Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately −5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.
We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt50) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt50 (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.
Citation: Koštál V, Korbelová J, Rozsypal J, Zahradníčková H, Cimlová J, Tomčala A, et al. (2011) Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster. PLoS ONE 6(9): e25025. https://doi.org/10.1371/journal.pone.0025025
Editor: Amit Singh, University of Dayton, United States of America
Received: June 27, 2011; Accepted: August 22, 2011; Published: September 21, 2011
Copyright: © 2011 Kostal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Czech Science Foundation grants 206/07/0269 and 203/09/2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Genus Drosophila comprises almost 1500 described species and is thought to be of tropical origin . The ancestral level of cold tolerance in this genus is suggested to be relatively low , and most recent species are chill susceptible . This is also true for a common model of modern biology, the fruit fly Drosophila (Sophophora) melanogaster. Its pre-adult development halts at temperatures below approximately 10°C , . The adults and pupae die when chilled to temperatures below −5°C even for just 2 h, and the larvae are even more chill susceptible . The following are the reasons for studying cold tolerance in this and other such non-cold-hardy organisms. First, the accumulated knowledge about D. melanogaster biology makes it a promising model to investigate detailed mechanisms at the suborganismal level. Second, different drosophilid species widely differ in their cold tolerance , . For instance, the larva of the temperate/subarctic drosophilid Chymomyza costata represents one of the most cold-hardy organisms on the earth as it survives cooling to a temperature as low as that of liquid nitrogen (−196°C) , . Third, the level of cold tolerance is a subject of significant phenotypic plasticity –. Thus, both short-term cold hardening and long-term cold acclimation considerably improve cold tolerance of D. melanogaster , –. Fourth, knowledge on cold tolerance may help in the development of techniques for long-term storage and/or cryopreservation of Drosophila strains for research and industry .
Previous studies on the cold tolerance of D. melanogaster focused mainly on the cold shock responses. Cold shock occurs on rapid cooling without ice formation  and induces a specific type of chilling injury (which will be described later). Thus, most previous studies assessed the lower lethal temperatures (LLTs) for relatively brief exposures (hours) to relatively severe cold (subzero temperatures close to the supercooling point [SCP]). In addition, many studies have investigated the influence of rapid cold hardening (RCH) on LLTs and cold shock survival , –, –. RCH is a quick cold acclimation response that occurs within minutes to hours of pre-exposure to a non-lethal low temperature and improves survival after subsequent cold shock . Cold shock response is just one aspect of cold tolerance, however. The injuries caused by low temperatures are likely numerous, heterogenous, and complex. The three basic types of cold injury generally distinguished  are freezing of body water, direct chilling injury, and indirect chilling injury. Freezing of body water may result in mechanical damage to delicate intracellular ultrastructures and the extracellular matrix. It also causes cellular dehydration and freeze concentration of solutes, likely causing them to reach toxic concentration levels . Direct chilling injury results from cold shock, which may cause dissociation of multimeric macromolecular complexes, protein denaturation , , membrane lipid phase transitions, massive ion leakage, and cell death , . Indirect chilling injuries accumulate over relatively long exposures (days to months) to relatively mild cold (temperatures well above the SCP, often around or above zero). Disturbed coordination of various metabolic pathways (disorder), excess production of reactive molecules (oxidative stress), disturbance of ion homeostasis, or depletion of available free chemical energy (basically adenosine triphosphate [ATP]) are the most likely causes of indirect chilling injury –.
In contrast to the wealth of studies on cold shock responses of D. melanogaster, our knowledge on how it copes with indirect chilling injuries and what is the role of long-term cold acclimation is considerably less. Previous studies have indicated that the ability of the third-instar larvae of D. melanogaster to survive at 0°C is very low. For instance, a study  reported less than 20% survival after 2-h-long exposure to 0°C. It is believed that long-term cold acclimation in insects is based, at least partly, on the accumulation of cryoprotective solutes and restructuring of biological membranes , –. Cold acclimation of the larvae of C. costata is associated with extensive remodeling of the lipid composition of their membranes  and with the accumulation of high amounts of proline, which is directly associated with the ability to survive freezing and cryopreservation in liquid nitrogen , .
The main objective of this study was to examine the influence of long-term cold acclimation on the development of indirect chilling injuries (mortality) in the third-instar larvae of D. melanogaster. The larvae were acclimated by rearing at different constant temperatures of 25°C, 15°C, and 15°C followed by 6°C. Survival tests were performed at constant 0°C. Third-instar larvae were selected in order to obtain the comparative data for C. costata, which attains its maximum cold tolerance (survival in liquid nitrogen) in this developmental stage . We tested two related hypotheses that long-term cold acclimation of D. melanogaster larvae (a) improves their cold tolerance (extends Lt50 at 0°C) and leads to the development of the ability to survive freezing injury and (b) modifies the metabolomic profiles of organic acids, free amino acids, free fatty acids, sugars, and polyols and stimulates restructuring of biological membranes.
Insect rearing and acclimation
The laboratory stock of fruit flies, Drosophila (Sophophora) melanogaster (Meigen, 1830), strain Oregon, was maintained at constant 18°C with 12-h/12-h light/dark (L/D) cycle on a diet containing agar (1%), sugar (5%), yeast (4%), cornmeal (8%), and methylparaben (0.2%). For experiments, approximately 30 female flies that were 5- to 10-d-old were allowed to lay eggs in a vial (40 mL) containing 5 mL of standard diet, for 24 h (one L/D cycle). Thereafter, the flies were removed from the vial, and the embryos and larvae developing in the diet were raised under the following three different acclimation treatment conditions.
- 25°C acclimation—rearing at constant 25°C with 12-h/12-h L/D cycle for 5 d
- 15°C acclimation—rearing at constant 15°C with 12-h/12-h L/D cycle for 14 d
- 15°C→6°C acclimation—rearing at constant 15°C with 12-h/12-h L/D cycle for 14 d, followed by rearing at constant 6°C and continuous darkness for 2 d.
We sampled pre-wandering larvae of similar physiological age (but different ages in days) in all the treatments. At the end of an acclimation treatment, when the first wandering larvae occurred, the largest specimens of third-instar larvae were collected from the diet and subjected to survival experiments or processed for biochemical analyses. To avoid thermal stress, the larvae were washed out of the diet by using water of the same temperature as that in the respective treatment.
