Multiple combinations of RDL subunits diversify the repertoire of GABA receptors in the honey bee parasite Varroa destructor.

In insects, γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and GABA-gated ion channels are the target of different classes of insecticides, including fipronil. We report here the cloning of six subunits (four RDL, one LCCH3, and one GRD) that constitute the repertoire of the GABA-gated ion channel family of the Varroa mite (Varroa destructor), a honey bee ectoparasite. We also isolated a truncated GRD subunit with a premature stop codon. We found that when expressed in Xenopus laevis oocytes, three of the four RDL subunits (VdesRDL1, VdesRDL2, and VdesRDL3) formed functional, homomultimeric anionic receptors, whereas GRD and LCCH3 produced heteromultimeric cationic receptors. These receptors displayed specific sensitivities toward GABA and fipronil, and VdesRDL1 was the most resistant to the insecticide. We identified specific residues in the VdesRDL1 pore-lining region that explain its high resistance to fipronil. VdesRDL4 did not form a functional receptor when expressed alone, but it assembled with VdesRDL1 to form a heteromultimeric receptor with properties distinct from those of the VdesRDL1 homomultimeric receptor. Moreover, VdesRDL1 physically interacted with VdesRDL3, generating a heteromultimeric receptor combining properties of both subunits. On the other hand, we did not detect any functional interaction between VdesLCCH3 and the VdesRDL subunits, an observation that differed from what was previously reported for Drosophila melanogaster In conclusion, this study provides insights relevant to improve our understanding of the precise role of GABAergic signaling in insects and new tools for the development of Varroa mite-specific insecticidal agents that do not harm honey bees.

The diamide insecticide chlorantraniliprole increases the single-channel current activity of the mammalian skeletal muscle ryanodine receptor.

Very recently, the diamide insecticide chlorantraniliprole was shown to induce Ca2+-release from sarcoplasmic reticulum (SR) vesicles isolated from mammalian skeletal muscle through the activation of the SR Ca2+ channel ryanodine receptor. As this result raises severe concerns about the safety of this chemical, we aimed to learn more about its action. To this end, single-channel analysis was performed, which showed that chlorantraniliprole induced high-activity bursts of channel opening that accounts for the Ca2+-releasing action described before.

Cardiotoxicity of the diamide insecticide chlorantraniliprole in the intact heart and in isolated cardiomyocytes from the honey bee.

In honey bees, circulation of blood (hemolymph) is driven by the peristaltic contraction of the heart vessel located in the dorsal part of the abdomen. Chlorantraniliprole (CHL) is an insecticide of the anthranilic diamide class which main mode of action is to alter the function of intracellular Ca2+ release channels (known as RyRs, for ryanodine receptors). In the honey bee, it was recently found to be more toxic when applied on the dorsal part of the abdomen, suggesting a direct cardiotoxicity. In the present study, a short-term exposure of semi-isolated bee hearts to CHL (0.1-10 µM) induces alterations of cardiac contraction. These alterations range from a slow-down of systole and diastole kinetics, to bradycardia and cardiac arrest. The bees heart wall is made of a single layer of semi-circular cardiomyocytes arranged concentrically all along the long axis of tube lumen. Since the heart tube is suspended to the cuticle through long tubular muscles fibers (so-called alary muscle cells), the CHL effects in ex-vivo heart preparations could result from the modulation of RyRs present in these skeletal muscle fibers as well as cardiomyocytes RyRs themselves. In order to specifically assess effects of CHL on cardiomyocytes, for the first time, intact heart cells were enzymatically dissociated from bees. Exposure of cardiomyocytes to CHL induces an increase in cytoplasmic calcium, cell contraction at the highest concentrations and depletion of intracellular stores. Electrophysiological properties of isolated cardiomyocytes were described, with a focus on voltage-gated Ca2+ channels responsible for the cardiac action potentials depolarization phase. Two types of Ca2+ currents were measured under voltage-clamp. Exposure to CHL was accompanied by a decrease in voltage-activated Ca2+ currents densities. Altogether, these results show that chlorantraniliprole can cause cardiac defects in honey bees.

Honeybee CaV4 has distinct permeation, inactivation, and pharmacology from homologous NaV channels.

