Physiological mechanisms of seasonal and rapid cold-hardening in insects

Authors

  • NICHOLAS M. TEETS,

    Corresponding author
    • Department of Entomology, Ohio State University, Columbus, Ohio, U.S.A.
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  • DAVID L. DENLINGER

    1. Department of Entomology, Ohio State University, Columbus, Ohio, U.S.A.
    2. Department of Evolution, Ecology, and Organismal Biology, Ohio State University, Columbus, Ohio, U.S.A.
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Correspondence: Nicholas M. Teets, Department of Entomology, 300 Aronoff Laboratory, 318W. 12th Avenue, Columbus, Ohio 43210, U.S.A. Tel.: +1 614 292 4477; e-mail: teets.23@osu.edu

Abstract

Insects have evolved a number of physiological mechanisms for coping with the detrimental effects of low temperature. As autumn progresses, insects use environmental signals such as shortening day lengths and gradually decreasing temperatures to trigger seasonal cold-hardening adaptations. These mechanisms include dramatic changes in biochemistry, cell function and gene expression that permit improved cell function and viability at low temperature. Insects are also capable of enhancing cold tolerance on a much shorter time scale, in a process called rapid cold-hardening (RCH). Rapid cold-hardening allows insects to improve cold tolerance almost instantaneously (i.e. within minutes to hours) to cope with sudden cold snaps and regularly-occurring diurnal drops in temperature. Initially, it was assumed that RCH would share many of the same basic mechanisms as seasonal cold-hardening, albeit on a shorter time scale. Although there is some evidence supporting this, recent work has called into question some of the original hypotheses concerning the mechanisms of RCH. Also, some mechanisms important for seasonal cold-hardening, such as up-regulation of stress proteins, are unlikely to function at the temperatures and time scales at which RCH occurs. In the present review, the current understanding of the physiological mechanisms governing both seasonal cold-hardening and RCH are summarized. A synthesis of the current literature suggests that these two forms of cold-hardening may be more mechanistically distinct than originally anticipated.

Introduction

Low temperature is one of the greatest challenges facing insects in temperate and polar regions. As ectotherms with small body sizes, insects readily lose heat to their environment and thus must be able to tolerate exposure to sub-optimal temperatures. Because insects are rarely cold-hardy year around, they rely on various forms of cold-hardening to prepare for sub-zero temperatures (Salt, 1961). Much of the work to date has focused on seasonal cold-hardening, in which insects use environmental stimuli such as decreasing temperature and photoperiod to trigger physiological changes enhancing cold tolerance (Denlinger, 1991). However, insects are also capable of cold-hardening on a much shorter time scale, in a process known as rapid cold-hardening (RCH; Lee et al., 1987). In this process, brief exposure to nonlethal low temperatures (of the order of minutes to hours) significantly enhances cold shock tolerance. Both types of cold-hardening clearly have ecological relevance; seasonal cold-hardening governs the gradual, predictable transition to the overwintering phenotype, whereas RCH permits survival during sudden cold-snaps, when the insect is not in a cold-hardy state.

In this review, the underlying mechanisms of both seasonal cold-hardening and RCH are summarized. Most physiological studies of cold-hardening address one type or the other, with very few including seasonal cold-hardening and RCH in the same study. The goal of this review is to compare and contrast the underlying mechanisms of seasonal and rapid responses to low temperature and provide some future directions for understanding these distinct forms of cold-hardening.

Basics principles of insect cold tolerance and mechanisms of cold injury

To appreciate fully the physiological mechanisms governing cold-hardening, it is necessary to understand the basic principles of insect cold tolerance and the nature of cold injury. Although comprehensive reviews on the principles of insect cold tolerance (Sinclair et al., 2003b; Lee, 2010) and cold injury (Denlinger & Lee, 1998; MacMillan & Sinclair, 2011) are available, a few of these basic points are briefly summarized.

Most insects can be classified as either freeze-intolerant or freeze-tolerant (Hawes & Bale, 2007). Freeze-intolerant (also referred to in the literature as freeze-avoidant or chill-susceptible) insects, which make up the majority of insects categorized thus far, succumb to internal ice formation and therefore must remain in a supercooled state at sub-zero temperatures. By contrast, freeze-tolerant insects can survive internal ice formation, provided that the ice crystals are restricted to the extracellular spaces. Typically, freeze-tolerant insects have relatively high supercooling points, which prevents mechanical damage from rapid, uncontrolled ice formation at extreme low temperatures (Lee, 2010). In recent years, a third overwintering strategy, termed cryoprotective dehydration, has been identified in desiccation-tolerant arthropods with highly permeable cuticles (Holmstrup et al., 2002; Elnitsky et al., 2008). During cryoprotective dehydration, environmental ice creates a vapour pressure gradient that drives a loss of body water to the surrounding ice, thereby depressing the haemolymph melting point and preventing internal freezing. Although the factors that determine whether a particular species is freeze-intolerant or freeze-tolerant are largely unknown, freeze-intolerance appears to be basal in arthropods because a majority of freeze-tolerant insects are found in higher orders, Diptera, Coleoptera, Lepidoptera and Hymenoptera (Sinclair et al., 2003a). Sinclair et al. (2003a, 2005) postulate that hemispheric differences in climatic variability may drive the evolution of freeze tolerance. It has long been known that both freeze-intolerant and freeze-tolerant insects are capable of seasonal cold-hardening (Salt, 1961), whereas RCH was assumed to be restricted to freeze-intolerant species. However, RCH has recently been described in three freeze-tolerant species (Lee et al., 2006b; Everatt et al., 2012; Levis et al., 2012), suggesting that it is a general adaptation for insects living in thermally variable environments.

