Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Most insects have limited capacity for thermoregulation, so their body temperature closely matches that of the environment (Heinrich, 2013; Lahondère, 2023). Their physiology is therefore intimately tied to external temperature, making performance and survival in extreme or variable thermal environments a key challenge (Harrison et al., 2012). This is particularly true for temperate, alpine and polar insect species, which often need to survive through extended periods of sub-zero temperatures during winter, a period that can represent a large proportion of their life span (Sinclair et al., 2003; Williams et al., 2015). For these reasons, low temperature survival and overwintering success are continuously highlighted as key factors in determining insect abundance and distribution (Bale, 1987; Addo-Bediako et al., 2000; Ramløv, 2000; Bale, 2002; Marshall et al., 2020). This is also true for the mountain pine beetle, Dendroctonus ponderosae, a pest native to western North America, which has spread beyond its native range and invaded regions east of the Rocky Mountains that experience more severe winter conditions than its native range (Safranyik et al., 2010; de la Giroday et al., 2012; Creeden et al., 2014). Thus, exploring the cold tolerance physiology of D. ponderosae is critical to understanding the processes that promote or limit its continued invasion into the eastern boreal forest.

Insects have acquired a wide range of adaptations that allow them to adjust to, thrive in, or at the very least survive low temperature environments. However, the majority of insects are characterized as chill-susceptible, meaning they succumb to relatively mild cold at temperatures above those that cause extracellular freezing (Overgaard and MacMillan, 2017). For these insects, survival depends on overcoming the direct, disruptive effects of low temperature on homeostasis, cellular integrity and molecular processes. Central to the current conceptual model of chill tolerance physiology is a substantial, cold-induced disruption of extracellular ion homeostasis, referred to as an ‘ionoregulatory collapse’ (MacMillan, 2019). Under benign conditions, the renal system (Malpighian tubules and hindgut) maintains hemolymph ionic and osmotic balance through a continuous cycle of secretion and reabsorption, which counteracts transepithelial and cellular leakage (Edney, 1977; Phillips, 1981; Dow, 2017). However, at low temperature, the activity of ion-motive transporters responsible for secretion and reabsorption are slowed below the level required for maintenance of homeostasis (MacMillan and Sinclair, 2011; Overgaard et al., 2021). Thus, prolonged exposure to stressful cold leads to a debilitating increase in hemolymph K+ concentration (hyperkalemia), which, in combination with cold, depolarizes excitable tissues, triggers an intracellular calcium overload, and leads to cell death and organismal injury (MacMillan et al., 2015c; Bayley et al., 2018). Subsequently, recovery from cold exposure depends on the ability to either restore hemolymph ion concentrations upon return to permissive temperatures or prevent ionoregulatory collapse altogether (MacMillan et al., 2014). However, within the group of chill-susceptible insects, there is substantial variation in the amount of cold that species or populations can handle, which has prompted the establishment of a vaguely defined separation of insects that are truly chill susceptible from those that are chill tolerant, where the latter group is able to either prevent or mitigate the loss of ion homeostasis via a wide range of ionoregulatory adaptations and therefore tolerate slightly more severe cold exposures (Sinclair, 1999; Overgaard and MacMillan, 2017; Overgaard et al., 2021). For example, the chill-tolerant fruit fly Drosophila montana is able to maintain ion balance and renal function under low temperature conditions where less tolerant congeners lose renal function and suffer cold- and hyperkalemia-induced cell depolarization and injury (MacMillan et al., 2015a; Andersen and Overgaard, 2019).

For other insects, cold survival is determined below or at the melting point of their extracellular fluids (Denlinger and Lee, 2010; Lee, 2012). These species are divided into two main strategies: freeze tolerant and freeze avoidant (Bale, 1987; Lee, 1991; Bale, 1996). As the name suggests, freeze-tolerant insects can survive, and even promote, freezing of the extracellular fluids and initiate freezing at relatively high sub-zero temperatures. This slows ice formation and helps limit ice to the extracellular space (Salt, 1961; Zachariassen, 1985). Thus, these insects have a relatively high supercooling point (SCP), the temperature at which freezing occurs spontaneously during a cooling challenge (Sinclair, 1999; Sinclair et al., 2015). Freeze-avoidant insects, in contrast, survive substantial cold by possessing the ability to supercool to extremely low temperatures (often lower than −30°C) (Sinclair et al., 2003). These species have adapted to survive through harsh winters without freezing by, for example, accumulation of cryoprotectants (i.e. compatible osmolites and anti-freeze proteins), removal of ice-nucleating agents and cryoprotective dehydration (Salt, 1961; Sømme, 1982; Graham et al., 1997; Holmstrup et al., 2002). Each of these processes effectively lowers the SCP of the insects but is rarely found alone, meaning that the impressive supercooling capacity of freeze-avoidant insects is achieved via a combination of colligative and non-colligative factors (Zachariassen, 1985; Block et al., 1990; Sinclair et al., 2003; Lee, 2012; Storey and Storey, 2012). For example, freeze-avoidant beetles accumulate low molecular weight polyols in preparation for overwintering and produce a wide range of anti-freeze proteins, which lower the limit for freezing by preventing the formation of nucleating ice crystals (Sømme, 1964; Storey and Storey, 1983; Gehrken, 1984; Graham et al., 2007).

Dendroctonus ponderosae typically overwinter as freeze-avoidant larvae and possess a remarkable ability to lower their SCP in this life stage (Régnière and Bentz, 2007; Rosenberger et al., 2017). This reduction in SCP is mainly achieved through accumulation of substantial amounts of glycerol in response to emerging winter conditions (Bentz and Mullins, 1999; Robert et al., 2016; Thompson et al., 2020). This ability to survive severe cold is thought to contribute to the recent eastward spread of D. ponderosae from its native range across the Rocky Mountains, and into Alberta and northeastern British Columbia (Canada) (Safranyik et al., 1975; de la Giroday et al., 2012; Creeden et al., 2014), where it has devastated substantial areas of coniferous forest and required over $500M to manage its eastern spread (Cullingham et al., 2011; Hodge et al., 2017). However, despite an impressive capacity for freeze avoidance, cold mortality remains a key factor in determining population abundance of D. ponderosae (Renault et al., 2002; Régnière and Bentz, 2007). Thus, D. ponderosae represents an interesting and valuable system to study the physiological mechanisms underlying pre-freeze mortality in freeze-avoidant insects, which are generally thought to survive unless frozen (Salt, 1961).

Little is known about the processes that limit cold survival of freeze-avoidant insects, independent of freezing, during prolonged cold exposures, although everything from dysregulation of metabolism to disrupted development has been suggested (Bale, 1987, 1991; Sømme, 1999). That being said, earlier studies have brought up the notion that ion balance regulation during cold exposure might be important for survival in freeze-avoidant insects (Koštál et al., 2004; Zachariassen et al., 2004). Thus, for freeze avoidance to be a viable cold tolerance strategy, its evolution must have included an ancestral or convergent ability to avoid ionoregulatory collapse or tolerate substantial changes in transmembrane ion concentrations. If this is the case, we expect that freeze-avoidant species can maintain ion balance during exposure to very low temperatures just above the lethal SCP. However, if ion homeostasis is disrupted in a freeze-avoidant insect, this could indicate an unappreciated role for ionoregulatory collapse in limiting cold survival, which could indicate that, much like in chill-susceptible insects, variation in ionoregulatory capacity dictates cold survival. Interestingly, phytophagous bark beetles, such as D. ponderosae, possess unique hemolymph ion compositions with very low concentrations of Na+ and elevated concentrations of K+, Mg2+ and Ca2+ (Duchǎteau et al., 1953; Jeuniaux, 1971). In other insects, low extracellular Na+ is associated with increased cold tolerance via mitigation of ionoregulatory collapse (MacMillan et al., 2015b; Lebenzon et al., 2020). Transmembrane concentrations of Na+ can play a role in freeze tolerance and avoidance; however, the evidence for this remains scarce (Zachariassen et al., 2004). Thus, exploring ion balance and its regulation in the freeze-avoidant D. ponderosae larvae might provide insight into evolutionarily conserved cold tolerance mechanisms.

In the present study, we investigated whether ion balance plays a role in limiting the cold tolerance of the freeze-avoidant D. ponderosae and asked the following main question: do D. ponderosae suffer from ionoregulatory collapse when exposed to prolonged periods of stressful cold at temperatures immediately above those that cause extracellular freezing? If this extremely cold-hardy insect suffers from ionoregulatory collapse in the cold, renal mechanisms of failure may be critical to fully understanding overwintering success of this and other freeze-avoidant species. If, in contrast, D. ponderosae do not suffer from ionoregulatory collapse, mechanisms that serve to defend against this disruption in the cold represent undescribed means by which freeze-avoidant insects acquire cold tolerance (Overgaard and MacMillan, 2017; MacMillan, 2019; Overgaard et al., 2021). Given that non-freezing cold mortality occurs during overwintering in this species (Régnière and Bentz, 2007), we hypothesized that this could be linked to a gradual loss of ion balance (i.e. hemolymph hyperkalemia). Here, we exposed D. ponderosae that were (1) in preparation for overwintering, (2) in the middle of overwintering and (3) emerging from overwintering to extended periods of cold stress (above their SCP). We obtained parallel measurements of cold tolerance phenotypes and hemolymph ion concentrations at select time points that yielded a dataset that demonstrates mountain pine beetles can partly, but not completely, mitigate ionoregulatory collapse in the cold during overwintering.