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (NaV), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca2+). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca2+, while excluding sodium ions (Na+), thus defining a new family of Ca2+ channels, known as CaV4. In this study, we characterize CaV4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like NaV and CaV1 channels, respectively. CaV4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as NaV but permeates Ca2+ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium-dependent modulation of voltage-gated Ca2+ and Na+ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of CaV4 and defines this channel as a novel class of voltage-gated Ca2+ channels.

Characterization of the first honeybee Ca²âº channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation.

The honeybee is a model system to study learning and memory, and Ca(2+) signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca(2+) channel subunit. We identified two splice variants of the Apis CaVß Ca(2+) channel subunit (Am-CaVß) and demonstrated expression in muscle and neurons. Although AmCaVß shares with vertebrate CaVß subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVßa and AmCaVßb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVß2a subunit does. However, as opposed to CaVß2a, slow inactivation induced by Am-CaVßa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba(2+) currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVß variants may be important to couple cell signaling to Ca(2+) entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVßa and AmCaVßb N termini, respectively, suggest that AmCaVß splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca(2+) channel activity by CaVß subunit.

Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle.

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.

Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA-A Receptors.

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α2ß2γ2 and α2ß2γ2). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.

Elementary calcium release events in the skeletal muscle cells of the honey bee Apis mellifera.

Calcium sparks are involved in major physiological and pathological processes in vertebrate muscles but have never been characterized in invertebrates. Here, dynamic confocal imaging on intact skeletal muscle cells isolated enzymatically from the adult honey bee legs allowed the first spatio-temporal characterization of subcellular calcium release events (CREs) in an insect species. The frequency of CREs, measured in x-y time lapse series, was higher than frequencies usually described in vertebrates. Honey bee CREs had a larger spatial spread at half maximum than their vertebrate counterparts and a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs' duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation-contraction coupling specificities in honey bee skeletal muscle fibres. The first characterization of CREs from an arthropod which shows strong genomic, ultrastructural and physiological differences with vertebrates may help in improving the research field of sparkology and more generally the knowledge in invertebrates cell Ca2+ homeostasis, eventually leading to a better understanding of their roles and regulations in muscles but also the myotoxicity of new insecticides targeting ryanodine receptors.

Flubendiamide, the first phthalic acid diamide insecticide, impairs neuronal calcium signalling in the honey bee's antennae.

Calcium is an important intracellular second messenger involved in several processes such as the transduction of odour signals and neuronal excitability. Despite this critical role, relatively little information is available with respect to the impact of insecticides on the dynamics of intracellular calcium homeostasis in olfactory neurons. For the first time here, physiological stimuli (depolarizing current or pheromone) were shown to elicit calcium transients in peripheral neurons from the honey bee antenna. In addition, neurotoxic xenobiotics (the first synthetic phthalic diamide insecticide flubendiamide or botanical alkaloids ryanodine and caffeine) do interfere with normal calcium homeostasis. Our in vitro experiments show that these three xenobiotics can induce sustained abnormal calcium transients in antennal neurons. The present results provide a new insight into the toxicity of diamides, showing that flubendiamide drastically impairs calcium homeostasis in antennal neurons. We propose that a calcium imaging assay should provide an efficient tool dedicated to the modern assessment strategies of insecticides toxicity.

Heterogeneous expression of GABA receptor-like subunits LCCH3 and GRD reveals functional diversity of GABA receptors in the honeybee Apis mellifera.

BACKGROUND AND PURPOSE: Despite a growing awareness, annual losses of honeybee colonies worldwide continue to reach threatening levels for food safety and global biodiversity. Among the biotic and abiotic stresses probably responsible for these losses, pesticides, including those targeting ionotropic GABA receptors, are one of the major drivers. Most insect genomes include the ionotropic GABA receptor subunit gene, Rdl, and two GABA-like receptor subunit genes, Lcch3 and Grd. Most studies have focused on Rdl which forms homomeric GABA-gated chloride channels, and a complete analysis of all possible molecular combinations of GABA receptors is still lacking. EXPERIMENTAL APPROACH: We cloned the Rdl, Grd, and Lcch3 genes of Apis mellifera and systematically characterized the resulting GABA receptors expressed in Xenopus oocytes, using electrophysiological assays, fluorescence microscopy and co-immunoprecipitation techniques. KEY RESULTS: The cloned subunits interacted with each other, forming GABA-gated heteromeric channels with particular properties. Strikingly, these heteromers were always more sensitive than AmRDL homomer to all the pharmacological agents tested. In particular, when expressed together, Grd and Lcch3 form a non-selective cationic channel that opens at low concentrations of GABA and with sensitivity to insecticides similar to that of homomeric Rdl channels. CONCLUSION AND IMPLICATIONS: For off-target species like the honeybee, chronic sublethal exposure to insecticides constitutes a major threat. At these concentration ranges, homomeric RDL receptors may not be the most pertinent target to study and other ionotropic GABA receptor subtypes should be considered in order to understand more fully the molecular mechanisms of sublethal toxicity to insecticides.

