Honey Bees: Estimating the Environmental Impact of Chemicals - Chapter 6 pps

In preliminary experiments, we defined the IMI dose that did not induce modifications of the gustatory threshold or a perturbation of motor activity.. To evaluate the gustatory threshold

6 Effects of imidacloprid on the

neural processes of memory

in honey bees

C Armengaud, M Lambin, and

M Gauthier

Summary

The cholinergic system in insects is the main target of insecticides One class of molecules, the neonicotinoids, induces direct activation of the neu-ronal nicotinic acetylcholine receptors (nAChRs) In the honey bee these receptors are mainly distributed in the olfactory pathways that link sensory neurons to antennal lobes and mushroom bodies These structures seem to play an important role in olfactory conditioning We have previ-ously shown that cholinergic antagonists injected in different parts of the brain impaired the formation and retrieval of olfactory memory We then advanced the hypothesis that, through the activation of the nAChR, the neonicotinoid imidacloprid (IMI) would lead to facilitation of the memory trace

To test this hypothesis, IMI was applied topically upon the thorax and the effects were tested on the habituation of the proboscis extension reflex induced by repeated sugar stimulation of the antennae Animals treated with IMI to a dose that did not affect sensory or motor functions needed fewer trials than nontreated animals to show a reflex inhibition This effect can be interpreted as a learning facilitation

We developed a functional histochemistry of cytochrome oxidase (CO)

to reveal the brain targets of the drug in the honey bee brain Following IMI injection, a CO staining increase, probably linked to an increase in metabolism, was observed in the antennal lobes In integrative structures,

in particular the calyces of mushroom bodies, IMI exerted a facilitatory or inhibitory effect on neuronal metabolism depending on the dose The brain targets of nicotinic ligands, including pesticides, can be compared by using this technique

Introduction

Two of the three main classes of insecticides exert their neurotoxic effects through action on the cholinergic system This is the case for the new class

of neonicotinoids, which are known to act on the nicotinic acetylcholine

receptor (nAChR) channel Imidacloprid (IMI)

these new molecules (Figure 6.1) According to the literature, IMI in insects acts at three pharmacologically distinct acetylcholine receptor sub-types inducing a dose-dependent depolarization [1] Other electrophysio-logical effects of IMI have been described in different models The single patch-clamp technique applied to the rat pheochromocytoma (PC 12) cells showed that the molecule may have both agonist and antagonist effects on the nAChR [2–4] Binding experiments of [3H]IMI to membranes from different species showed high-affinity binding sites in house fly head [5],

and high- and low-affinity binding sites in the aphid Myzus persicae [6].

The nicotinic receptor subunit composition seems to exert a profound influence upon IMI binding affinity [7] This brief review of the literature underlines the complex action of IMI on the nAChR

The neurotransmitter acetylcholine (ACh) is distributed largely in the honey bee brain [8] Acetylcholinesterase and ACh receptors have been identified in the antennal lobes and in the mushroom bodies (MBs), particularly in the calycal part [9, 10] In addition, Kenyon cells, which fill

the calyces, express functional nAChRs in vitro [11] The involvement of

the cholinergic system in memory processes in the honey bee has been demonstrated by intracranial injections of cholinergic antagonists using a classical conditioning procedure [12–14] Local brain injections have shown that the nAChR antagonist mecamylamine impaired the recall or the formation of the memory trace depending on the brain site injection and

Figure 6.1 Chemical structures of acetylcholine and nicotinic cholinoceptor ligands

used in this study.

the moment of the injection relative to the conditioning trial From these experiments, we postulated that ACh, as in vertebrates, exerts a facilitatory effect on memory processes We made the hypothesis that activation of the cholinergic pathways with agonists like those molecules belonging to the neonicotinoids would facilitate the formation and/or the recall of memory

To test this hypothesis, we submitted honey bees to the habituation of the proboscis extension reflex (PER) This nonassociative learning para-digm can be easily used to detect the behavioral effect of different kinds of molecules The PER is induced by antennal sucrose stimulation and involves activation of motor neurons situated in the subesophageal gan-glion and driving the mouthpart muscles The repetition of this non-noxious stimulation leads to a decrease in the response occurrence We postulated that IMI could reduce the number of stimulations needed to observe the response decrease However, given the neurotoxic action of IMI, the absence of the PER could indicate a problem of gustatory per-ception or a central motor disruption In preliminary experiments, we defined the IMI dose that did not induce modifications of the gustatory threshold or a perturbation of motor activity

