Overwintering adaptations of the stag beetle,Ceruchus piceus: removal of ice nucleators in the winter to promote supercooling

Summary Overwintering larvae and adults of the stag beetle,Ceruchus piceus, are freeze sensitive (i.e. cannot survive internal freezing). The most commonly described cold adaptation of freeze susceptible insects involves the production of antifreezes to promote supercooling, butCeruchus piceus larvae produced only low levels of antifreezes in the winter. However, by removing ice nucleators from the gut and hemolymph in the winter the larvae were able to depress their supercooling points from approximately –7°C in the summer to near –25°C in mid-winter. The ice nucleators present in the non-winter hemolymph were identified as lipoproteins. One of these lipoproteins with ice nucleator activity was purified using flotation ultracentrifugation and anion exchange (DEAE-Sephadex) chromatography.Removal of ice nucleators to promote supercooling in winter may be energetically preferable to costly production and maintenance of high, of-ten molar, concentrations of antifreeze. Obviously the ice nucleator must normally perform a function which the insect can spare over the winter. Hemolymph lipoproteins, which generally function in lipid transport, may fit this criterion during the winter period of reduced metabolic activity.Abbreviations LP I very low density lipoprotein - LP II low density lipoprotein - PAGE polyacrylamide gel electrophoresis - SCP supercooling point     

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996     

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. ¹³NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be ∼8.5 °C.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Insect antifreezes and ice-nucleating agents

Cold-tolerant, freeze-susceptible insects (those which die if frozen) survive subzero temperatures by proliferating antifreeze solutes which lower the freezing and supercooling points of their body fluids. These antifreezes are of two basic types. Lowmolecular-weight polyhydroxy alcohols and sugars depress the freezing point of water on a colligative basis, although at higher concentrations these solutes may deviate from linearity. Recent studies have shown that these solutes lower the supercooling point of aqueous solutions approximately two times more than they depress the freezing point. Consequently, if a freeze-susceptible insect accumulates sufficient glycerol to lower the freezing point by 5 °C, then the glycerol should depress the insect's supercooling point by 10 °C.Some cold-tolerant, freeze-susceptible insects produce proteins which produce a thermal hysteresis (a difference between the freezing and melting point) of several degrees in the body fluids. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins of polar marine teleost fishes. The THPs lower the freezing, and presumably the supercooling, point by a noncolligative mechanism. Consequently, the insect can build up these antifreezes, and thereby gain protection from freezing, without the disruptive increases in osmotic pressure which accompany the accumulation of polyols or sugars. Therefore the THPs can be more easily accumulated and maintained during warm periods in anticipation of subzero temperatures. It is not surprising then that photoperiod, as well as temperature, is a critical environmental cue in the control of THP levels in insects.Some species of freeze-tolerant insects also produce THPs. This appears somewhat odd, since most freeze-tolerant insects produce ice nucleators which function to inhibit supercooling and it is therefore not clear why such an insect would produce antifreeze proteins. It is possible that the THPs have an alternate function in these species. However, it also appears that the THPs function as antifreezes during those periods of the year when these insects are not freeze tolerant (i.e., early autumn and spring) but when subzero temperatures could occur. In addition, at least one freeze-tolerant insect which produces THPs, Dendroides canadensis, typically loses freeze tolerance during midwinter thaws and then regains tolerance. The THPs could be important during those periods when Dendroides loses freeze tolerance by making the insect less susceptible to sudden temperature decreases.Comparatively little is known of the biochemistry of insect THPs. However, comparisons of those few insect THPs which have been purified with the THPs of fishes show some interesting differences. The insect THPs lack the large alanine component commonly found in the fish THPs. In addition, the insect THPs generally contain greater percentages of hydrophilic amino acids than do those of the fish. Perhaps the most interesting insect THPs are those from Tenebrio molitor which have an extremely large cysteine component (28% in one THP). Studies on the primary and higher-order structure of the insect THPs need to be carried out so that more critical comparisons with the fish THPs can be made. This may provide important insights into the mechanisms of freezing point and supercooling point depression exhibited by these molecules. In addition, comparative studies of the freezing and supercooling point depressing activities of the various THPs, in relation to their structures, should prove most interesting.It has become increasingly apparent over the last few years that most freeze-tolerant insects, unlike freeze-susceptible species, inhibit supercooling by accumulating ice-nucleating agents in their hemolymph. These nucleators function to ensure that ice formation occurs in the extracellular fluid at fairly high temperatures, thereby minimizing the possibility of formation of lethal intracellular ice. Little is known of the nature of the insect ice-nucleating agents. Those few which have been studied are heat sensitive and nondialyzable and are inactivated by proteolytic enzymes, thus indicating that they are proteinaceous. Studies on the structure-function relationships of these unique molecules should be done.     

