Inoculative Freezing Research Articles

Antifreeze protein complements cryoprotective dehydration in the freeze-avoiding springtail Megaphorura arctica

The springtail, Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.

Supercooling and freezing as eco-physiological alternatives rather than mutually exclusive strategies: A case study in Pyrrhocoris apterus

Overwintering insects are categorized either as freeze tolerant or freeze avoiding (supercooling) based on their ability or inability, respectively, to tolerate the formation of ice in their body. The freeze tolerant insects set their supercooling point (SCP) higher for winter to stimulate freezing at higher temperatures, while freeze avoiding insects survive winter in a supercooled state by depressing their SCP. Some supercooling insects, however, were found to survive in frozen state when freezing occurred through inoculation by external ice at mild subzero temperatures. Here, we assessed the potential relevance of inoculative freezing and freeze tolerance strategy in an insect that was so far considered as a classical example of a ‘supercooler’, the linden bug (Pyrrhocoris apterus). Microclimatic conditions of the overwintering microhabitat of P. apterus (leaf litter layer with buffered temperature fluctuations, mild sub-zero extremes, high humidity, and presence of ice) present a potentially high risk of inoculative freezing. We found that P. apterus is highly susceptible to inoculation by external ice. The temperature at which inoculative freezing occurred (above −3°C) was much higher compared to SCP (−16 °C to −20 °C in winter). The insects were inoculated through body openings and across cuticle and were able to survive after freezing. There was, however, a distinct critical ice fraction, corresponding to 38.7–42.8% of total body water, beyond which survival rapidly decreased to zero. We found that P. apterus adaptively reduces the actual ice fraction below critical ice fraction for winter season. Since many insect species overwinter in habitats similar to that of P. apterus, the ability to tolerate freezing after inoculation by external ice crystals could be much more common among ‘supercooling’ insects than it is currently appreciated.

Reconstruction of the repetitive antifreeze glycoprotein genomic loci in the cold-water gadids Boreogadus saida and Microgadus tomcod

Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses. The BAC library construction encountered poor cloning efficiency initially, which we resolved by pretreating the agarose-embedded erythrocyte DNA with a cationic detergent, a method that may be of general use to BAC cloning for teleost species and/or where erythrocytes are the source of input DNA. The polar cod BAC library encompassed 92,160 clones with an average insert size of 94.7 kbp, and the Atlantic tomcod library contained 73,728 clones with an average insert size of 89.6 kbp. The genome sizes of B. saida and M. tomcod were estimated by cell flow cytometry to be 836 Mbp and 645 Mbp respectively, thus their BAC libraries have approximately 10- and 9.7-fold genome coverage respectively. The inclusiveness and depth of coverage were empirically confirmed by screening the libraries with three housekeeping genes. The BAC clones that mapped to the AFGP genomic loci of the two gadids were then isolated by screening the BAC libraries with gadid AFGP gene probes. Eight minimal tiling path (MTP) clones were identified for B. saida, sequenced, and assembled. The B. saida AFGP locus reconstruction produced both haplotypes, and the locus comprises three distinct AFGP gene clusters, containing a total of 16 AFGP genes and spanning a combined distance of 512 kbp. The M. tomcod AFGP locus is much smaller at approximately 80 kbp, and contains only three AFGP genes. Fluorescent in situ hybridization with an AFGP gene probe showed the AFGP locus in both species occupies a single chromosomal location. The large AFGP locus with its high gene dosage in B. saida is consistent with its chronically freezing high Arctic habitats, while the small gene family in M. tomcod correlates with its milder habitats in lower latitudes. The results from this study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms and history of gadid AFGP gene evolution driven by northern hemisphere glaciation.

Thermal analysis of ice and glass transitions in insects that do and do not survive freezing.

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to the solid phase and cells undergo freeze-induced dehydration. Here, we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IFmax) ranged between 67.9% and 77.7% total body water (TBW). Chymomyza costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IFmax) upon entry into diapause, cold acclimation and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol kg-1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.

