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Abstract The response of ectotherms to temperature stress is complex, non-linear, and is influenced by life stage and previous thermal exposure. Mortality is higher under constant low temperatures than under a fluctuating thermal regime (FTR) that maintains the same low temperature but adds a brief, daily pulse of increased temperature. Long term exposure to FTR has been shown to increase transcription of genes involved in oxidative stress, immune function, and metabolic pathways, which may aid in recovery from chill injury and oxidative damage. Previous research suggests the transcriptional response that protects against sub-lethal damage occurs rapidly under exposure to fluctuating temperatures. However, existing studies have only examined gene expression after a week or over many months. Here we characterize gene expression during a single temperature cycle under FTR. Development of pupating alfalfa leafcutting bees (Megachile rotundata) was interrupted at the red-eye stage and were transferred to 6°C with a 1-h pulse to 20°C and returned to 6°C. RNA was collected before, during, and after the temperature pulse and compared to pupae maintained at a static 6°C. The warm pulse is sufficient to cause expression of transcripts that repair cell membrane damage, modify membrane composition, produce antifreeze proteins, restore ion homeostasis, and respond to oxidative stress. This pattern of expression indicates that even brief exposure to warm temperatures has significant protective effects on insects exposed to stressful cold temperatures that persist beyond the warm pulse. Megachile rotundata’s sensitivity to temperature fluctuations indicates that short exposures to temperature changes affect development and physiology. Genes associated with developmental patterning are expressed after the warm pulse, suggesting that 1 h at 20°C was enough to resume development in the pupae. The greatest difference in gene expression occurred between pupae collected after the warm pulse and at constant low temperatures. Although both were collected at the same time and temperature, the transcriptional response to one FTR cycle included multiple transcripts previously identified under long-term FTR exposure associated with recovery from chill injury, indicating that the effects of FTR occur rapidly and are persistent. 

Exposure to stressful low temperatures during development can result in the accumulation of deleterious physiological effects called chill injury. Metabolic imbalances, disruptions in ion homeostasis, and oxidative stress contribute to the increased mortality of chill-injured insects. Interestingly, survival can be significantly increased when chill susceptible insects are exposed to a daily warm-temperature pulse during chilling. We hypothesize that warm pulses allow for the repair of damage associated with chill-injury. Here, we describe transcriptional responses during exposure to a fluctuating thermal regime (FTR), relative to constant chilled temperatures, during pupal development in the alfalfa leafcutting bee, Megachile rotundata using a combination of RNA-seq and qPCR. Pupae were exposed to either a constant, chilled temperature of 6°C, or 6°C with a daily pulse of 20°C for seven days. RNA-seq after experimental treatment revealed differential expression of transcripts involved in construction of cell membranes, oxidation-reduction and various metabolic processes. These mechanisms provide support for shared physiological responses to chill injury across taxa. The large number of differentially expressed transcripts observed after seven days of treatment suggests that the initial divergence in expression profiles between the two treatments occurred upstream of the time point sampled. Additionally, the differential expression profiles observed in this study show little overlap with those differentially expressed during temperature stress in the diapause state of M. rotundata. While the mechanisms governing the physiological response to low-temperature stress are shared, the specific transcripts associated with the response differ between life stages.