Subzero temperature tolerance in spiders: The role of thermal-hysteresis-factors

Summary The immature stages of two species of spiders which overwinter under the bark of standing dead trees survive subzero temperatures by depressing their supercooling points in winter. These are a crab spider,Philodromus sp. (Philodromidae), and a sac spider,Clubiona sp. (Clubionidae). The solutes which are at least partially responsible for the decrease in supercooling points in winter are: (1) proteins which produce a thermal hysteresis (a difference between the freezing and melting points) of approximately 2°C in the hemolymph and (2) glycerol. The thermal-hysteresis-factors and glycerol are only found in the spiders in winter. Acclimation of winter spiders to warm temperatures, at either long or short photoperiods, results in loss of the thermal hysteresis within two weeks. These thermal-hysteresis-factors appear to be similar to protein and glycoprotein antifreezes previously found in polar marine fishes and certain overwintering insects.     

Freezing avoidance of the Antarctic icefishes (Channichthyidae) across thermal gradients in the Southern Ocean

Biogeographic studies separate the Antarctic Notothenioid fish fauna into high- and low-latitude species. Past studies indicate that some species found in the high-latitude freezing waters of the High-Antarctic Zone have low-serum hysteresis freezing points, while other species restricted to the low-latitude seasonal pack ice zone have higher serum hysteresis freezing points above the freezing point of seawater (−1.9°C), but the relationship has not been systematically investigated. Freeze avoidance was quantified in 11 species of Antarctic icefishes by determining the hysteresis freezing points of their blood serum, in addition, the freezing point depression from serum osmolytes, the antifreeze activity from serum antifreeze glycoproteins (AFGPs), and the antifreeze activity from serum antifreeze potentiating protein were measured for each species. Serum hysteresis freezing point, a proxy for organismal freeze avoidance, decreased as species were distributed at increasing latitude (linear regression r ² 0.66, slope −0.046°C °latitude⁻¹), which appeared largely independent of phylogenetic influences. Greater freeze avoidance at high latitudes was largely a result of higher levels of antifreeze activity from serum AFGPs relative to those in species inhabiting the low-latitude waters. The icefish fauna could be separated into a circum High-Antarctic Group of eight species that maintained serum hysteresis freezing points below −1.9°C even when sampled from less severe habitats. The remaining three species with low-latitude ranges restricted to the waters of the northern part of the west Antarctic Peninsula and Scotia Arc Islands had serum hysteresis freezing points at or above −1.9°C due to significantly lower combined activity from all of their serum antifreeze proteins than found in the High-Antarctic Zone icefish.     

Activation of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Purified antifreeze proteins (AFPs) from the larvae of the beetle Dendroides canadensis do not produce the high levels of antifreeze activity seen in the hemolymph of overwintering larvae, even when the purified AFPs are assayed at very high concentrations. However, addition of certain proteins or agar (at concentrations sufficiently low that the gel state does not result) to the Dendroides AFP resulted in a 2–3-fold increase in activity. A 70-kDa protein with AFP-activating capabilities was purified from Dendroides larvae. Addition of this endogenous activator protein to a 4 mg·ml^-1 solution of AFP increased the activity of the AFPs to values comparable to those of the hemolymph of overwintering larvae. Data derived from a modified immunoblot technique demonstrate that the activators bind to the AFP, or vice versa. Formation of this association must allow the AFP to block ice crystal growth by binding to the surface of potential seed crystals in the normal fashion. However, because the AFP-activator complex is much larger than the AFP alone, the complex probably blocks a greater surface area of the crystal and is thus a more efficient antifreeze.Abbreviations AFP antifreeze protein - BSA bovine serum albumine - DEAE diethylaminoethyl - Ig immunoglubolin - LPIN lipoprotein ice nucleator - PIN protein ice nucleator - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TH thermal hysteresis     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

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.     

Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.     

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (∼2 °C) and the gut contents somewhat lower levels (∼0.6 °C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14 kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.     

A thaumatin-like protein from larvae of the beetle Dendroides canadensis enhances the activity of antifreeze proteins

The levels of thermal hysteresis (antifreeze activity) produced by purified antifreeze proteins (DAFPs) from the larvae of the beetle Dendroides canadensis at endogenous concentrations are lower than what are present in the hemolymph of overwintering larvae. Thermal hysteresis activity of DAFPs is dependent not only on AFP concentration but also on the presence of enhancers that may be either proteins (including other hemolymph DAFPs) or low-molecular mass enhancers such as glycerol. The purpose of this study was to identify endogenous protein enhancers using yeast two-hybrid, co-immunoprecipitation, and finally the enhancement of antifreeze activity. Here we show that a thaumatin-like protein from D. canadensis, until recently known only from plants, significantly enhances the thermal hysteresis of DAFP-1 and -2. Glycerol can further this enhancement, presumably by promoting the interaction of the DAFPs and thaumatin-like protein.     

