昆虫抗冻机理研究进展(英文)

大多数冰冻耐受性昆虫具有蛋白质／脂蛋白质或非溶性的晶体,它们相对地在较高温度下具有激活体内冰核的作用。最近已确证,许多昆虫肠道中正常的细菌和真菌是冰核激活菌丛。而对于非冰冻耐受性的昆虫,其存活是不允许体内冰的形成。它们在过冬过程中,关键是要调节体液的过冷却点,避免结冰。为了增加抗冻能力,非冰冻耐受性的过冬昆虫通过去除内源性冰核、积累低分子量的多元醇和糖类以及血淋巴中抗冻蛋白或抗冻肽的合成来降低体液的过冷却点。本文详尽综述了过冬昆虫抗冻机理的研究进展。     

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

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Reduction of insect cold-hardiness using ice-nucleating active fungi and surfactants

The supercooling point (SCP) of an insect model, the lady beetle Hippodamia convergens Guérin-Menéville (Coleoptera, Coccinellidae) was markedly elevated by treatment with aqueous suspensions of the filamentous, ice nucleation active (INA) fungi Fusarium avenaceum and slightly elevated by Fusarium acuminatum. Addition of the surfactant Tween 80 to the fungal suspensions further reduced the supercooling capacity of adult beetles. When used alone the surfactant Triton X-100 produced a greater SCP elevation than Tween 20 or Tween 80. The emulsifier gum arabic was ineffective in elevating beetle SCPs when applied alone and when added to INA fungal preparations it decreased their efficacy. Aqueous suspensions of both viable sporulating and viable pleomorphic (a permanent, degenerative, nonsporulating cultural state) forms of both fungal species were more effective in elevating the SCP than killed preparations except for the pleomorphic F. acuminatum suspension in which the killed form was slightly more active. Application of INA fungi applied in combination with surfactants may be useful in the development of methods for the biological control of overwintering freeze-susceptible insect pests by decreasing their capacity to avoid lethal freezing by supercooling.     

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.     

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     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

细菌冰核提高印度谷螟过冷却点的研究

印度谷螟(Plodia interpunctella)是一种不耐结冰的昆虫,在冬季它通过降低过冷却 点以避免结冰。现已查明,冰核活性细菌能显著提高植物的过冷却点,导致许多作物在较高 的温度下发生霜冻害。本文也证明细菌冰核能显著提高印度谷螟虫的过冷却点。对照的平均过冷却点是-17.6℃;分别用0.1g和1g细菌冰核与1kg面粉混合后进行处理,平均过冷却点分别比对照提高了12.8℃和13.6℃。研究结果支持这样的观点：细菌冰核有可能成为一种在冬季使用的、杀灭不耐结冰害虫的生物制剂。     

昆虫耐寒性研究

昆虫是变温动物,气候变化是造成种群季节消长的基本原因之一。尤其在不良的低温环境中,昆虫耐寒力的高低是其种群存在与发展的种要前提,昆虫对低温的适应能力及其机理也因而成为昆虫生态学和生物进化研究中的一个深受重视的问题,本文论述了与耐寒性直接相关的过冷却点昆虫的抗寒对策,明确了昆虫耐寒性的一些基本概念,一方面从环境影响昆虫的角度对耐寒性的一般规律,如季节性变化,地理变异快速冷驯化的作用等做了简要的概念括,另一方面阐述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等做了简要的概括,另一方面简单述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等。强调昆虫与环境相互作用过程中的生态生理适应,并指出昆虫耐寒性应当与生活史中别的因素联系起来,这样才能对耐寒性有一个更加全面的理解。     

