Ice-nucleating Agents Research Articles

ICE-nucleating activity in the freeze-tolerant tardigrade Adorybiotus coronifer

1. 1. The ice-nucleating activity in the cold-hardy tardigrade Adorybiotus coronifer was studied by differential scanning calorimetry. A crystallization temperature of − 6.7°C of intact animals indicates the presence of ice-nucleating agents (INA). Such compounds have not previously been described in this phylum. 2. 2. The ice-nucleating activity in body fluid from A. coronifer is reduced by 50% following ca 7 × 10 3 times dilution. Heating to temperatures above 68°C induces an abrupt decrease in the activity, suggesting that the nucleators are proteinaceous. 3. 3. Gel filtration of homogenized animals indicates that the ice-nucleating activity is confined to molecules with a molecular weight above 200,000.

Ice crystallization induced by silver iodide and bacteria in microsize droplets dispersed within emulsions

Abstract

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.

Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles

Winter survival for various species of amphibians and reptiles that hibernate on land depends on freeze tolerance, the ability to survive for long periods of time with up to 65% of total body water as extracellular ice. Freeze tolerance has been described for four species of frogs, one salamander, and hatchlings of the painted turtle. A very limited tolerance also occurs in garter snakes. Studies of freeze tolerance in vertebrates have primarily focused on the wood frog Rana sylvatica and have assessed the regulation of cryoprotectant synthesis, cryoprotectant action in freezing preservation of isolated cells and tissues, metabolism and energetics under the ischemic conditions imposed by freezing, and the role of ice-nucleating agents in blood. The adaptations that preserve life at subzero temperatures for these animals illustrate the principles of vertebrate organ cryopreservation and may have important applications in the development of technology for the freezing preservation of transplantable human organs.

The effect of ice-nucleating agents on ice-nucleating activity

The effect of ice-nucleating agents on nucleating activity was studied by the use of nucleators from the hemolymph of freeze-tolerant E. blanchardi beetles and from the central fluid of the alpine plant L. telekii. The nucleators from the plant were found to have a higher specific activity than those from the beetle. For samples containing nucleators with a given specific activity, the content of nucleators appears to be the parameter which determines ice-nucleating activity.

Juvenile hormone modulation of insect cold hardening: Ice-nucleating activity

Data are presented offering the first evidence for probable endocrine involvement in the control of cold hardening in Eurosta solidaginis. Juvenile hormone (JH) deprivation experiments in which the corpora allata were removed by head ligation resulted in a loss of supercooling (SC) capacity in larvae collected over 2 years. This loss of supercooling capacity is indicative of synthesis of organismal pools of ice-nucleating agents (INA). Larval sensitivity to JH removal (ligation) on SC is seasonally dependent. For example, in 1983, larvae were most sensitive in October, secondarily so in September, and relatively insensitive in December regardless of acclimation temperature. While in 1984, larvae acclimated to +5 °C were most sensitive in November, secondarily so in October, and relatively insensitive in December, and larvae acclimated to +15 °C were most sensitive in October and December and least so in November. Supercooling point elevations as great as 7 °C over controls were observed with maximal responses occurring within 1 day following ligation. In 1983, juvenile hormone replacement following ligation generally resulted in an expansion of supercooling capacity when compared to controls. As with ligation, the sensitivity to JH replacement on SC was seasonally dependent: September and October larvae being the most sensitive with December larvae being insensitive. Larvae collected in 1984 were given a greater dose of JH than those in the previous year and showed no significant change in SC over ligated-acetone controls. Hormone analog potentiation experiments in which unligated larvae collected in 1983 and acclimated to +5 °C were given methoprene resulted in depression of supercooling points for September and October larvae. JH titres appear to play an important role in the regulation of SC capacity in E. solidaginis larvae.

Effects of ingestion of liquids on the cold tolerance of an Antarctic mite

Samples of a cryptostigmatid mite, Alaskozetes antarcticus, were collected during summer and winter on Signy Island, South Orkney Islands, in the maritime Antarctic. In four experiments, adult mites were maintained at 4°C and 100% r.h. for periods of 14, 28 and 32 days. In two of these experiments the mites received cold-hardening pre-treatments at −15 and −20°C (56 and 21 days respectively). The experiments compared: (1) The effects of acclimation with and without access to distilled water, (2) the effects of liquid glucose (10% solution) and algal diets, in terms of cold hardiness (supercooling points and cryoprotectant levels). In one of the experiments, changes in live weight and body water content were determined. For cold-hardy mites, with high glycerol levels and low supercooling points, the distilled water and glucose treatments produced an extreme loss (about 20–25°C) of supercooling ability. When no liquid was available, only a small (about 4°C) loss occurred. Glycerol concentrations decayed in both cases. Non cold-hardy mites, with low glycerol levels and a wide range of supercooling points, did not show differing responses when acclimated at 4°C with and without distilled water, but exposure to glucose solution did result in extreme supercooling point elevations. These results are related to seasonal changes in Alaskozetes cold hardiness in the field, and are discussed in terms of hypotheses advocating external (bacterial and mineral) and internal (organic) sources of ice-nucleating agents.

