Physical and chemical changes associated with seasonal alterations in freezing tolerance in the adult northern tenebrionid, Upis ceramboides

The adult tenebrionid beetle Upis ceramboides overwinters in the northern taiga forests of North America in a hibernaculum typically just beneath loose tree bark above the snowline. The beetles may be exposed to temperatures as low as ?55°C, which is approximately the lower limit of cold tolerance found in specimens collected in mid-winter. Supercooling points average ?6.3°C throughout the year and, contrary to expectation, show no seasonal variation in spite of major alterations in haemolymph composition and freezing tolerance. Summer beetles are incapable of withstanding temperatures below the supercooling point but freezing tolerance increases during the fall (September–November) and the lower lethal temperature (LLT) is maintained at ca. ?55°C until March, after which it gradually rises to the summer level of ?6°C. Changes in freezing tolerance are closely associated with seasonal alterations in the polyhydric alcohols sorbitol and threitol. Neither polyol is present in measureable amount during summer; sorbitol accumulates to an average haemolymph concentration of 0.44 M/l in winter and threitol reaches 0.25 M/l. Summer beetles contain about 14% more water than beetles collected during the other seasons. Upis ceramboides thus undergoes unique seasonal changes in physical and chemical characteristics that enable it to tolerate severe, prolonged subfreezing temperatures.     

Cold hardiness in the Alpine beetles Patrobus septentrionis and Calathus melanocephalus

Alpine Patrobus septentrionis and Calathus melanocephalus (Col., Carabidae) were found to be susceptible to freezing. In the summer, the supercooling points were about ?5 to ?6°C. They were lowered during acclimation in the field and in the laboratory at 0 or ?3°C. Cold hardiness was correlated to, but not determined by, haemolymph osmolality. Thermal hysteresis was not detected. Increase in cold hardiness was concluded to be mainly a result of the influence of acclimation conditions on ice-nucleating compounds. In P. septentrionis, the results indicate that different compounds with ice-nucleation activity at different temperatures determine the limit of supercooling at different times. The haemolymph of both species supercooled well below the intact beetles at all seasons. Changes in haemolymph supercooling points could be ascribed to inactivation of ice nucleators in early autumn and to the effect of changes in solute concentration. In P. septentrionis, myo-inositol increased during cold-acclimation to 80–120 mMol concentrations, whereas C. melanocephalus produced 40–60 mMol trehalose.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration

Eggs of the stonefly, Arcynopteryx compacta, that overwinter in the alpine region of Norwegian mountains, increase their cold-hardiness by dehydration. Eggs enclosed in ice at −22°C survive the loss of about two-thirds of their total water content by shrinkage due to passive diffusion of body water along the concentration gradient. Fully hydrated eggs are killed by freezing at their supercooling point of −26°C, and by direct cooling to −30°C. Dehydrated eggs have a mean supercooling point of −31°C, and survive exposure at −27 and −29°C in ice. Judged from their melting points the eggs do not accumulate low-molecular-weight cryoprotective substances. The difference between freezing and melting points corresponds to a thermal hysteresis of up to 1.8°C. The presence of thermal hysteresis antifreezes may stabilize their supercooled state when enclosed by ice during overwintering. The eggs enter diapause in the autumn, and diapause completion is enhanced both by temperature and time during enclosure in ice.     

Cold tolerance of a New Zealand alpine cockroach, Celatoblatta quinquemaculata (Dictyoptera, Blattidae)

Abstract. Ecophysiological features, including survival and recovery from freezing and determination of the freezable water content, are reported for a cold-adapted cockroach Celatoblatta quinquemaculata Johns 1966 (Dictyoptera, Blattidae) inhabiting alpine communities at altitudes greater than 1300 m a.s.l. in mountains of Central Otago, New Zealand. Nymphs ranged from 15 to 51 mg live weight of which 67% was water. Cockroaches had a mean supercooling point temperature of ?5.4 ± 0.1°C; with recovery from freezing close to this temperature being rapid, but no recovery was observed when frozen at ?9 to ?10°C. The duration of exposure to freezing conditions and the time allowed for recovery (24–96 h) both influenced individual recovery and subsequent survival. Comparison of supercooling point data and survival shows that this species possesses a few degrees of freeze tolerance, and individuals have been found frozen in the field when subzero temperatures occur. Differential scanning calorimetry showed ≈ 74% of body water froze during cooling and between 24 and 27% of total body water was osmotically inactive (unfreezable under the experimental conditions). Carbohydrates, other than glucose at 7.5μg/mg fresh weight, were in low concentrations in the body fluids, suggesting little cryoprotection. No thermal hysteresis from antifreeze protein activity was detected in haemolymph samples using calorimetric techniques. It is suggested that slow environmental cooling rates, together with high individual supercooling points, confer a small amount of freezing tolerance on this species enabling it to survive low winter temperatures. This has allowed it to colonize and maintain populations in alpine habitats > 1300 m a.s.1. in New Zealand.     

