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

说明（由申请人提供）：疟蚊要成功传播疟原虫，必须在按蚊体内发展疟原虫。虽然最大的疾病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。在已知疟蚊传播媒介的地理分布中，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，间日疟原虫具有最广泛的地理分布，有25亿人面临该疾病的风险，每年有8 000至3亿临床病例，包括严重疾病和死亡。尽管有巨大的公共卫生负担，间日疟原虫的研究得到的关注和支持远远少于以恶性疟原虫为中心的研究。最近有一个高度重新强调的研究，旨在阐明间日疟原虫的传播生物学。不幸的是，间日疟原虫的分子和基因组信息很少，在SSA以外的一些流行地区，间日疟原虫也是恶性疟原虫的载体。此外，尽管“模式”非洲恶性疟媒介冈比亚按蚊基因组的分子功能研究相对较好，但在按蚊中观察到的大量进化差异限制了其作为整个谱系参考的效用，特别是在几种间日疟媒介方面。间日疟原虫和恶性疟原虫的主要传播媒介之一是西太平洋的法劳按蚊1号（FAR1/AF1）及其相关物种复合体，这是了解人类疟原虫通过蚊子传播生物学的一个潜在的重要模型系统。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与蚊子组织，特别是蚊子中肠之间的相互作用。在这个项目中，我们的首要问题是“什么是介导卵胞体疟原虫-按蚊中肠相互作用的分子决定因素？”因此，我们将对蚊子中肠腔中表达的免疫相关基因进行转录组学和蛋白质组学比较分析，这些基因来自于模型（冈比亚蚊）、最近测序的“新兴模型”按蚊载体（FAR1）菌落和野生型法罗蒂蚊（AF1），以及非模型但相关的物种（冈比亚蚊）。punctulatus,美联社)。我们的重点将放在免疫相关基因的亚群上，这些基因在血液摄食和疟原虫入侵的反应中作为转录物和蛋白质的差异表达。我们将首先关注实验室恶性疟原虫的FAR1感染，然后用FAR1/AF1/AP进行间日疟原虫膜喂养试验来补充这些研究。值得注意的是，本项目中描述的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增强媒介生物学界最近对该物种的FAR1测序和基因组注释，而且还将极大地有助于我们对间日疟-按蚊相互作用的了解，从而使两种研究系统“相互照亮”。

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.