Pirenzepine Binding Sites in the Brain of the Honeybee Apis mellifera: Localization and Involvement in Non-Associative Learning.

Muscarinic acetylcholine receptors (mAChRs) play a central role in learning and memory in mammals as in honeybees. The results obtained in the honeybee Apis mellifera are based on the detrimental effects of the mAChR antagonists, atropine and scopolamine, on olfactory associative memory. Binding sites for the mAChR antagonist BODIPY® FL pirenzepine were localized in the brain of the honeybee forager. Pirenzepine binding sites were detected indifferently in several somata and neuropilar areas. The highest binding site densities were present in the central complex and in somata of the dorsomedial border of the antennal lobes. An additional binding pattern was found in somata of the subesophageal ganglion. By contrast, Kenyon cell (KC) somata were not stained. Pirenzepine (PZ) effects on non-associative learning were evaluated. Treated animals required more trials for the habituation of the proboscis extension reflex (PER) than controls, and the duration of the PER increased after PZ brain injection. These results suggest that the network mediating habituation of the PER involves PZ binding sites that are not necessarily present on the circuitry mediating olfactory conditioning of the PER.

Amelα8 subunit knockdown in the mushroom body vertical lobes impairs olfactory retrieval in the honeybee, Apis mellifera.

We studied the involvement of the α8 subunit of nicotinic acetylcholine receptors (nAChRs) in olfactory learning and memory in Apis mellifera. We have previously shown, by injecting different nicotinic antagonists into the bee brain, that pharmacologically different subtypes of nAChRs are important for honeybee memory -α-bungarotoxin-sensitive receptors are necessary for memory consolidation and mecamylamine-sensitive receptors are involved in retrieval processes. Here, we took advantage of the honeybee genome sequencing and the development of a small interfering RNA (siRNA) tool to focus on the role of the α8 subunit, which has been shown to be expressed in brain areas important for olfactory learning, such as the antennal lobes and mushroom bodies. We first demonstrated the efficacy of the siRNA tool by showing a decrease of the α8 protein level at 6 h after brain injection of α8 siRNA. We then tested the general role of this subunit in olfactory conditioning, using brain systemic or localized siRNA injections in the antennal lobes or the calyces and vertical lobes of the mushroom bodies. These injections were performed at either 6 h before the learning acquisition or 6 h before the memory test. The most prominent result was that 6-h pre-test injection of siRNA in the mushroom body vertical lobes impaired memory retrieval at 24 and 48 h post-training. This indicated the importance of cholinergic extrinsic neurons and nAChRs containing the α8 subunit for this process.

Insights from honeybee (Apis mellifera) and fly (Drosophila melanogaster) nicotinic acetylcholine receptors: from genes to behavioral functions.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system of insects where they supply fast synaptic excitatory transmission and represent a major target for several insecticides. The unbalance is striking between the abundant literature on nAChR sensitivity to insecticides and the rarity of information regarding their molecular properties and cognitive functions. The recent advent of genome sequencing disclosed that nAChR gene families of insects are rather small-sized compared to vertebrates. Behavioral experiments performed in the honeybee demonstrated that a subpopulation of nAChRs sensitive to the venom α-bungarotoxin and permeant to calcium is necessary for the formation of long-term memory. Concomitant data in Drosophila reported that repetitive exposure to nicotine results in a calcium-dependent plasticity of the nAChR-mediated response involving cAMP signaling cascades and indicated that ACh-induced Ca++ currents are modulated by monoamines involved in aversive and appetitive learning. As in vertebrates, in which glutamate and NMDA-type glutamate receptors are involved in experience-associated synaptic plasticity and memory formation, insects could display a comparable system based on ACh and α-Bgt-sensitive nAChRs.

Expression patterns of nicotinic subunits α2, α7, α8, and ß1 affect the kinetics and pharmacology of ACh-induced currents in adult bee olfactory neuropiles.

