The ionoregulatory mechanisms of insect chill tolerance

我的长期目标是对温度如何限制动物的表现有一个综合的理解，我将开始关注耐寒性。大多数昆虫都是对寒冷敏感的，这意味着它们在相对温和的低温下会受到非冷冻伤害。对这些昆虫来说，冷暴露的主要后果是破坏离子和水的稳态;冷诱导的细胞外[K+]的升高使细胞去极化，导致损伤和死亡。然而，通过热适应或可塑性，昆虫可以在寒冷中更好地维持离子稳态，从而避免伤害。因此，离子调节器官的生理变化可能是昆虫耐寒性变化的基础。昆虫的离子平衡是通过马氏管和肠道的协调运输来维持的。这些器官与耐寒性高度相关，但低温损害其功能的机制尚不清楚。在我的第一个资助周期中，我打算用果蝇、蝗虫和蟋蟀来研究：1）低温对离子调节器官的运输生理的影响，2）低温对细胞旁上皮屏障的影响，以及3）耐低温的内分泌控制。在我的第一个目标中，飞蝗将被用来研究温度如何直接影响MT和肠上皮细胞中的跨细胞离子转运。为了实现这一目标，我将使用多种技术来检查温度对离子和水运输的影响，并量化单个转运蛋白对维持寒冷中离子平衡的相对贡献。这些技术将适用于蟋蟀物种的耐寒性不同，以检查上皮细胞的热adaptation.Molecules可以通过不仅跨细胞，但也paracellular路线，其紧密性介导的隔膜连接（SJ）。在我的第二个目标，我将确定的作用，SJS和它们的组成蛋白质在果蝇耐寒性。我将开始通过研究如何热适应调节肠道屏障特性和SJ蛋白在果蝇的组织特异性表达模式。与此同时，我将使用蝗虫来确定是否冷诱导SJ中断是一个共同的挑战所面临的昆虫，并本地化的屏障中断的关键organs.Within昆虫的一系列神经肽调节跨上皮细胞和细胞旁运输。在我的第三个目标中，我将筛选神经肽对耐寒性的影响，并确定耐寒性的诱导变化是否与果蝇在寒冷中维持离子稳态的能力改变有关。此外，我将使用组织特异性RNA-seq确定哪些神经肽和受体最有可能与耐寒适应和可塑性相关。

My long-term goal is to develop an integrative understanding of how temperature sets limits to animal performance, and I will begin with a focus on chill tolerance. The majority of insects are chill-susceptible, meaning they suffer non-freezing injury at relatively mild low temperatures. A primary consequence of cold exposure for these insects is a disruption of ion and water homeostasis; a cold-induced rise in extracellular [K+] depolarizes cells, causing injury and death. Through thermal adaptation or plasticity, however, insects can better maintain ion homeostasis in the cold, and thereby avoid injury. Thus, modifications to the physiology of ionoregulatory organs likely underlie variation in insect chill tolerance. Insect ion balance is maintained by the coordinated transport properties of the Malpighian tubules (MT) and gut. These organs are highly relevant to chill tolerance, but the mechanisms by which chilling impairs their function are unknown. In my first grant cycle I propose to use fruit flies, locusts, and crickets to examine: 1) the effects of chilling on the transport physiology of the ionoregulatory organs, 2) the effects of chilling on paracellular epithelial barriers, and 3) endocrine control of chill tolerance.In my first objective, the migratory locust will be used to examine how temperature directly impacts transcellular ion transport in the MT and gut epithelia. To accomplish this, I will use a variety of techniques to examine temperature effects on ion and water transport, and quantify the relative contributions of individual transporters to maintaining ion balance in the cold. These techniques will then be adapted to cricket species that vary in chill tolerance to examine epithelial thermal adaptation.Molecules can cross epithelia through not only transcellular, but also paracellular routes, the tightness of which are mediated by the septate junctions (SJ). In my second objective, I will determine the role of SJs and their component proteins in Drosophila chill tolerance. I will begin by examining how thermal acclimation modulates gut barrier properties and tissue-specific expression patterns of SJ proteins in Drosophila. In parallel, I will use locusts to determine whether cold-induced SJ disruption is a common challenged faced by insects, and to localize barrier disruption to key organs.Within insects a range of neuropeptides regulate transcellular and paracellular transport across epithelia. In my third objective, I will screen neuropeptides for effects on chill tolerance, and determine whether induced variation in chill tolerance is associated with an altered ability of flies to maintain ion homeostasis in the cold. Further, I will determine which neuropeptides and receptors are most likely to be relevant to chill tolerance adaptation and plasticity, using tissue-specific RNA-seq.