The transcriptional responses of insects to long-term, ecologically relevant temperature stress are poorly understood. Long-term exposure to low temperatures, commonly referred to as chilling, can lead to physiological effects collectively known as chill injury. Periodically increasing temperatures during long-term chilling has been shown to increase survival in many insects. However, the transcripts responsible for this increase in survival have never been characterized. Here, we present the first transcriptome-level analysis of increased longevity under fluctuating temperatures during chilling. Overwintering post-diapause quiescent alfalfa leafcutting bees (Megachile rotundata) were exposed to a constant temperature of 6°C, or 6°C with a daily fluctuation to 20°C. RNA was collected at two different time points, before and after mortality rates began to diverge between temperature treatments. Expression analysis identified differentially regulated transcripts between pairwise comparisons of both treatments and time points. Transcripts functioning in ion homeostasis, metabolic pathways and oxidative stress response were up-regulated in individuals exposed to periodic temperature fluctuations during chilling. The differential expression of these transcripts provides support for the hypotheses that fluctuating temperatures protect against chill injury by reducing oxidative stress and returning ion concentrations and metabolic function to more favorable levels. Additionally, exposure to fluctuating temperatures leads to increased expression of transcripts functioning in the immune response and neurogenesis, providing evidence for additional mechanisms associated with increased survival during chilling in M. rotundata.

‘Omics’ methods, such as transcriptomics, proteomics, lipidomics or metabolomics, yield simultaneous measurements of many related molecules in a sample. These approaches have opened new opportunities to generate and test hypotheses about the mechanisms underlying biochemical and physiological phenotypes. In this Commentary, we discuss general approaches and considerations for successfully integrating omics into comparative physiology. The choice of omics approach will be guided by the availability of existing resources and the time scale of the process being studied. We discuss the use of whole-organism extracts (common in omics experiments on small invertebrates) because such an approach may mask underlying physiological mechanisms, and we consider the advantages and disadvantages of pooling samples within biological replicates. These methods can bring analytical challenges, so we describe the most easily analyzed omics experimental designs. We address the propensity of omics studies to digress into ‘fishing expeditions’ and show how omics can be used within the hypothetico-deductive framework. With this Commentary, we hope to provide a roadmap that will help newcomers approach omics in comparative physiology while avoiding some of the potential pitfalls, which include ambiguous experiments, long lists of candidate molecules and vague conclusions.

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.

Diapause is a highly advantageous strategy for winter survival for insects living in temperate environments. However, insects typically do not feed during diapause and are therefore presented with a complicated energetics problem. The fall webworm, Hyphantria cunea, overwinter as diapausing pupae, but adults of this species lose their ability to feed due to the degeneration of their mouthparts. Thus, the energy reserves stored before diapauses contribute to the survival rate and fitness of the adults after emergence. In this study, we tested the hypothesis that diapause-destined larvae of H. cunea reserve more energy by increasing feeding rate, feeding efficiency, or both, during the diapause preparation phase compared with non-diapause-destined larvae. We observed higher digestive efficiency, increased lipase and amylase activity, and lower protease activity in diapause-destined larvae compared to non-diapause-destined larvae. These differences in digestive physiology during diapause preparation lead to greater body size and mass, increased lipid and carbohydrate content, and lower soluble protein content in diapausing pupae, relative to non-diapause pupae – results consistent with our hypothesis. We conclude that diapause-destined fall webworm reserve more energy than non-diapause-destined individuals by increasing feeding efficiency, and that this increase in efficiency is at least partially driven by increased lipase and amylase activities in the midgut. This is in contrast to non-diapause-destined larvae, which likely reserve greater protein than diapause-destined larvae to help maintain their physiological function.