Global climate change has the potential to negatively impact biological systems as organisms are exposed to novel temperature regimes. Increases in annual mean temperature have been accompanied by disproportionate rates of change in temperature across seasons, and winter is the season warming most rapidly. Yet, we know relatively little about how warming will alter the physiology of overwintering organisms. Here, we simulated future warming conditions by comparing diapausing Pieris rapae butterfly pupae collected from disparate thermal environments and by exposing P. rapae pupae to acute and chronic increases in temperature. First, we compared internal freezing temperatures (supercooling points) of diapausing pupae that were developed in common-garden conditions but whose parents were collected from northern Vermont, USA, or North Carolina, USA. Matching the warmer winter climate of North Carolina, North Carolina pupae had significantly higher supercooling points than Vermont pupae. Next, we measured the effects of acute and chronic warming exposure in Vermont pupae and found that warming induced higher supercooling points. We further characterized the effects of chronic warming by profiling the metabolomes of Vermont pupae via untargeted LC-MS metabolomics. Warming caused significant changes in abundance of hundreds of metabolites across the metabolome. Notably, there were warming-induced shifts in key biochemical pathways, such as pyruvate metabolism, fructose and mannose metabolism, and β-alanine metabolism, suggesting shifts in energy metabolism and cryoprotection. These results suggest that warming affects various aspects of overwintering physiology in P. rapae and may be detrimental depending on the frequency and variation of winter warming events. Further research is needed to ascertain the extent to which the effects of warming are felt among a broader set of populations of P. rapae, and among other species, in order to better predict how insects may respond to changes in winter thermal environments.

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