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忍受低温，促进它们在温带气候下越冬的能力。研究人员已经知道一些昆虫具有抗冻性近300年，但我们对内部冷冻如何导致损害以及抗冻昆虫用来对抗这种损害的机制的了解有限。我目前的研究重点是耐寒性的细胞生物学，以春田蟋蟀Gryllus veletis为模型系统。这些蟋蟀可以大大改变它们的耐寒性，当暴露（适应）六周的类似秋天的条件（低温，短日）时，它们从不耐冻转变为耐冻。我的学生和我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抗冻性的细胞机制。对于目标1，我们将在细胞水平上描述冷冻诱导的损伤。我们将结合细胞生物学和生物化学分析来确定蟋蟀细胞及其大分子（蛋白质、膜、DNA）是否在冷冻过程中受损，以及这种损伤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，以测试特定分子或基因是否能防止特定类型的损害。对于目标2，我们将确定驯化如何改变细胞结构和功能。在秋季驯化过程中基因表达的变化表明，抗冻蟋蟀通过几种方式调节其细胞生物学。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设。这些实验将确定秋季驯化是否会引起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期间保护细胞。我们将首先使用RNA干扰（RNAi）来敲低目标2中涉及细胞变化的基因的表达。然后，我们将使用类似于目标1的方法测试阻止目标基因的功能如何影响整个动物和细胞的抗冻能力。这将为基因和相关细胞过程提供急需的信息，这些信息对抗冻性机制至关重要。我提出的研究项目应用了在昆虫抗冻性领域缺乏的技术（细胞生物学，RNAi）。这些实验有可能大大提高我们对昆虫如何在内部冰形成中生存的理解。更广泛地说，这项工作具有潜在的应用于害虫管理，生物医学和食品工业的低温保存协议，以及确定越冬昆虫如何应对气候变化。

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.