisition rate, when R-linalool is used as CS and bees are tested right away after exposure (Table 2, GLMM 'O3 mixing ratio' factor, O3 direct effect, p = 0.121; also see Fig. 2A), although the tendency follows the same pattern, i.e., a lower learning rate for 80ppb O3-exposed bees compared to control bees. Interestingly, in almost every condition, the learning rate of honey bees exposed to 200 ppb of O3 does not significantly differ from the one of control bees nor bees exposed to 80 ppb and 120 ppb, independent on VOC or exposure protocol (except for eucalyptol used as CS under O3 direct effect; see Supplementary Table 2A). In summary, O3 does not affect the ability to acquire the association, but limits the PER rate of exposed bees: olfactory learning is less efficient after O3 exposure.

Effect on recall

One hour after the last acquisition trial, recall trials are performed; the CS presented alone is tested along with a new odor (NO) to test for olfactory memory specificity. For each O3 exposure protocol and each VOC used as CS, independently of O3 mixing ratios, the global PER response rate to CS is always significantly higher than the response rate to the NO (GLMM 'odor tested' factor; Table 1, O3 direct effect, p < 0.001, see also Fig 1B ; O3 delayed effect, p = 0.003, see also Fig. 1E; Table 2, O3 direct effect, p = 0.002, see also Fig 2B ; O3 delayed effect, p = 0.009, see also Fig 2E;). The O3 mixing ratios only impact significantly the CS recall of bees trained when eucalyptol is used as CS, right after O3 exposure (GLMM 'O3 mixing ratio' factor; Table 1, O3 direct effect, p = 0.006, see also Fig. 1B). Contrast comparisons show that the group exposed to 80 ppb of O3 has a significantly lower PER rate to CS compared to the control group, 15.8% vs 78.9% respectively (GLMM post-hoc CS recall comparisons; 80 ppb vs control, p < 0.001). In short, depending on the nature of the VOC, O3 affects the CS recall or not (eucalyptol vs R-linalool, see Figs 1B/1E & 2B/2E, respectively).

Effect on new odors presentation

While the specific response to CS is altered, we also observed an increased response rate to the new odor presentation. More specifically, under O3 direct effect, with eucalyptol used as CS, bees exposed to 200 ppb of O3 have a significantly higher response rate than bees exposed to control bees (GLMM post-hoc NO response comparisons; 200 ppb vs control, p = 0.006). For this specific condition, the interaction 'O3 mixing ratio * odor tested' was close to significance (Table 1, p = 0.095), meaning the effect of O3 can depend on the type of test performed (here, CS vs NO). But it was the only instance where it was this close, all the other interactions being not significant for the other conditions (Tables 1&2).

Moreover, as all the 'odor tested' factors were statistically significant in the memory analyses, this meant that the overall responses to CS were higher than the overall responses to NO. As a follow-up, we performed post-hoc contrasts to test which specific groups showed significant difference between CS and NO responses. As a result, only the control groups showed ). Also, the difference between the PER rates to CS and NO for the control groups is around 55% in average (ranging from 44.4 % to 78.9 % difference), while for the O3-exposed groups the CS-NO difference is around 17.5% (ranging from 5.3% to 31.6% difference). Given all these points, this shows that O3 decreases the difference between learned odor and new odor response rates.

These observations were corroborated with the frequency analysis of the distribution of PER response categories. In two conditions, we observe significant global differences in honey bee response's distributions. The first condition is when eucalyptol is used as CS, under O3 direct effect exposure protocol (Fig. 1C; Pearson χ², p < 0.001). Pairwise comparisons against control group reveal different observations: each O3-exposed group significantly differs in response distribution compared to control (Fig. 1C). Distribution of PER responses in the control group was 78.9% of response to the learned odor (CS + category) and 21.1% of no response to any odor ('no PER' category). For the O3-exposed groups, there are two types of difference from control distribution: for the 80-ppb group, a decrease of responses to CS alone (15.8% CS response rate) with an increase of no responses (84.2% 'no PER' response rate) (χ², p < 0.001) resulting in an opposite shift in distribution compared to control; while for the 120-ppb and 200-ppb groups, again a decrease of responses to CS alone (25.0% and 31.6% respectively) but here associated with an increase of all responses (20.0% and 36.8% respectively), not observed in control group, when eucalyptol is used as CS (120-ppb: χ², p = 0.003; 200-ppb: χ², p = 0.004).

