domain: sde sdv

text:
Séverine Suchail
email: severine.suchail@univ-avignon.fr

Adrien Le Navenant

Yvan Capowiez

Alain Thiéry

Magali Rault

L Séverine Suchail

An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia L

Keywords: earwig, energy reserves, glycogen, lipid, protein, biometry, pest management strategy Highlights

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The United Nations Food and Agricultural Organization reported that the world's population will reach to nearly 10 billion by 2050. This will lead to an increase in world food production in order to keep pace with the demand of this growth [START_REF] Gill | Pesticides : Environmental impacts and management strategies[END_REF]. Agricultural chemicals can enhance crop productivity. However, pesticides can also have negative effects on the environment, ecosystem and biodiversity. Pesticides can have an impact on non-target and beneficial species through direct or indirect routes, such as water contamination and runoff, pesticide residues, and by consuming food that has been sprayed [START_REF] Martinou | Lethal and behavioral effects of pesticides on the insect predator Macrolophus pygmaeus[END_REF]. The destruction of these beneficial arthropods can exacerbate pest problems as they play an important role in regulating pest population levels. In France, apple orchards are heavily treated with, on average, more than 30 pesticide applications per year [START_REF] Sauphanor | Ecophyto R&D: Towards crop management systems that save crop protection products[END_REF][START_REF] Mazzia | The impacts of management strategies in apple orchards on the structural and functional diversity of epigeal spiders[END_REF]. Two pest management strategies are generally identified in commercial apple orchards: Integrated Pest Management (IPM) and organic farming. The European earwig, Forficula auricularia L. (Dermaptera: Forficulidae) is a common, sub-social and omnivorous insect in agro-ecosystems worldwide. These arthropods are commonly found in apple orchards. Adults and nymphs are nocturnal and hide during the day under shelters such as bark or in holes in the ground. They can be found in high abundance in artificial shelters and traps [START_REF] Sauphanor | An aggregation pheromone in the European earwig Forficula auricularia L[END_REF][START_REF] Fountain | Non-target consequences of insecticides used in apple orchards on Forficula auricularia L. (Dermaptera: Forficulidae)[END_REF]. In apple orchards, earwigs are a key biological control agent against numerous insect pests such as aphids, psyllids and spider mites [START_REF] Dib | The role of natural enemies on the population dynamics of the rosy apple aphid, Dysaphis plantaginea Passerini (Hemiptera: Aphididae) in organic apple orchards in south-eastern France[END_REF][START_REF] Debras | Prospective research on the "source" role of hedges in integrated fruit production. The case of earwigs: Forficula auricularia L. et Forficula pubescens L. Gené[END_REF][START_REF] Romeu-Dalmau | Friend or foe? The role of earwigs in a Mediterranean organic citrus orchard[END_REF]. Their ease of capture and identification make them an interesting potential bioindicator of the intensity of pesticide use in orchards [START_REF] Dib | The role of natural enemies on the population dynamics of the rosy apple aphid, Dysaphis plantaginea Passerini (Hemiptera: Aphididae) in organic apple orchards in south-eastern France[END_REF].

Earwigs are sensitive to different management strategies in IPM apple orchards where several pesticides can be applied, e.g., organophosphates [START_REF] Ffrench-Constant | Soil contact toxicity of insecticides to the European earwig Forficula auricularia (Dermaptera)[END_REF]Malagnoux et al. 2015a), pyrethroids (Badjii 2004) or neonicotinoids [START_REF] Shaw | Susceptibility of the European earwig, Forficula auricularia, to insecticides residues on apple leaves[END_REF]Malagnoux et al. 2015b). Other agricultural practices such as tillage and the presence and quality of hedges can also influence earwig abundance [START_REF] Sharley | The effects of soil tillage on beneficial invertebrates within the vineyard[END_REF][START_REF] Moerkens | Natural and human causes of earwig mortality during winter: temperature, parasitoids and soil tillage[END_REF]) and the presence and quality of hedges [START_REF] Debras | Prospective research on the "source" role of hedges in integrated fruit production. The case of earwigs: Forficula auricularia L. et Forficula pubescens L. Gené[END_REF]. Management strategies in apple orchards also influence diversity in the earwig community, with F. auricularia being less sensitive to insecticides than F. pubescens (Malagnoux et al. 2015b). Studies concerning F. auricularia focused on its ecological role in orchards [START_REF] Kölliker | Maternal attendance and the maintenance of family groups in common earwigs (Forficula auricularia ): a field experiment[END_REF]Malagnoux et al. 2015a) or the direct impact of commonly used pesticides on its populations [START_REF] Ffrench-Constant | Soil contact toxicity of insecticides to the European earwig Forficula auricularia (Dermaptera)[END_REF][START_REF] Campos | Insecticide selectivity and behavioral response of the earwig Doru luteipes[END_REF]Malagnoux et al. 2015a;[START_REF] Fountain | Non-target consequences of insecticides used in apple orchards on Forficula auricularia L. (Dermaptera: Forficulidae)[END_REF]. However, there is, to our knowledge, little available data on the negative physiological effects of pesticides on energy reserves in these earwigs.

Glycogen and the lipid, triacylglycerol, are the predominant energy reserves stored in the fat bodies distributed throughout the body of the insect (Arrese and Soulage 2010). This compartment plays an essential role in energy storage and has important functions throughout the insect's life cycle including growth, timing of metamorphosis and egg development. In addition, the fat body also synthesizes most of the hemolymph proteins and circulating metabolites. Glycogen can be rapidly hydrolyzed to glucose in the haemolymph of insects to be used as a glycolytic fuel [START_REF] Steele | Glycogen phosphorylase in insects[END_REF]. The glycogen content may vary depending on physiological state [START_REF] Anand | Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilisation by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus[END_REF] or environmental conditions [START_REF] Chowanski | Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana[END_REF].

Triacylglycerol is formed by fatty acids that can be used for energy production through b-oxidation.

Lipids are always the main component of the fat body which, in insect, represents more than 50% of their dry weight [START_REF] Ziegler | Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta[END_REF]. Lipids provide energy during diapause [START_REF] Hahn | Meeting the energetic demands of insect diapause: nutrient storage and utilization[END_REF], for embryo development [START_REF] Ziegler | Lipid uptake by insect oocytes[END_REF] and sustained flight activity (Van der Horst 2003).

Proteins can also supply energy through catabolism and are essential in tissue building and repair. For this reason, it is also relevant to study the variability of insect protein content under different conditions.

Living organisms use reserves to maintain their bodies, growth, development, and reproduction (Arrese and Soulage 2010). Under stressful conditions, there is an increased expenditure associated with detoxification which can have a negative impact on life-history traits in the life cycle. After exposure to pesticides, it has been shown that energy reserves decreased in several insects [START_REF] Ribeiro | Effect of Endosulfan and Parathion on Energy Reserves and Physiological Parameters of the Terrestrial Isopod Porcellio dilatatus[END_REF][START_REF] Nath | Shifts in glycogen metabolism in hemolymph and fat body of the silkworm, Bombyx mori (Lepidoptera: Bombycidae) in response to organophosphorus insecticides toxicity[END_REF]Da Silva 2004;[START_REF] Rharrabe | Effects of azadirachtin on post-embryonic development, energy reserves and a-amylase activity of Plodia interpunctella Hübner (Lepidoptera: Pyralidae)[END_REF].

A low level of energy reserves could potentially influence the population dynamics of earwigs and decrease their role in pest management in apple orchards. Markaw (1995) and [START_REF] Nestel | Resource allocation and compensation during development in holometabolus insects[END_REF] showed that ecological stressors can affect developmental instability, which leads to reduced fitness.

Morphological parameters e.g. mass or insect size can be used to detect the effects of environmental stress on an organism.

The aim of our study was to assess the effects of two orchard management strategies, organic and IPM practices, on earwig (F. auricularia) weight, energy reserves (glycogen, lipid and protein contents) and two morphometric parameters (inter-eye and prothorax widths).

Materials and methods

Chemical compounds

Sodium chloride, Tris-HCl, trichloroacetic acid, ethanol, Oyster glycogen, glucose, sodium acetate, amyloglucosidase from Aspergillus Niger, phosphovanillin, sulfuric acid and bovin serum albumin were purchased from Sigma-Aldrich ® (Saint Quentin-Fallavier, France) while glucose RTU TM was purchased from bioMérieux SA ® (Geneva, Suisse).

Earwigs

Male and female adults of F. auricularia L. earwigs were caught in July 2015 using cardboard traps placed on apple tree branches in five orchards located in Noves, near Avignon (south-eastern France).

The landscape around these orchards is similar and characterized by the presence of a dense network of windbreak hedgerows, which protects orchards against the prevailing northern winds. Since this species is univoltine, these adults were of roughly the same age. The five orchards had two management strategies: three were under IPM management and thus applied synthetic plant protection products and two were under organic farming and used a restricted range of products (such as carpovirusine, spinosad, copper, sulfur…). Twenty male and 20 female earwigs were collected from each management system (i.e. 40 and 60 earwigs per sex for organic and IPM strategies, respectively), frozen and stored at -20 °C until biochemical and morphometry measurements were carried out.