For cold-hardening to be successful, it must protect against or mitigate the negative effects of low temperature. Depending on the severity and duration of cold exposure, insects experience several levels of cold injury (Fig. 1). One of the first symptoms of low temperature injury is an inability to regulate ion balance, which impairs neuromuscular function and leads to the onset of chill coma (Koštál et al., 2004, 2006; MacMillan & Sinclair, 2011; Armstrong et al., 2012; MacMillan et al., 2012). Although chill coma is reversible, insects are at risk of indirect chilling injury if these conditions continue over an extended period (days to weeks). Indirect chilling injury is a result of prolonged impairment of cellular metabolism, leading to irreversible ion imbalance, depletion of cellular ATP and the build up of toxic metabolic end products (Dollo et al., 2010; Koštál et al., 2011a). At more severe temperatures, insects succumb to direct chilling injury, also known as cold shock injury (Lee, 2010). Although the exact mechanisms of cold shock injury are still under investigation, injury appears to be largely a consequence of membrane damage resulting from phase transition (i.e. from a liquid crystalline to a gel state) (Steponkus, 1984). Additionally, cold causes actin depolymerization, leading to reorganization of the cytoskeleton at low temperatures (Kim et al., 2006). Finally, cold shock directly denatures proteins, thereby inhibiting function and causing harmful aggregations of denatured proteins (Feder & Hofmann, 1999). When freezing occurs, additional injury can follow as a result of freeze concentration (Lee, 2010). Because only water molecules join the growing ice lattice, extracellular solutes become concentrated, driving water out of the cell and causing cell shrinkage. The growing ice crystals can also cause physical damage to cells by fracturing cell membranes (Tursman & Duman, 1995).

Figure 1.

Summary of the major sources of cold injury and the underpinning cold-hardening mechanisms that mitigate these damages. Solid boxes indicate cellular targets of cold injury, whereas dashed boxes indicate mechanisms of cold-hardening. After each mechanism, ‘S’ indicates mechanisms that have been associated with seasonal cold-hardening, whereas ‘R’ indicates mechanisms associated with rapid cold-hardening. The question mark in the ‘Cryoprotectant Synthesis’ box denotes that there is discrepancy over whether cryoprotectant synthesis is an essential component of rapid cold-hardening.

Mechanisms of seasonal cold-hardening

In the present review, the physiological mechanisms associated with seasonal cold-hardening are described. Here, seasonal cold-hardening is defined as cold-hardening that requires at least days to weeks for induction. Under the umbrella of seasonal cold-hardening, both cold acclimation and photoperiodic diapause are included, at least in cases where there is a clear link between diapause induction and enhanced cold tolerance. Although many studies on cold acclimation and diapause cover time periods much shorter than natural seasonal changes, they are labelled generically as ‘seasonal cold-hardening’ because these experiments attempt to simulate seasonal shifts in temperature and/or photoperiod as summer transitions to winter. It is acknowledged that this definition of ‘seasonal cold-hardening’ is broad and encompasses numerous time scales, although the primary purpose of the present review is to compare and contrast cold-hardening processes that require days to weeks for induction and those that only require minutes to hours for induction (i.e. rapid cold-hardening).

Cold acclimation is defined as an enhancement of cold tolerance after prolonged (i.e. days to weeks) exposure to mild, sub-lethal low temperatures (Colinet et al., 2012). Colinet & Hoffmann (2012) further distinguish between cold acclimation that occurs within a single developmental stage (i.e. ‘gradual acclimation’) and that which occurs across multiple developmental stages (i.e. ‘developmental acclimation’). Cold acclimation is observed both in the field and in the laboratory. For example, larvae of the goldenrod gall fly Eurosta solidaginis are unable to survive freezing at −20 °C in early autumn but, in response to gradually decreasing temperatures in the autumn, larvae are fully freeze tolerant at −20 °C by the onset of winter (Williams et al., 2004). In the laboratory, nondiapausing Drosophila melanogaster raised at 19 °C are significantly more cold-tolerant than those raised at 25 °C (Rako & Hoffmann, 2006). In many cases, cold-hardening is a component of overwintering diapause; an environmentally-programmed period of dormancy characterized by developmental arrest and reduced oxygen consumption rates (Denlinger et al., 2005). Although diapause typically enhances stress tolerance, in some cases, diapause and cold acclimation are independent (Denlinger, 1991). For this reason, only physiological mechanisms of diapause that are clearly associated with enhanced stress tolerance are considered in the present review.

The earliest-described mechanism of seasonal cold-hardening is the accumulation of low-molecular weight cryoprotectants (Salt, 1961; Lee, 2010). The most common cryoprotectants observed in overwintering insects are low molecular weight sugar alcohols, such as glycerol, sorbitol and inositol. The functions of these compounds include stabilizing membranes and proteins, enhancing supercooling capacity, and preventing osmotic damage to cells (Yancey, 2005). However, the types and amounts of cryoprotective polyols vary significantly by species; E. solidaginis accumulates approximately equal amounts of glycerol and sorbitol (Storey et al., 1981), whereas closely-related Drosophila montana synthesizes large amounts of myo-inositol as the primary osmoprotectant (Vesala et al., 2012a).

