Prolonged effects of in-hive monoterpenoids on the honey bee Apis mellifera.

Honey bees are exposed in their environment to contaminants but also to biological stressors such as Varroa destructor that can weaken the colony. Preparations containing monoterpenoids that are essential oil components, can be introduced into hives to control Varroa. The long-term sublethal effects of monoterpenoids used as miticides have been poorly investigated. Analysis of behavior of free-moving bees in the laboratory is useful to evaluate the impact of chemical stressors on their cognitive functions such as vision function. Here, the walking behavior was quantified under a 200-lux light intensity. Weeks and months after introduction of the miticide (74 % thymol) into the hives, decreases of phototaxis was observed with both summer and winter bees. Curiously, in spring, bees collected in treated hives were less attracted by light in the morning than control bees. The survival of bees collected in spring was increased by treatment. After a 1-year period of observation, the colony losses were identical in treated and non-treated groups. Colony loss started earlier in the non-treated group. In public opinion, natural substances as essential oils are safer and more environmentally friendly. We demonstrated that a monoterpenoid-based treatment affects bee responses to light. The latter results have notable implications regarding the evaluation of miticides in beekeeping.

Varroa destructor relies on physical cues to feed in artificial conditions.

Olfaction is a major sense in Varroa destructor. In natural conditions, it is known that this honey bee parasite relies on kairomones to detect its host or to reproduce. Yet, in artificial conditions, the parasite is able to feed and survive for a few days even though most honey bee pheromones are lacking. Other key cues are thus probably involved in V. destructor perception of its close environment. Here, we used several artificial feeding designs to explore the feeding behaviour of the parasite when it is deprived of olfactory cues. We found that V. destructor is still able to feed only guided by physical cues. The detection of the food source seems to be shape-related as a 3D membrane triggers arrestment and exploration more than a 2D membrane. The tactile sense of V. destructor could thus be essential to detect a feeding site, although further studies are needed to assess the importance of this sense combined with olfaction in natural conditions.

Title: Varroa destructor s'appuie sur des signaux physiques pour se nourrir dans des conditions artificielles. Abstract: L'olfaction est un sens prépondérant chez Varroa destructor. En conditions naturelles, ce parasite de l'abeille domestique dépend en effet de kairomones qui lui permettent de détecter son hôte ou de se reproduire. Pourtant, lorsqu'il se retrouve en conditions artificielles, le parasite se nourrit et survit plusieurs jours malgré l'absence de la majorité des phéromones émises par l'abeille. Des indices clés autres qu'olfactifs sont donc très probablement impliqués dans la perception de l'environnement de V. destructor. Dans cette étude, plusieurs dispositifs d'alimentation artificielle ont été testés afin d'explorer le comportement de nourrissage du parasite lorsqu'il est privé d'indices olfactifs. Les résultats montrent que V. destructor est tout à fait capable de se nourrir en étant uniquement guidé par des indices physiques. En l'occurrence, la détection de la source nutritive semble être liée à sa forme puisqu'une membrane 3D provoque des comportements exploratoires plus prononcés qu'une membrane plane (2D). Le sens du toucher serait donc essentiel à V. destructor pour trouver son site de nourrissage. Des études complémentaires permettraient néanmoins d'évaluer les importances relatives des sens olfactif et tactile en conditions naturelles.

Identification, localization and function of glutamate-gated chloride channel receptors in the honeybee brain.

Glutamate-gated chloride channels (GluCls) are members of the cys-loop ligand-gated ion channel superfamily whose presence has been reported in a variety of invertebrate tissues. In the honeybee, a single gene, amel_glucl, encoding a GluClα subunit, was found in the genome but both the pattern of expression of this gene in the bee brain and its functional role remained unknown. Here we localised the expression sites of the honeybee GluClα subunit at the mRNA and protein levels. To characterise the functional role of GluCls in the honeybee brain, we studied their implication in olfactory learning and memory by means of RNA interference (RNAi) against the GluClα subunit. We found that the GluClα subunit is expressed in the muscles, the antennae and the brain of honeybees. Expression of the GluClα protein was necessary for the retrieval of olfactory memories; more specifically, injection of dsRNA or siRNA resulted in a decrease in retention performances ∼24 h after injection. Knockdown of GluClα subunits impaired neither olfaction nor sucrose sensitivity, and did not affect the capacity to associate odor and sucrose. Our data provide the first evidence for the involvement of glutamate-gated chloride channels in olfactory memory in an invertebrate.