Ice nucleation studies of two beetles from sub-antarctic South Georgia

Summary Supercooling points of adults and larvae of the coleopterans Hydromedion sparsutum and Perimylops antarcticus at South Georgia ranged from -3.0 to -5.4°C with Perimylops freezing at c.1.6°C lower than Hydromedion. Intact excised guts from adults of both species froze c. 1°C lower than the adult insects. Ice nucleating activity of homogenized faeces from larvae and adults of both species and excised guts were compared with three potential food plants using an ice nucleation spectrometer. Mean supercooling points of the insect materials at four concentrations in distilled water (range from 0.01 to 10 g 1^–1) were significantly different (P<0.01) within species, and within life stages between species. Differences in the supercooling points of suspensions of Polytrichum alpinum (moss) and Usnea fasciata (lichen) were not significant. In general, differences between supercooling points were greater at the higher concentrations. Histograms of the supercooling points showed unimodal distributions particularly at high concentrations and greater dispersion with increased dilution. Spectra showing the concentration of active ice nucleators over the temperature range 0 to -20°C were developed. These showed that nucleation occurred as high as -2°C in faecal material and all insect samples nucleated above -3°C, whereas the plant materials nucleated between -4 and -5°C. The calculated number of ice nucleators for each material in suspension revealed low values (5.3 to 5.8 × 10³) for the plants, but a greater abundance (1.3 × 10⁵ to 1.3 × 10⁶) in the insect samples. It is concluded that c.1000 active nucleators g^–1 are required for ice nucleation to occur in these suspensions. Ice nucleator activity of a suspension of Hydromedion faeces was much reduced by heating to 75°C, suggesting a proteinaceous structure. These results are discussed in relation to ice nucleation in other insects, and it is concluded that bacteria may be responsible for the high nucleation temperatures, and hence poor supercooling, in these South Georgia insects. An empirical model is developed for ice nucleation spectra based on these data.     

Cold-hardiness of the arctic beetle, Pytho americanus Kirby Coleoptera,Pythidae (Salpingidae)

The arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages. This is the first demonstration that an insect can tolerate freezing in more than one life stage, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold hardening process of low temperature acclimation are increasing glycerol and decreasing glycogen concentrations. Glycerol is the only polyol to be synthesized during acclimation, and it accumulates to a maximum of 8.2 and 12.2% of the fresh body weight in larvae and adults respectively. This coincides with the peak of frost tolerance. In addition to its normally assumed roles in cryoprotection it is suggested that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of ‘bound’ water. Evidence is presented that indicates that glycerol is synthesized mainly from carbohydrate reserves, especially glycogen, but it does not rule out the possibility that a proportion of free glycerol comes from glyceride sources.P. americanus larvae and adults have low supercooling potential and maintain their supercooling points in the region of ?4° to ?8°C. It is hypothesized that these elevated supercooling points are a result of the presence in the haemolymph of nucleating agents which ensure ice formation at high sub-zero temperatures. It is believed that this beetle overwinters in a frozen state within its microhabitat, which is under bark of fallen spruce which is, in turn, covered by an insulating blanket of snow. The advantages of this overwintering strategy are discussed.     

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.     

Comparative analysis of overwintering physiology in nine species of semi‐aquatic bugs (Heteroptera: Gerromorpha)

In semi‐aquatic bugs (Heteroptera: Gerromorpha), the strategies of overwintering in a cryothermic state (i.e. at body temperatures below the equilibrium freezing point) remain largely unexplored. The present study provides an analysis of the ecophysiological aspects of overwintering in nine gerromorphan species. All nine species avoid ice formation by means of a more or less extensive supercooling of their body fluids. There is a tight correlation between the supercooling point (SCP) and the lower lethal temperature. Different species use different physiological adjustments to increase the likelihood of survival in a supercooled state. These include stabilization of the supercooled state by active antifreeze factors that cause thermal hysteresis between equilibrium melting and freezing points, the accumulation of low‐molecular weight sugars and polyols with putative cryoprotective functions, or by having a relatively high body fluid osmolality, combined with a low level of hydration. The majority of species under study overwinter only as adults, whereas Velia caprai Tamanini can overwinter either as an adult or in the egg stage. The supercooling capacity of V. caprai adults is insufficient to prevent the risk of lethal freezing. The adults therefore survive only opportunistically in suitable microhabitats, and/or during mild winters. The survival of V. caprai in winter is assured by extensive supercooling and having overwintering eggs that are highly cold tolerant.     