The second condition is when R-linalool is used as CS, under O3 delayed effect exposure protocol (Fig. 2F; Pearson χ², p = 0.041). Distribution of PER responses in the control group was, in order, 50.0% of response to CS alone, 13.6% to both CS and NO ('All odors' category), and 36.4% of no response to any odor ('no PER' category). Here, pairwise comparisons against control group reveal only one O3-exposed group differing significantly from control group in terms of response distribution: the 80-ppb group has fewer response to CS alone (5.3%) and an increase of responses to all odors (63.2%) (Fig. 2F; 80-ppb: χ², p = 0.020). For the groups exposed to higher mixing ratios of O3, although a few bees appear to respond to the NO only (CS -/NO + category), which is never observed in the control group, their overall response distributions are not statistically different from the control group (120-ppb: χ², p = 0.137; 200ppb: χ², p = 0.249). For the other two conditions, the same pattern 'decreased response to CS alone with increased response to all odors' was consistent with O3 mixing ratios but the response distributions were not statistically different from their respective control groups (Pearson χ²; CS: eucalyptol, O3 delayed effect, p = 0.198; CS: R-linalool, O3 direct effect, p = 0.505; see Supplementary Table 2C).

Ozone alters VOC detection

The electrophysiological recordings of antennal activity reveal significant effects of O3 exposure in three conditions over four, either decreasing or increasing compared to control, depending on the exposure protocol (Table 3; VOC: eucalyptol, O3 direct effect, p < 0.001; VOC: R-linalool, O3 direct effect, p < 0.001; VOC: R-linalool, O3 delayed effect, p = 0.049).

When testing the effect of O3 treatment directly after exposure, and stimulated with eucalyptol, we measured a significant decrease of EAG activity for 80-ppb and 200-ppb treated bees (0.72 mV and 0.73 mV, respectively) compared to control bees (0.98 mV amplitude) (Fig. 3A; VOC: eucalyptol; control vs 80 ppb, p = 0.002; control vs 200 ppb, p = 0.002). We measured the same significant decrease with R-linalool stimulation, where EAG activity for 80-ppb and 200-ppb treated bees (1.97 mV and 2.39 mV, respectively) was lower than the one of control bees (2.85 mV) (Fig. 3C; VOC: R-linalool; control vs 80 ppb, p < 0.001; control vs 200 ppb, p = 0.022).

In contrast, the delayed effect of O3 increases the EAG activity of treated bees compared to control, but only significantly for antennas tested with R-linalool: the higher the O3 mixing ratio, the higher the amplitude of the recorded activity. In this case, the bees treated with 120 ppb of O3 show a tendency of increased activity (3.65 mV), while bees treated with 200 ppb of O3 display a significant increase in amplitude (3.75 mV) compared to control (2.93 mV) (Fig. 3D; control vs 120 ppb, p = 0.071; control vs 200 ppb, p = 0.039). Finally, the only condition where O3 does not statistically affect the EAG response amplitude of the treated bees is when we stimulated the antenna with eucalyptol after the two-hour rest (Table 3; O3 delayed effect, p = 0.430; see Fig. 3 and Supplementary Table 2D). While R-linalool and eucalyptol display the same pattern of response within the same exposure protocol, the biological effect of the increased EAG activity with the increase in O3 mixing ratios is only statistically significant with R-linalool (Figs 3B & 3D). We hypothesize that this is due to the difference in absolute amplitude between R-linalool stimulations and eucalyptol stimulations, around 3 mV and 1 mV, respectively. Overall, these EAG findings highlight the fact that the effects of O3 on antennae depend more on physiological dynamics (i.e. direct vs delayed) rather than VOC nature.

Discussion / Conclusion.

In this study, under controlled conditions, we reported for the first time the effects of O3 exposures on honey bees' olfactory learning and memory, along with its impacts on antennal activity and responses. We tested the olfactory modality by using common VOCs found in flowers, R-linalool and eucalyptol [START_REF] Knudsen | Diversity and Distribution of Floral Scent[END_REF]. We found that O3 affects both detection at the antenna level (Fig. 3) and perception at a more global level (Figs 1&2).