Survival at low temperatures
For survival experiments, groups of approximately 20 pre-wandering larvae were placed in 1 g of larval diet in a plastic tube (diameter, 1 cm; length, 5 cm), which was plugged with artificial cotton. The larvae were then exposed to either (a) constant 5°C or 6°C (maintained in a programmable thermostat, F32-ME; Julabo, Seelbach, Germany); or (b) constant 0°C (maintained in melting ice); or (c) a temperature program (set in the programmable thermostat F32-ME in combination with a temperature probe, TC-08; Pico Technology, St. Neots, United Kingdom). The temperature program started at −1°C and comprised three steps: (i) cooling to −2°C at a rate of 0.033°C min−1 (30 min); (ii) cooling to −5°C at a rate of 0.1°C min−1 (30 min); and (iii) heating to +5°C at a rate of 0.33°C min−1 (30 min). Thus, the larvae spent a total of 75 min at subzero temperatures. At the start of the program, a small ice crystal was either added on the surface of diet (freezing condition) or not added (supercooling condition). Adding the ice crystal resulted in an almost immediate freezing of water in the diet and, probably, ice inoculation and freezing of larval body fluids. When no ice was added, the diet did not freeze (no freeze exotherm observed), and the larvae probably supercooled. After cold exposure, the tubes with the larvae were kept at 18°C with 12-h/12-h L/D cycle for 1 week, and pupariation was scored as a criterion of survival.
Individual fresh mass (FM) of 20 larvae in each acclimation treatment was measured using a Sartorius electronic balance (precision, 0.01 mg). The weighed specimens were dried at 60°C for 3 d, and their dry mass (DM) was measured. Water mass (WM, in mg) and hydration (H, in mg; water·mg−1 DM) were calculated from the gravimetric data.
A total of 5 larvae in 4 replications were processed from each acclimation treatment to determine the basic biochemical parameters. Total proteins were measured by the bicinchoninic acid protein assay  after extraction of total water-soluble proteins by using 50 mM Tris, pH 6.8, followed by re-extraction of detergent-soluble proteins from a centrifugation pellet in the same buffer by the addition of 0.5% deoxycholate and 0.1% sodium dodecyl sulfate (SDS). Total lipid content was measured by spectrophotometric analysis with phosphoric acid-vanillin solution  after extraction of lipids by using chloroform∶methanol solution (2∶1, v/v) . Glycogen content was measured by colorimetric determination by using phenol and concentrated sulfuric acid  after extraction of glycogen in hot alkali .
SCP refers to the temperature at which spontaneous freezing of body water occurs during gradual cooling of insect specimen. A constant cooling rate of 0.2°C min−1 was used and the minimum temperature was set to −30°C. The individual larvae were attached to thermocouples (type K, Pico Technology) using thermally conductive paste Cooler Master HTK-002 (Sundial Micro, Ontario, CA, USA).We measured the SCP of 16 larvae in each acclimation treatment by recording the exotherms associated with water-ice phase transition, using the programmable thermostat F32-ME (Julabo) in combination with the temperature data logger TC-08 (Pico Technology).
The metabolomic profiles were extensively investigated by a set of targeted and nontargeted mass spectrometry (MS)-based analytical methods. Whole larvae (10 larvae in 4 replications from each acclimation treatment) were homogenized and extracted in 70% ethanol. Low-molecular-weight sugars and polyols were quantitatively determined in the ethanolic extracts after o-methyloxime trimethylsilyl derivatization and subsequent analysis by gas chromatography (GC) coupled to MS (GC/MS), as described previously . Nontargeted metabolomic profiling was done by performing a combination of GC/MS and liquid chromatography (LC) coupled to MS (LC/MS) in the same ethanolic extracts after their treatment with ethyl chloroformate under pyridine catalysis and simultaneous extraction in chloroform , .
GC/MS metabolite profiles were obtained on a VF-17 capillary column (Agilent, Santa Clara, CA, USA) coupled to a dual-stage quadrupole (DSQ) mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an electron impaction ion source and operated in the full-scan mode from 40 to 500 amu. A Thermo Trace gas chromatograph with a programmable injector and interface hold at 230°C was directly coupled to the mass spectrometer via an interface held at 250°C. A 0.5-µL aliquot of the chloroform extract was injected in the splitless mode into the GC/MS column. Oven temperature was initially maintained at 50°C for 1 min. Thereafter, it was raised to 302°C at a rate of 12°C min−1 and maintained for 2 min. Helium was used as the carrier gas and delivered at a constant flow rate of 1.2 mL min−1.
LC/MS metabolite profiles were measured after evaporating a 30-µL aliquot of the chloroform extract to dryness by using a mild stream of nitrogen. After dissolution in 200 µL of the LC mobile phase, a 5-µL aliquot was injected into and separated on a Kinetex C18 column (150×2 mm; internal diameter [ID], 2.6 µm; Phenomenex, Torrance, CA, USA) at 35°C at a flow rate of 200 µL min−1, using a gradient elution with the mobile phase consisting of (A) 5 mM ammonium formate in methanol and (B) 5 mM ammonium formate in water. The gradient elution program was linear from 30% to 100% A for 12 min, then held at 100% A for14 min, and finally equilibrated for 5 min.
The nontargeted GC/MS and LC/MS data were processed with the Thermo Scientific Xcalibur 2.1 software and an in-house developed Metabolite Mapper platform, which provides automated peak detection and metabolite deconvolution by employing retention time and mass spectral and detector response features, followed by time alignment of the data obtained in each particular analysis for a defined experimental sample set and generation of data matrix, which is automatically exported to a predefined Microsoft Excel™ spreadsheet for further statistical processing. The 39 major metabolites were identified against relevant standards and further subjected to quantitative analysis by using an internal standard calibration method. All chemicals used were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), except the isotope-labeled metabolites used as internal calibration standards, which were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Whole-body concentrations of the metabolites were recalculated as mmol·L−1 of whole body water (mM).
