Hanine Almasri

Antonia Daiana

Maryline Tavares

Deborah Pioz

Sylvie Sene

Marianne Tchamitchian

Jean-Luc Cousin

Luc P Brunet

Belzunces
email: luc.belzunces@inrae.fr

Sylvie Sené

Luc P Belzunces Inrae

Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic physiological disturbances in winter Apis mellifera honey bees

Keywords: winter honey bee, pesticide mixtures, synergy, cocktail effects, physiological state

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite the 45% global increase in managed honey bee colonies since 1961 [START_REF] Aizen | The global stock of domesticated honey bees is growing slower than agricultural demand for pollination[END_REF][START_REF] Faostat | The longevity of worker honey bees (Apis mellifera)[END_REF], regional colony losses have been reported in different areas, such as the United States of America (USA) and Europe. In the USA, 31.3% of colonies were lost between 2007 and 2008, while in central Europe, a significant decrease of 25% took place between 1985 and 2005 [START_REF] Potts | Declines of managed honey bees and beekeepers in Europe[END_REF][START_REF] Vanengelsdorp | A survey of honey bee colony losses in the US, fall 2007 to spring 2008[END_REF]. The reduction in managed beehives is accompanied by a global decrease in the number and diversity of other animal pollinators [START_REF] Ollerton | Pollinator diversity: distribution, ecological function, and conservation[END_REF]. It has been attributed to multiple factors, including the decline in diversity and abundance of flowers, the lack of natural habitat, the presence of parasites and pathogens and exposure to pesticides [START_REF] Goulson | Bee declines driven by combined stress from parasites, pesticides, and lack of flowers[END_REF][START_REF] Vanengelsdorp | A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them[END_REF].

Field surveys have confirmed a transfer from crops to beehive matrices of applied pesticides belonging to the three main classes of insecticides, fungicides and herbicides [START_REF] Piechowicz | Transfer of the active ingredients of some plant protection products from raspberry plants to beehives[END_REF][START_REF] Pohorecka | Residues of neonicotinoid insecticides in bee collected plant materials from oilseed rape crops and their effect on bee colonies[END_REF][START_REF] Skerl | Residues of pesticides in honeybee (Apis mellifera carnica) bee bread and in pollen loads from treated apple orchards[END_REF]. Scientists were interested in knowing the effects of insecticides on honey bees, as these products are considered the most potentially dangerous pesticides to beneficial insects [START_REF] Brandt | The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.)[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] Glavan | The synergy of xenobiotics in honey bee Apis mellifera: mechanisms and effects[END_REF][START_REF] Gregorc | Cell death localization in situ in laboratory reared honey bee (Apis mellifera L.) larvae treated with pesticides[END_REF][START_REF] Guez | Contrasting effects of imidacloprid on habituation in 7-and 8-day-old honeybees (Apis mellifera)[END_REF][START_REF] Kessler | Bees prefer foods containing neonicotinoid pesticides[END_REF][START_REF] Yang | Abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (Hymenoptera: Apidae)[END_REF]. Fungicides and herbicides are considered harmless to honey bees due to their low acute toxicity. Nevertheless, an increasing number of studies are addressing their actual effects [START_REF] Christen | Fungicides chlorothanolin, azoxystrobin and folpet induce transcriptional alterations in genes encoding enzymes involved in oxidative phosphorylation and metabolism in honey bees (Apis mellifera) at sublethal concentrations[END_REF][START_REF] Cousin | Size changes in honey bee larvae oenocytes induced by exposure to paraquat at very low concentrations[END_REF][START_REF] Jaffe | Effect of fungicide on pollen foraging by honeybees (Hymenoptera: Apidae) in cranberry differs by fungicide type[END_REF][START_REF] Ladurner | Assessing delayed and acute toxicity of five formulated fungicides to Osmia lignaria Say and Apis mellifera[END_REF][START_REF] Moffett | Toxicity of some herbicidal sprays to honey bees Hymenoptera-Apidae[END_REF]. In beehive matrices, the phytopharmaceutical products of three main classes can coexist with acaricides used to control infestation by Varroa destructor [START_REF] Chauzat | Influence of pesticide residues on honey bee (Hymenoptera: Apidae) colony health in France[END_REF][START_REF] Chauzat | A survey of pesticide residues in pollen loads collected by honey bees in France[END_REF][START_REF] Mullin | High levels of miticides and agrochemicals in north american apiaries: implications for honey bee health[END_REF]. Therefore, honey bees could be continuously exposed to mixtures of pesticides that may exhibit similar or completely different modes of action.

Despite the high probability of honey bee exposure to mixtures of pesticides, only a few studies have focused on their effects on honey bees, and most of them were restricted to the interactions between insecticides (pyrethroids and neonicotinoids) and fungicides (ergosterol biosynthesis inhibitor (EBI) family) [START_REF] Bjergager | Determining lower threshold concentrations for synergistic effects[END_REF][START_REF] Colin | Evidence of synergy between prochloraz and deltamethrin in Apis mellifera L. -a convenient biological approach[END_REF][START_REF] Iwasa | Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera[END_REF][START_REF] Meled | Seasonal variations in susceptibility of Apis mellifera to the synergistic action of prochloraz and deltamethrin[END_REF][START_REF] Schmuck | Field relevance of a synergistic effect observed in the laboratory between an EBI fungicide and a chloronicotinyl insecticide in the honeybee (Apis mellifera L, Hymenoptera)[END_REF][START_REF] Thompson | Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops[END_REF]Zhu et al., 2017a;Zhu et al., 2017b). Effects varied from no effects to synergism, depending on the pesticides used, the method and duration of exposure, and the concentrations in food.

Therefore, there is a large gap in the assessment of pesticide risk in the registration procedure because the mixtures were never investigated, and further studies are urgently needed in this field.

The losses of honey bee colonies are mostly seen at the end of the winter season [START_REF] Genersch | The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies[END_REF][START_REF] Guzmán-Novoa | Varroa destructor is the main culprit for the death and reduced populations of overwintered honey bee (Apis mellifera) colonies in Ontario, Canada[END_REF], with approximately 20 to 30% losses in Canada, Europe and the USA (van der [START_REF] Van Der Zee | Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008-9 and 2009-10[END_REF]. During this period, beehive tasks are performed by a specific category of workers known as winter honey bees. These honey bees can survive up to 6 months (Free and Spencer-booth, 1959), and they rely on the consumption of stored honey and bee bread for survival, exposing them to pesticides for a relatively long period.