Respectively, these effects of O3 strongly depend on the time elapsed since O3 exposure on one hand, and the VOCs tested on the other hand.

Ozone affects olfactory conditioning and memory recall of VOCs

Ozone exposure had a significant effect on olfactory conditioning, during acquisition (olfactory learning) and recall (olfactory memory), which is in line with previous results: a decrease in olfactory learning and CS recall due to iron-induced oxidative stress [START_REF] Farooqui | Iron-induced oxidative stress modulates olfactory learning and memory in honeybees[END_REF]). Right after exposure, for O3 direct effect, the mixing ratio of 80 ppb of O3 significantly decreased the acquisition rate and the recall rate of eucalyptol used as CS (60 % difference compared to control, Figs 1A &1B). For the delayed effect, this decrease after 80 ppb of O3 was still observable on both acquisition and recall of eucalyptol as CS (respectively 50 % and 30% difference compared to control, Figs 1D &1E). For olfactory conditioning using R-linalool as CS, the acquisition rate right after O3 exposure was not significantly impaired, but slightly decreased for the O3-exposed groups (around 25% difference compared to control for 80-ppb and 120-ppb groups, Fig. 2A), and the recall was not significantly affected (less than 20% difference with control group response rate, Fig. 2B). For the O3 delayed effect, the acquisition rate of R-linalool used as CS was significantly decreased for groups exposed to 80 ppb & 120 ppb, compared to control group (30% difference, Fig. 2D). The recall rate was decreased for these two groups as well, compared to control, but not significantly (25% difference, Fig. 2E).

Overall, the CS acquisition rate and recall rate were mostly impacted by the lower mixing ratio tested (80 ppb), and less by the other mixing ratios, independent of the exposure protocols used.

On one hand, the specific response to the learned VOC during recall was decreased; on the other hand, the response to a new odor presented during recall was increasing with the increase of O3 mixing ratios (Figs 1&2). There was a significantly higher response to the new odor when eucalyptol was used as CS, testing O3 direct effect, in parallel with a statistically different distribution of PER response types between control bees and 200-ppb exposed bees (Fig. 1C).

These results strongly indicate a generalization of the new odor response to the learned odor (CS response). This is also underlined by the limited difference between CS-NO response rates for O3-exposed groups (17.5% average difference) compared to control (55% average difference). All in all, this means that the PER responses are less specific to the only reinforced VOC. While responses to new odor always increase with increasing mixing ratios of O3, it is only statistically different with eucalyptol as CS, testing O3 direct effect, suggesting the generalization might be dependent on VOCs used as CS and on exposure protocols.

The decrease of CS recall in parallel with the increased responses to new odor is reported for the first time for the effect of O3 on the honey bee's olfactory memory. But a similar effect has been reported when measuring the impacts of certain pesticides on honey bee behavior, e.g. the effect of the phenylpyrazole fipronil [START_REF] Hassani | Glutamatergic and GABAergic effects of fipronil on olfactory learning and memory in the honeybee[END_REF]), but also when inhibiting the glutamate chloride (GluCl) neurotransmission [START_REF] Boumghar | Evidence for a role of GABA-and glutamate-gated chloride channels in olfactory memory[END_REF][START_REF] Démares | Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera[END_REF].

Fipronil targets the GABA chloride channels and the GluCl channels [START_REF] Cole | Action of Phenylpyrazole Insecticides at the GABA-Gated Chloride Channel[END_REF][START_REF] Barbara | Acetylcholine, GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera[END_REF][START_REF] Janssen | Dorsal Unpaired Median Neurons of Locusta migratoria Express Ivermectin-and Fipronil-Sensitive Glutamate-Gated Chloride Channels[END_REF][START_REF] Wolstenholme | Glutamate-gated Chloride Channels[END_REF], which are expressed in insect interneurons in all areas of the honey bee brain, and are involved in inhibitory neurotransmission [START_REF] Bicker | Histochemistry of classical neurotransmitters in antennal lobes and mushroom bodies of the honeybee[END_REF][START_REF] Bicker | Histochemistry of classical neurotransmitters in antennal lobes and mushroom bodies of the honeybee[END_REF][START_REF] Démares | Expression and localization of glutamategated chloride channel variants in honeybee brain (Apis mellifera)[END_REF]. At sublethal doses, the oxidative damages of fipronil have been observed at the enzymatic level on different species including honey bees: more specifically, fipronil modulates the activity of catalase, alkaline phosphatases and carboxylesterases (Carvalho et al. 2013; see [START_REF] Wang | Fipronil insecticide toxicology: oxidative stress and metabolism[END_REF] for review) thus we hypothesize a possible correlation between oxidative stress, interneurons functions, and olfactory memory.