Animal collection sites and husbandry

All specimens of Dendroctonus ponderosae (Hopkins 1902) used in this study were collected from a population from its expanded range in central Alberta. To collect experimental animals, adult D. ponderosae were lured to lodgepole pine trees (Pinus contorta var. latifolia) at a field site in the expanded range of D. ponderosae outside of Cynthia, Alberta, Canada (53.34 N, −115.48W; see Fig. 1). Beetles were attracted to trees at the field site by four funnel traps containing aggregation pheromones (trans-verbenol and exo-brevicomin) and host tree volatiles (myrcene and terpinolene) (product 3093, Synergy Semiochemical, Delta, Canada) and trees were mass attacked over a 5-week period during July and August 2022. Mass-attacked trees were left on the landscape for egg laying and brood development until harvest. In October 2022, seven trees were harvested and cut into 50 logs (∼40–­50 cm long and ∼30–­50 cm in diameter), and the ends were sealed with paraffin wax to prevent desiccation. Infested logs were transported to the Department of Biological Sciences at the University of Alberta for outdoor storage and overwintering. Temperature loggers (HOBO 2x External Temperature Data Logger, Onset, Bourne, MA, USA) were implanted underneath the bark of three separate logs to monitor the thermal environment of the larvae during the overwintering period. Under-bark recordings could only be started after trees were harvested and the logs positioned for storage overwinter, so the prior thermal history of the trees was approximated by obtaining and averaging temperature data from three weather stations located near the field site (https://climate.weather.gc.ca/index_e.html, see Fig. 1 and Table S1 for locations). At three points during the overwintering period, logs were transported (by air, overnight and in double-walled secure containers with export authorization from Alberta Agriculture, Forestry and Rural Economic Development, permit #UofA-03-2022) to the Insect Production and Quarantine Laboratory at the Great Lakes Forestry Centre (GLFC) in Sault Ste. Marie, Ontario (part of Natural Resources Canada, Canada), where experiments on live animals were carried out in a plant pest containment and quarantine facility (level 2A, see Canadian Food Inspection Agency, 2014). The time points were chosen such that they included: (1) larvae from mid-autumn at the very beginning of overwintering (25 October 2022), (2) overwintering larvae from mid-winter (24 January 2023) and (3) larvae towards the end of overwintering in spring (12 April 2023). Ten, 17 and 23 logs were transported to GLFC in October, January and April, respectively, and after their arrival they were stored in the dark at 6°C until extraction for experiments (within 9 days). Extraction was carried out at room temperature (21–22°C) and larvae (only 4th instar) were used in experiments within an hour of being removed from the log.

Fig. 1.

Map overview of the field site, and experimental locations. The experimental field site was just outside of Cynthia (Alberta, Canada; green circle). Here, trees were baited, monitored for beetle attack, and finally harvested and brought to the Department of Biological Science at the University of Alberta (Edmonton, Alberta, Canada; blue circle) for storage. At specific time points, subsamples of logs were transported to the quarantine facilities at the Great Lakes Forestry Centre (Natural Resources Canada, Sault Ste. Marie, Ontario, Canada; orange circle), where experiments on live animals were carried out with permission. Hemolymph samples collected here were transported to the Department of Biology at Carleton University (Ottawa, Ontario, Canada), where ion concentrations were measured. The bark temperature of the logs stored at University of Alberta was measured continuously, and temperature at the field site was approximated using hourly temperature measurements from three nearby field stations (smaller black circles) (see Fig. 2 for temperature profiles).

Fig. 1.

Map overview of the field site, and experimental locations. The experimental field site was just outside of Cynthia (Alberta, Canada; green circle). Here, trees were baited, monitored for beetle attack, and finally harvested and brought to the Department of Biological Science at the University of Alberta (Edmonton, Alberta, Canada; blue circle) for storage. At specific time points, subsamples of logs were transported to the quarantine facilities at the Great Lakes Forestry Centre (Natural Resources Canada, Sault Ste. Marie, Ontario, Canada; orange circle), where experiments on live animals were carried out with permission. Hemolymph samples collected here were transported to the Department of Biology at Carleton University (Ottawa, Ontario, Canada), where ion concentrations were measured. The bark temperature of the logs stored at University of Alberta was measured continuously, and temperature at the field site was approximated using hourly temperature measurements from three nearby field stations (smaller black circles) (see Fig. 2 for temperature profiles).

Fig. 2.

Temperature profiles and supercooling capacities of overwintering Dendroctonus ponderosae larvae from late 2022 to early 2023. (A) During the overwintering of D. ponderosae from autumn 2022 to spring 2023, temperature was tracked at their collection site using historical climate data from Environment Canada (grey line), and monitored directly under the bark of logs that were moved into outdoor storage for experiments (colored line). Note that the trees that were cut into logs and stored for subsequent larvae extraction and experiments appear to have been sheltered from the most severe winter conditions (i.e. the coloured line is always above the grey line). (B) At three time points during this period, larvae were extracted from the logs to measure their supercooling points. Small, translucent points denote individual data points, while the large opaque point denotes the mean. Error bars not visible are obscured by the symbols, and groups not sharing a letter are statistically different. For sample sizes, see Table S2.

Fig. 2.

Temperature profiles and supercooling capacities of overwintering Dendroctonus ponderosae larvae from late 2022 to early 2023. (A) During the overwintering of D. ponderosae from autumn 2022 to spring 2023, temperature was tracked at their collection site using historical climate data from Environment Canada (grey line), and monitored directly under the bark of logs that were moved into outdoor storage for experiments (colored line). Note that the trees that were cut into logs and stored for subsequent larvae extraction and experiments appear to have been sheltered from the most severe winter conditions (i.e. the coloured line is always above the grey line). (B) At three time points during this period, larvae were extracted from the logs to measure their supercooling points. Small, translucent points denote individual data points, while the large opaque point denotes the mean. Error bars not visible are obscured by the symbols, and groups not sharing a letter are statistically different. For sample sizes, see Table S2.

Cold tolerance measurements

At each of the three seasonal sampling points, we started by measuring SCPs of a subset of extracted D. ponderosae larvae before sampling larvae for all other experiments. SCPs were measured following the approach outlined by Sinclair et al. (2015). First, type-K thermocouples were gently attached to the outside of each larva with a small amount of vacuum grease, after which individual larvae were put into 200 µl microcentrifuge tubes. These were then gently stopped with cotton and placed into a custom-built aluminum casing through which a cooling bath (Proline RP855; Lauda, Würzburg, Germany) circulated a 1:1 water:methanol solution. From here, larvae were kept at 6°C for 10 min, after which the temperature was lowered by 0.5°C min−1 until the temperature reached −35°C to ensure that all animals froze. At the end of the experiment, temperature traces were analyzed for each animal and the temperature at which the freezing exotherm occurred was recorded as the SCP. In this and subsequent experiments, all frozen animals were confirmed dead. A total of 53 larvae were used for this experiment, with 19, 22 and 12 in autumn, winter and spring, respectively.

After establishing the supercooling capacity of the larvae, we chose exposure temperatures that we expected to be stressful, but also minimized the risk of freezing. We chose (1) −10°C in the autumn, (2) −10°C and −20°C in the winter and (3) −10°C in the spring, which represented 0.07%, 0.01%, 7.33% and 7.59% risk of reaching the SCP, respectively (based on standard deviations and assumed normally distributed datasets). This approach does not account for the stochastic nature of freezing in cold-exposed insects (Ditrich, 2018); however, frozen larvae were easy to detect and were discarded (0, 0 and 17 larvae froze in the autumn, winter and spring, respectively, representing 0, 0 and ∼6.9% of larvae in these experiments). The −10°C exposure in the winter-acclimatized animals was included to allow for direct comparisons across seasons. From here, larvae were exposed to their respective temperature treatments by first holding them at 6°C for 10 min, before lowering the temperature by 0.5°C min−1 until −10°C or −20°C was reached and holding at that temperature for up to 168 h (7 days). During this cold exposure, larvae were removed from the cold at pre-determined time points at 0 (immediately upon reaching the exposure temperature), 4, 16, 24, 48, 96 and 168 h.

At the specified time points, larvae were removed from the cold treatments and returned to room temperature (21–22°C) to estimate chill coma recovery time and survival as described for other insect larvae (Sinclair et al., 2015); any frozen larvae were discarded. Briefly, we removed larvae rapidly from tubes and placed them on a piece of paper at room temperature to observe and time the onset of movement for each larva. During this time, larvae were encouraged to move by gently poking them with a fine brush. Larvae were omitted from the data analyses if they did not move within 90 min. After the 90 min observation period, larvae were moved back into their tubes, which were gently stopped with cotton, and placed in the dark for 24 h at room temperature. Subsequently, their survival was estimated after the 24 h period by again placing them on a piece of paper and scoring them based on the ability to move as follows: 2=spontaneous movements (alive, uninjured), 1=only moving when stimulated (alive, injured) and 0=no movements (dead). A total of 390 larvae were used in these experiments (see Dataset 1 for distribution between seasons and treatments).

Hemolymph collection and ion concentrations

In parallel with the experiment described above, larvae exposed to the same conditions were sampled for hemolymph after 0, 4, 16, 24, 48, 96 and 168 h after being held at either −10°C or −20°C. Additional hemolymph samples were collected immediately after beetle extraction from the logs at room temperature (21–22°C) and after 24 h at 6°C following extraction. To sample hemolymph, we followed the procedure described for larvae by Campbell et al. (2018). Briefly, after removal from the cold treatments, larvae were rapidly submerged in hydrated paraffin oil in a Petri dish. A 26G needle or a pair of micro scissors was used to puncture the dorsal cuticle of the prothorax or metathorax. The extruding hemolymph droplets from individual larvae were collected with a micropipette, transferred to a round-bottomed 96-well PCR plate, and stored under hydrated paraffin oil at −80°C. Hemolymph samples were shipped to the Department of Biology at Carleton University in Ottawa on dry ice and stored at −80°C until hemolymph ion concentrations were measured. A total of 413 hemolymph samples were collected in these experiments (see Dataset 1 for distribution between seasons and treatments).

