Diapause in Culex Mosquitoes

描述(申请人提供)：蚊媒疾病是大多数发展中国家的主要健康问题，已成为发达国家疫情暴发的重大威胁。加剧这些问题的是蚊子种群出现抗药性、寄生虫种群出现抗药性、疫苗开发进展缓慢、缺乏对蚊子控制项目的支持以及全球旅行增加。库蚊复合体中的蚊子是淋巴丝状线虫寄生虫、班克罗夫氏舞蹈菌和几种虫媒病毒的主要媒介。这包括最近在北美引入的西尼罗河病毒。拟议研究的长期目标是确定、分离和表征与滞育相关的遗传因素，这一特征强烈影响库蚊复合体内物种的地理分布和生存。目前，关于库蚊滞育的分子决定因素尚不清楚。这项应用的目的是基于这样一个普遍假设，即与滞育表型相关的基因可以通过位置克隆技术进行识别和分离。决定滞育的基因的识别和对相关生化途径的详细了解可能有几个潜在的应用，包括：促进种群替换后改变和疾病限制行为的转基因构建，开发靶向生物杀虫剂，或识别生物相关的拮抗剂，扰乱对病原体传播或生存至关重要的行为。该项目的具体目标是：1)精细绘制基因组区域图，确定包含决定滞育表型的基因；2)确定决定滞育的候选基因；3)对决定滞育的候选基因进行功能分析。来自基因杂交的信息将被用来对抗CX。Ququefasciatus基因组计划，以确定离散基因组区域内的候选基因。候选基因将使用微阵列进行评估，然后进行实时定量聚合酶链式反应分析，以确认和扩展微阵列结果以寻找有前景的基因。阳性候选基因将通过RNA干扰介导的基因敲除或使用重组Sindbis转导病毒进行基因过度表达来检测其生物学相关性。实验室实验的结果将在代表CX的自然种群中进行评估。罗布麻属(Piniens S.S.)和CX.[医]五倍子属公共卫生相关性：库蚊复合体中的蚊子是人类淋巴丝虫病和包括西尼罗河病毒在内的几种虫媒病毒的主要媒介。没有疫苗可用，疾病预防在很大程度上依赖于避免接触受感染的蚊子。这项拟议的研究将提供有关蚊子生物学的有价值的新信息，这些信息与成年雌性蚊子在温和气候的冬季滞育状态下生存的能力有关。

DESCRIPTION (provided by applicant): Mosquito-borne diseases represent major health problems in most developing countries and have emerged as significant threats for epidemic outbreaks in developed countries. Exacerbating these problems are the emergence of insecticide resistance in mosquito populations, drug resistance among parasite populations, delayed progress in vaccine development, lack of support for mosquito control programs, and increased global travel. Mosquitoes in the Culex pipiens complex are primary vectors for the lymphatic filarioid nematode parasite, Wuchereria bancrofti and several arboviruses. This includes the recently introduced West Nile virus in North America. The long- term objectives of the proposed research are to identify, isolate and characterize genetic factors associated with diapause, a trait that strongly impacts the geographic distribution and survival of species within the Culex pipiens complex. At present, no knowledge exists on the molecular determinants of diapause in Culex. The objectives of this application are based on the general hypothesis that genes associated with the diapause phenotype can be identified and isolated by positional cloning techniques. Identification of genes determining diapause and the knowledge from the detailed understanding of the associated biochemical pathways could have several potential applications including: transgene constructs that promote altered and disease limiting behaviors following population replacement, development of targeted biopesticides, or identification of biologically relevant antagonists that disrupt behaviors critical to pathogen transmission or survival. The specific aims of the project are to: 1) fine-scale map genome regions identified as containing genes that determine the diapause phenotype, 2) identify candidate genes determining diapause, and 3) conduct functional analysis of candidate genes determining diapause. Information from genetic crosses will be leveraged against the Cx. quinquefasciatus genome project to identify candidate genes within discrete genome regions. Candidate genes will be evaluated using microarrays, followed by quantitative real time PCR assays to confirm and expand microarray results for promising genes. Positive candidate genes will be assayed for biological relevance using RNA interference mediated gene knockdowns or gene overexpression using recombinant Sindbis transducing viruses. Results from laboratory experiments will then be evaluated among natural populations representing Cx. pipiens s.s. and Cx. quinquefasciatus. PUBLIC HEALTH RELEVANCE: Mosquitoes in the Culex pipiens complex are primary vectors for human lymphatic filariasis and several arboviruses, including the West Nile virus. No vaccines are available and disease prevention is largely dependent on avoiding contact with infected mosquitoes. The proposed research will provide valuable new information on the biology of the mosquito relating to the ability of adult females to survive temperate climate winter in a diapause state.