Monique Gauthier

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.

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.

The insecticide imidacloprid is a partial agonist of the nicotinic receptor of honeybee Kenyon cells.

The main targets of the insecticide imidacloprid are neuronal nicotinic acetylcholine receptors (nAChRs) within the insect brain. We tested the effects of imidacloprid on ligand-gated ion channels of cultured Kenyon cells of the honeybee, Apis mellifera. Kenyon cells build up the mushroom body neuropils, which are involved in higher order neuronal processes such as olfactory learning. We measured whole-cell currents through nicotinic and γ-aminobutyric acid (GABA) receptors using patch-clamp techniques. Pressure applications of imidacloprid elicited inward currents, which were irreversibly blocked by α-bungarotoxin. Imidacloprid was a partial nicotinic agonist, since it elicited only 36% of ACh-induced currents and competitively blocked 64% of the peak ACh-induced currents. GABA-induced currents were partially blocked when imidacloprid was coapplied and this block was independent upon activation of nAChRs. Our results identify the honeybee nAChR as a target of imidacloprid and an imidacloprid-induced inhibition of the insect GABA receptor.

Contrasting Effects of Imidacloprid on Habituation in 7- and 8-Day-Old Honeybees (Apis mellifera)

We examined the effects of sublethal doses (0.1, 1, and 10 ng per animal) of a new neonicotinoid insecticide, Imidacloprid, on habituation of the proboscis extension reflex (PER) in honeybees (Apis mellifera) reared under laboratory conditions. In untreated honeybees, the habituation of the proboscis extension reflex is age-dependent and there is a significant increase in the number of trials required for habituation in older bees (8-10 days old) as compared to very young bees (4-7 days old). Imidacloprid alters the number of trials needed to habituate the honeybee response to multiple sucrose stimulation. In 7-day-old bees, treatment with Imidacloprid leads to an increase in the number of trials necessary to abolish the response, whereas in 8-day-old bees, it leads to a reduction in the number of trials for habituation (15 min and 1 h after treatment), and to an increase 4 h after treatment. The temporal effects of Imidacloprid in both 7- and 8-day-old bees suggest that 4h after treatment the observed effects are due to a metabolite of Imidacloprid, rather than to Imidacloprid itself. Our results suggest the existence of two distinct subtypes of nicotinic receptors in the honeybee that have different affinities to Imidacloprid and are differentially expressed in 7- and 8-day-old individuals.

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.

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-β-erythroidine and α-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.

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 GABAB 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.

Antennal tactile learning in the honeybee: Effect of nicotinic antagonists on memory dynamics

Restrained worker honeybees (Apis mellifera L.) are able to learn to associate antennal-scanning of a metal plate with a sucrose reinforcement delivered to the mouthparts. Learning occurs reliably in a single association of the two sensory stimuli. The involvement of nicotinic pathways in memory formation and retrieval processes was tested by injecting, into the whole brain through the median ocellus, either mecamylamine (0.6 microg per bee) or alpha-bungarotoxin (2.4 ng per bee). Saline served as a control. Mecamylamine injected 10 min before the retrieval test impairs the retention level tested 3 h and 24 h after single- or multi-trial learning. Retrieval tests performed at various times after the injection show that the blocking effect of mecamylamine lasts about 1 h. The drug has no effect on the reconsolidation or extinction processes. Mecamylamine injected 10 min before conditioning impairs single-trial learning but has no effect on five-trial learning and on the consolidation process. By contrast, alpha-bungarotoxin only impairs the formation of long-term memory (24 h) induced by the five-trial learning and has no effect on medium-term memory (3 h), on single-trial learning or on the retrieval process. Hence, owing to previous data, at least two kinds of nicotinic receptors seem to be involved in honeybee memory, an alpha-bungarotoxin-sensitive and an alpha-bungarotoxin-insensitive receptor. Our results extend to antennal mechanosensory conditioning the role of the cholinergic system that we had previously described for olfactory conditioning in the honeybee. Moreover, we describe here in this insect a pharmacological dissociation between alpha-bungarotoxin sensitive long-term memory and alpha-bungarotoxin insensitive medium-term memory, the last one being affected by mecamylamine.

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%).

Nicotine injected into the antennal lobes induces a rapid modulation of sucrose threshold and improves short-term memory in the honeybee Apis mellifera

In honeybee, although it is known that the food perception and the olfactory memory can be modulated by many environmental and biochemical factors, nothing is known on the effects of nicotine on these processes. Using microinjections of nicotine in the antennal lobes, we show that nicotine at 10(-3) M and 10(-4) M but not at 10(-5) M induced an increase of sucrose sensitivity and that post-training injection of 10(-5) M nicotine improved retention of olfactory learning. These results demonstrate that potentiation of the cholinergic system in the honeybee enhances sucrose perception and facilitates olfactory memory.