Acetylcholine (ACh) is the main excitatory neurotransmitter of the insect brain, where nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee Apis mellifera, nAChRs are expressed in diverse structures including the primary olfactory centers of the brain, the antennal lobes (ALs) and the mushroom bodies (MBs), where they participate in olfactory information processing. To understand the nature and properties of the nAChRs involved in these processes, we performed a pharmacological and molecular characterization of nAChRs on cultured Kenyon cells of the MBs, using whole cell patch-clamp recordings combined with single-cell RT-PCR. In all cells, applications of ACh as well as nicotinic agonists such as nicotine and imidacloprid induced inward currents with fast desensitization. These currents were fully blocked by saturating doses of the antagonists α-bungarotoxin (α-BGT), dihydroxy-ß-erythroidine (DHE), and methyllycaconitine (MLA) (MLA ≥ α-BGT ≥ DHE). Molecular analysis of ACh-responding cells revealed that of the 11 nicotinic receptor subunits encoded within the honeybee genome, α2, α8, and ß1 subunits were expressed in adult Kenyon cells. Comparison with the expression pattern of adult AL cells revealed the supplementary presence of subunit α7, which could be responsible for the kinetic and pharmacological differences observed when comparing ACh-induced currents from AL and Kenyon cells. Together, our data demonstrate the existence of functional nAChRs on adult MB Kenyon cells that differ from nAChRs on AL cells in both their molecular composition and pharmacological properties, suggesting that changing receptor subsets could mediate different processing functions depending on the brain structure within the olfactory pathway.

Honeybee tracking with microchips: a new methodology to measure the effects of pesticides.

Losses of foraging bees are sometimes attributed to altered flight pattern between a meliferous plant treated with an insecticide and the hive. Only a limited number of studies has investigated the impact of pesticides on homing flight due to the difficulty of measuring the flight time between the food source and the hive. Monitoring the flights of the foraging bees needs their individual identification. The number of bees monitored simultaneously and the time span during which observations can be made limit most of the monitoring techniques. However, techniques of automatic tracking and identification of individuals have the potential to revolutionize the study of the ecotoxicological effects of xenobiotics on the bee behaviors. Radio Frequency Identification (RFID) offer numerous advantages such as an unlimited number of codes, a large number of simultaneous recording, and a quick reading, especially through materials (e.g., wood). The aim of this study was to show how the RFID device can be used to study the effects of pesticides on both the behavioral traits and the lifespan of bees. In this context, we have developed a method under tunnel to automatically record the displacements of foragers individualized with RFID tags and to detect the alteration of the flight pattern between an artificial feeder and the hive. Fipronil was selected as test substance due to the lack of information on the effects of this insecticide on the foraging behavior of free-flying bees. We showed that oral treatment of 0.3 ng of fipronil per bee (LD50/20) reduced the number of foraging trips. The strengths of our approach were briefly discussed.

State of the art on insect nicotinic acetylcholine receptor function in learning and memory.

Acetylcholine is the most abundant excitatory neurotransmitter in the insect brain and the most numerous acetylcholine receptors are the nicotinic ones (nAChRs). The genome sequencing of diverse insect species has demonstrated the existence of at least 10 nAChR genes coding for alpha and beta subunits, suggesting the existence in the insect CNS of several subtypes ofnAChRs whose molecular composition and pharmacological properties are still unknown. Insect nAChRs have given rise to an abundance of literature about their sensitivity to neonicotinoid insecticides but only limited data are available on the functional role of nAChRs in insect cognitive functions. The data we have collected on honeybees are the only data that shed light on the role of nAChRs in learning and memory processes. The behavioral response of proboscis extension (PER), which appears when the honeybee perceives sugar, was used to quantify learning and memory performances in associative and non-associative learning procedures. Habituation of the PER, which consists in ceasing to respond to sucrose upon repetitive antennal sucrose stimulation, was facilitated by the injection into the brain of one of the nicotinic antagonists mecamylamine, alpha-bungarotoxin (alpha-BGT) or methyllycaconitine (MLA). Pavlovian associative protocol was used to condition the PER to olfactory or tactile stimulus after single- or multiple-trial training. Localized brain injections of the nicotinic antagonist mecamylamine were performed before or after one-trial olfactory learning in the mushroom bodies (MB), the integrative structures of the insect brain. The results showed that the calical input structures of the MB are necessary for the acquisition processes and the output a-lobe regions are involved in retrieval processes. Brain injection of one of the three nicotinic antagonists mecamylamine, alpha-BGT and MLA was combined with single- and multiple-trial olfactory and tactile learning and memory performances were evaluated at long- or short-term intervals. Mecamylamine impaired the acquisition of one-trial learning and the retrieval of information, regardless of the number of trials during training and the learning modality (olfactory or tactile cues used as conditioned stimulus). Memory performance evaluated at long-term intervals was decreased by injection of alpha-BGT and MLA in multiple-trial olfactory and tactile experiments. We conclude from these results that at least two subtypes of nAChRs exist in the honeybee brain. The alpha-BGT-sensitive nAChRs are necessary for the formation of long-term memory and the alpha-BGT-insensitive nAChRs are involved in one-trial acquisition and in retrieval processes. The hypothesis is put forward that multiple-trial associative learning triggers activation of the alpha-BGT-sensitive nAChRs that, in turn, activate intracellular events leading to LTM formation.