Whole larvae (5 larvae in 8 replications from each acclimation treatment) were homogenized and extracted in ice-cold chloroform∶methanol solution (2∶1, v/v) by using a previously described method , . After extraction, the solvents were evaporated under a stream of nitrogen, and lipids were stored at −80°C until analysis. High-performance LC (HPLC) combined with electrospray ionization MS (ESI-MS) ,  was performed on an LC quadrupole (LCQ) ion-trap mass spectrometer (Thermo Fisher Scientific) coupled to a Rheos 2000 ternary HPLC system (Flux Instruments, Basel, Switzerland) equipped with a FAMOS autosampler and Thermos thermostat. The stored dry samples were dissolved in 1 mL of methanol, and 5-µL aliquots were injected into a Synergi Polar HPLC column (150×2 mm; ID, 3.5 µm; Phenomenex). The mobile phase consisted of (A) 10 mM ammonium acetate in methanol, (B) 10 mM ammonium acetate in water, and (C) isopropanol. A linear gradient of A∶B∶C changing from 90∶10∶0 to 70∶0∶30 within 14 min was applied with a flow rate of 300 µL min−1. The column temperature was maintained at 30°C. The mass spectrometer was operated either in the positive or the negative ion detection mode at +4 kV or −3.6 kV, respectively. Capillary temperature was 240°C, and nitrogen was used as both the sheath and the auxiliary gas. For MS2 and MS3 fragmentations, ion isolation windows were 5 Da and 2 Da, respectively. The maximum ion injection time was 100 ms; collision energies were 30% (MS2) or 35% (MS3); and mass range of 600 Da to 800 Da was scanned every 0.5 s. The basic chemicals used for extraction and analysis were purchased from Sigma-Aldrich Co. Calibration standards were obtained from Avanti Polar Lipids (Alabaster, AL, USA).
The GPL analysis results were expressed in relative values, i.e., relative proportion of each GPL species from the total of 100%. Our analysis was focused on the major glycerophosphoethanolamines (GPEtns), glycerophosphocholines (GPChols), and glycerophosphoserines (GPSers). The relative proportions of individual fatty acyls (FAs) were calculated from the GPL data.
Survival data (proportions of pupariated larvae) were fitted to exponential decay curves, using the following formula.where Top is survival in a control (untreated) group of larvae and K denotes the slope of decay. The K parameters in the different acclimation treatments were compared using the F-test. The time of exposure to low temperature that was lethal for 50% of the larvae in a sample (Lt50) was calculated from the exponential curves.
One-way analysis of variance (ANOVA) tests were used to analyze whether the acclimation treatments influenced the physiological and biochemical parameters. Bonferroni post-hoc tests were applied to identify the differences among the acclimation treatments. The analyses were performed using Prism v. 4 (GraphPad Software, San Diego, CA, USA).
The complex association of metabolomic changes with acclimation treatments was determined by principal component analysis (PCA) with Canoco v. 4.52 for Windows (Biometris-Plant Research International, Wageningen, The Netherlands).
Long-term cold acclimation significantly improved survival at low temperatures
We observed a clear acclimation effect of relatively low rearing temperature of 15°C on subsequent survival at low temperatures in the third-instar larvae of D. melanogaster. While Lt50 at 0°C was as short as 0.137 h (8 min 13 s) for the larvae acclimated at 25°C, it increased to 3.165 h (3 h 9 min 54 s) for those acclimated at 15°C. Survival of the 15°C-acclimated larvae further improved on exposure to 6°C for 2 d. After this treatment, Lt50 at 0°C increased to 86.658 h (3 d 14 h 39 min 29 s) (Fig. 1). The K parameters of the survival curves differed significantly among the three acclimation treatments (F-test: F = 154.0, degrees of freedom of the numerator [dfN] = 2, degrees of freedom of the denominator [dfD] = 15; P<0.0001). We used the temperature of 6°C for the acclimation treatment because this was the lowest temperature at which no significant mortality was observed within the 2 d-long exposure of the 15°C-acclimated larvae. In contrast, Lt50 was as short as 32.356 h at 5°C (Fig. 1, inset).
The three acclimation treatments (25°C, 15°C, and 15°C→6°C) are explained in the text. Each point shows the mean ± standard error of mean (SEM) of survival at 0°C in a group of approximately 20 larvae (replications, 3–8; total, n). Survival data were fitted to exponential decay curves. The inset shows the survival of 15°C-acclimated larvae at two different temperatures, 6°C and 5°C.
The larvae did not show survival ability in conditions favorable for external ice inoculation and partial freezing of their body fluids. None of the larvae survived cooling to −5°C when freezing of the surrounding diet was stimulated by adding a small ice crystal. In contrast, relatively high proportions of the larvae survived cooling to −5°C under the supercooling condition. However, acclimation at 15°C or 15°C→6°C was a prerequisite for their survival in supercooled state (Table 1).
Physiological and biochemical changes associated with cold acclimation
Although the physiological ages of the larvae reared at 25°C and those reared at 15°C were similar (the FM of the larvae was consistently within 85%–95% of the “final” FM of the wandering larvae in the respective treatment), the latter grew bigger and had larger reserves of total lipids than the former (see Dataset S1 for detailed results and statistical analysis). Hydration, total protein content, and glycogen levels were similar in the larvae in the two acclimation treatments. Further acclimation of the 15°C-reared larvae at 6°C for 2 d resulted in significant reduction in FM, DM, and glycogen levels, while hydration, total protein content, and total lipid content remained constant. SCPs of the larvae in all the three acclimation treatments were similar and relatively low (means ranged between −19.9°C and −20.2°C; Dataset S1).
We investigated the changes in the profiles of 39 major metabolites that were present in detectable amounts in most samples and the identities of which were verified by MS. The concentrations of approximately two-thirds of the metabolites were significantly influenced by the acclimation treatments (see Dataset S2 for a complete list of metabolites and statistical analyses). PCA identified a group of metabolites that showed an increase in concentration during the cold acclimation process (Fig. 2). This group included citrate (no. 4), alpha-aminobutyric acid (no. 7), proline (no. 14), asparagine (no. 15), glutamate (no. 18), tryptophan (no. 26), putrescine (no. 28), and trehalose (no. 39) (the numbers in parentheses correspond to the numbers shown in Fig. 2; more details in Dataset S2). The details of the five most abundant metabolites in this group are shown in Fig. 3. The sum concentration of sugars plus polyols and that of free amino acids increased during cold acclimation, while the sum concentration of detectable organic acids decreased. The sum concentration of free fatty acids remained unchanged (Fig. 2).
Red point, 25°C; green point, 15°C; blue point, 15°C→6°C. The numbers (metabolites) are partially decoded in the text and completely listed in Dataset S2. PC1 axis explains 47.0% of the variation and PC2 axis explains additional 31.5% of the variation (Monte Carlo test: F = 20.508; P = 0.0010). The eigenvectors extending beyond the inner and outer dashed circles represent the compounds that fit the model by more than 60% and 90%, respectively. OA organic acids; FFA, free fatty acids; FAA, free amino acids; PolyOH, sugars and polyols.
