Fall-like acclimation alters mitochondrial function and substrate use in freeze-tolerant crickets.

Acclimation to low temperatures in insects involves complex metabolic adjustments to ensure survival. Notably, cryoprotectants including glycerol, trehalose and proline can accumulate in insects to promote freeze tolerance. Interestingly, these metabolites can also be converted to metabolic fuels sustaining mitochondrial ATP production. This study investigates the metabolic shifts in the freeze-tolerant cricket Gryllus veletis during fall-like acclimation, focusing on mitochondrial function and its possible link with cryoprotectant accumulation. We hypothesized that fall-like acclimation promotes a shift in mitochondrial substrate utilization within the fat body tissue of crickets, transitioning from a reliance on substrates that donate electrons to complex I (e.g., carbohydrates and fatty acids) toward increased utilization of alternative substrates, such as glycerol-3-phosphate (G3P), which can be derived from glycerol. We demonstrated that acclimation leads to lower glycogen content in the fat body, concomitant with increased pyruvate kinase activity, suggesting increased carbon flux through glycolysis. Moreover, we observed decreased overall mitochondrial oxygen consumption and enzymatic activity of cytochrome c oxidase. Interestingly, while overall mitochondrial capacity decreased, the relative contribution of G3P to mitochondrial metabolism increased, supported by increased enzymatic activity of mitochondrial G3P dehydrogenase. Thus, fall-like acclimation appears to induce a metabolic shift in crickets, redirecting metabolites away from the tricarboxylic acid cycle and towards the production of G3P, which subsequently sustains mitochondrial respiration. This study reveals a key mechanism associated with the development of freeze tolerance in crickets, which could also be important for other cold- and freeze-tolerant insect species.