Physiology of diapause in the adult bark beetle, Ips acuminatus Gyll., studied in relation to cold hardiness

The ovarioles of the bark beetle, Ips acuminatus are telotrophic. Ovarian development is suppressed at an immature stage with primary germ cells present in the germaria. Lower oxygen consumption is found in beetles during autumn and early winter, and a substantial rise in mean respiration rates occurs in the beginning of January paralleled by a resumption of pre-vitellogenesis in all females maintained either at 3°C or out of doors after return to 21°C for 2 weeks. It is concluded that I. acuminatus enters faculative diapause soon after enclosure to the adult, and that diapause is terminated by mid-winter in beetles kept for 18 weeks at either 3°C or out of doors. The specimens remain thereafter in reproductive quiescence until ovarian development can proceed.Photic cues are neither involved in the elevation of mean respiration rates, nor needed to abolish the inhibition of ovarian maturation in beetles kept at 3°C or in those returned to 21°C. However, follicle formation in ovarioles is only seen in positive phototactic females reared during “long-day” conditions, suggesting a photoperiodic regulation of the later stages of vitellogenesis.Detectable amounts of ethylene glycol are found at the beginning of November in freezing-susceptible I. acuminatus hibernating in its galleries underneath bark of Scots pine (Pinus silvestris) at 3°C. The gradual catabolism of the cryoprotective solute at 3°C through December occurred at a time when individuals achieved the competence to resume ovarian maturation during 2 weeks at 21°C, but prior to the substantial rise in their mean respiration rates. However, resumption of ovarian development in spring had no effect on the capability of outdoor beetles to enhance their supercooling capacity when subjected to sub-zero temperatures. Since the ability to respond to temperature changes occurred in post-diapause I. acuminatus as well, the maintenance of prolonged cold hardiness in specimens could not be related to diapause itself. Apparently, the ability of beetles to resynthesize ethylene glycol when a detectable level is present in the organism remains unaltered during overwintering.     

Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis

Two populations of the gall fly Eurosta solidaginsis utilize different strategies to endure seasonal exposure to temperatures below freezing. Both populations are freezing tolerant. In north temperate populations, supercooling points rise from ?10.2°C to ?6.2°C following exposures to temperatures below freezing. This level is maintained throughout winter and ensures frequent and prolonged periods of tissue freezing. South temperate populations depress the supercooling point to ?14.2°C during autumn and early winter, and this depression precludes extracellular ice formation during periods of supra-optimal temperature fluctuations. During mid-winter, supercooling points rise to the same level as in northern groups.Both populations accumulate three principal cryoprotective agents following first frost exposures (glycerol, sorbitol and trehalose). Cryoprotectants levels do not peak in northern populations until 4–6 weeks after first frost. In southern populations the accumulation profile is characterized by a high initial rate of synthesis, a protective overshoot and pronounced seasonal fluctuations. The relative survival advantages of each strategy are discussed.     

Supercooling ability and cold hardiness of the pollen beetle Meligethes aeneus

Supercooling point (SCP) and cold‐hardiness of the pollen beetle Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae) were investigated. Mature eggs from the oviduct were supercooled on average to ?28.0 °C and from oilseed rape buds to ?24.4 °C; first instars were supercooled to ?21.0 °C and second instars to ?16.8 °C. Despite their high supercooling ability, none of the eggs survived 24 h exposure to ?2.5 °C. The supercooling ability of adults varied significantly among feeding and non‐feeding beetles: high SCPs prevailed during the whole warm period, being about ?12 °C; low values of SCP of ?20 °C dominated in non‐feeding beetles. In spring and autumn, beetles displayed the same acclimation efficiency: after 1 week of exposure at 2.0 °C with no access to food their SCPs were depressed equally by about 3 °C. Meligethes aeneus beetles have a different response to low temperatures depending on the season. The lowest tolerance was found in reproductively active beetles after emergence from overwintering sites; the time needed to kill 50% of individuals (Ltime₅₀) was 56.2 h at ?7 °C and the lower lethal temperature needed to kill 50% (Ltemp₅₀) after 24 h exposure was ?8.6 °C. Cold hardiness increased from midsummer to midwinter; Ltime₅₀ was 80 h in August, 182.8 h in September, and 418.1 h in January. Lethal temperature after 24 h exposure was ?9.1 °C in August and ?9.8 °C in September. In February, after diapause, the beetles started to loose their cold tolerance, and Ltemp₅₀ was slightly increased to ?9.5 °C. Hibernating beetles tolerated long exposure at ?7 °C well, but mortality was high after short exposure if the temperature dropped below ?9 °C for 24 h. Despite the season, the beetles died at temperatures well above their mean SCP; consequently, SCP is not a suitable index for cold hardiness of M. aeneus.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Ecophysiological strategies of Antarctic intertidal invertebrates faced with freezing stress

