Apis mellifera ion channels and tools to study agroecosystems contaminants

世界各地昆虫数量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多万个物种在濒危名单上。昆虫种群受到的打击更大，在过去的25-40年里，昆虫的生物量下降了75%到98%。虽然有许多人为原因，但长期接触杀虫剂是农业当务之急与环境问题之间冲突的核心，这是政治舞台和大多数媒体的中心。尽管目前在现代农业中使用的杀虫剂对人类相对安全，但它们可以严重影响目标害虫和有益昆虫。杀虫剂包括杀虫剂、除草剂和杀菌剂。使用最广泛的杀虫剂是神经毒性杀虫剂。最活跃的物质属于4个主要的家族和靶突触离子通道：GABA和尼古丁受体(NAChR)，它是氟虫腈和新烟碱的靶标；NAV通道，它是拟除虫菊酯和其他NAV阻滞剂(如甲呋喃酮)的靶标；胆碱酯酶，它是有机磷和甲基氨基甲酸酯的靶标。它们的生态毒理效应包括显著增加蜜蜂群体的损失。我们将对蜜蜂大脑突触中表达的几种离子通道上的一系列常用杀虫剂进行体外分析，并将在表达系统中克隆和表征这些通道。我们假设蜜蜂离子通道对杀虫剂很敏感，这可能解释了它们对这些所需传粉者的有害影响。我们的研究计划假设将通过三个目标进行验证：目标1：鉴定、克隆和测序在蜜蜂突触表达的GABA受体亚单位基因，并分析它们在成年蜜蜂和幼虫组织中的表达模式。在非洲爪哇卵母细胞中表达后，表征这些形成孔的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杀虫剂的影响。目的#2：鉴定、克隆和测序蜜蜂突触表达的nAChR受体亚单位基因，鉴定它们在非洲爪哇卵母细胞中的表达，并研究它们的生物物理和药理特性以及目前用于农业和家庭用途的农药，包括霓虹灯农药的影响。目的#3：建立每种通道类型的分子模型，有效分子可以对接到该模型上，以更好地了解离子通道调节的分子机制，并通过结构/序列变化识别可能的物种特异性。影响：该项目最终将为深入了解主要植物制药产品的毒性提供一个宝贵的工具，并将构成对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.