Search

Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags ( v i = 0 ) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E′ of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (τmax) to reach the steady-state were 800 and 1000 s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the τmax were high and similar to human AChE and BuChE.

National audience

International audience; The European honey bee, Apis mellifera, is the most economically valuable pollinator of agricultural crops worldwide. Bees are also crucial in maintaining biodiversity by pollinating numerous plant species whose fertilisation requires an obligatory pollinator. Apis mellifera is a species that has shown great adaptive potential, as it is found almost everywhere in the world and in highly diverse climates. In a context of climate change, the variability of the honey bee's life history traits as regards tempe rature and the environment shows that the species possesses such plasticity and genetic variability that this could give rise to the selection of development cycles suited to new environmental conditions. Although we do not know the precise impact of potential environmental changes on honey bees as a result of climate change, there is a large body of data at our disposal indicating that environmental changes have a direct influence on honey bee development. In this article, the authors examine the potential impact of climate change on honey bee behaviour, physiology and distribution, as well as on the evolution of the honey bee's interaction with diseases. Conservation measures will be needed to prevent the loss of this rich genetic diversity of honey bees and to preserve ecotypes that are so valuable for world biodiversity

Diplôme : DUT

Diplôme : DUT

Social insects live in colonies; each colony, composed of up to hùndreds of thousands or evenmillions of individuals, called workers, behaves as a single integratedentity. A colony reproduces as a unit, and its properties are believed to result from selectionmainly at the level ofthe whole colony (25).Although social insects possess brains capable of sophisticated cognitive functions (22), it is unlikely that an individual colony member has the capacity to acquire and integrate information on the global state ofits colony and how that changes as a result of changing internaI and external conditions (28). One ofthe central problems in social insect biology, therefore, is that ofcolony integration: how the activities of thousands of individual workers are integrated to form a productive colony (37). Colony integration in the insect societies has been studied· intensively, and much has been learned about the mechanisms by which it is governed (4, 5, 32). Several different types of group decision-making activities in insect societies appear to be regulated by processes similar to quorum sensing in bacteria, includingdecisionsonnest sites (32a) and the allocation of labor to different activities.. Identifyingcommonalities · in the social behavior of social insects and microbes might provide ideas on social regulation in general. Studies of sorne species of microorganisms have revealed that the core elements of sociality-altruism and division oflabor-are possible without a brain at aIl (7,29). This chapter teviews our understanding of the regulation of division of labor in honeybee colonies from the perspective of quorum sensing.

International audience; Bee pollinators are currently recorded with many different sampling methods. However, the relative performances of these methods have not been systematically evaluated and compared. In response to the strong need to record ongoing shifts in pollinator diversity and abundance, global and regional pollinator initiatives must adopt standardized sampling protocols when developing large-scale and long-term monitoring schemes. We systematically evaluated the performance of six sampling methods (observation plots, pan traps, standardized and variable transect walks, trap nests with reed internodes or paper tubes) that are commonly used across a wide range of geographical regions in Europe and in two habitat types (agricultural and seminatural). We focused on bees since they represent the most important pollinator group worldwide. Several characteristics of the methods were considered in order to evaluate their performance in assessing bee diversity: sample coverage, observed species richness, species richness estimators, collector biases (identified by subunit-based rarefaction curves), species composition of the samples, and the indication of overall bee species richness (estimated from combined total samples). The most efficient method in all geographical regions, in both the agricultural and seminatural habitats, was the pan trap method. It had the highest sample coverage, collected the highest number of species, showed negligible collector bias, detected similar species as the transect methods, and was the best indicator of overall bee species richness. The transect methods were also relatively efficient, but they had a significant collector bias. The observation plots showed poor performance. As trap nests are restricted to cavity-nesting bee species, they had a naturally low sample coverage. However, both trap nest types detected additional species that were not recorded by any of the other methods. For large-scale and long-term monitoring schemes with surveyors with different experience levels, we recommend pan traps as the most efficient, unbiased, and cost-effective method for sampling bee diversity. Trap nests with reed internodes could be used as a complementary sampling method to maximize the numbers of collected species. Transect walks are the principal method for detailed studies focusing on plant–pollinator associations. Moreover, they can be used in monitoring schemes after training the surveyors to standardize their collection skills.

Après la France et l’Europe, l’Amérique du Nord subit des problèmes de mortalités massives d’abeilles. Un déficit d’abeilles pour la pollinisation et les résultats d’enquêtes auprès des apiculteurs attestent clairement que ce phénomène est très préoccupant pour l’apiculture américaine. Le déclin de ce pollinisateur peut constituer un problème environnemental grave pour les écosystèmes.

Honeybees take part in 80% of Angiosperms pollination. With pollen transport and transfer from flower to flower, they increase fruit and seed set compared to hand pollination. Nevertheless, it was shown in some species that bee contact decreases pollen viability. The aim of this study was to assess the bee contact effect on Cucurbitaceae pollen viability during different times. The studied model is cantaloupe melon (Cucumis melo L.), an economically important crop of Southern France. Pollen was collected and artificially applied to honeybee body hairs. The pollen viability was determined after 1 h; 2.5 h and 4 h of bee contact by fluorochromatic reaction (FCR). After the first hour, the viability of pollen in contact with honeybee body hairs was null whereas the viability of pollen without contact (control) was 45 %. These results show c1early that honeybee body hairs affect the cantaloupe pollen viability since 1 hour of contact. This would be due to substances present on body hairs like cuticular hydrocarbons or secretions of mandibular and labial glands deposited on body hairs by brushing. Studies are in progress (i) to identify the biochemical nature of the substances affecting pollen viability and (ii) to determine the period during which bee can transport viable pollen able to fertilize plant after harvesting.