Excitation-contraction coupling in skeletal muscle fibers from adult domestic honeybee.

Excitation-contraction coupling was characterized in enzymatically isolated adult honeybee skeletal muscle fibers. The voltage-dependent Ca(2+) current (I(Ca)) underlies action potential (AP) depolarization phase in honeybee muscle. A single AP leads to rapid and transient cytoplasmic Ca(2+) increase ("Ca(2+) transient"), which afterwards returns toward baseline following an exponential time course. Trains of APs elicit a staircase increase of Ca(2+), as a result of multiple Ca(2+) transient summation. Surprisingly, the nifedipine-sensitive I(Ca) is blocked by allethrin, a pyrethroid insecticide, revealing myotoxic effects of this neurotoxic insecticide for honeybees. Ca(2+) transients are under the control of Ca(2+) entry through voltage-activated Ca(2+) channels. Indeed, Ca(2+) transient amplitude depends on extracellular Ca(2+) concentration, and bell-shaped relationships are obtained for both I(Ca) integral and the Ca(2+) transient peak in response to depolarizations of increasing amplitude. The slow inactivation kinetics of I(Ca) induces long-lasting Ca(2+) transients that tend to reach a plateau and to return toward a resting level after the end of the stimulation. A Ca(2+)-induced Ca(2+) release mechanism is suggested by two results. First, caffeine (>or=5 mM) and 4-cmc (>0.4 mM), two activators of the sarcoplasmic reticulum Ca(2+) release channels (CRCs), induce Ca(2+) elevations in the absence of extracellular Ca(2+). Second, ryanodine (5 microM) a plant alkaloid that binds specifically to CRCs, depresses voltage-induced Ca(2+) transients. Honeybee muscle fibers represent a valuable model to study invertebrate excitation-contraction coupling and insecticide myotoxicity toward useful insects.

Honey bees long-lasting locomotor deficits after exposure to the diamide chlorantraniliprole are accompanied by brain and muscular calcium channels alterations.

Diamides belong to one of the newest insecticides class. We characterized cellular effects of the first commercialized diamide, chlorantraniliprole (ChlorAnt). ChlorAnt not only induces a dose-dependent calcium release from internal stores of honey bee muscle cells, but also a dose-dependent blockade of the voltage-gated calcium current involved in muscles and brain excitability. We measured a long lasting impairment in locomotion after exposure to a sublethal dose and despite an apparent remission, bees suffer a critical relapse seven days later. A dose that was sublethal when applied onto the thorax turned out to induce severe mortality when applied on other body parts. Our results may help in filling the gap in the toxicological evaluation of insecticides that has recently been pointed out by international instances due to the lack of suitable tests to measure sublethal toxicity. Intoxication symptoms in bees with ChlorAnt are consistent with a mode of action on intracellular calcium release channels (ryanodine receptors, RyR) and plasma membrane voltage-gated calcium channels (CaV). A better coupling of in vitro and behavioral tests may help in more efficiently anticipating the intoxication symptoms.

Voltage-gated sodium channels from the bees Apis mellifera and Bombus terrestris are differentially modulated by pyrethroid insecticides.

Recent experimental and in-field evidence of the deleterious effects of insecticides on the domestic honey bee Apis mellifera have led to a tightening of the risk assessment requirements of these products, and now more attention is being paid to their sublethal effects on other bee species. In addition to traditional tests, in vitro and in silico approaches may become essential tools for a comprehensive understanding of the impact of insecticides on bee species. Here we present a study in which electrophysiology and a Markovian multi-state modelling of the voltage-gated sodium channel were used to measure the susceptibility of the antennal lobe neurons from Apis mellifera and Bombus terrestris, to the pyrethroids tetramethrin and esfenvalerate. Voltage-gated sodium channels from Apis mellifera and Bombus terrestris are differentially sensitive to pyrethroids. In both bee species, the level of neuronal activity played an important role in their relative sensitivity to pyrethroids. This work supports the notion that honey bees cannot unequivocally be considered as a surrogate for other bee species in assessing their neuronal susceptibility to insecticides.

