Overwintering under the ice: seasonal shifts in cold and hypoxia stress physiology of three winter-active pond insects

Winter poses harsh physiological challenges for insects living in temperate ponds due to the combination of low temperatures and reduced oxygen levels (hypoxia). Despite these stressors, water boatmen ( Hesperocorixa sp.), backswimmers ( Notonecta sp.) and diving beetles ( Laccophilus maculosus ) remain active year-round in eastern Nova Scotia, including in ice- covered ponds. However, the underlying mechanisms that allow pond insects to survive overwintering have not been widely studied. We hypothesized that cold and hypoxia tolerance would improve from September to March in these insects, with changes in whole-animal and biochemical correlates of tolerance to both stressors. We collected insects from the field and stocked them in outdoor freshwater mesocosms. Every two months, between September 2023 and April 2024, we characterized whole animal responses and biochemical changes. Whole insect cold tolerance assays indicated a trend of improved ability to sustain voluntary muscle control (CT _min ) at lower temperatures in winter-collected insects and higher internal fluid freezing temperatures (SCP). There were no changes in whole animal correlates of hypoxia tolerance over time (surface respiration frequency, submersion and surface time). Potential cryoprotectants like proline, myo -inositol and trehalose increased in concentration in multiple insect taxa during winter but were relatively low compared to terrestrial insect studies. As lactate dehydrogenase activity did not change, there is little evidence that the insects experienced functional hypoxia sufficient to induce anaerobic metabolism. Overall, this research has established a baseline cold and hypoxia tolerance dataset in understudied pond insects.