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Towards the development of an in vitro test to evaluate the toxicity of insecticides to bees

Insecticides are incriminated as being one of the factors responsible for the decline of bee populations. Numerous laboratory and field studies have proved the susceptibility of insect pollinators to some of these substances, even at low levels of exposure. In order to better anticipate the toxicity of plant health products, and to contribute to developing alternative solutions, scientists from the Research Unit on Bees and the Environment (at the PACA INRA Centre) have for several years been carrying out work on the neuronal ion channels of bees. The results of their studies have provided foundations for the development of an in vitro test that can determine whether chemical compounds are toxic to bees.  This test, developed in collaboration with CNRS in Montpellier and a Canadian university, has resulted in the filing of two patents.

Sous les sensilles olfactives (ovales), visibles au microscope électronique à l’extrémité de l’antenne, se trouvent des neurones qui détectent les odeurs et qui sont sensibles aux insecticides.. © INRA, Claude COLLET
By Irène TROIN
Updated on 01/30/2017
Published on 09/28/2016

The neurotoxicity of insecticides: a story of channels

The susceptibility of the domestic honeybee Apis mellifera to plant health products is an issue that is of considerable concern to scientists at INRA and other research organisations. In the Research Unit on Bees and the Environment, based in Avignon, scientists specialised in neurophysiology and toxicology have studied in the honeybee the functioning of the ion channels that are the principal targets of neurotoxic insecticides.  Ion channels control the transport of ions through the membranes of several cell types, and particularly neurons, where sodium channels (the main target of pyrethroid insecticides) trigger nerve signals.  Several other types of ion channel involved in synaptic neurotransmission are also targeted by insecticides (acetylcholine, GABA or glutamate receptor channels). By disturbing the functioning of these channels, insecticides can compromise physiological processes such as olfaction or taste, which are essential to the survival of the insects. For example, the scientists have shown that even at a low, non-fatal dose, a pyrethroid insecticide (that affects the functioning of sodium channels), or a neonicotinoid (that disturbs the receptor channels for a neurotransmitter called acetylcholine), can cause a locomotor deficit in young bees that persists for several hours after exposure.

In the context of the Bee-Channels project funded by the French National Research Agency (ANR), intended to improve methods to evaluate the neurotoxicity of insecticides to bees, the scientists have succeeded in developing an in vitro test that could prove valuable in the context of the Ecophyto 2 plan to reduce insecticide use, initiated by the Ministry of the Environment, Energy and the Seas.  This work was carried out in collaboration with the CNRS Centre for Research in Macromolecular Biochemistry (CRBM*) and Université Laval in Quebec**.

An INRA innovation at the service of bees

In the same way as the in vitro method that has been used for several years in human pharmacology to predict the cardiotoxic risk of candidate drugs (hERG test), the new test developed by these scientists may revolutionise the toxicology of pollinating insects. The scientists have succeeded in inducing, in vitro, the expression of neuronal sodium and calcium channels from the honeybee, in order to test their functionality following exposure to insecticides.  At present, the expression of other types of ion channel is also under study. Thanks to this method, scientists can quantify the impairment of channels by studying various nerve signal parameters. All these effects provide a predictive indication of the in vivo neurotoxic effect of the substances under test. Candidate compounds could thus be screened using a robotic system in order to select those which are effective against crop pests but harmless to pollinating insects.

This functional test should also have the advantage of reducing the number of experiments required on live bees, by screening compounds in vitro well upstream of classical toxicology tests, and thus complying with the "3R rule"  aimed at Reducing, Refining and Replacing which provides the ethical framework for modern animal experimentation. In order to exploit the two patents filed jointly by INRA, the technology could in the future be the subject of a valorisation partnership with an established firm or start-up. The test developed by INRA scientists could be a valuable tool in the fight to prevent the disappearance of bees.
* Pharmacology of Synaptic Transmission and Neuroprotection Team at the Institut des Biomolécules Max Mousseron, in Montpellier, which was joined by the CRBM researchers involved. ** Molecular and Cellular Neurosciences Team at Université Laval in Quebec.

Contact(s)
Scientific contact(s):

References

Kadala, A., Charreton, M., Jakob, I., Le Conte, Y. and Collet, C. (2011). A use-dependent sodium current modification induced by type I pyrethroid insecticides in honeybee antennal olfactory receptor neurons. Neurotoxicology 32 : 320-330.

ANR Bee-Channels (ANR-13-BSV7-0010)
Susceptibility of sodium and calcium channels in the bee to pyrethroids: molecular aspects and effects of foraging behaviour

Brevets

Calcium channels:
French Patent application published as no. FR3003863 in the names of CNRS, UM, INRA – N° WO2014155021
"Modèle in vitro d’expression des canaux calciques d’abeilles comme outil d’évaluation de la toxicité potentielle de produits phytosanitaires" (In vitro model of the expression of calcium channels in bees as a tool to evaluate the potential toxicity of plant health products).
Sodium channels:
Invention which was the subject of a priority European filing as no. EP 3040345 A1 in the names of CNRS, UM, Université Laval, INRA.
« Sodium channel subunits of pollinator insects and uses thereof ».