The possibility to use acetylcholinesterase as biomarker of exposure to deltamethrin insecticide in the honeybee, Apis mellifera were considered. Joined actions of deltamethrin and pirimicarb (carbamate), alone or in association (dual treatment), were investigated on AChE activity in surviving and dead honeybees in order to test its reliability as biomarker. All treatments induced a reduction in tissue AChE activity in dead bees. In surviving bees, deltamethrin treatment induced an important increase of AChE activity that is not abolished by pirimicarb treatment. The analysis of AChE forms revealed an increase in the soluble form in surviving and dead bees and an increase of the membrane form in surviving bees. No direct effect of deltamethrin on soluble and membrane AChE was observed in vitro. The important increase in AChE activity in response to deltamethrin, not altered by pirimicarb treatment, suggests that AChE activity could represent a robust biomarker specific to deltamethrin exposure in living bees.

National audience; New generations of insecticides targeting intracellular Ca2+ release channels (e.g. ryanodine receptors) are considered to be possible alternative tools to overcome resistance to insecticides developed by crop pests. Conversely, honeybees play a major role in plant pollination and in the stability of environmental vegetal biodiversity. For this reason, deleterious ecotoxological effects of insecticides must be as much as possible avoided on these useful insects. We explored here excitation-contraction coupling properties of enzymatically isolated adult honeybee skeletal muscle fibres, i.e., the mechanisms linking electrical excitation of the muscle fibre membrane to the release of Ca2+ by intracellular stores. We used similar experimental conditions as those previously established to characterize electrophysiological properties of this cell preparation with the whole-cell patch-clamp technique. Here, fibres were loaded with the fluorescent Ca2+ indicator Fluo-3, added directly in the patch-clamp pipette and this configuration allowed us to simultaneously monitor Ca2+ variations and electrophysiological responses to various stimuli. Ca2+ transients were both captured under voltage- and current-clamp with a synchronized cooled CCD camera. Ca2+ transients are under the control of Ca2+ entry through voltage-activated Ca2+ channels. This is the first time calcium transients are monitored from honeybee muscle fibres, allowing to explore physiological aspects of excitation-contraction coupling and calcium release from stores through bee ryanodine receptors. These results settle the basis for future prospective ecotoxicological experiments intended for studying the effects of the new generations of insecticides targeting intracellular calcium channels in useful insects.

International audience; Adult honeybee glutamatergic neuromuscular transmission properties have been explored at the macroscopic and single-channel level with the patch-clamp technique on single muscle fibers enzymatically isolated from metathoracic leg tibias. In the whole-cell configuration, dose-responses curves obtained with a computer-operated and pressure-driven fast perfusion system indicate that the two kinetically distinct components activated by L-glutamate have different affinity for the physiological agonist. Moreover, a component of the current, sustained throughout the perfusion with L-glutamate reverses close to 0 mV, while a fast inactivating component of the current reverses at more negative potentials. The glutamate channels agonists and antagonists classically known to affect neuromuscular transmission in skeletal muscle from other invertebrates (quisqualic acid, ibotenic acid and picrotoxin) were used to characterize the glutamatergic transmission pharmacology in honeybee. Single channel currents were also recorded from cell-attached and outside-out patches. The data obtained suggest that both qGluR and GluCl ion channels are present in the membrane of adult honeybee tibia muscle fibers. The single muscle fiber preparation described here provides a convenient way to study adult neuromuscular transmission in invertebrates, as well as its perturbation by pesticides targeting junctional or extrajunctional ionotropic glutamate receptors.