In addition to the sugar alcohols, other classes of compounds are recognized as cryoprotectants. Trehalose, the blood sugar of insects, functions as a seasonally-accumulated cryoprotect in many species (Thompson, 2003), whereas the amino acid proline is the primary cryoprotectant in the freeze-tolerant drosophilid Chymomyza costata (Koštál et al., 2011a). Proline appears to be a particularly potent cryoprotectant because diet supplementation permits C. costata to survive freezing in liquid nitrogen (Koštál et al., 2011b) and allows D. melanogaster, which is normally freeze-intolerant, to become mildly freeze-tolerant (Koštál et al., 2012).

The discovery of additional biochemical changes accompanying seasonal cold-hardening has been facilitated by recent advances in metabolomics. In the flesh fly Sarcophaga crassipalpis, diapausing pupae have elevated levels of known cryoprotectants such as glycerol, alanine and glucose, as well as reduced levels of key Krebs cycle intermediates (Michaud & Denlinger, 2007). However, in this case, it has not been possible to determine which changes are adaptive for cold-hardening and which are merely components of the diapause phenotype. Two metabolomic studies of cold acclimation in D. melanogaster, one using constant acclimation temperatures in larvae (Koštál et al., 2011a) and the other using thermoperiods in adults (Colinet et al., 2012), both detected an increase in trehalose and a decrease in Krebs cycle activity in cold-acclimated flies. However, there were also several discrepancies between the two studies; for example, constant-temperature acclimation in larvae increased the concentration of proline significantly (Koštál et al., 2011a), whereas thermoperiodic acclimation in adults failed to elicit an increase in proline (Colinet et al., 2012). This suggests that metabolic changes during cold acclimation may be dependent on the acclimation regime and/or developmental stage, implying that caution should be used when extrapolating these results to natural populations. Vesala et al. (2012a) circumvent this by sampling field populations of the northern drosophilid D. montana. Seasonal accumulation of trehalose and proline supports the important role of these osmoprotectants in this overwintering insect. This species also shows a 400-fold accumulation of myo-inositol over the course of the autumn, the largest noted for any of the cryoprotectants.

Because cell membranes are particularly susceptible to cold, a number of seasonal cold-hardening mechanisms involve membrane modifications. A hypothesis termed homeoviscous adaptation proposes that organisms adjust their membrane composition at low temperatures to maintain a consistent level of membrane fluidity (Sinensky, 1974; Koštál, 2010). Although homeoviscous adaptation is a widely-accepted cold-hardening mechanism, there are several biochemical mechanisms by which it can be achieved. First, membrane fluidity can be enhanced by increasing the proportion of unsaturated fatty acids in the cell membrane. For example, in E. solidaginis, the proportion of unsaturated membrane fatty acids increases by 50% over the course of autumn (Bennett & Lee, 1997). Second, because short-chain fatty acids have lower melting points than long-chain fatty acids, some species increase the ratio of 16-carbon fatty acids relative to 18-carbon fatty acids (Michaud & Denlinger, 2006; Tomčala et al., 2006). Third, in addition to simply increasing the amount of unsaturated and/or short-chain fatty acids in the membrane, the position of these moieties within glycerophospholipids impacts membrane fluidity. The sn-2 position, which penetrates deeper into the lipid bilayer, has a greater impact on membrane fluidity than the sn-1 position. Accordingly, developmentally cold-acclimated adult D. melanogaster have a higher proportion of glycerophosphoethanolamines with the unsaturated linoleic acid at the sn-2 position (Overgaard et al., 2008). Finally, membrane fluidity can be enhanced by a restructuring of the polar head groups of membrane phospholipids; glycerophosphoethanolamines, at the expense of glycerophosphocholines, promote membrane disorder at low temperature (Koštál, 2010). This principle is demonstrated in diapausing flesh flies S. crassipalpis, which increase the ratio of glycerophosphoethanolamines by 16% (Michaud & Denlinger, 2006). Aside from modifications to phospholipids, cholesterol may also be involved in insect cold-hardening because membrane-bound cholesterol can prevent coalescing of adjacent fatty acyl chains (Shreve et al., 2007). Thus, homeoviscous adaptation plays a central role in cold-hardening, although the underpinning mechanisms by which it is achieved vary significantly between species. The prominent role of the cell membrane during seasonal cold-hardening responses is further reflected by alterations in membrane transport properties. Gradual cold acclimation reduces or eliminates ion imbalance in response to low temperature, thereby permitting faster recovery from chill coma (Koštál et al., 2004, 2006).

At the molecular level, a number of genes and proteins have been implicated as key components of diapause and cold acclimation. Perhaps best documented are the heat shock proteins, comprising molecular chaperones that refold denatured proteins during periods of stress (Feder & Hofmann, 1999). Heat shock proteins are up-regulated during diapause in a number of insects, including the egg diapause of silk moths (Moribe et al., 2010), pupal diapause in flesh flies (Yocum et al., 1998; Rinehart et al., 2000) and adult diapause in Colorado potato beetles (Yocum, 2001). Functional experiments have demonstrated that not only are these genes correlated with cold-hardening, but also they are essential in conferring cold tolerance. In S. crassipalpis, knocking down either hsp70 or hsp23 with RNA interference reduces acute cold tolerance of overwintering pupae (Rinehart et al., 2007), whereas knocking down small heat shock protein genes in D. melanogaster increases chill coma recovery time (Colinet et al., 2010). However, not all diapause-associated cold hardening is associated with increased heat shock protein expression; for example, the blowfly Lucilia sericata (Tachibana et al., 2005) and mosquito Culex pipiens (Rinehart et al., 2006) both increase cold tolerance during diapause with no concurrent changes in heat shock protein transcript abundance.