Low and high thermal tolerance characteristics for unfed larvae of the winter tick Dermacentor albipictus (Acari: Ixodidae) with special reference to moose

We report that larvae of the winter tick Dermacentor albipictus, the only stage that will quest for a host, can tolerate short-term cold shock down to −25°C and short-term heat shock as high as 46°C. Unlike a three host-tick, larvae of D. albipictus have no preconditioning response to low or high temperature exposure by rapid cold hardening (RCH) or heat hardening, and poor ability to acclimate to low and high temperature extremes. Thermal tolerance limits were not improved as the result of larval clustering, and there was only a minimal effect due to changes in photoperiod. These larvae are freeze intolerant and die at higher temperatures (−5 to −10°C) from contact with ice by inoculative freezing. In absence of cold-associated resistance mechanisms, winter survival requires that larvae procure a host before the first snow cover. Their low and high temperature tolerance, however, is a key survival element that adapts them for off-host periods during summer, which in the arctic could allow for northern expansion.

Cold hardiness and influences of hibernaculum conditions on overwintering survival of American dog tick larvae

Understanding how ticks survive the multitude of stresses experienced during winter is integral to predicting population dynamics and transmission of tick-borne pathogens. The American dog tick (Dermacentor variabilis), a predominant vector of Rocky Mountain spotted fever, overwinters in any of its post-egg life stages. In this study, we characterized the cold tolerance of larval D. variabilis and examined the effect of various acclimatory conditions on cold hardiness. Cold-shock survival during 2h exposure to various subzero temperatures was assessed and a lower lethal temperature of −20°C and a 50% mortality temperature near −16°C was established. Larvae exposed to −5°C showed high survival (∼70%) after 14 d and near 50% for up to 56d at −5°C. Larvae cycled between supra- and subzero temperatures showed better long-term survival than at constant −5°C. The temperature of crystallization (Tc) was ∼−23°C and no larvae survived freezing after reaching their Tc. Larvae exposed to inoculative freezing survived brief, mild treatments (70% survival of −5°C for 2h) but survival was reduced compared to larvae cooled in the absence of ice. Reduced photophase, rapid cold hardening, dehydration, and long-term thermal acclimation all improved larval cold hardiness to varying degrees. Survival data were compared to measurements of hibernacula conditions, and we conclude that larvae face little threat from cold-induced mortality but inoculative freezing does pose a risk, and the geographic distribution of D. variabilis is only minimally influenced by the ability of larvae to survive low temperature exposure.

Efficiency of beetle (Dendroides canadensis) recombinant antifreeze protein for buffalo semen freezability and fertility

Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo (Bubalus bubalis) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 106 sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 μg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concentrations did not affect sperm progressive motility or plasma membrane integrity (PMI), either before or after the semen was cooled to 4 °C in 2 hours. However, after 24 hours of cryostorage at −196 °C, followed by thawing at 37 °C for 30 seconds, sperm progressive motility and PMI were higher (P < 0.05) in extender containing DAFP at 10 μg/mL compared with control. The in vitro–fertilizing ability of cryopreserved (−196 °C) sperm supplemented with DAFP (10 μg/mL) was slightly higher (P = 0.098) compared with control, as assessed through in vitro cleavage rate of in vitro matured buffalo oocytes. Also, the in vivo fertility rate was evaluated by inseminating 100 buffaloes (50 inseminations per extender) 12 hours after standing heat. The fertility rate of cryopreserved buffalo bull sperm in terms of positive pregnancy at 90 days after insemination was clinically higher but remained statistically nonsignificant in extender containing DAFP at 10 μg/mL (52.0%) compared with control (43.8%). In conclusion, supplementation of 10 μg/mL of DAFP in the extender improved the motility and PMI of Nili-Ravi buffalo sperm after freeze-thawing, and yielded numerically higher, although statistically nonsignificant, in vitro cleavage, and in vivo fertility rate.

Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming.

Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals.

