Freeze Avoidance Research Articles

The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Constraints on habitat possibilities: overwintering of a micro snail species facing climate change consequences in a harsh environment

The aim of this study was to establish the overwintering strategy and cold tolerance in V. moulinsiana. We observed a seasonal change in the SCP measurements, indicating that the species' preparation for winter results in a decrease in the SCP. Along with the results obtained from the frost survival experiment, this suggests the presence of a freezing avoidance strategy in this species. It overwinters in a buffered zone, separated from harsh conditions by a layer of leaf litter and snow, which protects it from temperature fluctuations and severe frost. The results of our research may indicate a possible poor resistance of these snails to snow-free conditions. Therefore, climate change-induced alterations could profoundly impact this species, especially considering the predicted disappearance of snow before the frost.

Effects of rapid cold-hardening and cold acclimation on egg survival and cryoprotectant contents in Ceracris kiangsu (Orthoptera: Arcypteridae)

Abstract The bamboo grasshopper, Ceracris kiangsu Tsai, is a serious pest of bamboo in China and Southeast Asia, and it overwinters as eggs for a period of up to 9 mo. Despite its importance, cold tolerance has not been thoroughly examined in this species. Thus, the present study investigated the low-temperature tolerance of overwintering eggs collected in the field, particularly the effects of rapid cold-hardening (RCH) and cold acclimation (ACC) on egg survival, and changes in the cryoprotectant contents. The supercooling point of overwintering eggs collected from the field was −23.9 °C, and their survival rate after exposure to −22 °C for 6 h was 26.6%, thereby suggesting that the cold tolerance strategy employed by C. kiangsu is freeze avoidance. After exposure to 0 and 4 °C for 12 and 24 h, or 0, 4, and 8 °C for 5 days, the survival rate of eggs subjected to −22 °C increased to approximately 50%, with significant effects of RCH and ACC. The glycerol levels in RCH and ACC treated eggs were also significantly higher than those in the control group, but the protein and trehalose contents were either reduced or not significantly altered. These results indicate that glycerol functions as a low molecular weight cryoprotectant in C. kiangsu eggs, and that RCH and ACC treatment facilitate its accumulation, thereby enhancing cold tolerance. Furthermore, the correlation between RCH and ACC was investigated.

Osmotic and metabolic responses to cold acclimation and acute cold challenge in a freeze avoidant lizard, Podarcis siculus

Ectotherms survive exposure to subzero temperatures through freeze tolerance or freeze avoidance. Among vertebrate ectotherms, glucose is commonly used as a cryoprotectant in freeze tolerant strategies and as an osmolyte in freeze avoidant strategies, while also functioning as a metabolic substrate. Whereas some lizard species are capable of both freeze tolerance and freeze avoidance, Podarcis siculus is limited to freeze avoidance through supercooling. We hypothesized that, even in a freeze-avoidant species such as P. siculus, plasma glucose would accumulate with cold acclimation and would increase in response to acute exposure to subzero temperatures. To investigate this, we tested whether plasma glucose concentration and osmolality would increase in response to a subzero cold challenge before and after cold acclimation. In addition, we examined the relationship between metabolic rate, cold acclimation, and glucose by measuring metabolic rate during the cold challenge trials. We found that plasma glucose increased during the cold challenge trials, and that the increase was more pronounced after cold acclimation. However, baseline plasma glucose decreased throughout cold acclimation. Interestingly, total plasma osmolality did not change, and the increase in glucose only slightly altered freezing point depression. Metabolic rate during the cold challenge decreased after cold acclimation, and changes in respiratory exchange ratio suggest an increased relative use of carbohydrates. Overall, our findings demonstrate an important role for glucose in the response of P. siculus to an acute cold challenge, thus adding evidence for glucose as an important molecule for overwintering ectotherms that use freeze avoidant strategies.

Biophysical characterization and applications of an insect antifreeze protein.

Nature employs a variety of compounds and strategies for freeze avoidance and freeze tolerance that enhance the survival of organisms in extreme cold environments. Antifreeze proteins have evolved in numerous species to protect from cellular damage at temperatures below freezing, and they function by adsorbing to the ice surface and inhibiting the growth of ice crystals. The goal of this project is to investigate the mechanism by which antifreeze proteins protect against the damage typically inflicted by the cold, including the underlying molecular mechanism of ice-binding. Here we are presenting structural and functional characterization of an antifreeze protein, ApAFP752 from the desert beetle Anatolica polita, utilizing nuclear magnetic resonance (NMR) spectroscopy and other biophysical methods, and we are also highlighting a few applications of this protein.