Each column represents the mean ± standard deviation (SD) of 4 independent replications (10 larvae each). Influence of acclimation treatment on metabolite concentration was tested by ANOVA followed by Bonferroni post-hoc test (means indicated with different letters are significantly different). Pro, proline; Asn, asparagine; His, histidine; Glu, glutamate.
We identified 41 different GPLs (list and statistical analysis in Dataset S3). Most of the GPL species were present in relatively small proportions, not exceeding 1% of the total GPL pool. Almost 50% of the GPL species exhibited statistically significant acclimation-related changes (Dataset S3). PCA identified two GPEtns, GPEtn 16∶0/18∶2 (no. 18) and GPEtn 18∶1/18∶2 (no. 21), which showed a close association with the most cold-hardy group of larvae (Fig. 4). Detailed results for these two compounds are shown in Fig. 5, which also illustrates some of the changes in parameters calculated from the GPL composition data. The relative proportion of total GPEtns was slightly lower in the 15°C-acclimated larvae than in the 25°C-acclimated larvae, but it significantly increased (from 57.0% to 65.2%) in the 15°C→6°C-acclimated larvae. The relative proportions of total unsaturated FAs and total 18-carbon FAs did not change significantly with cold acclimation (Fig. 5).
Red point, 25°C; green point, 15°C; blue point, 15°C→6°C. The numbers (glycerophospholipids) are partially decoded in the text and completely listed in Dataset S3. PC1 axis explains 32.3% of the variation and PC2 axis explains additional 31.3% of the variation (Monte Carlo test: F = 8.471; P = 0.0020). The eigenvectors extending beyond the inner and outer dashed circles represent the compounds that fit the model by more than 60% and 90%, respectively. GPEtns, glycerophosphoethanolamines; GPChols, glycerophosphocholines; GPSers, glycerophosphoserines; UFAs, unsaturated fatty acyls; 18C FAs, 18-carbon fatty acyls.
Each column represents the mean ± SD of 4 independent replications (5 larvae each). The influence of acclimation treatment on relative proportions was tested by ANOVA, followed by Bonferroni post-hoc test when significant influence was detected (means indicated with different letters are significantly different). GPEtn 16∶0/18∶2, 1-palmityl-2-linoleyl-sn-glycerophosphoethanolamine; GPEtn 18∶1/18∶2, 1-oleyl-2-linoleyl-sn-glycerophosphoethanolamine; UFAs, unsaturated fatty acyls; 18C FAs, 18-carbon fatty acyls.
We found that long-term cold acclimation considerably improved cold tolerance in the third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the survival time (or Lt50) at constant 0°C as much as 630-fold (from 0.137 h to 86.658 h) (Fig. 1). Our data indicate that LLT was also shifted by cold acclimation, although this was not focused upon in our study. About 50% of the larvae survived supercooling to −5°C for a brief period when acclimated at 15°C→6°C, while none of the larvae survived the same treatment when acclimated at 25°C (Table 1).
Nature of cold injury and effect of cold acclimation
It is not easy to directly compare our results with those of other studies on cold tolerance of D. melanogaster because, as pointed earlier , various authors have used at least 27 different tests of cold tolerance and assessed different metrics of response (survival, chill coma onset or recovery, reproductive success). Most previous studies have dealt with cold shock situation, and one clear commonality can be derived: LLT is close to −5°C and is relatively fixed , –, . This means that relatively low variability or plasticity, spanning a range of only a few degrees, has been observed for LLT at the evolutionary or physiological level, respectively. Thus, LLT is similar in closely related Drosophila species  in different populations of D. melanogaster ,  and in different generations of experiments on the selection for cold shock tolerance (low adaptive evolutionary variability) , , . Similarly, both RCH and long-term cold acclimation have shown relatively weak effects on LLT, shifting it by only a few degrees at the most (low physiological plasticity associated with acclimation) –.
In contrast to the relatively fixed LLT, Lt50 at 0°C has shown dramatic plasticity in response to long-term cold acclimation in D. melanogaster larvae (this study) and adults . LLT suitably describes the effect of cold shock, while Lt50 is much better suited for describing the effect of indirect chilling injury. This is because the physiological bases of cold shock injury and indirect chilling injury are likely different , , , . Acute direct chilling injury during cold shock is probably related mainly to protein denaturation and membrane lipid phase transitions –. Acclimation mechanisms (associated with both RCH and long-term cold acclimation) apparently have only a limited ability to prevent/repair this type of injury, i.e., to shift the LLT by more than a few degrees. On the other hand, chronic (cumulative) indirect chilling injury is probably related to the inability to maintain homeostatic processes, which results in a lack of free chemical energy, metabolic disorder, oxidative stress, and disturbance of ion homeostasis –. The deleterious effects of this type of injury can relatively be well prevented by acclimation processes, especially long-term cold acclimation. This is why Lt50 shows such marked plasticity. Long-term cold acclimation involves numerous adjustment processes that require considerable time to be executed properly, such as gradual cessation of cold-sensitive processes (cell cycle, morphogenesis, development, reproduction) , regulated metabolic suppression , bolstering the antioxidative potential , accumulation of cryoprotective solutes , synthesis of specific proteins with antifreeze or ice nucleation activity –, membrane restructuring , stimulation of protein stabilization/refolding machinery , , and channel arrest , . In contrast, the physiological nature of acclimation processes beyond RCH remains largely unknown, but these processes may involve accumulation of cryoprotective solutes such as glycerol, rapid membrane restructuring, and stimulation of repair mechanisms , , .