Choice or constraint: Plasticity in overwintering sites of the European spruce bark beetle

Winter mortality is an important driver in bark beetle population dynamics. Overwintering site – be it in the soil or within the tree – can influence mortality, because temperature dynamics can differ between soil and tree at the same site. For Ips typographus L. (Coleoptera, Curculionidae, Scolytinae), it is not yet definitely resolved which of the two aforementioned is its overwintering site. Both sites, sometimes even mixed within the same population, have been reported in earlier studies. Here we studied the underlying mechanism of soil overwintering in I. typographus. We used soil emergence traps at the base of infested trees with variable amounts of fallen bark during winter. The number of soil overwintering beetles increased with increasing amount of fallen bark and decreased with increasing distance from the stem base of infested trees. Removing the bark beneath soil emergence traps resulted in a smaller number of emerged beetles, indicating that a majority of beetles was located in the fallen bark and not in the litter. Thus, soil hibernation in our study area in Central Europe occurred passively, mediated through falling bark.Furthermore, we studied post-diapause fitness in terms of body weight and lipid reserves in dependence of overwintering site (tree vs. soil). Fresh and dry masses of beetles were higher before than after winter, but not different after winter between overwintering sites. Lipid reserves were more depleted in tree overwintering beetles. Thus, the soil seems to be more advantageous as overwintering site from an energetic point of view. However, both possible overwintering sites have different other advantages and mortality risks regarding onset of flight in spring, inoculative freezing, fungal infection or predation risk.The mixed overwintering sites, caused by individuals’ active movement to the drier part of the bark and remaining in the stem or a passive dropping to the soil via fallen bark, spread winter mortality risks within populations and thus may contribute to local population stability. This phenomenon complicates forest management measures because a considerable number of beetles may be hibernating within fallen bark on the ground. Thus, when trees infested during the summer swarming period are not removed as long as bark is still in close contact with the stem, a great number of beetles can remain in the forest within fallen bark, even when trees are removed at a later point.

Wet hibernacula promote inoculative freezing and limit the potential for cryoprotective dehydration in the Antarctic midge, Belgica antarctica

The terrestrial midge, Belgica antarctica, occupies a diverse range of microhabitats along the Antarctic Peninsula. Although overwintering larvae have the physiological potential to survive by freezing or cryoprotective dehydration, use of the latter strategy may be constrained by inoculative freezing within hibernacula. To investigate the influence of microhabitat type on larval overwintering, we selected four substrate types that differed markedly in their composition and hydric characteristics. Organic content of these substrates ranged from 14 to 89 %. High organic content was associated with higher values for saturation moisture content (up to 2.0 H2O g−1 dry mass) as well as elevated levels of field moisture content. Seasonal values of field moisture content remained near or above the saturation moisture value for each microhabitat type, and when larvae were cooled in substrates rehydrated to field-based levels, they were unable to avoid inoculation by environmental ice, regardless of substrate type. Consequently, our data suggest that wet hibernacula would force most larvae to overwinter in a frozen state. Yet, dehydrated larvae were collected in April during the seasonal transition to winter suggesting that spatial and temporal variations in precipitation and microhabitat conditions may expose larvae to dehydration and promote larval overwintering in a cryoprotectively dehydrated state.

Avoidance and tolerance of freezing in ectothermic vertebrates

Ectothermic vertebrates have colonized regions that are seasonally or perpetually cold, and some species, particularly terrestrial hibernators, must cope with temperatures that fall substantially below 0°C. Survival of such excursions depends on either freeze avoidance through supercooling or freeze tolerance. Supercooling, a metastable state in which body fluids remain liquid below the equilibrium freezing/melting point, is promoted by physiological responses that protect against chilling injury and by anatomical and behavioral traits that limit risk of inoculative freezing by environmental ice and ice-nucleating agents. Freeze tolerance evolved from responses to fundamental stresses to permit survival of the freezing of a substantial amount of body water under thermal and temporal conditions of ecological relevance. Survival of freezing is promoted by a complex suite of molecular, biochemical and physiological responses that limit cell death from excessive shrinkage, damage to macromolecules and membranes, metabolic perturbation and oxidative stress. Although freeze avoidance and freeze tolerance generally are mutually exclusive strategies, a few species can switch between them, the mode used in a particular instance of chilling depending on prevailing physiological and environmental conditions.