This was not the aim of that study, but future studies will enable us to provide a basis for this claim; for instance, by studying the effects of antioxidant treatments on olfactory recall of honey bees undergoing oxidative stress. Interneurons are also present in the optic lobes and other brain areas of the honey bee [START_REF] Strausfeld | Organization of the honey bee mushroom body: Representation of the calyx within the vertical and gamma lobes[END_REF][START_REF] Démares | Expression and localization of glutamategated chloride channel variants in honeybee brain (Apis mellifera)[END_REF]). Since O3 exposure may affect the neurotransmission in interneurons, we can suspect effects on the visual system as well. Here we report O3 effects at a global physiological level, encompassing learning, memory, olfaction, and potential metabolic effects due to oxidative stress: we cannot dissociate them only through a behavioral basis, even though odor generalization potentially suggests an effect at the central level. In order to begin to disentangle each effect, we measured and reported effects at the peripheral level on the antennas.

Ozone affects olfactory detection of VOCs at the antennal level

Ozone exposure had a biphasic effect on the antennal responses to VOCs stimulation, depending on the exposure protocol. With the first protocol, the direct effect of O3 decreased the amplitude of VOC-evoked antennal activity, for both eucalyptol and R-linalool (Fig. 3A&3C). Surprisingly, the antennal responses were statistically lower than control with the 80-ppb and 200-ppb O3 mixing ratios, but not with 120 ppb, which was not different from any other group. While the global effect is in line with previous results reported on bumble bees [START_REF] Vanderplanck | Ozone Pollution Alters Olfaction and Behavior of Pollinators[END_REF], we can only hypothesize that this effect of slight rebound with 120ppb O3 direct exposure is either species-specific or protocol-dependent. When tested with the second protocol, the O3 delayed effect increased the amplitude of antennal responses with increasing mixing ratios for both eucalyptol and R-linalool, the latter being statistically different from the control group responses (Fig. 3D). We hypothesize that this is due to the difference in absolute amplitude between R-linalool stimulations and eucalyptol stimulations, around 3 mV and 1 mV, respectively. Overall, these EAG findings highlight the fact that the effects of O3 on antennae depend more on physiological dynamics (i.e. direct vs delayed) rather than VOC nature. This delayed effect operated a reversal from decreased response to increased response.

We hypothesize that this biphasic effect could be due to two elements: a direct mechanistic effect of O3 on the antenna decreasing its activity, and a delayed activation threshold acting more globally on the whole bee physiology and counteracting O3-induced damages, eventually leading to increased antennal activity. It is possible that the activation threshold might be related to anti-oxidant pathways, which we will test in future works. Additionally, since we observed O3 direct effects starting at 80 ppb on perception and detection, we will also need to test lower mixing ratios to test the sensitivity thresholds from which the honey bees show behavioral and electrophysiological changes, from 'no observable adverse effect' to 'lowest observable adverse effect level'.

Ozone potentially disrupts the link between detection and perception

The observable effects between exposure protocols, O3 direct effect vs. delayed effect, independent of VOCs used CS, are: (1) a decrease of PER acquisition rate; (2) a decrease of response to CS and a slight increase of response to all odors, on the PER response distribution; and not least (3), an increase of the antennal activity, with the response amplitudes increasing with increasing O3 mixing ratios (Fig. 3). While the direct effect of O3 decreases the antennal activity and can affect the learning rate of eucalyptol as CS, the delayed effect of O3 seems to correlate with both increased antennal activity and increased generalization response: indeed, when exposed to O3 and conditioned right away with associative learning, exposed bees are tested one hour after for recall, which can relate to a transition from direct to delayed O3 effect (Fig. 3). While the delayed effect of O3 correlates with an increased generalization, we are aware that we cannot properly conclude about discrimination between CS and NO responses: a discriminative CS + /CS -associative learning would have been more appropriate [START_REF] Sandoz | Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee ( Apis mellifera L.)[END_REF]. Nonetheless, this simple conditioning already offers a good basis for future studies on the effects of O3 on learning and memory.