Imidacloprid (insecticide), difenoconazole (fungicide) and glyphosate (herbicide) are among the pesticides that are frequently detected in beehive matrices [START_REF] Berg | Glyphosate residue concentrations in honey attributed through geospatial analysis to proximity of large-scale agriculture and transfer offsite by bees[END_REF][START_REF] Chauzat | An assessment of honeybee colony matrices, Apis mellifera (hymenoptera apidae) to monitor pesticide presence in continental france[END_REF][START_REF] Mullin | High levels of miticides and agrochemicals in north american apiaries: implications for honey bee health[END_REF]. Imidacloprid, together with its metabolite 6-chloronicotinic acid, was the most abundant pesticide in beehive matrices in French apiaries, with a mean concentration of 0.7 µg/kg in honey and 0.9 µg/kg in pollen [START_REF] Chauzat | An assessment of honeybee colony matrices, Apis mellifera (hymenoptera apidae) to monitor pesticide presence in continental france[END_REF]. However, concentrations of 0.14-0.275 µg/kg in honey, 1.35 µg/kg in pollen and 3-5.09 µg/kg in wax comb were found in other studies [START_REF] Lambert | Widespread occurrence of chemical residues in beehive matrices from apiaries located in different landscapes of western France[END_REF][START_REF] Lopez | Screening of pesticide residues in honeybee wax comb by LC-ESI-MS/MS. A pilot study[END_REF][START_REF] Nguyen | Does imidacloprid seed-treated maize have an impact on honey bee mortality?[END_REF]. Imidacloprid belongs to the neonicotinoid family and acts as an agonist of the nicotinic acetylcholine receptors, leading to the disruption of the nervous system through impaired cholinergic neurotransmission [START_REF] Casida | Neuroactive insecticides: targets, selectivity, resistance, and secondary effects[END_REF]. Glyphosate is the most dominant herbicide worldwide. Its use has increased 15-fold since the introduction of genetically engineered glyphosate-tolerant crops in 1996 [START_REF] Benbrook | Trends in glyphosate herbicide use in the United States and globally[END_REF], and it was detected in beehive matrices at concentrations ranging between 17 to 342 µg/kg in honey and 52.4 to 58.4 µg/kg in beebread [START_REF] Berg | Glyphosate residue concentrations in honey attributed through geospatial analysis to proximity of large-scale agriculture and transfer offsite by bees[END_REF][START_REF] El Agrebi | Honeybee and consumer's exposure and risk characterisation to glyphosate-based herbicide (GBH) and its degradation product (AMPA): Residues in beebread, wax, and honey[END_REF][START_REF] Rubio | Survey of glyphosate residues in honey, corn, and soy products[END_REF]. It acts by inhibiting the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), an enzyme necessary for the biosynthesis of aromatic amino acids in plants and some microorganisms, which leads to cell death [START_REF] Amrhein | The site of the inhibition of the shikimate pathway by glyphosate. II. Interference of glyphosate with chorismate formation in vivo and in vitro[END_REF]. Difenoconazole, a curative and preventive fungicide of the triazole family, is authorized for use during full bloom. It has been found at mean concentrations of 0.6 µg/kg in honey, 43 µg/kg in pollen, 270 µg/kg in beebread and 1 µg/kg in wax comb [START_REF] Kubik | Residues of captan (contact) and difenoconazole (systemic) fungicides in bee products from an apple orchard[END_REF][START_REF] Lopez | Screening of pesticide residues in honeybee wax comb by LC-ESI-MS/MS. A pilot study[END_REF]. It belongs to the ergosterol biosynthesis inhibitor (EBI) fungicides and acts by inhibiting the demethylation of lanosterol [START_REF] Zarn | Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase[END_REF].

To understand the effects of pesticide mixtures on winter honey bees, we conducted a study investigating the effects of the insecticide imidacloprid, the fungicide difenoconazole and the herbicide glyphosate alone or in combinations in winter bees orally exposed at concentrations found in honey and pollen [START_REF] Berg | Glyphosate residue concentrations in honey attributed through geospatial analysis to proximity of large-scale agriculture and transfer offsite by bees[END_REF][START_REF] Chauzat | An assessment of honeybee colony matrices, Apis mellifera (hymenoptera apidae) to monitor pesticide presence in continental france[END_REF][START_REF] Kubik | Residues of captan (contact) and difenoconazole (systemic) fungicides in bee products from an apple orchard[END_REF][START_REF] Nguyen | Does imidacloprid seed-treated maize have an impact on honey bee mortality?[END_REF][START_REF] Thompson | Determination of glyphosate, AMPA, and glufosinate in honey by online solid-phase extraction-liquid chromatography-tandem mass spectrometry[END_REF]. Attention was focused on survival and physiology. The effects on physiological functions were assessed by analyzing the modulation of five physiological markers involved in the nervous system, detoxification, oxidative stress, metabolism and immunity.

2.

Materials and Methods France). The colonies were continuously checked for their health status. The honey bees were mixed together, slightly anesthetized with carbon dioxide and then placed, in groups of 30 honey bees, in plastic cages (6 x 8.5 x 10 cm) with a sheet of filter paper placed on the bottom and replaced daily to maintain hygiene. The honey bees were placed in the dark in incubators at 30°C ± 2°C and 60% ± 10% relative humidity. During the first day, the bees were fed water and candy (Apifonda®) ad libitum. The following day, the few dead bees were removed and replaced, and the chronic exposure to pesticides for 20 days was begun.

Chronic exposure to pesticides

The bees were exposed to the insecticide imidacloprid (I), the fungicide difenoconazole (F) DMSO. These primary solutions were used to generate the mother solutions of the individual pesticides or were mixed to obtained the mother solutions of the pesticide mixtures. The mother solutions of the pesticides were prepared by serial dilution of the primary mother solutions to obtain 1% (v/v) DMSO and stored at -20°C. The sucrose solutions used for exposure to pesticides were prepared daily by 10-fold dilution of the mother pesticide solutions in sucrose solution to obtain final concentrations of 60% (m/v) sucrose, 1% (m/v) proteins and 0.1% (v/v) DMSO. The pesticide concentrations were checked by GC-MS/MS according to two analytical methods with RSD < 10% [START_REF] Paradis | Sensitive analytical methods for 22 relevant insecticides of 3 chemical families in honey by GC-MS/MS and LC-MS/MS[END_REF][START_REF] Wiest | Multi-residue analysis of 80 environmental contaminants in honeys, honeybees and pollens by one extraction procedure followed by liquid and gas chromatography coupled with mass spectrometric detection[END_REF]. The control bees were fed a sucrose solution devoid of pesticides. For each modality of exposure (including the controls), 14 cages of 30 bees were used. Each day, the bee mortality and food consumption were recorded, the dead bees were discarded, and the filter paper placed at the bottom of the cage was replaced. For the analysis of the physiological markers, the bees were sampled 10 and 20 days after the beginning of chronic exposure.