Overwintering Physiology and Microhabitat Use of Phyllocnistis populiella (Lepidoptera: Gracilliariidae) in Interior Alaska

We investigated the overwintering physiology and behavior of Phyllocnistis populiella Chambers, the aspen leaf miner, which has caused severe and widespread damage to aspen in Alaska over the past 10 yr. Active P. populiella moths caught in spring and summer supercooled to an average temperature of -16°C, whereas dormant moths excavated from hibernacula in the leaf litter during fall and winter supercooled to an average of -32°C. None of the moths survived freezing in the laboratory. Counts of overwintering moths in leaf litter across microhabitats in interior Alaska demonstrated that moths occurred at significantly higher density beneath white spruce trees than beneath the aspen host, several other hardwood species, or in open areas among trees. During winter, the temperature 1-2 cm below the surface of the leaf litter beneath white spruce trees was on average 7-9°C colder than beneath aspen trees, and we estimate that during at least one period of the winter the temperature under some white spruce trees may have been cold enough to cause mortality. However, the leaf litter under white spruce trees was significantly drier than the litter from other microhabitats, which may assist P. populiella moths to avoid inoculative freezing because of physical contact with ice. We conclude that in interior Alaska, P. populiella overwinter in a supercooled state within leaf litter mainly under nonhost trees, and may prefer relatively dry microhabitats over moister ones at the expense of lower environmental temperature.

False codling moth Thaumatotibia leucotreta (Lepidoptera, Tortricidae) larvae are chill‐susceptible

Abstract This study reports on the low temperature tolerance and cold hardiness of larvae of false codling moth, Thaumatotibia leucotreta. We found that larvae have mean critical thermal minima (lower limits of activity) of 6.7°C which was influenced by feeding status. The effects of low temperature exposure and duration of exposure on larval survival were assessed and showed that the temperature at which 50% of the population survives is −11.5 ± 0.3°C after 2 h exposure. The supercooling point (SCP, i.e., freezing temperature) was investigated using a range of cooling rates and under different conditions (feeding and hydration status) and using inoculative freezing treatments (in contact with water or orange juice). The SCP decreased significantly from −15.6°C to −17.4°C after larvae were fasted for 24 h. Twenty‐four hour treatments at either high or low relative humidity (95.9% or 2.4%) also significantly decreased SCP to −17.2°C and −18.2°C respectively. Inoculative freezing (by water contact) raised SCP from −15.6°C to −6.8°C which could have important implications for post‐harvest sterilization. Cooling rates did not affect SCP which suggests that there is limited phenotypic plasticity of SCP during the larval life‐stage, at least over the short time‐scales investigated here. In conclusion, larvae of T. leucotreta are chill‐susceptible and die upon freezing. These results are important in understanding this pest's response to temperature variation, understanding pest risk status and improving post‐harvest sterilization efficacy.

Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica: a comparison between frozen and supercooled larvae

In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at -5°C in the presence of water during RCE and those that were supercooled at -5°C in a dry environment. Although >95% of larvae survived a single 12 h bout of freezing at -5°C, after five cycles of RCE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during RCE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during RCE had higher expression of hsp70 than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60 h of freezing at -5°C over five cycles of RCE and those that were frozen continuously for 60 h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during RCE.

Cold tolerance in sealworm (Pseudoterranova decipiens) due to heat-shock adaptations

Third-stage larvae of Pseudoterranova decipiens commonly infect whitefish such as cod, and the parasite can be transferred to humans through lightly prepared (sushi) meals. Because little is known about the nematode's cold tolerance capacity, we examined the nematode's ability to supercool, and whether or not cold acclimation could induce physiological changes that might increase its ability to tolerate freezing conditions. Even if third-stage Pseudoterranova decipiens larvae have some supercooling ability, they show no potential for freezing avoidance because they are not able to withstand inoculative freezing. Still, they have the ability to survive freezing at high subzero temperatures, something which suggests that these nematodes have a moderate freeze tolerance. We also show that acclimation to high temperatures triggers trehalose accumulation to an even greater extent than cold acclimation. Trehalose is a potential cryoprotectant which has been shown to play a vital role in the freeze tolerance of nematodes. We suggest that the trehalose accumulation observed for the cold acclimation is a general response to thermal stress, and that the nematode's moderate freeze tolerance may be acquired through adaptation to heat rather than coldness.

