A New Genetic Expression System to Determine the Odor Tuning of Insect Vector Ionotropic Receptors Sensitive to Human-Derived Odorants

项目摘要/摘要 蚊子是全世界人类健康的主要威胁。被感染的蚊子叮咬后， 传播导致疟疾、寨卡和西尼罗河等疾病的各种病原体。最致命的 蚊子种类是冈比亚按蚊，是导致疟疾的疟原虫寄生虫的主要媒介; 在2020年，超过60万人死于这种昆虫的叮咬。为了识别和定位人类， 蚊子利用它们的嗅觉来识别源自人类的气味。人体皮肤上的羧酸， 如乳酸、丙酸、丁酸、庚酸、辛酸和十四烷酸，起到 在吸引寻找宿主的蚊子和宿主偏好方面都起着关键作用。嗅觉家族 检测这种人源性酸性气味的受体可能是嗅觉离子型受体（IR）。然而，在这方面， 先前尝试使用表达系统来表征蚊子IR的气味响应谱， 成功有限。在这里，我们提出了一种遗传方法在果蝇的发展 果蝇，这将有助于按蚊IR的功能表征。为了实现这一目标， 我们将产生一个“红外解码系统”苍蝇，它允许按蚊IRX异源 在缺乏内源性气味结合IrX的果蝇神经元中表达。具体目标是：1） 在果蝇中开发用于在感器中表达按蚊调谐IrX受体的遗传方法 缺乏内源性气味反应，以及2）鉴定冈比亚按蚊IrX， 羧酸气味剂，特别是已知吸引蚊子的那些。我们将首先生成一个“空 果蝇的三个IrX基因（Ir 41 a，Ir 75 a，Ir 75 d）在果蝇的三个嗅区中天然表达， ac 2感受器中的神经元在Ir 41 a-GAL 4转基因和Phi-C31 attP 2对接的背景下突变 绝佳的价钱我们将为在按蚊触角中大量表达的所有AgIrX生成attB-UAS-AgIrX构建体。 将attB-UAS-AgIrX插入空感器果蝇的attP 2位点将产生IR解码器系统 其中AgIrX在单个ac 2神经元中异位表达。AgIrX的气味响应特性- 表达神经元将通过对60种气味剂的一组的单感器记录来定量分析， 集中在那些人源羧酸和/或蚊子引诱剂。这些实验将 确定AgIrX对有吸引力的羧酸气味剂如乳酸最敏感，并且可以 提示了用于蚊子控制的合适分子靶标。这项研究将产生一个有价值的新的遗传工具， 研究了蚊虫嗅觉反应，为进一步研究蚊虫嗅觉反应奠定了基础 来自多种其他昆虫疾病媒介的嗅觉IrX的功能。

Project Abstract/Summary Mosquitoes are a major threat to human health throughout the world. From an infected bite, mosquitoes can transmit a variety of pathogens that lead to such diseases as malaria, Zika, and West Nile. The deadliest of all mosquito species is Anopheles gambiae, the predominant vector for Plasmodium parasites that lead to malaria; in 2020, over 600,000 people died as a result of bites from this insect. To identify and navigate towards humans, mosquitoes use their sense of smell to recognize human-derived odorants. Carboxylic acids on human skin, such as lactic acid, propanoic acid, butanoic acid, heptanoic acid, octanoic acid, and tetradecanoic acid, play a critical role in both attracting host-seeking mosquitoes as well as in host-preference. The family of olfactory receptors that detect such human-derived acid odorants are likely olfactory ionotropic receptors (IRs). However, previous attempts to use expression systems to characterize the odor-response profiles of mosquito IRs met with limited success. Here, we propose the development of a genetic method in Drosophila melanogaster flies that will facilitate the functional characterization of Anopheles mosquito IRs. To accomplish this goal, we will generate a “IR Decoder System” fly which allows Anopheles mosquito IrXs to be heterologously expressed in Drosophila neurons lacking their endogenous odor-binding IrX. The specific aims are: 1) to develop a genetic method in Drosophila for expressing an Anopheles tuning IrX receptor in a sensillum devoid of endogenous odorant responses, and 2) to identify the Anopheles gambiae IrXs that detect carboxylic acid odorants, particularly those known to attract mosquitoes. We will first generate an “Empty Sensillum” fly in which the three Drosophila IrXs (Ir41a, Ir75a, Ir75d) natively expressed in the three olfactory neurons in ac2 sensilla are mutated in the background of a Ir41a-GAL4 transgene and Phi-C31 attP2 docking site. We will generate attB-UAS-AgIrX constructs for all AgIrXs abundantly expressed in the Anopheles antenna. Insertion of the attB-UAS-AgIrX into the attP2 site of a Empty Sensillum fly will generate an IR Decoder System fly in which the AgIrX is ectopically expressed in a single ac2 neuron. The odor-response properties of the AgIrX- expressing neuron will be quantitatively analyzed by single sensillum recordings to a panel of 60 odorants, focused on those that are human-derived carboxylic acids and/or mosquito attractants. These experiments will identify the AgIrXs that are the most sensitive to attractive carboxylic acid odorants such as lactic acid, and could suggest suitable molecular targets for mosquito control. This study will generate a valuable new genetic tool for the investigation of olfactory responses of mosquito IrXs, and could further serve as the foundation to investigate the function of olfactory IrXs from a variety of other insect disease vectors.