Survival rate and food consumption

In each cage, the survival rate was recorded daily and expressed as a ratio of the initial population. Every morning, the dead bees were removed for sanitary considerations.

Food consumption was recorded for 20 days by measuring the food consumed daily by the bees in each cage. Individual daily food consumption was calculated by dividing the food consumed per cage by the number of bees that remained alive each day in each cage.

Choice of physiological markers

The effects of the pesticide combinations on honey bee physiology were assessed by analyzing the modulation of five physiological markers. The markers were chosen to distinguish the systemic and tissue-specific actions of the pesticides alone and in combination.

The following two markers common to the three biological compartments (head, midgut and abdomen) were analyzed: CaE-3 and GST. In contrast, one specific physiological marker was chosen in each compartment as follows: AChE in the head, G6PDH in the abdomen and ALP in the midgut. These five markers have been found to be relevant in assessing the effects of pesticides on honey bees in different biological compartments [START_REF] Badiou-Beneteau | Honeybee biomarkers as promising tools to monitor environmental quality[END_REF][START_REF] Badiou-Beneteau | Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam[END_REF][START_REF] Boily | Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments[END_REF][START_REF] Carvalho | Enzymatic biomarkers as tools to assess environmental quality: A case study of exposure of the honeybee Apis mellifera to insecticides[END_REF][START_REF] Kairo | Nosema ceranae, fipronil and their combination compromise honey bee reproduction via changes in male physiology[END_REF]Zhu et al., 2017a;Zhu et al., 2017b).

Tissue preparation and marker extraction

At days 10 and 20, the surviving bees were sampled. To avoid animal suffering, the bees were anesthetized with carbon dioxide, the heads were separated from the rest of the body using a scalpel, and the midguts were obtained by pulling the stinger. The heads, midguts and abdomens (with the intestinal tract removed) were placed in 2 mL microfuge tubes, weighed and stored at -80°C until analysis. For each treatment modality and each type of tissue, 3 tissues were used and pooled to prepare the sample. From this sample, the tissues were homogenized to prepare a single tissue extract. Seven tissue extracts (7 ⨯ 3 tissues) were prepared (n=7) for each treatment modality. Each sample was assayed in triplicate. The tissues were homogenized in the extraction medium [10 mM sodium chloride, 1% (w/v) Triton X-100, 40 mM sodium phosphate pH 7.4 and protease inhibitors (2 µg/ml of pepstatin A, leupeptin and aprotinin, 0.1 mg/ml soybean trypsin inhibitor and 25 units/ml antipain)] to make 10% (w/v) extracts. Homogenization was achieved by grinding tissues with a highspeed Qiagen TissueLyser II at 30 Hz for 5 periods of 30 seconds at 30 second intervals. The extracts were centrifuged at 4°C for 20 min at 15000 × g av. and the supernatants were kept on ice for further enzyme assays. Carboxylesterase para (CaE-3) and glutathione-S-transferase (GST) were extracted from the head, midgut and abdomen; acetylcholinesterase (AChE) from the head; glucose-6-phosphate dehydrogenase (G6PDH) from the abdomen; and alkaline phosphatase (ALP) from the midgut.

Enzyme assays

CaE-3 was assayed in a medium containing the tissue extract, 10 µM BW284C51 (acetylcholinesterase inhibitor), 0.1 mM p-NPA as the substrate and 100 mM sodium phosphate pH 7.0. The reaction was monitored at 410 nm [START_REF] Badiou-Beneteau | Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam[END_REF][START_REF] Gomori | Human esterases[END_REF][START_REF] Renzi | Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined[END_REF]. GST was assayed at 340 nm by measuring the conjugation of GSH to CDNB. The extract was incubated in a medium containing 1 mM EDTA, 2.5 mM GSH as the cosubstrate, 1 mM CDNB as the substrate and 100 mM disodium phosphate pH 7.4 [START_REF] Carvalho | Enzymatic biomarkers as tools to assess environmental quality: A case study of exposure of the honeybee Apis mellifera to insecticides[END_REF]. AChE was assayed at 412 nm in a medium containing the tissue extract, 1.5 mM DTNB, 0.3 mM AcSCh as the substrate and 100 mM sodium phosphate pH 7.0 [START_REF] Belzunces | Properties of acetylcholinesterase from Apis mellifera heads[END_REF]. G6PDH was measured by following the formation of NADPH at 340 nm in a medium containing the tissue extracts, 1 mM G6P as the substrate, 0.5 mM NADP + as the coenzyme, 10 mM MgCl 2 and 100 mM Tris-HCl pH 7.4 [START_REF] Renzi | Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined[END_REF].

ALP was assayed at 410 nm in a medium containing the tissue extract, 20 µM MgCl 2 , 2 mM p-NPP as the substrate and 100 mM Tris-HCl pH 8.5 [START_REF] Bounias | Toxicology of cupric salts on honeybees .V. Gluconate and sulfate action on gut alkaline and acid phosphatases[END_REF]. All reactions started after adding the substrate, and the activity was assessed by determining the initial velocity of the enzymatic kinetics, which corresponded to the slope of the tangent at the origin. All enzymatic reactions were followed using a TECAN F500 spectrophotometer.

Mode of interaction between pesticides

The interaction ratio (IR) was used to define the mode of interaction between pesticides (additive, antagonistic and synergistic) [START_REF] Colin | Evidence of synergy between prochloraz and deltamethrin in Apis mellifera L. -a convenient biological approach[END_REF][START_REF] Piggott | Reconceptualizing synergism and antagonism among multiple stressors[END_REF]:

where Mix represents the crude mortality of the mixture (binary or ternary), C the mortality of the control, and (Mix -C) the mortality of the pesticide mixture corrected by the control mortality.

represents the sum of the mortalities induced by each pesticide (n) in the mixture corrected by the control mortality, which corresponds to the theoretical expected mortality of the mixture. A value of IR = 1 reflects a pure additive effect. However, considering the variation in the effects, an IR is considered equal to 1 when 0.95 ≤ IR ≤ 1.05.