Morphometric measurements

Frozen earwigs were slightly thawed to avoid change to morphometric measurements. All morphometric measurements of earwigs were carried out using an eyepiece micrometer on a Nikon ® binocular microscope. Measurements were first recorded as units of the ocular micrometer scale to the nearest 1µm and were then converted into mm (± 0.1 mm). Each earwig was positioned carefully to avoid bias in measuring and to achieve the same plane of view. A single person carried out all the measurements to avoid user bias. We measured the maximum prothorax and inter-eye (i.e., distance between the eyes) widths. The inter-eye width was measured in a straight line between the two eyes and the prothorax width between two reference points (Fig. 1).

Scanning electron microscopy

We used scanning electron microscope photography (Philips XL 30 ESEM with detector SE) to obtain images (e.g. Figure 1) of dried specimens coated with a gold layer of 10 -12 m. The equipment was set to operate at 20KV acceleration voltage, at 35 for the magnification and at 28.9 mm for the working distance.

Crude extract preparation

After morphometric measurements, earwigs (male and female) were weighed individually using a Precisa ® XT120A electronic precision balance with a readability of 0.1 mg. The whole body of the earwig was homogenized on ice in 10% (w/v) low-salt buffer containing 10 mM Tris-HCl (pH 7.3) and 10 mM NaCl and centrifugated for 10 min at 3000 g. This supernatant was the crude extract on which further glycogen, lipid or protein measurements were carried out.

Biochemical measurements Determination of glycogen content

The method, based on enzymatic hydrolysis of glycogen by amyloglucosidase (EC 3.2.1.3), was used according to [START_REF] Parrou | A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells[END_REF]. Solution trichloroacetic 4% acid was added to 250 µl of crude extract (v/v) for deproteinization and the solution was centrifuged at 3000 g for 1 min at 4 °C.

After centrifugation, two volumes of 95% ethanol were added to precipitate the glycogen, which was present in the supernatant. Glycogen was finally pelleted by centrifugation at 5000 g for 5 min at 4 °C.

Ethanol was removed and the pellet was dried at room temperature. The dried pellet was incubated for 2 h at 60 °C in 500 µL of 0.2 M sodium acetate, pH 5.2, containing 7 UI of amyloglucosidase. After incubation, the solution was cooled on ice for 5 min and the amount of glucose generated from glycogen was determined using the Glucose RTU TM method adapting to 96-well microplate format.

The reaction mixture (275 µL final volume), containing 250 µL Glucose RTU TM and 25 µL glucose produced above, was left to stand for 20 min at room temperature, before glucose detection by measuring the absorbance at 505 nm. The amount of glucose was calculated from a standard curve (A 505 = f [glucose]) containing pure glucose as a standard treated within the same conditions. Because the final values included the amount of glycogen was corrected for the glucose content in samples that were not incubated with amyloglucosidase.

Determination of lipid content

Total lipids were determined using a sulphophosphovanillin reaction according to the method of [START_REF] Frings | Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction[END_REF] adapted to a 96-well microplate format. 294 µL concentrated sulfuric acid was added either to 6 µL of earwig crude extract or to 6 µl of a standard solution (olive oil). The solutions were homogenized and placed in boiling water for 10 min and cooled on ice for 5 min. A 700 µL of the phosphovanillin reagent was added to each extract or lipid standard, homogenized and incubated at 37 °C in a water bath for 15 min. After the samples were allowed to cool for 5 min the absorbance was recorded at 540 nm.

Determination of protein content

Total protein content was determined by the Lowry method modified according to [START_REF] Markwell | A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples[END_REF], with bovine serum albumin as the standard.

The energy reserve values were derived directly from a standard curve prepared with known concentrations of bovine serum albumin, glycogen and lipid. All assays were run in triplicate.

All the above components were expressed as milligrams per g of earwig.

Statistical analysis

For all the parameters (energy reserves and morphometric measurements), the effects of orchard management (organic vs IPM) and earwig sex were included in a mixed model with management and sex as fixed factors and apple orchard as a random factor. The assumptions of normality and homogeneity of variance were first tested, and log-transformation was applied when required (weight and morphometric measurements). We compared all the means for both factors using the 'lsmeans' package. The effects of the factor were thus assessed with the general model but sometimes also on simplified models (one factor only) depending on the significance of the interaction between the factors. All computations were carried out using R (version 2.15.3).

Results

Weight

Both earwig sex and orchard management had a significant effect on earwig weight (p<0.001) and no significant interaction was observed between the two factors (p=0.37). Regardless of orchard management strategy, male earwigs were significantly heavier than females. The weight of both sexes of earwigs sampled in organic orchards was also higher than those sampled in IPM orchards (Fig. 2).

Morphometric parameters

Prothorax and inter-eye widths Similar observations to above were obtained with the morphometric analysis of the earwigs. For both, prothorax and inter-eye widths measurements, sex did not have a significant effect, and the interaction between sex and management strategy was also not significant. Only management strategy had a significant effect with significantly smaller prothorax (p=0.032) and inter-eye widths (p=0.026) for earwigs sampled in IPM orchards compared to those sampled in organic orchards (Fig. 3). The decrease in inter-eye width was about 3% and between 2 to 5% for the prothorax width.

Biochemical analysis

Glycogen

The interaction between sex and management strategy was highly significant (p<0.001) for the glycogen content. For both management practices (p<0.001) sex had a significant effect with females having higher glycogen content than males. Management practices had no overall effect but when only females were considered, higher glycogen contents were observed in earwigs from organic compared to IPM orchards. No significant difference was observed for males explaining the significant interaction.

Lipids

Sex and the interaction between sex and management strategy had no significant effect on the earwig lipid contents. However, management strategy had a significant effect (p=0.008) with lower lipid content in earwigs sampled in IPM orchards than those sampled in organic orchards (23.49 mg ± 10.57 vs 32.10 mg ± 10.39 and 24.23 mg ± 11.12 vs 29.49 mg ± 10.57 respectively for females and males (Fig. 4 B)). On average, earwigs sampled in IPM orchards had 25 to 42 % less triacylglycerol than those under organic management.

Proteins

Sex and the interaction between sex and management strategy had no significant effect on earwig protein contents. However, management strategy had a significant effect (p=0.016) with higher body protein content in earwigs from IPM orchards than organic orchards (Fig. 4 C). Earwigs in IPM orchards had 70% more protein than those under organic management for both female and male.

Discussion and conclusion

It is well recognized that agricultural management practices including the use of pesticides affects non-target species and leads to a decline in biodiversity in agroecosystems (Mac Laughlin and Mineau 1995;[START_REF] Kromp | Carabid beetles in sustainable agriculture: A review on pest control efficacy, cultivation impacts and enhancement[END_REF][START_REF] Nash | Effect of remnant vegetation, pesticides, and farm management on abundance of the beneficial predator Notonomus gravis (Chaudoir) (Coleoptera: Carabidae)[END_REF]. The hazard of pesticide usage depends on the application rate, intrinsic toxicity and the level of uptake, persistence, volatilization, metabolization and bioavailability of the active ingredient (Sanchez-Bayo 2009). The exposure time is also a critical variable in risk assessments of agrochemicals. Organisms may develop a number of strategies to minimize the negative toxic effects induced by chemical stress. All responses to resist toxicants are costly for the organism in terms of metabolic resources and energy demand [START_REF] Calow | Physiological costs of combating chemical toxicants: ecological implications[END_REF]. Because the energy resources of organisms are limited and used for growth, reproduction and basal metabolism, the additional metabolic costs due to pesticide management results in a reallocation of energy resources, and can increase energy consumption leading to reduced energy reserves [START_REF] Congdon | Resource allocation-based life histories : a conceptual basis for studies of ecological toxicology[END_REF][START_REF] Souza | Effects of pesticides on the growth and reproduction of earthworm: A review Applied and[END_REF]. Resource allocation and life history strategies are inevitably interconnected for all organisms because of the competition and the limitation of available energy reserves either in the environment or within the individual itself.

Our results confirm that pest management strategy can result in changes to energy reserves in earwigs.