Meanings of upper limit of cold injury zone and SCP
Theoretical modeling and fitting of experimental data for various insects have revealed that an upper threshold temperature may exist for indirect chilling injury. The lowest temperature that causes no significant mortality during prolonged exposure to cold is referred to as upper limit of cold injury zone (ULCIZ) , . We did not collect sufficient data in our study to estimate the value of ULCIZ precisely. The Lt50 of the 15°C-acclimated D. melanogaster larvae was 3.2 h at 0°C, extended to 32.4 h at 5°C, and further to >48 h at 6°C (Fig. 1). We suppose, however, that cold-related mortality occurs even above 6°C. The lower threshold temperature for larval development (LDT) is approximately 10°C , . Hence, at temperatures below 10°C, the larvae cannot continue to develop and become fated to die. If we accept that the main cause of their death is the inability to maintain the balance between energy demand and supply and thus support other homeostatic functions that depend on access to free energy, then the death is due to accumulation of typical indirect chilling injuries. This suggests that ULCIZ and LDT are, in fact, the same thresholds in D. melanogaster larvae. This may be a rule for other chill-susceptible, quiescent insects. On the other hand, the concept of ULCIZ is useful for chill-tolerant, diapausing insects. Rich empirical knowledge of many researchers shows that these insects can be stored for very long periods (months to years) at low temperatures (typically around 0°C) without much loss of viability, i.e., above ULCIZ .
Is it theoretically possible to identify a lower threshold temperature for indirect chilling injury, analogous to ULCIZ? In experimental setting, it would mean to identify a threshold temperature at which acute direct chilling injuries start to occur and prevail over the chronic effects of indirect chilling injuries. Perhaps, −2°C can be close to such a threshold temperature for D. melanogaster, as some experiments have indicated that survival of various developmental stages rapidly declines when exposure temperature drops below −2°C , .
Another threshold temperature, SCP, can be measured easily and precisely. In our experiments, it was close to −20°C in all the acclimation treatments. The ecophysiological meaning of SCP, however, is limited in D. melanogaster larvae, as in the other chill-susceptible insects . SCP represents the temperature at which ice crystallization of body fluids occurs when larvae are in a dry environment, i.e., without any surrounding ice. External ice crystals may stimulate freezing of body fluids at temperatures close to 0°C (inoculation by external ice). In fact, such inoculation is a prerequisite of freeze tolerance in the larvae of C. costata and some other insects . Therefore, we tested whether our acclimation treatments could stimulate development of freeze tolerance in D. melanogaster larvae. None of the larvae survived in our freezing tests despite that the conditions were relatively mild (slow cooling and melting rates, inoculation by external ice at −1°C, minimum temperature of −5°C, and total 75 min spent at subzero temperatures) (Table 1). We have no direct evidence that the mortality observed in the freezing tests was indeed caused by freezing injuries. Nevertheless, we consider it highly probable because the larvae survived the same temperature program under the supercooling condition (i.e., without ice nucleation). Our data are supported by previous studies, in which no ability to tolerate freezing injury has been observed in the larvae of 22 different species of Drosophila .
Metabolomic profiling and long-term cold acclimation
Almost two-thirds of the 39 major metabolites identified in this study exhibited statistically significant concentration changes in response to long-term cold acclimation. Most of the changes, however, were relatively small and rarely reached a several-fold magnitude. For instance, the concentration of putrescine in the 15°C→6°C-acclimated larvae was approximately 4.5-fold that in the 25°C-acclimated larvae. This difference was approximately 3-fold for the concentrations of the amino acids asparagine and histidine. Most metabolites were present in relatively low concentrations (<10 mM), which makes their effective contribution to cryoprotective functions unlikely.
Two compounds, trehalose and proline, were present in relatively high amounts (>10 mM) and showed a positive association with increasing cold acclimation (Figs. 2 and 3). Both these compounds have received much attention in previous studies as they belong to a group of compatible solutes, i.e., they are accumulated in relatively high concentrations in variously stressed organisms and play different protective roles in these organisms –. Previous studies have observed accumulations up to several hundred mmol·L−1 in many overwintering insects, for both trehalose  and proline –. When accumulated in relatively high amounts (which is not the case of D. melanogaster larvae), these compounds can colligatively contribute to the extension and stabilization of supercooling  or can stimulate vitrification, i.e., transition of body water from the liquid phase to an amorphous, glass-like phase during drying or freezing –. At relatively low concentrations, they may non-colligatively protect the native macromolecular structures such as proteins and biological membranes by preferential exclusion from their hydration shells , . In addition to such non-specific cryoprotective mechanisms, some specific roles have been attributed to trehalose and proline. Trehalose can assist refolding of unfolded proteins by molecular chaperones , ; serve as a scavenger of oxygen radicals ; and directly replace missing water molecules in the hydration shells of proteins and phospholipid membranes during desiccation –. Amphipathic proline molecules can intercalate between the headgroups of membrane phospholipids during freeze dehydration and alleviate mechanical stresses in the membranes or can disturb the membranes, making them less prone to the liquid crystalline-to-gel transition .
In comparison to other cold-acclimated insects, the larvae of D. melanogaster in our study showed relatively low levels of accumulated trehalose and proline. We therefore consider it premature to speculate whether these compounds causally contributed to cold tolerance of the larvae and/or which of the abovementioned mechanisms was involved. Nevertheless, three aspects of our metabolomic analysis are quite interesting when compared with the literature data. First, several previous studies have investigated RCH-induced changes in putative cryoprotectants in D. melanogaster adults and obtained mixed results. Targeted analysis of glycerol did not confirm glycerol accumulation , ; nontargeted proton nuclear magnetic resonance (1H-NMR) metabolomic profiling showed increase in trehalose and glucose levels and no change in proline levels ; and targeted analysis of glucose failed to confirm glucose accumulation in a slightly modified RCH protocol . These observations indicate that (a) metabolomic profiles are sensitive to small modifications in acclimation conditions, which themselves have little or no effect on cold tolerance and (b) some facets of acclimation response are shared between RCH and long-term cold acclimation (such as trehalose accumulation), while some others differ (such as proline accumulation occurring only during long-term cold acclimation). Second, in a previous study, a 3- to 6-fold increase was found in the proline levels in D. melanogaster strains selected for resistance to chilling injury at 0°C for 30–60 h or to cold shock at −7°C for 2–3 h . This observation supports our results, suggesting that there is a close association between proline level and cold tolerance. Third, trehalose and proline are two compounds that exhibit the clearest accumulation responses to diapause transition and long-term cold acclimation in C. costata, reaching the levels of 56 mM (trehalose) and 147 mM (proline) in diapausing cold-acclimated larvae , . In addition, a direct evidence of the essential role of proline in high freeze tolerance, including survival in liquid nitrogen, has been obtained by artificially increasing the levels of proline in the tissues of C. costata larvae . Thus, the larvae of D. melanogaster possess the metabolic ability to accumulate proline, the same as that critical for the development of high cold tolerance in the larvae of the related species C. costata. These results open avenues for further investigating the role of proline in the cold tolerance of D. melanogaster.
