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     

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     

Hormonal control of hemolymph lipoprotein ice nucleators in overwintering freeze-susceptible larvae of the stag beetle Ceruchus piceus: adipokinetic hormone and juvenile hormone

Summary Freeze-resistant overwintering larvae of the stag beetle Ceruchus piceus do not produce antifreezes in winter, but instead lower their supercooling points by seasonal removal of lipoprotein ice nucleators (LPINs) from the hemolymph. The normal lipid transport function of these lipoproteins becomes less essential during winter because of the low temperatures and the diapause state of the larvae. Adipokinetic hormone (AKH) and juvenile hormone (JH) were shown to be involved in the control of supercooling abilities and LPIN levels. Treatment of midwinter larvae with AKH resulted in an increase in ice nucleator activity within 2 h, associated with elevated levels of LPINs, as demonstrated by Western blots derived from native PAGE gels probed with polyclonal antibodies to the LPINs. AKH also stimulated the release of LPIN in vitro from cultured fat bodies. In contrast, JH treatments of larvae with high hemolymph ice nucleator contents (either autumn or spring larvae) caused a decrease in ice nucleator activity and supercooling points. However, Western blots showed increased LPIN levels in these JH treated larvae. Apparently, this JH-induced, inactive form of LPIN lacks some component(s) essential for ice nucleator activity.Abbreviations AKH Adipokinetic hormone - Apo-I Apolipoprotein-I - Apo-II Apolipoprotein-II - JH Juvenile hormone - LPIN Lipoprotein ice nucleator - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecyl sulfate - SCP Supercooling point - THP Thermal hysteresis protein - HDLp High density lipophorin - VHDLp Very high density lipophorin     

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

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

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.     

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.     

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.     

Cold hardiness adaptations of codling moth, cydia pomonella

The cold hardiness adaptations of natural and laboratory reared populations of the codling moth, Cydia pomonella, were examined. Hemolymph, gut, and whole body supercooling points (SCPs), 24-h LT50s, polyhydroxy alcohol concentrations, hemolymph freezing points, and hemolymph melting points were determined. Nondiapausing codling moth larvae do not have appreciable levels of ice nucleators in the hemolymph or gut. Whole body supercooling points were higher than hemolymph supercooling points. For nondiapausing larvae, LT50s were significantly higher than both the whole body and the hemolymph supercooling points, indicating the presence of chill sensitivity. As the larvae left the food source and spun a cocoon, both hemolymph and whole body SCPs decreased. Diapause destined larvae had significantly lower hemolymph SCPs than nondiapausing larvae, but whole body SCPs were not significantly different from nondiapausing larvae of the same age. The LT50s of diapause destined and diapausing larvae were significantly lower than that of nondiapausing larvae. Codling moths are freezing intolerant, with LT50s close to the average whole body supercooling point in diapause destined and diapausing larvae. The overwintering, diapausing larvae effectively supercool to avoid lethal freezing by removal of ice nucleators from the gut and body without appreciable increase of antifreeze agents such as polyols or antifreeze proteins.     

Cryoprotective mechanisms in subtidally cultivated and intertidal blue mussels (mytilus edulis) from the Magdalen Islands, Québec

Centrifuged extracts from subtidally cultivated and intertidal blue mussels have higher supercooling points (up to -5.5°C) in winter than in summer (up to -12.5°C). The concentration of nucleators (as estimated by the dilution factor) is greater in winter than in summer in both groups. The nucleator concentration in the extracts of winter mussels is one to two orders of magnitude higher than that in the haemolymph of Norwegian mussels. Although these extracts show spilule-like growth of ice crystals, they caused no thermal hystersis. The seasonal variation of these cryoprotective mechanisms is similar for intertidal and cultivated mussels. However, in the spring, cultivated mussels have a lower supercooling point and a lower concentration of nucleators than their intertidal counterparts. This suggests that cultivated mussels decrease their cryoprotective capacity earlier than intertidal mussels.     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

