Critical thermal limits,temperature tolerance and water balance of a sub-Antarctic caterpillar,Pringleophaga marioni (Lepidoptera: Tineidae)

Thermal tolerance, supercooling point, water balance and osmoregulatory ability of Pringleophaga marioni Viette (Lepidoptera: Tineidae) are investigated in this study. Field-fresh larvae had a mean CT(Min) (cold stupor) of -0.6 degrees C and a mean CT(Max) (heat coma) of 38.7 degrees C. The mean supercooling point of field-fresh individuals was -5.0 degrees C. Caterpillars showed 100% survival of freezing to -6.5 degrees C, but at -12 degrees C mortality rose to 100%. Survival of a 30h exposure to -6.0 degrees C was 80%, but declined to 30% in the 6-12h interval at -7.5 degrees C. No caterpillars survived for longer than 12h at -9.0 degrees C. Survival of high temperatures (35 degrees C and above) was poor. Tolerance of water loss (46% of fresh mass) and rates of water loss (1% fresh massh(-1)) were similar to those found in other mesic insects. P. marioni larvae were incapable of metabolizing lipids to replenish lost water and showed no haemolymph osmoregulatory ability. It is suggested that the preponderance of freeze tolerance in high-latitude southern hemisphere species may be associated with their occurrence in moist habitats, and that the "freeze tolerance" category be re-examined in the light of the range of strategies adopted by such arthropods.     

Desiccation stress at sub-zero temperatures in polar terrestrial arthropods

Cold tolerant polar terrestrial arthropods have evolved a range of survival strategies which enable them to survive the most extreme environmental conditions (cold and drought) they are likely to encounter. Some species are classified as being freeze tolerant but the majority of those found in the Antarctic survive sub-zero temperatures by avoiding freezing by supercooling. For many arthropods, not just polar species, survival of desiccating conditions is equally important to survival of low temperatures. At sub-zero temperatures freeze avoiding arthropods are susceptible to desiccation and may lose water due to a vapour diffusion gradient between their supercooled body fluids and ice in their surroundings. This process ceases once the body fluids are frozen and so is not a problem for freeze tolerant species. This paper compares five polar arthropods, which have evolved different low temperature survival strategies, and the effects of exposure to sub-zero temperatures on their supercooling points (SCP) and water contents. The Antarctic oribatid mite (Alaskozetes antarcticus) reduced its supercooling point temperature from -6 to -30 degrees C, when exposed to decreasing sub-zero temperatures (cooled from 5 to -10 degrees C over 42 days) with little loss of body water during that period. However, Cryptopygus antarcticus, a springtail which occupies similar habitats in the Antarctic, showed a decrease in both water content and supercooling ability when exposed to the same experimental protocol. Both these Antarctic arthropods have evolved a freeze avoiding survival strategy. The Arctic springtail (Onychiurus arcticus), which is also freeze avoiding, dehydrated (from 2.4 to 0.7 g water g(-1) dry weight) at sub-zero temperatures and its SCP was lowered from c. -3 to below -15 degrees C in direct response to temperature (5 to -5.5 degrees C). In contrast, the freeze tolerant larvae of an Arctic fly (Heleomyza borealis) froze at c. -7 degrees C with little change in water content or SCP during further cold exposure and survived frozen to -60 degrees C. The partially freeze tolerant sub-Antarctic beetle Hydromedion sparsutum froze at c. -2 degrees C and is known to survive frozen to -8 degrees C. During the sub-zero temperature treatment, its water content reduced until it froze and then remained constant. The survival strategies of such freeze tolerant and freeze avoiding arthropods are discussed in relation to desiccation at sub-zero temperatures and the evolution of strategies of cold tolerance.     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.     

Freezing survival, body ice content and blood composition of the freeze-tolerant European common lizard, Lacerta vivipara

To investigate the freeze tolerance of the European common lizard, Lacerta vivipara, we froze 17 individuals to body temperatures as low as -4 degrees C under controlled laboratory conditions. The data show that this species tolerates the freezing of 50% of total body water and can survive freezing exposures of at least 24-h duration. Currently, this represents the best known development of freeze tolerance among squamate reptiles. Freezing stimulated a significant increase in blood glucose levels (16.15+/- 1.73 micromol x ml(-1) for controls versus 25.06 +/- 2.92 micromol x ml(-1) after thawing) but this increase had no significant effect on serum osmolality which was unchanged between control and freeze-exposed lizards (506.0 +/- 23.8 mosmol x l(-1) versus 501.0 +/- 25.3 mosmol x l(-1), respectively). Tests that assessed the possible presence of antifreeze proteins in lizard blood were negative. Recovery at 5 degrees C after freezing was assessed by measurements of the mean time for the return of breathing (5.9 +/- 0.5 h) and of the righting reflex (44.8 +/- 4.5 h). Because this species hibernates in wet substrates inoculative freezing may frequently occur in nature and the substantial freeze tolerance of this lizard should play a key role in its winter survival.     

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35 degrees C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 microg trehalose/mg dry weight, respectively) during acclimation at 5 and 35 degrees C, respectively. Warm and cold acclimation enhanced heat (40 degrees C for 8h) and freezing (-20 degrees C for 4h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.     