Diplôme : Master Recherche; Over the past years, decline in domestic honeybee populations have been reported in the hives, in France and all over the world. To date, causes responsible for honeybee decline are uncertain although among possible hypothesis, insecticides have been pointed out as possible contributors to the decline of those crucial pollinators. Several insecticides have neurotoxic effects, but very often neurotoxicity alone cannot explain the whole observed phenomenon. In an ecotoxicological approach intended for contributing to the understanding of honeybee decline phenomenon, we explored possible myotoxic effects of a pyrethroid insecticide. We 1 used the patch-clamp electrophysiological method on isolated muscle fibers. Current-clamp results show that allethrin affects honeybee muscle fibers action potentials. In voltage-clamp experiments, voltage-dependent calcium channels appear as the primary molecular target for allethrin. Using allethrin as a pharmacological tool, we also obtained results suggesting the presence of an Ic transient potassium current in honeybee fibers. Experiments confirm that cellular approaches are essential in ecotoxicological studies made on pollinators.; Ces dernières années, des phénomènes de dépopulations ont été constatés dans les cheptels d’abeilles domestiques, en France et partout dans le monde. Les causes de la disparition des abeilles sont à l’heure actuelle incertaines, mais l’hypothèse a été émise que certains insecticides pourraient être en partie responsables du déclin de ces pollinisateurs de premier plan. Plusieurs insecticides ont une action neurotoxique sur l’abeille, mais la neurotoxicité ne peut cependant, à elle seule expliquer les effets observés sur cet insecte. Dans le cadre d’une démarche écotoxicologique visant à contribuer à la compréhension des phénomènes de déclin, nous avons exploré les effets potentiellement myotoxiques d’un insecticide de la classe des pyréthrinoïdes par la technique électrophysiologique de patch-clamp, dans des cellules musculaires isolées. Les résultats obtenus en conditions de courant imposé montrent que l’alléthrine a un effet sur le potentiel d’action de la fibre musculaire d’abeille. Au niveau moléculaire, en condition de potentiel imposé, le canal calcique activé par la dépolarisation semble être la cible préférentielle de l’alléthrine. Par ailleurs, en utilisant l’alléthrine comme un outil pharmacologique, nous avons obtenu des résultats permettant de suggérer la présence d’une composante de courant potassique transitoire de type Ic dans la fibre d’abeille. Ces expériences confirment que l’approche cellulaire est une composante essentielle de l’écotoxicologie lorsque l’on s’intéresse au devenir des pollinisateurs.

International audience; (Introduction) In Europe, 80% of crop species are pollinated mostly by bees (e.g., Fig 1). Bee populations are declining worldwide (Biesmeijer et al. 2006), and this loss may alter the stability of the pollination service they provide (e.g. Chapin et al. 2001). It is therefore urgent to better understand the relationship between bee populations and biogeographic conditions. To explain the abundance and diversity of bees in agro-ecosystems, we looked at the landscape context around the fields

Diplôme : Master

International audience; We document the ability of a population of honey bee colonies to survive in France without Varroa suppression measures. We compared the mortality of collected Varroa surviving bee (VSB) stock with that of miticide-treated Varroa-susceptible colonies. Varroa infestation did not induce mortality in the VSB colonies. Some of the original colonies survived more than 11 years without treatment and the average survival of the experimental colonies was 6.54 ± 0.25 years. Swarming was variable (41.50 ± 9.94%) depending on the year. Honey production was significantly higher (1.7 times) in treated than in VSB colonies. For the first time since Varroa invaded France, our results provide evidence that untreated local honey bee colonies can survive the mite, which may be the basis for integrated Varroa management.

Diplôme : Dr. d'Université; Acetylcholinesterase (AChE, EC 3.1.1.7) is a biological marker of environmental contamination that is widely used in aquatic ecosystems. The necessity of developing tools to monitor the quality of ground and air ecosystems, prompted us to investigate the fundamental properties of AChE in honeybees, Apis mellifera. The identification and the purification of the various forms of honeybee AChE revealed the existence of two membrane forms, AChEm1 and AChEm2, and a majority soluble form, AChEsol1. The two membrane forms represent 95% of the total activity and their physio-chemical properties were determined. The kinetic characterization of all forms in the steady and pre-steady states provides useful information on their catalytic properties as well as on their conformationnal dynamics. A slow equilibrium between several conformational pre-existing states was observed in AChE from Apis mellifera and Drosophila melanogaster. This equilibrium could be the basis of a protective mechanism against toxins. Finally, a comparative study (in vivo/in vitro) of deltamethrin (a pyrethroid) effects on the various forms of AChE is presented. The results suggest a new perspective on the use of AChE as a biomarker.; L’acétylcholinesterase (AChE, EC 3.1.1.7) est un marqueur biologique de contamination environnementale très étudiée et très utilisée dans les milieux aquatiques. La nécessité de développer des outils de surveillance de la qualité des milieux terrestres et aériens nous a conduit à approfondir les connaissances de l’AChE de l’abeille, Apis mellifera. L’identification et la purification des différentes formes d’AChE présentes ont révélé l’existence de deux formes membranaires, AChEm1 et AChEm2, et d’une forme soluble majoritaire, AChEsol1. Les deux formes membranaires représentent 95% de l’activité totale et leurs propriétés physcico-chimiques ont été déterminées. La caractérisation cinétique, à l’état stationnaire et pré-stationnaire, de l’ensemble des formes a fourni des renseignements sur leurs propriétés catalytiques ainsi que sur leur dynamique conformationnelle. Un équilibre lent entre plusieurs états conformationnels pré-existants a été mis en évidence pour chacune des formes d’AChE ainsi que pour la forme soluble de Drosophila melanogaster. Cet équilibre pourrait être à la base d’un mécanisme de protection aux toxiques. Enfin, une étude comparée (in vivo/in vitro) de l’effet de la deltaméthrine (pyréthrinoïde) sur les différentes formes de l’AChE est présentée et permet de proposer une nouvelle perspective d’utilisation de l’AChE en tant que biomarqueur.