Microcurvature Controllable Metal-Organic Framework Nanoagents Capable of Ice-Lattice Matching for Cellular Cryopreservation.

Ice-binding proteins (IBPs) produced by psychrophilic organisms to adapt for the survival of psychrophiles in subzero conditions have received illustrious interest as a cryopreservation agent required for cells and tissues to completely recover after freezing/thawing. Depressing water-freezing point and avoiding ice-crystal growth affect their activities which are closely related to the presence of ice crystal well-matched binding moiety. The interaction of IBPs with ice and water is critical in enhancing their freeze avoidance against cell or tissue damage. Metal-organic frameworks (MOFs) with a controllable lattice at the molecular level and a size at the nanometer scale can offer periodically ordered ice-binding sites by modifying organic linkers and controlling microcurvature at the ice surface. Herein, zirconium (Zr)-based MOF-801 nanoparticles (NPs) with good biocompatibility were used as a cryoprotectant that is well dispersed and colloidal-stable in an aqueous solution. The MOF NP size was precisely controlled, and 10, 35, 100, and 250 nm NPs were prepared. The specific IBPs-mimicking pendants (valine and threonine) were simply introduced into the MOF NP-surface through the acrylate-based functionalization to endow with hydrophilic and hydrophobic dualities. When small-sized MOF-801 NPs were attached to ice, they confined ice growth in high curvature between the adsorption sites because of the decreased radius of the convex area of the growth region, leading to highly enhanced ice recrystallization inhibition (IRI). Surface-functionalized MOF NPs could increase the number of anchored clathrate water molecules with hydrophilic/hydrophobic balance of the ice-binding moiety, effectively inhibiting ice growth. The MOF-801 NPs were biocompatible with various cell lines regardless of concentration or NP surface-functionalization, whereas the smaller-sized surface-functionalized NPs showed a good cell recovery rate after freezing/thawing by induction of IRI. This study provides a strategy for the fabrication of low-cost, high-volume antifreeze nanoagents that can extend useful applications to organ transplantation, cord blood storage, and vaccines/drugs.

Preferential freezing avoidance localised in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualised by magnetic resonance imaging.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

Role of MicroRNAs in Extreme Animal Survival Strategies.

The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to play a major role. In this chapter, we discuss the activation, expression, biogenesis, and unique attributes of miRNA regulation required to facilitate profound metabolic rate depression and implement stress-specific metabolic adaptations. We examine the role of miRNA in strategies of biochemical adaptation including mammalian hibernation, freeze tolerance, freeze avoidance, anoxia and hypoxia survival, estivation, and dehydration tolerance. By comparing these seemingly different adaptive programs in traditional and exotic animal models, we highlight both unique and conserved miRNA-meditated mechanisms for survival. Additional topics discussed include transcription factor networks, temperature dependent miRNA-targeting, and novel species-specific and stress-specific miRNAs.

Delaying Budbreak to Reduce Freeze Damage: Seasonal Vine Performance and Wine Composition in Two Vitis vinifera Cultivars

Spring freeze events pose a threat to vineyard productivity worldwide. We compared two methods to delay grapevine budbreak for freeze avoidance and evaluated their effects on phenology, yield components, fruit composition, and postharvest parameters, including wine chemistry, carbohydrate storage, and bud freeze tolerance. The two methods to delay budbreak were a vegetable oil-based adjuvant (Amigo) applied to dormant buds at 8% and 10% (v/v) and late pruning applied when apical buds reached approximately Eichhorn-Lorenz stage 7. Treatments were applied in 2018 and 2019 on two <i>Vitis vinifera</i> cultivars, Lemberger and Riesling, and compared to a control treatment with no delayed budbreak strategy. Amigo and late pruning delayed budbreak compared to control vines in both years and cultivars. The delay in budbreak varied from three to six days for Amigo 8%, five to eight days for Amigo 10%, and 10 to 11 days later for late pruning. In 2019, there was a freezing event near budbreak. Compared to control vines, late-pruned Lemberger vines had less shoot damage when measured during the growing season and greater yield at harvest. Delayed budbreak treatments did not influence wine chemistry either year or consistently affect carbohydrate storage or bud freeze tolerance in the following dormant season. However, in Riesling, late pruning reduced cluster and berry weight by up to 34 and 22%, respectively, compared to control vines. Furthermore, Amigo 10% may decrease bud survival when applied to Riesling vines. In general, late pruning delayed budbreak more effectively and mitigated freeze damage better than Amigo application without negatively affecting vine health or wine composition; however, the cultivar-dependent effect of late pruning on cluster weight is a consideration prior to adoption.