Respiration of individual honeybee larvae in relation to age and ambient temperature

The CO₂ production of individual larvae of Apis mellifera carnica, which were incubated within their cells at a natural air humidity of 60–80%, was determined by an open-flow gas analyzer in relation to larval age and ambient temperature. In larvae incubated at 34 °C the amount of CO₂ produced appeared to fall only moderately from 3.89±1.57 µl mg^–1 h^–1 in 0.5-day-old larvae to 2.98±0.57 µl mg^–1 h^–1 in 3.5-day-old larvae. The decline was steeper up to an age of 5.5 days (0.95±1.15 µl mg^–1 h^–1). Our measurements show that the respiration and energy turnover of larvae younger than about 80 h is considerably lower (up to 35%) than expected from extrapolations of data determined in older larvae. The temperature dependency of CO₂ production was determined in 3.5-day-old larvae, which were incubated at temperatures varying from 18 to 38 °C in steps of 4 °C. The larvae generated 0.48±0.03 µl mg^–1 h^–1 CO₂ at 18 °C, and 3.97±0.50 µl mg^–1 h^–1 CO₂ at 38 °C. The temperature-dependent respiration rate was fitted to a logistic curve. We found that the inflection point of this curve (32.5 °C) is below the normal brood nest temperature (33–36 °C). The average Q₁₀ was 3.13, which is higher than in freshly emerged resting honeybees but similar to adult bees. This strong temperature dependency enables the bees to speed up brood development by achieving high temperatures. On the other hand, the results suggest that the strong temperature dependency forces the bees to maintain thermal homeostasis of the brood nest to avoid delayed brood development during periods of low temperature.Abbreviations m body mass - R rate of development or respiration - T_I inflexion point of a logistic (sigmoid) curve - T_L lethal temperature - T_O temperature of optimum (maximum) developmentCommunicated by G. Heldmaier     

Cold hardiness in Entomobrya nivalis (Collembola, Entomobryidae): annual cycle of polyols and antifreeze proteins, and antifreeze triggering by temperature and photoperiod

In the Swiss Prealps Entomobrya nivalis hibernates in an inactive state, hidden under bark flakes on spruce. For freeze avoidance it relies on thermal hysteresis proteins (THPs) and polyols (mainly ribitol, with small amounts arabitol and threitol). Polyols are present only during the inactive state, THPs additionally protect during the transition phase in spring and autumn, when animals are still active but frosts may occur. Peak values were recorded in February/March for THPs (3.5 °C hysteresis between melting and freezing point) and for polyols (26 μg mg⁻¹ FW; hemolymph osmolality 680 mosmol l⁻¹). E. nivalis is able to control its hemolymph osmolality independently of body water content. Mean osmolality in summer was 350– 440 mosmol l⁻¹, in winter it was elevated to 650 mosmol l⁻¹, due to a synthesis mainly of ribitol. Body water content varied between 1.8 and 3.3 mg H₂O mg⁻¹ DW, depending on humidity conditions. Experiments on triggering of antifreeze synthesis showed the action of temperature and photoperiod as cues, but there was also evidence for an endogenous rhythm. No clear correlation between antifreeze concentration and supercooling ability could be established, suggesting that gut content or other parameters also play an inportant role. Accepted: 18 November 1995     

Glycerol metabolism in a freeze-tolerant arctic insect: an in vivo13C NMR study

Summary Freeze-tolerance in larvae ofGynaephora groenlandica is enhanced by the accumulation of glycerol in the winter. Since summer larvae remain freeze-tolerant despite the lack of glycerol, we investigated glycerol metabolism as a function of acclimation and body temperature using non-invasive¹³C NMR spectroscopy. Major constituents of hemolymph isolated from cold- and warm-acclimated larvae were identified with the aid of standard NMR spectra and confirmed by TLC and GLC. Spectra obtained on live, warm-acclimated larvae showed the presence of lipids, glycogen, glucose, trehalose and amino acids. Similar spectra of cold-acclimated or previously frozen larvae showed the additional presence of glycerol. In vitro time-lapse¹³C spectra ofd-[1-¹³C]glucose added separately to hemolymph or extracted fat body tissue showed that glycerol is synthesized from glucose in the fat body tissue and distributed to the peripheral tissue via hemolymph. In vivo time-lapse¹³C spectra of cold- and warm-acclimated larvae were obtained after injection withd-[1-¹³C]glucose to monitor the production of labeled metabolic intermediates and end-products. [¹³C]Glycerol was produced between –30°C and 30°C but accumulated only below 5°C. Above 5°C glycerol was degraded and the¹³C label incorporated mainly into glycogen. The mechanism underlying temperature control of glycerol biosynthesis and degradation may provide a clue to the role of glycerol in enhancing freeze-tolerance in these insects.     

