Development of optogenetically controlled gene expression tools for the characterization of neuronal circuits involved in insect reproduction

生殖行为及其调控是所有动物最基本的行为，但在昆虫种群控制中却很少被利用。由于它们是硬连接到大脑中的，我们可以了解这种行为程序是如何在大脑中编码的，以及如何通过感知和决策过程来塑造。了解行为是如何在大脑中编码的是生物学中的一个重大挑战，需要一个行为和遗传上易于处理的模型生物，但也需要操纵局部神经元的工具。实现这一目标的关键工具之一是可光操纵的分子，如离子通道，其中光可用于控制神经元在空间和时间上的活动。然而，由于昆虫的体积小，这种技术有其局限性。在这里，我们想适应光诱导转录因子来自细菌和植物已经用于哺乳动物细胞培养果蝇的特征参与生殖的神经元回路。在这项分析中，我们将利用已知的基因表达调控序列来表征参与生殖的神经元群体，但这些基因表达模式是复杂的。因此，为了能够将功能分配给局部神经元，需要对这些基因表达模式进行空间解剖，这可以通过光控转录因子来实现。为了开发这种光控工具来操纵基因表达，我们将利用果蝇的强大交配后反应（PMR）。在这里，雄性交配时转移的性肽（SP）是导致拒绝再交配和诱导产卵的关键分子。果蝇雌性对性肽的强烈行为反应为定位SP反应神经元并最终了解诸如交配选择和产卵控制等复杂行为是如何在大脑中编码的提供了必要的先决条件。我们目前还不知道这些神经元在果蝇中的位置，但是，我们可以证明这两种交配后的反应是可以分开的。SP诱导的交配后反应的候选神经元包括生殖道和腿中的感觉神经元，但也包括腹神经节和中枢脑中的神经元。为了确定性肽反应的神经元电路，我们将使用光诱导的基因表达针对神经元在特定部位的雌性苍蝇身体表达膜系SP。这种光遗传操纵的基因表达的优势是完全可控的空间和时间。通过这些实验，我们将检验对SP的反应是由单个元素的模块化组装组成的假设，例如拒绝重新交配或诱导产卵。与先前的模型相比，主张所有PMR的中央诱导，一个模块化的组装个人PMR持有进化的灵活性，在物种形成和适应不同的栖息地，但可以保持基本的监管原则，如控制产卵。因此，我们预计，从我们的研究中获得的知识将适用于广泛的害虫精确定位到新的策略，害虫管理，以保护作物和控制昆虫传播的疾病，通过干扰产卵。特别是，我们的研究结果可以直接转移到近亲Suzukii果蝇，这是少数几个能够将卵产在水果中的物种之一，目前正在入侵包括英国在内的欧洲，并对水果生产造成价值数十亿英镑的损失。

Reproductive behaviors and their regulation are most fundamental to all animals, but have been exploited little for population control in insects. Since they are hard-wired into the brain we can learn how this behavioral program is encoded in the brain and shaped by perception and decision-making processes. Understanding how behavior is encoded in the brain is one of the big challenges in biology and requires a behaviorally and genetically tractable model organism, but also tools to manipulate localized neurons. One of the key tools to achieve this aim are light-manipulateable molecules, such ion channels, where light can be used to control neuronal activity in space and time. Due to the small size of insects, however, this technology has its limitation. Here, we want to adapt light-inducible transcription factors derived from bacteria and plants already used in mammalian cell culture to Drosophila to characterize the neuronal circuits involved in reproduction. For this analysis we will capitalize on gene expression regulatory sequences known to characterize neuronal populations involved in reproduction, but these gene expression patterns are complex. To be able to assign functions to localized neurons therefore requires spatial dissection of these gene expression patterns, which can be achieved by light-controlled transcription factors.To develop such light-controlable tools to manipulate gene expression, we will capitalize on the robust post-mating responses (PMRs) of the fruit fly Drosophila melanogaster. Here, male-derived sex-peptide (SP) transferred during mating is the key molecule leading to refusal to remate and induction of egg laying. The very robust behavioral response of Drosophila females to sex-peptide provides the essential prerequisites to map SP responsive neurons and eventually learn how complex behaviors such as mating choice and control of egg laying are encoded in the brain.Our recent studies showed that there are several distinct neuronal populations that can via exposure to SP induce refusal to remate and egg laying. We currently do not know where in the fly these neurons are located, however, we could show that these two post-mating responses can be separated. Candidate neurons for SP induced post-mating responses include sensory neurons in the genital tract and in the legs, but also neurons in the abdominal ganglion and the central brain. To identify the neuronal circuitry underlying the sex-peptide response, we will use light induced gene expression directed to neurons in specific parts of the female fly body to express membrane-tethered SP. Such optogentic manipulation of gene expression has the advantage to be fully controllable in space and time. With these experiments we will test the hypothesis that the response to SP is comprised of a modular assembly of individual elements, e.g. refusal to remate or induction of egg laying. Compared to the previous model arguing for central induction of all PMRs, a modular assembly of individual PMRs holds evolutionary flexibility during speciation and adaptation to diverse habitats, but can maintain basic regulatory principles such as the control of egg laying. We therefore anticipate that the knowledge obtained from our studies will be applicable to a wide range of pest insects pinpointing towards novel strategies for pest management to protect crop and control insect born diseases by interfering with egg laying. In particular, our findings are directly transferable to the close relative Drosophila suzukii, one of the few species able to lay eggs into fruits, which is currently invading Europe including the UK and causing damage worth billions of pounds to fruit production.