Membrane restructuring in response to long-term cold acclimation
Long-term cold acclimation stimulated small but statistically significant changes in the lipid composition of biological membranes in D. melanogaster larvae (Figs. 4 and 5). Membrane restructuring in response to cold has been documented in various poikilotherms, including insects. Several basic patterns have been repeatedly reported such as FA desaturation, shortening of average FA chain length, increase in the relative proportion of GPEtns, and reshuffling of FAs , –. We found that the relative proportion of unsaturated FAs and the length of FA chains did not change significantly with cold acclimation in D. melanogaster larvae. The relative proportion of GPEtns was significantly lower in the 25°C- and 15°C-acclimated larvae than in the 15°C→6°C-acclimated larvae, which is in agreement with the generally expected trend. The ethanolamine moiety is less hydrated and occupies a smaller area than the choline moiety. As a result, GPEtns assume a more conical conformation and pack less efficiently into the lipid bilayers than GPChols, thus decreasing their order. Increase in the relative proportion of GPEtns in cold thus counteracts the ordering effects of low temperatures . In this study, 50% of all the molecular species of GPLs were slightly but significantly influenced by cold acclimation. Most of the changes, however, were of so small a magnitude that it would be too speculative to explain them on an adaptive basis. We reached a similar conclusion in our previous analysis of membrane lipids in D. melanogaster adults . One change, however, merits special attention. In both the larvae and the adults of D. melanogaster, the most prominent change related to cold acclimation was the increase in the relative proportion of GPEtns with linoleic acid (FA 18∶2) esterified at the sn-2 position of glycerol. In a study , the level of GPEtn 16∶0/18∶2 in C. costata larvae was also elevated by almost 20% during cold acclimation. A similar response has been reported in some other insects , . It has been proposed that the adaptive value of specific pairing of palmitic and linoleic acids in a single GPL molecule may be related to the widening of the window between the temperatures of gel phase transition (Tm) and hexagonal phase transition (Th), thus increasing the span of environmental temperatures at which the membrane remains fluid and functional . Verification of this hypothesis requires further study.
Overgaard and coworkers observed very small changes in the FA composition of membrane GPLs in D. melanogaster in response to RCH , . The response was characterized by an increase in the proportion of linoleic acid (FA 18∶2) at the expense of palmitic acid (FA 16∶0) and oleic acid (FA 18∶1). Typically, all the changes were in the order of less than 1 molar percent, but together they caused a slight increase in the proportion of the unsaturated FAs and a decrease in the average FA chain length. Slow cooling rates (i.e., longer RCH) resulted in more pronounced changes, which indicated that membrane restructuring requires some time. Both slow and fast rates of RCH showed positive effects on survival after cold shock, despite that they differed in the absence and presence of membrane remodeling response, respectively. Influence of RCH on membrane remodeling was re-assessed later  and no significant effects of RCH on the molar percent composition of FAs or on the relative proportion of unsaturated FAs was found. These results suggest that membrane remodeling is not a significant mechanistic explanation of RCH response in D. melanogaster.
Basic physiological parameters of variously acclimated 3rd instar larvae of Drosophila melanogaster .
Concentrations of metabolites in body fluids of variously acclimated 3rd instar larvae of Drosophila melanogaster .
We thank our students Marie Doležalová and Jan Fesl, and technicians Irena Vacková, Marie Texlová, Petra Berková and Pavla Kružberská, for assistance with insect rearing, sample preparations and analyses.
Conceived and designed the experiments: VK. Performed the experiments: VK JK JR HZ JC AT. Analyzed the data: VK HZ AT PS. Contributed reagents/materials/analysis tools: PS. Wrote the paper: VK.
- 1. Throckmorton LH (1975) The phylogeny, ecology, and geography of Drosophila. In: King RC, editor. Handbook of Genetics: Invertebrates of Genetic Interest. New York: Plenum. pp. 421–469.
- 2. Strachan LA, Tarnowski-Garner HE, Marshall KE, Sinclair BJ (2011) The evolution of cold tolerance in Drosophila larvae. Physiol Biochem Zool 84: 43–53.
- 3. Bale JS (1993) Classes of insect cold hardiness. Funct Ecol 7: 751–753.
- 4. Loeb J, Northrop JH (1917) On the influence of food and temperature upon the duration of life. J Biol Chem 32: 103–121.
- 5. Bliss CI (1927) Temperature characteristics for prepupal development in Drosophila melanogaster. J Gen Physiol 9: 467–495.
- 6. Czajka MC, Lee RE Jr (1990) A rapid cold-hardening response protecting against cold shock injury in Drosophila melanogaster. J Exp Biol 148: 245–254.
- 7. Hoffmann AA, Watson M (1993) Geographical variation in the acclimation responses of Drosophila to temperature extremes. Amer Natur 142: S93–S113.
- 8. Kellermann V, van Heerwaarden B, Sgro CM, Hoffmann AA (2009) Fundamental evolutionary limits in ecological traits drive Drosophila species distributions. Science 325: 1244–1246.
- 9. Moon I, Fujikawa S, Shimada K (1996) Cryopreservation of Chymomyza larvae (Diptera: Drosophilidae) at −196°C with extracellular freezing. CryoLett 17: 105–110.
- 10. Koštál V, Zahradníčková H, Šimek P (2011) Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen. Proc Nat Acad Sci USA 108: 13041–13046.
- 11. Sinclair BJ, Roberts SR (2005) Acclimation, shock and hardening in the cold. J Thermal Biol 30: 557–562.
- 12. Rako L, Hoffmann AA (2006) Complexity of the cold acclimation response in Drosophila melanogaster. J Insect Physiol 52: 94–104.
- 13. Jensen D, Overgaard J, Sørensen JG (2007) The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster. J Insect Physiol 53: 179–186.
- 14. Chen C-P, Walker VK (1994) Cold-shock and chilling tolerance in Drosophila. J Insect Physiol 40: 661–669.
- 15. Rajamohan A, Sinclair BJ (2008) Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae. J Insect Physiol 54: 708–718.
- 16. Rajamohan A, Sinclair BJ (2009) Hardening trumps acclimation in improving cold tolerance of Drosophila melanogaster larvae. Physiol Entomol 34: 217–223.
- 17. Overgaard J, Tomčala A, Sørensen JG, Holmstrup M, Krogh PH, et al. (2008) Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster. J Insect Physiol 54: 619–629.
