Sensitivity of honeybee sodium and calcium channels to pyrethroids : molecular aspects and consequences on foraging behaviour – BEE-CHANNELS

This integrative project brings together 4 partners with combined expertise in apidology, behavioural and cellular neurophysiology, toxicology, molecular cloning and electrophysiology. It aims to improve our understanding of the neurobiology of sensory and cognitive functions in honeybees and to clarify the sublethal effects of pyrethroids on these processes. The project will also provide screening tools for evaluating the toxicity of phytopharmaceutical products toward these key channels.

We have identified in the honeybee genome the pore-forming subunit genes of the voltage–gated sodium channels (1 gene) and calcium channels (3 genes) as well as respectively 5 and 4 genes for regulatory subunits. All these subunits have been cloned, integrated into expression vectors and expressed in heterologous systems. The tissue expression patterns were determined during honeybee development, from larva to adult. The electrophysiological effects of three pyrethroids were investigated on bee neurons in culture, and differential effects of these insecticides have been shown on sodium currents, especially on their inactivation and deactivation kinetics. At the behavioral level, sublethal doses of three pyrethroids induced clear effects on bees’ locomotor activity. Even lower doses showed significant effects on non-associative learning performance

Currently, we are initiating the characterization of biophysical and pharmacological properties of muscle calcium channels and the study of gene expression in bees exposed or not to pyrethroids. The study of sublethal effects of pyrethroids on honeybee behavior will continue by addressing olfactory perception and learning. Further experiments in semi-natural conditions will measure the effects of these insecticides on the foraging activity of bees.
In the context of the moratorium for other insecticide classes, pyrethroids may represent attractive alternatives and see their use increase. The results already achieved in the project clearly show that these insecticides have deleterious sublethal effects on important functions such as locomotion and learning. These elements must be considered in the context of a rational use of these products.

Restrained honeybee for the study of pyrethroids’ effects on sucrose sensitivity and olfactory perception, learning and memory. B) Honeybee voltage-gated calcium channel subunit (Cens et al. 2015). C) Expression of variants of the CaVß regulatory subunit during development (Cens et al. 2013). D) Effect of permethrin on sodium currents in central neurons (Kadala et al. 2014). Credit : JC Sandoz, Claude Collet, Pierre Charnet, Mohamed Chahine

The crucial role of the pollination activity of domestic honeybees is at risk because of an alarming increase in numbers of colony losses in the last years. Among the multiple factors that impact colony fitness, the sensitivity of honeybees to sublethal doses of insecticides, affecting crucial functions involved in foraging, including sensory processing, learning, memory and motor functions have been revealed. Acute toxicity of pyrethroids, the most commonly used insecticides, mainly occurs via binding to voltage-gated sodium (Na+) channels. However, it is clear now that calcium (Ca2+) channels are affected at similar doses. Because Na+ and Ca2+ channels are key actors of neuronal and muscular excitability, they are involved in many neural processes such as locomotion, sensory processing, learning and memory formation in vertebrates and invertebrates. They are thus both suspected of participating in sublethal toxicity pyrethroids. Oddly, the biophysical properties, regulation and pharmacology of Na+ and Ca2+ channels are still poorly studied in honeybees. Moreover, their subunit composition and molecular identity in the tissues potentially affected by these insecticides (muscle, central and peripheral nervous system) is largely unknown.
We have identified in the genomic honeybee database the pore-forming subunit genes of the voltage–gated Na+ channels (2 genes? and Ca2+ channels (3 genes) as well as respectively 5 and 4 genes for regulatory subunits. Our preliminary cloning (for all Na+ and Ca2+ channels subunits) and expression studies (for one regulatory subunit) have revealed unique amino-acids sequences and specific biophysical and regulatory properties in honeybees.

In this integrative project, we propose to analyse the role of these genes in honeybee physiology and behaviour (locomotion, olfactory perception, orientation, learning, memory) and in the sensitivity of this insect to sublethal doses of pyrethroids.

• Task 1 will characterize the effects of sublethal doses of type I and type II pyrethroids on honeybee physiology. We will determine sublethal doses of pyrethroids on honeybees. We will then characterize their effects on Na+ and Ca2+ currents in honey bee muscle cells and central and peripheral neurons in vitro, as well as their effects and dependence on calcium homeostasis. We will also analyze the effect of pyrethroids on gene expression.
• Task 2 will address the molecular characterization of Na+ and Ca2+ channel subunits identified in honeybees. We will define their expression profiles, biophysical properties and sensitivity to pyrethroids using heterologous expression. Their roles in Na+ and Ca2+ influxes in muscle cells and neurons will be challenged using siRNA and specific antibodies. Cell lines expressing actual combinations of subunits found in honeybees will be produced as screening tools for in vitro toxicological tests.
• Task 3 will unravel the role of Na+ and Ca2+ channel subunits in sensory, cognitive and behavioural tasks, so that the sublethal toxicity of pyrethroids can be understood. The impact of siRNA and of sublethal doses of pyrethroids will be evaluated on olfactory processing, learning and memory, locomotor activity and free-flying foraging behaviour.

This integrative approach brings together 4 partners with combined expertise in apidology, behavioural neurophysiology, toxicology, molecular cloning, electrophysiology and heterologous expression. It will improve our understanding of the neurobiology of sensory and cognitive functions in honeybees and clarify acute and chronic toxicity of pyrethroids and their effects on these processes. This project will also provide screening tools for evaluating the toxicity of phytopharmaceutical products toward these key channels.