Cholinergic Pesticides Cause Negative Neuronal Effects in Honeybees

by Lia Metzger

Recently, pesticides that target cholinergic neurotransmission have been found to aid in the decline of insect pollinators. In particular, neonicotinoids (nicotinic receptor agonists) and organophosphate miticides (acetylcholinesterase inhibitors) are commonly used and thus, frequently come in contact with honey bees. Palmer et al. (2013) investigated how these pesticides affect the neurophysiology of honey bees by using recordings from mushroom body Kenyon cells. Instead of studying the learning and behavior of honey bees that are exposed to neonicotinoids and organophosphate miticides, the authors used whole-cell recordings from Kenyon cells in honey bee brains, and assessed the native connectivity and nAChR expression in KCs. They found that the two neonicotinoids, imidacloprid and clothianidin, and coumaphos oxon decreased the KC excitability by inhibiting action-potential firing and reduced KC responsiveness to ACh. When the honey bee brains were exposed to both neonicotinoids and miticides as is common in large crops, the combined exposure added to the effects on KC excitability and nAChR-mediated responses. The honey bees are usually exposed to much higher concentrations of cholinergic pesticides, which indicates that the negative effects on the neurophysiological responses of the mushroom body cells would be heightened in reality.

In order to study neurophysiological effects of pesticides on honey bees, Palmer et al. analyzed the mushroom body Kenyon cells in the honey bee brains for action-potential effects and inhibition of membrane activity. They studied KCs in particular because they are the most significant cells in the honey bee brains for neurological function. In addition, KCs from live tissue were used instead of cultured KCs because cultured KCs do not give full readings of the effects of cholinergic pesticides, according to their image comparisons. Imidacloprid and clothianidin were used because they are the most common neonicotinoids that honey bees are exposed to, and coumaphos oxon was used because it is produced from metabolizing Coumaphos, a common organphosphate miticide that is becoming a frequent subsititute for neonicotinoids.

Specifically, the authors anaesthetized adult worker honey bees, isolated the intact brain, and removed the tissue and membranes to obtain whole-cell recordings from KCs. While taking recordings, the brain was in a recording chamber, where it was secured with a mesh weight and continuously perfused with extracellular solution at room temperature. Recordings of whole-cell voltage-clamp and current-clamp of the mushroom body KCs were taken. The authors used an EPC-10 patch-clamp amplifier controlled by Patchmaster software to record the membrane currents (IM) and the resting membrane potential (VM). To find the transient nAChR-mediated responsed, the authors used pressure application of ACh 25−50 μM from the KC. In addition, the Bradford assay was used to determine protein concentrations in the brains and the Ellman assay was used to determine AChE activity. Using appropriate concentrations, the AChE inhibitors were incubated in honeybee brain lysates for 20 minutes, and then samples were incubated with a color-indicator reaction mix and monitored by absorbance for AChE activity. Graphs representing the KC current effects and AChE inhibitor effects on the current and membrane potential were included for both neonicotinoids, coumaphos oxon, and the combined pesticides. Images of KCs from live tissues and comparisons of currents between cultured KCs and live-tissue KCs were also included.

Palmer et al. found that neonicotinoids cause rapid, concentration-dependent depolarization of KC membrane potential. Action potential firing under neonicotinoids occurred during the initial development of the depolarization but they did not continue during the plateau phase. Similarly, coumaphos-oxon caused a concentration-dependent depolarization of the membrane potential and a lack of AP firing during the plateau stage, although the depolarization occurred more slowly. As far as the effects on KC ACh responses, neonicotinoids that were applied by batch to the KCs caused a tonic inward current, which indicates a sustained activation and desensitization of KC nAChRs. This means that imidacloprid and clothianidin reduce KC responsiveness to ACh. For coumaphos oxon, AChE activity was initially potentiated, but with continued exposure, the tonic inward current was developed, meaning that AChE was inhibited. With higher doses of coumaphos oxon, potentiation and inhibition occurred more rapidly. Finally, when simultaneously exposing KCs to neonicotinoids and coumaphos oxon, the effects on KC function were additive, causing a sustained depolarization and inhibition of ACh responses.

The effects of these pesticides on KCs indicate that the neurophysiological function of honey bees is severely impaired, causing multisensory integration, associative learning and memory, and spatial orientation to be impaired because these functions are dependent on the mushroom cloud KCs. While many studies have shown that the memory, learning, foraging, and navigation behavior of honey bees have been negatively impacted by neonicotinoids and coumaphos, this study links the behavior to concrete neurological activity.

Palmer, M.J., Moffat, C., Saranzewa, N., Harvey, J., Wright, G.A., Connolly, C.N., 2013. Cholinergic pesticides cause mushroom body neuronal inactivation in honeybees. Nature communications 4, 1634.

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