National audience; Le tournesol sécrète, notamment au niveau du capitule, des gouttelettes résineuses riches en composés allélopathiques. Ces substances naturelles ont des activités biologiques et participent aux mécanismes de défense contre les insectes phytophages. La présence de sécrétions à proximité des fleurs suggère un contact des abeilles avec des composés allélopathiques pendant la collecte de nectar et de pollen. Dans l’objectif de mieux appréhender la nature des relations tournesol-abeille, la toxicité aiguë de contact ainsi que les effets des sécrétions de tournesol (ST) sur les comportements de l’abeille domestique ont été évalués en laboratoire, en conditions semicontrôlées et au champ.

SUMMARYIn the hive, a wide range of honeybees tasks such as cell cleaning,nursing, thermogenesis, flight, foraging and inter-individual communication(waggle dance, antennal contact and trophallaxy) depend on proper muscle activity. However, whereas extensive electrophysiological studies have been undertaken over the past ten years to characterize ionic currents underlying the physiological neuronal activity in honeybee, ionic currents underlying skeletal muscle fibre activity in this insect remain, so far, unexplored. Here, we show that, in contrast to many other insect species, action potentials in muscle fibres isolated from adult honeybee metathoracic tibia,are not graded but actual all-or-none responses. Action potentials are blocked by Cd2+ and La3+ but not by tetrodotoxin (TTX) in current-clamp mode of the patch-clamp technique, and as assessed under voltage-clamp, both Ca2+ and K+ currents are involved in shaping action potentials in single muscle fibres. The activation threshold potential for the voltage-dependent Ca2+ current is close to–40 mV, its mean maximal amplitude is –8.5±1.9 A/F and the mean apparent reversal potential is near +40 mV. In honeybees, GABA does not activate any ionic membrane currents in muscle fibres from the tibia, but L-glutamate, an excitatory neurotransmitter at the neuromuscular synapse induces fast activation of an inward current when the membrane potential is voltage clamped close to its resting value. Instead of undergoing desensitization as is the case in many other preparations, a component of this glutamate-activated current has a sustained component, the reversal potential of which is close to 0 mV, as demonstrated with voltage ramps. Future investigations will allow extensive pharmacological characterization of membrane ionic currents and excitation–contraction coupling in skeletal muscle from honeybee, a useful insect that became a model to study many physiological phenomena and which plays a major role in plant pollination and in stability of environmental vegetal biodiversity.

Diplôme : IUT; The use of honeybee (Apis mellifera L.) colonies for crop pollination is now a common practice with clear and well known advantages, both from a quantitative and a qualitative standpoint. However, the bases for this practice are still largely lacking in order to optimize colony use within the context of sustainable agriculture and the common interest of beekeepers and farmers. The complexity of honeybees social organisation has led to consider their colonies as true superorganisms made of closely interacting individuals. With this approach in mind, the objective of our assay was to compare in the set environment of an insectproof tunnel the pollinating activity of an 8,000 bee-strong colony with that of two 4,000 bee-strong colonies placed together. We used cantaloupe as a crop model and a total of 5 adjacent tunnels as replicates. The recordings were done daily for 10 days starting with the colony introduction in the tunnels. We assessed the evolution of available resources for bees (nectar and pollen) and recorded visually the foraging activity every 2 hours between 1000 and 1600 h. The pollinating activity was measured by analyzing the stigmatic pollen load on a daily sample of open-pollinated flowers. The fruit set of these flowers was monitored and the fertilization efficiency, measured by the fruit content in filled seeds, was measured. Based on the results from two dates including the day of colony introduction, the number of colonies per tunnel did not affect forager density nor the resulting pollination intensity that averaged 5,200 melon pollen grains per stigma. Fruit set over the 10 days of recording was not homogeneous among the tunnels and it was higher in the presence of two colonies than with a single one. Seed content per fruit was not affected by the colony treatment. The implications of these results are discussed.; L’utilisation de colonies d’abeilles domestiques (Apis mellifera L.) pour la pollinisation des cultures est aujourd’hui une pratique courante dont les avantages, tant sur le plan quantitatif que qualitatif, sont largement reconnus. Cependant les bases de cette pratique font encore largement défaut pour optimiser l’utilisation des colonies dans un contexte d’agriculture durable et dans l’intérêt commun des apiculteurs et des agriculteurs. La complexité de l’organisation sociale chez A. mellifera a conduit à considérer ses colonies comme de véritables super-organismes constitués d’individus vivant en interdépendance. Dans cette optique, notre essai avait pour objectif de comparer, dans l’environnement d’un tunnel « insectproof », l’activité pollinisatrice d’une colonie de 8 000 abeilles à celle de deux colonies de 4 000 abeilles disposées ensemble. Nous avons utilisé le melon comme modèle végétal et un total de 5 tunnels pour nos répétitions. Les relevés ont été effectués pendant 10 jours à partir de l’introduction des colonies dans les tunnels. Nous avons suivi l’évolution des ressources disponibles pour les abeilles (pollen et nectar) et mesuré visuellement l’activité de butinage toutes les 2 heures entre 10h00 et 16h00. L’activité pollinisatrice globale a été mesurée par l’analyse de la charge en pollen de melon des stigmates d’un échantillon quotidien de fleurs en pollinisation libre. Le devenir de ces fleurs (taux de fructification) a été suivi et on a évalué l’efficacité de fécondation par le nombre de graines pleines par fruit. D’après les résultats sur deux dates dont le jour d’introduction des colonies, la charge en colonies par tunnel n’a pas eu d’effet sur l’activité de butinage des abeilles et sur l’activité pollinisatrice qui en a résulté avec une moyenne de 5 200 grains de pollen de melon par stigmate. Sur les 10 jours de suivi, le taux de fructification n’a pas été homogène entre tous les tunnels et il était plus élevé dans les tunnels avec deux colonies que dans ceux avec une seule colonie. Au contraire, le nombre de graines pleines par fruit n’a pas varié avec le traitement. Les implications de ces résultats sont discutés