This report shows a clear effect of O3 on detection and perception of VOCs by honey bees. The surprising aspect of the O3 direct effect is the asymmetrical effect between eucalyptol and Rlinalool. This can be interpreted as a subtle effect on olfactory coding, at the antennal lobe level [START_REF] Sachse | The spatial representation of chemical structures in the antennal lobe of honeybees: steps towards the olfactory code: Glomerular representation of chemical structures[END_REF][START_REF] Paoli | Olfactory coding in honeybees[END_REF]. Every VOC detected at the antennal level activates a different set of olfactory receptor neurons, which in turn will activate specific sets of glomeruli [START_REF] Deisig | Neural representation of olfactory mixtures in the honeybee antennal lobe[END_REF]: each VOC has its own activation pattern at the antennal lobe level. The biphasic effect of O3 (direct and delayed) on the antennal activity can be transferred onto the glomeruli activity. The generalization of odors is asymmetrical, meaning that responding to odor A while being conditioned to odor B is not equal to responding to odor B while being conditioned to odor A [START_REF] Sandoz | Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee ( Apis mellifera L.)[END_REF][START_REF] Paoli | Olfactory coding in honeybees[END_REF]. Hence, we can underline an asymmetrical effect of generalization between eucalyptol and R-linalool, which could be related to an O3-altered glomerular pattern activation during acquisition. Exposed honey bees can learn, even an altered odor, but the least resilient pattern (the most sensitive to O3 alteration) will be generalized during memory recall.

The reported generalization effect of O3 on olfaction, coupled with a possible effect on vision of the forager honey bee, can impact foraging activity and success, as this has been reported with the effects of pesticides [START_REF] Weick | Effects of Acute Sublethal Exposure to Coumaphos or Diazinon on Acquisition and Discrimination of Odor Stimuli in the Honey Bee (Hymenoptera: Apidae)[END_REF][START_REF] Decourtye | Effects of imidacloprid and deltamethrin on associative learning in honeybees under semi-field and laboratory conditions[END_REF][START_REF] Mustard | Honeybees fail to discriminate floral scents in a complex learning task after consuming a neonicotinoid pesticide[END_REF].

Since foragers are the honey bees that will be the most likely exposed to tropospheric O3, two directions can stem from this: (1) we need to characterize how forager honey bees are resilient to oxidative stress and air pollution; and (2) other castes of the honey bee colonies, living inside hives, may be much less exposed to ozone. Interestingly, while 500-ppm O3, up to 1000 ppm, was suggested as a viable inside-hive treatment to control pests and decrease pesticides residues [START_REF] James | Potential of Ozone as a Fumigant to Control Pests in Honey Bee (Hymenoptera: Apidae) Hives[END_REF], the naturally-occurring mixing ratio of O3 inside hives has never been tested nor reported yet. Forager bees will rely on multiple signals and cues, not only olfactory ones such as VOCs. While the effects of O3 on plants and VOCs can lead to disturbed emissions and alter the integrity of the chemical signal [START_REF] Peñuelas | BVOCs and global change[END_REF][START_REF] Jürgens | Changing odour landscapes: the effect of anthropogenic volatile pollutants on plant-pollinator olfactory communication[END_REF][START_REF] Dubuisson | Tropospheric ozone alters the chemical signal emitted by an emblematic plant of the Mediterranean region: the true lavender (Lavandula angustifolia Mill[END_REF], bees may still be able to forage relying on different salient cues such as shapes and colors for navigation, flower approach and recognition (Chittka & [START_REF] Chaitanya | Oxidative Stress in Invertebrate Systems[END_REF][START_REF] Raine | The Adaptive Significance of Sensory Bias in a Foraging Context: Floral Colour Preferences in the Bumblebee Bombus terrestris[END_REF][START_REF] Dötterl | The chemical ecology and evolution of bee-flower interactions: a review and perspectivesThe present review is one in the special series of reviews on animal-plant interactions[END_REF][START_REF] Blande | Effects of air pollution on plant-insect interactions mediated by olfactory and visual cues[END_REF]. The association between floral bouquet and nectar/pollen reward can be affected as the O3 may level all olfactory cues. From the forager standpoint, the olfactory landscape under tropospheric O3 exposure can appear "flatten", that is to say, less salient and less heterogenous [START_REF] Jürgens | Changing odour landscapes: the effect of anthropogenic volatile pollutants on plant-pollinator olfactory communication[END_REF][START_REF] Conchou | Insect Odorscapes: From Plant Volatiles to Natural Olfactory Scenes[END_REF]. To put it another way, every odor will smell the same for the foragers affected by O3. Although this situation should not prevent honey bees from foraging, they might do so less efficiently.

