Freeze-avoiding Insects Research Articles

Supercooling points of freeze-avoiding bumble bees vary with caste and queen life stage

Bumble bees thrive in cold climates including high latitude and high altitude regions around the world, yet cold tolerance strategies are largely unknown for most species. To determine bumble bee cold tolerance strategy, we exposed bumble bees to a range of low temperatures and measured survival 72 h post-exposure. All bees that froze died within 72 h while only one bee died without freezing, suggesting that bumble bees are generally freeze-avoiding insects and may be slightly chill susceptible. We then assessed whether temperatures that cause internal ice formation (supercooling points, SCP) varied among bumble bee castes (drones, workers, and queens), or across queen life stages, collection elevation, species, or season. Males froze at significantly lower temperatures than workers or queens. Queens in pre-overwintering or overwintering states froze at significantly lower temperatures than queens stimulated to initiate ovary development by CO2 narcosis (i.e., “spring” queens). We also tested whether the presence of water (i.e., wet or dry) or ramping rate affected SCP. As expected, queens inoculated with water froze at significantly higher temperatures than dry queens. SCP tended to be lower, but not significantly so, at faster ramping rates (0.5 °C/min vs 0.25 °C/min). We also found no differences in SCP between queen bumble bees collected in spring and fall, between queens collected at two sites differing in elevation by 1100 m, or between three field-caught bumble bee species. Bumble bees appear to have relatively high, invariable SCPs, likely making them highly susceptible to freezing across all seasons. As bumble bees are not freeze-tolerant and appear to lack the ability to prevent freezing at temperatures much below 0 °C, they may rely on season- and caste-specific micro-habitat selection to thrive in cold climates.

Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications.

Identification and profiling of miRNAs in the freeze-avoiding gall moth Epiblema scudderiana via next-generation sequencing.

The rapid development of high-throughput next-generation sequencing approaches in recent years has facilitated large-scale discovery and expression analysis of non-coding RNAs, including miRNAs, in traditional and non-traditional animal models. Such an approach has been leveraged to amplify, identify, and quantify miRNAs in several models of cold adaptation. The present study is the first to investigate the status of these small RNAs in an insect species that uses the freeze avoidance strategy of cold hardiness, the gall moth Epiblema scudderiana. To characterize the overall miRNA expression profile and to identify cold-modulated miRNAs in control (5 °C) and cold-exposed (-15 °C) E. scudderiana larvae, a next-generation sequencing-based approach was undertaken. A total of 44 differentially expressed miRNAs were identified between the two conditions; 21 up-regulated miRNAs and 23 down-regulated miRNAs in -15 °C-exposed larvae as compared with controls. Among the most significant changes observed in miRNAs with potential relevance to cold adaptation were elevated miR-1-3p, miR-92b-3p, and miR-133-3p levels as well as reduced miR-13a-3p and miR-13b-3p levels in E. scudderiana larvae exposed to cold temperatures. Expression values obtained from next-generation sequencing were also validated by a quantitative PCR approach for five miRNAs; miR-34-5p, miR-274-5p, miR-275-3p, miR-307a-3p, and miR-316-5p. Overall, this work provides the first description of a miRNA signature for subzero survival by a freeze-avoiding insect using a high-throughput approach and positions a new group of miRNAs at the forefront of the molecular changes underlying cold adaptation.

Probability of freezing in the freeze-avoiding beetle larvae Cucujus clavipes puniceus (Coleoptera: Cucujidae) from interior Alaska

Freeze-avoiding insects must resist freezing or die. A suite of adaptations to low temperatures, including the production of antifreeze proteins, colligative antifreezes (polyols), and dehydration allows most individuals to prevent freezing below the lowest ambient temperatures experienced in situ; however, there can be a wide variance in the minimum temperatures that individuals of freeze-avoiding species reach before freezing. We used logistic regression to explore factors that affect this variance and to estimate the probability of freezing in larvae of the freeze-avoiding beetle Cucujus clavipes puniceus. We hypothesized that water content ≤ 0.5 mg mg −1 dry mass would lead to deep supercooling (avoidance of freezing below −58 °C). We found a significant interaction between water content and ambient below-snow temperature and a significant difference between individuals collected from two locations in Alaska: Wiseman and Fairbanks. Individuals collected in Wiseman deep supercooled with greater water content and to a greater range of ambient temperatures than individuals collected in Fairbanks, leading to significantly different lethal water contents associated with 50% probability of freezing.