When IR > 1, the interaction is synergistic. For IR < 1, three cases were distinguished: (i)

when the mortality of the mixture was lower than the mortality of the lowest toxic substance alone, the interaction was considered purely antagonistic. (ii) When the toxicity of the mixture was higher than the mortality of the most toxic substance but below the expected mortality, the interaction was considered subadditive. In this case, it was not possible to speak in terms of antagonism because the effect of the mixture was higher than the effect of each substance.

(iii) When the effect of the mixture was between the effect of the least toxic substance and the effect of the most toxic substance, the interaction was also considered subadditive. In this case, it was also not possible to speak in terms of antagonism because, compared to the most toxic substance, antagonism could be considered, but compared to the least toxic substance, synergy could also be considered. (iv) The effect of the mixture was judged independent when the mixture induced a mortality similar to that of each pesticide.

2.9.Statistical analyses

The statistical analyses were performed using R software (Rstudio Version 1.1.463). The bee survival was analyzed by the Kaplan-Meier method (log-rank test), followed by a post hoc test to compare survival and treatments. The effects of the treatments on food consumption were investigated by comparing the individual cumulative sucrose consumption during the exposure period using the Kruskal-Wallis test, followed by pairwise comparisons using the Wilcoxon rank sum test with a Benjamini-Hochberg correction. The effects of the treatments on the physiological markers were determined by ANOVA, followed by Tukey's HSD test, when the data followed a normal distribution or a Kruskal-Wallis test, followed by a post hoc Dunn test (with Benjamini-Hochberg correction), when the data followed a non-normal distribution.

Results

Honey bee survival

Exposure to pesticides significantly decreased the survival rate of honey bees at 20 days, except for I0.1, I10 and F0.1, for which no significant difference from the control (20.0 ± 2.7%) was observed (p > 0.05) (Fig. 1A, 1B, 1C and Table S1). Based on mortality rates, the toxicities of pesticides could be ranked as follows: at 0. Based on the interaction ratio (IR), which corresponds to the ratio between the obtained mortality of the mixture and the expected mortality (sum of the obtained mortalities of the substances in the mixture), the interaction effects between the pesticides could be grouped into 5 different categories (Table S1): additive, synergistic, subadditive, antagonistic and independent effects. (i) A synergistic effect was observed for all the binary mixtures and the ternary mixture at 0.1 µg/L and for IF1 and IH10. (ii) An additive effect was observed for IF10. (iii) A subadditive effect was observed for IH1, IHF1 and IHF10. (iv) An independent effect was observed for HF1. (v) An antagonistic effect was observed for HF10. The five most toxic pesticide mixtures were ranked as follows based on mortality rates: IF10 (41.0%) < IHF1 (43.3%) = IHF10 = IH0.1 (43.3%) < IH10 (45.7%) < HF0.1 (49.1%) < IF1 (52.9%).

[2-column fitting color image]

Effects of exposure to pesticides on food consumption behavior

Food consumption was monitored daily. In general, at the end of the exposure period, it appeared that the food consumption was higher in the exposed bees (Fig. 2 and Table S2).

This higher consumption was significant for all exposure conditions except F1, I1, F10 and I10 for pesticides alone, and HF10 and IHF10 for the mixtures. Information). Therefore, for difenoconazole and glyphosate at 0.1, 1 and 10 µg/L, each honey bee ingested 1/1.6x10 6 , 1/1.7x10 5 and 1/1.8x10 4 of the LD 50 , respectively (Table S2).

[2-column fitting image]

Effect of exposure to pesticides on the physiological status of honey bees

The physiological status of the honey bees was examined by studying the modulation of physiological markers in different compartments to distinguish the local from the systemic effects of the pesticides (Table 1). The responses of the honey bee markers to the exposure to the pesticides alone or in combination were analyzed after 10 and 20 days of chronic exposure to concentrations of 0.1 µg/L and 1 µg/L (Fig. 3, Fig. 4, Table S3 and Table S4). The lowest concentrations were chosen because they are particularly environmentally relevant. To render the data comparable, the enzymatic activities are expressed as percentages of the control values (Zhu et al., 2017a). Repartitioning of physiological markers across honey bee compartments. The following three tissues were investigated: head, abdomen and midgut. In each tissue, 1 specific marker (AChE in the head, G6PDH in the abdomen and ALP in the midgut) and 2 common markers (CaE-3 and GST) were considered.

[2-column fitting image] At 0.1 µg/L, head, midgut and abdomen CaE-3 and midgut GST were not modulated by all types of exposure at day 10 and day 20. Head AChE was not modulated at day 10. However, at day 20, its activity was 119% of the control activity (127.5 ± 16.0 mUA.min -1 .mg of tissue - 1 ) for H, 126% for HF and 141% for IHF. Head GST, abdomen G6PDH, and midgut ALP underwent modulation at day 10. For IHF, these modulations corresponded to a decrease in head GST (82% of control activity (115.3 ± 7.5 mUA.min -1 .mg of tissue -1 )) and a decrease in abdomen G6PDH (48% of control activity (2.07 ± 0.53 mUA.min -1 .mg of tissue -1 )). For IH, midgut ALP increased to 199% of the control activity (10.86 ± 2.75 mUA.min -1 .mg of tissue -1 ). Conversely, no modulation was observed at day 20 for any of these latter enzymes.

A decrease in abdomen GST was observed at 10 and 20 days. At 10 days, GST decreased to 57% of the control activity (116.1 ± 33.3 mUA.min -1 .mg of tissue -1 ) for H. At day 20, GST decreased to 48% of the control activity (83.0 ± 28.7 mUA.min -1 .mg of tissue -1 ) for IH and 49% for HF.

At 1 µg/L, head, midgut and abdomen CaE-3 and midgut ALP were not modulated for all types of exposure at day 10 and day 20. Head and abdomen GST underwent modulation at day 10. Head GST decreased to 75% of the control activity (115.3 ± 7.5 mUA.min -1 .mg of tissue -1 ) for H and 93% for IHF. Abdomen GST decreased for all types of exposure except IH: 49% of the control activity for I; 44% for H; 36% for F; 35% for IF; 51% for HF and 18%

for IHF (116.1 ± 33.3 mUA.min -1 .mg of tissue -1 for the control). Conversely, head and abdomen GST were not modulated at day 20. Abdomen G6PDH decreased at day 10 for all types of exposure: 56% of the control activity for I; 44% for H; 41% for F; 46% for IH; 38%

for IF; 55% for HF and 44% for IHF (12.1 ± 0.5 mUA.min -1 .mg of tissue -1 for the control).