Our measurements of energy reserves indicated a lower lipid and glycogen contents in earwig bodies collected from IPM compared to organic orchards. The major differences between the organic and IPM strategies were lower total Treatment Frequency Index (TFI: total number of pesticide treatment weighted by the ratio of the dose used to the recommended dose [START_REF] Jørgensen | Denmark's action plans for pesticides: status and role of research[END_REF], and agricultural practices such as tillage, total weed removal or the presence and quality of hedges in the organic orchard. In organic strategy, pest control is based on a greater use of mineral fungicides (copper and sulphur) and natural insecticides (granulovirus, mineral oils and spinosad). In contrast, non-organic orchards mainly used synthetic pesticides which are known to be more efficient against pests. Beside the fact that the total TFI, was slightly but significantly lower in organic orchards (22.2), than in IPM orchards (26.6) it has been previously shown that the earwig community of F. auricularia was lower in IPM orchards compared to organic orchards and was correlated to a higher insecticide use (4.9 in organic vs 9.2 in IPM) (Malagnoux et al. 2015b). By their toxicity, insecticides increase lethality and/or affect the behavior of non-target organisms. For example, high sensitivity of F. auricularia to many insecticides was observed after exposure to organophosphates [START_REF] Ffrench-Constant | Soil contact toxicity of insecticides to the European earwig Forficula auricularia (Dermaptera)[END_REF]Malagnoux et al. 2015a), pyrethrinoids [START_REF] Badji | Impact of deltamethrin on arthropods in maize under IPM and no-tillage cultivation[END_REF]) and neonicotinoids [START_REF] Shaw | Susceptibility of the European earwig, Forficula auricularia, to insecticides residues on apple leaves[END_REF]Malagnoux et al. 2015b). Moreover, insecticide exposure induced disturbed behavior in predation activity of earwigs (Malagnoux et al. 2015a), cast production of earthworms (Jouni et al. 2018), feeding rate of the white shrimp (Garcia de La [START_REF] Parra | Effects of methamidophos on acetylcholinesterase activity, behavior, and feeding rate of the white shrimp (Litopenaeus vannamei)[END_REF] or locomotion of zebrafish [START_REF] Tilton | Swimming impairment and acetylcholinesterase inhibition in zebrafish exposed to copper or chlorpyrifos separately, or as mixtures[END_REF]. Because the process of food detection involves complex nervous activity that can be disrupted by neurotoxic pesticides [START_REF] Desneux | The sublethal effects of pesticides on beneficial arthropods[END_REF], impairments on behavior modify the feeding ability of the organisms and leads to a decrease in their energy reserves. Then, despite the possible loss in prey availability induced by higher TFI in IPM orchards [START_REF] Sauphanor | Evaluation au champ des effets secondaires des pesticides sur Forficula auricularia et Anthocoris nemoralis: validation des résultats de laboratoire[END_REF][START_REF] Altieri | The ecological role of biodiversity in agroecosystems[END_REF][START_REF] Pekar | Effect of IPM practices and IPM spraying on spider population dynamics in an apple orchard[END_REF][START_REF] Dib | Effect of management strategies on arthropod communities in the colonies of rosy apple aphid, Dysaphis plantaginea Passerini (Hemiptera: Aphididae) in southeastern France[END_REF], we assumed that the main factor responsible for depletion in energy reserves lies to a direct effect of insecticides on the organisms rather than on environmental modification (i.e. food availability). Our assumption was supported by several studies that have been conducted in laboratory experiments without food limitation. These studies have shown that exposure to organophosphorus insecticides, either severely disturbed glycogen metabolism in the Lepidoptera Bombyx mori [START_REF] Nath | Shifts in glycogen metabolism in hemolymph and fat body of the silkworm, Bombyx mori (Lepidoptera: Bombycidae) in response to organophosphorus insecticides toxicity[END_REF], or induced a significant depression of glycogen, lipid, and protein contents in the terrestrial isopod Porcellio dilatatus [START_REF] Ribeiro | Effect of Endosulfan and Parathion on Energy Reserves and Physiological Parameters of the Terrestrial Isopod Porcellio dilatatus[END_REF].

Glycogen is a major glucose reserve and can be rapidly mobilized whereas lipids and proteins are considered as long-term reserves (Arrese and Soulage 2010). In our study, the protein values were higher in male and female earwigs sampled from the orchard under IPM. Our results are in agreement with those of [START_REF] Jeon | Charaterization of acetylcholinesterase inhibition and energy allocation in Daphnia magna exposed to carbaryl[END_REF] who observed an increase in protein of Daphnia magna after carbaryl exposure, associated to a decrease in lipid and carbohydrate contents, or with the results of Staempfli et al. (2007) in the soil arthropod, Folsomia candida after dinoseb exposure. Reallocation of energy reserves to maintain homeostasis under pesticide exposure mainly focuses on protein induction to improve defense mechanisms, and they often involve overproduction of detoxification enzymes [START_REF] Calow | Physiological costs of combating chemical toxicants: ecological implications[END_REF]. According to those results and ours, it is unlikely that the increase in protein levels in IPM practices may offset the reduction in glycogen and lipid but, on the contrary, the consumption of glycogen and lipid content allow protein synthesis, which is highly cost effective. According to the thermodynamics of each nutrient (17.5 kJ/g glycogen, 24 kJ/g protein, and 39.5 kJ/g lipids) [START_REF] Gnaiger | Calculation of energetic and biochemical equivalents of respiratory oxygen consumption[END_REF]), total energy reserves are higher in term of global kJ in IPM orchards by 19% and 28% for male and female earwigs, respectively. However, this amount of energy is not available fast enough. When the organisms become unable to cope with stress or pollutants, the energy budget results of imbalance between the rate of synthesis and rate of degradation. Then, after the use of glycogen and lipids, proteins constitute the last energy reserve to change or be used upon stress exposure [START_REF] Ferreira | Biomarkers and energy reserves in the isopod Porcellionides pruinosus: The effects of long-term exposure to dimethoate[END_REF]. In this context, studies have reported that a decrease of protein content is associated to an increase in mortality. It was the case observed by Staempfli et al. (2007) where a biphasic concentration-response linked to the phenomenon of hormesis was observed, showing that after having produced an effort to increase growth and reproduction, lethality increases. [START_REF] Rharrabe | Effects of azadirachtin on post-embryonic development, energy reserves and a-amylase activity of Plodia interpunctella Hübner (Lepidoptera: Pyralidae)[END_REF] demonstrated that for lepidoptera Plodia interpunctella Hübner, the insecticide azadirachtin caused a severe reduction in protein levels and led larval weight loss, developmental delay followed by high lethality. These authors postulated that azadirachtin exerts its effect on insects by modifying the protein synthesis capacity of the fat body as also observed for S. litura [START_REF] Huang | Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin[END_REF]). Moreover, Ribero et al. (2001) observed a decrease in protein content in parathion-intoxicated isopods along with an increase in mortality, indicating that physiological adaptability to compensate pesticide stress requires high-energy but ineffective demand associated with.

In addition to agricultural practices, other factor can have an impact on rates of energy consumption, e.g. age and environmental conditions including temperature or dehydrating state. Anurag and Lorenz (2008) reported that the cricket, Gryllus bimaculatus, fat body energy reserves were age-dependent.

Climatic conditions also change abundance of energy reserves [START_REF] Chowanski | Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana[END_REF]. F. auricularia is univoltine, and in south of France, earwigs reach the adult stage in early summer. It is noteworthy that because all the organisms analyzed were caught in July, they were of roughly the same age.

Moreover this study was carried out on a same town and thus a reduced geographic area (less than 10 km radius), climatic conditions and landscape type are very similar. In this region, all orchards are surrounded by hedges due to the prevailing wind. Moreover, earwigs are nocturnal, inactive and hidden during the day inside the cardboard traps. When they are captured, we can assumed they are all in the same diet stage with an almost empty gut that will limit differences in energy reserves. Thus, in the studied apple orchards, the main factor governing energy budget in F. auricularia was assumed to be management strategy, and suggested that insecticide use, quantitatively and qualitatively, is able to decrease F. auricularia energy reserves. Other pollutant molecules can also lead to changes in energy reserves under natural conditions. Indeed, studies of chronically polluted with metals effects on the bettle Pterostichus oblongopunctus, showed a significant decrease in energy reserves [START_REF] Bednarska | Energy reserves and accumulation of metals in the ground beetle Pterostichus oblongopunctatus from two metal-polluted gradients[END_REF]. [START_REF] Givaudan | Impact of orchard management strategies on earthworm (Allobophora chlorotica) energy reserves[END_REF] showed higher lipid and glycogen contents in earthworms collected in abandoned and organic orchards compared to IPM orchards. Thus, the depletion of such biochemical constituents can be due to the major mobilization of these nutrients in response to the toxic effect of the pesticides.

Biochemical parameters appear to be good biomarkers for monitoring environmental management strategies but they need to be complemented with morphological parameters to confirm the impact of orchard management practices. Weight and morphometric traits in our earwigs were lower in IPM than in organic orchards. Earwigs from IPM orchards have to make trade-offs between maintenance, growth, storage and resistance to agrochemicals so as to maximize their fitness. The observed reduction in their prothorax and inter-eye widths is likely to be the consequence of these trade-offs induced by pesticides. A lot of studies in other insects showed that pesticides can lead to morphometric variations. [START_REF] Marcus | Atrazine exposure affects longevity, development time and body size in Drosophila melanogaster[END_REF] showed that exposure to atrazine, the one of the most widely used herbicides significantly affected male and female adult body size of Drosophila melanogaster. In Pterostichus melasitalicus, a carabidae, morphometric analyses also showed that dimethoate application caused a reduction in body or elytra lengths in females [START_REF] Giglio | Effects of the pesticide dimethoate on a nontarget generalist carabid, Pterostichus melas italicus (Dejean, 1828) (Coleoptera: Carabidae)[END_REF]. [START_REF] Giglio | Impact of agrochemicals on non-target species : Calthus fuscipes Goeze 1777 (Coleoptera : Carabidae) as model[END_REF] showed in another carabidae, Calathus fuscupes, that morphological parameters e.g. body and pronotum width decreased after an exposure to the larvicide lambda-cyhalothrin.

Moreover, studies of the effects of an inorganic insecticide (boric acid) against Blatella germanica showed a reduction in the size of basal oocytes, which affected its reproduction [START_REF] Habes | Effects of an inorganic insecticide (boric acid) against Blattella germanica: Morphometric measurements and biochemical composition of ovaries[END_REF].

All these studies suggest that agrochemical molecules can have an impact on the morphometric parameters of exposed organisms.