In addition to heat shock proteins, targeted studies have revealed other classes of genes involved in diapause and cold acclimation responses. Aquaporins, which are membrane water channels that promote water movement across cell membranes, appear to be essential for freeze-tolerant insects. In E. solidaginis, aquaporins increase in abundance in the weeks leading to winter (Philip & Lee, 2010) and blocking aquaporins with mercuric chloride reduces tissue freeze tolerance (Philip et al., 2008). These channels are considered to be essential for the rapid movement of water between intracellular and extracellular spaces, permitting necessary adjustments in cell volume and osmotic conditions during freezing. In D. melanogaster, senescence marker protein-30, a calcium binding protein, is elevated during gradual cold acclimation, perhaps to maintain intracellular calcium concentrations at low temperature (Goto, 2000). Additionally, noncoding polymorphisms in this gene correlate with cold tolerance in a panel of inbred D. melanogaster lines that differ in susceptibility to cold (Clowers et al., 2010). The onion maggot Delia antiqua up-regulates a desaturase gene in response to gradual cold acclimation, which likely contributes to membrane restructuring in preparation for winter (Kayukawa et al., 2007). Finally, there is growing evidence that antioxidant enzymes, which allow insects to cope with the cold induced disruption of aerobic respiration, are an essential component of the overwintering toolkit. Various antioxidant enzymes are up-regulated during diapause in the goldenrod gall moth Epiblema scudderiana (Joanisse & Storey, 1996), the flesh fly S. crassipalpis (Ragland et al., 2010) and the mosquito C. pipiens (Sim & Denlinger, 2011). However, in some species, antioxidant defences are instead down-regulated during diapause (Joanisse & Storey, 1996), presumably because diapause-induced hypometabolism sufficiently prevents the accumulation of reactive oxygen species in these insects (Storey & Storey, 2011).

In some cases, seasonal cold-hardening involves proteins and molecules that regulate ice formation. Antifreeze proteins accumulate in select freeze-intolerant insects, particularly species that rely on deep supercooling, and interact with ice crystals to inhibit growth of the ice lattice (Duman, 2001; Duman et al., 2010). There are a few cases in which antifreeze proteins are also found in freeze-tolerant insects (Duman, 1979), although the adaptive function in this case is unclear. More recently, a nonprotein xylomannan glycolipid antifreeze compound was identified (Walters et al., 2009) and appears to be present in numerous insect taxa (Walters et al., 2011). For freeze-tolerant insects, it is advantageous to have a high supercooling point so that ice formation is slow and controlled. As such, a number of protein ice nucleating agents have been identified in freeze-tolerant insects; these proteins are secreted into the extracellular space to promote ice crystallization (Duman, 2001; Duman et al., 2010). Nonprotein ice nucleators are also sometimes used; for example, calcium phosphate stored in the Malpighian tubules of E. solidaginis initiates ice crystallization (Mugnano et al., 1996).

Although the above targeted studies have yielded several important candidate genes involved in diapause and seasonal cold-hardening, recent transcriptomics and proteomics experiments have revealed many additional players. Using proteomic analysis, Li et al. (2007) reported 80 proteins that are differentially regulated in brains of diapausing flesh fly pupae S. crassipalpis. A number of these are the well-known heat shock proteins, whereas additional differentially-regulated proteins are either unidentified or have unknown function. At the mRNA level, changes are even more conspicuous. In S. crassipalpis, > 50% of the entire transcriptome is differentially expressed during diapause (Ragland et al., 2010), including an abundance of stress-related proteins and metabolic genes. For example, diapausing pupae up-regulate a number of genes associated with thermal and oxidative stress, whereas metabolic adjustments supporting reduced oxygen consumption and mobilization of carbohydrates are also evident in the transcriptome. Additional studies are needed to distinguish between expression changes that are essential for enhanced stress tolerance and those associated with other aspects of diapause, such as developmental arrest. To date, the only transcriptomic study of cold acclimation per se is a candidate gene microarray (containing 219 genes) of gradual cold acclimation in D. montana and Drosophila virilis (Vesala et al., 2012b). That study detected the up-regulation of a few heat shock proteins, as well as the down-regulation of several genes involved in central metabolism. Interestingly, in both species, cold acclimation caused down-regulation of desaturase genes, despite the presumed importance of membrane desaturation during cold acclimation (Overgaard et al., 2008)

Mechanisms of rapid cold-hardening

Although the mechanisms of seasonal cold-hardening have been relatively well studied, the physiological underpinnings of RCH are not as well-established, owing to its more recent discovery. Because seasonal cold acclimation and RCH result in similar endpoints (i.e. enhanced survival and performance at low temperature), it is reasonable to suspect they share many of the same mechanisms. Thus, as a starting point, many of the mechanisms governing seasonal cold acclimation have been examined in the context of RCH. Indeed, some of the ‘usual suspects’ appear to be involved during RCH. In response to RCH, pharate adults of S. crassipalpis synthesize glycerol, increasing the concentration by almost four-fold to approximately 80 mm (Chen et al., 1987). Whereas this concentration of glycerol is modest compared with some overwintering insects, later experiments demonstrate that injection of small amounts of glycerol can cause immediate increases in cold tolerance (Yoder et al., 2006).

