Bernd Grünewald

RFID Tracking of Sublethal Effects of Two Neonicotinoid Insecticides on the Foraging Behavior of Apis mellifera

The development of insecticides requires valid risk assessment procedures to avoid causing harm to beneficial insects and especially to pollinators such as the honeybee Apis mellifera. In addition to testing according to current guidelines designed to detect bee mortality, tests are needed to determine possible sublethal effects interfering with the animals vitality and behavioral performance. Several methods have been used to detect sublethal effects of different insecticides under laboratory conditions using olfactory conditioning. Furthermore, studies have been conducted on the influence insecticides have on foraging activity and homing ability which require time-consuming visual observation. We tested an experimental design using the radiofrequency identification (RFID) method to monitor the influence of sublethal doses of insecticides on individual honeybee foragers on an automated basis. With electronic readers positioned at the hive entrance and at an artificial food source, we obtained quantifiable data on honeybee foraging behavior. This enabled us to efficiently retrieve detailed information on flight parameters. We compared several groups of bees, fed simultaneously with different dosages of a tested substance. With this experimental approach we monitored the acute effects of sublethal doses of the neonicotinoids imidacloprid (0.15–6 ng/bee) and clothianidin (0.05–2 ng/bee) under field-like circumstances. At field-relevant doses for nectar and pollen no adverse effects were observed for either substance. Both substances led to a significant reduction of foraging activity and to longer foraging flights at doses of ≥0.5 ng/bee (clothianidin) and ≥1.5 ng/bee (imidacloprid) during the first three hours after treatment. This study demonstrates that the RFID-method is an effective way to record short-term alterations in foraging activity after insecticides have been administered once, orally, to individual bees. We contribute further information on the understanding of how honeybees are affected by sublethal doses of insecticides.

Dual olfactory pathway in the honeybee, Apis mellifera

The antennal lobes (ALs) are the primary olfactory centers in the insect brain. In the AL of the honeybee, olfactory glomeruli receive input via four antennal sensory tracts (T1–4). Axons of projection neurons (PNs) leave the AL via several antenno‐cerebral tracts (ACTs). To assign the input–output connectivity of all glomeruli, we investigated the spatial relationship of the antennal tracts and two prominent AL output tracts (medial and lateral ACT) mainly formed by uniglomerular (u) PNs using fluorescent tracing, confocal microscopy, and 3D analyses. Furthermore, we investigated the projections of all ACTs in higher olfactory centers, the mushroom‐bodies (MB) and lateral horn (LH). The results revealed a clear segregation of glomeruli into two AL hemispheres specifically supplied by PNs of the medial and lateral ACT. PNs of the lateral ACT innervate glomeruli in the ventral‐rostral AL and primarily receive input from T1 (plus a few glomeruli from T2 and T3). PNs of the medial ACT innervate glomeruli in the dorsal‐caudal hemisphere, and mainly receive input from T3 (plus a few glomeruli from T2 and T4). The PNs of the m‐ and l‐ACT terminate in different areas of the MB calyx and LH and remain largely segregated. Tracing of three mediolateral (ml) ACTs mainly formed by multiglomerular PNs revealed terminals in distinct compartments of the LH and in three olfactory foci within the lateral protocerebrum. The results indicate that olfactory input in the honeybee is processed via two separate, mainly uPN pathways to the MB calyx and LH and several pathways to the lateral protocerebrum. J. Comp. Neurol. 499:933–952, 2006.

