Midgut Transcriptome and Proteome Analyses: Non-model Anopheline Malaria Vectors

描述(申请人提供)：若要成功传播疟疾寄生虫，必须在按蚊体内培育出疟原虫。虽然疾病的最大负担发生在撒哈拉以南非洲，但疟疾发病率和死亡率远远超出了非洲大陆。在已知的疟疾按蚊媒介的地理分布中，发病率和死亡率都是由恶性疟原虫和间日疟原虫引起的。事实上，间日疟原虫的地理分布最广，有25亿人面临这种疾病的风险，每年有8000万至3亿临床病例，包括严重疾病和死亡。尽管造成了巨大的公共卫生负担，但与以恶性疟原虫为中心的研究相比，间日疟原虫的研究受到的关注和支持要少得多。最近，人们高度重视旨在阐明间日疟原虫传播生物学的研究。不幸的是，关于间日疟原虫媒介的分子和基因组规模的信息很少，在SSA以外的一些流行区也是恶性疟原虫的媒介。此外，尽管“模式”非洲恶性疟原虫媒介冈比亚按蚊基因组的分子功能得到了较好的研究，但在按蚊线内观察到的实质性进化差异限制了其作为整个谱系的参考作用，特别是对于几个间日疟原虫媒介。西太平洋法氏按蚊(Anopheles farauti 1，FAR1/AF1)及相关物种复合体成员是间日疟原虫和恶性间日疟原虫的主要媒介之一，也是了解人类疟原虫通过蚊虫传播生物学的潜在模式系统。为了更好地了解疟疾传播生物学，必须更彻底地研究寄生虫和蚊子组织之间的相互作用，特别是蚊子中肠。在这个项目中，我们的首要问题是“是什么分子决定因素调节着疟原虫--按蚊中肠的相互作用？”因此，我们将对蚊子中肠腔表达的免疫相关基因进行比较转录和蛋白质组学分析，这些基因来自模式(冈比亚)、最近测序的“新兴模式”按蚊载体(FAR1)群体和野生型法拉蒂(AF1)蚊子，以及非模式但相关物种(AN)。美联社)。我们的重点将放在免疫相关基因的子集上，这些基因在血液喂养和疟原虫入侵时以转录本和蛋白质的形式差异表达。我们将首先关注实验室恶性疟原虫的FAR1感染，并用FAR1/AF1/AP补充这些研究。值得注意的是，这个项目中描述的FAR1/AF1蚊子中肠的转录组组装和蛋白质组学分析不仅将有力地补充和加强媒介生物界最近对该物种的FAR1测序工作和基因组注释，而且还将极大地有助于使我们对间日疟原虫-按蚊相互作用的了解与恶性疟原虫-冈比亚蚊的相互作用平起平坐，从而使这两个研究系统能够相互“相互启发”。

DESCRIPTION (provided by applicant): Development of Plasmodium parasites in the Anopheles mosquito is required for successful malaria parasite transmission. Although the greatest burden of disease occurs in Sub-Saharan Africa (SSA), malaria morbidity and mortality extends far beyond the African continent. Across the geographical distribution of known anopheline vectors of Plasmodium, morbidity and mortality are caused by both Plasmodium falciparum and Plasmodium vivax. In fact, P. vivax has the widest geographic distribution with 2.5 billion people at risk of the disease, and between 80 - 300 million clinical cases every year, including severe disease and death. Despite this tremendous public health burden, P. vivax research has received far less attention and support than efforts centered on P. falciparum. There is a recent heightened re-emphasis on studies aimed at elucidating the transmission biology of P. vivax. Unfortunately, little molecular and genome-scale information exists for vectors of P. vivax, which in some endemic regions outside of SSA are also vectors of P. falciparum. Moreover, although the molecular functions of the genome of the "model" African P. falciparum vector, Anopheles gambiae is relatively well studied, the substantial evolutionary divergence observed within anophelines limits its utility as the reference for the entire lineage, especially with respect to several P. vivax vectors. One of the major vectors of P. vivax and P. falciparum, and a potentially great emerging model system to understand the transmission biology of human Plasmodia through mosquitoes, is Anopheles farauti 1 (FAR1/AF1) and related species complex members in the Western Pacific. To better understand malaria transmission biology, it is imperative to more thoroughly examine the interaction between the parasite and mosquito tissues, particularly the mosquito midgut. In this project our overarching question is "What are the molecular determinants mediating Plasmodium ookinete-Anopheles midgut interactions?" Thus, we will perform comparative transcriptomic and proteomic analyses of mosquito midgut lumen expressed immune- related genes from the model (gambiae), the recently sequenced "emerging model" anopheline vector (FAR1) colony and wild-type farauti (AF1) mosquitoes, as well as the non-model but related species (An. punctulatus, AP). Our focus will be on the subset of immune-related genes that are differentially expressed as both transcript and protein in response to blood feeding and Plasmodium invasion. We will first focus on FAR1 infections with laboratory P. falciparum and complement these studies with subsequent P. vivax membrane feeding assays with FAR1/AF1/AP. It should be noted that the transcriptome assembly and proteomics analysis of the FAR1/AF1 mosquito midgut described in this project will not only strongly complement and enhance the vector-biology community's recent FAR1 sequencing effort and genome annotation for this species but will also greatly contribute to bringing our knowledge on vivax-anopheles interactions on par with that of falciparum-gambiae, thus allowing the two study systems to "reciprocally illuminate" one another.