However, no modulation was observed at day 20. Midgut GST was not modulated at day 10 but was modulated at day 20. Its activity decreased with all exposure types except IH and HF:

95% of the control activity for I; 88% for H; 96% for F; 93% for IF and 88% for IHF (147.9 ± 18.8 mUA.min -1 .mg of tissue -1 for the control). At day 10, head AChE increased to 128% of the control activity (127.7 ± 18.5 mUA.min -1 .mg of tissue -1 ) for HF and 134% of the control activity for IHF. At day 20, the activity of AChE increased to 124% of the control (127.5 ± 16.0 mUA.min -1 .mg of tissue -1 ) for HF, 127% of the control for IHF and 119% of the control for IF.

When comparing the dose effect of each type of exposure on physiological markers (comparison of the effects at 0.1 and 1 µg/L), no dose effect could be observed for I alone.

The effects of H on all markers were similar at both concentrations except for AChE at day 20 and head GST at day 10 (H0.1 > H1). F had the same effect on all markers at both concentrations except for AChE at day 20 (F0.1 > F1). The effect of IH on CaE-3, ALP, and abdomen GST was not similar at both concentrations. The effect of IH on head CaE-3 at day 10 and on abdomen CaE-3 and GST at day 20 was lower at 0.1 µg/L than at 1 µg/L.

Conversely, the effect of IH on midgut CaE-3 at days 10 and 20 and on abdomen CaE-3 and midgut ALP at day 10 was higher at 0.1 µg/L than at 1 µg/L. The effect of IF on midgut GST at day 20 was higher at 0.1 µg/L than at 1 µg/L. Depending on the concentration, the IF mixture modulated abdomen GST at day 10 (IF0.1 > IF1) and abdomen G6PDH at day 10 (IF0.1 > IF1). The effect of HF was dose-dependent only on the activity of GST in the abdomen at day 20 (HF0.1 < HF1). The effect of the ternary mixture IHF on abdomen GST at day 10 and on midgut GST at day 20 was higher at 0.1 µg/L than at 1 µg/L (IHF0.1 > IHF1) (Table S5).

4.

Discussion

Honey bees that emerge at the end of the summer are considered winter bees. These bees can live up to 6 months (Free and Spencer-booth, 1959) and, therefore, are chronically exposed to pesticide residues throughout the winter. In this study, the mixtures induced relatively high toxicity even though the winter honey bees were exposed for only 20 days to these three pesticides, alone or in binary and ternary mixtures, at concentrations equal to or even less than the environmental concentrations detected in beehive matrices. Thus, determining the effect of these pesticides on colony winter survival is highly important.

Pesticide combinations are more toxic to honeybees than individual pesticides

In this study, these three pesticides alone or in combination affected the survival of winter honey bees at all tested exposure concentrations, except for I0.1, I10 and F0.1. Concerning imidacloprid, the toxicity was less pronounced than that previously observed at the same concentrations on summer bees, where 50% mortality was reached after 8 days of chronic exposure at all concentrations [START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF]. In contrast, imidacloprid toxicity was much more pronounced than that observed in young summer bees after 14 days of exposure at 1 µg/L [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF]. The differences in imidacloprid toxicity could be attributed to seasonal variations [START_REF] Decourtye | Learning performances of honeybees (Apis mellifera L) are differentially affected by imidacloprid according to the season[END_REF][START_REF] Meled | Seasonal variations in susceptibility of Apis mellifera to the synergistic action of prochloraz and deltamethrin[END_REF][START_REF] Piechowicz | Circadian and seasonal changes in honeybee (Apis Mellifera) worker susceptibility to pyrethroids[END_REF], genetic differences [START_REF] Smirle | Intercolony variation in pesticide detoxification by the honey-bee (hymenoptera, apidae)[END_REF], the age of the bees or the exposure duration.

Herbicides and fungicides were considered nontoxic to honey bees for a long time.

Concentrations of imidazole fungicides and glyphosate up to 0.084 and 35 mg/L, respectively (Zhu et al., 2017a), were shown to be nonlethal. However, in this study, chronic exposure to glyphosate and difenoconazole (except for F0.1) was lethal. All pesticide combinations alter honey bee survival and are more toxic than pesticides alone, except HF10, which exhibits an antagonistic effect. Thus, the tier approach implemented in the pesticide registration procedure, which is first based on acute toxicity, shows great limits in detecting pesticides toxic to bees.

Increased concentrations of pesticides are not always linked to increased toxicity

In terms of dose-effect relationships, in general, it appears that the highest concentration was not the most dangerous, and the highest mortalities were observed at the intermediate concentration of 1 µg/L. This bell-shaped non-monotonic dose response relationship (NMDR)

(high response at intermediate doses and lower responses at low and high doses) was previously observed for imidacloprid and glyphosate [START_REF] Boily | Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments[END_REF][START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF][START_REF] Vazquez | Glyphosate affects the larval development of honey bees depending on the susceptibility of colonies[END_REF]. Three main hypotheses might explain this profile [START_REF] Lagarde | Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment[END_REF]. The first is the plurality of molecular targets, i.e., each xenobiotic has several molecular targets of different affinities that may induce opposite effects across the range of the tested concentrations. The second hypothesis is the metabolic hypothesis [START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF], which proposes that detoxification enzymes are induced at high but not at low concentrations. This hypothesis is consistent with the action of glyphosate, whose main metabolite, aminomethylphosphonic acid (AMPA), was shown to be nontoxic to honey bees [START_REF] Blot | Glyphosate, but not its metabolite AMPA, alters the honeybee gut microbiota[END_REF]. However, the metabolic hypothesis is not consistent with the action of imidacloprid because all metabolites were shown to be toxic to honey bees after chronic exposure [START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF]. The third hypothesis is receptor desensitization, where at high concentrations, numerous receptors are bound to xenobiotics, leading to a downregulation phenomenon [START_REF] Lagarde | Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment[END_REF].

The mixture of EBI fungicides with imidacloprid or glyphosate was shown in different studies to have no synergistic action [START_REF] Iwasa | Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera[END_REF][START_REF] Thompson | Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops[END_REF]Zhu et al., 2017b) or to induce a synergistic effect [START_REF] Biddinger | Comparative toxicities and synergism of apple orchard pesticides to Apis mellifera (l.) and Osmia cornifrons (Radoszkowski)[END_REF]. However, these studies were based on acute contact exposure. Therefore, it is not possible to directly compare these results with those of our study in which the mixtures induced an increase in mortality after chronic oral exposure. On the other hand, in two studies based on chronic oral exposure, the imidaclopridfungicide and/or imidacloprid-glyphosate mixture did not show a synergistic or additive effect [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF]Zhu et al., 2017a). The differences in the mixture effects between the different studies could be attributed to multiple factors: (i) The age of exposed honey bees, with newly emerged honey bees in the studies of [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF] and Zhu et al. (2017b), and adult honey bees in our study. (ii) The duration of exposure, which did not exceed 14 days in the studies of [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF] and Zhu et al. (2017b) but was 20 days in our study. (iii) The type of exposure, with the active ingredient in our study and in the study of [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF] and with the formulated products in the study of Zhu et al. (2017b). (iv) Seasonal variability, which could be reflected by the use of winter honey bees in our study and summer or spring honey bees in the two previously cited studies.