Type I Antifreeze Proteins Enhance Ice Nucleation above Certain Concentrations

In this study, we examined the effects that antifreeze proteins have on the supercooling and ice-nucleating abilities of aqueous solutions. Very little information on such nucleation currently exists. Using an automated lag time apparatus and a new analysis, we show several dilution series of Type I antifreeze proteins. Our results indicate that, above a concentration of ∼8 mg/ml, ice nucleation is enhanced rather than hindered. We discuss this unexpected result and present a new hypothesis outlining three components of polar fish blood that we believe affect its solution properties in certain situations.

Expression of two self-enhancing antifreeze proteins from the beetle Dendroides canadensis in Drosophila melanogaster

Antifreeze proteins (AFPs) lower the freezing point of water without affecting the melting point. This difference between melting point and freezing point has been termed thermal hysteresis. Antifreeze protein genes, dafp-1 and/or dafp-4, from the freeze-avoiding insect, Dendroides canadensis, were transferred to Drosophila melanogaster via P-element-mediated transformation. The Northern and Western blots showed expression of DAFP(s) at both transcript and protein levels. The highest thermal hysteresis activity of 6.78 ± 0.12 °C was detected in 5-day adult flies containing two copies of each of the dafp-1 and dafp-4 genes, while flies with two copies of either dafp-1 or dafp-4 had less activity, 5.52 and 3.24 °C, respectively (measured by nanoliter osmometer). This suggests synergistic enhancement of thermal hysteresis activity between DAFP-1 and DAFP-4 in transgenic D. melanogaster containing both DAFPs. Supercooling points without ice in contact with the insects were lowered in all 5 transgenic lines compared with controls, however, when ice was in contact with the flies, supercooling points were lowered only in the heterozygous <DAFP-1> + < DAFP-4> transgenic line. Also, transgenic D. melanogaster exhibited higher survivorship compared with controls when placed at low non-freezing temperatures (0 and 4 °C), however, DAFP-1 and DAFP-4 did not display any synergistic enhancement in these non-freezing survival experiments.

Responses of protein phosphatases and cAMP-dependent protein kinase in a freeze-avoiding insect,Epiblema scudderiana

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of winter cold hardiness and show multiple metabolic adaptations for subzero survival including accumulation of large amounts of glycerol as a colligative antifreeze. Induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. Changes in the activities of several signaling enzymes including cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) were monitored over the winter and during experimental exposures of larvae to subzero temperatures (-4 degrees C, a temperature that triggers rapid glycerol synthesis, or -20 degrees C, a common midwinter ambient temperature) or anoxia. A strong increase in the amount of active PP1 in the latter part of the winter may be responsible for shutting off glycogenolysis once glycerol levels are maximized. There appears to be a limited role for PKA in overwintering but PP2A and PP2C activities rose when larvae were exposed to -20 degrees C and PTP activities rose significantly over the winter months and also in response to laboratory subzero (-20 degrees C) and anoxia exposures. The strong responses by PTPs suggest that these may be involved in cell cycle and growth arrest during winter diapause.

Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause

The beetle Cucujus clavipes is found in North America over a broad latitudinal range from North Carolina (latitude approximately 35 degrees N) to near tree line in the Brooks Range in Alaska (latitude, approximately 67 degrees 30' N). The cold adaptations of populations from northern Indiana (approximately 41 degrees 45' N) and Alaska were compared and, as expected, the supercooling points (the temperatures at which they froze) of these freeze-avoiding insects were significantly lower in Alaska insects. Both populations produce glycerol, but the concentrations in Alaska larvae were much higher than in Indiana insects (approximately 2.2 and 0.5 mol l(-1), respectively). In addition, both populations produce antifreeze proteins. Interestingly, in the autumn both populations have the same approximate level of hemolymph thermal hysteresis, indicative of antifreeze protein activity, suggesting that they synthesize similar amounts of antifreeze protein. A major difference is that the Alaska larvae undergo extreme dehydration in winter wherein water content decreases from 63-65% body water (1.70-1.85 g H2O g(-1) dry mass) in summer to 28-40% body water (0.40-0.68 g H2O g(-1) dry mass) in winter. These 2.5-4.6-fold reductions in body water greatly increase the concentrations of antifreeze in the Alaska insects. Glycerol concentrations would increase to 7-10 mol l(-1) while thermal hysteresis increased to nearly 13 degrees C (the highest ever measured in any organism) in concentrated hemolymph. By contrast, Indiana larvae do not desiccate in winter. The Alaska population also undergoes a diapause while insects from Indiana do not. The result of these, and likely additional, adaptations is that while the mean winter supercooling points of Indiana larvae were approximately -23 degrees C, those of Alaska larvae were -35 to -42 degrees C, and at certain times Alaska C. clavipes did not freeze when cooled to -80 degrees C.

Ice nucleation in solutions and freeze-avoiding insects—homogeneous or heterogeneous?

This article challenges the common view that solutions and cold-hardy freeze-avoiding insects always freeze by heterogeneous nucleation. Data are presented to show that the nucleation temperatures of a variety of solutions and freeze-avoiding insects are a function of the water volume as described by the data previously published by Bigg in 1953. The article also points out that the relationships between melting point depression and depression of nucleation temperature are different for samples undergoing homogeneous nucleation and those undergoing heterogeneous nucleation. Aqueous solutions and freeze-avoiding insects display a relationship like that of homogeneously nucleated samples. It is also argued that the identity of the “impurities” assumed to cause heterogeneous nucleation in aqueous solutions and insects is obscure and that the “impurities” have features which make their existence rather unlikely.

Climatic variability and the evolution of insect freeze tolerance.

Insects may survive subzero temperatures by two general strategies: Freeze-tolerant insects withstand the formation of internal ice, while freeze-avoiding insects die upon freezing. While it is widely recognized that these represent alternative strategies to survive low temperatures, and mechanistic understanding of the physical and molecular process of cold tolerance are becoming well elucidated, the reasons why one strategy or the other is adopted remain unclear. Freeze avoidance is clearly basal within the arthropod lineages, and it seems that freeze tolerance has evolved convergently at least six times among the insects (in the Blattaria, Orthoptera, Coleoptera, Hymenoptera, Diptera and Lepidoptera). Of the pterygote insect species whose cold-tolerance strategy has been reported in the literature, 29% (69 of 241 species studied) of those in the Northern Hemisphere, whereas 85 % (11 of 13 species) in the Southern Hemisphere exhibit freeze tolerance. A randomization test indicates that this predominance of freeze tolerance in the Southern Hemisphere is too great to be due to chance, and there is no evidence of a recent publication bias in favour of new reports of freeze-tolerant species. We conclude from this that the specific nature of cold insect habitats in the Southern Hemisphere, which are characterized by oceanic influence and climate variability must lead to strong selection in favour of freeze tolerance in this hemisphere. We envisage two main scenarios where it would prove advantageous for insects to be freeze tolerant. In the first, characteristic of cold continental habitats of the Northern Hemisphere, freeze tolerance allows insects to survive very low temperatures for long periods of time, and to avoid desiccation. These responses tend to be strongly seasonal, and insects in these habitats are only freeze tolerant for the overwintering period. By contrast, in mild and unpredictable environments, characteristic of habitats influenced by the Southern Ocean, freeze tolerance allows insects which habitually have ice nucleators in their guts to survive summer cold snaps, and to take advantage of mild winter periods without the need for extensive seasonal cold hardening. Thus, we conclude that the climates of the two hemispheres have led to the parallel evolution of freeze tolerance for very different reasons, and that this hemispheric difference is symptomatic of many wide-scale disparities in Northern and Southern ecological processes.

Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect

Phosphofructokinase (PFK) from larvae of the freeze-avoiding gall moth Epiblema scudderiana was purified 711-fold using ATP-agarose affinity chromatography to a final specific activity of 23 U/mg protein. The native molecular mass of the enzyme was 420 000 ± 20 000 Da. The enzyme showed an optimum pH of 8.13 ± 0.21 at22 °Cand 8.19 ± 0.11 at5 °C. Arrhenius plots of PFK activity showed a sharp break at 9 °C. S0.5 values for fructose 6-phosphate showed positive thermal modification, decreasing with decreasing assay temperature; the opposite was true for ATP-Mg2+. PFK was activated by fructose 2,6-bisphosphate, AMP, and inorganic phosphate; activator effects were temperature-dependent. The enzyme was inhibited by ATP-Mg2+, citrate-Mg2+, and phosphoenolpyruvate. The positive effects of low temperature on enzyme kinetic properties would promote PFK activity to channel glycolytic carbon flow into the production of glycerol during cold-stimulated cryoprotectant synthesis.

Biochemical adaptation for cold hardiness in insects

Specific biochemical adaptations permit winter survival at subzero temperatures by both freeze-tolerant and freeze-avoiding insects. Common to both survival strategies is the accumulation of high concentrations of polyols, providing deep supercooling point depression for freeze-avoiding forms and regulating cell volume reduction during extracellular freezing in freeze-tolerant insects. Studies in my laboratory have elucidated the molecular mechanisms (temperature effects on enzyme properties, allosteric regulation, reversible protein phosphorylation) that control the massive conversion of glycogen to polyols and, in some species, regulate the differential synthesis of dual polyols. New studies have highlighted the importance of aerobic ATP production for glycerol biosynthesis, suggested the importance of microcompartmentation for optimal conversion efficiency, documented seasonal changes in the capacity for polyol synthesis versus reconversion to glycogen and analysed the role of protein phosphorylation in enzyme regulation during polyol synthesis.

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

The course of glycerol biosynthesis, initiated by exposure to −4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to −4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 μmol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at −4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.

Physiology of cold tolerance in insects.

From the available experimental data a relatively clear picture can be established with regard to the physiological importance of some of the mechanisms involved in insect cold hardening. In freeze-avoiding insects, all potent ice-nucleating agents are removed or inactivated, leading to a depression of the supercooling points to about 20 degrees C. Accumulation of polyols causes a further depression with a magnitude of about twice the corresponding melting-point depression. Production of thermal hysteresis factors causes a stabilization of the supercooled state. In freeze-tolerant insects, potent ice-nucleating agents are produced in the extracellular body fluid, ensuring a protective extracellular freezing at a few degrees below zero. Accumulation of polyols causes a steep drop in the lethal temperature, due to a reduction of the amount of ice by a colligative mechanism. However, there is still much to be learned about the mechanisms by which ice-nucleating agents, polyols, and thermal hysteresis agents are acting. Furthermore, the regulatory mechanisms involved in the production and elimination of these components from the body fluid of the insects are not understood. Also, when it comes to the influence of environmental factors, like photoperiod and temperature, there is much to be learned. In addition to giving attention to these topics, future research should be focused on the possible role of other factors in cold hardening such as bound water, dehydration, low-molecular-weight solutes other than polyols, and the biochemical mechanisms forming the basis of the seasonal changes in the cold hardiness of insects.