Cold-regulated proteins with potent antifreeze and cryoprotective activities in spruces (Picea spp.)

Antifreeze proteins (AFPs) were obtained from intercellular spaces of spruce needles Picea abies (L.) Karst. and Picea pungens Engelm. by vacuum infiltration with ascorbic acid, followed by centrifugation to recover the infiltrate. As shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), apoplastic proteins are accumulated in these spruce species as a group of 5–9 polypeptide bands. These proteins have a molecular mass of 7–80 kDa. The spruce AFPs have the ability to modify the growth of ice and thermal hysteresis, TH, caused by these AFPs was close to 2.0 °C at a concentration of 400 μg/ml. The antifreeze activity of proteins from these winter-hardy coniferous species showed a positive correlation with the concentration of proteins after cold acclimation of needle tissues. Apoplastic proteins from winter spruce needles exhibited antifreeze activity, whereas no such activity was observed in extracts from summer needles. When we examined the possible role of spruce AFPs in cryoprotection, we found that lactate dehydrogenase, LDH, activity was higher after freezing in the presence of AFPs compared with bovine serum albumin. Amino-terminal sequence comparisons indicated that a 27-kDa protein from both P. abies and P. pungens was similar to some pathogenesis-related proteins namely chitinases, also from conifer species. These results show that spruces produce AFPs that are secreted into the apoplast of needles. The accumulation of AFPs in extracellular spaces caused by seasonal cold acclimation during winter indicates that these proteins may play a role in the acquisition of freezing tolerance of needle cells in coniferous species.     

Adaptations to terrestrial overwintering of hatchling northern map turtles,<Emphasis Type="Italic"> Graptemys geographica</Emphasis>

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle (Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to –5.4 °C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g^–1 day^–1), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at –2.5 °C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to –14.8 °C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to –8 °C. Others, held at –6 °C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N₂ gas for 8 days, may promote survival during exposure to subfreezing temperatures.Abbreviations EWL evaporative water loss - FP_eq equilibrium freezing point - INA ice-nucleating agents - T_c temperature of crystallizationCommunicated by L.C.-H. Wang     

Freeze or dehydrate: only two options for the survival of subzero temperatures in the arctic enchytraeid<Emphasis Type="Italic"> Fridericia ratzeli</Emphasis>

Hygrophilic soil animals, like enchytraeids, overwintering in frozen soil are unlikely to base their cold tolerance on supercooling of body fluids. It seems more likely that they will either freeze due to inoculative freezing, or dehydrate and adjust their body fluid melting point to ambient temperature as has been shown for earthworm cocoons and Collembola. In the present study we tested this hypothesis by exposing field-collected adult Fridericia ratzeli from Disko, West Greenland, to freezing temperatures under various moisture regimes. When cooled at –1 °C min^–1 under dry conditions F. ratzeli had a mean temperature of crystallisation (T_c) of –5.8 °C. However, when exposed to temperatures above standard T_c for 22 h, at –4 °C, most individuals (90%, n= 30) remained unfrozen. Slow cooling from –1 °C to –6 °C in vials where the air was in equilibrium with the vapour pressure of ice resulted in freezing in about 65% of the individuals. These individuals maintained a normal body water content of 2.7–3.0 mg mg^–1 dry weight and had body fluid melting points of about –0.5 °C with little or no change due to freezing. About 35% of the individuals dehydrated drastically to below 1.1 mg mg^–1 dry weight at –6 °C, and consequently had lowered their body fluid melting point to ca. –6 °C at this time. Survival was high in both frozen and dehydrated animals at –6 °C, about 60%. Approximately 25% of the animals (both frozen and dehydrated individuals) had elevated glucose concentrations, but the mean glucose concentration was not increased to any great extent in any group due to cold exposure. The desiccating potential of ice was simulated using aqueous NaCl solutions at 0 °C. Water loss and survival in this experiment were in good agreement with results from freezing experiments. The influence of soil moisture on survival and tendency to dehydrate was also evaluated. However, soil moisture ranging between 0.74 g g^–1 and 1.15 g g^–1 dry soil did not result in any significant differences in survival or frequency of dehydrated animals even though the apparent wetness and structure of the soil was clearly different in these moisture contents.Abbreviations DW dry weight - FW fresh weight - MP melting point - RH relative humidity - Tc crystallisation temperatures - WC water contentCommunicated by I.D. Hume     

Expression of Two Self-enhancing Antifreeze Proteins from the Beetle <Emphasis Type="Italic">Dendroides canadensis</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T ₃ DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.