(v) The concentrations of the active ingredients constituting the mixtures, which were lower in our study when compared to the studies of Zhu et al. (2017b) and [START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF].

In this study, all binary mixtures had a differential effect on mortality in terms of both dose dependence and number of substances present in the mixture. Regarding the differential dose effect, HF induced a synergistic effect at 0.1 µg/L, an independent effect at 1 µg/L and an antagonistic effect at 10 µg/L. IF induced a synergistic effect at 0.1 and 1 and an additive effect at 10 µg/L. IH induced a synergistic effect at 0.1 and 10 µg/L and a subadditive effect at 1 µg/L. The ternary mixture induced a subadditive effect at 1 and 10 µg/L and a synergistic effect at 0.1 µg/L. Interactions between substances can occur not only through the primary biological targets responsible for the expected effect (insecticide, herbicide or fungicide) and common metabolic pathways, if they exist in the honey bee, but also through secondary targets responsible for non-intentional effects. Because primary and secondary targets may have different affinities for these substances, the effects induced could depend on the internal body concentration and, therefore, the exposure level. Hence, substances may interfere by blocking or activating metabolic pathways triggered by the substances in the mixtures, which explains why the nature and importance of the effects vary with the doses [START_REF] Lagarde | Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment[END_REF]. However, at 0.1 µg/L, the mortality induced by IHF was lower than those induced by IH and IF, leading us to conclude that increasing concentration or number of substances does not always increase the toxicity of a mixture. This finding exemplifies that the toxicity of a mixture is not merely the sum of the toxicity of the substances or the basic sum of the individual modes of actions.

Pesticides modulate feeding behavior through an increase in food consumption

Bees exposed to imidacloprid, difenoconazole and glyphosate, alone or in mixtures, consume more food than unexposed bees. Different hypotheses could explain this high consumption. (i)

A higher food consumption could be triggered by energetic stress due to an increase in intermediary metabolism induced by the pesticides or the spoliation of energetic resources as has been shown for pyrethroids [START_REF] Bounias | Sublethal effects of a synthetic pyrethroid, deltamethrin, on the glycemia, the lipemia, and the gut alkaline-phosphatases of honeybees[END_REF]. (ii) Honey bees could display a preference for sucrose solutions containing glyphosate and imidacloprid, as previously shown [START_REF] Kessler | Bees prefer foods containing neonicotinoid pesticides[END_REF][START_REF] Liao | Behavioral responses of honey bees (Apis mellifera) to natural and synthetic xenobiotics in food[END_REF]. In contrast, a study has shown a decrease in food consumption after exposure to mixtures of the formulated products of imidacloprid with tetraconazole and of imidacloprid with glyphosate (Zhu et al., 2017a). This finding suggests that the decrease in food consumption could be attributed to adjuvants present in the formulated products that might have a repellent feeding effect. However, the effect on food consumption could also depend on the concentration of the pesticides to which honey bees are exposed. In our study, the presence of pesticides elicited a higher food consumption, whereas in the study conducted by Zhu et al. (2017b), at higher concentrations, the pesticides elicited a lower food consumption. Thus, active substances, adjuvants or both could induce concentration-dependent effects on food consumption depending on their affinities to the biological target.

The honey bees received a cumulative dose of imidacloprid equivalent to 1/60, 1/6 and 1/0.6 of the LD 50 at 0.1, 1 and 10 µg/L, respectively. However, for glyphosate and difenoconazole, the cumulative quantity ingested was, at least, equivalent to 1/1.52x10 6 , 1/1.57x10 5 and 1/1.65x10 4 of the LD 50 at 0.1, 1 and 10 µg/L. Despite cumulative exposure ratios of difenoconazole and glyphosate at least 10 000 times less than the LD 50 , these two pesticides caused significant increases in mortality except for F0.1. Therefore, pesticides that are considered harmless to honey bees (high LD 50, superior to 100 µg/bee) can become dangerous even at very low concentrations after long-term exposure. This highlights the importance of an in-depth revision of the current risk assessment schemes used in the pesticide registration procedure [START_REF] Sgolastra | Bees and pesticide regulation: Lessons from the neonicotinoid experience[END_REF].

4.4. Pesticides induce perturbations in the detoxification process, nervous system, defense against oxidative stress, metabolism and immunity CaE-3, along with the other carboxylesterases, is involved in the metabolism of xenobiotics by catalyzing the hydrolysis of substrates containing amide, ester and thioester bonds. It is also involved in lipid metabolism [START_REF] Badiou-Beneteau | Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam[END_REF][START_REF] Ross | Carboxylesterases: Dual roles in lipid and pesticide metabolism[END_REF]. In our study, head, midgut and abdomen CaE-3 were not significantly modulated by any type of exposure. However, the activity of this enzyme was reported to decrease after acute exposure to 2.56 ng bee -1 thiamethoxam (neonicotinoid) [START_REF] Badiou-Beneteau | Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam[END_REF] and at LD 50 /20 of fipronil [START_REF] Carvalho | Enzymatic biomarkers as tools to assess environmental quality: A case study of exposure of the honeybee Apis mellifera to insecticides[END_REF]. Several studies have shown differential expression of carboxylesterases (CaEs) after exposure to pesticides [START_REF] Badiou-Beneteau | Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam[END_REF][START_REF] Zhu | Long-term risk assessment on noneffective and effective toxic doses of imidacloprid to honeybee workers[END_REF]Zhu et al., 2017a;Zhu et al., 2017b). Thus, measuring only overall CaE activity with nonspecific substrates could mask the differential modulation of several isoforms, including CaE-3.

