Local thermal environment and warming influence supercooling and drive widespread shifts in the metabolome of diapausing Pieris rapae butterflies

Emily E. Mikucki, Brent L. Lockwood

Preprint posted on 3 July 2021

Warming Alert! Winter warming may hamper diapause of cabbage white butterfly pupae by affecting their supercooling point and metabolomics. For details:

Selected by Fouzia Haider

Categories: ecology, molecular biology


As compared to the other seasons, winter is the most impacted by climate change in terms of higher mean temperatures and fewer days below the freezing point. This is particularly detrimental to organisms that go dormant during winter (diapause). During diapause, overwintering organisms endure the cold using a ‘supercooling’ method, where the temperature of their body fluids drops below 0°C without forming ice, offering an effective strategy to avoid freezing. Diapause is supported by several drivers, both internal (ice binding protein formation, cryoprotective dehydration) and external (temperature). Therefore, an increase in external temperature during winter or of their internal supercooling points can negatively affect the diapause of these organisms, and hence disrupt their life-cycle. Many insects are vulnerable to winter warming, and this is reflected in the evidence of local adaptation of cold tolerance in several species according to their local thermal environment.

Various studies have investigated the relationship between thermal environment and overwintering physiology in a targeted manner, such as by studying the effect of temperature on cryoprotectant metabolites. Although targeting specific metabolites provides us with important discoveries by regarding the molecular basis of diapause and overwintering physiology of insects, it also comes with ascertainment bias. To avoid this bias and understand the influence of temperature on the general metabolome and supercooling process, the authors of this preprint studied the thermal effects on supercooling points and metabolome on two different populations (Vermont and North Carolina) of North American Pieris rapae (cabbage white butterflies). They investigated the effect of acute (hours) and chronic (weeks) increase in temperature on supercooling, and the effect of chronic temperature increase on untargeted metabolomics in P. rapae. They also correlated metabolomics to supercooling.

The thermal exposure was designed as follows: for the control group, a daily fluctuation of 4 – 8°C was administered, the chronic group was exposed to 7 – 11°C (to mimic the local winter warming pattern noticed from 1940 till 2020) and acute group was exposed to 4 – 8°C (as the control group) with 24-hour warming events of fluctuating 18 – 23°C on days 25, 50 and 75 days (to mimic the recorded high local winter temperatures). The authors compared the supercooling points of (1) Vermont vs. North Carolina populations (under control conditions), (2) Vermont control vs. Vermont chronic warmed, and (3) Vermont control vs. Vermont acute warmed. Metabolomics were only investigated in the Vermont population between control and chronic warming groups.


The supercooling point of P. rapae pupae was lower in the Vermont population (-26.3 ± 0.3°C) compared to the North Carolina population (-19.8 ± 4.0°C), which corresponds to the extreme low in local temperature (approximately -26°C and -15°C, respectively). Both chronic and acute temperature increase caused a higher supercooling point on day 50 in diapause. This effect was not observed on day 75 in diapause (Fig. 1; used per authors’ consent).

Figure 1: Effect of chronic and acute winter warming on the supercooling points of Vermont population of Pieris rapae pupae.

Diapause caused significant changes to the abundance of 1,370 metaboites, of which 443 responded significantly to chronic warming and 16 changed significantly in response to diapause and warming. Principal component analysis showed that supercooling point explains the largest variation (27%), while the second major axis of variation is the days of diapause (10%) (Fig. 2; used per authors’ consent).

Figure 2: Principal Component analysis (PCA) of whole metabolomics of Pieris rapae pupae. The first component (PC1) is supercooling point which explains 26.9% of variation and the second component (PC2) is the days in diapause which explains 9.97%. The shapes of the points depict days in diapause, the size of the points depicts temperature treatment and the color shade depicts supercooling points.

Chronic warming showed significantly increased enrichment in 1 biochemical pathway, and significantly decreased enrichment in 7 others (Fig. 3; used per authors’ consent). The increase was observed in arachidonate metabolism, indicating that winter warming could affect the usage of energy storage. The pathways demonstrating a decrease in response to chronic warming are well-known cryoprotectants – ß-alanine metabolism, fructose and mannose metabolism, and glycine, serine and threonine metabolism.

Fig. 3: Effect of winter warming on different biochemical pathways in Pieris rapae pupae. Positive value in X axis indicates higher abundance and negative values indicate lower abundances of metabolites relative to control group. The shape of the points depicts days in diapause and the color shade depicts normalized enrichment score (NES).

What I like about this preprint

The authors of this preprint highlighted a crucial biological process being impacted by global warming: diapause in organisms that undergo a metabolic depression during winter. Temperature is an important external factor moderating diapause in these organisms, and winter warming could affect their diapause, and life cycle. This could occur by affecting the supercooling point of these organisms or by altering other physiological mechanisms, such as biochemical pathways. Although previous research has focused on certain metabolites under different circumstances, this study took an untargeted approach to understand the effect of winter warming on metabolomics. Thus, we can identify, biochemical pathway(s) that have been otherwise overlooked.


Posted on: 11 August 2021 , updated on: 12 August 2021


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Author's response

Emily E. Mikucki shared

The increase in arachidonate metabolism in response to winter warming is an interesting finding. Since this pathway is related to the energy storage and its usage, was there any change in the energy storage in these pupae?

E.E.M: We did not measure energy storage or usage due to warming in this experiment; however, our results suggest that metabolism could change due to warming while in diapause. Another experiment we did showed that warmed-diapausing individuals use different energy stores than diapausing individuals who are not warmed. We are currently preparing that manuscript for publication, so stay tuned for those results!

Results show chronic and acute warming have a similar effect on the supercooling point in these pupae. Did the authors investigate a nested effect of acute warming on chronic temperature rise?

E.E.M: We did not analyze the data with a nested analysis because the two warming conditions (chronic and acute) were completely different such that the acute exposures were not nested in the chronic design. But it is an interesting suggestion!

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