Two independent genome projects for the honey bee, a microsatellite linkage map and a genome sequence assembly, have interactively produced an almost complete organization of the euchromatic genome., Two independent genome projects for the honey bee, a microsatellite linkage map and a genome sequence assembly, interactively produced an almost complete organization of the euchromatic genome. Assembly 4.0 now includes 626 scaffolds that were ordered and oriented into chromosomes according to the framework provided by the third-generation linkage map (AmelMap3). Each construct was used to control the quality of the other. The co-linearity of markers in the sequence and the map is almost perfect and argues in favor of the high quality of both.

F1000 Factor 8.0 - Exceptional Faculty of 1000 Biology: Evaluated by Taylor Ricketts 14 nov.2006, Gary Luck 9 jan.2007; The extent of our reliance on animal pollination for world crop production for human food has not previously been evaluated and the previous estimates for countries or continents have seldom used primary data. In this review, we expand the previous estimates using novel primary data from 200 countries and found that fruit, vegetable or seed production from 87 of the leading global food crops is dependent upon animal pollination, while 28 crops do not rely upon animal pollination. However, global production volumes give a contrasting perspective, since 60% of global production comes from crops that do not depend on animal pollination, 35% from crops that depend on pollinators, and 5% are unevaluated. Using all crops traded on the world market and setting aside crops that are solely passively self-pollinated, wind-pollinated or parthenocarpic, we then evaluated the level of dependence on animal-mediated pollination for crops that are directly consumed by humans. We found that pollinators are essential for 13 crops, production is highly pollinator dependent for 30, moderately for 27, slightly for 21, unimportant for 7, and is of unknown significance for the remaining 9. We further evaluated whether local and landscape-wide management for natural pollination services could help to sustain crop diversity and production. Case studies for nine crops on four continents revealed that agricultural intensification jeopardizes wild bee communities and their stabilizing effect on pollination services at the landscape scale.

We present a method of statistical inference of demographic and historic parameters in evolutionary scenarios. It is based on the ApproximateBayesian Computation (ABC) approach and deals with complex scenarios involving population divergence and admixture. It has been applied to a honeybee population data set including eight microsatellite loci. The evolutionary scenario involves four populations linked through two divergence and two admixture events. Four groups of populations have been analysed twice to evaluate estimation stability. In addition, mean relative bias and errors have been computed from 500 data sets simulated with known values of parameters (close to estimates on real data), showing that the order of magnitude of all parameters is correctly estimated. Time estimates of divergences are compatible with previous estimates: -0.6 My for lineages M and C divergence and –0.2 My for French and Italian M lineage divergence. The estimated proportion of lineage M alleles in subspecies ligustica, amounting to 0.12, is intermediate between estimates obtained by two different methods. Furthermore, this analysis allows to decompose the previous estimate of 0.35 of lineage M alleles in the population of Courmayeur (Aosta Valley) as 0.23 from the local mellifera and 0.77*0.12 (0.092) from the imported ligustica, making a total of 0.322. The most unexpected result concerns the time of the admixture of lineages M and C that gave rise to the subspecies ligustica. It is estimated at -2,000 years with an approximate credibility interval of (-1,000, -8,000)., Nous présentons une méthode d'inférence statistique sur des paramètres démographiques et historiques de scénarios évolutifs. Cette méthode, fondée sur l'approche Approximate Bayesian Computation (ABC), aborde des scénarios complexes incluant des divergences et des remises en contact de populations. Nous l'avons appliquée à un jeu de données d'abeilles domestiques caractérisées à huit locus microsatellites. Le scénario concerne quatre populations reliées par deux divergences et deux remises en contact. Quatre groupes de populations ont subi chacun une double analyse afin d'évaluer la stabilité des estimations. De plus, les erreurs et biais relatifs moyens, calculés sur 500 jeux de données simulés avec des valeurs de paramètres voisines des estimations obtenues, montrent que l'ordre de grandeur est correctement estimé pour tous les paramètres. Les temps de divergence obtenus sont compatibles avec les estimations antérieures : -0,6 Ma pour la divergence des lignées M et C et -0,2 Ma pour celle des lignées M française et italienne. La proportion d'allèles de la lignée M chez ligustica, 0,12, est intermédiaire entre des estimations obtenues antérieurement par deux méthodes différentes. Notre analyse permet en outre de décomposer l'estimation de cette proportion dans la population de Courmayeur : une partie (0,23) provient de la race locale, l'autre (0,77 ×0,12 = 0,092) provenant des importations de reines ligustica, pour un total de 0,32, voisin de l'estimation antérieure (0,35). Le résultat le plus inattendu concerne la datation de la remise en contact des lignées M et C à l'origine de la race ligustica, qui est estimée à seulement -2000 ans (intervalle de crédibilité à 90 % : -1000, -8000 ans).