- 18. Leopold RA, Rinehart JP (2010) A template for insect cryopreservation. In: Denlinger DL, Lee RE Jr, editors. Low Temperature Biology of Insects. Cambridge: Cambridge University Press. pp. 325–341.
- 19. Morris GJ, Watson PF (1984) Cold shock injury – a comprehensive bibliography. CryoLett 5: 352–372.
- 20. Novitski E, Rush G (1949) Viability and fertility od Drosophila exposed to sub-zero temperatures. Biol Bull Mar Biol Lab, Woods Hole 97: 150–157.
- 21. Chiang HC, Benoit D, Maki J (1962) Tolerance of adult Drosophila melanogaster to sub-freezing temperatures. Can Entomol 94: 722–727.
- 22. Tucic N (1979) Genetic capacity for adaptation to cold resistance at different developmental stages of Drosophila melanogaster. Evolution 33: 350–358.
- 23. Kelty JD, Lee RE Jr (1999) Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster. J Insect Physiol 45: 719–726.
- 24. Kelty JD, Lee RE Jr (2001) Rapid cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. J Exp Biol 204: 1659–1666.
- 25. MacMillan HA, Guglielmo CG, Sinclair BJ (2009) Membrane remodeling and glucose in Drosophila melanogaster: A test of rapid cold-hardening and chilling tolerance hypotheses. J Insect Physiol 55: 243–249.
- 26. Lee RE Jr, Chen CP, Denlinger DL (1987) A rapid cold-hardening process in insects. Science 238: 1415–1417.
- 27. Lee RE Jr (2010) A primer on insect cold-tolerance. In: Denlinger DL, Lee RE Jr, editors. Low Temperature Biology of Insects. Cambridge: Cambridge University Press. pp. 3–34.
- 28. Muldrew K, Acker JP, Elliott JAV, McGann LE (2004) The water to ice transition: Implications for living cells. In: Fuller B, Lane N, Benson EE, editors. Life in the Frozen State. Boca Raton: CRC Press. pp. 67–108.
- 29. Privalov P (1990) Cold denaturation of proteins. Crit Rev Biochem Mol Biol 25: 281–305.
- 30. Tsai CJ, Maizel JV, Nussinov R (2002) The hydrophobic effect: A new insight from cold denaturation and two-state water structure. Crit Rev Biochem Mol Biol 37: 55–69.
- 31. Knight JD, Bale JS, Franks F, Mathias SF, Baust JG (1986) Insect cold hardiness – supercooling points and prefreeze mortality. CryoLett 7: 194–203.
- 32. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, et al. (1993) Cold shock damage is due to lipid phase transition in cell membranes: a demonstration using sperm as a model. J Exp Zool 265: 432–437.
- 33. Hochachka PW (1986) Defense strategies against hypoxia and hypothermia. Science 231: 234–241.
- 34. Rojas RR, Leopold RA (1996) Chilling injury in the housefly: evidence for the role of oxidative stress between pupariation and emergence. Cryobiol 33: 447–458.
- 35. Koštál V, Vambera J, 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.
- 36. Koštál V, Yanagimoto M, Bastl J (2006) Chill-injury and disturbance of ion homeostasis in the coxal muscle of the tropical cockroach (Nauphoeta cinerea). Comp Biochem Physiol B 143: 171–179.
- 37. Kostal V, Renault D, Mehrabianova A, Bastl J (2007) Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: role of ion homeostasis. Comp Biochem Physiol A 147: 231–238.
- 38. Sömme L (1982) Supercooling and winter survival in terrestrial arthropods. Comp Biochem Physiol A 73: 519–543.
- 39. Storey KB, Storey JM (1991) Biochemistry of cryoprotectants. In: Lee RE Jr, Denlinger DL, editors. Insects at Low Temperature. New York and London: Chapman and Hall. pp. 64–93.
- 40. Kostal V (2010) Cell structural modifications in insects at low temperatures. In: Denlinger DL, Lee RE Jr, editors. Low Temperature Biology of Insects. Cambridge: Cambridge University Press. pp. 116–140.
- 41. Koštál V, Berková P, Šimek P (2003) Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae). Comp Biochem Physiol B 135: 407–419.
- 42. Shimada K, Riihima A (1990) Cold-induced freezing tolerance in diapausing and non-diapausing larvae of Chymomyza costata (Diptera: Drosophilidae) with accumulation of trehalose and proline. CryoLett 11: 243–250.
- 43. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85.
- 44. Gessner MO, Neumann PTM (2005) Total lipids. In: Graça MAS, Bärlocher F, Gessner MO, editors. Methods to study litter decomposition: A practical guide. Berlin: Springer. pp. 103–107.
- 45. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350–356.
- 46. Bueding E, Orrell SA (1964) A mild procedure for the isolation of polydisperse glycogen from animal tissue. J Biol Chem 239: 4018–4020.
- 47. Koštál V, Zahradníčková H, Šimek P, Zelený J (2007) Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus. J Insect Physiol 53: 580–586.
- 48. Hušek P (1997) Urine organic acid profiling by capillary gas chromatography after a simple sample pretreatment. Clin Chem 43: 1999–2001.
- 49. Hušek P, Šimek P (2001) Advances in amino acid analysis. LC-GC North Am 19: 986–999.
- 50. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497–509.
- 51. Koštál V, Šimek P (1998) Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa. J Comp Physiol B 168: 453–460.
- 52. Tomčala A, Tollarová M, Overgaard J, Šimek P, Koštál V (2006) Seasonal acquisition of chill-tolerance and restructuring of membrane glycerophospholipids in an overwintering insect: triggering by low temperature, desiccation and diapause progression. J Exp Biol 209: 4102–4114.
- 53. Hoffmann AA, Anderson A, Hallas R (2002) Opposing clines for high and low temperature resistance in Drosophila melanogaster. Ecol Lett 5: 614–618.
- 54. Gilchrist GW, Huey RB, Partridge L (1997) Thermal sensitivity of Drosophila melanogaster: Evolutionary responses of adults and eggs to laboratory natural selection at different temperatures. Physiol Zool 70: 403–414.
- 55. Powell SJ, Bale JS (2005) Low temperature acclimated populations of the grain aphid Sitobion avenae retain abiltiy to rapidly cold harden with enhanced fitness. J Exp Biol 208: 2615–2620.
