Animals that overwinter in temperate climates must prevent or repair damage to their cells to survive winter, but we know little about how they protect cellular structure at the cytoskeletal level. Both chilling (no ice formation) and freezing (ice formation) are hypothesized to cause substantial challenges to cell structure and the cytoskeleton. The spring field cricket Gryllus veletis becomes freeze tolerant following a 6-week acclimation to fall-like conditions, during which they differentially express multiple cytoskeleton-related genes. We tested the hypothesis that G. veletis alter their cytoskeleton during acclimation to support maintenance of cytoskeletal structure during freezing and thawing. We used immunocytochemistry and confocal microscopy to characterize changes in microfilaments (F-actin, a polymer of G-actin) and microtubules (a polymer of ��- and ��-tubulin) in three tissues. While we saw no effect of acclimation on microtubules, crickets increased the abundance of microfilaments in fat body and Malpighian tubules. When we chilled or froze these freeze-tolerant crickets, there was no apparent damage to the actin or tubulin cytoskeleton in fat body, but there was decreased cytoskeleton abundance in Malpighian tubules. When we froze freeze-intolerant (unacclimated) crickets, microfilament abundance decreased in fat body tissue, while microfilaments were unaffected by chilling to the same subzero temperature. Our study shows that freeze-tolerant crickets are able to prevent or rapidly repair ice-induced damage to the actin cytoskeleton in fat body, likely due to preparatory changes in advance of freezing - i.e., during acclimation. We suggest future directions examining the mechanisms that underlie these structural changes.