International audience; Electrical properties of adult honeybee skeletal muscle fibres enzymatically isolated from the metathoracic leg tibia were explored with the whole-cell patch-clamp technique. In a physiological-like extracellular solution, pressure perfusion (2 psi) with L-glutamate, an excitatory neurotransmitter at the neuromuscular synapse of invertebrates, induced fast activation of an inward current in fibres voltage clamped at a resting membrane potential of -80 mV. During the course of a 3 s application of L-glutamate (1 mM), half of the fibres responded with an initial peak showing fast inactivation, followed by a sustained component, while the other half of the fibres apparently lacked the initial peak. On average, the mean relative amplitude of the sustained component was 49 ± 8% of the initial peak one (n=23, mean ± S.E.M.). Responses to voltage ramps indicated that the sustained current component had a reversal potential around 0 mV. In another set of experiments, single action potentials or trains of action potentials overshooting to ~ +30 mV, were recorded in current-clamp mode in response to current steps from a membrane potential adjusted to -80 mV with slight basal current injection (on average, the resting potential of fibres before adjustment was -47 ± 5 mV, n=9). While the voltage-dependant sodium channel blocker tetrodotoxin (TTX, 1 μM) did not affect action potential responses, cadmium and lanthanum applied together (0.5 and 0.3 mM, respectively) induced disappearance of this all-or-none response. In voltage-clamp, the inward and outward currents underlying this action potential activity were recorded in response to controlled membrane depolarizations from a resting potential of -80 mV, in a physiological-like extracellular solution. In an extracellular solution devoid of sodium but containing 2 mM calcium and the potassium channel blockers tetraethylammonium and 4-aminopyridine, a current with an L-type calcium channel profile was recorded. This current was not affected by TTX, but was completely blocked by Cd-La. The activation threshold potential for this calcium current was ~ - 40 mV and its mean maximal amplitude, reached at ~ 0 mV, was -8.5 ±1.9 A/F (n=13). Individual I-V curves were fitted with equation I(V)=G(V-V)/({1+exp[(V-V)/k]}) and mean values for G, V, V and k were 217 ± 35 S/F, +39.8 ± 4.1 mV, -13.7 ± 1.8 mV and 5.4 ± 0.5 mV, respectively (n=13). This original cell preparation appears particularly suitable for patchclamp studies and future investigations will allow a complete characterization of the pharmacological sensitivity of its membrane ionic currents as well as its calcium release apparatus properties, in order to explore excitation-contraction coupling in this insect model.

Fink, Friedich (eds) ; International audience; Adult honeybee skeletal muscle fibers were enzymatically isolated from the metathoracic tibia (third pair of legs). Their electrophysiological properties were explored with the whole-cell patch-clamp technique. In a physiological-like extracellular solution, both inward and outward currents underlying an action potential activity were recorded in response to controlled membrane depolarizations from a resting potential of -80 mV. In an extracellular solution devoid of sodium but containing 2 mM calcium and the potassium channel blockers TEA and 4-AP, a current with an L-type calcium channel profile was recorded. The activation threshold potential for this current was ~ -40 mV and its mean maximal amplitude, reached at ~ 0 mV, was -8.5 ±1.9 A/F (n=13, mean ± S.E.M). Individual I/V curves were fitted with equation I(V)=Gmax(V-Vrev)/({1+exp[(V0.5-V)/k]}) and mean values for Gmax, Vrev, V0.5 and k were 217 ± 35 S/F, +39.8 ± 4.1 mV, -13.7 ± 1.8 mV and 5.4 ± 0.5 mV respectively (n=13). Responses to L-glutamate, an excitatory neurotransmitter at the neuromuscular synapse of invertebrates were also recorded in the whole-cell configuration. This cell preparation appears quite suitable for patch-clamp studies, and further work will be made on the pharmacological sensitivity of ionic currents in this preparation, including their possible perturbations by insecticides compounds.