- 56. Shintani Y, Ishikawa Y (2007) Relationship between rapid cold-hardening and cold acclimation in the eggs of the yellow-spotted longicorn beetle, Psacothea hilaris. J Insect Physiol 53: 1055–1062.
- 57. Koštál V (2006) Eco-physiological phases of insect diapause. J Insect Physiol 52: 113–127.
- 58. Duman JG (1979) Thermal hysteresis factors in overwintering insects. J Insect Physiol 25: 805–810.
- 59. Duman JG (2001) Antifreeze and ice nuclator proteins in terrestrial arthropods. Annu Rev Physiol 63: 327–357.
- 60. Zachariassen KE, Kristiansen E (2000) Ice nucleation and antinucleation in nature. Cryobiol 41: 257–279.
- 61. Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SAL, et al. (2007) Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proc Natl Acad Sci U S A 104: 11130–11137.
- 62. Koštál V, Tollarová-Borovanská M (2009) The 70 kDa heat shock protein assists during the reparation of chilling injury in the insect, Pyrrhocoris apterus. PLoS One 4: e4546.
- 63. Nedvěd O (1998) Modelling the relationship between cold injury and accumulated degree days in terrestrial arthropods. CryoLett 19: 267–274.
- 64. Nedvěd O, Lavy D, Verhoef HA (1998) Modelling the time-temperature relationship in cold injury and effect of high-temperature interruptions on survival in a chill-sensitive collembolan. Funct Ecol 12: 816–824.
- 65. Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6: 431–438.
- 66. Kandror O, DeLeon A, Goldberg AL (2002) Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc Nat Acad Sci USA 99: 9727–9732.
- 67. Hincha DK, Hagemann M (2004) Stabilization of model membranes during drying by compatible solutes involved in the stress tolerance of plants and microorganisms. Biochem J 383: 277–283.
- 68. Hengherr S, Heyer AG, Köhler H-R, Schill RO (2007) Trehalose and anhydrobiosis in tardigrades – evidence for divergence of responses to dehydrarion. FEBS J 275: 281–288.
- 69. Schill RO, Mali B, Dandekar T, Schnölzer M, Reuter D, et al. (2009) Molecular mechanisms of tolerance in tardigrades: New perspectives for preservation and stabilization of biological material. Biotechnol Adv 27: 348–352.
- 70. Storey KB, Baust JG, Storey JM (1981) Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J Comp Physiol 144: 183–190.
- 71. Morgan TD, Chippendale GM (1983) Free amino acids of the haemolymph of the Southwestern corn borer and the European corn borer in relation to their diapause. J Insect Physiol 29: 735–740.
- 72. Fields PG, Fleurat-Lessard F, Lavenseau L, Febvay G, Peypelut L, et al. (1998) The effect of cold acclimation and deacclimation on cold tolerance, trehalose and free amino acid levels in Sitophilus granarius and Cryptolestes ferrugineus (Coleoptera). J Insect Physiol 44: 955–965.
- 73. Renault D, Bouchereau A, Delettre YR, Hervant F, Vernon P (2006) Changes in free amino acids in Alphitobius diaperinus (Coleoptera: Tenebrionidae) during thermal and food stress. Comp Biochem Physiol A 143: 279–285.
- 74. Zachariassen KE (1985) Physiology of cold tolerance in insects. Physiol Rev 65: 799–832.
- 75. Sakurai M, Furuki T, Akao K, Tanaka D, Nakahara Y, et al. (2008) Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplancki. Proc Natl Acad Sci USA 105: 5093–5098.
- 76. Hengherr S, Worland MR, Reuner A, Brümmer F, Schill RO (2009) High temperature tolerance in anhydrobiotic tardigrades is limited by glass transition. Physiol Biochem Zool 82: 749–755.
- 77. Sformo T, Walters K, Jeannet K, Wowk B, Fahy GM, et al. (2010) Deep supercooling, vitrification and limited survival to −100 degrees C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. J Exp Biol 213: 502–509.
- 78. Arakawa T, Timasheff SN (1983) Preferential interactions of proteins with solvent components in aquaeous amino acid solutions. Arch. Biochem Biophys 224: 169–177.
- 79. Arakawa T, Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47: 411–414.
- 80. Lee DH, Goldberg AL (1998) Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Sacharomyces cerevisiae. Mol Cell Biol 18: 30–38.
- 81. Viner RI, Clegg JS (2001) Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/α-crystallin protein. Cell Stress Chaperones 6: 126–135.
- 82. Benaroudj N, Lee DH, Goldberg AL (2001) Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. J Biol Chem 29: 24261–24267.
- 83. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: Evolution of osmolyte systems. Science 217: 1214–1222.
- 84. Crowe JH, Crowe LM, Carpenter JF, Wistrom AC (1987) Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem J 242: 1–10.
- 85. Crowe JH (2002) Lessons from nature: The role of sugars in anhydrobiosis. Comp Biochem Physiol A 131: 505–513.
- 86. Overgaard J, Malmendal A, Sørensen JG, Bundy JG, Loeschke V, et al. (2007) Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster. J Insect Physiol 53: 1218–1232.
- 87. Misener SR, Chen C-P, Walker VK (2001) Cold tolerance and proline metabolic gene expression in Drosophila melanogaster. J Insect Physiol 47: 393–400.
- 88. Sinensky M (1974) Homeoviscous adaptation – a homeostatic process that regulates viscosity of membrane lipids in Escherichia coli. Proc Nat Acad Sci USA 71: 522–525.
- 89. Cossins AR, Prosser CL (1978) Evolutionary adaptation of membranes to temperature. Proc Nat Acad Sci USA 75: 2040–2043.
- 90. Hazel JR (1995) Thermal adaptation in biological membranes: Is homeoviscous adaptation the explanation? Annu Rev Physiol 57: 19–42.
- 91. Hazel JR (1989) Cold adaptation in ectotherms: Regulation of membrane function and cellular metabolism. Adv Comp Environ Physiol 4: 1–50.
- 92. Hodková M, Berková P, Zahradníčková H (2002) Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum. J Insect Physiol 48: 1009–1019.
- 93. Overgaard J, Sørensen JG, Petersen SO, Loeschke V, Holmstrup M (2005) Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster. J Insect Physiol 51: 1173–1182.
- 94. Overgaard J, Sørensen JG, Petersen SO, Loeschke V, Holmstrup M (2006) Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster. Physiol Entomol 31: 328–335.