In this changing environment, the resilience to oxidative stress from air pollution may play a key role in the adaptation of pollinators' behaviors. While different sensory modalities may be affected, such as olfaction -as reported in this study-and perhaps vision as well, pollinators will either rely on different cues, reinforcing the prevalence of one unaffected modality, or will adapt to an equally changing environment [START_REF] Leonard | Acute Exposure to Urban Air Pollution Impairs Olfactory Learning and Memory in Honeybees[END_REF][START_REF] Blande | Effects of air pollution on plant-insect interactions mediated by olfactory and visual cues[END_REF]. The pollination services provided by pollinators are already affected by different factors [START_REF] Goulson | Bee declines driven by combined stress from parasites, pesticides, and lack of flowers[END_REF][START_REF] Potts | Safeguarding pollinators and their values to human well-being[END_REF]. Now, the follow-up questions are: how fast does the pollution arising from human activities affect the environment? And from that, how resilient the pollinators can be, to continue providing such services? Ryalls and colleagues (2022) recently provided insights for the latter, reporting a striking decrease of in-field pollinator counts and flower visits linked to air pollution from ozone and diesel exhaust. This might reflect the effects of air pollution on pollinators' olfaction and vision, or more broadly the ability to navigate their environment. Air pollution affects the in-field presence of the main groups of pollinators [START_REF] Ryalls | Anthropogenic air pollutants reduce insect-mediated pollination services[END_REF], potentially leading to harmful effects to pollination services. Table 1. Results of the GLMMs with binomial distribution, performed on acquisition and recall phases of PER conditioning with Eucalyptol used as CS, and separated by exposure protocols (direct vs delayed effect). For the acquisition phase, 'O3 mixing ratio' and 'Acquisition trial' were used as GLMM factors, along with their interaction 'Mix ratio * Trial'. For the recall phase, 'O3 mixing ratio' and 'Odor tested' were used as GLMM factors, along with their interaction 'Mix ratio * Odor'. F-values correspond to statistical indicators related to inter-and intra-group degrees of freedom (df), giving a calculated p-value for each factor and interaction. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, ** < 0.010, *** < 0.005, ns > 0.050), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure 1.

Contributions

Table 2. Results of the GLMMs with binomial distribution, performed on acquisition and recall phases of PER conditioning with R-linalool used as CS, and separated by exposure protocols (direct vs delayed effect). For the acquisition phase, 'O3 mixing ratio' and 'Acquisition trial' were used as GLMM factors, along with their interaction 'Mix ratio * Trial'. For the recall phase, 'O3 mixing ratio' and 'Odor tested' were used as GLMM factors, along with their interaction 'Mix ratio * Odor'. F-values correspond to statistical indicators related to inter-and intra-group degrees of freedom (df), giving a calculated p-value for each factor and interaction. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, ** < 0.010, *** < 0.005), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure 2. Table 1. Results of the GLMMs with binomial distribution, performed on acquisition and recall phases of PER conditioning with Eucalyptol used as CS, and separated by exposure protocols (direct vs delayed effect). For the acquisition phase, 'O3 mixing ratio' and 'Acquisition trial' were used as GLMM factors, along with their interaction 'Mix ratio * Trial'. For the recall phase, 'O3 mixing ratio' and 'Odor tested' were used as GLMM factors, along with their interaction 'Mix ratio * Odor'. F-values correspond to statistical indicators related to inter-and intra-group degrees of freedom (df), giving a calculated p-value for each factor and interaction. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, ** < 0.010, *** < 0.005, ns > 0.050), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure 1. 2. Results of the GLMMs with binomial distribution, performed on acquisition and recall phases of PER conditioning with R-linalool used as CS, and separated by exposure protocols (direct vs delayed effect). For the acquisition phase, 'O3 mixing ratio' and 'Acquisition trial' were used as GLMM factors, along with their interaction 'Mix ratio * Trial'. For the recall phase, 'O3 mixing ratio' and 'Odor tested' were used as GLMM factors, along with their interaction 'Mix ratio * Odor'. F-values correspond to statistical indicators related to inter-and intra-group degrees of freedom (df), giving a calculated p-value for each factor and interaction. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, ** < 0.010, *** < 0.005), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure 2.