Thermal adaptations of adults and eggs in the Arctic seed bug Nysius groenlandicus (Insecta: Hemiptera) from South Greenland

Temperatures in Arctic and sub-Arctic regions are highly variable, with long, and cold winters, and short summers. Studies on thermal biology of ectotherms from these latitudes have mostly focused on how organisms cope with cold temperatures. However, temperatures in microhabitats such as south-facing slopes can occasionally reach high and potentially stressful temperatures. Such microclimatic conditions are challenging for organisms, since they need to time their development to coincide with the short summer, while coping with thermal extremes during summer and winter. In the present study, we investigate upper and lower thermal limits of adults and eggs in Nysius groenlandicus, a univoltine seed bug widely distributed in Greenland and alpine areas of Scandinavia and with preference for dry and sunny habitats. Secondly, we investigate the survival strategy of the overwintering egg stage during cold exposure. We found a clear association between the microhabitat temperatures experienced by each specific life stage and their heat and cold tolerance. Thus, adults and eggs, which are both exposed to high summer temperatures, showed a high heat tolerance. Adults which do not overwinter had very poor cold tolerance, whereas eggs produced in autumn were very cold hardy (with some survival at − 21 °C). Cold tolerance of the eggs of N. groenlandicus was based on freeze avoidance through extensive supercooling and by conserving water. These results suggest life stage-specific adaptation to heat and cold stress in Arctic and sub-Arctic insects, and highlight new research avenues addressing adaptation to higher and more variable temperatures in Arctic and sub-Arctic regions.

MiR-31-5p regulates cold acclimation of the wood-boring beetle Monochamus alternatus via ascaroside signaling

BackgroundSurvival to cold stress in insects living in temperate environments requires the deployment of strategies that lead to physiological changes involved in freeze tolerance or freeze avoidance. These strategies may consist of, for instance, the induction of metabolic depression, accumulation of cryoprotectants, or the production of antifreeze proteins, however, little is known about the way such mechanisms are regulated and the signals involved in their activation. Ascarosides are signaling molecules usually known to regulate nematode behavior and development, whose expression was recently found to relate to thermal plasticity in the Japanese pine sawyer beetle Monochamus alternatus. Accumulating evidence also points to miRNAs as another class of regulators differentially expressed in response to cold stress, which are predicted to target genes involved in cold adaptation of insects. Here, we demonstrate a novel pathway involved in insect cold acclimation, through miRNA-mediated regulation of ascaroside function.ResultsWe initially discovered that experimental cold acclimation can enhance the beetle’s cold hardiness. Through screening and functional verification, we found miR-31-5p, upregulated under cold stress, significantly contributes to this enhancement. Mechanistically, miR-31-5p promotes production of an ascaroside (asc-C9) in the beetle by negatively targeting the rate-limiting enzyme, acyl-CoA oxidase in peroxisomal β-oxidation cycles. Feeding experiments with synthetic asc-C9 suggests it may serve as a signal to promote cold acclimation through metabolic depression and accumulation of cryoprotectants with specific gene expression patterns.ConclusionsOur results point to important roles of miRNA-mediated regulation of ascaroside function in insect cold adaptation. This enhanced cold tolerance may allow higher survival of M. alternatus in winter and be pivotal in shaping its wide distribution range, greatly expanding the threat of pine wilt disease, and thus can also inspire the development of ascaroside-based pest management strategies.

Cryo-scanning electron microscopy reveals that supercooling of overwintering buds of freezing-resistant interspecific hybrid grape ‘Yamasachi’ is accompanied by partial dehydration

Dormant compound buds of grapevines adapt to subfreezing temperatures through a freezing avoidance mechanism. One still-unclear question, however, is whether supercooled water in primordial cells of dormant grape buds are partially dehydrated under subfreezing temperatures. In this study, we used differential thermal analysis (DTA) and cryo-scanning electron microscopy (cryo-SEM) to look for partial dehydration of primordial cells of the freezing-resistant interspecific hybrid cultivar ‘Yamasachi’. According to DTA, the freezing temperature of supercooled water in primary buds was not significantly affected by cooling rates between 2 and 5 °C/h; however, maintaining the bud temperature at −15 °C for 12 h followed by cooling at a rate of 5 °C/h depressed the freezing temperature. As revealed by cryo-SEM observation, many wrinkles were present on inner surfaces of walls and outer surfaces of plasma membranes of leaf primordial cells in dormant buds frozen to −15 °C. These results suggest the existence of partial dehydration in dormant-bud primordial cells under subfreezing temperatures. The apparent absence of extracellular ice crystals in bud primordial tissues under subfreezing temperatures suggests that Yamasachi dormant buds adapt to subfreezing temperatures by extraorgan freezing. When we coated primary buds with silicone oil to inhibit freeze dehydration of primordial cells, the freezing temperature of buds was slightly but significantly increased. This result suggests that the partial dehydration of cells promotes bud supercooling capability and has an important role in the freezing adaptation mechanism of grapevines.