AChE is a neural enzyme hydrolyzing the neurotransmitter acetylcholine in cholinergic synapses [START_REF] Badiou | Existence of two membrane-bound acetylcholinesterases in the honey bee head[END_REF]. AChE was found to be involved in learning and memory processes [START_REF] Gauthier | Modulatory effect of learning and memory on honey-bee brain acetylcholinesterase activity Comp[END_REF][START_REF] Guez | Enhanced cholinergic transmission promotes recall in honeybees[END_REF]. Its activity was significantly increased for HF1 and IHF1 at day 10 and for IF, HF and IHF at 0.1 and 1 µg/L at day 20. Therefore, the increase in AChE activity is closely related to the duration of exposure and the concentrations of the pesticides forming the mixture. This reflects a delayed effect of the pesticide combinations on the nervous system and reveals the importance of studies on the effects of these pesticide combinations on the behavior and cognitive functions of honey bees.

Glyphosate increased AChE activity in the bees exposed to 0.1 µg/L. This finding contradicts the results showing that both newly emerged and adult honey bees exposed for up to 14 days during the summer period to glyphosate or its formulated product Roundup, at concentrations ranging from 2.5 to 10 ng/bee [START_REF] Boily | Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments[END_REF] and 35 mg/L, exhibit a decrease in AChE activity (Zhu et al., 2017a). The difference in the effect of glyphosate between our study and the previously cited studies could be attributed to seasonal variability. This hypothesis is supported by studies showing that the adverse effects of pesticides may be higher in summer bees than in winter bees. This higher sensitivity of summer bees has been shown in terms of the effects of imidacloprid on learning performance [START_REF] Decourtye | Learning performances of honeybees (Apis mellifera L) are differentially affected by imidacloprid according to the season[END_REF] and the synergistic effect of the pyrethroid insecticide deltamethrin and the azole fungicide prochloraz [START_REF] Meled | Seasonal variations in susceptibility of Apis mellifera to the synergistic action of prochloraz and deltamethrin[END_REF]. These alterations in AChE activities might explain, at least in part, the impairment of cognitive behaviors, sucrose responsiveness and olfactory learning observed in honey bees after exposure to glyphosate [START_REF] Balbuena | Effects of sublethal doses of glyphosate on honeybee navigation[END_REF][START_REF] Gonalons | Impaired associative learning after chronic exposure to pesticides in young adult honey bees[END_REF][START_REF] Herbert | Effects of field-realistic doses of glyphosate on honeybee appetitive behaviour[END_REF].

GST is a multifunctional enzyme involved in protection against oxidative stress and is a phase II enzyme involved in the detoxification of xenobiotics. It can also contribute to phase I detoxification by sequestering toxicants [START_REF] Berenbaum | Xenobiotic detoxification pathways in honey bees[END_REF][START_REF] Du Rand | Detoxification mechanisms of honey bees (Apis mellifera) resulting in tolerance of dietary nicotine[END_REF]. GST activity was mainly decreased after exposure to pesticides in the head, abdomen and midgut. This decrease could hypothetically be due either to inhibition of this enzyme or to a downregulation by these pesticides. However, noncovalent inhibition could not be detected because of the dilution of the tissue components during the step of tissue homogenization and the assay procedure (at least 1/200-fold final dilution). In addition, a covalent inhibition of GST by pesticides has never been reported, even with electrophilic pesticides such as organophosphorus insecticides or herbicides that include glyphosate. Thus, the decrease in GST activity, associated with the absence of inhibition, is consistent with GST downregulation, which is also consistent with the 4-fold downregulation of GST S1, which is responsible for fighting against oxidative stress, in the heads of honey bee larvae exposed to imidacloprid [START_REF] Wu | Gene expression changes in honey bees induced by sublethal imidacloprid exposure during the larval stage[END_REF]. Furthermore, no phase II metabolites in imidacloprid metabolism, including those that could be conjugated to glutathione, were found in the honey bee [START_REF] Suchail | In vivo distribution and metabolisation of C-14imidacloprid in different compartments of Apis mellifera[END_REF]. This could be explained either by an absence of conjugation with GST, by the production of GST conjugates at undetectable levels, or by drastic downregulation of GST by imidacloprid. Thus, the decrease in GST activity may indicate a decrease in the honey bee capacities to detoxify these pesticides and to fight against oxidative stress that takes place after exposure to imidacloprid and glyphosate [START_REF] Contardo-Jara | Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes[END_REF][START_REF] Gauthier | Chronic exposure to imidacloprid or thiamethoxam neonicotinoid causes oxidative damages and alters carotenoid-retinoid levels in caged honey bees (Apis mellifera)[END_REF][START_REF] Jasper | Evaluation of biochemical, hematological and oxidative parameters in mice exposed to the herbicide glyphosate-Roundup®[END_REF][START_REF] Lushchak | Low toxic herbicide Roundup induces mild oxidative stress in goldfish tissues[END_REF].

G6PDH is the primary enzyme of the pentose phosphate pathway that generates NADPH and is involved, among other things, in the regeneration of reduced glutathione, which contributes to the fight against oxidative stress [START_REF] Thomas | Identification of the structural gene for glucose-6phosphate-dehydrogenase in yeast -inactivation leads to a nutritional-requirement for organic sulfur[END_REF]. G6PDH activity decreased after 10 days of exposure to all modalities at 1 µg/L. However, it is improbable that this decrease is due to oxidative stress. Indeed, in the presence of oxidative stress, glyceraldehyde-3phosphate dehydrogenase (GAPD) is inhibited [START_REF] Chuang | Glyceraldehyde-3-phosphate dehydrogenase, apoptosis and neurodegenerative diseases[END_REF], which induces a deviation of glycolysis towards the pentose phosphate pathway and an increase in G6PDH activity [START_REF] Nicholls | GAPDH: A common enzyme with uncommon functions[END_REF][START_REF] Renzi | Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined[END_REF].

ALP is an enzyme of the digestive tract involved in adsorption and transport mechanisms through the gut epithelium [START_REF] Vlahović | Plastic responses of larval mass and alkaline phosphatase to cadmium in the gypsy moth larvae[END_REF] and in immune response (Chen et al., indicating that the honey bees were sensitive to the pesticides and were unable to compensate for the increase in exposure duration.

Conclusion

This study demonstrates that chronic exposure to insecticides, herbicides and fungicides, alone or in combination, may induce high toxicity via systemic action in winter honey bees and constitutes a threat to these workers in two ways. The first is a direct drastic effect on survival, with a mortality that exceeded 50% after only 20 days of exposure, which can endanger the colony. The second involves a systemic action of these pesticides that alters honey bee physiology through metabolism, immunity, the nervous system, detoxification and antioxidant defenses. A severe loss of the winter bee population may compromise colony development during the spring, which might explain the high winter losses encountered in many regions. If such cocktail effects occurred in summer bees, this would have drastic impacts on colonies that could largely explain the bee population decline, especially because summer bees are more susceptible to pesticides and pesticide combinations than winter bees.

