The cellular mechanisms of insect physiological plasticity and freeze tolerance

我的研究计划的长期目标是了解昆虫如何耐受低温，促进它们在温带气候中越冬的能力。研究人员已经知道一些昆虫耐冻近300年了，但我们对内部冻结是如何造成损害的了解有限，以及耐冻昆虫用来对抗这种损害的机制。我目前的重点是耐冻性的细胞生物学，使用春季田野蟋蟀Gryllus veletis作为模型系统。这些蟋蟀可以在很大程度上改变它们的耐寒性，在暴露(驯化)到六周的类似秋天的条件(低温、短日)时，从不耐冻转变为耐冻。我和我的学生将追求三个相互关联的目标，以确定驯化如何诱导促进昆虫耐冻性的细胞机制。对于目标1，我们将在细胞水平上描述冷冻诱导的损伤。我们将使用细胞生物学和生化分析相结合的方法来确定板球细胞及其大分子(蛋白质、膜、DNA)在冷冻过程中是否受到损害，以及这种损害是否可以在冷冻后修复。这将增加我们对与冷冻相关的挑战的理解，并促进进一步的研究，以测试特定的分子或基因是否对特定类型的损害具有保护作用。对于目标2，我们将确定驯化如何改变细胞结构和功能。秋季驯化过程中基因表达的变化表明，耐寒蟋蟀在几个方面调整了它们的细胞生物学。我们将使用免疫组织化学、膜脂组学和冷冻保护剂定量分析来检验这些假说。这些实验将确定秋季驯化是否会导致细胞结构和功能的变化，并将加深我们对减轻冰冻造成的损害的潜在机制的了解。对于目标3，我们将测试哪些分子和细胞过程有助于在冷冻过程中保护细胞。我们将首先使用RNA干扰(RNAi)来下调与目标2的细胞变化相关的基因的表达。然后，我们将使用与目标1类似的方法来测试阻止目标基因的功能如何影响整个动物和细胞的耐冻性。这将提供关于对耐冻性的机制至关重要的基因和相关细胞过程的急需的信息。我提出的研究计划应用了昆虫耐冻性领域一直缺乏的技术(细胞生物学，RNAi)。这些实验有可能极大地提高我们对昆虫如何在内部结冰中生存的理解。更广泛地说，这项工作在害虫管理、生物医学和食品行业的超低温保存协议以及确定越冬昆虫可能如何应对气候变化方面具有潜在的应用价值。

The long-term objective of my research program is to understand how insects tolerate low temperatures, facilitating their ability to overwinter in temperate climates. Researchers have known that some insects are freeze-tolerant for almost 300 years, yet we have limited understanding of the how internal freezing causes damage and the mechanisms that freeze-tolerant insects use to combat that damage. My current focus is on the cell biology of freeze tolerance, using the spring field cricket Gryllus veletis as a model system. These crickets can substantially modify their cold tolerance, transitioning from freeze-intolerant to freeze-tolerant when exposed (acclimated) to six weeks of fall-like conditions (low temperatures, short days). My students and I will pursue three linked objectives to determine how acclimation induces cellular mechanisms that facilitate insect freeze tolerance. For objective 1, we will characterize freeze-induced damage at the cellular level. We will use a combination of cell biology and biochemical assays to determine whether cricket cells and their macromolecules (proteins, membranes, DNA) are damaged during freezing, and whether that damage can be repaired post-freeze. This will increase our understanding of the challenges associated with freezing, and facilitate additional investigations that test whether specific molecules or genes protect against particular types of damage. For objective 2, we will determine how acclimation alters cell structure and function. The changes in gene expression during fall acclimation suggest that freeze-tolerant crickets adjust their cell biology in several ways. We will test these hypotheses using immunohistochemistry, membrane lipidomics, and cryoprotectant quantitation assays. These experiments will determine whether fall acclimation induces changes to cell structure and function and will deepen our knowledge of potential mechanisms that can mitigate damage caused by freezing. For objective 3, we will test which molecules and cellular processes help protect cells during freezing. We will first use RNA interference (RNAi) to knock down expression of genes involved in the cellular changes from objective 2. We will then test how preventing the function of target genes affects whole animal and cellular freeze tolerance using methods similar to objective 1. This will provide much-needed information on the genes and associated cellular processes that are important for the mechanisms underlying freeze tolerance. My proposed research program applies techniques (cell biology, RNAi) that have been lacking in the field of insect freeze tolerance. These experiments have potential to substantially advance our understanding of how insects survive internal ice formation. More broadly, this work has potential applications to insect pest management, cryopreservation protocols in the biomedical and food industries, and determining how overwintering insects may respond to climate change.