Recall tests Ozone mixing ratios

Eucalyptol as CS

R-

  statistically higher CS response compared to NO responses (Fig. 1B, O3 direct effect, CS = eucalyptol, CS vs NO rate, p < 0.001; Fig. 1E, O3 delayed effect, CS = eucalyptol, CS vs NO rate, p = 0.001; Fig. 2B, O3 direct effect, CS = R-linalool, CS vs NO rate, p = 0.002; Fig. 2E, O3 delayed effect, CS = R-linalool, CS vs NO rate, p < 0.001). All the O3-exposed groups did not have statistically different response rates between CS and NO (see Supplementary Table

2B

,Figure

  Figure 1. Olfactory learning and recall tests of O 3 -exposed bees, with eucalyptol used as

Figure 2 .

 2 Figure 2. Olfactory learning and recall tests of O 3 -exposed bees, with R-linalool used as

Figure 3 .

 3 Figure 3. Electroantennographic recordings of O 3 -exposed bees, with eucalyptol stimulations (A-

  Figure 1.

  Figure 3.

Table 3 . Results of ANOVAs performed on EAG antennal activity responses following eucalyptol or R-linalool stimulations.

 3 For each VOC tested, the 'O3 mixing ratio' factor was tested. Each ANOVA returned a statistical indicator (F-value) related to inter-and intra-group degrees of freedom (df), giving a calculated p-value. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, *** < 0.005), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure3.

	Linalool as CS		Acquisition (Learning Phase)		Recall (Memory Phase)
	O3 Exposure	GLMM factors	F value	df	p-value	GLMM factors	F value	df	p-value
	One hour	O3 Mixing Ratio	1.951	3.360	0.121 ns	O3 Mixing Ratio	1,323	3,144	0.269 ns
	(direct effect)	Acquisition Trial	10.038	4,360	< 0.001 ***	Odor tested	10,282	1,144	0.002 ***
		Mix Ratio * Trial	0.174	12,360	0.999 ns	Mix Ratio * Odor	0,924	3,144	0.431 ns
	One hour	O3 Mixing Ratio	3.225	3,370	0.023 *	O3 Mixing Ratio	0,869	3,148	0.459 ns
	+ 2h rest	Acquisition Trial	11.670	4,370	< 0.001 ***	Odor tested	6,997	1,148	0.009 **
	(delayed effect)	Mix Ratio * Trial	0.213	12,370	0.998 ns	Mix Ratio * Odor	1,870	3,148	0.137 ns

Olfactory Learning Olfactory Memory & Discrimination T1 PER rate 100% 0% 50% 50% 100% Learned VOC New Odor PER rate PER rate

International Research Project (IRP)-CNRS-MOST and National Research Agency (ANR) under the program "Investissements d'Avenir" (ANR-16-IDEX-0006).

Table 3. Results of ANOVAs performed on EAG antennal activity responses following eucalyptol

or R-linalool stimulations. For each VOC tested, the 'O3 mixing ratio' factor was tested. Each ANOVA returned a statistical indicator (F-value) related to inter-and intra-group degrees of freedom (df), giving a calculated p-value. P-values lower than the 0.05 alpha-level are statistically significant and indicated by the following symbols (* ≤ 0.050, *** < 0.005), while values higher than 0.05 are not statistically significant (ns > 0.050). This table is related to results reported in Figure 3.

Supplementary Information

Supplementary Table 1

Supplementary Table 2

Complete statistical numbers for GLMM on acquisition (sheet A) and on recall (sheet B), for PER response distribution (sheet C), and for EAG analyses (sheet D).