Our results underline the possible modifications in biochemical and morphometric parameters in earwigs due to orchard management strategies. Body energy reserves and prothorax and inter-eye widths in earwigs may be interesting biomarkers to characterize changes induced by agricultural farming both with pesticides and for other practices in orchards. This study shows more widely that F. auricularia, along with some other characteristics (ease of capture, identification, univoltine species) is an interesting potential bioindicator of the orchard management practices, especially in pip-fruit orchards, where it is considered an active natural enemy of aphids. In order to characterize the specific effects of pest management strategy on earwigs (direct and indirect effects), future studies should also be performed in abandoned orchards to assess the consequence of a free-pesticide environment. Other morphometric parameters could additionally be investigated to further determine if agricultural strategies influence other earwig life-history traits and further weakens these non-target species. In conclusion, this present study highlights the need to develop an integrated approach to assess both the physiological and biochemical modifications induced by pest management strategies on natural enemies.

Figure captions

Figure 1. Scanning electron microscopy dorsal view of head and prothorax of a male earwig showing the measurement method applied to selected body parts: the inter-eye (A) and the prothorax (B).

Figure 2 .

 2 Figure2. Mean weight (+SE) of the female and male adults of earwigs depending on the management strategy in apple orchards. (n=40 for male and n=40 for females for each strategy).

Figure 3 .

 3 Figure 3. Measurements of morphometric parameters: prothorax and inter-eye widths (means + SE) according to the management strategy in apple orchards. For each morphometric parameter, bars with the same letters are not significantly different form each other (n=40 for males and females for each strategy).

Figure 4 .

 4 Figure 4. Energy reserves content of the female and male of earwigs for each management strategy in apple orchards. A: Glycogen, B: lipid and C: protein. Data represent the means (+SE). Bars with the same letters are not significantly different form each other (n=40 for males and females for each strategy).

Acknowledgments

We thank Alain Tonetto (Pratim, Aix Marseille University) for assistance in taking the electronic microscopy photos. We are grateful to the Rovaltain Fundation for its financial support of the I-ResPect project.


text:
Séverine Suchail
email: suchail@avignon.inra.fr

Georges De Sousa

Roger Rahmani

Luc P Belzunces

In vivo distribution and metabolisation of [ 14 C]-imidacloprid in different compartments of Apis mellifera

Keywords: Imidacloprid, Neonicotinoid, Metabolite, Honeybee, Metabolisation, Compartment, Apis

In vivo distribution of imidacloprid, an insecticide that belongs to the neonicotinoid family, was followed during 72 h in 6 biological compartments of Apis mellifera: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 µg of [ 14 C]-imidacloprid per kg of bee, a dose close to the median lethal dose. Elimination half-life of total radioactivity in honeybee was 25 h.

Haemolymph was the compartment with the lower amount of total radioactivity whereas rectum had the higher level of total radioactivity during the whole study, with a maximum 24 h after treatment. Elimination half-life of imidacloprid in whole honeybee was 5 h. Imidacloprid was readily distributed and metabolised only by Phase I enzymes into five metabolites: 4/5-hydroxyimidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin and urea derivatives.

Guanidine derivative was not detected. Urea derivative and 6-chloronicotinic acid were the main metabolites and appeared particularly in midgut and rectum. Olefin derivative and 4/5-hydroxyimidacloprid preferentially occurred in head, thorax and abdomen that are nAChR-rich tissues.

Moreover, they presented a peak value around 4 h after imidacloprid ingestion. These results explain persistence of imidacloprid action in bees, and particularly the discrepancy between rapid intoxication symptoms and late mortality.

INTRODUCTION

Imidacloprid is the first active substance of the chemical class of neonicotinoid insecticides. This systemic insecticide is known to be extremely effective against a wide variety of insects [START_REF] Leicht | Imidacloprid a chloronicotinyl insecticide, biological activity and agricultural significance[END_REF]. The mode of action of imidacloprid was described by Liu and Casida (1993) as an agonistic effect on the cholinergic synapses that resulted in a consequent postsynaptic blockage in the nervous system. Imidacloprid binds to the nicotinic acetylcholine receptors (nAChR) with higher affinity for the insect than the mammalian site [START_REF] Yamamoto | Structural factors contributing to insecticidal and selective actions of neonicotinoids[END_REF]). Nevertheless, [START_REF] Tomizawa | Imidacloprid, thiacloprid, and their imine derivatives up-regulate the α4β2 nicotinic acetylcholine receptor in M10 cells[END_REF] indicated the possibility that some neonicotinoid insecticides or their metabolites, on accidental human exposure or when used for flea control on dogs, may also up-regulate the nAChR expression in mammals. The metabolism of imidacloprid has been studied in mammals, plants and soils, but there is little information about imidacloprid metabolism in insects for which the more in-depth works consist of the study of the metabolic fate in the whole body and the feces [START_REF] Mota-Sanchez | Metabolism and fate od 14 C-imidacloprid in Colorado potato beetle resistant to imidacloprid[END_REF]. In mammalians, [START_REF] Klein | The metabolism of imidacloprid in animals[END_REF] and [START_REF] Thyssen | Nicotinoïd insecticides and the nicotinic acetylcholine receptor[END_REF] reported the existence of two major metabolic pathways. The first is the oxidative cleavage of the molecule into the imidazolidine part, which is excreted directly via the urine, and the nicotinic moiety, which is subsequently modified by conjugation enzymes. The second important pathway is the hydroxylation of the parent molecule on the imidazolidine ring by the addition of water on the unsaturated metabolite olefin. These corresponding metabolic pathways permit to eliminate as metabolites more than 90 % of the given dose within 24 h and after 48 h imidacloprid was entirely excreted. In plants, biotransformation pathways are qualitatively similar to those of mammals [START_REF] Araki | Metabolism of imidacloprid in plants[END_REF].

Main imidacloprid metabolites identified in mammals and plants are the 5-hydroxy-imidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin guanidine and urea derivatives, and all contain the 2-chloropyridine moiety. Some of imidacloprid metabolites exhibit a toxicity to invertebrates, in particular olefin derivative and 5-hydroxy-imidacloprid. In aphid species and in the cotton whitefly, Bemisia tabaci, olefin is about 16 and 10 times more active than imidacloprid, respectively [START_REF] Nauen | Whitefly-active metabolites of imidacloprid: biological efficacy and translocation in cotton plants[END_REF]). In Apis mellifera, acute toxicity studies of imidacloprid have revealed important toxicological characteristics [START_REF] Suchail | Characteristics of imidacloprid toxicity in two Apis mellifera subspecies[END_REF][START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF]: (i) With oral and contact exposures, an unusual mortality profile is observed: mortality rises with low doses, falls with intermediate doses, and rises again with high doses, (ii) the kinetics of mortality is delayed as doses increased, (iii) neurotoxicity symptoms appear rapidly whereas mortality occurs 4 h after acute imidacloprid intoxication and is prolonged during more than 96 h. Thus, imidacloprid has either prolonged action or delayed action, but the symptoms are always immediate. As concerns metabolites, out of the 6 imidacloprid metabolites tested (5-hydroxy-imidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin guanidine and urea derivatives) only two, similar in chemical structure to imidacloprid, exhibit an acute toxicity close to that of the parent compound with a higher toxicity for olefin derivative and a lower toxicity for 5-hydroxy-imidacloprid. In contrast, in chronic toxicity studies, all imidacloprid metabolites were found equally toxic to bees, but the total dose ingested by the bees was about 3000-100 000 times lower than the doses needed to produce the same effect after an acute intoxication [START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF]). To explain the discrepancy between the kinetics of mortality and the rapid appearance of neurotoxicity symptoms and to discern a possible relation between the metabolite occurrence and the appearance of mortality, we have focused our investigation of imidacloprid biotransformation in whole honeybees into its two toxic metabolites, 5-hydroxy-imidacloprid and olefin derivative (Suchail et al. submitted). This study has shown that imidacloprid has a half-life of approximately 5 h and is rapidly metabolised into 5-hydroxy-imidacloprid and olefin derivative whose presented a peak value 4 h after intoxication. Thus, the appearance of 5-hydroxyimidacloprid and olefin derivative coincides with the appearance of mortality induced after acute oral intoxication by imidacloprid. These results suggest that 5-hydroxy-imidacloprid and/or olefin derivative take part in extending the action of imidacloprid in honeybees. In contrast to plants, this study has shown that, 5-hydroxy-imidacloprid and olefin derivative could not be the major metabolites in worker bees.

The objective of the present study is a better characterization of the imidacloprid pharmacokinetics after oral exposure to a dose close to the median lethal dose to understand its mode of action in the honeybees. The identification of all imidacloprid metabolites and their distribution in six biological compartments of honeybees were conducted. This study represents an original approach because imidacloprid metabolisation has never been studied in honeybees.

In addition, the more in-depth studies performed in insects, and particularly in honeybees, to have taken into account only the metabolic fate of pesticides in the whole animal and in the feces but not in different biological compartments, as in the present study (Mota-Sanchez et al. 2001; [START_REF] Pilling | Mechanism of synergism between the pyrethroid insecticide l-cyhalothrin and the imidazole fungicide prochloraz, in the honeybee (Apis mellifera L.)[END_REF].

MATERIALS AND METHODS

Chemical Compounds

We used [pyridinyl-14 C-methylene]-imidacloprid (1-(6-chloro-3-pyridylmethyl)-Nnitroimidazolidin-2-ylideneamine). The radiolabeled compound had a specific radioactivity of 3.7 MBq/mg (0.1 mCi mg -1 ), and a radiochemical purity of 99% determined by TLC and HPLC.