Morphology of Feedback Neurons in the Mushroom Body of the Honeybee, Apis mellifera

The anatomy of gamma-aminobutyric acid (GABA)-immunoreactive, recurrent feedback neurons in the mushroom body (MB) of the honeybee, Apis mellifera, was investigated by using intraneuropilar injections of cobalt ions and light microscopic techniques. Each MB contains approximately 110 GABA-immunoreactive neurons, and approximately 50% of them are feedback neurons, i.e., they connect the MB output regions--the alpha-lobe, beta-lobe, and pedunculus--with its input regions--the calyces. Their somata are located in the lateral protocerebral lobe, and their primary neurites project medially and bifurcate near the alpha-lobe. In the alpha-lobe feedback neurons form narrow banded, horizontal arborizations in the dorsal and median alpha-lobe; each cell innervates a certain alpha-lobe layer. The neurons form additional branches in the pedunculus and the beta-lobe. All calycal subcompartments--the lip, collar, and basal ring--are innervated by feedback neurons. However, individual feedback neurons innervate exclusively a certain subcompartment in both the median and lateral calyx. Due to the arrangement of intrinsic Kenyon cells, each calycal subcompartment is connected to its specific, corresponding layer in the alpha-lobe. Feedback neurons interconnect the alpha-lobe and the calyces in either a corresponding or a noncorresponding fashion. With respect to their branching pattern in the alpha-lobe, the basal ring and the collar neuropil receive input from feedback neurons innervating the corresponding dorsal and median alpha-lobe layers. By contrast, the lip region, which receives olfactory antennal input, is innervated by feedback neurons with arborizations in a noncorresponding dorsal alpha-lobe layer.

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.

Neonicotinoids interfere with specific components of navigation in honeybees

Three neonicotinoids, imidacloprid, clothianidin and thiacloprid, agonists of the nicotinic acetylcholine receptor in the central brain of insects, were applied at non-lethal doses in order to test their effects on honeybee navigation. A catch-and-release experimental design was applied in which feeder trained bees were caught when arriving at the feeder, treated with one of the neonicotinoids, and released 1.5 hours later at a remote site. The flight paths of individual bees were tracked with harmonic radar. The initial flight phase controlled by the recently acquired navigation memory (vector memory) was less compromised than the second phase that leads the animal back to the hive (homing flight). The rate of successful return was significantly lower in treated bees, the probability of a correct turn at a salient landscape structure was reduced, and less directed flights during homing flights were performed. Since the homing phase in catch-and-release experiments documents the ability of a foraging honeybee to activate a remote memory acquired during its exploratory orientation flights, we conclude that non-lethal doses of the three neonicotinoids tested either block the retrieval of exploratory navigation memory or alter this form of navigation memory. These findings are discussed in the context of the application of neonicotinoids in plant protection.

Nicotinic acetylcholine currents of cultured Kenyon cells from the mushroom bodies of the honey bee Apis mellifera

1 Acetylcholine‐induced currents of mushroom body Kenyon cells from the honey bee Apis mellifera were studied using the whole‐cell configuration of the patch clamp technique. Pressure application of 1 mM acetylcholine (ACh) induced inward currents with amplitudes between ‐5 and ‐500 pA. 2 The cholinergic agonists ACh and carbamylcholine had almost equal potencies of current activation at concentrations between 0·01 and 1 mM; nicotine was less potent. The muscarinic agonist oxotremorine did not elicit any currents. 3 Approximately 80 % of the ACh‐induced current was irreversibly blocked by 1 μM α‐bungarotoxin. Atropine (1 mM) did not block the ACh‐induced current. 4 Upon prolonged ACh application the current desensitized with a time course that could be approximated by the sum of two exponentials (τ1= 276 ± 45 ms (mean ± s.e.m.) for the fast component and τ2= 2·4 ± 0·7 s for the slow component). 5 Noise analyses of whole‐cell currents yielded elementary conductances of 19·5 ± 2·4 pS for ACh and 23·7 ± 5·0 pS for nicotine. The channel lifetimes, calculated from the frequency spectra, were τo= 1·8 ms for ACh and τo= 2·5 ms for nicotine. 6 Raising the external calcium concentration from 5 to 50 mM shifted the reversal potential of the ACh‐induced current from +4·6 ± 0·9 to +37·3 ± 1·3 mV. The calcium‐to‐sodium permeability ratio (PCa : PNa) was 6·4. 7 In high external calcium solution (50 mM) the ACh‐induced current rectified in an outward direction at positive membrane potentials. 8 We conclude that Kenyon cells express nicotinic ACh receptors with functional profiles reminiscent of the vertebrate neuronal nicotinic ACh receptor subtype.