By contrast to the major shifts in gene expression that accompany seasonal cold-hardening (Ragland et al., 2010, 2011), there is very little evidence that RCH requires the synthesis of new gene products. Sinclair et al. (2007) measured the expression of five candidate genes during RCH and recovery from cold shock and, although some are differentially expressed during recovery from cold, none were differentially regulated during the hardening period. In D. montana and D. virilis, RCH differentially regulated 3 and 0 genes (out of 219 tested), respectively, and only one of these was verified successfully by the quantitaive polymerase chain reaction (Vesala et al., 2012b). On a larger scale, the expression of 15 000 expressed sequence tags has been measured using a cDNA microarray in the flesh fly Sarcophaga bullata (Teets et al., 2012). Although recovery from cold elicited significant changes in gene expression (approximately 10% of all genes were differentially expressed), not a single transcript was differentially expressed during the RCH period. Even more unexpectedly, RCH has very little impact on the transcript signature during recovery from subsequent cold stress. Compared with flies directly exposed to cold shock, only five transcripts are differentially expressed in flies pre-treated with RCH, and none of these differed in expression by more than 33%. Indeed, RCH can still occur when protein synthesis is blocked with cycloheximide (Misener et al., 2001), indicating that it comprises a cellular-driven process not requiring new gene products. It is suspected that very little transcription can occur at RCH temperatures (typically around 0 °C), especially within the time frame for RCH (less than 30 min in some cases).

Recent studies using metabolomics have provided further evidence that metabolic adjustments are a key component of RCH. In S. crassipalpis, metabolomics has confirmed the accumulation of glycerol during RCH and also revealed accumulation of two amino acids (alanine and glutamine), an additional polyol (sorbitol) and a sugar (glucose) (Michaud & Denlinger, 2007). Also, an accumulation of pyruvate, in conjunction with elevated glucose levels and increased synthesis of other metabolites shunted from the glycolytic pathway (e.g. glycerol and sorbitol), suggests an increased reliance on glycolysis during RCH, which may provide necessary energy and substrates for downstream physiological functions. Similar experiments in D. melanogaster indicated RCH-induced glucose accumulation, suggesting that the rapid mobilization of glucose may be a common theme during RCH (Overgaard et al., 2007). An additional consequence of RCH is to minimize metabolic perturbations during recovery from a subsequent cold shock (Overgaard et al., 2007; Teets et al., 2012). For example, in the flesh fly S. bullata, flies pre-exposed to RCH conditions had significantly lower levels of several sugars and polyols during recovery from cold shock, indicating that RCH minimizes the impact of cold shock on metabolic homeostasis (Teets et al., 2012). Whether this function is a direct mechanism of RCH or simply a consequence of reduced cell damage is unknown. As with cold acclimation, the metabolic effects of RCH appear to be highly dependent on the exact parameters of the RCH treatment. For example, although 8 h at 4 °C elevates several sugars and polyols in S. crassipalpis (Michaud & Denlinger, 2007), this pattern is not evident in the congeneric S. bullata when 2 h at 0 °C is used for the hardening treatment (Teets et al., 2012), despite both RCH-regimes resulting in a similar degree of cryoprotection (Chen et al., 1987; Teets et al., 2012). This suggests that elevation of sugars and polyols may not be essential for RCH (MacMillan et al., 2009).

As with seasonal acclimation, the cell membrane is also a target for RCH. In S. crassipalpis, RCH causes elevation of oleic acid in the cell membrane (Michaud & Denlinger, 2006), which leads to a measurable increase in membrane fluidity at low temperature (Lee et al., 2006a). Additional chemical modifications to phospholipid head groups in response to RCH may also enhance membrane fluidity (Michaud & Denlinger, 2006). Membrane restructuring also occurs during RCH in D. melanogaster, although, in this species, it is linoleic rather than oleic acid that is responsible for increased membrane fluidity (Overgaard et al., 2005). Furthermore, the rate of cooling impacts the exact composition of the cell membrane, which may in part explain why RCH is most effective within a specific range of cooling rates (Overgaard et al., 2006). Membrane cholesterol may also be important during RCH; augmentation of cholesterol in the diet not only enhances baseline cold tolerance, but also increases the capacity to undergo RCH (Shreve et al., 2007). Nonetheless, there have been cases where RCH is observed in the absence of detectable changes in membrane composition, once again calling into question the essentiality of these canonical cold-hardening pathways during RCH (MacMillan et al., 2009). In terms of membrane function, a single study has examined the effects of RCH on potassium distribution in the brain of D. melanogaster (Armstrong et al., 2012). Unexpectedly, flies exposed to RCH have larger perturbations of potassium homeostasis during chill coma, although these flies recover faster than those directly exposed to chill coma.