Radioactive imidacloprid was diluted in 100% dimethylsulfoxide (DMSO) to give a stock solution with a concentration of 5 g a.s. L -1 . Unlabelled imidacloprid and its metabolites were of the highest purity available, i.e. at least 97%. Imidacloprid and its metabolites were obtained from Bayer AG (Leverkussen, Germany). All other chemicals and organic solvents used were of analytical grade.

Biological materials

For all bioassays, worker honeybees (Apis mellifera) were collected from honey and pollen combs in a healthy queen-right colony (drones were discarded). To avoid intercolony and seasonal variations, bees were taken from the same colony. Immediately before treatment, bees were anaesthetised with carbon dioxide and kept in cages (10.5 x 7.5 x 11.5 cm) by groups of 20.

They were stored in a temperature-controlled chamber in the obscurity at 25 ± 1.5°C and 65%

relative humidity where they remained quiet and protected from stress-induced biochemical changes. Bees were fed a 50% (w/v) sucrose solution ad libitum (EPPO 1992). Experiments were performed in triplicate and repeated at least three times.

Oral application

The honeybees were deprived of food for 2 h before administration of [ 14 C]-imidacloprid.

Radioactive imidacloprid solution was diluted in the 50% (w/v) feeding sucrose solution. The final concentration of DMSO in the sucrose solutions of control and assay tests was 0.02% (v/v).

The dosing solution was prepared freshly for each test. Each bee received 10 µl of 50% sucrose solution (vehicle) containing 10 ng of radiolabeled imidacloprid (100 µg.kg -1 of bee), a dose close to the median lethal dose [START_REF] Suchail | Characteristics of imidacloprid toxicity in two Apis mellifera subspecies[END_REF] or the dosing vehicle alone (control). After consuming this solution, bees had access to 50% straight sucrose solution ad libitum. Metabolic study was performed only on surviving worker bees. Twenty honeybees were sacrificed at specific time: 0, 2, 4, 6, 10, 15, 24, 30, 48 and 72 hours following oral administration. Time 0 corresponded to the end of total ingestion of the imidacloprid dose by honeybees, which took about 20 min.

Extraction of residues from different biological compartments

All tissues and organ were recovered after bees had been anaesthetised with carbon dioxide. After recovery, all tissues and organs were immediately dipped in liquid nitrogen and stored at -80°C, until analysis.

Haemolymph

Haemolymph was collected by puncturing the dorsal aorta with a 5 µL Hamilton microsyringe.

The volume removed per bee was 5 ± 0.1 µL( mean ± SE). To each sample of haemolymph from 20 bees, 4 volumes of an acetonitrile/water (2:1, v/v) solution were added.

Midgut and rectum

The last segment of abdomen was pulled out to extract midgut and rectum. Midgut (mean ± SE:

12 ± 0.1 mg) and rectum (20 ± 0.1 mg) were separated. At each time, midguts and rectums from 20 bees were supplemented with 400 µL of 100 mM Phosphate buffer pH 7.4 and immediately grinded at 0°C. Membrane solubilisation were achieved by adding to 1 volume of extract 4

volumes of 1 M NaOH. After stirring, each sample was stored in the dark during 3 hours at 37°C.

The medium of resulting sample was neutralised with 3 M HCl and supplemented with 1/100 volume of hydrogen peroxide (110 volumes) to reduce the quenching of radioactivity. After an overnight incubation at 37°C, samples were centrifuged at 13000 g for 5 min at room temperature and the supernatant was supplemented with 4 volumes of an acetonitrile/water (2:1, v/v) solution.

The sample was concentrated to obtain 1 mL of final extract.

Head, thorax and abdomen

After extraction of haemolymph, midgut and rectum, the head (mean ± SE:14.4 ± 0.4 mg), thorax (32.4 ± 1 mg) and abdomen (21.9 ± 1.2 mg) were recovered, weighed, plunged in liquid nitrogen and stored at -80°C. Heads and abdomens, and thoraxes of 20 bees were grinded in 4 mL and 5 mL of an acetonitrile/water (2:1, v/v) solution, respectively. Tissue extracts were centrifuged at 25000 g for 30 min at 4°C and the resulting supernatant was concentrated to obtain 1 mL of final extract.

Radioactivity of samples from haemolymph, midgut, rectum, head, thorax and abdomen were measured with a scintillation counter (Tri-Carb 2100 TR, Packard Co.) using scintillation liquid (Ultima Gold, Packard Co.).

Thin-Layer Chromatography and radioanalysis

Silica gel 60F 254 (0.25 mm thickness, Merck) thin layer chromatography (TLC) plates (20x20 cm) were used for analytical separation and identification of imidacloprid and its metabolites.

One hundred µL of samples from head, thorax, abdomen, haemolymph, midgut and rectum extracts were loaded on TLC plates along with reference metabolites.

Total radioactivity half-life

Half-life was determined by plotting ln (I/Io) versus time according to the formula ln (I/Io) = -k . t, where Io and I represent initial and residual imidacloprid concentrations, respectively. Half-life was calculated with the equation t 1/2 = (ln2) / k, where t 1/2 is the half-life and k is the apparent elimination constant.

RESULTS

Total radioactivity

The distribution kinetics of total radioactivity in compartments indicated the existence of two different kinetic profiles (figure 1). In thorax and abdomen, total radioactivity was maximum 20 min after the beginning of exposure and then rapidly decreased with time. In contrast, in haemolymph, head, midgut and rectum, the highest total radioactivity was reached only 2, 6, 10 and 24 h after intoxication of bee, respectively. It was observed that radioactivity level in haemolymph was very low, thus showing that haemolymph was rather a transfer compartment than a stocking compartment. Rectum had the highest level of total radioactivity during the whole kinetic with a maximum 24 h after oral treatment. Elimination of total radioactivity in the whole honeybee follows a first-order kinetic (ρ 2 = 0.8483) and the elimination half-life of radioactivity, which represents the sum of radioactivity of all detected compounds in all honeybee compartments, was about 25 h.

Distribution and metabolism

To understand the role of the metabolites in imidacloprid toxicity, the quantitative and qualitative analysis of each imidacloprid metabolite was performed in 6 biological compartments of honeybee. Our TLC method permitted to separate distinctly imidacloprid, 4,5-dihydroxy-imidacloprid, mono-hydroxy-imidacloprid, 6-chloronicotinic acid, and olefin, guanidine and urea derivatives. However, in honeybees, guanidine and metabolites different from those cited above were never detected. The retention factors (R f ) of imidacloprid and its metabolites are summarized in Table 1. The 5-hydroxy and 4-hydroxy-imidacloprid will be noted 4/5-hydroxy-imidacloprid because it was not possible to separate 5-hydroxy from 4-hydroxyimidacloprid.

In head

The metabolic pattern in head showed 5 metabolites that were detected during the 72-h observation period: imidacloprid, 4/5-hydroxy-imidacloprid, olefin derivative, 4,5-dihydroxyimidacloprid and 6-chloronicotinic acid (figure 2 A). Imidacloprid was distributed very rapidly through the whole bee. Imidacloprid level was about of 5% of the ingested dose during the first 4 hours, 2% between 6 and 24 h, and under the detection threshold after 24 h. Olefin derivative and 4/5-hydroxy-imidacloprid, the two metabolites toxic to honeybees regardless of the dose and mode of treatment, were the main imidacloprid metabolites in this compartment and were detected during 24 and 30 h for 4/5-hydroxy-imidacloprid and olefin derivative, respectively.

However, the level of olefin derivative was always higher than that of 4/5-hydroxy-imidacloprid.

In thorax

In thorax, the metabolites detected were the same than those identified in head, but in higher amounts (figure 2 B). In this compartment, kinetic profiles also showed a rapid distribution and metabolisation of imidacloprid and its metabolites. Imidacloprid appeared in the first 20 min, then decreased and was not detected after 30 h, as in the head. Olefin derivative and 4/5-hydroxyimidacloprid were prevalent during the first 15 h. In contrast with the head, however, 4,5-dihydroxy-imidacloprid and 6-chloronicotinic acid were present in significant amounts.

Moreover, 30 h after intoxication of honeybees, metabolites remained in important proportions.

In abdomen

Distribution profile of imidacloprid and its metabolites was similar to that of thorax (figure 2 C).

Imidacloprid was the main compound on this compartment and represented 38% of ingested radioactivity at the initial time (i.e., 20 min after imidacloprid ingestion). Metabolites were in lower proportions than that of imidacloprid but in similar amounts as in head and thorax.

In haemolymph

Haemolymph was the compartment with the lowest quantity of radioactivity and was the only one without detectable amounts of imidacloprid (figure 2 D). Four metabolites were detected:

4/5-hydroxy-imidacloprid, olefin, 4,5-dihydroxy-imidacloprid and 6-chloronicotinic acid.

Metabolite amounts fluctuated over time, which suggested that transfers of metabolites occurred between compartments.

In midgut

In midgut, 3 compounds were detected: imidacloprid, 6-chloronicotinic and urea derivative (figure 2 E). Urea derivative was never detected in the other compartments and represented a high proportion of the ingested radioactivity (15% of the ingested dose).

