Characterization of the sodium channel mutations involved in insecticide resistan

描述（由申请人提供）：蚊子传播的疾病，包括疟疾，是全世界人类的头号杀手。控制这些疾病的一个主要障碍是蚊子对杀虫剂产生了抗药性。拟议研究的长期目标是确定控制蚊子对杀虫剂反应的分子机制，并利用这些信息开发新的，更有效的疗法，以防止抗性发展，控制抗性蚊子，并最终减少蚊子传播的疾病及其影响。大量研究表明，钠通道的点突变降低了钠通道对拟除虫菊酯杀虫剂的敏感性，反过来，这种突变赋予拟除虫菊酯抗性。尽管有这样的发现，仍然没有一个全面的，全球性的图片涉及整个钠通道基因的突变，有助于任何昆虫，包括蚊子的杀虫剂抗性。这项提案正在寻求一个问题的答案：在整个钠通道中，有多少同义和非同义突变参与了蚊子的杀虫剂抗性？初步研究已经从致倦库蚊S-Lab和HAmCqG 0、HAmCqG 8、MoAmCqG 0和MoAmCqG 8蚊子中分离了全长钠通道的cDNA，并且已经鉴定了25个突变（7个非同义突变和18个同义突变）。拟议研究的目的是表征7种蚊子品系、2种蚊子品系和16组将用不同浓度的氯菊酯处理的蚊子中的25种钠通道突变。该表征将确定拟除虫菊酯杀虫剂涉及哪些突变或突变组合。我们的假设是：（a）拟除虫菊酯类杀虫剂抗药性所涉及的突变（"关键突变"）在杀虫剂选择后的蚊子种群中普遍存在，以及（B）"关键"突变的普遍性与杀虫剂抗药性的水平有关。我们预测，将突变或突变组合引入野生型（易感）蚊子钠通道将降低钠通道对拟除虫菊酯治疗的敏感性。我们的具体目标是：1）确定杀虫剂抗性水平与钠通道突变和突变组合的流行率之间的关系，2）确定关键突变对钠通道对拟除虫菊酯敏感性的影响，3）确定蚊子钠通道中关键突变的等位基因表达在DNA水平和RNA水平之间的关系。拟议的研究独特地集中在整个蚊子钠通道中自然发生的所有突变上，这直接反映了已知天然具有多个钠通道突变的蚊子的野外种群。这项研究的独特之处还在于，它不仅表征了非同义突变，而且还表征了同义突变对拟除虫菊酯钠通道敏感性的影响。表征整个蚊子钠通道中的非同义和同义突变或突变组合的作用是理解钠通道不敏感介导的抗性所涉及的分子机制的重要的早期步骤，并且最终为控制蚊子和预防蚊媒疾病（特别是疟疾）的新型治疗方法的开发提供信息。 公共卫生相关性：拟议的项目旨在表征钠通道突变，包括非同义突变和同义突变，这些突变涉及蚊子媒介致倦库蚊中的拟除虫菊酯杀虫剂抗性。我们将确定杀虫剂抗性水平和钠通道突变流行率之间的关系，并在这种蚊子载体的突变组合。我们将使用非洲爪蟾卵母细胞表达系统确定关键突变和突变组合对钠通道对拟除虫菊酯敏感性的影响。我们还将通过在DNA水平上比较每个关键突变的等位基因，在RNA水平上比较每个关键突变的等位基因，来确定蚊子钠通道中关键突变的转录后调控机制。

DESCRIPTION (provided by applicant): Mosquito-borne diseases, including malaria, are the number-one killers of humans worldwide. A major obstacle in controlling these diseases is that mosquitoes have developed resistance to insecticides. The long-term goals of the proposed research are to determine molecular mechanisms that control the mosquito's response to insecticides and to use this information to develop novel, more effective therapies to prevent resistance development, to control resistant mosquitoes and, ultimately, to reduce mosquito-borne diseases and their impact. A large number of studies showed that point mutations of sodium channels reduce sodium channel sensitivity to pyrethroid insecticides and that, in turn, such mutations confer pyrethroid resistance. Despite such findings, there is still no comprehensive, global picture of the mutations involved in an entire sodium channel gene that contributes to insecticide resistance in any insect, including the mosquito. This proposal is seeking an answer to the question: How many mutations, both synonymous and nonsynonymous, in an entire sodium channel are involved in insecticide resistance in the mosquito? Preliminary research has isolated cDNAs of the full-length sodium channels from Culex quinquefasciatus S- Lab and the HAmCqG0, HAmCqG8, MoAmCqG0, and MoAmCqG8 mosquitoes, and 25 mutations (7 nonsynonymous and 18 synonymous) have been identified. The objective of the proposed research is to characterize the 25 sodium channel mutations in 7 mosquito strains, in 2 mosquito lines, and in 16 groups of mosquitoes that will be treated with different concentrations of permethrin. This characterization will determine which mutations or mutation combinations are involved in pyrethroid insecticide. Our hypotheses are that: (a) mutations involved in pyrethroid insecticide resistance ("key mutations") are prevalent in mosquito populations following insecticide selection and (b) the "key" mutation prevalence is related to the level of insecticide resistance. We predict that the introduction of the mutations or mutation combinations into the wild (susceptible)-type mosquito sodium channel will decrease the sensitivity of the sodium channel, in response to pyrethroid treatment. Our specific aims are to: 1) Define the relationship between the level of insecticide resistance, and the prevalence of sodium channel mutations and mutation combinations, 2) Determine the effects of key mutations on sodium channel sensitivity to pyrethroids, and 3) Determine the relationship between the allelic expression of key mutations in the mosquito sodium channel at the DNA level and at the RNA level. The proposed research uniquely focuses on all of the mutations that naturally occur in an entire mosquito sodium channel, which directly reflects field populations of mosquitoes that are known to naturally have multiple sodium channel mutations. This study is also unique in its characterizing the effects not only of nonsynonymous but also of synonymous mutations on sodium channel sensitivity to pyrethroids. Characterizing the roles of nonsynonymous and synonymous mutations or mutation combinations in an entire mosquito sodium channel is an important, early step in understanding molecular mechanisms involved in sodium channel insensitivity-mediated resistance and, ultimately, in informing the development of novel treatments to control mosquitoes and to prevent mosquito-borne diseases, in particular malaria. PUBLIC HEALTH RELEVANCE: The proposed project seeks to characterize sodium-channel mutations, both nonsynonymous and synonymous mutations, that are involved in pyrethroid insecticide resistance in the mosquito vector Culex quinquefasciatus. We will define the relationship between the level of insecticide resistance and prevalence of sodium channel mutations and mutation combinations in this mosquito vector. We will determine the effects of key mutations and mutation combinations on sodium channel sensitivity to pyrethroids, using the Xenopus oocyte expression system. We also will determine the post-transcriptional regulation mechanism for the key mutations in the mosquito sodium channel by comparing the alleles of each key mutation at the DNA level, with the alleles of each key mutation at the RNA level.