This study also reveals that the standard 10-day chronic toxicity test, used during pesticide risk assessment procedures, may not always be reliable in detecting the potential toxicities of pesticides. In addition, this study highlights the difficulty in predicting the cocktail effects of pollutants because the toxicity of the mixture is not always directly linked to the number of substances or the exposure level.

Fig. 1 .

 1 Fig. 1. Effects of pesticides alone or in combination on honey bee longevityFor 20 days, winter honey bees were fed sucrose solutions containing no pesticides (Control), difenoconazole (F), glyphosate (H), glyphosate + difenoconazole (HF), imidacloprid (I), imidacloprid + difenoconazole (IF), imidacloprid + glyphosate (IH) or imidacloprid + glyphosate + difenoconazole (IHF), at 0.1 µg/L (A), 1 µg/L (B) and 10 µg/L (C). The data represent the proportion of surviving honeybees exposed to these pesticides.Numbers after the abbreviations of each treatment refer to the concentrations of the pesticides in the sucrose solution. Treatments with different letters are significantly different (p < 0.05).

Fig. 2 .

 2 Fig. 2. Effects of pesticides alone or in combination on food consumption For 20 days, winter honey bees were fed sucrose solutions containing no pesticide (C, control), difenoconazole (F), glyphosate (H), glyphosate + difenoconazole (HF), imidacloprid (I), imidacloprid + difenoconazole (IF), imidacloprid + glyphosate (IH) or imidacloprid + glyphosate + difenoconazole (IHF), at 0.1 µg/L, 1 µg/L, and 10 µg/L. Food consumption was followed during the 20 days of exposure by measuring the food consumed daily by the bees alive in each cage. Box plots represent the cumulated individual consumption (mg/bee) for 7 cages of 30 bees per treatment. Statistical analyses were performed using the Kruskal-Wallis test followed by pairwise comparisons using the Wilcoxon rank sum test with the Benjamini-Hochberg correction. The numbers after the abbreviations of each treatment refer to the concentrations of the pesticides in the sucrose solution. Asterisks indicate significant differences from the control group (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Fig. 3 .

 3 Fig. 3. Physiological impacts of pesticides alone or in combination in winter bees after 10 days of exposure For 20 days, winter honey bees were fed sucrose solutions containing no pesticides (C, control), imidacloprid (I), glyphosate (H), difenoconazole (F), imidacloprid + glyphosate (IH), imidacloprid + difenoconazole (IF),glyphosate + difenoconazole (HF), or imidacloprid + glyphosate + difenoconazole (IHF). The impact of the exposure to pesticides on the physiology of the surviving honey bees at day 10 was investigated through an analysis of 2 common markers in the head, abdomen and midgut (GST and CaE-3) and 3 specific markers

Table 1 .

 1 Distribution of common and specific physiological markers across honey bee tissues

		Head	Abdomen	Midgut
	Common	CaE-3	CaE-3	CaE-3
	markers	GST	GST	GST
	Specific markers	AChE	G6PDH	ALP

Acknowledgments

This study was supported in part by the recurrent funding of the Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE) and by the Agence Nationale de la Recherche (ANR) (grant ANR-15-CE34-0004-01). Hanine ALMASRI was supported by grants from Lebanese University (grant number 2364) and the PACA region. The authors thank Jacques Sénéchal and Alexandre Gorit, beekeepers of the UR 406 INRAE Research Unit, for their expert beekeeping.

2011

). The activity of ALP was not modulated after 10 and 20 days of exposure. Thus, imidacloprid, glyphosate and difenoconazole did not affect the activity of ALP. This finding strongly contrasts with the results of other studies that showed a modulation of ALP in bees exposed to other pesticides, such as fipronil and spinosad, and following infection by Nosema [START_REF] Carvalho | Enzymatic biomarkers as tools to assess environmental quality: A case study of exposure of the honeybee Apis mellifera to insecticides[END_REF][START_REF] Dussaubat | Gut pathology and responses to the microsporidium Nosema ceranae in the honey bee Apis mellifera[END_REF][START_REF] Kairo | Nosema ceranae, fipronil and their combination compromise honey bee reproduction via changes in male physiology[END_REF]. Thus, the apparent absence of ALP modulation in our study could reflect either an absence of effect or the occurrence of a compensatory phenomenon. 4.5. The effect of exposure to pesticides is systemic and tissue-specific By comparing the dose effect of IH on CaE-3, it is possible to notice that for the same exposure duration, the effect of IH on CaE-3 at 0.1 and 1 µg/L differed among the biological compartments. For the modulations of CaE-3 at day 10, IH0.1 < IH1 in the head and IH0.1 > IH1 in the midgut and abdomen. For the modulations of CaE-3 at day 20, IH0.1 > IH1 in the gut and IH0.1 < IH1 in the abdomen. This complex profile of modulations was also found for both head and midgut GST after exposure to Bt spores and to Nosema-fipronil combination [START_REF] Kairo | Nosema ceranae, fipronil and their combination compromise honey bee reproduction via changes in male physiology[END_REF][START_REF] Renzi | Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined[END_REF], thus confirming a spatially differential response due to the specificity of each tissue and to the occurrence of pesticide metabolism not only in the gut but also in other honey bee compartments [START_REF] Suchail | In vivo distribution and metabolisation of C-14imidacloprid in different compartments of Apis mellifera[END_REF].

GST activity was modulated in the head, midgut and abdomen. In addition, AChE was modulated in the head, G6PDH in the abdomen and ALP in the midgut. These results indicate that the effects of the exposure to pesticides are not localized in the midgut (and in turn in the abdomen), which is considered the primary site of interaction with the ingested pesticide, but are spread across all biological compartments, leading to a systemic response that could explain the severe impact on honey bee survival.

The effects of the pesticides on physiological markers were determined in surviving bees after 10 and 20 days of daily exposure. The results at day 10 revealed a massive modulation of all physiological markers except CaE-3 and midgut GST. However, a less pronounced effect was detected at day 20 with a higher number of non-modulated enzymes (CaE-3, head GST, ALP and G6PDH were not modulated). This lower effect at day 20 suggests that the honey bee population at day 10 was composed of both sensitive and resistant individuals, while the population that survived until the twentieth day mainly contained honey bees that were more resistant to these pesticides alone or in combination. However, this hypothesis could be ruled out because the progression of mortality during this period was approximately linear,