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.

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.

Thymol Affects Congruency Between Olfactory and Gustatory Stimuli in Bees.

Honey bees learn to associate sugars with odorants in controlled laboratory conditions and during foraging. The memory of these associations can be impaired after exposure to contaminants such as pesticides. The sub-lethal effects of acaricides such as 5-methyl-2-(propan-2-yl)-phenol (thymol) introduced into colonies to control varroa mites are of particular concern to beekeeping, due to detrimental effects of some acaricides on bees. Here we assess whether various odorant/sugar pairs are identically memorized in a differential appetitive olfactory conditioning experiment and whether this learning is affected by thymol exposure. Responses to odorants in retrieval tests varied according to the sugar they were paired with, a property called congruency. Interestingly, congruency was altered by pre-exposure to some thymol concentrations during retrieval tests, although electroantennography recordings showed it left odorant detection intact. This highlights the importance of taking into account subtle effects such as odor/sugar congruency in the study of the effect of pesticides on non-target insects, in addition to the simpler question of memory impairment.

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.

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.

Monoterpenoid-based preparations in beehives affect learning, memory, and gene expression in the bee brain.

Bees are exposed in their environment to contaminants that can weaken the colony and contribute to bee declines. Monoterpenoid-based preparations can be introduced into hives to control the parasitic mite Varroa destructor. The long-term effects of monoterpenoids are poorly investigated. Olfactory conditioning of the proboscis extension reflex (PER) has been used to evaluate the impact of stressors on cognitive functions of the honeybee such as learning and memory. The authors tested the PER to odorants on bees after exposure to monoterpenoids in hives. Octopamine receptors, transient receptor potential-like (TRPL), and γ-aminobutyric acid channels are thought to play a critical role in the memory of food experience. Gene expression levels of Amoa1, Rdl, and trpl were evaluated in parallel in the bee brain because these genes code for the cellular targets of monoterpenoids and some pesticides and neural circuits of memory require their expression. The miticide impaired the PER to odors in the 3 wk following treatment. Short-term and long-term olfactory memories were improved months after introduction of the monoterpenoids into the beehives. Chronic exposure to the miticide had significant effects on Amoa1, Rdl, and trpl gene expressions and modified seasonal changes in the expression of these genes in the brain. The decrease of expression of these genes in winter could partly explain the improvement of memory. The present study has led to new insights into alternative treatments, especially on their effects on memory and expression of selected genes involved in this cognitive function. Environ Toxicol Chem 2017;36:337-345. © 2016 SETAC.

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.

Effect of a thymol application on olfactory memory and gene expression levels in the brain of the honeybee Apis mellifera.

Essential oils are used by beekeepers to control the Varroa mites that infest honeybee colonies. So, bees can be exposed to thymol formulations in the hive. The effects of the monoterpenoid thymol were explored on olfactory memory and gene expression in the brain of the honeybee. In bees previously exposed to thymol (10 or 100 ng/bee), the specificity of the response to the conditioned stimulus (CS) was lost 24 h after learning. Besides, the octopamine receptor OA1 gene Amoa1 showed a significant decrease of expression 3 h after exposure with 10 or 100 ng/bee of thymol. With the same doses, expression of Rdl gene, coding for a GABA receptor subunit, was not significantly modified but the trpl gene was upregulated 1 and 24 h after exposure to thymol. These data indicated that the genes coding for the cellular targets of thymol could be rapidly regulated after exposure to this molecule. Memory and sensory processes should be investigated in bees after chronic exposure in the hive to thymol-based preparations.

Regional brain variations of cytochrome oxidase staining during olfactory learning in the honeybee (Apis mellifera).