On the day of ion measurement, hemolymph samples were thawed on ice and transferred to a glass Petri dish (60 mm diameter) with an elastomer covering the bottom (Sylgard 184, Dow Corning Corporation, Midland, MI, USA) containing hydrated paraffin oil. Here, the concentrations of Na+ and K+ were measured using ion-selective microelectrodes as described in MacMillan et al. (2015a). The Na+ concentration was measured only at a subset of time points. Ion-selective electrodes were fashioned from borosilicate glass (TW150-4; World Precision Instruments, Sarasota, FL, USA), pulled on a Flaming-Brown P-1000 micropipette puller (Sutter Instruments, Novato, CA, USA) to a tip diameter of ∼1–3 µm, and silanized at 300°C in an atmosphere of N,N-dimethyltrimethyl silylamine (Sigma-Aldrich, St Louis, MO, USA). From here, electrodes were back-filled with 100 mmol l−1 KCl or NaCl, depending on which ion was being measured, and front-filled with the associated ionophore. For K+ measurements we used K+ ionophore I, cocktail B (Sigma-Aldrich), and for Na+ measurements we used a Na+ ionophore X (Sigma-Aldrich) cocktail designed to optimize Na+ selectivity (Messerli et al., 2008). Once filled, electrode tips were dipped in a solution of polyvinyl-chloride in tetrahydrofuran (10 mg in 3 ml; Sigma-Aldrich) to prevent ionophore displacement. Raw voltage outputs from the ion-selective electrodes were connected to a pH Amp (ADInstruments, Colorado Springs, CO, USA), digitized using a PowerLab 4SP A/D converter (ADInstruments), and read by a computer running LabChart 4 software (ADInstruments). The circuit was completed with a thinly pulled borosilicate glass electrode (1B200F-4, WPI) filled with 500 mmol l−1 KCl. Voltages were converted to ion concentrations by comparison to standard with a 10-fold difference in concentration of the target ion (K+: 10 and 100 mmol l−1, Na+: 2.5 and 25 mmol l−1; difference made up with LiCl) and the following formula:
formula
(1)
where [ion] is the concentration of the target ion, [c] is the ion concentration in one of the standards, ΔV is the difference in voltage between the sample and the standard, and S is the difference in voltage between the two standards. The latter voltage response should be close to Nernstian (58.2 mV per 10-fold change in concentration) and was 55.1±1.7 mV (N=46) and 55.6±1.7 mV (N=37) for K+ and Na+ electrodes (means±s.d.), respectively.

Statistical analyses

All data analyses were performed in R software (v4.2.2, https://www.r-project.org/). Data normality was tested with Shapiro–Wilk tests, and homoscedasticity was tested for normally distributed datasets with F-tests. SCPs were compared between seasonal sampling points with a Kruskal–Wallis rank sum test followed by pairwise Mann–Whitney–Wilcoxon tests with a Benjamini–Hochberg correction. The effects of season, exposure time and exposure temperature on chill coma recovery times and survival were analyzed using linear models. Concentrations of Na+ and K+ were first compared with control groups (24 h at 22°C, 24 h at 6°C and 0 h at each exposure temperature) with one-way ANOVAs. These analyses found no differences, so the effects of season, exposure time and exposure temperature were analyzed with linear models while excluding the room temperature and 6°C controls for each ion. The relationships between chill coma recovery time, survival and hemolymph K+ concentration were all analyzed with linear regression, linear regression on log-transformed data (i.e. an exponential relationship), or non-linear regression to a sigmoidal model using the nls() function. The best fitting models were chosen based on AIC scores. For all analyses, the level of statistical significance was 0.05, and values reported represent means±s.e.m. unless stated otherwise. For sample sizes, see Tables S2–S4.

Overwintering conditions and cold tolerance of Dendroctonus ponderosae

To simulate overwintering, tree logs containing D. ponderosae larvae were kept in an outdoor enclosure at the Department of Biological Sciences at University of Alberta. Here, they experienced a natural winter and a range of temperatures from ∼10°C in mid-autumn to a minimum temperature of −26.3°C in mid-late December and a gradual climb back to ∼20°C towards mid-April with several cold snaps in between (Fig. 2A). During this time, larval SCPs changed significantly (χ2=40.4, d.f.=2, P<0.001) from −15.7±0.4°C in the autumn, down to −26.7±1.0°C in mid-winter and back to −12.6±0.5°C in the spring (Fig. 2B).

At the same time, we estimated the ability of larvae to tolerate and survive prolonged exposure to extreme cold by measuring the chill coma recovery times and subsequent survival scores after each exposure (Fig. 3). The exposure temperatures were designed to be stressful while minimizing the risk of freezing; however, a few animals did freeze during the experiments performed in the spring, and these were removed from later analyses. We found that the recovery times increased with time exposed to cold stress (F1,357=616.2, P<0.001), decreasing temperatures (estimated only in winter; F1,357=4.7, P=0.031) and their interaction (F1,357=7.6, P=0.006) (Fig. 3A–C). At the same time, we found a clear effect of season on recovery times (i.e. autumn versus winter versus spring; F2,358=14.2, P<0.001), which also shared a significant interaction with exposure time (F2,357=45.5, P<0.001). Specifically, after having just reached their exposure temperatures at the end of the ramp-down (i.e. the 0 h exposure), the recovery time was similar between all larvae exposed to −10°C (2.2±0.1, 1.9±0.3 and 2.4±0.1 min, for autumn-, winter- and spring-acclimatized larvae) with winter-acclimatized larvae exposed to −20°C taking slightly longer (4.8±0.8 min). From here, recovery times increased with exposure time, and more so at lower temperatures (measured in winter) and in the spring, such that after 168 h these values had grown to 26.7±3.5, 24.2±3.8 and 51.3±2.7 min for −10°C exposed autumn-, winter- and spring-acclimatized larvae, respectively, and to 44.1±5.9 for winter-acclimatized larvae exposed to −20°C. The proportion of larvae that did not start moving within the 90 min time frame was generally low but tended to be higher in the spring group compared with the autumn and winter groups (Table 1, data not analyzed).

Fig. 3.

Cold-tolerance measures of overwintering Dendroctonus ponderosae larvae. The cold tolerance of D. ponderosae larvae was estimated at three time points during the overwintering period from autumn 2022 to spring 2023 (see Fig. 1 for dates). This was done by exposing them to periods of stressfully low, non-freezing, coma-inducing temperatures (–10°C in circles with a solid line, and −20°C in winter in triangles with a dashed line) up to 168 h (7 days) and estimating (A–C) how long it took them to recovery mobility and (D–F) whether they accrued cold-related injuries (see Materials and Methods). Note that some larvae did not recovery function within the 90 min observation period (horizontal dashed line) and were removed from this dataset (see Table 1); however, unless frozen by the exposure, they were kept to assess their survival after the full recovery period. Overall, recovery times increased the longer larvae were exposed to cold stress; however, this was further prolonged if the exposure temperature was lowered or if the larvae had exited their overwintering stage (i.e. in spring). Survival, in contrast, was only slightly decreased by extended periods of cold stress and was similar across seasons and temperature exposures. Small, translucent points denote individual data points, whereas the large opaque point denotes the mean. Individual data points in D–F are jittered along the ordinal y-axis for clarity. Error bars not visible are obscured by the symbols. For sample sizes, see Table S3.

Fig. 3.

Cold-tolerance measures of overwintering Dendroctonus ponderosae larvae. The cold tolerance of D. ponderosae larvae was estimated at three time points during the overwintering period from autumn 2022 to spring 2023 (see Fig. 1 for dates). This was done by exposing them to periods of stressfully low, non-freezing, coma-inducing temperatures (–10°C in circles with a solid line, and −20°C in winter in triangles with a dashed line) up to 168 h (7 days) and estimating (A–C) how long it took them to recovery mobility and (D–F) whether they accrued cold-related injuries (see Materials and Methods). Note that some larvae did not recovery function within the 90 min observation period (horizontal dashed line) and were removed from this dataset (see Table 1); however, unless frozen by the exposure, they were kept to assess their survival after the full recovery period. Overall, recovery times increased the longer larvae were exposed to cold stress; however, this was further prolonged if the exposure temperature was lowered or if the larvae had exited their overwintering stage (i.e. in spring). Survival, in contrast, was only slightly decreased by extended periods of cold stress and was similar across seasons and temperature exposures. Small, translucent points denote individual data points, whereas the large opaque point denotes the mean. Individual data points in D–F are jittered along the ordinal y-axis for clarity. Error bars not visible are obscured by the symbols. For sample sizes, see Table S3.

Table 1.

Number of Dendroctonus ponderosae larvae not moving within 90 min of being removed from the cold in the experiment where recovery times and survival were investigated (see Fig. 3)

Number of Dendroctonus ponderosae larvae not moving within 90 min of being removed from the cold in the experiment where recovery times and survival were investigated (see Fig. 3)
Number of Dendroctonus ponderosae larvae not moving within 90 min of being removed from the cold in the experiment where recovery times and survival were investigated (see Fig. 3)

After recovery from the cold exposure, larvae were left to recover at room temperature for 24 h, after which their survival scores were estimated based on their ability to move. We saw very little evidence of injury in the larvae left to recover at room temperature for 24 h (Fig. 3D–F). Nonetheless, we identified a significant effect of exposure time on the survival score, as longer exposures slightly reduced the survival score (F1,382=6.6, P=0.011). In addition, there was a marginally significant trend for exposure to lower temperatures to further reduce survival (F1,382=3.5, P=0.061). Seasonality did not affect survival (F2,382=2.2, P=0.107), and there were no interactions between exposure time and other fixed factors (exposure time×temperature: F1,382=2.6, P=0.107; exposure time×season: F2,382=1.5, P=0.219).