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.

Scorpion Cold Hardiness

The scorpion Centruroides vittatus is periodically exposed to freezing temperatures in its natural habitat. It survives such stress by employing physiological mechanisms characteristic of the freeze-tolerant survival strategy used by some insects. Studies over a 12-mo period revealed that scorpions collected in the field and exposed to freezing temperatures in the laboratory froze at high subzero temperatures. High percentages of these animals recovered after freezing. Neither polyols nor thermal hysteresis proteins, which may serve as antifreezes, were found in sufficiently high levels in the hemolymph to substantially lower body supercooling points. Instead, potent ice-nucleating activity compartmentalized in the gut induced ice formation. Ice-nucleating activity peaks in winter months and appears to be proteinaceous. This is the first reported observation of freeze tolerance and ice-nucleating agents in scorpions.

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.

A method for quantitative determination of ice nucleating agents in insect hemolymph.

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 103, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph. This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.

Insect anti-freezes and ice-nucleating agents

Insect anti-freezes and ice-nucleating agents

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.

Factors involved in overwintering survival of the freeze tolerant beetle,Dendroides canadensis

Overwintering larvae of the beetle,Dendroides canadensis, from northern Indiana were able to survice freezing at temperatures down to −28°C. During a late winter thaw or upon warm acclimation the larvae became freeze susceptible, but cold acclimation or acclimatization restored freeze tolerance. Concentrations of the cryoprotectants glycerol and sorbitol, ice nucleating agents (probably proteins) and thermal hysteresis proteins increase in the autumn, peak in winter and decline in spring. These and other, as yet unknown, factors are responsible for the capacity ofDendroides to survice subzero temperatures. The hemolymph ice nucleating agents limit the amount of undercooling to approximately 1°C below the hemolymph freezing point and thereby prevent lethal intracellular ice formation. Therefore, the freezing point depression provided by the thermal hysteresis proteins becomes essential to the survival of the larvae during periods such as winter thaws, autumn, and spring when the larvae are freeze susceptible.

Environmental impacts of artificial ice nucleating agents. Ed. by D. A. Klein, Dowden. Hutchinson and Ross, Inc. 1978. Pp. 256. £13.65

Quarterly Journal of the Royal Meteorological SocietyVolume 105, Issue 445 p. 728-728 Book Review Environmental impacts of artificial ice nucleating agents. Ed. by D. A. Klein, Dowden. Hutchinson and Ross, Inc. 1978. Pp. 256. £13.65 P. R. Jonas, P. R. JonasSearch for more papers by this author P. R. Jonas, P. R. JonasSearch for more papers by this author First published: July 1979 https://doi.org/10.1002/qj.49710544517Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume105, Issue445July 1979Pages 728-728 RelatedInformation

The role of ice nucleators in the frost tolerance of overwintering queens of the bald faced hornet

1. 1. Overwintering queens of the bald faced hornet, Vespula maculata, are tolerant of ice formation in their body fluids down to temperatures of approximately — 14°C. 2. 2. Contributing to this frost tolerance is a high concentration of the cryoprotectant glycerol in the overwintering queens. An additional factor is the presence in the hemolymph of macromolecular ice nucleating agents which function to induce harmless extracellular ice formation at comparatively high temperatures (−4.6°C), and thereby prevent lethal intracellular ice formation. 3. 3. These ice nucleating agents have molecular weights greater than 3500 and are probably proteinaceous.

A Supersonic Expansion Method of Ice Nuclei Generation for Weather Modification

Abstract Aerosols of organic ice nucleating agents were formed by rapid adiabatic expansion, by passing superheated steam laden with the organic vapors through a supersonic nozzle. The effect of the Mach number of the flow through the nozzle on the aerosol particle size and number concentration was investigated. It was found that the number of particles formed increased by a factor of 103 as the Mach number increased from 1 to 2, but further increases in Mach number had little effect. The ice nuclei activity spectra of the organic aerosols thus generated showed plateaus at colder temperatures, with approximately 1014 nuclei per gram below −9°C for 1,5-dihydroxynaphthalene and 1012 g−1 below −7°C for metaldehyde. Coagulation rates of both organic smokes were determined. Coagulation of 1,5-dihydroxynaphthalene aerosols followed the Smoluchowski equation. Coagulation of metaldehyde aerosols was not only extremely rapid, but in addition the coagulation constant increased with time. The latter effect was attri...