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.     

Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 °C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 °C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival. Accepted: 7 March 2000     

Cold acclimation and lipid composition in the earthworm Dendrobaena octaedra

We have investigated the lipid chemistry during cold acclimation in the freeze tolerant earthworm Dendrobaena octaedra. The dominant phospholipid fatty acids (PLFA) of D. octaedra were 20:4, 20:5 and 20:1 (50% of total PLFA) followed by 18:0, 18:1 and 18:2omega6,9 (25% of total PLFA). The ability to tolerate freezing in this species was acquired after acclimation at low temperature for 2-4 weeks. During this period one particular membrane PLFA, 18:2omega6,9, increased significantly and there was a good correlation between the proportion of this PLFA and the survival of freezing. The composition of neutral lipid fatty acids (NLFA), most likely representing storage lipids (triacylglycerides), also changed during cold acclimation so that the overall degree of unsaturation increased. Using a common-garden experiment approach, we compared lipid composition of three genetically different populations (Denmark, Finland and Greenland) that differed in their freeze tolerance. Inter-populational differences and differences due to cold acclimation in overall fatty acid composition were evident in both PLFAs and NLFAs. Specifically, the PLFAs, 20:4 and 20:5, were considerably more represented in worms from Greenland, and this contributed to a higher UI of PLFAs in this population.     

Small Dendrobaena earthworms survive freezing better than large worms

Dendrobaena octaedra is a freeze tolerant earthworm widely distributed in boreal regions. Specimens collected in Sweden were cold acclimated and then frozen at -7 degrees C to examine the influence of body mass on survival of freezing. Results showed that survival was negatively correlated to body mass. Glycogen content of the worms was variable and seemed to decrease with increasing body mass consistent with the hypothesis that freeze survival is dependent on the ability to rapidly break down glycogen and accumulate high concentrations of glucose. The results suggest that large worms (subadults and adults) invest energy in production of cocoons at the expense of glycogen storage for cryoprotectant production, whereas juvenile worms increase their survival chances by investing energy in glycogen storage at the expense of growth as a preparation for winter.     

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

Repeated freezing induces oxidative stress and reduces survival in the freeze-tolerant goldenrod gall fly,Eurosta solidaginis

Freeze tolerant insects must not only survive extracellular ice formation but also the generation of reactive oxygen species (ROS) during oxygen reperfusion upon thawing. Furthermore, diurnal fluctuations in temperature place temperate insects at risk of being exposed to multiple freeze–thaw cycles, yet few studies have examined metrics of survival and oxidative stress in freeze-tolerant insects subjected to successive freezing events. To address this, we assessed survival in larvae of the goldenrod gall fly Eurosta solidaginis, after being subjected to 0, 5, 10, 20, or 30 diurnally repeated cold exposures (RCE) to −18 °C or a single freeze to −18 °C for 20 days. In addition, we measured indicators of oxidative stress, levels of cryoprotectants, and total aqueous antioxidant capacity in animals exposed to the above treatments at 8, 32, or 80 h after their final thaw. Repeated freezing and thawing, rather than time spent frozen, reduced survival as only 30% of larvae subjected to 20 or 30 RCE successfully pupated, compared to those subjected to fewer RCE or a single 20 d freeze, of which 82% pupated. RCE had little effect on the concentration of the cryoprotectant glycerol (4.26 ± 0.66 μg glycerol·ng protein⁻¹ for all treatments and time points) or sorbitol (18.8 ± 2.9 μg sorbitol·mg protein⁻¹ for all treatments and time points); however, sorbitol concentrations were more than twofold higher than controls (16.3 ± 2.2 μg sorbitol·mg protein⁻¹) initially after a thaw in larvae subjected to a single extended freeze, but levels returned to values similar to controls at 80 h after thaw. Thawing likely produced ROS as total aqueous antioxidant capacities peaked at 1.8-fold higher than controls (14.7 ± 1.6 mmol trolox·ng protein⁻¹) in animals exposed to 5, 10, or 20 RCE. By contrast, aqueous antioxidant capacities were similar to controls in larvae subjected to 30 RCE or the single 20 d freeze regardless of time post final thaw, indicating these animals may have had an impaired ability to produce primary antioxidants. Larvae lacking an antioxidant response also had elevated levels of oxidized proteins, nearly twice that of controls (21.8 ± 3.2 mmol chloramine-T·mg protein⁻¹). Repeated freezing also lead to substantial oxidative damage to lipids that was independent of aqueous antioxidant capacity; peroxides were, on average, 5.6-fold higher in larvae subjected to 10, 20 or 30 RCE compared to controls (29.1 ± 7.3 mmol TMOP·μg protein⁻¹). These data suggest that oxidative stress due to repeated freeze–thaw cycles reduces the capacity of E. solidaginis larvae to survive freezing.     