Hemolymph ion composition during prolonged non-freezing cold exposure

To investigate whether the observed increase in chill coma recovery time and reduction in survival score could be the result of an ionoregulatory collapse, as seen in other insects, we took hemolymph samples from animals exposed to the same time and temperature treatments and measured the concentrations of K+ and Na+ with ion-sensitive electrodes (Fig. 4).

Fig. 4.

Prolonged exposure to stressful, non-freezing cold leads to moderate hemolymph hyperkalemia and hyponatremia in overwintering Dendroctonus ponderosae. After establishing that concentrations of K+ and Na+ in the hemolymph of larval D. ponderosae were unaffected by exposure to benign temperatures (squares in shaded area; C1=24 h at 21–22°C and C2=24 h at 6°C), larvae were again exposed to stressful cold (–10°C in circles with a solid line, and −20°C in winter in triangles with a dashed line) for up to 168 h during which hemolymph was sampled. (A–C) During the exposure, hemolymph [K+] gradually increased, until an apparent asymptote was reached after a ∼80–82% increase above the control concentration in larvae held at −10°C. In −20°C-exposed larvae (winter), hemolymph [K+] increased further to ∼123% above baseline. (D–F) Conversely, hemolymph [Na+] decreased gradually and generally reached concentrations ∼30–38% below baseline regardless of exposure temperature. Small, translucent points denote individual data points, whereas the large opaque point denotes the mean. Error bars not visible are obscured by the symbols. For sample sizes, see Table S4.

Fig. 4.

Prolonged exposure to stressful, non-freezing cold leads to moderate hemolymph hyperkalemia and hyponatremia in overwintering Dendroctonus ponderosae. After establishing that concentrations of K+ and Na+ in the hemolymph of larval D. ponderosae were unaffected by exposure to benign temperatures (squares in shaded area; C1=24 h at 21–22°C and C2=24 h at 6°C), larvae were again exposed to stressful cold (–10°C in circles with a solid line, and −20°C in winter in triangles with a dashed line) for up to 168 h during which hemolymph was sampled. (A–C) During the exposure, hemolymph [K+] gradually increased, until an apparent asymptote was reached after a ∼80–82% increase above the control concentration in larvae held at −10°C. In −20°C-exposed larvae (winter), hemolymph [K+] increased further to ∼123% above baseline. (D–F) Conversely, hemolymph [Na+] decreased gradually and generally reached concentrations ∼30–38% below baseline regardless of exposure temperature. Small, translucent points denote individual data points, whereas the large opaque point denotes the mean. Error bars not visible are obscured by the symbols. For sample sizes, see Table S4.

At each seasonal sampling point (autumn, winter and spring), we first compared the hemolymph ion concentration in samples from control animals that were kept at room temperature (21–22°C), at 6°C for 24 h, and immediately upon reaching the exposure temperature (either −10 or −20°C for 0 h). For Na+, however, we did not include the room temperature control. For hemolymph K+ concentration, the values never differed between control groups (autumn: F2,30=0.8, P=0.464; winter: F2,37=1.9, P=0.148; spring: F2,43=0.5, P=0.583) and always stayed at ∼32 mmol l−1 across seasons (mean±s.d.=32.3±5.0 mmol l−1). The same was true for Na+ concentrations (autumn: F1,19=0.4, P=0.545; winter: F2,26=0.1, P=0.889; spring: F1,29=0.1, P=0.709), where the concentration stayed constant at ∼6 mmol l−1 (mean±s.d.=5.8±1.1 mmol l−1).

After confirming that our control conditions and very short cold exposure resulted in no change to hemolymph K+ and Na+ concentrations, we tested whether these were altered by prolonged exposure to stressful, non-freezing cold at −10°C, and at −20°C in winter. Hemolymph K+ concentration (Fig. 4A–C) increased with exposure time (F1,335=257.0, P<0.001), a lower exposure temperature (F1,335=35.4, P<0.001) and their interaction (F1,335=9.7, P=0.002), with no effect of seasonality (F2,335=2.1, P=0.126) or interaction between seasonality and exposure time (F2,335=0.4, P=0.646). Specifically, after 168 h at −10°C it increased from the control value of ∼32 mmol l−1 to 56.5±1.5, 56.2±3.5 and 58.9±1.5 mmol l−1 in the autumn, winter and spring, respectively, in a seemingly asymptotical manner, whereas it increased further to 69.9±3.2 mmol l−1 in the winter group exposed to −20°C. Conversely, the hemolymph Na+ concentration (Fig. 4D–F) decreased as exposure time increased (F1,219=63.3, P<0.001), with no effects of exposure temperature (F1,219=2.3, P=0.130), season (F2,219=1.1, P=0.320) or any interactions (time×temperature: F1,219=0.1 and P=0.810, time×season: F2,219=0.5, P=0.606). Specifically, over the time course of 168 h, it decreased from the control value of ∼6 mmol l−1 and stabilized at 4.0±0.1, 4.3±0.2 and 3.8±0.1 mmol l−1, in the autumn, winter and spring, respectively, and 3.6±0.2 in the winter group exposed to −20°C.

Correlations between hemolymph K+ and cold tolerance measures

Because we collected measurements of cold tolerance and hemolymph ion concentrations at the same time points throughout seasons and exposures, we can investigate the putative relationship between these parameters based on group means to investigate whether this matches patterns observed for less cold tolerant, chill-susceptible insects (Fig. 5).

Fig. 5.

Magnitude of hemolymph hyperkalemia correlates with chill coma recovery times and survival outcome in cold-exposed, overwintering Dendroctonus ponderosae larvae. Correlations were made using the matching hemolymph [K+] and cold tolerance measurements at each time points across seasons and temperature exposures. Overall, there was a strong exponential relationship between hemolymph hyperkalemia and the ability to recover movement after cold exposure (chill coma recovery time, A–D), and this relationship was right-shifted in larvae exposed to −20°C in mid-winter (B,D), indicating a faster rate of recovery at a similar degree of hyperkalemia. Exposure to −10°C for extended periods of time did not lead to any significant injury in the autumn (E) or winter (F). However, if larvae exposed to −20°C in winter were combined with those exposed to −10°C, the beginning of a sigmoidal dose–response curve emerged (F), and predicted that that the average larvae would be expected to have accrued injury at a 170.4±27.1% increase in hemolymph [K+] (LD50, based on extrapolation). In spring, the relationship between survival score and hemolymph [K+] revealed a similar, yet statistically non-significant, sigmoidal curve with an LD50 of 110.3±28.5% above control (G). With all datasets combined, we saw a significant relationship between hemolymph hyperkalemia and the decrease in survival score during the exposure to stressful cold (H), which also appeared sigmoidal and with a LD50 at a 180.8±28.3% increase hemolymph [K+]. Full lines and dashed black lines denote a significant relationship between the cold tolerance measure and the proportional increase in hemolymph [K+]; no line indicates the absence of a statistically significant relationship. A grey line denotes that the relationship is based on multiple treatment or seasonal groups (e.g. −10°C exposed groups across all seasons in D), whereas a dotted line represents a model-based extrapolation (in F–H).

Fig. 5.

Magnitude of hemolymph hyperkalemia correlates with chill coma recovery times and survival outcome in cold-exposed, overwintering Dendroctonus ponderosae larvae. Correlations were made using the matching hemolymph [K+] and cold tolerance measurements at each time points across seasons and temperature exposures. Overall, there was a strong exponential relationship between hemolymph hyperkalemia and the ability to recover movement after cold exposure (chill coma recovery time, A–D), and this relationship was right-shifted in larvae exposed to −20°C in mid-winter (B,D), indicating a faster rate of recovery at a similar degree of hyperkalemia. Exposure to −10°C for extended periods of time did not lead to any significant injury in the autumn (E) or winter (F). However, if larvae exposed to −20°C in winter were combined with those exposed to −10°C, the beginning of a sigmoidal dose–response curve emerged (F), and predicted that that the average larvae would be expected to have accrued injury at a 170.4±27.1% increase in hemolymph [K+] (LD50, based on extrapolation). In spring, the relationship between survival score and hemolymph [K+] revealed a similar, yet statistically non-significant, sigmoidal curve with an LD50 of 110.3±28.5% above control (G). With all datasets combined, we saw a significant relationship between hemolymph hyperkalemia and the decrease in survival score during the exposure to stressful cold (H), which also appeared sigmoidal and with a LD50 at a 180.8±28.3% increase hemolymph [K+]. Full lines and dashed black lines denote a significant relationship between the cold tolerance measure and the proportional increase in hemolymph [K+]; no line indicates the absence of a statistically significant relationship. A grey line denotes that the relationship is based on multiple treatment or seasonal groups (e.g. −10°C exposed groups across all seasons in D), whereas a dotted line represents a model-based extrapolation (in F–H).

