Neuronal basis of pheromone perception in mosquitoes

项目的动机。按蚊是影响世界一半人口的疟疾媒介（世卫组织，2019年）。蚊子在很大程度上依赖于它们的嗅觉来寻找人类的血餐（Konopka et al, 2021）。令人惊讶的是，关于按蚊之间化学交流的报道非常少，而且是最近才出现的，而且信息素的使用也没有普遍适用于任何蚊子。与蚊子相反，众所周知，大多数其他昆虫都使用挥发性和非挥发性信息素（Blomquist和bagner<s:1>, 2010）。我们对蚊子信息素的明显缺乏很感兴趣，因此隐含的假设是，与其他昆虫相比，蚊子不产生和使用化学线索来进行种内交流。本研究采用新颖的方法和技术，在冈比亚按蚊中鉴定出17种潜在的雄性和雌性信息素候选者。该项目将验证和加强我们的初步发现，并将探索冈比亚按蚊在细胞、器官和整个生物体水平上对候选信息素的反应。我们将采用昆虫神经科学和基因工程的尖端技术来实现我们的目标。目的：1)验证候选信息素，鉴定雌雄蚊子中检测候选信息素的嗅觉受体；2)创造一种转基因蚊子系，靶向对信息素有特异性反应的神经元；3)通过基因调控信息素神经元，测试其对蚊子交配行为的影响。方法。候选信息素将通过GC/MS结合热脱附进行验证。嗅觉受体将通过活钙成像从蚊子和果蝇的嗅觉神经元鉴定。利用CRISPR/Cas9将候选OR基因的终止密码子替换为T2A-QF2构建体，生成新的转基因系。将对雄性和雌性冈比亚蚊进行嗅觉偏好、飞行活动、交配和行为分析。时间表。第一年：达勒姆和维尔茨堡的第一个目标；欧洲ECRO/ESITO会议；二年级：目标1和目标2在达勒姆；SfN会议/神经行为学大会；巴黎神经科学学院光学成像与电生理记录；三年级：纽卡斯尔和达勒姆三年级；pip值位置;ESA会议;四年级：MBL课程“神经系统与行为”，伍兹霍尔，美国；完成实验并写报告。新鲜事物。该项目将涉及昆虫神经行为学、神经科学和遗传学的前沿技术，导师在这些领域处于世界领先地位。此外，了解蚊子的性交流对蚊子和疟疾的控制具有深远的意义。

Motivation for the project. Anopheles mosquitoes are the vectors of malaria that affects half of the world population (WHO 2019). Mosquitoes heavily rely on their sense of smell to find a human for a blood meal (Konopka et al, 2021). Surprisingly, reports of chemical communication in Anopheles are very scarce and recent, and the use of pheromones is not universally established for any mosquitoes. In contrast to mosquitoes, most other insects are well known to use volatile and non-volatile pheromones (Blomquist and Bagnerés, 2010). We were intrigued by the obvious lack of information about mosquito pheromones, and thus the implicit assumption that mosquitoes, in contrast to other insects, don't produce and use chemical cues to communicate intraspecifically. We employed novel methods and techniques for our pilot studies, and identified 17 potential male and female pheromone candidates in Anopheles gambiae. This project will verify and strengthen our preliminary findings, and will explore how Anopheles gambiae respond to pheromone candidates at the levels of cells, organs and the entire organism. We will employ cutting edge techniques of insect neuroscience and genetic engineering to achieve our aims. Aims. 1) To verify pheromone candidates and identify olfactory receptors that detect pheromone candidates in male and female mosquitoes; 2) To create a transgenic mosquito line targeting neurons that specifically respond to the pheromones; 3) To genetically manipulate the pheromone neurons and test their effect on mosquito mating behaviours. Methodology. Pheromone candidates will be verified by GC/MS coupled with thermodesorption. Olfactory receptors will be identified via live calcium imaging from mosquito and Drosophila olfactory neurons. New transgenic lines will be generated by using CRISPR/Cas9 to replace the stop codon of the candidate OR genes with a T2A-QF2 construct. Panel of olfactory preference, flight activity and mating and behavioural assays will be developed and conducted on male and female An.gambiae. Timetable. Year1: Aim 1 in Durham and Wuerzburg; conference ECRO/ESITO in Europe; Year2: Aims 1 and 2 in Durham; SfN conference/Neuroethology Congress; Optical Imaging and Electrophysiological Recordings in Neuroscience School, Paris; Year3: Aim 3 in Newcastle and Durham; PIPS placement; ESA conference; Year4: MBL course "Neural System and Behavior", Woods Hole, USA; finishing up experiments and writing up. Novelty. This project will involve cutting edge techniques of insect neuroethology, neuroscience and genetics that the supervisors are world leaders in. In addition, understanding mosquito sexual communication has profound implications for mosquito and malaria control.