Honeybees Exposed to Acetylcholinesterase Inhibitors Exhibit Impaired Motor Function

Stress from parasites, pathogens, and pesticides have been contributing to the global decline of populations of honeybees and many pollinators for the past two decades. Specifically, the use of pesticides that affect neuromuscular functioning and kill parasitic mites have caused the accumulation of acaricides, or mite pesticides, in the wax combs of bees’ hives. To investigate the possibility of this accumulation contributing to the decline of bee populations, Williamson et al. (2013) studied the effects of prolonged exposure to pesticides that inhibit acetylcholinesterase (AChE) on the physiology and behavior of bees. Adult worker bees were fed sub-lethal concentrations of four AChE inhibitors in sucrose solutions and then were observed for walking, stopped, grooming, and upside down behavior. After the behavioral study, the bees were dissected to confirm that the four compounds they assayed or their metabolites were responsible for the change in behavior by testing for transcript expression levels of two honeybee AChE inhibitors and through biochemical assays. All AChE inhibitors caused increased grooming behavior, but coumaphos in particular caused more grooming and symptoms of sickness as the concentration increased. The authors found that the effects of pesticides that inhibit AChE on the motor functioning of bees could reduce their survival and contribute to the decline of bee colonies. —Lia Metzger

                  Williamson, S.M., Moffat, C., Gomerall, M.A., Saranzewa, N., Connolly, C.N., Wright, G.A., 2013. Exposure to acetylcholinesterase inhibitors alters the physiology and motor function of honeybees. Frontiers in physiology 4.

                  Numerous studies have focused on the effect of pesticides on bees, with uncertain conclusions as to how chronic exposure to acaricides in hives may affect the behavior of foraging worker bees. Williamson et al. chose to experiment on adult foraging worker bees in both the winter and summer with four compounds of AChE insulators in order to consider prolonged exposure to pesticides that affect motor function. The authors compared the effects of 10 nM of the four AChE inhibitors—coumaphoes, chlorphyrifos, aldicarb, and donepezil—in 1 M of sucrose solution with the control of 1 M of sucrose and investigated the effects of three different doses of coumaphos on bees.
                  Bees were caught, placed in boxes, and fed the different solutions from feeding tubes at liberty for 24 hours and the feeding tubes were weighed before and after the feeding time to determine drug consumption. After feeding, the authors observed each individual bee for 15 minutes for walking, flying, remaining still, falling upside down, grooming the head, and unusual abdominal spasms, noting frequency and time intervals for each movement. To test for IC50 values for AChE, the bees were dissected after observation, and subsequently, the Bradford assay was used to determine protein concentrations and the Ellman’s assay was used to assay AChE activity. Using semi-quantitative PCR amplification, the authors tested for AChE gene transcript levels in the brain and gut of the honeybee to find what the AChE inhibitors effected the transcript levels. A principal components method of factor analysis was used to find correlations between the behaviors for time intervals. Comparative graphs and tables between winter and summer for each type of AChE inhibitor were created using a multivariate general linear model and least square differences.
                  The authors found that four factors accounted for 82.8% of the variation in the data, each of which indicated how the bees’ expression of behavior was affected by exposure to the AChE inhibitors. Overall, bees did not walk as much, could not right themselves well if they fell over, and had abdominal spasms that the control group did not have. In the summer, the bees treated with chlorpyrifos significantly exhibited less walking behavior, more time upside down, and abdominal spasm compared to the control bees. In the winter, the bees did not show a difference in walking behavior, but in the summer, the bees walked less. In both seasons, there was an increase in grooming behavior. In addition, bees did not exhibit significant differences in their behavior of remaining still or flying with exposure to AChE inhibitors.
                  The authors further separated the behavior of grooming into the head and body because the AChE inhibitors had the most significant effect on this behavior. The total time spent grooming the head but not the body was increased, and aldicarb and coumaphos contributed to the increased time spent head grooming the most. Both heads and bodies were more frequently groomed in all treatment groups than the control. In particular, for the AChE inhibitor coumaphos, the highest concentration (1μM) treatment group expressed head and body grooming behavior and abdominal spasms more than the controls in the winter but not in the summer.
                  Biochemical assays found that chlorpyifos oxon and coumaphos oxon, and aldicarb sulfoxide, metabolites of AChE inhibitors, showed greater inhibition of the enzyme AChE than parent compounds. Chlorpyrifos oxon was the most potent inhibitor of AChE for both gut and brain tissues, but the AChE inhibitors were more potent for gut tissues than for those of the brain (lower IC50 values in gut). AChE gene transcription also increased in both the brain and the gut for AChE-2 transcripts, mostly in response to coumaphos and aldicarb in the brain tissues and in response to coumaphos and chlorpyrifos in the gut tissues.

                  The increase in the frequency of grooming behavior, the impaired ability to flip over, and the exhibition of abdominal spasms indicate that AChE inhibiting pesticides alter the behavior of honey bees. Bees exhibited lower levels of AChE in the brain, which could indicate that the behavior of the bees would be affected long-term. It is possible that the increased grooming behavior could be helpful to ridding mites by using coumaphos, but coumaphos also increased abdominal spasms. The spasms could suggest pain, disruption of gut function, or even a gut parasite that has been correlated in high levels with acaricides in combs. Moreover, the metabolites of coumaphos, chlorpyrifos, and aldicarb inhibit AChE in both the brain and gut tissue, which suggests a correlation between the environmental levels of the parent compound with the active metabolite. This correlation could help predict the percentage of AChE inhibitors in comb wax that contaminate the bees. Additionally, the increase of AChE-2 transcripts suggests that long-term contamination may result in increased AChE levels as a heritable trait, which could help the bees adapt to the inhibition of AChE-1 by pesticides. These possibilities, however, do not overshadow the evidence of pesticides affecting the behavior of honey bees, and potentially contributing to the decline of the bees by changes in foraging behavior and gut disruption.

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