We found a strong link between chill coma recovery time and hemolymph hyperkalemia, which closely matched an exponential relationship in the autumn (F1,5=67.8, P<0.001), winter (F1,10=94.0, P<0.001) and spring (F1,5=57.9, P<0.001), as well as throughout the overwintering season when combined into one dataset (F1,24=227.6, P<0.001) (Fig. 5A–D). Interestingly, we also found that a lower exposure temperature (in mid-winter) resulted in improved recovery rates, but only at higher degrees of hyperkalemia (hyperkalemia×temperature interaction: F1,10=5.2, P=0.045) (effect of temperature: F1,24=2.6, P=0.135; Fig. 5B). When datasets were combined, the faster recovery rate after exposure to lower temperature became statistically significant (F1,24=10.8, P=0.003), and the effect remained larger at more severe hyperkalemia (F1,24=9.7, P=0.005) (Fig. 4D).

The link between hemolymph hyperkalemia during cold exposure and survival outcome after recovery was less clear (Fig. 5E–H). In the autumn and spring, there were no apparent relationships (autumn: F1,5=0.2, P=0.655, Fig. 5E; spring: F1,5=3.4, P=0.123, Fig. 5G); however, a significant relationship emerged in winter (F1,12=6.9, P=0.022; Fig. 5F). When combining data across seasons, the overall relationship between hemolymph hyperkalemia and survival was supported (F1,26=9.8, P=0.004; Fig. 5H). The sigmoidal regressions fit to the statistical significant relationships showed that the degree of hemolymph hyperkalemia expected to injure all animals was an increase of ∼170–180% above control (i.e. ∼86–90 mmol l−1; Fig. 4F,H).

Similar significant relationships occurred between cold tolerance measures and hemolymph hyponatremia (Fig. S1). Specifically, we found an exponential relationship between chill coma recovery and hyponatremia (F1,16=53.0, P<0.001), which was season-specific (effect of season: F2,16=5.1, P=0.020), and a sigmoidal, dose-response-like relationship with the survival score (F1,18=5.4, P=0.031) which indicated an onset of injury at a 72.0±19.2% decrease in hemolymph Na+ concentration.

In the present study, we examined how the cold tolerance of D. ponderosae larvae changed during overwintering and how potential non-freezing mortality might relate to ion balance disruption as seen in less tolerant insects. We show that overwintering larvae exposed to extremely low temperatures, just above those that cause them to spontaneously freeze, for prolonged periods experience physiological stress and sustain injury which manifested as increased recovery times and reduced survival. Stressed larvae experience a loss of ion balance, characterized by hyperkalemia and hyponatremia, which correlates strongly with the degree of cold-induced injury.

Survival of overwintering mountain pine beetle larvae is only compromised after prolonged exposure to severe non-freezing cold

Previous studies demonstrated that overwintering larvae of D. ponderosae are freeze avoiding with the ability to substantially lower their SCP during winter (Régnière and Bentz, 2007; Rosenberger et al., 2017). Here, we reaffirm that overwintering larvae in the expanded range are able to lower their supercooling points, and also show that dysregulation of ion balance and non-freezing chilling injury occur during prolonged chilling at ecologically relevant temperatures in this species.

The SCPs we observed in mid-winter were well below the lowest observed temperature of the stored logs (see Fig. 2). This extensive SCP depression is a defining characteristic of freeze-avoidant species (Zachariassen, 1985), and the lowest SCP observed here is similar to that found in another beetle, the emerald ash borer (Agrilus planipennis; see Crosthwaite et al., 2011; Duell et al., 2022). During our experiments, we noted that fall-collected larvae had food in their guts, whereas those extracted in winter and spring had empty guts. This is a common strategy for freeze-avoidant insects, as this removes most internal ice nucleating agents. We also noted that some fall-collected larvae held for prolonged periods at –10°C evacuated their guts during the experiment.

Mechanisms that suppress the SCP, and thereby reduce the risk of freezing, have historically been considered as the primary means by which freeze-avoidant insects survive winter. The potential for non-freezing injury has received considerably less attention in freeze-avoidant species. For less cold-tolerant (chill-susceptible) insects, survival is determined by physiological effects of low temperature in the absence of freezing. In these species, low temperature tolerance is estimated by assessing the capacity to recover from and survive a non-freezing cold exposure (Sinclair et al., 2015; Overgaard and MacMillan, 2017). Our findings reveal that larvae of D. ponderosae exposed to prolonged periods of low temperatures just above their SCP were cold-stressed; recovery times gradually increased with exposure time and survival decreased slightly after prolonged exposure. These effects of cold exposure were exacerbated at lower temperatures and during seasons of reduced cold tolerance (i.e. spring, see Fig. 3). These trends are remarkably similar to those observed for chill-susceptible insects, where both cold acclimation and milder exposure temperatures improve recovery times and survival outcomes (Andersen et al., 2017a; Yerushalmi et al., 2018; Tarapacki et al., 2021). Similarly, recovery times in other chill-susceptible insects reach an apparent plateau as injury and mortality begin to set in (MacMillan et al., 2012). That being said, the progression of adverse effects in D. ponderosae is markedly slower than that for chill-susceptible insects, even though the larvae were exposed to temperatures that would rapidly kill less tolerant insects (Koštál et al., 2004, 2006; Findsen et al., 2013; Andersen et al., 2015). Interestingly, chill-tolerant Drosophila montana recover rapidly following prolonged cold stress without injury, which could suggest shared physiological mechanisms to survive low temperatures (MacMillan et al., 2015a; Andersen and Overgaard, 2019). However, despite D. ponderosae larvae being able to mitigate the cascade of events that prolongs recovery times, they eventually suffer injury and mortality, indicating that they are unable to completely counteract the negative effects of the cold stress.

Ion balance and partial ionoregulatory collapse during stressful cold exposure in mountain pine beetle larvae

In the ‘ionoregulatory collapse’ model of insect cold tolerance, increased recovery times and decreased survival are linked to a progressive, debilitating cold-induced hemolymph hyperkalemia (Overgaard and MacMillan, 2017; MacMillan, 2019; Overgaard et al., 2021). The hemolymph composition of phytophagous coleopterans has previously been described as ‘unconventional’; they often have high concentrations of K+ (>30 mmol l−1) and Mg2+ (>30 mmol l−1), and unusually low concentrations of Na+ (<15 mmol l−1) (Duchǎteau et al., 1953; Jeuniaux, 1971). This is also the case in D. ponderosae. Hemolymph concentrations of K+ and Na+ were ∼32 and ∼6 mmol l−1, respectively, for larvae kept under benign conditions (Mg2+ was not measured) (Fig. 4). As in chill-susceptible species, these values remained unchanged during the cooling process (MacMillan et al., 2014), but prolonged exposure caused hemolymph hyperkalemia and hyponatremia. Further, the degree of change in the concentration of these ions strongly correlated with the severity of the cold exposure and relative cold tolerance of the larvae (compare Figs 3 and 4). Cold-induced hemolymph hyperkalemia is a hallmark of the ionoregulatory collapse model derived mainly from the study of dipteran and orthopteran species that are chill susceptible (Overgaard and MacMillan, 2017; Overgaard et al., 2021). The same phenomenon has occurred in a chill-susceptible coleopteran, Alphitobius diaperinus (Koštál et al., 2007), and chill-susceptible lepidopterans with an ion homeostasis similar to that of D. ponderosae (Natochin and Parnova, 1987; Andersen et al., 2017b). Thus, despite being categorized as a freeze-avoidant insect during short cold exposures with acute endpoints (Régnière and Bentz, 2007; Rosenberger et al., 2017), D. ponderosae appears to be limited in its non-freezing cold tolerance during prolonged exposures via mechanisms that closely resemble those limiting chill-susceptible insects. Indeed, when the degree of hemolymph hyperkalemia is correlated with recovery times and survival outcomes at the different time points, the emerging relationships resemble those found for chill-susceptible insects (Fig. 5). Specifically, there is a strong exponential correlation between the increase in hemolymph [K+] and recovery times, whereas the relationship with survival appears sigmoidal in nature (yet we caution that the fitted sigmoidal relationships extrapolate beyond the data). This relationship between cold-induced hemolymph hyperkalemia and injury is remarkably similar to that observed for the fire bug (Pyrrhocoris apterus), which is characterized as chill susceptible despite a substantial capacity for supercooling, survival and mitigation of ionoregulatory collapse during month-long exposures to temperature just above their SCP (Koštál et al., 2004). Thus, the ability to mitigate, or delay, ionoregulatory collapse during prolonged exposure to severe cold is not unique to freeze-avoidant insects, indicating shared cold-tolerance mechanisms between freeze-avoidant and chill-tolerant insects.

In chill-susceptible insects, the exponential relationship between recovery times and hemolymph hyperkalemia has been suggested to stem from the need for water redistribution after the cold-induced osmotic disruption (MacMillan et al., 2015a,b). In the present study, we did not investigate water balance; however, the cold-induced osmotic disruption in fruit flies is caused by a steep hemolymph-to-gut Na+ gradient that is unlikely to exist in D. ponderosae larvae as control concentrations are ∼6 mmol l−1. Nonetheless, hemolymph hyperkalemia does occur, and the approximate LD50 found here (∼170–180% increase, see Fig. 5) closely matches that for chill-susceptible insects (∼200% increase; see Overgaard and MacMillan, 2017). Thus, even if different upstream homeostatic disruptions cause hyperkalemia in this species, the mechanism(s) by which increased hemolymph [K+] causes injury and cell death during a cold exposure might be shared.