Isolated tissues of S. crassipalpis are capable of RCH ex vivo (Yi & Lee, 2004), and the same is true for tissues from a freeze-tolerant species Belgica antarctica (Teets et al., 2008). Although the presence of the brain enhances the RCH response (Yoder et al., 2006), the fact that the brain and neurohormones are not absolutely essential for RCH implies that cell-mediated signalling pathways are capable of inducing RCH. This was an important development because it directed focus to second messenger pathways as key mediators of RCH. To date, three signalling pathways have been implicated in the RCH response: mitogen-activated protein (MAP) kinase signalling, apoptosis signalling and calcium signalling. MAP kinases are a group of serine/threonine protein kinases that are activated by diverse cell stimuli, most notably stress and immune signals (Pearson et al., 2001). In S. crassipalpis, a specific MAP kinase, p38 MAP kinase, is phosphorylated immediately upon transfer to RCH-inducing conditions (Fujiwara & Denlinger, 2007). Activation of p38 is maximal at 0 °C (the optimal temperature for RCH) and is independent of the brain, making it a strong candidate for a mediator of RCH. In the silverleaf whitefly Bemisia tabaci, chilling similarly activates p38 MAP kinase, with activation occurring in as little as 3 min after transfer to 0 °C (Li et al., 2012). However, the upstream signals that activate p38 during RCH are unknown, as are the downstream targets and effectors.

One deleterious effect of cold shock is activation of apoptotic cell death pathways (Yi et al., 2007; Yi & Lee, 2011). Although the underlying mechanisms of cold-induced apoptosis are unknown, it may be a consequence of calcium imbalance and mitochondrial malfunction, both of which are known to trigger apoptosis in a range of animal systems (Kroemer et al., 1998). In D. melanogaster and S. crassipalpis, RCH inhibits cold-induced apoptosis (Yi et al., 2007; Yi & Lee, 2011), permitting cell survival during otherwise lethal conditions. Initially, these results appeared counterintuitive because apoptosis is considered to be a protective mechanism that safely eliminates damaged cells (Kroemer et al., 1998). However, it appears that, during cold shock, the apoptotic pathway is overactive, resulting in > 75% of cells dying via apoptosis (Yi & Lee, 2011). Thus, the inhibition of apoptosis by RCH is beneficial in this case, as a result of preventing unnecessary apoptotic cell death. The mechanism by which RCH inhibits apoptosis likely involves inhibition of caspase activity, as well as the accumulation of anti-apoptotic protein bcl-2 (Yi et al., 2007; Yi & Lee, 2011). However, as with p38, the upstream mechanisms that target the apoptosis pathway during RCH remain unidentified.

A third signalling pathway implicated in RCH is calcium signalling (Teets et al., 2008). Isolated tissue of B. antarctica loaded with either the calcium chelator 1,2-Bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA) or the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) fail to undergo RCH ex vivo. Thus, both intracellular calcium and activation of calmodulin appear to be essential for RCH. Indirect evidence for a role of calcium during cold-hardening is provided from experiments on a D. melanogaster mutant strain lacking dystroglycan, an extracellular matrix protein (Takeuchi et al., 2009). Mutants lacking functional dystroglycan have elevated levels of intracellular calcium, which leads to enhanced cold tolerance and a preference for lower temperatures. Also, transient receptor potential channels in specific neurones of larval D. melanogaster respond directly to low temperature by conducting an inward calcium current (Rosenzweig et al., 2008), although the function of these calcium channels during RCH or in peripheral tissues has not been addressed.

Based on the results described above, it is proposed that calcium signalling may be the master regulator by which cells can sense changes in temperature directly and trigger downstream cold-hardening pathways. Cold acclimation in plants, which occurs in the order of days to weeks (Thomashow, 1999), is calcium-regulated, and it is hypothesized that calcium similarly governs rapid responses to low temperature in insects. The immediate disintegration of ion gradients in response to low temperature (Koštál et al., 2004, 2006; Armstrong et al., 2012) provides a clear mode of entry for calcium in response to low temperature. Additionally, in non-insect systems, calcium is known to influence a number of physiological pathways important for stress tolerance, including stimulation of mitochondrial energy metabolism by activating mitochondrial dehydrogenase (Denton, 2009; Takeuchi et al., 2009); activation of glycogen phosphorylase kinase, which in turn activates glycogen phosphorylase and stimulates glycogenolysis and osmoprotectant mobilization (Johnson, 1992); cross-talk with MAP kinase (Takeda et al., 2004) and apoptosis pathways (Yano et al., 1998) to promote cell survival; and trafficking and/or activation of proteins such as aquaporins (Chou et al., 2000), which may need to be rapidly activated during cold exposure. Current and future efforts in the authors' group aim to clarify the role of calcium signalling during RCH, with the ultimate goal of mapping the entire pathway, beginning with the detection of cold and continuing through the downstream effectors conferring enhanced cold tolerance.

Seasonal versus rapid cold-hardening: similarities and differences

Because both seasonal cold-hardening and RCH result in dramatic increases in cold tolerance, current hypotheses suggest they share many of the same mechanisms. Indeed, studies on the role of cryoprotectants (Chen et al., 1987) and membrane modifications (Overgaard et al., 2005; Michaud & Denlinger, 2006; Overgaard et al., 2006) reflect this line of thinking. However, the degree to which seasonal and rapid cold-hardening rely on the same mechanisms is largely unknown. In the present review, similarities and differences are summarized between seasonal and rapid cold-hardening that are reasonably well-supported by the literature. Figure 1 and Table 1 summarize the major mechanisms associated with both types of cold-hardening. Despite the obvious connection between seasonal cold-hardening and RCH, few studies have compared the two directly. In all cases examined, RCH offers additional protection against low temperature stress beyond that afforded by cold acclimation alone. In D. melanogaster, Rajamohan & Sinclair (2009) reported no improvement in cold tolerance after gradual cold acclimation, whereas RCH was still effective at enhancing acute cold tolerance. With the same species, Colinet & Hoffmann (2012) demonstrated improved acute cold tolerance after both gradual acclimation and RCH. However, the survival benefits of these two acclimation regimes are additive, suggesting different mechanisms are involved. Similarly, in both the grain aphid Sitobion avenae (Powell & Bale, 2005) and eggs of the beetle Psacothea hilaris (Shintani & Ishikawa, 2007), RCH further improved cold tolerance of cold-acclimated individuals. These studies at the organismal level suggest that RCH uses either different mechanisms from cold acclimation or it provides a ‘boost’ to cold acclimation mechanisms already in place.