Topical Application of Ice-Nucleating-Active Bacteria Decreases Insect Cold Tolerance

The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ice spontaneously nucleates within their body fluids. We examined the effect of ice-nucleating-active bacteria on the cold-hardiness of the lady beetle, Hippodamia convergens, a freeze-intolerant species that overwinters by supercooling to ca. −16°C. Topical application of the ice-nucleating-active bacteria Pseudomonas syringae increased the supercooling point to temperatures as high as −3°C. This decrease in cold tolerance was maintained for at least 3 days after treatment. Various treatment doses (10⁸, 10⁶, and 10⁴ bacteria per ml) and modes of action (bacterial ingestion and topical application) were also compared. At the highest concentration of topically applied P. syringae, 50% of the beetles froze between −2 and −4°C. After topical application at the lowest concentration, 50% of the individuals froze by −11°C. In contrast, beetles fed bacteria at this concentration did not begin to freeze until −10°C, and 50% were frozen only at temperatures of −13°C or less. In addition to reducing the supercooling capacity in H. convergens, ice-nucleating-active bacteria also significantly reduced the cold-hardiness of four additional insects. These data demonstrate that ice-nucleating-active bacteria can be used to elevate the supercooling point and thereby decrease insect cold tolerance. The results of this study support the proposition that ice-nucleating-active bacteria may be used as a biological insecticide for the control of insect pests during the winter.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Ice nucleation and freezing tolerance in New Zealand alpine and lowland weta, Hemideina spp. (Orthoptera; Stenopelmatidae)

Abstract.The alpine tree weta Hemidiena maori Pictet et Saussure (Orthoptera: Stenopelmatidae) is a large, flightless insect found above the treeline on many of the mountain ranges of the South Island of New Zealand. The population found on the Rock and Pillar Range, Central Otago has been identified as freezing tolerant with a haemolymph ice nucleating agent. The ability of H. maori to survive freezing is compared to the lowland weta Hemideina thoracica Walker and H. crassidens Blanchard, both of which are able to survive the formation of some ice in their bodies. Mortality is associated with time spent frozen in H. thoracica , and it is hypothesized that this species is killed when a critical proportion of its body water is frozen. All five subalpine and alpine populations of H. maori surveyed were found to be freezing tolerant.
Comparison of temperatures of first nucleation and mean supercooling point of haemolymph droplets suggest that haemolymph ice nucleating activity varies between populations of H. maori. Hemideina maori collected from the Mt Cook region appear to lack a haemolymph ice nucleator. This population is nevertheless freezing tolerant, suggesting that the haemolymph ice nucleating agent described in H. maori is not essential for freezing tolerance. Hemideina crassidens and H. ricta Hutton, both of which are found in lowland habitats, also had high mean supercooling point and temperatures of first nucleation of haemolymph droplets, suggesting that these species also have a haemolymph ice nucleator.
Comparison of ice nucleation characteristics of haemolymph and faecal material (representing gut contents) suggests that gut nucleators in H. maori may be at least as efficient as the haemolymph nucleator. It is concluded that freezing tolerance is probably not an adaptation to the alpine environment. This highlights the need for inter- and intraspecific comparative studies if physiological data are to be used to draw evolutionary conclusions.     

Ice nucleating activity in the blood of the freeze-tolerant frog, Rana sylvatica

Although the presence of antifreeze and ice nucleating agents in the hemolymph of insects has been well documented, there have been no reports of either of these types of agent in vertebrates. The technique of differential scanning calorimetry was used to examine the blood, serum, and plasma of a freeze-tolerant frog, Rana sylvatica, for the presence of antifreeze protein activity. Results demonstrate the absence of antifreeze protein but the presence of an ice nucleating agent that may serve as a functional component of the overwintering strategy of this species. Ice nucleating activity was detected in samples of cell-free blood, serum, and plasma, suggesting that the agent is a soluble component and possibly plasma protein. To our knowledge, the identification of ice nucleating activity in this freeze-tolerant vertebrate is novel.     