Regional brain variations of cytochrome oxidase (CO) staining were analyzed in the honeybee (Apis mellifera) after olfactory conditioning of the proboscis extension reflex. Identification of brain sites where stimuli converge was done by precise image analysis performed in antennal lobes (AL) and mushroom bodies (MB). In Experiment 1, bees received 5 odorant stimulations that induced a transient decrease of CO activity in the lateral part of the AL. In Experiment 2, bees were trained with 5-trial olfactory conditioning. CO activity transiently increased in the lips of the MB calyces. There was also a delayed increase in the lateral part of the AL. An olfactory stimulus presented alone and an odor paired to a sucrose stimulation are treated by different pathways, including both AL and MB.

Thymol as an alternative to pesticides: persistence and effects of Apilife Var on the phototactic behavior of the honeybee Apis mellifera.

Thymol is a natural substance increasingly used as an alternative to pesticides in the fight against the Varroa destructor mite. Despite the effectiveness of this phenolic monoterpene against Varroa, few articles have covered the negative or side effects of thymol on bees. In a previous study, we have found an impairment of phototaxis in honeybees following application of sublethal doses of thymol-lower or equal to 100 ng/bee-under laboratory conditions. The present work shows the same behavioral effects on bees from hives treated with Apilife Var®, a veterinary drug containing 74 % thymol, with a decrease in phototactic behavior observed 1 day after treatment. Thus, thymol causes disruption of bee phototactic behavior both under laboratory conditions as well as in beehives. The bee exposure dose in treated hives was quantified using gas chromatography coupled to mass spectrometry (GC-MS), giving a median value of 4.3 µg per body 24 h after treatment, with 11 ng in the brain. The thymol level in 20 organic waxes from hives treated with Apilife Var® was also measured and showed that it persists in waxes (around 10 mg/kg) 1 year after treatment. Thus, in the light of (1) behavioral data obtained under laboratory conditions and in beehives, (2) the persistence of thymol in waxes, and (3) the high load on bees, it would appear important to study the long-term effects of thymol in beehives.

Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera.

Glutamate-gated chloride channels (GluCl) belong to the cys-loop ligand-gated ion channel superfamily and their expression had been described in several invertebrate nervous systems. In the honeybee, a unique gene amel_glucl encodes two alternatively spliced subunits, Amel_GluCl A and Amel_GluCl B. The expression and differential localization of those variants in the honeybee brain had been previously reported. Here we characterized the involvement of each variant in olfactory learning and memory processes, using specific small-interfering RNA (siRNA) targeting each variant. Firstly, the efficacy of the two siRNAs to decrease their targets' expression was tested, both at mRNA and protein levels. The two proteins showed a decrease of their respective expression 24h after injection. Secondly, each siRNA was injected into the brain to test whether or not it affected olfactory memory by using a classical paradigm of conditioning the proboscis extension reflex (PER). Amel_GluCl A was found to be involved only in retrieval of 1-nonanol, whereas Amel_GluCl B was involved in the PER response to 2-hexanol used as a conditioned stimulus or as new odorant. Here for the first time, a differential behavioral involvement of two highly similar GluCl subunits has been characterized in an invertebrate species.

Expression and localization of glutamate-gated chloride channel variants in honeybee brain (Apis mellifera).

Due to its specificity to invertebrate species, glutamate-gated chloride channels (GluCls) are the target sites of antiparasitic agents and insecticides, e.g. ivermectin and fipronil, respectively. In nematodes and insects, the GluCls diversity is broadened by alternative splicing. GluCl subunits have been characterized according to their sensitivity to drugs, and to their anatomical localization. In the honeybee, the GluCl gene can encode different alpha subunits due to alternative splicing of exon 3. We examined mRNA expression in brain parts and we confirmed the existence of two GluCl variants with RT-PCR, Amel_GluCl A and Amel_GluCl B. Surprisingly, a mixed isoform not yet described in insect was obtained, we called it Amel_GluCl C. We determined precise immunolocalization of peptide sequence corresponding to Amel_GluCl A and Amel_GluCl B in the honeybee brain. Amel_GluCl A is mainly located in neuropils, whereas Amel_GluCl B is mostly expressed in cell bodies. Both proteins can also be co-localized. According to their anatomical localization, different GluCl variants might be involved in olfactory and visual modalities and in learning and memory.

Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

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.

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.