植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants

A study was performed to examine whether or not betaine (glycinebetaine), a compatible solute, is accumulated in response to cold stress and is involved in mechanisms that protect plants from freezing injury. For this purpose, we used near-isogenic lines of barley, with each line differing only in a single gene for the spring type of growth habit; the various lines were produced by back-crosses to a recurrent cultivar of the winter type. The winter type of growth habit requires a low temperature for triggering of flower development (vernalization), whereas the spring type does not. Betaine was accumulated to five times the basal level over the course of 3 weeks at low temperature (5 °C) in the winter-type cultivar and in a spring-sh line having the sh gene for the spring-type growth habit, but the level was only doubled in the spring-Sh3 line, which carried the Sh₃ gene for the spring-type growth habit. Among near-isogenic lines of the same cultivar, the levels of betaine accumulated in leaves at low temperature were well correlated with the percentages (on a dry weight basis) of green leaves that survived freezing injury (-5 °C). This observation indicates the possibility, separate from the recognized role of betaine in the response to salinity and/or drought, that betaine accumulates in response to cold stress and that the accumulation of betaine during cold acclimation is associated to some extent with freezing tolerance in leaves of barley plants.     

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999     

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.     

Annual variation in glycerol mobilization and effect of freeze rigor on post-thaw locomotion in the freeze-tolerant frog <Emphasis Type="Italic">Hyla versicolor</Emphasis>

This study documents post-thaw recovery of jump distance and cryoprotectant mobilization in the freeze-tolerant frog Hyla versicolor over two successive years. Cold acclimated frogs had plasma glycerol levels near 1.0 mM in 2004 but it was nearly 70× higher during 2005. Freezing of frogs induced nearly identical levels of plasma glycerol (ca. 177 mM) during 2004 and 2005. Plasma glucose was only mobilized upon somatic freezing, with averages ranging between 21 and 36 mM. Control jump distance showed no difference between the two years of the study. The post-thaw jump response was identical during the first 2 years despite large differences in glycerol mobilization between these 2 years. Recovery proceeded much faster in 2005 when frogs mobilized glycerol prior to freeze exposure. Frogs were more impaired in their locomotion performance during the initial stages of recovery period when they were frozen at a lower temperature (−3 vs. −1.5°C) but they eventually recovered. Moderate lengthening of the freeze duration (3 vs. 7 days) with the 2004 collection group did not affect recovery of jump distance when frogs were frozen at −1.5°C. Hence, postfreeze impairment of locomotion is dependent of the intensity of the freeze temperature but it is a reversible process that is mitigated when glycerol is more freely distributed to body tissues.     

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.     

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.     

The role of ABA in freezing tolerance and cold acclimation in barley

The role of ABA in freezing resistance in nonacclimated and cold‐acclimated barley ( Hordeum vulgare L.) was studied. Eleven nonacclimated cultivars differed in their LT₅₀, ranging from −10.8 to −4.8°C. Sugars, free proline, soluble proteins and ABA were analyzed in nonacclimated cultivars and during cold acclimation of one cultivar. There was an inverse correlation between LT₅₀ and both ABA and sucrose contents. Exogenous ABA caused a decrease in the freezing point of leaf tissue in the cultivar with the lowest level of endogenous ABA, but not in the cultivar with the highest level, suggesting that ABA in the latter may be near the optimum endogenous level to induce freezing tolerance. Plants of cv. Aramir treated with ABA or allowed to acclimate to cold temperature increased their soluble sugar content to a similar level. The LT₅₀ of leaves of cold‐acclimated cv. Aramir decreased from −5.8 to −11.4°C, with biphasic kinetics, accumulating proline and soluble sugars with similar kinetics. The biphasic profile observed during cold acclimation could be a direct consequence of cryoprotectant accumulation kinetics. ABA and soluble protein accumulation showed a single step profile, associated mainly with the second phase of the LT₅₀ decrease. Thus, a significant increase in endogenous ABA is part of the response of barley to low temperature and may be required as a signal for the second phase of cold acclimation. Endogenous ABA contents in the nonacclimated state may determine constitutive freezing tolerance.