Table 1. Mechanisms associated with seasonal and rapid cold-hardening (RCH)
MechanismAssociated with seasonal cold-hardening?Associated with RCH?Key references
Photoperiodic inductionYesNo

Seasonal: Denlinger (1991)

RCH: Lee & Denlinger (2010)

Triggered by low temperatureYesYes

Seasonal: Salt (1961), Koštál et al. (2011a) and Colinet et al. (2012)

RCH: Lee et al. (1987) and Lee & Denlinger (2010)

Cryoprotectant synthesisYesAmbiguous

Seasonal: Salt (1961), Storey & Storey (1991), Koštál et al. (2011a) and Colinet et al. (2012);

RCH: Lee et al. (1987), Michaud & Denlinger (2007) and Overgaard et al. (2007); c.f. MacMillan et al. (2009); c.f. Teets et al. (2012)

Membrane restructuringYesYes, with one exception

Seasonal: Koštál & Šimek (1998), Koštál et al. (2003), Michaud & Denlinger (2006), Overgaard et al. (2006) and Overgaard et al. (2008);

RCH: Overgaard et al. (2005), Michaud & Denlinger (2006) and Lee et al. (2006a); c.f. MacMillan et al. (2009)

Adjustments to ion transport mechanismsYesYes

Seasonal: Koštál et al. (2004, 2006)

RCH: Armstrong et al. (2012)

Up-regulation of stress-related genes (e.g. heat shock proteins)YesNo

Seasonal: Rinehart et al. (2007), Ragland et al. (2010) and Vesala et al. (2012b)

RCH: Sinclair (2007), Teets et al. (2012) and Vesala et al. (2012b)

Synthesis of antifreeze proteins and/or ice nucleating agentsYesUnknown but unlikely (given lack of gene expression observed during RCH)Seasonal: Duman (1979), Mugnano et al. (1996), Duman (2001) and Walters et al. (2009);
Inhibition of apoptotic cell deathUnknownYesRCH: Yi et al. (2007) and Yi & Lee (2011)
MAP kinase signallingUnknownYesRCH: Fujiwara & Denlinger (2007) and Li et al. (2012)
Calcium signallingUnknownYesRCH: Teets et al. (2008)

The additive effects of RCH and cold acclimation on acute cold tolerance suggest different underlying mechanisms. In addition, the effects of these acclimation regimes depend on the metric of cold tolerance used. Both seasonal cold-hardening and RCH improve tolerance of acute cold shock (see above), although RCH does not appear to protect against indirect chilling injury (Lee & Denlinger, 2010). For example, in eggs of P. hilaris, RCH improves survival after brief cold-shock near the supercooling point but fails to enhance survival after longer exposures to milder sub-zero conditions (Shintani & Ishikawa, 2007). Furthermore, only cold acclimation (and not RCH) appears to influence chill coma recovery time (Rako & Hoffmann, 2006). Thus, although cold acclimation generally improves every metric of cold tolerance (Rako & Hoffmann, 2006; Koštál et al., 2011a; Colinet & Hoffmann, 2012), RCH appears to offer specific protection against cold shock injury, with minimal impact on other metrics of cold tolerance.

One clear difference between seasonal cold-hardening and RCH is the environmental cues that initiate these responses. Seasonal cold-hardening is often associated with diapause, which is typically triggered by photoperiod (Denlinger, 1991). Although environmental temperature can modify the cold tolerance of diapausing insects, in many cases, photoperiodic diapause enhances cold tolerance irrespective of temperature (Lee & Denlinger, 1985). By contrast, RCH is typically triggered by low temperature alone (Lee & Denlinger, 2010). In the laboratory, this is usually accomplished by stepwise transfer to RCH conditions, although an increasing number of studies are using ecologically-relevant thermoperiods and cooling rates to induce RCH (Kelty & Lee, 2001; Powell & Bale, 2005). Also, the induction temperatures for RCH are much lower than those required for cold acclimation. Cold acclimation often occurs at temperatures that permit growth and reproduction, whereas RCH typically occurs at approximately 0 °C (Lee & Denlinger, 2010), at temperatures well below the developmental threshold of most species. Although low temperature is the most common (and most ecologically relevant) cue for RCH, in some instances, brief conditioning to other types of environmental stress can induce RCH. For example, RCH can be induced by heat shock in flesh flies (Chen et al., 1987), anoxia in house flies (Coulson & Bale, 1991) and brief desiccation in goldenrod gall flies (Levis et al., 2012).

