Apis mellifera ion channels and tools to study agroecosystems contaminants

研究农业生态系统污染物的意大利蜜蜂离子通道和工具

• 批准号: RGPIN-2020-06359
• 负责人: CHAHINE, Mohamed
• 金额: $ 2.62万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760583
• 关键词: Apis; mellifera; ion; channels; tools

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.

世界各地昆虫和许多其他物种数量的急剧下降和消失现在是一个科学事实，超过100万种物种被列入濒危物种名单。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%到98%。虽然有许多人为原因，但长期接触农药是农业迫切需要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前沿和中心。尽管现代农业中使用的农药对人类来说是相对安全的，但它们对目标害虫和有益昆虫都有严重的影响。杀虫剂包括杀虫剂、除草剂和杀菌剂。最广泛使用的杀虫剂是神经毒性的。最活跃的物质属于4大家族，并靶向突触离子通道：氟虫腈和新烟碱类靶向GABA和烟碱受体（nAChR），拟除虫菊酯和其他Nav阻滞剂（如甲氟咪唑）靶向Nav通道，有机磷酸盐和氨基甲酸甲酯靶向胆碱酯酶。它们的生态毒理学影响包括蜜蜂种群损失的显著增加。我们将对一组常用杀虫剂在蜜蜂（Apis mellifera）大脑突触中表达的几种离子通道进行体外分析，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敏感，这可能解释了它们对这些需要的传粉媒介的有害影响。我们的研究计划假设将通过3个目标进行验证：目标1：鉴定、克隆和测序蜜蜂突触中表达的GABA受体亚基基因，并分析它们在成年蜜蜂和幼虫组织中的表达模式。描述在非洲爪蟾卵母细胞中表达后，这些孔隙形成亚基的生物物理和药理学特性，以及目前用于农业或家庭用途的一组杀虫剂的影响。目标#2：鉴定、克隆和测序蜜蜂突触中表达的nAChR受体亚基基因，表征它们在非洲爪蟾卵母细胞中的表达，研究它们的生物物理和药理学特性，以及目前用于农业或家庭用途的农药（包括新农药）的影响。目标#3：建立每个通道类型的分子模型，有效分子可以停靠在上面，以更好地理解离子通道调节的分子机制，并通过结构/序列变化识别可能的物种特异性。影响：该项目将为深入了解主要植物药物的毒性提供宝贵的工具，并将构成完整突触通道体的第一个全面的分子和功能表征。