A distinct pattern of ion balance disruption in mountain pine beetles leads to new paths of inquiry on the mechanisms of seasonal plasticity and cold-tolerance evolution

Intriguingly, the level of hemolymph hyperkalemia we observed in D. ponderosae plateaus at a level just below those that start to cause extensive mortality in other chill-susceptible species (i.e. they stabilize below a ∼150% increase whereas a ∼200% increase leads to injury; see Overgaard and MacMillan, 2017). That being said, the absolute concentrations of hemolymph K+ experienced by D. ponderosae here (55–70 mmol l−1) would be detrimental to other chill-susceptible insects. Regardless, this suggests that this freeze-avoidant species has previously undescribed mechanisms that help prevent extensive cell depolarization, cell death and organismal mortality in the cold. These mechanisms could already be established through seasonal plasticity before cold stress (i.e. seasonal plasticity or acclimatization), could be induced rapidly during cold stress (i.e. rapid hardening), or a combination of both.

In chill-tolerant Drosophila species or cold-acclimated D. melanogaster, concentration gradients for the prevailing cation in the hemolymph (Na+) are reduced, and other compatible osmolytes are more abundant and serve to reduce the driving force for Na+ leak (MacMillan et al., 2015b; Olsson et al., 2016). Similar osmolyte activity occurs in some freeze-avoidant beetles (Hanzal et al., 1992). In Drosophila, these changes occur in response to environmental change but before a stressful cold exposure. This modification of the ‘resting’ state is thought to limit the extent of ion balance disruption during the chilling event. The osmolytes that take the place of these extracellular ions include classical cryoprotectants such as trehalose and proline, which led to the hypothesis that their accumulation may protect against ionoregulatory collapse regardless of the risk of freezing (Olsson et al., 2016). This mechanism of protection is compelling, because it would provide an evolutionary stepping-stone toward the role of cryoprotectants in SCP depression and support the notion of chill susceptibility, chill tolerance and freeze avoidance as a continuum of thermal tolerance strategies. Indeed, chill-susceptible insects accumulate cryoprotectants in preparation for overwintering (Koštál and Šimek, 2000; Koštál et al., 2004) or when given mild cold treatments (Wang and Kang, 2005; Overgaard et al., 2007). This putative role for cryoprotectants relies on far lower concentrations of these solutes to provide protection against the cold, and prevention of ionoregulatory collapse could therefore represent a prerequisite for acquiring either freeze avoidance or freeze tolerance as a survival strategy. If cryoprotectants play this role in D. ponderosae, seasonal accumulation of glycerol and other cryoprotectants in the hemolymph may be critical to establishing a favorable equilibrium of water balance during chilling that passively protects against catastrophic hyperkalemia. By contrast, rapid production of cryoprotectants during chilling in this species (Sømme, 1964; Thompson et al., 2020) could actively prevent further progression of ionoregulatory collapse during cold stress and lead to the plateau in [K+] we observed here. Most likely, these means of altering osmotic gradients are combined with extensive changes to the renal system that allow for maintenance of ion homeostasis at extreme low temperatures (MacMillan et al., 2015a; Yerushalmi et al., 2018; Andersen and Overgaard, 2020). In less tolerant insects, renal plasticity plays a critical role in chill tolerance, and we expect that maintenance of ion balance in the cold is achieved by D. ponderosae through complementary means.

An alternative explanation for the observed plateau in hemolymph ion concentrations could be that these reflect a change in steady-state conditions for D. ponderosae rather than a physiological failure leading to reduced fitness (i.e. survival). This would switch the survival strategy from one of resisting cold-induced ionoregulatory dysfunction to one of tolerating cold-induced suppression of ionoregulatory mechanisms or similar. In fact, such a strategy has been suggested in terms of abiotic stress tolerance in insects and ectotherms and has been hypothesized to conserve energy during exposure to severely unfavorable conditions via mechanisms such as channel or spike arrest (Boutilier, 2001; Campbell et al., 2018; Robertson et al., 2020) or diapause (Hahn and Denlinger, 2011; Sinclair, 2015). Energy conservation during winter is critical for spring emergence and life history of D. ponderosae; however, whether they enter a true diapause remains a topic of debate (Lester and Irwin, 2012; Bentz and Hansen, 2018). More research is needed to examine which ‘strategy’ is utilized, whether these strategies come with potential trade-offs in terms of energy conservation, and what that means in terms of physiological mechanisms of injury and injury prevention.

Conclusions

In conclusion, this study unveils unique cold-tolerance mechanisms in overwintering D. ponderosae larvae. Specifically, we show that despite possessing an impressive capacity for freeze-avoidance, these cold-hardy larvae experience physiological stress and sustain injury during prolonged exposure to severe, stressful cold, which is linked to a gradual disruption of hemolymph ion balance (hyperkalemia) in a manner similar to that of less tolerant, chill-susceptible insects. The asymptotical nature of the ion balance disruption and the ability of D. ponderosae to stabilize hemolymph ion concentrations to just below levels causing extensive injury suggest previously unknown mechanisms of ion balance regulation and protection, which we speculate might be related to the production and accumulation of cryoprotectants. These mechanisms may stem from seasonal or rapid changes to their physiology, or a combination of both. Further research into renal and epithelial function is needed to elucidate the molecular and cellular mechanisms that promote this physiological capacity. Indeed, gaining a deeper understanding of the complex, integrative biology and physiology of overwintering in D. ponderosae is likely to affect future management efforts. Lastly, our findings challenge the current and historical division of cold-tolerance strategies and mechanisms, and we posit that chill susceptibility, chill tolerance and freeze avoidance represent a continuum of thermal tolerance strategies. Thus, drawing parallels between the physiological mechanisms that limit the cold tolerance of chill-susceptible insects and those that permit winter survival of more tolerant insects, such as D. ponderosae, might promote rapid advances in our understanding of cold-tolerance physiology.

We extend our utmost gratitude to Ashlyn Wardlaw and Dr Anna Turbelin for their help in running experiments in the quarantine facilities at the Great Lakes Forestry Centre in Sault Ste. Marie (where both are affiliated). We furthermore acknowledge Caroline Whitehouse, Glenn Dobransky and Andrea Sharpe for their help with providing permits and guidance in collecting the beetle-infested tree logs, as well as Leanne Petro for fieldwork associated with baiting of adult beetles to the field site and trees. Lastly, we are thankful for the assistance of Colleen Fortier, Taylor Volappi, Marion Mayerhofer and the High Country Arborists for coordinating and assisting with the tree harvest.

Author contributions

Conceptualization: M.K.A., A.D.R., H.A.M.; Methodology: M.K.A., A.D.R., A.E.M., M.L.E., H.A.M.; Validation: M.K.A.; Formal analysis: M.K.A.; Investigation: M.K.A., A.D.R., A.L., S.F., F.H.; Resources: A.D.R., A.E.M., M.L.E., H.A.M.; Data curation: M.K.A.; Writing - original draft: M.K.A.; Writing - review & editing: M.K.A., A.D.R., A.L., A.E.M., S.F., F.H., M.L.E., H.A.M.; Visualization: M.K.A.; Supervision: M.K.A., A.D.R., M.L.E., H.A.M.; Project administration: M.K.A., A.D.R., H.A.M.; Funding acquisition: A.D.R., M.L.E., H.A.M.

Funding

Funding for this research has been provided through grants to the TRIA-FoR Project (https://tria-for.ualberta.ca/) to A.D.R., M.A. and H.A.M. from Genome Canada (project no. 18202), the Government of Alberta through Genome Alberta (project ID L20TF), and the Ontario Research Fund – Ontario Ministry of Colleges and Universities through Ontario Genomics (File No. 18202), with contributions from the University of Alberta, Carleton University, and the Great Lakes Forestry Centre – Natural Resources Canada. Further funding for this research has been provided by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-05322) to H.A.M. Equipment used in this study was purchased with support from the Canadian Foundation for Innovation (project no. 37721). Open access funding provided by Carleton University. Deposited in PMC for immediate release.

Data availability

All relevant data can be found within the article and its supplementary information.