A comparison of low temperature biology of Pieris rapae from Ontario, Canada, and Yakutia, Far Eastern Russia

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. −9.3 °C and were freeze-tolerant in 2002–2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< −20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to −15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

Internal Seed Structure of Alpine Plants and Extreme Cold Exposure

Cold tolerance in seeds is not well understood compared to mechanisms in aboveground plant tissue but is crucial to understanding how plant populations persist in extreme cold conditions. Counter-intuitively, the ability of seeds to survive extreme cold may become more important in the future due to climate change projections. This is due to the loss of the insulating snow bed resulting in the actual temperatures experienced at soil surface level being much colder than without snow cover. Seed survival in extremely low temperatures is conferred by mechanisms that can be divided into freezing avoidance and freezing tolerance depending on the location of ice crystal formation within the seed. We present a dataset of alpine angiosperm species with seed mass and seed structure defined as endospermic and non-endospermic. This is presented alongside the locations of temperature minima per species which can be used to examine the extent to which different seed structures are associated with snow cover. We hope that the dataset can be used by others to demonstrate if certain seed structures and sizes are associated with snow cover, and if so, would they be negatively impacted by the loss of snow resulting from climate change.

Overwintering Red Velvet Mites Are Freeze Tolerant.

Although many arthropods are freeze tolerant (able to withstand internal ice), small-bodied terrestrial arthropods such as mites are thought to be constrained to freeze avoidance. We field-collected active adult red velvet mites, Allothrombium sp. (Trombidiidae), in winter in Southwestern Ontario, Canada, where temperatures drop below -20°C. These mites froze between -3.6° and -9.2°C and survived internal ice formation. All late-winter mites survived being frozen for 24 h at -9°C, and 50% survived 1 wk. The lower lethal temperature (LLT50; low temperature that kills 50% of mites) was ca. -20°C in midwinter. Hemolymph osmolality and glycerol concentration increased in midwinter, accompanied by decreased water content. Thus, this species is freeze tolerant, demonstrating that there is neither phylogenetic nor size constraint to evolving this cold tolerance strategy.

The frequency of freeze events in subtropical blueberry growing regions in Florida

Freeze events during January and February can result in major crop and economic losses for growers of low-chill, early-ripening cultivars of blueberry (Vaccinium spp.) in Florida. Overhead irrigation for freeze protection is commonly used by growers to protect their crop from freeze events. Freezes are typically categorized as either radiative or advective, each with their own set of risk parameters. The objective of this research was to use historical weather data to provide growers with additional information for their selection of passive and active freeze protection strategies to mitigate risk. Three sites in Florida were chosen as representative of major blueberry growing regions and weather data from 2000-2015 were downloaded from the Florida Automated Weather Network (FAWN) and analyzed. Hourly temperature, wind speed, and relative humidity were used to determine the average of number of days per month with sub-zero temperatures, the average duration of freeze events, and to classify each event as a radiation, advective, or mixed freeze. Radiation freezes were the most common type of freeze event in Alachua County, Florida, whereas Polk County has fewer freeze events including some years without sub-zero temperatures, without a clear prevalent freeze event type. Ultimately, the frequency and severity of freeze events, based on historical data, can be used to further inform grower investments in freeze avoidance and protection.

Physiological and Biochemical Differences in Diapausing and Nondiapausing Larvae of Eurytoma plotnikovi (Hymenoptera: Eurytomidae).