Homomeric RDL and heteromeric RDL/LCCH3 GABA receptors in the honeybee antennal lobes: two candidates for inhibitory transmission in olfactory processing.

gamma-Aminobutyric acid (GABA)-gated chloride channel receptors are abundant in the CNS, where their physiological role is to mediate fast inhibitory neurotransmission. In insects, this inhibitory transmission plays a crucial role in olfactory information processing. In an effort to understand the nature and properties of the ionotropic receptors involved in these processes in the honeybee Apis mellifera, we performed a pharmacological and molecular characterization of GABA-gated channels in the primary olfactory neuropile of the honeybee brain-the antennal lobe (AL)-using whole cell patch-clamp recordings coupled with single-cell RT-PCR. Application of GABA onto AL cells at -110 mV elicited fast inward currents, demonstrating the existence of ionotropic GABA-gated chloride channels. Molecular analysis of the GABA-responding cells revealed that both subunits RDL and LCCH3 were expressed out of the three orthologs of Drosophila melanogaster GABA-receptor subunits encoded within the honeybee genome (RDL, resistant to dieldrin; GRD, GABA/glycine-like receptor of Drosophila; LCCH3, ligand-gated chloride channel homologue 3), opening the door to possible homo- and/or heteromeric associations. The resulting receptors were activated by insect GABA-receptor agonists muscimol and CACA and blocked by antagonists fipronil, dieldrin, and picrotoxin, but not bicuculline, displaying a typical RDL-like pharmacology. Interestingly, increasing the intracellular calcium concentration potentiated GABA-elicited currents, suggesting a modulating effect of calcium on GABA receptors possibly through phosphorylation processes that remain to be determined. These results indicate that adult honeybee AL cells express typical RDL-like GABA receptors whose properties support a major role in synaptic inhibitory transmission during olfactory information processing.

Glutamatergic and GABAergic effects of fipronil on olfactory learning and memory in the honeybee.

We investigated here the role of transmissions mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees, both of these channels being a target for fipronil. To do so, we combined olfactory conditioning with injections of either the GABA- and glutamate-interfering fipronil alone, or in combination with the blocker of glutamate transporter L-trans-Pyrrolidine-2,4-Dicarboxylicacid (L-trans-PDC), or the GABA analog Trans-4-Aminocrotonic Acid (TACA). Our results show that a low dose of fipronil (0.1 ng/bee) impaired olfactory memory, while a higher dose (0.5 ng/bee) had no effect. The detrimental effect induced by the low dose of fipronil was rescued by the coinjection of L-trans-PDC but was rather increased by the coinjection of TACA. Moreover, using whole-cell patch-clamp recordings, we observed that L-trans-PDC reduced glutamate-induced chloride currents in antennal lobe cells. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory memory induced by fipronil.

A new family of receptor tyrosine kinases with a venus flytrap binding domain in insects and other invertebrates activated by aminoacids.

BACKGROUND: Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. METHODS AND FINDINGS: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA(B) receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. CONCLUSION: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.

Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior.

Laboratory bioassays were conducted to evaluate the effects on honeybee behavior of sublethal doses of insecticides chronically administered orally or by contact. Emergent honeybees received a daily dose of insecticide ranging from one-fifth to one-five-hundredth of the median lethal dose (LD50) during 11 d. After exposure to fipronil (0.1 and 0.01 ng/bee), acetamiprid (1 and 0.1 microg/bee), or thiamethoxam (1 and 0.1 ng/bee), behavioral functions of honeybees were tested on day 12. Fipronil, used at the dose of 0.1 ng/bee, induced mortality of all honeybees after one week of treatment. As a result of contact treatment at 0.01 ng/bee, honeybees spent significantly more time immobile in an open-field apparatus and ingested significantly more water. In the olfactory conditioning paradigm, fipronil-treated honeybees failed to discriminate between a known and an unknown odorant. Thiamethoxam by contact induced either a significant decrease of olfactory memory 24 h after learning at 0.1 ng/bee or a significant impairment of learning performance with no effect on memory at 1 ng/bee. Responsiveness to antennal sucrose stimulation was significantly decreased for high sucrose concentrations in honeybees treated orally with thiamethoxam (1 ng/bee). The only significant effect of acetamiprid (administered orally, 0.1 microg/bee) was an increase in responsiveness to water. The neonicotinoids acetamiprid and thiamethoxam tested at the highest dose (one-tenth and one-fifth of their oral LD50, respectively) and fipronil at one-five-hundredth of LD50 have limited effects on the motor, sensory, and cognitive functions of the honeybee. Our data on the intrinsic toxicity of the compounds after chronic exposure have to be taken into account for evaluation of risk to honeybees in field conditions.

Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning.

Although the forelegs of honeybees are one of their main gustatory appendages, tarsal gustation in bees has never been systematically studied. To provide a more extensive account on honeybee tarsal gustation, we performed a series of behavioral experiments aimed at characterizing (1) tarsal sucrose sensitivity under different experimental conditions and (2) the capacity of tarsal sucrose stimulation to support olfactory conditioning. We quantified the proboscis extension reflex to tarsal sucrose stimulation and to odors paired with tarsal sucrose stimulation, respectively. Our experiments show that tarsal sucrose sensitivity is lower than antennal sucrose sensitivity and can be increased by starvation time. In contrast, antennae amputation decreases tarsal sucrose sensitivity. Furthermore, we show that tarsal sucrose stimulation can support olfactory learning and memory even if the acquisition level reached is relatively low (40%).

In situ hybridization assays for localization of the chronic bee paralysis virus in the honey bee (Apis mellifera) brain.

Chronic bee paralysis virus (CBPV) is a common single-stranded RNA virus which may cause significant losses in honey bee colonies. As this virus seems to exhibit neurotropism, an in situ hybridization based method was developed to localize the genomic and antigenomic CBPV RNAs in infected honey bee brains. Double-stranded cDNA probes as well as genomic and antigenomic-specific single-stranded cDNA probes were prepared, using the polymerase chain reaction in presence of labelled d-UTP with non-radioactive digoxigenin. Both genomic and antigenomic RNAs were detected the brain of honey bee infected naturally or artificially. Hybridization signals were obtained in some somata and neuropile regions of the brain. In particular, high signals were observed at the level of the mushroom bodies and central complex, regions that are known to be engaged in higher neuronal functions and in the optic and antennal lobes that are sensorial neuropiles. Thus, the presence of virus at these levels may explain the nervous symptoms observed in infected bees. The in situ hybridization procedure proved to be a useful tool to localize specifically CBPV and may be helpful for understanding the observed symptoms.

Inhibitory neurotransmission and olfactory memory in honeybees.

In insects, gamma-aminobutyric acid (GABA) and glutamate mediate fast inhibitory neurotransmission through ligand-gated chloride channel receptors. Both GABA and glutamate have been identified in the olfactory circuit of the honeybee. Here we investigated the role of inhibitory transmission mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees. We combined olfactory conditioning with injection of ivermectin, an agonist of GluCl receptors. We also injected a blocker of glutamate transporters (L-trans-PDC) or a GABA analog (TACA). We measured acquisition and retention 1, 24 and 48 h after the last acquisition trial. A low dose of ivermectin (0.01 ng/bee) impaired long-term olfactory memory (48 h) while a higher dose (0.05 ng/bee) had no effect. Double injections of ivermectin and L-trans-PDC or TACA had different effects on memory retention, depending on the doses and agents combined. When the low dose of ivermectin was injected after Ringer, long-term memory was again impaired (48 h). Such an effect was rescued by injection of both TACA and L-trans-PDC. A combination of the higher dose of ivermectin and TACA decreased retention at 48 h. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory long-term memory induced by ivermectin. These results illustrate the diversity of inhibitory transmission and its implication in long-term olfactory memory in honeybees.

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains.

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-beta-erythroidine and alpha-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.

Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera).