In rectum

In rectum, detected compounds were the same than in the midgut (figure 2 F). Imidacloprid, 6-chloronicotinic acid and urea derivative increased during the first 15 hours with a maximum of radioactivity at 15 h, then they decreased until 48 h and were not detected at 72 h. Urea derivative appeared during the first 20 min, following intoxication whereas 6-chloronicotinic acid was detected only 4 h after intoxication. Urea derivative and 6-chloronicotinic acid occurred in rectum at high amounts and represented approximately 15% and 10% of the ingested radioactivity, respectively 15 h after intoxication.

Metabolites in the whole honeybee

The kinetic of imidacloprid and metabolites in the whole honeybee is shown in figure 3. Values represented the sum of the radioactivity amounts detected for each compound in compartments at each time. Imidacloprid corresponded to 55% of ingested radioactivity at the initial time. The guanidine derivative was never detected in the analysed compartments. Elimination half-life of imidacloprid was about 5 h in the whole honeybee. On the basis of the radiolabeling studies, the major metabolites of imidacloprid identified in the whole honeybee were the 6-chloronicotic acid and the urea derivative with 10 and 20% of the ingested radioactivity, respectively, from 2 h to 24 h after intoxication. Moreover, olefin derivative and 4/5-hydroxy-imidacloprid were at maximum 4 h after intoxication. Machemer 1999). In contrast, in honeybees, no metabolites produced by Phase II enzymes were detected either because they occur at very low levels or are not produced by honeybee, or because imidacloprid is able to inhibit Phase II enzymes. Another difference between plants, mammals and honeybees is the lack of guanidine derivative in honeybee. Guanidine derivative is either not formed or formed but quickly transformed because of a rapid turnover, or is in too low quantity to be detected. The last difference between honeybees and plants is that 6-chloronicotinic acid and urea derivatives are the main metabolites in honeybee instead of 5-hydroxy-imidacloprid and olefin derivative in plant. The preferential appearance of these two major metabolites in midgut and rectum suggests that they might be late metabolites in honeybee.

DISCUSSION

On the basis of the metabolites identified and their appearance kinetic, the metabolic pathway of imidacloprid in honeybees was summarised (figure 4). Three major routes of metabolism can be distinguished in honeybee. The first concern the oxidative cleavage of the methylene bond of imidacloprid and/or its metabolites followed by an oxidation to form 6-chloronicotinic acid. The second involves the removal of the nitro group followed by an oxidation to form urea derivative. The third concerns the hydroxylation of the imidazolidine ring to form 4/5-hydroxy-imidacloprid and further 4,5-dihydroxy-imidacloprid. In plants, soils and mammals, it has been proposed that olefin derivative is preferentially formed by the dehydratation of the 4-hydroxy-imidacloprid or 5-hydroxy-imidacloprid [START_REF] Araki | Metabolism of imidacloprid in plants[END_REF][START_REF] Klein | The metabolism of imidacloprid in animals[END_REF]). However, the results obtained in bees show that the appearance of olefin derivative and 4/5-hydroxy-imidacloprid coincides but olefin derivative amounts are always higher. This suggests either that olefin results from the dehydratation of 4/5-hydroxy-imidacloprid and that 4/5-hydroxy-imidacloprid has a very high transformation turnover, or that olefin derivative results from dehydrogenation of imidacloprid, which is subsequently transformed into 4/5hydroxy-imidacloprid after hydratation. In this metabolic scheme, we propose the existence of an intermediary epoxy metabolite that could explain the formation of mono-and dihydroxyimidacloprid. The formation of such mono-and dihydroxylated compounds from an epoxy derivative formed from desaturated molecules has already been demonstrated for various xenobiotics such as aldrin or halobenzene derivatives [START_REF] Woolf | Rat liver cytochrome P-450 isoenzymes as catalysts of aldrin epoxidation in reconstituted monooxygenase systems and microsomes[END_REF][START_REF] Rietjens | Cytochrome P450-catalysed oxidation of halobenzene derivatives[END_REF]).

The results obtained in this metabolic study could explain in part the action of imidacloprid in bees and particularly the discrepancy between rapid intoxication symptoms and late mortality, and the reasons of the evolution of mortality during more that 96 h [START_REF] Suchail | Characteristics of imidacloprid toxicity in two Apis mellifera subspecies[END_REF][START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF]. First, the rapid imidacloprid disappearance and its biotransformation into olefin derivative and 5-hydroxy-imidacloprid, during the first 6 h confirm previous results. In fact, study of imidacloprid biotransformation in whole honeybee by means HPLC-MS/MS shows that olefin derivative and 5-hydroxy-imidacloprid present peak values 4 h after oral intoxication (Suchail et al. submitted). This time period coincides with the appearance of mortality induced by imidacloprid after acute oral intoxication [START_REF] Suchail | Characteristics of imidacloprid toxicity in two Apis mellifera subspecies[END_REF]. This suggests strongly that olefin derivative and/or 5-hydroxy-imidacloprid might be involved in the toxicity of imidacloprid.

However, two experimental results strongly suggest that the involvement of olefin derivative in the imidacloprid toxicity is prevalent. (i) Olefin derivative is 2-time more toxic than imidacloprid and 10-time more toxic than 5-hydroxy-imidacloprid [START_REF] Suchail | Characteristics of imidacloprid toxicity in two Apis mellifera subspecies[END_REF] and (ii) in nAChR-rich tissues such as head and thorax, olefin derivative amounts are about to half of imidacloprid amounts during the first 6 h and are higher than that of imidacloprid after 6 h. In addition, in abdomen, olefin derivative and 4/5-hydroxy-imidacloprid were also in high amounts during a relatively long period. Furthermore, in head, thorax and abdomen, olefin derivative presented a long persistence and was in significant amounts after 30 h following intoxication whereas imidacloprid was no longer detected after 24 h. These results are consistent with a prolonged action of imidacloprid due to the appearance of the most toxic metabolite olefin.

The privileged occurrence of olefin and 4/5-hydroxy-imidacloprid in nAChR-rich tissues suggests that they could interact with the nAChR, probably because these metabolites have a chemical structure very close to that of imidacloprid, and induce the honeybee mortality. In to enhance their affinity for nAChR. Thus, we can assume that the high affinity of olefin and 5-hydroxy-imidacloprid for honeybee nAChR and their strong presence in nAChR-rich tissues confer them an important toxicity to honeybee. Therefore, all results obtained in this present study tend to show that the immediate neurotoxicity symptoms observed in honeybee in our previous toxicological studies are due to the action of imidacloprid, whereas the lethal effect involve 5-hydroxy-imidacloprid and even more so olefin derivative. These two imidacloprid metabolites could act either alone or together with low residual amounts of the parent compound.

The knowledge of in vivo metabolisation pathways of imidacloprid in honeybees helps us to better understand the unusual characteristics of imidacloprid toxicity. This study has shown that olefin and 5-hydroxy-imidacloprid, two minor metabolites in honeybee, might be strongly involved in imidacloprid toxicity. Therefore, it is necessary now to consider as pertinent metabolites not only the main metabolites but also minor metabolites to assess the risk of this compound for honeybee and other non-target-insects. Indeed, in the risk assessment methods of future neonicotinoids, it would be important to take into account, in addition to the parent compound, all metabolites able to induce mortality of non-target-insects. Therefore, the introduction of the new generation of neonicotinoid compounds on the market needs the development of an in-depth understanding of imidacloprid action to better evaluate the potential impact of future neonicotinoids on honeybee colonies as well as on bees in general.

Results indicate that imidacloprid is readily distributed and metabolised into 5 metabolites: 4 / 5 -

 545 hydroxy-imidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin and urea derivatives. The rapid appearance of metabolites in the different biological compartments and the variation of their kinetic profiles suggest the existence of an imidacloprid metabolisation not only in midgut, which is the main place of metabolisation, but also in the other honeybee compartments. Conversely, haemolymph is the compartment with the lowest amount of radioactivity, which confirms that haemolymph is a transfer compartment rather than a storage compartment. It is noteworthy that metabolites detected in these different biological compartments are similar to those found in plants, soils and mammals, but their relative importance are different[START_REF] Araki | Metabolism of imidacloprid in plants[END_REF][START_REF] Thyssen | Nicotinoïd insecticides and the nicotinic acetylcholine receptor[END_REF] Machemer 1999; Robert and Hutson 1999,[START_REF] Rouchaud | Imidacloprid insecticide soil metabolism in sugar beet field crops Bull[END_REF]. All metabolites in honeybee result from imidacloprid biotransformation by mixed function oxidases. These enzymes permit to convert imidacloprid into more polar hydrophilic metabolites, which are more readily excreted, by means of several reactions including hydroxylation, epoxidation and other oxidations. In plants and mammals, imidacloprid biodegradation involves conjugation pathways[START_REF] Araki | Metabolism of imidacloprid in plants[END_REF][START_REF] Thyssen | Nicotinoïd insecticides and the nicotinic acetylcholine receptor[END_REF]

  honeybees,[START_REF] Nauen | Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae)[END_REF] have shown that imidacloprid and only metabolites that contain the nitroguanidine pharmacophore (olefin, 5-hydroxy-and 4,5-dihydroxy-imidacloprid) can displace [ 3 H]-imidacloprid from its receptor binding site. In contrast, only olefin derivative and 5-hydroxy-imidacloprid are able to act agonistically on neurons isolated from the antennal lobe of A. mellifera and to induce mortality. In the housefly Musca domestica, interactions between olefin derivative, 5-hydroxy-imidacloprid and nAChR have been reported by[START_REF] Nauen | Efficacy of plant metabolites of imidacloprid against Myzus persicae and Aphis gossypii (Homoptera: Aphididae)[END_REF].Compared to imidacloprid, olefin derivative exhibits higher affinity to M. domestica head nicotinic receptor, whereas 5-hydroxy-imidacloprid is less specific than imidacloprid but shows high insecticidal activity. Conversely,Liu et al. (1993) and[START_REF] Buckingham | Actions of nitromethylenes on an alpha-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor[END_REF] have shown that the toxicity of imidacloprid and its related compounds correlates closely with their relative affinity for insect nAChRs.[START_REF] Kagabu | Chloronicotinyl insecticides. 8. Crystal and molecular structures of imidacloprid and analogous compounds[END_REF] have shown that the existence of the 2chloropyridine moiety in imidacloprid and in metabolites is particularly important and contributes