At the metabolite level, there is evidence that both seasonal cold-hardening and RCH involve the synthesis of cryoprotectants. Specifically, in S. crassipalpis, both diapause and RCH increase the concentrations of three cryoprotectants, alanine, glycerol, and glucose (Michaud & Denlinger, 2007). Also, in D. melanogaster, there is evidence that trehalose is involved in both cold acclimation (Koštál et al., 2011a; Colinet et al., 2012) and RCH (Overgaard et al., 2007). However, as discussed above, there is discrepancy regarding whether cryoprotectant synthesis is an essential component of RCH. For example, within the flesh flies, RCH in S. crassipalpis involves the synthesis of a modest level of glycerol (Lee et al., 1987; Michaud & Denlinger, 2007), whereas metabolomics experiments in closely-related S. bullata, which has an equally robust RCH response, failed to detect cryoprotectant synthesis (Teets et al., 2012). Similarly, in D. melanogaster, RCH has been reported both in the presence (Overgaard et al., 2007) and absence (Kelty & Lee, 2001; MacMillan et al., 2009) of carbohydrate synthesis. Furthermore, the concentration of cryoprotectants accumulated during RCH is typically much lower than the accumulation found in overwintering insects. Thus, it appears that cryoprotectant synthesis during RCH may be species and context specific, and not a general feature of RCH.

By contrast to cryoprotectant mobilization, there is fairly conclusive evidence that both seasonal cold-hardening and RCH involve cell membrane modifications. In S. crassipalpis, both diapause and RCH increase the proportion of oleic acid in the cell membrane to a similar degree, although the effects of diapause and RCH are not additive (Michaud & Denlinger, 2006). Similarly, in D. melanogaster, long-term acclimation and RCH both increase membrane desaturation by elevating the levels of linoleic acid (Overgaard et al., 2005, 2008). However, RCH in D. melanogaster has been observed in the absence of membrane modifications (MacMillan et al., 2009), calling into question the essentiality of this process.

Perhaps the most significant mechanistic difference between seasonal cold-hardening and RCH is the role of gene expression. Although diapause and seasonal cold acclimation appear to involve dramatic changes in the transcriptome, RCH is primarily a cell-mediated event with little or no reliance on gene transcription. In the only gene expression study examining cold acclimation and RCH simultaneously, Vesala et al. (2012b) observed the differential expression of 14 and 18 genes (out of 219 measured) after cold acclimation in D. montana and D. virilis, respectively. However, in the same two species, only one gene was confirmed to be differentially expressed after RCH. In flesh flies, whereas diapause results in differential expression of >50% of the transcriptome (Ragland et al., 2010), RCH does not change the abundance of any transcripts (Teets et al., 2012). Thus, despite activating cell signalling pathways such as calcium signalling (Teets et al., 2008) and MAP kinase signalling (Fujiwara & Denlinger, 2007), RCH appears to have little impact on gene expression, both during the hardening period and during recovery from a subsequent cold shock.

Future directions

With advances in molecular biology and the ‘omics’ revolution, there has been an vast increase of studies in the last decade addressing the mechanisms of cold hardening. Whereas recent efforts have expanded the molecular toolkits for cold-hardy species (Hahn et al., 2009), much of what is known is based on research in D. melanogaster. Drosophila melanogaster is a great genetic model, although surviving at low temperature is not its strong suit. Thus, additional large-scale molecular studies in cold-hardy species are needed to grasp fully the mechanisms of cold-hardening across all insect taxa. Furthermore, studies that combine two or more ‘omics’ approaches (e.g. transcriptomics and metabolomics, see Teets et al., (2012)] are useful for deciphering which changes in gene expression are well-correlated with changes in phenotype. Also, additional functional studies are needed to assess which mechanisms and pathways are essential for cold-hardening and which merely correlate with enhanced cold tolerance.

In addition to shifting the focus to non-Drosophila species, future experiments should address seasonal cold-hardening and RCH simultaneously. Although cold acclimation and RCH have been nicely compared in terms of their effects on acute cold tolerance (Powell & Bale, 2005; Shintani & Ishikawa, 2007; Rajamohan & Sinclair, 2009), very few studies have directly compared the underlying physiological mechanisms. Both seasonal cold-hardening and RCH have clear ecological relevance, although the degree to which they rely on the same mechanisms is largely unknown. Part of the issue is that very little is known about the mechanisms conferring cold tolerance during RCH. Canonical cold-hardening mechanisms, such as membrane modifications, cryoprotectant synthesis and heat shock protein expression, are either not associated with RCH, or evidence for their involvement in RCH is inconclusive. Also, given the speed at which RCH occurs, it is difficult to imagine that RCH involves the same type of large-scale physiological overhaul as seen in seasonal cold-hardening. Rather, it is hypothesized that RCH is driven primarily by protein phosphorylation and other signalling events. For example, whereas heat shock proteins are not synthesized during RCH, they may still be involved. In humans, stress activation of MAP kinase signalling pathways phosphorylates small heat shock proteins, which influences their distribution and function within the cell (Landry et al., 1992). It is suspected that signalling events such as these that alter the cellular location and activities of proteins are the main drivers of RCH.

Understanding the limits of cold tolerance is essential for predicting the potential range of insects, particularly in a changing climate. Seasonal cold-hardening mechanisms, such as diapause and cold acclimation, allow insects to prepare for predictable decreases in daily temperature, whereas RCH allows insects to track daily fluctuations in temperature and cope with sudden cold snaps. It was initially assumed that RCH uses the same mechanisms as seasonal cold-hardening, albeit on a compressed time scale. However, the growing literature suggests this is not the case. A challenge moving forward is to determine whether seasonal cold-hardening and RCH are derived from a general, conserved response to low temperature or whether they are distinct adaptations that have arisen independently.

Acknowledgements

This work is supported by NSF grant IOS-0840772. We are grateful to two anonymous reviewers for their insightful comments.

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