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，我将从耐寒性开始。大多数昆虫对寒冷敏感，这意味着它们在相对温和的低温下遭受非冰冻伤害。对这些昆虫来说，寒冷暴露的主要后果是破坏离子和水的动态平衡；寒冷诱导的细胞外[K]升高会使细胞去极化，导致伤害和死亡。然而，通过热适应或可塑性，昆虫可以更好地在寒冷中保持离子动态平衡，从而避免伤害。因此，离子调节器官生理学的改变可能是昆虫耐寒性变化的基础。 昆虫的离子平衡是由马氏管(MT)和肠道的协调运输特性维持的。这些器官与耐寒性高度相关，但寒冷损害它们功能的机制尚不清楚。在我的第一个赠款周期中，我建议使用果蝇、蝗虫和蟋蟀来检查：1)冷藏对离子调节器官运输生理的影响，2)冷藏对细胞旁上皮屏障的影响，以及3)耐寒性的内分泌控制。 在我的第一个目标中，蝗虫将被用来研究温度如何直接影响MT和肠道上皮细胞内的跨细胞离子运输。为了做到这一点，我将使用各种技术来研究温度对离子和水运输的影响，并量化单个转运体在寒冷条件下维持离子平衡的相对贡献。然后，这些技术将被适应于耐寒性不同的板球物种，以检查上皮细胞的热适应。 分子不仅可以通过跨细胞途径，也可以通过细胞旁途径跨越上皮，这些途径的紧密程度由间隔连接(SJ)调节。在我的第二个目标中，我将确定SJS及其组成蛋白在果蝇耐寒性中的作用。我将从研究热习服如何调节果蝇肠道屏障特性和SJ蛋白的组织特异性表达模式开始。同时，我将使用蝗虫来确定寒冷诱导的SJ破坏是否是昆虫面临的常见挑战，并定位关键器官的屏障破坏。 在昆虫体内，一系列神经肽调节跨细胞和旁细胞跨上皮运输。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苍蝇在寒冷中维持离子动态平衡的能力改变有关。此外，我将使用组织特异性RNA-seq来确定哪些神经肽和受体最有可能与耐寒适应和可塑性有关。

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.