The pistachio seed wasp, Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae), is one of the main pests in various pistachio growing regions of Iran. This pest passes the winter as diapausing last instar larvae. In this study, the relationship between diapause and cold hardiness and also the physiological and biochemical characteristics the diapausing and nondiapausing larvae of E. plotnikovi were investigated. Digestive α-amylase enzyme showed a high activity (70.41 ± 2.36 µg maltose/min per mg protein) in nondiapausing larvae, but its activity vigorously decreased during the diapause period. Glycogen declined at the beginning of diapause until March. Decrease in glycogen content was proportional to increase in total simple body sugars, trehalose, myo-inositol, and sorbitol contents. Lipid accumulated from the onset of diapause in September until January reaching a high concentration of 28.74 mg/g fresh body weight, but then declined from March to end of diapause in April. The supercooling points were decreased from August (-17.68 ± 0.14°C) to January and reached to its lowest point in January (-23.14 ± 0.27°C), the coldest month of the year, then gradually increased through April (-21.38 ± 0.32°C). The survival rates at low temperature indicate that last instar larvae of E. plotnikovi are most cold tolerant in December-February when total body sugars, trehalose, myo-inositol, and sorbitol concentration is high, suggesting an alternative cryoprotective role for these compounds. The experimental data show that E. plotnikovi is freeze avoidance insect.

Elevated chaperone proteins are a feature of winter freeze avoidance by larvae of the goldenrod gall moth, Epiblema scudderiana

Winter survival for many insect species includes a need to maintain metabolic homeostasis and structural/functional integrity of macromolecules not only over a wide range of cold temperatures but also in response to rapid temperature change. Chaperones are well-known to protect/stabilize protein structure with regard to heat stress but less is known about their potential involvement in long-term protection of the proteome at subzero temperatures. The present study assessed the participation of chaperone proteins in the cold hardiness of larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae), monitoring changes in nine proteins over the winter months as well as their responses to laboratory cold acclimation or anoxia exposure. Four heat shock proteins (HSPs: Hsp110, Hsp70, Hsp60, Hsp40), three glucose-regulated proteins (GRPs: Grp78, Grp 94, Grp170) and the tailless complex polypeptide 1 (TCP-1) as well as the heat shock transcription factor (HSF1) were investigated. In general, all were significantly elevated in larvae collected from an outdoor site between October and March, as compared with September values, and chaperone levels were reduced again in April. The October to March interval also includes the period of diapause followed by cold quiescence in the species. Relative expression of Hsp70, Hsp60 and Hsp40 rose by 2–3-fold, GRPs increased 1.5–3-fold, and levels of active (hyperphosphorylated) HSF1 increased by 4–4.8-fold over the midwinter months. Chilling from 15°C to 4°C in the laboratory upregulated Grp78 protein content that remained high as temperature was further reduced to −4°C and then −20°C whereas Hsp110, Hsp70 and HSF1 levels increased when larvae were exposed to −4°C and −20°C. Grp170 (also known as oxygen-regulated protein 150) was the only chaperone that increased significantly in the larvae in response to anoxia exposure at 4°C. The data also indicated that multiple subcellular compartments received enhanced protection for their proteome since upregulation of chaperones included proteins known to occur in cytosolic (Hsp40, Hsp70), mitochondrial (Hsp60) and endoplasmic reticulum (Grp170) locations. Overall, the data indicate that chaperones have a significant role to play in the winter cold hardiness of E. scudderiana and identify declining temperatures (and perhaps also oxygen restriction) as potential modulators of chaperone production. The data add support to a relatively understudied area of insect cold hardiness – the long-term protection and stabilization of the proteome over the winter months.

Insect cold hardiness: the role of mitogen-activated protein kinase and Akt signalling in freeze avoiding larvae of the goldenrod gall moth, Epiblema scudderiana.

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of cold hardiness to survive the winter. Here we report that protein kinase-dependent signal transduction featuring mitogen-activated protein kinase (MAPK) signalling cascades (extracellular signal regulated kinase, c-jun N-terminal kinase and p38 MAPK pathways) and the Akt (also known as protein kinase B, or PKB) pathway could be integral parts of the development of cold hardiness by E. scudderiana. We used Luminex technology to assess the protein levels and phosphorylation status of key components and downstream targets of those pathways in larvae in response to low temperature acclimation. The data showed that MAPK pathways (both total protein and phosphorylated MAPK targets) were inhibited after 5°C acclimation, but not -15°C exposure, as compared with the 15°C control group. However, total heat shock protein 27 (HSP27) levels increased dramatically by ∼12-fold in the -15°C acclimated insects. Elevated HSP27 may facilitate anti-apoptotic mechanisms in an Akt-dependent fashion. By contrast, both 5 and -15°C acclimation produced signs of Akt pathway activation. In particular, the inhibitor phosphorylated Glycogen Synthase Kinase 3a (p-GSK3) levels remained high in cold-exposed larvae. Additionally, activation of the Akt pathway might also facilitate inhibition of apoptosis independently of GSK3. Overall, the current study indicates that both MAPK and Akt signal transduction may play essential roles in freeze avoidance by E. scudderiana.

Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy

Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.