Addo-Bediako
,
A.
,
Chown
,
S. L.
and
Gaston
,
K. J.
(
2000
).
Thermal tolerance, climatic variability and latitude
.
Proc. R. Soc. B
267
,
739
-
745
.
Andersen
,
M. K.
and
Overgaard
,
J.
(
2019
).
The central nervous system and muscular system play different roles for chill coma onset and recovery in insects
.
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
233
,
10
-
16
.
Andersen
,
M. K.
and
Overgaard
,
J.
(
2020
).
Maintenance of hindgut reabsorption during cold exposure is a key adaptation for Drosophila cold tolerance
.
J. Exp. Biol.
223
,
jeb213934
.
Andersen
,
J. L.
,
Manenti
,
T.
,
Sørensen
,
J. G.
,
MacMillan
,
H. A.
,
Loeschcke
,
V.
and
Overgaard
,
J.
(
2015
).
How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
.
Funct. Ecol.
29
,
55
-
65
.
Andersen
,
M. K.
,
Folkersen
,
R.
,
MacMillan
,
H. A.
and
Overgaard
,
J.
(
2017a
).
Cold acclimation improves chill tolerance in the migratory locust through preservation of ion balance and membrane potential
.
J. Exp. Biol.
220
,
487
-
496
.
Andersen
,
M. K.
,
Jensen
,
S. O.
and
Overgaard
,
J.
(
2017b
).
Physiological correlates of chill susceptibility in Lepidoptera
.
J. Insect Physiol.
98
,
317
-
326
.
Bale
,
J.
(
1987
).
Insect cold hardiness: freezing and supercooling—an ecophysiological perspective
.
J. Insect Physiol.
33
,
899
-
908
.
Bale
,
J.
(
1991
).
Implications of cold hardiness for pest management
. In
Insects at Low Temperature
(ed. R. E. Lee and D. L. Denlinger), pp.
461
-
498
.
Springer
.
Bale
,
J. S.
(
1996
).
Insect cold hardiness: a matter of life and death
.
Eur. J. Entomol.
93
,
369
-
382
.
Bale
,
J.
(
2002
).
Insects and low temperatures: from molecular biology to distributions and abundance
.
Phil. Trans. R. Soc. B
357
,
849
-
862
.
Bayley
,
J. S.
,
Winther
,
C. B.
,
Andersen
,
M. K.
,
Grønkjær
,
C.
,
Nielsen
,
O. B.
,
Pedersen
,
T. H.
and
Overgaard
,
J.
(
2018
).
Cold exposure causes cell death by depolarization-mediated Ca2+ overload in a chill-susceptible insect
.
Proc. Natl Acad. Sci. USA
115
,
E9737
-
E9744
.
Bentz
,
B. J.
and
Hansen
,
E. M.
(
2018
).
Evidence for a prepupal diapause in the mountain pine beetle (Dendroctonus ponderosae)
.
Environ. Entomol.
47
,
175
-
183
.
Bentz
,
B.
and
Mullins
,
D.
(
1999
).
Ecology of mountain pine beetle (Coleoptera: Scolytidae) cold hardening in the intermountain west
.
Environ. Entomol.
28
,
577
-
587
.
Block
,
W.
,
Baust
,
J.
,
Franks
,
F.
,
Johnston
,
I.
and
Bale
,
J.
(
1990
).
Cold tolerance of insects and other arthropods
.
Phil. Trans. R. Soc. B
326
,
613
-
633
.
Boutilier
,
R. G.
(
2001
).
Mechanisms of cell survival in hypoxia and hypothermia
.
J. Exp. Biol.
204
,
3171
-
3181
.
Campbell
,
J. B.
,
Andersen
,
M. K.
,
Overgaard
,
J.
and
Harrison
,
J. F.
(
2018
).
Paralytic hypo-energetic state facilitates anoxia tolerance despite ionic imbalance in adult Drosophila melanogaster
.
J. Exp. Biol.
221
,
jeb177147
.
Canadian Food Inspection Agency
(
2014
). . Government of Canada.
Creeden
,
E. P.
,
Hicke
,
J. A.
and
Buotte
,
P. C.
(
2014
).
Climate, weather, and recent mountain pine beetle outbreaks in the western United States
.
For. Ecol. Manag.
312
,
239
-
251
.
Crosthwaite
,
J. C.
,
Sobek
,
S.
,
Lyons
,
D. B.
,
Bernards
,
M. A.
and
Sinclair
,
B. J.
(
2011
).
The overwintering physiology of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)
.
J. Insect Physiol.
57
,
166
-
173
.
Cullingham
,
C. I.
,
Cooke
,
J. E.
,
Dang
,
S.
,
Davis
,
C. S.
,
Cooke
,
B. J.
and
Coltman
,
D. W.
(
2011
).
Mountain pine beetle host–range expansion threatens the boreal forest
.
Mol. Ecol.
20
,
2157
-
2171
.
de la Giroday
,
H. M. C.
,
Carroll
,
A. L.
and
Aukema
,
B. H.
(
2012
).
Breach of the northern Rocky Mountain geoclimatic barrier: initiation of range expansion by the mountain pine beetle
.
J. Biogeogr.
39
,
1112
-
1123
.
Denlinger
,
D. L.
and
Lee
,
R. E.
Jr
. (
2010
).
Low Temperature Biology of Insects
.
Cambridge University Press
.
Ditrich
,
T.
(
2018
).
Supercooling point is an individually fixed metric of cold tolerance in Pyrrhocoris apterus
.
J. Therm. Biol.
74
,
208
-
213
.
Dow
,
J. A.
(
2017
).
The essential roles of metal ions in insect homeostasis and physiology
.
Curr. Opin. Insect Sci.
23
,
43
-
50
.
Duchǎteau
,
G.
,
Florkin
,
M.
and
Leclercq
,
J.
(
1953
).
Concentrations des bases fixes et types de composition de la base totale de l'hémolymphe des insectes
.
Arch. Int. Physiol.
61
,
518
-
549
.
Duell
,
M. E.
,
Gray
,
M. T.
,
Roe
,
A. D.
,
MacQuarrie
,
C. J.
and
Sinclair
,
B. J.
(
2022
).
Plasticity drives extreme cold tolerance of emerald ash borer (Agrilus planipennis) during a polar vortex
.
Curr. Res. Insect Sci.
2
,
100031
.
Edney
,
E. B.
(ed.)
(
1977
).
Excretion and osmoregulation
. In
Water Balance in Land Arthropods
, pp.
96
-
171
.
Springer
.
Findsen
,
A.
,
Andersen
,
J. L.
,
Calderon
,
S.
and
Overgaard
,
J.
(
2013
).
Rapid cold hardening improves recovery of ion homeostasis and chill coma recovery time in the migratory locust, Locusta migratoria
.
J. Exp. Biol.
216
,
1630
-
1637
.
Gehrken
,
U.
(
1984
).
Winter survival of an adult bark beetle Ips acuminatus Gyll
.
J. Insect Physiol.
30
,
421
-
429
.
Graham
,
L. A.
,
Liou
,
Y.-C.
,
Walker
,
V. K.
and
Davies
,
P. L.
(
1997
).
Hyperactive antifreeze protein from beetles
.
Nature
388
,
727
-
728
.
Graham
,
L. A.
,
Qin
,
W.
,
Lougheed
,
S. C.
,
Davies
,
P. L.
and
Walker
,
V. K.
(
2007
).
Evolution of hyperactive, repetitive antifreeze proteins in beetles
.
J. Mol. Evol.
64
,
387
-
398
.
Hahn
,
D. A.
and
Denlinger
,
D. L.
(
2011
).
Energetics of insect diapause
.
Annu. Rev. Entomol.
56
,
103
-
121
.
Hanzal
,
R.
,
Bjerke
,
R.
,
Lundheim
,
R.
and
Zachariassen
,
K. E.
(
1992
).
Concentration of sodium and free amino acids in the haemolymph and other fluid compartments in an adephagous and a polyphagous species of beetle
.
Comp. Biochem. Physiol. A Physiol.
102
,
741
-
744
.
Harrison
,
J. F.
,
Woods
,
H. A.
and
Roberts
,
S. P.
(
2012
).
Ecological and Environmental Physiology of Insects
.
Oxford University Press
.
Heinrich
,
B.
(
2013
).
The Hot-blooded Insects: Strategies and Mechanisms of Thermoregulation
.
Springer Science & Business Media
.
Hodge
,
J.
,
Barry
,
C.
and
Rory
,
M.
(
2017
).
A strategic approach to slow the spread of mountain pine beetle across Canada
.
Report
.
Forest Pest Working Group of the Canadian Council of Forest Ministers
.
Holmstrup
,
M.
,
Bayley
,
M.
and
Ramløv
,
H.
(
2002
).
Supercool or dehydrate? An experimental analysis of overwintering strategies in small permeable arctic invertebrates
.
Proc. Natl. Acad. Sci. USA
99
,
5716
-
5720
.
Jeuniaux
,
C.
(
1971
).
Hemolymph–Arthropoda
. In
Chemical Zoology
, Vol.
6
(ed.
M.
Florkin
and
B.
Scheer
), pp.
62
-
118
. NY:
Academic Press
.
Koštál
,
V. R.
and
Šimek
,
P.
(
2000
).
Overwintering strategy in Pyrrhocoris apterus (Heteroptera): the relations between life-cycle, chill tolerance and physiological adjustments
.
J. Insect Physiol.
46
,
1321
-
1329
.
Koštál
,
V.
,
Vambera
,
J.
and
Bastl
,
J.
(
2004
).
On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus
.
J. Exp. Biol.
207
,
1509
-
1521
.
Koštál
,
V.
,
Yanagimoto
,
M.
and
Bastl
,
J.
(
2006
).
Chilling-injury and disturbance of ion homeostasis in the coxal muscle of the tropical cockroach (Nauphoeta cinerea)
.
Comp. Biochem. Physiol. B Biochemi. Mol. Biol.
143
,
171
-
179
.
Koštál
,
V.
,
Renault
,
D.
,
Mehrabianova
,
A.
and
Bastl
,
J.
(
2007
).
Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: role of ion homeostasis
.
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
147
,
231
-
238
.
Lahondère
,
C.
(
2023
).
Recent advances in insect thermoregulation
.
J. Exp. Biol.
226
,
jeb245751
.
Lebenzon
,
J. E.
,
Des Marteaux
,
L. E.
and
Sinclair
,
B. J.
(
2020
).
Reversing sodium differentials between the hemolymph and hindgut speeds chill coma recovery but reduces survival in the fall field cricket, Gryllus pennsylvanicus
