The Neuropeptide Control of Mosquito Host-Seeking Behavior

描述（由申请人提供）：埃及伊蚊是几种人类疾病的主要媒介，包括登革热，每年产生约5000万病例，使25亿人处于危险之中（17）。对埃及伊蚊以及疟疾媒介冈比亚按蚊等其他蚊子来说，迫切需要对抗蚊媒疾病的新策略。我们的目标是找到新的方法来干扰蚊子寻找人类宿主吸血的能力。然而，寄主寻找的机制基础及其调控尚不完全清楚。虽然已知蚊子需要人类气味线索来定位人类宿主，但尚未确定关键的气味成分和相关的嗅觉受体（12,23）。吸血后三天内，蚊子寻找宿主的行为受到抑制。这种对摄食行为的抑制部分是由神经肽头肽- 1的释放引起的，但这种肽改变蚊子行为的机制以及它所通过的信号受体尚不清楚(4)。头肽i抑制寄主寻找行为的一个可能机制是调节蚊子闻到人类寄主的能力。为了验证这一假设，我们建议通过去孤儿化和定位目前未知的Head Peptide-I受体来鉴定Head Peptide-I反应神经元。使用基于细胞的钙成像筛选，我们已经确定了g蛋白偶联受体npy样受体-1 （NPYLR1）作为头肽-1的候选受体。支持数据显示，多种NPYLR1激动剂在注射到非吸血雌性时能够抑制寻宿主行为。为了研究NPYLR1是否需要抑制头肽i，我们使用最近首创的锌指核酸酶靶向诱变来创建NPYLR1零突变蚊子。我们预测，这些突变体在血餐后不会再失去对人类气味线索的反应。为了定位头部肽i应答的NPYLR1神经元，我们将使用定制的NPYLR1抗体，并创建转基因蚊子，荧光标记表达NPYLR1的细胞。这一信息将阐明头部肽i抑制的神经机制，并为控制蚊子寻宿主行为的新策略奠定基础。

DESCRIPTION (provided by applicant): Aedes aegypti mosquitoes are the principal vectors for several human diseases including Dengue Fever, which generates ~50 million cases per year and places 2.5 billion people at risk (17). Novel strategies to combat mosquito-borne diseases are in high demand for A. aegypti as well as other mosquitoes such as the malarial vector Anopheles gambiae. Our goal is to discover new ways to interfere with the mosquito's ability to locate a human host for a blood meal. However, the mechanistic basis of host-seeking and its regulation remains incompletely understood. Although it is known that mosquitoes require human odor cues to locate a human host, the critical odor components and associated olfactory receptors have not been identified (12, 23). Mosquito host-seeking behavior is inhibited for three days following a blood meal. This suppression of feeding behavior is initiated in part by the release of the neuropeptide Head Peptide-I, but the mechanism by which this peptide alters mosquito behavior and the receptor through which it signals is unknown (4). One possible mechanism by which Head Peptide-I may inhibit host-seeking behavior is to modulate the mosquito's ability to smell a human host. To investigate this hypothesis, we propose to identify the Head Peptide-I responsive neurons by de-orphanizing and localizing the currently unknown Head Peptide-I receptor. Using a cell-based calcium-imaging screen, we have identified the G-protein coupled receptor NPY-Like Receptor-1 (NPYLR1) as a candidate Head Peptide-I receptor. Supporting data reveals that multiple NPYLR1 agonists are capable of inhibiting host-seeking behavior when injected into non-bloodfed females. To investigate whether NPYLR1 is required for Head Peptide-I inhibition, we are using recently pioneered targeted mutagenesis with zinc-finger nucleases to create NPYLR1 null-mutant mosquitoes. We predict that these mutants will no longer lose responsiveness to human odor cues after a blood meal. To localize Head Peptide-I responsive NPYLR1 neurons, we will use a custom NPYLR1 antibody and create transgenic mosquitoes that fluorescently mark NPYLR1 expressing cells. This information will clarify the neural mechanism of Head Peptide-I inhibition, and could form the basis of novel strategies to control mosquito host-seeking behavior.