Simultaneous freeze tolerance and avoidance in individual fungus gnats, Exechia nugatoria

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean +/- SEM: -31.5 +/- 0.2 degrees C, 70% survival and -50.7 +/- 0.4 degrees C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (-30.1 +/- 1.1 degrees C) and head/thorax freezing (-48.7 +/- 1.3 degrees C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 +/- 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 +/- 0.8% water).

Cryoprotective dehydration and the resistance to inoculative freezing in the Antarctic midge, Belgica antarctica

During winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7-8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to -3 degrees C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by approximately 40% and depressed the body fluid melting point more than threefold to -2.6 degrees C. This melting point depression was the result of the concentration of existing solutes (i.e. loss of body water) and the de novo synthesis of osmolytes. By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the insect's intimate contact with environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter.

Influence of soil moisture on egg cold hardiness in the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

Abstract The present study investigates the influence of environmental moisture on cold hardiness of the migratory locust, Locusta migratoria. The water content of locust eggs kept in soil at 30 °C varies according to the moisture content of the substrate. In turn, it can significantly affect the supercooling point of locust eggs (range from −26 to −14.8 °C) and the mortality when exposed to subzero temperatures. Environmental moisture influences the supercooling capacity of eggs and their survival at low temperature. When locust eggs of the same water content are exposed to subzero temperatures under different soil moistures, their mortality varies between short‐time exposure and long‐time exposure at subzero temperatures. Given a short‐time exposure, mortality in wet soil is lower than in dry soil due to the buffering effect of soil water against temperature change. The pattern of egg mortality is reversed after long‐time exposure at low temperature, suggesting that inoculative freezing may be an important mortality factor. It is suggested that interactions between soil moisture and low temperature can influence the cold hardiness of locust eggs, and partial dehydration is beneficial to over‐wintering eggs of the migratory locust.

Temperature, humidity and inoculative freezing are all signals for glucose accumulation in the freeze tolerant earthworm Dendrobaena octaedra

Temperature, humidity and inoculative freezing are all signals for glucose accumulation in the freeze tolerant earthworm Dendrobaena octaedra

Responses of springtail and mite populations to prolonged periods of soil freeze-thaw cycles in a sub-arctic ecosystem

The effect of temperature changes on soil communities is an important aspect when estimating the effects of a predicted climate change. The aim of this investigation was to increase knowledge on how freeze-thaw cycles alter the soil microarthropod community in the sub-arctic. The abundance of springtails and mites was investigated after three seasons of prolonged periods of freeze-thaw cycles in the field, and the presence or absence of migration barriers, at two different field sites. Dome shaped transparent plastic greenhouses were successfully used as a novel method to increase freeze-thaw cycle frequencies in the soil. At a fellfield site, freeze-thaw treatment did not lead to significant differences in the five main soil faunal groups, but increased abundance were seen in a number of separate taxa. There was no freeze-thaw treatment effect on soil microbial biomass or soil nutrients, although treatments interacted as inorganic N increased in the separate freeze-thaw and migration barrier treatments. By contrast, at a glade site responses were strong due to more pronounced increases in the number of freeze-thaw cycles. The highest numbers of Collembola after 2 years of treatment were found in the freeze-thaw plots, in combination with migration barriers. The freeze-thaw treatment here also resulted in more Oribatida, microbial biomass C and dissolved organic C. A common hypothesis is that an increased number of freeze-thaw cycles would result in elevated winter mortality in microarthropods due to increased risk of inoculative freezing. However, we observed no increased mortality due to freeze-thaw events. Rather, there was a stimulation of soil microarthropods and microbial biomass, perhaps due to a prolonged period of microbial and faunal activity when the soil is repeatedly frozen and thawed compared to a constantly frozen soil.