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。     

PROGRESS IN THE RESEARCH ON MECHANISM OF INSECT COLD-HARDINESS

Abstract A number of freeze-tolerant insect species contain proteins/lipoproteins or insoluble crystals that are ice nucleating active at relatively high subzero temperatures. Recently ice nucleating active bacteria and fungi have been identified as normal flora in insect guts. However, most insects are unable to survive internal ice formation and the key factor in their overwintering survival is the regulation of the temperature at which they spontaneously freeze. To enhance their supercooling capacity overwintering insects eliminate endogenous ice nucleators, accumulate low molecular weight polyols and sugars, and synthesize hemolymph antifreeze proteins or peptides. The factors affecting the supercooling capacity of overwintering insects or the mechanism of cold-hardiness are discussed.     

Supercooling phenomenon and water content independence in the overwintering beetle, Coleomegilla maculata

Insect supercooling phenomena observed during overwintering have been coupled with the seasonality of water and nucleator content and feeding behaviour. However, the strength of the conclusion has in most cases rested on presumption and model systems since nucleator content is not readily quantifiable. The ladybird, Coleomegilla maculata is an overwintering adult insect that fasts for weeks before hibernation, does not accumulate identifiable cryoprotectants during cold exposures, and is freezing intolerant. This species would be expected to overwinter with a constancy of ice nucleators subject to relative increases due to winter dehydration and decreases in supercooling ranges.However, acclimatization and laboratory acclimation experiments have demonstrated both temporal and temperature independence of water content while supercooling levels varied substantially. Supercooling ranges were narrow during warm exposures with a unimodal peak at −6·3°C (±0·2 S.E.). Progressive temperature reductions yielded phasic shifts to multimodal ranges until a low temperature peak of −18·4°C (±0·5 S.E.) was attained. Induced feedings of controlled nucleator substances resulted in predictable supercooling variations while ‘nucleator free’ diets yielded results consistent with natural populations.     

Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions

The ability of 11 different organic solutes in physical solution to mask the effect of nucleating agents from hemolymph of freezing tolerant insects was tested. The masking effect was tested by measuring the supercooling points of samples with various solute concentrations, with and without hemolymph. Hemolymph was obtained from freeze-tolerant Eleodes blanchardi tenebrionid beetles.The depressive effect of the solutes on the supercooling points was nearly equivalent to the corresponding melting point depression, indicating that the depression was due only to the colligative properties of the solutes. Thus, no ability for nucleator masking was demonstrated.     

Purification and characterization of an insect hemolymph lipoprotein ice nucleator: evidence for the importance of phosphatidylinositol and apolipoprotein in the ice nucleator activity

Summary A lipoprotein with ice nucleator activity was purified from the hemolymph of the freezetolerant larvae of the craneflyTipula trivittata. Characterization of this lipoprotein ice nucleator (LPIN) showed that it differed from other previously described insect hemolymph lipoproteins which lack ice nucleator activity, by the presence of phosphatidylinositol (PI) at 11.0% by weight of the total phospholipid content. The potential roles of PI and other lipoprotein components in the ice nucleating activity were examined using various phospholipases, proteases, LPIN antibodies, borate compounds and various lipid-protein reconstitutions. It was found that phosphatidylinositol specific phospholipase C was the most effective phospholipase in eliminating the activity of the LPIN. Borate compounds effectively depressed activity. Treatment of the LPIN with protease also eliminated ice nucleator activity but the binding of LPIN specific antibody did not. Reconstitutions consisting of the native LPIN lipids, PI specific phospholipase-treated native LPIN lipids, or pure standard phospholipids with the apolipoproteins of the LPIN andManduca sexta larval lipoproteins gave evidence that both the apolipoproteins of the LPIN and PI are necessary for the ice nucleating activity.Abbreviations LPIN polyclonal antibodies to lipoprotein ice nucleator - ANOVA analysis of variance - Apo-I apolipoprotein I - Apo-II apolipoprotein II - LPIN lipoprotein ice nucleator - PAGE polyacrylamide gel electrophoresis - PAS Periodoacetate-Schiff's base - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - SCP supercooling point (ice nucleation temperature) - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