Figure 1 :

 1 Figure 1: Distribution kinetic of total radioactivity in different biological compartments of

Figure 2 :

 2 Figure 2: Distribution kinetic of imidacloprid and its metabolites in head (A), thorax (B),

Figure 3 :

 3 Figure 3: Distribution kinetic of imidacloprid and its metabolites in the whole honeybee.

Figure 4 :

 4 Figure 4 : Proposed metabolic pathway of imidacloprid in honeybee. 1: Oxidative cleavage of

TABLE guanidine

 guanidine

			6-CNA	urea	olefin	imid.	4-OH	5-OH	4,5-OH
	R f	0.20	0.43	0.67	0.72	0.78	0.86	0.86	0.93

Table 1 :

 1 Retention factor (R f ) values of imidacloprid and its metabolites. Imidacloprid and its metabolites were separated by one-dimensional TLC. Relative migration of compounds were expressed by R f . 6-CNA, 4-OH, 5-OH, 4,5-OH were 6-chloronicotinic acid, 4-hydroxy-imidacloprid, 5-hydroxy-imidacloprid and 4,5-dihydroxy-imidacloprid, respectively.

ACKNOWLEDGMENTS

We are grateful to Bernard E. Vaissière for critical reading of the manuscript and to Alexandra Badiou and Clothilde Biscarat for their technical assistance. This work was supported in part by a CIFRE agreement between INRA and Bayer AG.


Keywords: imidacloprid, Apis mellifera, honey bees, toxicity, lethal dose, insecticide

Imidacloprid (1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine) belongs to a new chemical family of chloronicotinyl compounds whose mode of action on the insect nervous system differs from that of traditional neurotoxic products. Imidacloprid, a strong systemic compound, is effective against several sucking and mining pests. The acute toxicity of oral and contact applications on two Apis mellifera species, A. m. mellifera and A. m. caucasica, was investigated. The dose-effect relation revealed important characteristics. With low imidacloprid concentrations, a mortality peak appeared with both application modes, especially with the oral mode. With medium doses, mortality profiles at 24 h and 48 h were different only after oral application. The mortality kinetics showed that the higher the imidacloprid dose, the later the mortality. After oral intoxication, the LD50 values of imidacloprid at 24 h and at 48 h were about 5 ng.bee -1 for both A. m. mellifera and A. m. caucasica. After contact application, the LD50 values at 24 h and at 48 h were approximately 24 ng.bee -1 for A. m. mellifera and 14 ng.bee -1 for A. m. caucasica. Imidacloprid ranks among the more potent insecticides when in direct contact with the bees.

INTRODUCTION

The honey bees, Apis mellifera, is widely recognised as an insect of great agronomic, ecological and scientific importance. At the agronomic level, it produces valuable products (honey, pollen, royal jelly, propolis and wax) and plays a major role in crop protection [START_REF] Free | Insect pollination[END_REF].

One of the main problems currently hindering with crop pollination is pollinator losses after the bees contact with pollutants such as pesticides. Hence, because of agronomic and environmental problems efforts have been made to improve the assessment of pesticiderelated risks to bees [START_REF] Eppo | Decision-Making scheme for the environmental risk assessment of plant protection products[END_REF]3].

Recently, the chloronicotinyl molecules a novel class of selective insecticides was discovered, its leading first compound is imidacloprid, a nitroguanidine systemic molecule that has a new mode of molecular action by competiting agonistically with the nicotinic acetylcholine receptor (nAChR) of insects [START_REF] Soloway | Nitromethylene insecticides[END_REF][START_REF] Bai | Actions of imidacloprid and a related nitromethylene on cholinergic receptors of an identified insect motor neurone[END_REF][START_REF] Buckingham | Imidacloprid actions on insect neunoral acetylcholine receptors[END_REF][START_REF] Tomizawa | Pharmacological effects of imidacloprid and its related compouds on the nicotinic acetylcholine receptor with its ion channel from the Torpedo electric organ[END_REF]. Imidacloprid is extremely effective against sucking insects and some heteroptera, coleoptera, lepidoptera species, whereas vertebrates appear to be relatively insensitive to it [START_REF] Liu | High affinity binding of [3H] imidacloprid in the insect acetylcholine receptor[END_REF][START_REF] Yamamoto | Structural factors contributing to insecticidal and selective actions of neonicotinoids[END_REF]. In contrast with nicotine, imidacloprid is a striking example of a product with high insecticide activity and low mammalian toxicity; the oral lethal dose 50 (LD50) in rat was about 450 mg.kg -1 [START_REF] Moriya | 1-(6chloronicotinyl)-2-nitroimino-imidazolidines and related compounds as potential new insecticides[END_REF].

Major applications of imidacloprid include seed dressing, spraying, and the use of pills and granules [START_REF] Pflüger | Ecotoxicological profile of imidacloprid[END_REF]. With seed dressing, insects can be poisoned through the oral way by parent or metabolite compounds. With spraying, nectar can also be contaminated and bees are poisoned either through direct contact with the product or through contact with its residues.

The purpose of this research was to examine the acute effects of imidacloprid to foraging bees. To determinate the intrinsic toxicity of this insecticide to honey bees, studies of laboratory-based dose response were carried out on two Apis mellifera species, A. m. mellifera and A. m. caucasica to provide an estimate of the median lethal dose (LD50) after oral and contact applications. Mortality kinetics were also studied using different imidacloprid doses.

MATERIALS AND METHODS

Materials

The effects of technical grade (98% pure) imidacloprid (1-(6-chloro-3-pyridylmethyl)-Nnitroimidazolidin-2-ylideneamine) from Bayer AG (Leverkussen, Germany) was investigated from June to August. Two honey bees species (A. m. mellifera and A. m. caucasica) mainly foragers were captured from honey and pollen combs in a healthy queen-right colony.

Immediately before treatment, bees were anaesthetised with carbon dioxide and kept in cages (10.5 x 7.5 x 11.5 cm) placed in a temperature-controlled chamber at 25 ± 2°C with 60 ± 10% relative humidity. They were fed a 50% sucrose solution ad libitum [START_REF] Eppo | Guideline on test methods for evaluating the side-effects of plant protection products on honey bees[END_REF]. In each experiment, three cages of twenty bees were used for each dose of treatment. Experiments were conducted at least three times.

Mode of treatment

Oral application

The honey bees were deprived of food for 2 h before administration of imidacloprid.

Imidacloprid solutions were prepared in a 1% dimethylsulfoxide solution (DMSO) and then diluted 10-fold in the 50% (w/v) feeding sucrose solution. The final concentration of DMSO in all tests was 0.1%. The contaminated solutions were prepared extemporaneously for each test. Each bee received 10 µl of 50% sucrose solution containing or not (control group) imidacloprid. After consuming this solution, bees were fed 50% straight sucrose solution ad libitum. Bee mortality was recorded at 2, 4, 6, 10, 14, 20, 24 and 48 h.

Contact application

One µl of insecticide solution in 100% DMSO was applied with a microsyringe on the dorsal part of the bee's thorax. After the application, all bees were fed 50% sucrose solution ad libitum. Control bees received 1 µL of pure solvent. Bee mortality was recorded 24 and 48 h after topical application.

Data analysis

Mortality data were corrected according to Abbott [START_REF] Abbott | Actions of imidacloprid and a related nitromethylene on cholinergic receptors of an identified insect motor neurone[END_REF]. The usual log-probit representation was not used because the dose-effect relation did not systematically increase throughout the range of doses tested. A polynomial regression analysis was used to determine oral and contact LD50. One-way analysis of variance (ANOVA) was used to evaluate statistically significant differences between groups.

-6 -

RESULTS

The toxicity of imidacloprid to foraging bees was investigated with different application modes. For both A. m. mellifera or with A. m. caucasica, within 24 h following imidacloprid application, regardless of dose, most of the bees exhibited neurotoxic symptoms such as trembling, tumbling and lack of coordination.

The contact toxicity of imidacloprid was studied first. In A. m. mellifera, 24-h mortality increased for doses of between 1 and 7 ng.bee -1 and then decreased for doses ranging from 7 to 15 ng.bee -1 (Fig. 1A). At doses higher than 15 ng.bee -1 , the mortality rate increased steadily as the dose increased. This mortality profile was also noted at 48 h; it was less pronounced.