Physiological properties and response modulations of mushroom body feedback neurons during olfactory learning in the honeybee, Apis mellifera

Abstract Mushroom bodies are central brain structures and essentially involved in insect olfactory learning. Within the mushroom bodies γ-aminobutyric acid (GABA)-immunoreactive feedback neurons are the most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was investigated by analyzing modulations of odor responses of feedback neurons during olfactory learning in vivo. In the honeybee, Apis mellifera, feedback neurons were intracellularly recorded at their neurites. They produced complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials indicate that their synaptic input region lies within the lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of different neurons to the same odor were highly variable. Response modulations were determined by comparing odor responses of feedback neurons before and after one-trial olfactory conditioning or sensitisation. Shortly after pairing an odor stimulus with a sucrose reward, odor-induced spike activity of feedback neurons decreased. Repeated odor stimulations alone, equally spaced as in the conditioning experiment, did not affect the odor-induced excitation. A single sensitisation trial also did not alter odor responses. These findings indicate that the level of odor-induced inhibition within the mushroom bodies is specifically modulated by experience.

Acetylcholine, GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera

The honeybee, Apis mellifera, is a valuable model system for the study of olfactory coding and its learning and memory capabilities. In order to understand the synaptic organisation of olfactory information processing, the transmitter receptors of the antennal lobe need to be characterized. Using whole-cell patch-clamp recordings, we analysed the ligand-gated ionic currents of antennal lobe neurons in primary cell culture. Pressure applications of acetylcholine (ACh), γ-amino butyric acid (GABA) or glutamate induced rapidly activating ionic currents. The ACh-induced current flows through a cation-selective ionotropic receptor with a nicotinic profile. The ACh-induced current is partially blocked by α-bungarotoxin. Epibatidine and imidacloprid are partial agonists. Our data indicate the existence of an ionotropic GABA receptor which is permeable to chloride ions and sensitive to picrotoxin (PTX) and the insecticide fipronil. We also identified the existence of a chloride current activated by pressure applications of glutamate. The glutamate-induced current is sensitive to PTX. Thus, within the honeybee antennal lobe, an excitatory cholinergic transmitter system and two inhibitory networks that use GABA or glutamate as their neurotransmitter were identified.

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.

Using local anaesthetics to block neuronal activity and map specific learning tasks to the mushroom bodies of an insect brain

The formation of a stable olfactory memory requires activity within several brain regions. The honeybee provides a valuable model to map complex olfactory learning tasks onto certain brain areas. To this end, we used injections of the local anaesthetic procaine to reversibly block spike activity in a specific brain region, the mushroom body (MB). We first investigated the physiological effects of procaine on cultured MB neurons from adult honeybee brains. Using the whole‐cell configuration of the patch‐clamp technique, we show that procaine blocks voltage‐gated Na+ and K+ currents in a dose‐dependent manner between 0.1 and 10 mm. The effects are reversible within a few minutes of wash. Lidocaine acts similarly, but is less effective at the tested concentrations. We then studied the role of the MBs during reversal learning by blocking the neural activity within these structures by injecting procaine. During reversal learning bees learn to revert their responses to two odorants, one rewarded (A+) and one unrewarded (B–), if their contingencies are changed (A– vs B+). Injecting procaine into the MBs impaired reversal learning. Procaine treatment during acquisition left the later retention of the initial learning (A+ vs B–) intact. Similarly, a differential conditioning task involving novel odorants (C+ vs D–) was intact under procaine treatment. Our experiments show that local injections of procaine can be used to map learning tasks onto specific regions of the insect brain. We conclude that intact MB activity is required for the acquisition of reversal learning, but not for simple differential learning tasks.