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle.

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.

Whole-cell voltage clamp on skeletal muscle fibers with the silicone-clamp technique.

Control of membrane voltage and membrane current measurements are of strong interest for the study of numerous aspects of skeletal muscle physiology and pathophysiology. The silicone-clamp technique makes use of a conventional patch-clamp apparatus to achieve whole-cell voltage clamp of a restricted portion of a fully differentiated adult skeletal muscle fiber. The major part of an isolated muscle fiber is insulated from the extracellular medium with silicone grease, and the tip of a single microelectrode connected to the amplifier is then inserted within the fiber through the silicone layer. This method represents an alternative to the traditional vaseline-gap isolation and two or three microelectrode voltage-clamp techniques. This chapter reviews the main benefits of the silicone-clamp technique and provides detailed insights into its practical implementation.

Metaflumizone inhibits the honeybee NaV 1 channel by targeting recovery from slow inactivation.

Metaflumizone is the latest addition to the armamentarium of the Na+ channel inhibitor insecticide family. We used the Xenopus oocyte expression system and a Markovian model to assess the effect of metaflumizone on Apis mellifera Na+ channels (AmNaV 1). Our results reveal that metaflumizone inhibits AmNaV 1 channels by targeting the kinetics of recovery from slow inactivation. Multistate modeling of fast and slow inactivation of the AmNaV 1 channel made it possible to study the effects of metaflumizone on a set of rate constants underlying the transition between the open and inactivated conformations and provided insights into their specificity. We conclude that the methods we used could be extended to assessing the toxicity of other Na+ channel inhibitor insecticides.

Stress response in honeybees is associated with changes in task-related physiology and energetic metabolism.

In a rapidly changing environment, honeybee colonies are increasingly exposed to diverse sources of stress (e.g., new parasites, pesticides, climate warming), which represent a challenge to individual and social homeostasis. However, bee physiological responses to stress remain poorly understood. We therefore exposed bees specialised in different tasks (nurses, guards and foragers) to ancient (immune and heat stress) or historically more recent sources of stress (pesticides), and we determined changes in the expression of genes linked to behavioural maturation (vitellogenin - vg and juvenile hormone esterase - jhe) as well as in energetic metabolism (glycogen level, expression level of the receptor to the adipokinetic hormone - akhr, and endothermic performance). While acute exposure to sublethal doses of two pesticides did not affect vg and jhe expression, immune and heat challenges caused a decrease and increase in both genes, respectively, suggesting that bees had responded to ecologically relevant stressors. Since vg and jhe are expressed to a higher level in nurses than in foragers, it is reasonable to assume that an immune challenge stimulated behavioural maturation to decrease potential contamination risk and that a heat challenge promoted a nurse profile for brood thermoregulation. All behavioural castes responded in the same way. Though endothermic performances did not change upon stress exposure, the akhr level dropped in immune and heat-challenged individuals. Similarly, the abdomen glycogen level tended to decline in immune-challenged bees. Altogether, these results suggest that bee responses are stress specific and adaptive but that they tend to entail a reduction of energetic metabolism that needs to be studied on a longer timescale.

Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4.

Bilaterian voltage-gated Na(+) channels (NaV) evolved from voltage-gated Ca(2+) channels (CaV). The Drosophila melanogaster Na(+) channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca(2+)-permeable Na(+) channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca(2+) than Na(+) In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd(2+) and Zn(2+) These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca(2+) and Ba(2+) ions, marginal permeability to Li(+), and impermeability to Mg(2+) and Na(+) ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee's channel, namely slow activation and inactivation kinetics and strong selectivity for Ca(2+) ions.

A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera.

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee's locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD48h, i.e. causing no mortality at 48 h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.

Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels.

Voltage-gated Ca(2+) channels allow the influx of Ca(2+) ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca(2+) transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca(2+) channels, and several genes encoding the two auxiliary subunits, Cavß and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavß subunit (AmelCavßc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca(2+) channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavßc and AmCavα2δ1 produces High Voltage-Activated Ca(2+) channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca(2+) channels.