Conversely in A. m. caucasica, at 24 h and 48 h, the mortality peak was less pronounced and occurred at lower doses (Fig. 1B). In both species, there was no significant difference between the mortality rate at 24 h and 48 h.

Oral application, in contrast with contact application, triggered important differences of mortality between 24 and 48 h at intermediate doses. In A. m. mellifera, 24-h and 48-h mortality rates rose with doses up to approximately 50 ng.bee -1 (Fig. 2A). At higher doses, the mortality rate decreased slightly and then started climbing again. This phenomenon was more pronounced with A. m. caucasica (Fig. 2B). At 24 h, doses of up to approximately 20 ng.bee -1 caused a maximum mortality rate of about 90% while doses between 20 and 90 ng.bee -1 made the rate fall to about 60%. For doses between 90 and 200 ng.bee -1 , the mortality rate remained more or less stable, and gradually rose at doses above 200 ng.bee -1 . At 48 h, the mortality profile was different from that obtained at 24 h. There was only a slight decrease in the 48-h mortality rate with doses between 20 and 90 ng.bee -1 and then the mortality gradually went up for both species as the doses of imidacloprid were increased.

The kinetics of mortality in both species was studied using the oral mode of intoxication because (i) with treatment by seed-dressing, the main intoxication mode is the ingestion of contaminated nectar, (ii) a mortality difference only existed in the oral mode between 24 and 48 h and (iii) a mortality peak occurs after the oral application. In A. m. mellifera, with 1 ng.bee -1 , the maximum mortality level was reached within the first ten hours (Fig. 3A). For doses ranging from 5 to 50 ng.bee -1 , the mortality kinetics were similar during the first fourteen hours but at 50 ng.bee -1 mortality rates subsequently increased. At 200 ng.bee -1 , mortality appeared to be delayed since it and was lower than with doses at 5, 10 and 50 ng.bee -1 during at least 20 h, but continually increased after 24 h. A mortality delay was

DISCUSSION

Contact and oral intoxication by imidacloprid induces behavioural abnormalities in the two

Apis mellifera species. Most of the bees showed neurotoxic symptoms such as movement coordination problems, trembling and tumbling. Similar behaviour after imidacloprid application was described in a Coleoptera Diaprepes abbreviatus [START_REF] Quintela | Pathogenicity enhancement of Metarhizium anisopliae and Beauveria bassiana to first instars of Diaprepes[END_REF]. The biological activity of Heteroptera Podisus maculiventris after different imidacloprid applications has also been investigated [START_REF] Cock | Toxicity of diafenthiuron and imidacloprid to the predatory bug Podisus maculiventris (Heteroptera: Pentatomidae)[END_REF]. After 24 h, all pathways of exposure to imidacloprid caused neurotoxic symptoms in most of individuals.

For a given species, imidacloprid toxicity changes with the application mode. In P. maculiventris, the toxicity decreases in the following order: topical exposure > ingestion > residual contact. In A. mellifera, imidacloprid unlike most insecticides is more toxic in the oral mode than in the contact mode. The toxicity of organophosphate insecticides, such as chlorpyrifos, for instance, is four times higher by contact application than by oral application (contact LD50 = 59 ng.bee -1 , oral LD50 = 250 ng.bee -1 ). Similarly, contact application of bifenthrin pyrethroid is seven times more potent than oral application (contact LD50 = 15 ng.bee -1 , oral LD50 = 100 ng.bee -1 ).

The LD50 values of imidacloprid obtained in A. mellifera (LD50 ranging from 4 to 24 ng.bee -1 ) are very low compared with those of insecticides from other families with different modes of action. Three of the most toxic insecticides of different families, the triazophos organophosphate (contact LD50 = 55 ng.bee -1 ), and the cyhalothrin and deltamethrin pyrethroids (contact LD50 = 27 ng.bee -1 , LD50 = 51 ng.bee -1 ), are all less harmful to honey bees than imidacloprid [START_REF] Smart | Laboratory estimation of toxicity of pyrethroid insecticides to honey bees: relevance to hazard in the field[END_REF]. For these insecticides, the highest toxicity is obtained by contact treatment but not by oral treatment, as it is the case for imidacloprid.

Thus, imidacloprid is one of the most potent insecticides to honey bees and should not be applied during the flowering period [START_REF] Pflüger | Ecotoxicological profile of imidacloprid[END_REF]. Applications by seed dressing and granules could preclude a direct effect on honey bees.

Imidacloprid is very selective towards insect species. Imidacloprid has a low insecticidal activity towards Heliothis virescens and Spodoptera Littoralis, two polyphagous pests (at 48 h, contact LD50 = 350 ng.mg -1 of insect, for H. virescens, and LD50 = 650 ng.mg -1 of insect, for S. Littoralis) [START_REF] Lagadic | Topical and oral activities of imidacloprid and cyfluthrin against susceptible laboratory strains of Heliothis virescens and Spodoptera littoralis[END_REF]. Hence, for this species, imidacloprid is not potent enough to efficiently control cotton insect pest populations in the field. On the other hand, imidacloprid is extremely effective against sucking insects such as Myzus persicae (48 h oral LD50 = 3 pg.mg -1 insect) [START_REF] Yu | Detoxication capacity in the honey bee Apis mellifera L[END_REF]. Thus, honey bees have an intermediary sensitivity to imidacloprid (48 h oral LD50 = 50 pg.mg -1 insect) compared with these pest species. The differences in sensitivity between the honey bee and sucking insects is an important feature because in using field rates only toxic to M. persicae, it could be possible to protect honey bees life.

One of the most surprising characteristics of imidacloprid toxicity is the unusual mortality profile observed with the two application modes: honey bee mortality rates rise with low doses of imidacloprid, then, with medium doses fall off, but to rise again, an increase with high doses. Furthermore, with contact application, the 24-h and 48-h mortality rates are similar whereas with oral application, the mortality rate is higher at 48 h than at 24 h. Another interesting observation is that mortality kinetics show that the more the dose increases, the more the mortality is delayed. These particular features of imidacloprid toxicity suggest that metabolic pathways might be involved in the imidacloprid toxicity. With low imidacloprid doses, the few toxic metabolites produced together with the parent compound might be responsible for the high mortality during the first ten hours. Medium doses of imidacloprid may trigger an induction of detoxifying enzymes that reduces honey bees mortality. With higher doses, the increase of mortality could be due either to a high amount of toxic metabolites or to the saturation of the pesticide-metabolising enzymes. This is supported by the fact that honey bees, like other insects, possess various enzymes that can be induced by various chemicals (xenobiotic, plant chemicals…) and can metabolise pesticides [START_REF] Elbert | Imidacloprid -a new systemic insecticide[END_REF].

Induction of cytochrome P-450 has been noticed in honey bees after 2 days of exposure to fluvalinate pyrethroid, which demonstrates that induction of pesticide-metabolising enzymes can occur rapidly [START_REF] Kezic | Induction of mixed function oxidase activity in honey bee as a bioassay for detection of environmental xenobiotics[END_REF]. In polyphagous insects, small quantities of plant substances suffice to induce mixed function oxidases within 30 minutes following initiation of feeding [START_REF] Smirle | Intercolony variation in pesticide detoxification by the honey bee (Hymenoptera : Apidae)[END_REF]. Hence, it is not possible to rule out the possibility of a rapid metabolic activation of imidacloprid as already demonstrated for organophosphate compounds [START_REF] Klein | The metabolism of imidacloprid in plants and soil. Book of abstract[END_REF]. Thus, in imidacloprid toxicity, metabolisation might explain not only the shape of the dose-response curve but also the evolution of mortality between 24 h and 48 h.

Studies in rats show that imidacloprid metabolisation leads to the formation of different main metabolites such as mono-hydroxy-imidacloprid, guanidine-compound, olefin and 6-chloro nicotinic acid [START_REF] Smirle | Intercolony variation in pesticide detoxification by the honey bee (Hymenoptera : Apidae)[END_REF]. It would be interesting to study the toxicity of the main imidacloprid metabolites in A. mellifera to explain the occurrence of a mortality peak when doses are low and the mortality difference at 24 h and 48 h. To understand the mechanism of mortality induced by imidacloprid, this study should be expanded to include the kinetics of imidacloprid degradation and the appearance of metabolites.

ACKNOWLEDGEMENTS

This work is supported in part by the CIFRE convention between INRA and Bayer AG. We are very grateful to Dr. Magali Rault-Leonardon and Drs R. Nauen, H.W. Schmidt and R.

Schmuck for the carefully and critically reading this paper.

also observed in A. m. caucasica, (Fig. 3B). The mortality delay was more pronounced than in A. m. mellifera and systematically increased with the doses, throughout the tests. The higher the dose increased, the longer the mortality was delayed.

The LD50 values of imidacloprid in honey bee species obtained with contact and oral tests are summarised in Table 1. The LD50 values of imidacloprid in Apis mellifera were very low. In A. m. mellifera the LD50 mean at 24 h and 48 h were approximately 4.5 ng.bee -1 and 24 ng.bee -1 for oral and contact application, respectively. In A. m. caucasica, the LD50 means at 24 h and 48 h were approximately 6.5 ng.bee -1 and 14 ng.bee -1 for oral and contact application, respectively. ANOVA tests (p<0.05) indicated a significant difference of sensitivity to imidacloprid between A. m. mellifera and A. m. caucasica for contact application at 24 h but not at 48 h.