.
Comp. Biochem. Physiol. A: Mol. Integr. Physiol.
244
,
110699
.
Lee
,
R. E.
(
1991
).
Principles of insect low temperature tolerance
. In
Insects at Low Temperature
(ed. R. E. Lee and D. L. Denlinger), pp.
17
-
46
.
Springer
.
Lee
,
R.
(
2012
).
Insects at Low Temperature
.
Springer Science & Business Media
.
Lester
,
J. D.
and
Irwin
,
J. T.
(
2012
).
Metabolism and cold tolerance of overwintering adult mountain pine beetles (Dendroctonus ponderosae): evidence of facultative diapause?
J. Insect Physiol.
58
,
808
-
815
.
MacMillan
,
H. A.
(
2019
).
Dissecting cause from consequence: a systematic approach to thermal limits
.
J. Exp. Biol.
222
,
jeb191593
.
MacMillan
,
H. A.
and
Sinclair
,
B. J.
(
2011
).
Mechanisms underlying insect chill-coma
.
J. Insect Physiol.
57
,
12
-
20
.
MacMillan
,
H. A.
,
Williams
,
C. M.
,
Staples
,
J. F.
and
Sinclair
,
B. J.
(
2012
).
Reestablishment of ion homeostasis during chill-coma recovery in the cricket Gryllus pennsylvanicus
.
Proc. Natl. Acad. Sci. USA
109
,
20750
-
20755
.
MacMillan
,
H. A.
,
Findsen
,
A.
,
Pedersen
,
T. H.
and
Overgaard
,
J.
(
2014
).
Cold-induced depolarization of insect muscle: differing roles of extracellular K+ during acute and chronic chilling
.
J. Exp. Biol.
217
,
2930
-
2938
.
MacMillan
,
H. A.
,
Andersen
,
J. L.
,
Davies
,
S. A.
and
Overgaard
,
J.
(
2015a
).
The capacity to maintain ion and water homeostasis underlies interspecific variation in Drosophila cold tolerance
.
Sci. Rep.
5
,
18607
.
MacMillan
,
H. A.
,
Andersen
,
J. L.
,
Loeschcke
,
V.
and
Overgaard
,
J.
(
2015b
).
Sodium distribution predicts the chill tolerance of Drosophila melanogaster raised in different thermal conditions
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
308
,
R823
-
R831
.
MacMillan
,
H. A.
,
Baatrup
,
E.
and
Overgaard
,
J.
(
2015c
).
Concurrent effects of cold and hyperkalaemia cause insect chilling injury
.
Proc. R. Soc. B
282
,
20151483
.
Marshall
,
K. E.
,
Gotthard
,
K.
and
Williams
,
C. M.
(
2020
).
Evolutionary impacts of winter climate change on insects
.
Curr. Opin. Insect Sci.
41
,
54
-
62
.
Messerli
,
M. A.
,
Kurtz
,
I.
and
Smith
,
P. J.
(
2008
).
Characterization of optimized Na+ and Cl liquid membranes for use with extracellular, self-referencing microelectrodes
.
Anal. Bioanal. Chem.
390
,
1355
-
1359
.
Natochin
,
Y. V.
and
Parnova
,
R.
(
1987
).
Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects
.
Comp. Biochem. Physiol. A Physiol.
88
,
563
-
570
.
Olsson
,
T.
,
MacMillan
,
H. A.
,
Nyberg
,
N.
,
Stærk
,
D.
,
Malmendal
,
A.
and
Overgaard
,
J.
(
2016
).
Hemolymph metabolites and osmolality are tightly linked to cold tolerance of Drosophila species: a comparative study
.
J. Exp. Biol.
219
,
2504
-
2513
.
Overgaard
,
J.
and
MacMillan
,
H. A.
(
2017
).
The integrative physiology of insect chill tolerance
.
Annu. Rev. Physiol.
79
,
187
-
208
.
Overgaard
,
J.
,
Malmendal
,
A.
,
Sørensen
,
J. G.
,
Bundy
,
J. G.
,
Loeschcke
,
V.
,
Nielsen
,
N. C.
and
Holmstrup
,
M.
(
2007
).
Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster
.
J. Insect Physiol.
53
,
1218
-
1232
.
Overgaard
,
J.
,
Gerber
,
L.
and
Andersen
,
M. K.
(
2021
).
Osmoregulatory capacity at low temperature is critical for insect cold tolerance
.
Curr. Opin. Insect Sci.
47
,
38
-
45
.
Phillips
,
J.
(
1981
).
Comparative physiology of insect renal function
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
241
,
R241
-
R257
.
Ramløv
,
H.
(
2000
).
Aspects of natural cold tolerance in ectothermic animals
.
Hum. Reprod.
15
,
26
-
46
.
Régnière
,
J.
and
Bentz
,
B.
(
2007
).
Modeling cold tolerance in the mountain pine beetle, Dendroctonus ponderosae
.
J. Insect Physiol.
53
,
559
-
572
.
Renault
,
D.
,
Salin
,
C.
,
Vannier
,
G.
and
Vernon
,
P.
(
2002
).
Survival at low temperatures in insects: what is the ecological significance of the supercooling point?
CryoLetters
23
,
217
-
228
.
Robert
,
J. A.
,
Bonnett
,
T.
,
Pitt
,
C.
,
Spooner
,
L. J.
,
Fraser
,
J.
,
Yuen
,
M. M.
,
Keeling
,
C. I.
,
Bohlmann
,
J.
and
Huber
,
D. P.
(
2016
).
Gene expression analysis of overwintering mountain pine beetle larvae suggests multiple systems involved in overwintering stress, cold hardiness, and preparation for spring development
.
PeerJ
4
,
e2109
.
Robertson
,
R. M.
,
Dawson-Scully
,
K. D.
and
Andrew
,
R. D.
(
2020
).
Neural shutdown under stress: an evolutionary perspective on spreading depolarization
.
J. Neurophysiol.
123
,
885
-
895
.
Rosenberger
,
D. W.
,
Aukema
,
B. H.
and
Venette
,
R. C.
(
2017
).
Cold tolerance of mountain pine beetle among novel eastern pines: a potential for trade-offs in an invaded range?
For. Ecol. Manag.
400
,
28
-
37
.
Safranyik
,
L.
,
Shrimpton
,
D.
and
Whitney
,
H.
(
1975
).
An interpretation of the interaction between lodgepole pine, the mountain pine beetle and its associated blue stain fungi in western Canada
.
Manag. Lodgepole Pine Ecosyst.
1
,
406
-
428
.
Safranyik
,
L.
,
Carroll
,
A. L.
,
Régnière
,
J.
,
Langor
,
D.
,
Riel
,
W.
,
Shore
,
T. L.
,
Peter
,
B.
,
Cooke
,
B. J.
,
Nealis
,
V.
and
Taylor
,
S. W.
(
2010
).
Potential for range expansion of mountain pine beetle into the boreal forest of North America
.
Can. Entomol.
142
,
415
-
442
.
Salt
,
R.
(
1961
).
Principles of insect cold-hardiness
.
Annu. Rev. Entomol.
6
,
55
-
74
.
Sinclair
,
B. J.
(
1999
).
Insect cold tolerance: how many kinds of frozen?
Eur. J. Entomol.
96
,
157
-
164
.
Sinclair
,
B. J.
(
2015
).
Linking energetics and overwintering in temperate insects
.
J. Therm. Biol.
54
,
5
-
11
.
Sinclair
,
B. J.
,
Vernon
,
P.
,
Klok
,
C. J.
and
Chown
,
S. L.
(
2003
).
Insects at low temperatures: an ecological perspective
.
Trends Ecol. Evol.
18
,
257
-
262
.
Sinclair
,
B. J.
,
Alvarado
,
L. E. C.
and
Ferguson
,
L. V.
(
2015
).
An invitation to measure insect cold tolerance: methods, approaches, and workflow
.
J. Therm. Biol.
53
,
180
-
197
.
Storey
,
K. B.
and
Storey
,
J. M.
(
1983
).
Biochemistry of freeze tolerance in terrestrial insects
.
Trends Biochem. Sci.
8
,
242
-
245
.
Storey
,
K. B.
and
Storey
,
J. M.
(
2012
).
Insect cold hardiness: metabolic, gene, and protein adaptation
.
Can. J. Zool.
90
,
456
-
475
.
Sømme
,
L.
(
1964
).
Effects of glycerol on cold-hardiness in insects
.
Can. J. Zool.
42
,
87
-
101
.
Sømme
,
L.
(
1982
).
Supercooling and winter survival in terrestrial arthropods
.
Comp. Biochem. Physiol. A Physiol.
73
,
519
-
543
.
Sømme
,
L.
(
1999
).
The physiology of cold hardiness in terrestrial arthropods
.
Eur. J. Entomol.
96
,
1
-
10
.
Tarapacki
,
P.
,
Jørgensen
,
L. B.
,
Sørensen
,
J. G.
,
Andersen
,
M. K.
,
Colinet
,
H.
and
Overgaard
,
J.
(
2021
).
Acclimation, duration and intensity of cold exposure determine the rate of cold stress accumulation and mortality in Drosophila suzukii
.
J. Insect Physiol.
135
,
104323
.
Thompson
,
K. M.
,
Huber
,
D. P.
and
Murray
,
B. W.
(
2020
).
Autumn shifts in cold tolerance metabolites in overwintering adult mountain pine beetles
.
PLoS One
15
,
e0227203
.
Wang
,
H.-S.
and
Kang
,
L.
(
2005
).
Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs
.
Cryobiology
51
,
220
-
229
.
Williams
,
C. M.
,
Henry
,
H. A.
and
Sinclair
,
B. J.
(
2015
).
Cold truths: how winter drives responses of terrestrial organisms to climate change
.
Biol. Rev.
90
,
214
-
235
.
Yerushalmi
,
G. Y.
,
Misyura
,
L.
,
MacMillan
,
H. A.
and
Donini
,
A.
(
2018
).
Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster
.
J. Exp. Biol.
221
,
jeb174904
.
Zachariassen
,
K. E.
(
1985
).
Physiology of cold tolerance in insects
.
Physiol. Rev.
65
,
799
-
832
.
Zachariassen
,
K. E.
,
Kristiansen
,
E.
and
Pedersen
,
S. A.
(
2004
).
Inorganic ions in cold-hardiness
.
Cryobiology
48
,
126
-
133
.

Competing interests

The authors declare no competing or financial interests.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Supplementary information