Apis mellifera ion channels and tools to study agroecosystems contaminants

世界各地昆虫种群和许多其他物种的急剧减少和消失现在是一个科学事实，有100多万种物种在濒危名单上。昆虫种群受到的打击更大，在过去的25-40年里，昆虫生物量下降了75%至98%。尽管有许多人为原因，但长期接触农药是农业需要和环境问题之间冲突的核心，这是政治竞技场和大多数媒体的前沿和中心。尽管现代农业中目前使用的农药对人类相对安全，但它们会严重影响目标害虫和益虫。 农药包括杀虫剂、除草剂和杀真菌剂。最广泛使用的杀虫剂是神经毒性的。最具活性的物质属于4个主要家族，靶向突触离子通道：GABA和烟碱受体（nAChR），由非泼罗尼和类烟碱靶向; Nav通道，由拟除虫菊酯和其他Nav阻滞剂（如氰氟虫腙）靶向;胆碱酯酶，由有机磷酸酯和氨基甲酸甲酯靶向。它们的生态毒理学效应包括显著增加蜂群的损失。我们将进行体外分析的收集常用的农药在蜜蜂（蜜蜂）大脑的突触中表达的几个离子通道，并将克隆和表征这些通道的表达系统。我们假设蜜蜂离子通道对杀虫剂敏感，这可以解释它们对这些需要授粉的昆虫的有害影响。我们的研究计划假设将通过3个目标进行测试：目标#1：识别，克隆和测序GABA受体亚基基因在蜜蜂突触表达，并分析其在成年蜜蜂和幼虫组织中的表达模式。在非洲爪蟾卵母细胞中表达后，表征这些成孔亚基的生物物理和药理学特性以及目前用于农业或家庭目的的一组农药的影响。 目标二：识别，克隆和序列nAChR受体亚基基因在蜜蜂突触表达，表征其在非洲爪蟾卵母细胞中的表达，并研究其生物物理和药理学特性以及目前用于农业或家庭目的的农药，包括新农药的影响。目标3：产生每种通道类型的分子模型，有效分子可以对接到其上，以更好地理解离子通道调节的分子机制，并通过结构/序列变异识别可能的物种特异性。影响：该项目将提供一个非常宝贵的工具，用于深入了解主要植物药物产品的毒性，并将构成完整突触通道组的第一个全面的分子和功能表征。

The dramatic decline and disappearance of insect populations, and of many other species, around the world is now a scientific fact, with more than one million species on the endangered list. Insect populations have been harder hit, with a 75% to 98% decline in insect biomass in the last 25-40 years. Although there are many anthropogenic causes, chronic exposure to pesticides is central to the conflict between agricultural imperatives and environmental issues, which is front and centre in the political arena and most media. Despite the fact that the pesticides currently used in modern agriculture are relatively safe for humans, they can severely affect both target pests and beneficial insects. Pesticides include insecticides, herbicides, and fungicides. The most widely used insecticides are neurotoxic. The most active substances belong to 4 major families and target synaptic ion channels: GABA and nicotinic receptors (nAChR), which are targeted by fipronil and neonicotinoids, Nav channels, which are targeted by pyrethroids and other Nav blockers such as metaflumizone, and Choline esterase, which is targeted by organophosphates and methylcarbamates. Their ecotoxicological effects include a significant increase in the loss of honey bee colonies. We will perform in vitro analyses of a collection of commonly used pesticides on several ion channels expressed in the synapses of the honey bee (Apis mellifera) brain and will clone and characterize these channels in an expression system. We hypothesize that honey bee ion channels are sensitive to insecticides, which may explain their deleterious effect on these needed pollinators. Our research program hypothesis will be tested via 3 Aims: Aim #1: Identify, clone, and sequence GABA receptor subunit genes expressed at honeybee synapses and analyze their expression patterns in tissues from adult honey bees and larvae. Characterize, after expression in Xenopus oocytes, the biophysical and pharmacological properties of these pore-forming subunits as well as the effects of a panel of pesticides currently used for agricultural or domestic purposes. Aim #2: Identify, clone, and sequence nAChR receptor subunit genes expressed at honeybee synapses, characterize their expression in Xenopus oocytes, and study their biophysical and pharmacological properties well as the effects of pesticides currently used for agricultural or domestic purposes, including neonic pesticides. Aim #3: Produce a molecular model of each channel type onto which effective molecules can be docked to better understand the molecular mechanisms of ion channel regulation and identify possible species specificities through structural/sequence variations. Impact: This project will in fine provide an invaluable tool for providing an in-depth understanding of the toxicity of the main phytopharmaceutical products and will constitute the first comprehensive molecular and functional characterization of a complete synaptic channelome.