The involvement of mushroom bodies in memory processes is well established in insects Consequently imidacloprid brain targets were inves-tigated using cytochrome oxidase (CO) histochemistry CO activity is com-monly used in vertebrates as an endogenous metabolic marker of neuronal activity Energy demand due to neuronal activity increases the production

of oxidative energy [15] Classically, CO histochemistry is used in verte-brates to identify a pathological modification [16, 17] or the effect of chronic surgical and pharmacological treatments [18–20] We attempted to develop a functional histochemistry of CO in honey bee brain that allowed the analysis of the short-term effect of cholinergic ligands including IMI

on the metabolism of the different brain structures [21]

Materials and methods

Worker honey bees (Apis mellifera) were caught at the hive entrance and maintained with food and water ad libitum in small Plexiglas boxes until

the beginning of the experiments To evaluate the gustatory threshold and for learning and metabolism experiments, honey bees were immobilized individually in small plastic tubes with a drop of wax–collophane mixture laid between the dorsal part of the thorax and the tube The head and the prothoracic legs were free to move, allowing the honey bee to clean its antennae from the repeated sucrose stimulations Honey bees underwent a 2-hour starvation period before the beginning of the experiments

Imidacloprid (Cluzeaux, France; molecular weight: 255.7; degree of purity 98 percent) was dissolved in dimethyl sulfoxide (DMSO; Sigma)

successive dilutions in saline Control groups were treated with DMSO

dissolved in saline (vehicle) in the same proportions For behavioral

the thorax Doses ranging from 1.25 to 5 ng/bee were used which were below the DL50 value (10 to 20 ng/bee, defined for thoracic application to

Apis mellifera at 24 h; unpublished observations from L.P Belzunces) For

CO experiments, we tested the effect of intracranial injection of saline or

and antagonist, respectively

Behavioral tests

Gustatory threshold

The aim of this experiment was to study the effect of IMI on the gustatory perception The gustatory threshold was defined as the lowest concentra-tion of a sucrose soluconcentra-tion applied to the antennae able to elicit a proboscis extension The threshold was defined twice for each honey bee: first before any treatment and then after IMI or vehicle application Several doses of IMI were used with several time-intervals between the application of the drug and the test

The gustatory threshold was determined as follows Fasted honey bees were submitted to antennal stimulations (1-minute intertrial interval) with increasing concentrations of sucrose solutions ranging from M/1024 to 4 M and following a geometric progression (M/1024, M/512, M/256, etc.) The range of increasing sucrose concentrations was applied twice separated by

a 5-minute interval The lowest concentration of sucrose that elicits the PER was defined as the gustatory threshold Honey bees that failed to respond to one of the sucrose solutions were discarded The remaining honey bees were fed with two drops of 50 percent (w/v) sucrose solution and fasted for 2 hours This was done to ensure that the gustatory thresh-old determination under IMI application was made under the same moti-vational state The thoracic application of vehicle or IMI at a dose of 1.25, 2.50, or 5 ng/bee was performed during the starvation period, 15 min, 30 min or 60 min before the second gustatory threshold determination This second determination was done like the first one For data quantification, any modification of the gustatory threshold between the two determina-tions from one sucrose concentration to the one immediately lower or higher was respectively quantified as 1 or 1 arbitrary unit

Locomotion

Locomotion of honey bees was tested in an open-field-like apparatus con-sisting of a white Plexiglas box (30304cm) with a glass front for

obser-vation The back surface was divided into 5-cm2squares and the box was illuminated from above The box did not allow the honey bees to fly A hole made in the bottom right-hand side of the box allowed the introduc-tion of a single honey bee for a 5-min observaintroduc-tion period The posiintroduc-tion of the honey bee was recorded every 5 s The duration of successive 5-s periods in the same square was reported as immobility as the locomotor activity of the honey bee in the same square was very low, if nonexistent Otherwise, the honey bee was moving around The effect of the drug on locomotor activity was studied 15, 30, and 60 min after application of a dose of 1.25, 2.50, and 5 ng/bee and was compared to the effect of vehicle

Habituation

Fasted honey bees were stimulated repeatedly with a 50 percent (w/v) sucrose solution applied to one antenna at 1-min intervals The habitua-tion criterion was defined as three consecutive sucrose stimulahabitua-tions without proboscis extension When this criterion was reached, the sucrose solution was applied to the controlateral antenna to rule out the eventual-ity of motor tiredness Honey bees that did not respond to the 50 percent sucrose solution and to the restoration test of the reflex were discarded IMI was applied at 1.25 ng/bee and the drug effect was tested after 15 min,

30 min or 1 hour in three independent groups A group receiving no treat-ment and a solvent-treated group were also added

CO histochemistry

Thirty minutes after injection of the drug, the animals were killed by rapid

pre-pared for CO histochemistry, according to Wong-Riley [20] Quantifica-tion of staining was performed by computer-aided densitometry of CO histochemistry intensity We focused our investigations on antennal lobes,

after an injection of AChR antagonists in these structures, memory

per honey bee At higher doses IMI induced toxic and lethal effects

Data analysis

Data sets were analyzed using a two-population independent two-tailed

t-test or an analysis of variance (ANOVA) Figures show meanss.e.m In

all cases, P-values less than 0.05 were considered as significant.