Freezing induces a loss of freeze tolerance in an overwintering insect

Cold-hardy insects overwinter by one of two main strategies: freeze tolerance and freeze avoidance by supercooling. As a general model, many freeze-tolerant species overwinter in extreme climates, freeze above -10 degrees C via induction by ice-nucleating agents, and once frozen, can survive at temperatures of up to 40 degrees C or more below the initial freezing temperature or supercooling point (SCP). It has been assumed that the SCP of freeze-tolerant insects is unaffected by the freezing process and that the freeze-tolerant state is therefore retained in winter though successive freeze-thaw cycles of the body tissues and fluids. Studies on the freeze-tolerant larva of the hoverfly Syrphus ribesii reveal this assumption to be untrue. When a sample with a mean 'first freeze' SCP of -7.6 degrees C (range of -5 degrees C to -9.5 degrees C) were cooled, either to -10 degrees C or to their individual SCP, on five occasions, the mean SCP was significantly depressed, with some larvae subsequently freezing as low as -28 degrees C. Only larvae that froze at the same consistently high temperature above -10 degrees C were alive after being frozen five times. The wider occurrence of this phenomenon would require a fundamental reassessment of the dynamics and distinctions of the freeze-tolerant and freeze-avoiding strategies of insect overwintering.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Freeze tolerance in Aporrectodea caliginosa and other earthworms from Finland

Earthworms that live in subarctic and cold temperate areas must deal with frost even though winter temperatures in the soil are often more moderate than air temperatures. Most lumbricid earthworms can survive temperatures down to the melting point of their body fluids but only few species are freeze tolerant, i.e. tolerate internal ice formation. In the present study, earthworms from Finland were tested for freeze tolerance, and the glycogen reserves and glucose mobilization (as a cryoprotectant) was investigated. Freeze tolerance was observed in Aporrectodea caliginosa, Dendrobaena octaedra, and Dendrodrilus rubidus, but not in Lumbricus rubellus. A. caliginosa tolerated freezing at -5 degrees C with about 40% survival. Some individuals of D. octaedra tolerated freezing even at -20 degrees C. Glycogen storage was largest in D. octaedra where up to 13% of dry weight consisted of this carbohydrate, whereas the other species had only 3-4% glycogen of tissue dry weight. Also glucose accumulation was largest in D. octaedra which was the most freeze-tolerant species, but occurred in all four species upon freezing. It is discussed that freeze tolerance may be a more common phenomenon in earthworms than previously thought.     

Anoxia blocks thermotolerance and the induction of rapid cold hardening in the flesh fly, Sarcophaga crassipalpis

Abstract. Anoxia induced by nitrogen or carbon dioxide, or hypoxic/hypobaric conditions generated by a partial vacuum sensitizes red-eye pharate adults of Sarcophaga crassipalpis Macquart to a high temperature exposure that is normally nonlethal (40C for 2–3 h). Thermotolerance induced by a2h exposure to 40C (under aerobic conditions) doubles the pharate adults' tolerance to 45C but provides no protection against a combined exposure to 45C and anoxia, and only modest protection against a combined exposure to 40C and anoxia. Under aerobic conditions, exposing pharate adults to 0C for 2 h increases their tolerance to -10C (rapid cold hardening). Rapid cold hardening at 0C is not induced under anoxia. These results imply that tolerance to high temperatures and rapid cold hardening are dependent on aerobic processes and suggest that certain forms of temperature stress can be further exacerbated with anoxia.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.     

Comparison of the cold hardiness capacities of the oviparous and viviparous forms of Lacerta vivipara

The lizard Lacerta vivipara has allopatric oviparous and viviparous populations. The cold hardiness strategy of L. vivipara has previously been studied in viviparous populations, but never in oviparous ones. The present study reveals that both the oviparous and viviparous individuals of this species are able to survive in a supercooled state at -3 degrees C for at least one week when kept on dry substrates. The mean crystallisation temperatures of the body, around -4 degrees C on dry substrata and -2 degrees C on wet substrata, do not differ between oviparous and viviparous individuals. All the individuals are able to tolerate up to 48-50% of their body fluid converted into ice, but only viviparous individuals were able to stabilize their body ice content at 48%, and hence were able to survive even when frozen at -3 degrees C for times of up 24 hours. Ice contents higher than 51% have been constantly found lethal for oviparous individuals. This suggests that, in L. vivipara, the evolution towards a higher degree of freezing tolerance could parallel the evolution of the viviparous reproductive mode, a feature believed to be strongly selected under cold climatic conditions. This is the first report, among reptiles, of an intraspecific variation regarding the freeze tolerance capacities.     

Gene expression analysis of cold and freeze stress in Baker's yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10 degrees C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Factors affecting the freeze tolerance of the hoverfly Syrphus ribesii (Diptera: syrphidae)

Larvae of Syrphus ribesii collected from overwintering sites in the U.K. are strongly freeze tolerant with 70% survival at -35 degrees C. The cold tolerance of laboratory reared insects increased with increasing periods of acclimation at 0 degrees C, with a concurrent rise in the supercooling point (SCP) from -6.8+/-0.1 to -5.1+/-0.3 degrees C. There was 50% survival in the most cold-hardy group 72h after brief exposures to -30 degrees C. The retention of gut contents caused a decrease in cold hardiness, with only 13% of larvae surviving 72h after exposure to -15 degrees C, with no subsequent pupation or emergence. Wet larvae had a significantly higher SCP (-5.0+/-0.2 degrees C) compared to dry larvae (-7.8+/-0.4 degrees C), although survival of larvae was similar in both groups. There was no nucleator activity in the haemolymph of field collected larvae. The importance of these findings are discussed in relation to the freeze tolerance strategy of S. ribesii.     

Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S . tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes ( AtCBF1-3 ) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 °C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.