Adaptations to cold in Canadian arctic insects

The survival of insects that inhabit Canadian arctic regions depends on a number of factors which have important ecological, behavioral, physiological, and biochemical components. The ability to withstand low winter temperatures is one of the most conspicuous adaptations of northern insects and the one most closely studied in the laboratory. Most species studied so far conform to one or other of the two major overwintering strategies, namely, frost susceptibility, the ability to avoid freezing by supercooling to a considerable degree, or frost tolerance, the survival of actual ice formation within the body. The Arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold-hardening process in this species are increasing glycerol and decreasing glycogen concentrations. In addition to its normally assumed roles in cryoprotection there is evidence to suggest that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of bound water. P. americanus larvae and adults have narrow supercooling ranges and maintain their supercooling points in the region of ?4 to ?8 °C. It is hypothesized that these elevated supercooling points are a result of the presence in the hemolymph of nucleating agents which ensure ice formation at high subzero temperatures.Low temperature tolerance strategies of some other arctic and alpine species have been examined and compared with those of relatives from more southerly latitudes. P. americanus has been collected in the Canadian Rockies at elevations of over 6000′, and its frost-tolerant attributes are identical to those of the population collected in the Arctic. A closely related species, P. deplanatus, from the Rockies, however, although it too exhibits frost tolerance in the larval stage, differs markedly from P. americanus in its ability to depress its supercooling range to ?54 °C. It appears that P. deplanatus does not have the ability to synthesize ice-nucleating agents and, therefore, can overwinter in a supercooled condition. Two congeneric species of willow leaf gall sawflies (Pontania spp.), one from Tuktoyaktuk, N.W.T., and the other from southern Vancouver Island have also been compared and contrasted. Pontania sp. on Salix glauca (Tuk., ca. 70 °N) is frost tolerant in its larval stage, has relatively high supercooling points (ca. ?9.0 °C), but does not accumulate glycerol. Pontania sp. from Salix lasiandra (Victoria, ca. 48 °N) has almost identical overwintering properties, indicating the close phylogenetic affinities of cold tolerance in this genus rather than independent adaptation to widely different climatic conditions. Some of the lowest supercooling points ever recorded are from willow stem gall forming insects. Rhabdophaga sp. (Cecidomyiidae) forms potato galls on the stems of Salix lanata in the Inuvik area, N.W.T. After low temperature acclimation, supercooling points down to ?66 °C have been recorded from individual larvae. This is a record, and it indicates that we may be dealing with a system in which most water is in a metabolically bound state. Glycerol levels reach 20% of the fresh body weight during this period. Diastrophus kincaidii Cynipidae) forms stem galls on Thimble Berry (Rubus parviflorus) on southern Vancouver Island. Both of the forementioned species overwinter as larvae in their galls and are, therefore, exposed to ambient air temperatures. A more benign winter climate on Vancouver Island is reflected in the fact that D. kincaidii has supercooling points only in the ?30 to ?33 °C range at the peak of low temperature acclimation, and glycerol levels just below 4% of fresh body weight. Both species are frost susceptible and depend on their supercooling abilities to survive low winter temperatures.     

Seasonal changes of free amino acids and thermal hysteresis in overwintering heteropteran insect, Pyrrhocoris apterus

Overwintering adults of Pyrrhocoris apterus do not tolerate freezing of their body fluids and rely on a supercooling strategy and seasonal accumulation of polyols to survive at subzero body temperatures. We sampled the adults monthly in the field during the cold season 2008-2009 and found active thermal hysteresis factors (THFs) in hemolymph of winter-sampled adults. The hysteresis between the equilibrium melting and freezing points ranged from 0.18°C to 0.30°C. No signs of THFs activity were found in the autumn- and spring-sampled insects. The total free amino acid pool almost doubled during winter time. The sum concentrations of 27 free amino acids ranged between 35 and 40mM in whole body water and 40-45mM in hemolymph during December-February. Two amino acids, Pro and α-Ala most significantly contributed to the seasonal increase, while Gln showed the most dramatic seasonal decrease. Moderate levels of amino acid accumulation in overwintering P. apterus suggest that they are by-products of protein degradation and pentose pathway activity during the state of metabolic suppression imposed by diapause and low body temperature. Potential colligative effects of accumulated amino acids, extending the supercooling capacity of overwintering P. apterus, are negligible. Non-colligative effects require further study.     

Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia pomonella)

Background

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.

Principal Findings

We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately −15.3°C during summer to −26.3°C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to −15°C, even in partially frozen state.

Conclusion

Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).     

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.