In vivo metabolization of acetamiprid (AAP) was studied in the honeybee, Apis mellifera. The distribution of AAP and its metabolites was monitored during 72 h in six biological compartments: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 μg of [14C]-AAP per kg of bee, a dose about 1500 times lower than the median lethal dose at 72 h. Surprisingly, after 72 h, only 40% of the total radioactivity was eliminated, suggesting that AAP and its metabolites tended to persist in the honeybee. AAP was rapidly distributed in all compartments and metabolized. Just after AAP administration, radioactivity was mainly localized in the abdomen, and then in the rectum. After 72 h, the maximal amount of radioactivity (about 20% of the ingested dose) was recovered again in the abdomen, whereas the lowest amount of total radioactivity was recovered in the haemolymph. Radioactivity in the head did not exceed 7.6% of total ingested radioactivity. More than 50% of AAP was metabolized in less than 30 min indicating a very low half-life of AAP. During the first hours, AAP was mainly detected in nicotinic acetylcholine receptor-rich tissues. Among the 7 detected metabolites, the major metabolites were the 6-choronicotinic acid and an unknown metabolite called U1, present mainly in the rectum, the thorax and the head. Our results indicated that the low toxicity of AAP is a reflection of its rapid and efficient metabolization despite of the persistence of AAP metabolites in the honeybee., La métabolisation de l’acétamipride (AAP) a été étudiée chez l’abeille Apis mellifera. Le suivi de l’AAP et des ses métaboliques est réalisé durant 72 h dans six compartiments biologiques que sont l’intestin, l’hémolymphe, le thorax, la tête, l’abdomen et le rectum. Les abeilles ont ingérées une dose 1500 fois plus faible que la Dose Létale médiane (DL50) soit 100 μg de [14C]-AAP par kg, une dose comparable à celle à la DL50 de l’imidaclopride (IMI). Le fait le plus marquant de cette étude est de retrouver plus de 60% de la dose ingérée après 72h. Toutefois, l’AAP est très rapidement distribué et métabolisé dans l’organisme. Après l’ingestion, la radioactivité retrouvée est principalement localisée dans l’abdomen puis dans le rectum. A 72 h, 20% de la dose ingérée est retrouvée au niveau de l’abdomen. L’hémolymphe est le compartiment où la présence de radioactivité est constamment la plus faible. Au niveau de la tête, la quantité de radioactivité n’excède pas 7,6% de la quantité initiale. L’AAP présente une demi-vie très faible (inférieure à 30 min.) dans l’organisme. Toutefois, l’AAP est localisé dans les premiers temps au niveau des compartiments riches en cibles principales, les récepteurs nicotiniques à l’acétylcholine. Parmi les 7 métabolites détectés, deux métabolites sont majeurs : l’acide 6-chloronicotinique et un métabolite inconnu U1. Cette étude nous permet d’affirmer que la plus faible toxicité de l’AAP par rapport à celle de l’IMI est due à une détoxication plus efficace et ce, malgré la persistance de métabolites dans l’organisme.

National audience

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

Diplôme : DUT

Diplôme : Master

Honey bees undergo an age-related, socially regulated transition from working in the hive to foraging that has been previously associated with changes in the expression of thousands of genes in the brain. To understand the meaning of these changes, we conducted microarray analyses to examine the following: ( i ) the ontogeny of gene expression preceding the onset of foraging, ( ii ) the effects of physiological and genetic factors that influence this behavioral transition, and ( iii ) the effects of foraging experience. Although >85% of ≈5,500 genes showed brain differences, principal component analysis revealed discrete influences of age, behavior, genotype, environment, and experience. Young bees not yet competent to forage showed extensive, age-related expression changes, essentially complete by 8 days of age, coinciding with previously described structural brain changes. Subsequent changes were not age-related but were largely related to effects of juvenile hormone (JH), suggesting that the increase in JH that influences the hive bee–forager transition may cause many of these changes. Other treatments that also influence the onset age of foraging induced many changes but with little overlap, suggesting that multiple pathways affect behavioral maturation. Subspecies differences in onset age of foraging were correlated with differences in JH and JH-target gene expression, suggesting that this endocrine system mediates the genetic differences. We also used this multifactorial approach to identify candidate genes for behavioral maturation. This successful dissection of gene expression indicates that, for social behavior, gene expression in the brain can provide a robust indicator of the interaction between hereditary and environmental information.

L’abeille domestique, élément de la faune pollinisatrice endémique de France. Quel est le rayon d’action d’une colonie d’abeilles domestiques ? Quel est l’impact des abeilles domestiques dans le maintien de la biodiversité ? Accueillir l’abeille domestique dans les Parcs naturels, … … c’est reconnaître son impact positif sur la biodiversité et apprécier l’apiculture comme un élevage particulier. Un risque négligeable.