Recent studies have revealed a previously unanticipated level of biodiversity present in the Antarctic littoral. Here, we report research on the ecophysiological strategies adopted by intertidal species that permit them to survive in this environment, presenting cold-tolerance data for the widest range of invertebrates published to date from the Antarctic intertidal zone. We found significant differences in levels of cold tolerance between species within this zone. However, and contrary to expectations, intraspecific comparisons of subtidal and intertidal groups of eight species found significant differences between groups in only three species. One species, the nemertean Antarctonemertes validum, showed evidence of the presence of antifreeze proteins (thermal hysteresis proteins), with 1.4°C of thermal hysteresis measured in its haemolymph. We found a strong inverse relationship across species between mass and supercooling point, and fitted a power law model to describe the data. The scaling exponent (0.3) in this model suggests a relationship between an animal’s supercooling point and its linear dimensions.     

Antifreeze activity in the cerambycid beetle Rhagium inquisitor

The present study revealed that hibernating freeze-avoiding Rhagium inquisitor beetles have thermal hysteresis antifreeze agents in the intracellular fluid as well as in the intestinal fluid and the haemolymph. The antifreeze activity in all three compartments increased with diminishing size of the seeding ice crystal, suggesting that all three compartments are well protected against spontaneous ice nucleation at low sub-zero temperatures. Accepted: 4 October 1998     

Cold-hardiness of the arctic beetle, Pytho americanus Kirby Coleoptera,Pythidae (Salpingidae)

The arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages. This is the first demonstration that an insect can tolerate freezing in more than one life stage, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold hardening process of low temperature acclimation are increasing glycerol and decreasing glycogen concentrations. Glycerol is the only polyol to be synthesized during acclimation, and it accumulates to a maximum of 8.2 and 12.2% of the fresh body weight in larvae and adults respectively. This coincides with the peak of frost tolerance. In addition to its normally assumed roles in cryoprotection it is suggested that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of ‘bound’ water. Evidence is presented that indicates that glycerol is synthesized mainly from carbohydrate reserves, especially glycogen, but it does not rule out the possibility that a proportion of free glycerol comes from glyceride sources.P. americanus larvae and adults have low supercooling potential and maintain their supercooling points in the region of ?4° to ?8°C. It is hypothesized that these elevated supercooling points are a result of the presence in the haemolymph of nucleating agents which ensure ice formation at high sub-zero temperatures. It is believed that this beetle overwinters in a frozen state within its microhabitat, which is under bark of fallen spruce which is, in turn, covered by an insulating blanket of snow. The advantages of this overwintering strategy are discussed.     

The supercooling ability of ticks (Acari,Ixodoidea)

 The supercooling capacity of nine laboratory- held species of ticks originating from different geographical areas, as well as five field-collected species from Germany, was investigated. All but one tick species showed mean supercooling points between about −17 and −23 °C, suggesting that the capacity to supercool to temperatures of ≤−17 °C might be an inherent property of many tick species unrelated to their geographic origin. Photoperiod did not influence the mean supercooling point in any of the species and there was also no distinct seasonal pattern of supercooling in seasonally acclimatized Dermacentor marginatus. Thus, the supercooling ability was independent of the presence/absence of diapause. The finding of thermal hysteresis in D. marginatus hemolymph raises the question of whether or not anti-freeze proteins are involved in the supercooling capacity of that species. An interspecies comparison revealed a weak negative correlation between relative water content and supercooling point of the ticks and an even weaker correlation between body mass or body water mass and the supercooling point. Since the ticks exhibited low supercooling points both before and shortly after feeding, the blood used as food should lack potent ice nucleators. Accepted: 14 June 1996     

Experimental studies on the cold tolerance of Alaskozetes antarcticus

The cold tolerance mechanism of the Antarctic terrestrial mite Alaskozetes antarcticus (Michael) was investigated in cultured animals. Freezing is fatal in this species and winter survival occurs by means of supercooling, which is enhanced by the presence of glycerol in the body. There is an inverse, linear relationship between the concentration of glycerol and the supercooling point, which may be as low as ?30°C. Feeding detracts from supercooling ability by providing ice nucleators in the gut which initiate freezing at relatively high sub-zero temperatures. Experiments on the effects of various environmental factors showed that low temperature acclimation gave rise to increased glycerol concentrations and suppressed feeding, while desiccation also stimulated glycerol production. Photoperiod had no effect on cold tolerance in this species. The juvenile instars of A. antarcticus were found to possess a greater degree of low temperature tolerance than adults.     

Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions

The ability of 11 different organic solutes in physical solution to mask the effect of nucleating agents from hemolymph of freezing tolerant insects was tested. The masking effect was tested by measuring the supercooling points of samples with various solute concentrations, with and without hemolymph. Hemolymph was obtained from freeze-tolerant Eleodes blanchardi tenebrionid beetles.The depressive effect of the solutes on the supercooling points was nearly equivalent to the corresponding melting point depression, indicating that the depression was due only to the colligative properties of the solutes. Thus, no ability for nucleator masking was demonstrated.     

Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae)

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn. Accepted: 22 February 1999     

Thermal-hysteresis-factors in overwintering insects

The haemolymph of 9 species of insects found overwintering under the bark of dead trees in northern Indiana contained factors which produced a thermal hysteresis (a difference between the freezing and melting points) of several degrees. These thermal-hysteresis-factors were common in overwintering beetles, but rare in non-Coleoptera, and are similar to the macromolecular antifreezes of polar marine teleost fishes. The factors were found in both freeze-susceptible and freeze-tolerant species, and their function in freeze-susceptible insects appears to be to depress the supercooling points and therefore the lower lethal temperatures of the insects. However, the function of the factors in freeze-tolerant species is not clear. Possible functions are discussed.     

The ontogeny of cold tolerance in the gall fly, Eurosta solidagensis

The lower lethal temperature of many insects indicates an overwintering flexibility as a result of either extensive supercooling or production of cryoprotectants. Ontogenetically, the gall fly (Eurosta solidagensis) utilizes both means of seasonal cryoprotection. All stages except third instar larvae demonstrate supercooling points well below the lowest temperature normally experienced by that particular stage. The third instar larvae exhibit a high supercooling point but are well protected by a cryoprotectant system consisting of glycerol, sorbitol, and trehalose. Glycerol is accumulated, possibly from triglyceride sources, during early autumn and reaches plateau levels (0·6 M) by early winter. Sorbitol synthesis is delayed until freezing exposures and reaches a plateau with glycerol at 0·3 M. It is not until mid-winter that peak trehalose levels are reached (300 mg %). All cryoprotectant levels are a reflection of haemolymph concentrations.Laboratory acclimation experiments further quantify these results. Trehalose synthesis is time and temperature dependent and appears to be affected by developmental processes.     

Water Crystallisation of Model Sugar Solutions with Nanobubbles Produced from Dissolved Carbon Dioxide

This study was conducted to examine the influence of CO₂ nanobubbles on crystallisation behaviour of water during freezing of model sugar (2–5%w/v) solutions. CO₂ gas was dissolved at 0, 1000, and 2000-ppm concentrations before freezing. Carbonated sugar solutions in 50 mL plastic tubes were immersed in a pre-cooled (−15 °C) ethylene glycol bath and left to freeze at −15 °C for 90 min. When the temperature of the solutions reached 0 °C, ultrasound (US; 20 kHz) was emitted in the bath for 20 s duration through a metal horn transducer. The US wave applied in the ethylene glycol bath was expected to propagate to the sugar solutions in the tube and promote gas bubble formation from dissolved CO₂, which will trigger the ice nucleation. Obtained freezing curves were analysed for nucleation time and temperature, supercooling degree, and time taken for phase change. In general, the CO₂ gas promoted freezing of water, causing a noticeable shift in nucleation parameters. For example, nucleation time of 2000-ppm carbonated water coupled with sonication emission for 20 s (7.8 min) was much shorter than that of controls (pure water without any treatment = 19.1 min and US only = 14.3 min). The former initiated ice nucleation just below sub-zero temperature (−0.2 °C) whereas the onset temperature of controls (pure water without any treatment = −11.3 °C and the US only treatment = −10.3 °C). A similar effect was observed with different model sugar solutions. The current findings can be applied to refine the manufacturing process of ice-cream and frozen desserts by the food industries.