Neuronal basis of pheromone perception in mosquitoes

项目的动机。按蚊是疟疾的媒介，影响世界人口的一半(世卫组织2019年)。蚊子严重依赖它们的嗅觉来寻找人类作为血餐(Konopka等人，2021年)。令人惊讶的是，关于按蚊之间的化学交流的报道非常稀少，而且最近才有报道，而且信息素的使用并不普遍适用于任何蚊子。与蚊子不同，大多数其他昆虫使用挥发性和非挥发性信息素是众所周知的(Blomquist和BagneréS，2010)。我们对明显缺乏关于蚊子信息素的信息很感兴趣，因此隐含的假设是，与其他昆虫相比，蚊子不会产生和使用化学线索来进行种内交流。我们采用了新的方法和技术进行了初步研究，确定了冈比亚按蚊17个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如何在细胞、器官和整个有机体水平上对信息素候选做出反应。我们将利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。目标。1)验证信息素候选者，并确定在雄性和雌性蚊子中检测信息素候选者的嗅觉受体；2)创建针对特定对信息素做出反应的神经元的转基因蚊子系；3)对信息素神经元进行遗传操作，并测试它们对蚊子交配行为的影响。方法论。信息素候选将通过GC/MS和热解吸相结合的方法进行验证。嗅觉受体将通过蚊子和果蝇嗅觉神经元的活体钙成像来识别。通过使用CRISPR/Cas9将候选OR基因的终止密码子替换为T2A-QF2构建，将产生新的转基因株系。将开发嗅觉偏好、飞行活动以及交配和行为测试小组，并对雄性和雌性冈比亚按蚊进行测试。时间表。第一年：达勒姆和维尔茨堡的目标1；欧洲ECRO/ESITO会议；第二年：达勒姆的目标1和第二次会议；SFN会议/神经行为学大会；巴黎神经科学学院的光学成像和电生理记录；第三年：纽卡斯尔和达勒姆的目标3；PIPS安置；欧空局会议；第四年：MBL课程，美国伍兹霍尔；完成实验和写作。新奇。该项目将涉及昆虫神经行为学、神经科学和遗传学的尖端技术，这些技术的监督者在这方面处于世界领先地位。此外，了解蚊子的性行为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.