Supercooling temperature distribution curves as possible indicators of aphid food quality

Supercooling temperature distribution curves for three aphid species, Elatobium abietinum, Drepanosiphum platanoides and Eucallipterus tiliae, were compared. All showed a tendency towards bimodality, and changes in the form of the curves were interpreted in terms of changing nucleator concentration. In the cases of D. platanoides and E. tiliae the form of the distribution curves for adult samples differed from that for samples of larvae. This was attributed to differences exhibited by adults and larvae in their choice of feeding sites on the host leaf. The distribution curves for E. abietinum obtained from Sitka and Norway spruces differed according to host plant.Evidence is presented to suggest that ice nucleation in aphids normally occurs in the gut contents, and that the quality of ingested phloem sap influences the form of the supercooling temperature distributions.     

Experimental studies on the cold tolerance of Alaskozetes antarcticus

The cold tolerance mechanism of the Antarctic terrestrial mite Alaskozetes antarcticus (Michael) was investigated in cultured animals. Freezing is fatal in this species and winter survival occurs by means of supercooling, which is enhanced by the presence of glycerol in the body. There is an inverse, linear relationship between the concentration of glycerol and the supercooling point, which may be as low as ?30°C. Feeding detracts from supercooling ability by providing ice nucleators in the gut which initiate freezing at relatively high sub-zero temperatures. Experiments on the effects of various environmental factors showed that low temperature acclimation gave rise to increased glycerol concentrations and suppressed feeding, while desiccation also stimulated glycerol production. Photoperiod had no effect on cold tolerance in this species. The juvenile instars of A. antarcticus were found to possess a greater degree of low temperature tolerance than adults.     

The effect of feeding on specific soil algae on the cold-hardiness of two Antarctic micro-arthropods (Alaskozetes antarcticus and Cryptopygus antarcticus)

The effect of consuming terrestrial algae on the cold tolerance of two Antarctic micro-arthropods was examined. From the results of preferential feeding experiments, seven species of Antarctic terrestrial micro-algae were chosen and fed to two common, freeze-avoiding Antarctic micro-arthropods: the springtail Cryptopygus antarcticus (Collembola: Isotomidae), and the mite Alaskozetes antarcticus (Acari: Oribatida). Mites were very selective in their choice of food whereas the springtails were less discriminating. The ice nucleating activity of each species of alga was measured using an ice nucleator spectrometer and a differential scanning calorimeter. Pure cultures of individual species of algae had characteristic supercooling points ranging from ca. −5 to −18 °C. The effect of eating a particular alga on the supercooling point of individual micro-arthropods cultured at two different temperatures (0 and 10 °C) was examined. Neither species showed a preference for algae with low ice-nucleating activity and there was no clear correlation between the supercooling point of food material and that of the whole animal. However, feeding on certain algae such as Prasiola crispa, which contained the most active ice nucleators, decreased the cold tolerance of both species of arthropods. Accepted: 6 May 2000     

Cold hardiness in the Alpine beetles Patrobus septentrionis and Calathus melanocephalus

Alpine Patrobus septentrionis and Calathus melanocephalus (Col., Carabidae) were found to be susceptible to freezing. In the summer, the supercooling points were about ?5 to ?6°C. They were lowered during acclimation in the field and in the laboratory at 0 or ?3°C. Cold hardiness was correlated to, but not determined by, haemolymph osmolality. Thermal hysteresis was not detected. Increase in cold hardiness was concluded to be mainly a result of the influence of acclimation conditions on ice-nucleating compounds. In P. septentrionis, the results indicate that different compounds with ice-nucleation activity at different temperatures determine the limit of supercooling at different times. The haemolymph of both species supercooled well below the intact beetles at all seasons. Changes in haemolymph supercooling points could be ascribed to inactivation of ice nucleators in early autumn and to the effect of changes in solute concentration. In P. septentrionis, myo-inositol increased during cold-acclimation to 80–120 mMol concentrations, whereas C. melanocephalus produced 40–60 mMol trehalose.     

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。