National audience; Les canaux ioniques sous-tendent l’activité électrique physiologique de cellules telles que les neurones et les cellules musculaires. Par le passé, les expériences effectuées sur les invertébrés ont donné lieu à plusieurs découvertes fondamentales dans les domaines de la conduction axonale, la transmission synaptique mais aussi la neurobiologie intégrative et le comportement. Parmi les invertébrés, l’abeille (Apis mellifera) représente dans ces domaines l’un des modèles d’étude les plus pertinents car (i) la morphologie de son système neuro-musculaire est relativement simple et (ii) les interactions interindividuelles nécessaires à sa vie sociale hiérarchisée montrent qu’il existe une extrême complexité et une finesse de régulation des fonctions de locomotion, de thermogenèse, de vision, d’olfaction, de communication chimique ainsi que des processus impliqués dans l’apprentissage et la mémorisation. Outre son intérêt agro-économique et environnemental, l’abeille présente donc un grand intérêt scientifique fondamental. Au niveau environnemental, elle contribue notablement au maintien de la biodiversité floristique et pour cette raison, l’abeille est un insecte protégé par les lois Européennes : dans le cadre de l’homologation des nouvelles molécules insecticides, l’innocuité des produits doit être démontrée sur cet insecte utile. Plusieurs classes d’insecticides ont vraisemblablement un mode d’action commun sur les canaux ioniques des cellules des systèmes nerveux et neuro-musculaire. Nous avons pour la première fois ici mis au point une préparation de cellules musculaires isolées à partir d’abeilles adultes. Les propriétés d’excitabilité de ces cellules ont été explorées par la technique électrophysiologique de patch-clamp dans sa configuration cellule entière. Plusieurs canaux ioniques voltage-dépendants ou ligand-activés ont été identifiés. Electrophysiological study of macroscopic ionic currents in adult honeybee skeletal muscle fiber using the patch-clamp technique. Ion channels underlie the physiological activity of electrically excitable cells such as neurons or muscle cells. In the past, important discoveries concerning axonal conduction, synaptic transmission, integrative neurobiology and behavior were made using invertebrates. Among invertebrates, honeybee is one of the most useful models, owing to (i) the relative simplicity of its neuromuscular system and (ii) the great complexity and the fine tuning of physiological functions both needed for the social interactions to take place in the hive (locomotion, thermogenesis, vision, olfaction, learning and memory). Not only honeybee is important on agro-economical and environmental points of view, but also in scientific research. It also notably contributes to vegetal biodiversity stability and for those reasons, honeybee is protected by European laws: new insecticide compounds must prove to be non-toxic on this useful insect. Several insecticides likely affect ion channels of both neural and neuromuscular systems. Here, we have for the first time designed a procedure to isolate adult honeybee skeletal muscle fibers. Electrophysiological properties of those cells were explored with the whole-cell patch-clamp technique. Several voltage-dependant or ligand-activated ion channels have been identified.

Egalement publié dans : Santé de l'Abeille (La), 2007 (217, 15-17 Auteurs : Le Conte Y., Crauser D., Bécard, J.M. et dans : dans Abeille de France et l'Apiculteur, 2007 (932), 26 Auteurs : Y. Le Conte, D. Crauser, J.M. Bécard; De nombreux exemples montrent qu’à terme les acariens deviennent résistants aux acaricides utilisés pour contrôler leurs populations. L’amitraze est utilisée depuis plus de 23 ans pour lutter contre le varroa et il se pose régulièrement la question de l’apparition de populations résistantes. Cet article traite de l’efficacité de cette molécule sur des populations de varroas en Avignon.

A brood pheromone identified in honeybee larvae has primer and releaser pheromone effects on adult bees. Using gas chromatography-mass spectrometry (GC-MS) to evaluate fatty acid esters--the pheromonal compounds--in different parts of the larvae, we have localized the source of the esters as the larval salivary glands. A histochemical study describes the glands and confirms the presence of lipids in the glands. Epithelial cells of the gland likely secrete the fatty acids into the lumen of the gland. These results demonstrate the salivary glands to be a reservoir of esters, components of brood pheromone, in honeybee larvae.

Maîtres de stage : Le Conte, Y. et Costagliola, G. *INRA UMR 406 UAPV Ecologie des Invertébrés Avignon (FRA) Diffusion du document : INRA UMR 406 UAPV Ecologie des Invertébrés Avignon (FRA)

International audience; Using a conditioned proboscis extension response (PER) assay, honeybees (Apis mellifera L.) can be trained to associate an odor stimulus with a sucrose reward. Previous studies have shown that observations of conditioned PER were of interest for assessing the behavioral effects of pesticides on the honeybee. In the present study, the effects of sublethal concentrations of nine pesticides on learning performances of worker bees subjected to the PER assay were estimated and compared. Pesticides were tested at three concentrations. The highest concentration of each pesticide corresponded to the median lethal dose value (48-h oral LD50), received per bee and per day, divided by 20. Reduced learning performances were observed for bees surviving treatment with fipronil, deltamethrin, endosulfan, and prochloraz. A lack of behavioral effects after treatment with lambda -cyalothrin, cypermethrin, tau -fluvalinate, triazamate, and dimethoate was recorded. No-observed-effect concentrations (NOECs) for the conditioned PER were derived for the studied pesticides. Our study shows that the PER assay can be used for estimating sublethal effects of pesticides on bees. Furthermore, comparisons of sensitivity as well as the estimation of NOECs, useful for regulatory purposes, are possible.

Maître de stage : Belzunces, C. *UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon Diffusion du document : UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon Diplôme : Master Recherche

National audience

Maître de stage : Le Conte, Y. *UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon Diffusion du document : UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon Diplôme : Licence

Maîtres de stage : Le Conte, Y. et Costagliola, G. *UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon (FRA) Diffusion du document : UMR 406 INRA UAPV Ecologie des Invertébrés, Avignon (FRA) Diplôme : Master

Le laboratoire de l’United States Department of Agriculture à Baton Rouge possède une renommée internationale pour la qualité et la quantité des travaux menés sur l’élevage et la sélection des abeilles. Une collaboration entre un des chercheurs de ce laboratoire, John Harbo, et l’INRA a rendu possible une visite dans cet endroit mythique de l’abeille

A l’issue du Colloque européen d’apidologie, qui s’est tenu à Udine en septembre dernier, un certain nombre de groupes de travail se sont formés, fédérant des scientifiques de différents domaines, comme la pathologie ou la sélection des abeilles.

National audience

7. ed. Mention d'édition : 7. ed.