The Apis mellifera channelome: an invaluable tool to study agroecosystems contaminants – Synaptic-Bee

Several methods are used:
1. isolate ion channel genes and analyze their behavior in the face of insecticides by expressing them in «host« cells lacking such channels (in our case Xenopus oocytes)
2. analyze also these effects (a) on bee behavior (olfaction, memory, locomotion, (b) on brain slices or isolated bee hearts, (c) on neurons or muscle cells isolated from bee
3. Using structural modeling tools and mathematical numerical integration tools, try to model the behavior of these channels in the presence of insecticides as well as the structure of their binding sites, to predict the toxicity of the products on the beneficial insects and the occurrence of possible cocktail effects.

One of the most important results is undoubtedly the development of recordings of the effect of insecticides on isolated cardiac cells and on the brain, a first in bees but also in insects (a single article on Drosophila). This opens up really interesting perspectives for better understanding the cardio- and neurotoxicity of insecticides.
In combination with the recordings of action potentials in vitro on cells and in vivo on bees, they should make it possible to establish the link between the effects on the channels expressed in the oocyte and the effects recorded in vivo on living bee ( locomotion, memory, flight).
Several new ion channel genes have also been isolated and their products characterized. They are important tools for understanding the effects of insecticides at the molecular level, and knowledge of their structure will help to understand the specificity, or lack of specificity, of insecticides for different insect species. In general, this approach will therefore provide keys for designing toxicological tests to better characterize the toxicity of products on beneficial insects.

The characterization of new ion channels reinforces the panoply of tools necessary to understand the physiology of the bee and its disturbance by insecticides. Knowledge of their structure makes it possible to better understand the problems of specificity of insecticides towards different species of insects and opens the way to new, more discriminating tests, but perhaps also to a reasoned use confined to the most beneficial insect-friendly products.

A major milestone in our research program is the publication in 2022 of a book entitled «Les abeilles face au risque toxic« (CNRS-edition) reviewing the data currently available on the effects of neurotoxic insecticides on animals in vivo but also at the cellular and molecular level. Several lectures/debates for the general public were also given locally (Poitiers, Le Vigan, St Mathieu de Trviers). The work already carried out has led to the publication of 6 articles in international journals and 8 communications (oral or poster) at international congresses.

Pollinators’ disappearance is mainly due to chemical stressors found in the environment. The harmful effects of these contaminants (herbicides, fungicides, insecticides, antiparasitics) on beneficial insects are mainly assessed with lethality tests (LD50) at 24-48h, but their impact on insect physiology at low doses is largely unknown. The most prevalent insecticides are neurotoxic (neonicotinoids, pyrethroids, phenylpyrazoles) and directly impact ion channels or ionotropic receptors. In doing so, they modify the spread and the delivery of the nerve signals and can be lethal at high doses or pathologic severely deleterious to the physiology at lower doses. Moreover, the combined presence of multiple stressors such as pesticides at the same place and time, is largely documented and it has been shown that more than 50 distinct residues could be present in pollen, wax, and even in 75% of the tested honey worldwide. These mixtures are suspected to act synergistically (cocktail effect), thus increasing the toxic impact of the products taken individually. Multiple recent studies have analysed analyzed such effects on bee performances and survival, but the effects of these lower doses as well as the combined effects of several of these pesticides together are poorly understood at the molecular level. Previous studies have been very often limited to the dedicated target of each of these molecules (i.e. Na+ channels for pyrethroids), and very rarely on the combined action of several of these pesticides or on their effects on supposedly untargeted channels.
The Synaptic-Bee project (SBp) is the first initiative dedicated to the in vitro and in vivo analysis of a panel of routinely found pesticides on the entirety of ion channels expressed (the so-called channelome) in bee brain. By cloning and characterizing these channels in heterologous expression systems, SBp will draw a complete picture of bees’ synaptic sensitivity to these contaminants at molecular level and provide a unique tool to understand pesticide toxicity and specificity. With numerical simulations of these channels working alone or in combination, SBp will increase our understanding of the effects of channel modulation on honeybee action potential, synaptic transmission and behavior.
The SBp project proposes:
(1) to identify and clone all Apis mellifera ion channels/receptors in order, to characterize their biophysical and pharmacological properties and their sensitivity to pesticides. To construct, for each of themthese channels, a numerical model where intoxication can be reproducedsimulated;
(2) to evaluate their expression pattern in 4 types of neurones and muscular cells crucial for locomotion, flight and olfactory memory, and thus for foraging (projection and mushroom body neurons, Kenyon cells, skeletal muscle cells and cardiac myocytes). To use the model of the channels present in these cell types to reproduce mimic/model/simulate cellular excitability, as a combination of in the form of action potential and synaptic transmission signals;
(3) To compare real recordings of action potential and synaptic transmission on these cells with their numerical counterpart in control conditions and under intoxication by one or several pesticides with the goal of precisely identifying the role of each channel in toxicity and the existence and mechanisms of potential cocktail effects;
(4) When possible, to build a spatial/3D/structural molecular model of these channels where the pesticides will be tentatively docked, to extract information about pesticide-induced molecular dysfunction and potential cross selectivity between channels and species (targeted pest vs pollinators);
(5) and finally reconcile these data with the effects of these pesticides recorded on two key functions in honeybees’ biology: olfactory learning and memory and locomotion
SBp, a program without equivalent at this time, will provide unique knowledge and tools to better understand honey bee toxicology.