Behavioral tests

Gustatory threshold

An increase in the gustatory threshold was observed between the first and the second determinations whatever the treatment (Figure 6.2) A very slight increase of less than one-half unit was found for the vehicle and for the lowest doses of IMI (1.25 and 2.50 ng/bee) Animals treated with the vehicle (controls) were not different from those receiving no treatment (data not shown) Groups that received 1.25 and 2.50 ng IMI were not dif-ferent from controls, so in subsequent habituation experiments, both doses could have been used A loss of sensitivity was noticed for the dose of 5 ng after 1 hour This delayed effect seems not to be related to the time needed by imidacloprid to reach the brain from the thoracic application

15 min (n 20) 3

2

1

0

30 min (n 10)

60 min (n 10)

DMSO 1.25 ng 2.50 ng 5 ng

Treatment

*

Figure 6.2 Variations of the gustatory threshold (arbitrary units) 15 min, 30

min, or 1 h after thoracic application of DMSO (n20 for each time) or imidacloprid at different doses (1.25, 2.5, 5 ng/bee) The number of imidacloprid-treated animals is indicated on the graph.

*P 0.05.

site since the high dose of 20 ng induces the same sensitivity loss as soon as

15 min after application (data not shown)

Locomotion

Opposite effects of IMI on motor displacements were observed depending

on the dose (Figure 6.3) Compared to the vehicle, the lowest dose of IMI (1.25 ng/bee) induced an increase in displacements independently of time (shown as a decrease in immobility in Figure 6.3) A significant increase in locomotion was also observed for 2.5 ng/bee at 15 min With 5 ng, IMI induced a decrease in the honey bee displacements in the box as soon as

30 min after application The decrease in displacements was explained by a loss of motor coordination The honey bees fell down on their backs, showing leg movements and body and wing shaking Additional observa-tions up to 2 hours after drug application showed that there was no behav-ioral recovery

Unlike the previous experiment on gustatory perception, we did not observe a dose–effect relationship in this experiment, as there were more

0

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15 min

30 min

60 min

100

200

300

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*

DMSO 1.25 ng 2.50 ng 5 ng

Treatment

Figure 6.3 Time spent in immobility (seconds) in honeybees treated with

DMSO or imidacloprid at different doses (1.25, 2.5, 5 ng/bee), 15

numerous displacements at the lowest dose (1.25 ng/bee) This dose was retained to test the effect of IMI on habituation

Habituation

Under IMI treatment (1.25 ng/bee), honey bees needed fewer trials to display PER habituation than honey bees receiving the vehicle or receiv-ing no treatment (statistics highly significant in both cases, see Figure 6.4) There was no effect of time on the facilitating effect This observation is closer to the enhancing effect of 1.25 ng of IMI on displacements, which is also independent of time Dilute DMSO induced a slight but significant reduction in the number of trials compared to the groups receiving no treatment (statistics shown in Figure 6.4)

CO histochemistry

Histological modifications induced by IMI were of weak amplitude but

15 min 0

10

20

30

40

50

60

30 min 60 min Time interval between treatment and test













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*

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No treatment DMSO Imidacloprid

Figure 6.4 Number of trials required to reach habituation in animals receiving no

treatment or animals treated topically with DMSO or imidacloprid

staining increase in all the experiments performed The intensity of stain-ing was analyzed in the cortical layer and in the internal area of the

increase in staining was obtained for the two regions of the glomeruli The

The greatest modifications of CO labeling induced by IMI were

increment was significant in the dorsal, intermediate and ventral layers (B1, B2, and B3)

and lower (LD) divisions of the central body was significantly greater than

(Figure 6.5D)

In subsequent experiments other nAChR ligands were tested CO was stimulated by nicotine in a dose-dependent manner in many brain regions (data not shown) In particular, the internal part of the glomeruli

6.6A)

The effects of nicotine were statistically significant in the B1, B2, and

Moreover, for the ventral layer a significant increase was still present after

In calyces, whatever the concentration of nicotine tested, no significant differences were found between the saline and nicotine groups whereas

IMI injection, a decrease in brain metabolism was observed in the central

to the same concentration and at the same interval

Changes in the metabolic activity of the honey bee brain were exam-ined following nAChR antagonist (mecamylamine) administration to high

Comparison between saline- and antagonist-treated brains indicates that mecamylamine induced a significant decrease in neural metabolism in the

-lobe

(A) Antennal lobe: glomeruli cortical area, glomeruli internal

B3, ventral layer (C) Calyces: lip area, basal ring area (D) Central body: UD, upper division of central body; LD, lower division