Acetamiprid and thiamethoxam are insecticides introduced for pest control, but they can also affect non-target insects such as honeybees. In insects, these neonicotinoid insecticides are known to act on acetylcholine nicotinic receptors but the behavioral effects of low doses are not yet fully understood. The effects of acetamiprid and thiamethoxam were studied after acute sublethal treatment on the behavior of the honeybee (Apis mellifera) under controlled laboratory conditions. The drugs were either administered orally or applied topically on the thorax. After oral consumption acetamiprid increased sensitivity to antennal stimulation by sucrose solutions at doses of 1 microg/bee and impaired long-term retention of olfactory learning at the dose of 0.1 microg/bee. Acetamiprid thoracic application induced no effect in these behavioral assays but increased locomotor activity (0.1 and 0.5 microg/bee) and water-induced proboscis extension reflex (0.1, 0.5, and 1 microg/bee). Unlike acetamiprid, thiamethoxam had no effect on bees' behavior under the conditions used. Our results suggest a particular vulnerability of honeybee behavior to sublethal doses of acetamiprid.

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.

Involvement of alpha-bungarotoxin-sensitive nicotinic receptors in long-term memory formation in the honeybee (Apis mellifera).

In the honeybee Apis mellifera, multiple-trial olfactory conditioning of the proboscis extension response specifically leads to long-term memory (LTM) which can be retrieved more than 24 h after learning. We studied the involvement of nicotinic acetylcholine receptors in the establishment of LTM by injecting the nicotinic antagonists mecamylamine (1 mM), alpha-bungarotoxin (alpha-BGT, 0.1 mM) or methyllycaconitine (MLA, 0.1 mM) into the brain through the median ocellus 20 min before or 20 min after multiple-trial learning. The retention tests were performed 1, 3, and 24 h after learning. Pre-training injections of mecamylamine induced a lower performance during conditioning but had no effect on LTM formation. Post-training injections of mecamylamine did not affect honeybees' performances. Pre-training injections of MLA or post-training injection of alpha-BGT specifically induced LTM impairment whereas acquisition as well as memory retrieval tested 1 or 3 h after learning was normal. This indicates that brain injections of alpha-BGT and MLA did not interfere with learning or medium-term memory. Rather, these blockers affect the LTM. To explain these results, we advance the hypothesis that honeybee alpha-BGT-sensitive acetylcholine receptors are also sensitive to MLA. These receptors could be essential for triggering intracellular mechanisms involved in LTM. By contrast, medium-term memory is not dependent upon these receptors but is affected by mecamylamine.

Electrophysiological and behavioural characterization of gustatory responses to antennal 'bitter' taste in honeybees.

We combined behavioural and electrophysiological experiments to study whether bitter taste is perceived at the antennal level in honeybees, Apis mellifera. Our behavioural studies showed that neither quinine nor salicin delivered at one antenna at different concentrations induced a retraction of the proboscis once it was extended in response to 1 M sucrose solution delivered to the opposite antenna. Bees that extended massively their proboscis to 1 M sucrose responded only partially when stimulated with a mixture of 1 M sucrose and 100 mM quinine. The mixture of 1 m sucrose and 100 mM salicin had no such suppressive effect. No behavioural suppression was found for mixtures of salt solution and either bitter substance. Electrophysiological recordings of taste sensillae at the antennal tip revealed sensillae that responded specifically either to sucrose or salt solutions, but none responded to the bitter substances quinine and salicin at the different concentrations tested. The electrophysiological responses of sensillae to 15 mM sucrose solution were inhibited by a mixture of 15 mM sucrose and 0.1 mM quinine, but not by a mixture of 15 mM sucrose and 0.1 mM salicin. The responses of sensillae to 50 mM NaCl were reduced by a mixture of 50 mm NaCl and 1 mM quinine but not by a mixture of 50 mM NaCl and 1 mM salicin. We concluded that no receptor cells for the bitter substances tested, exist at the level of the antennal tip of the honeybee and that antennal bitter taste is not represented as a separate perceptual quality.

Effects of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera).

Fipronil is a phenylpyrazole insecticide introduced for pest control, but it can also affect non-target insects such as honeybees. In insects, fipronil is known to block GABA receptors and to inhibit ionotropic glutamate-gated chloride channels, but the behavioral effects of low doses are not yet fully understood. We have studied the effect of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera) under controlled laboratory conditions. The drug was either administered orally or applied topically on the thorax. A significant reduction of sucrose sensitivity was observed for the dose of 1 ng/bee 1 h after a thoracic application. No significant effect on sucrose sensitivity was obtained with acute oral treatment. A lower dose of fipronil (0.5 ng/bee applied topically) impaired the olfactory learning of the honeybees. By contrast, locomotor activity was not affected. Our results suggest a particular vulnerability of the olfactory memory processes and sucrose perception to sublethal doses of fipronil in the honeybee.