John G. Duman

Inhibition of recrystallization of ice by insect thermal hysteresis proteins: a possible cryoprotective role

Abstract Thermal hysteresis antifreeze proteins, first discovered in polar marine fishes, are fairly common in overwintering insects where they also help prevent freezing. However, a few species of insects that tolerate freezing also contain these proteins, and in these species the function of the proteins is uncertain. The studies outlined here demonstrate that the thermal hysteresis proteins from the overwintering larvae of the beetle Dendroides canadensis are extremely efficient in inhibiting ice recrystallization. Since recrystallization is a potential source of freezing damage, we suggest that this may be a role of the antifreeze proteins within freeze tolerant insects.

Plant thermal hysteresis proteins

Proteins which produce a thermal hysteresis (i.e. lower the freezing point of water below the melting point) are common antifreezes in cold adapted poikilothermic animals, especially fishes from ice-laden seas and terrestrial arthropods. However, these proteins have not been previously identified in plants. 16 species of plants collected from northern Indiana in autumn and winter had low levels of thermal hysteresis activity, but activity was absent in summer. This suggests that thermal hysteresis proteins may be a fairly common winter adaptation in angiosperms. Winter stem fluid from the bittersweet nightshade, Solanum dulcamara L., also showed the recrystallization inhibition activity characteristic of the animal thermal hysteresis proteins (THPs), suggesting a possible function for the THPs in this freeze tolerant species. Other potential functions are discussed. Antibodies to an insect THP cross reacted on immunoelectroblots with proteins in S. dulcamara stem fluid, indicating common epitopes in the insect and plant THPs.

Adaptations of Insects to Subzero Temperatures

Insects as a group have been especially successful in adapting to subzero temperatures. Typically, an integration of behavioral and developmental adaptations, as well as physiological and biochemical ones, are required to achieve overwintering success. In keeping with the tremendous adaptive radiation of the group, individual species often exhibit considerable variation in the particular set of adaptations by which they achieve the ability to survive subzero temperatures. Two fundamental physiological mechanisms are (1) freeze tolerance, adaptations that confer the abilty to survive extracellular ice formation, and (2) freeze avoidance (freeze resistance), adaptations that prevent freezing. Different populations of the same species, however, may exhibit different mechanisms, and even the same population may vary its overwintering mechanism from year to year. Feeze-avoidance adaptations usually involve production of antifreezes. These may be antifreeze proteins or colligative-type antifreezes, such as glycerol, which are often produced in molar concentrations. Antifreezes function not only to depress the freezing point, but also to extend the ability to supercool, an important factor since most species are protected by the cuticle from inoculation by external ice. Supercooling may also be extended by the removal of ice nucleators from the body fluids, either on an evolutionary-time scale or on a seasonal basis. Freeze-tolerance adaptations are likewise quite diverse. Most freeze-tolerant insects accumulate high levels of cryoprotectants, usually polyols (i.e., glycerol and sorbitol) and sugars (i.e., trehalose). The potential role of less traditional cryoprotectants, such as amino acids and methylamines, has not been properly investigated. Many freze-tolerant species produce extracellular ice nucleators, typically hemolymph proteins, which induce nucleation at fairly high subzero temperatures and thereby inhibit lethal intracellular ice formation. Some freeze-tolerant insects, however, have removed ice nucleators completely and supercool to - 50 to - 60circC, while other species require inoculative feezing by external ice at temperatures only a few degrees below zero. Other adaptations may involve an increase in unfreezeable water, recrystallization inhibition by antifreeze proteins, or vitrification (glass formation) of certain pools of body water. In spite of considerable progress in understanding subzero temperature adaptations in insects, it is certain that crucial adaptations remain unidentified, and a primary goal of future studies must be to elucidate these mechanisms. Emphasis should also be placed on providing an integrated understanding of the overall complement of adaptations used by particular species and how these adaptations impact, and are impacted by, the ecology of the organism. The great diversity of adaptations exhibited by different species should also be further investigated.

Long-range protein-water dynamics in hyperactive insect antifreeze proteins.

Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein–water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.

Insect antifreezes and ice-nucleating agents☆

Abstract 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 insects 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.

The role of macromolecular antifreeze in the darkling beetle,Meracantha contracta

SummaryMacromolecular antifreeze solutes are present in the hemolymph of the overwintering larvae of the darkling beetle,Meracantha contracta. These antifreeze solutes produce a thermal hysteresis in the hemolymph of overwinteringMeracantha larvae whereby the freezing point of the hemolymph may be 3–4 °C below the melting point. This thermal hysteresis is very similar to that produced by proteinaceous and glycoproteinaceous antifreezes which are used by many cold water, marine teleost fishes to prevent freezing. One function of the macromolecular antifreeze inMeracantha may be to hinder inoculative freezing which might otherwise occur because of the dampness of the hibernaculae. A probably more important function is to depress the supercooling point of the frost susceptibleMeracantha larvae, thereby preventing lethal ice formation in the larvaes body fluids down to temperatures of approximately −11 °C.

Thermal-hysteresis-factors in overwintering insects

Abstract The haemolymph of 9 species of insects found overwintering under the bark of dead trees in northern Indiana contained factors which produced a thermal hysteresis (a difference between the freezing and melting points) of several degrees. These thermal-hysteresis-factors were common in overwintering beetles, but rare in non-Coleoptera, and are similar to the macromolecular antifreezes of polar marine teleost fishes. The factors were found in both freeze-susceptible and freeze-tolerant species, and their function in freeze-susceptible insects appears to be to depress the supercooling points and therefore the lower lethal temperatures of the insects. However, the function of the factors in freeze-tolerant species is not clear. Possible functions are discussed.

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

SummaryOverwintering 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.

Hemolymph Proteins Involved in Insect Subzero-Temperature Tolerance: Ice Nucleators and Antifreeze Proteins

Prior to 1976, the majority of published studies dealing with the mechanisms of adaptation to subzero temperatures in cold-tolerant insects were concerned with the roles of low-molecular-weight solutes, mainly polyols and sugars. Since that time numerous examples of the importance of hemolymph proteins in insect cold adaptation have been determined. In this chapter, we discuss two types of hemolymph proteins with functionally opposite effects on the physical state of water at subzero temperatures. These are antifreeze proteins, which inhibit freezing, and ice nucleating proteins, which inhibit supercooling and induce ice formation at subzero temperatures above those at which freezing would normally take place in their absence.

Antifreeze agents of terrestrial arthropods

Abstract 1. 1. Many insects and other terrestrial arthropods which overwinter in exposed sites in temperate and polar regions are freeze-susceptible (unable to survive freezing) and therefore the seasonal production of antifreezes is critical for their survival. 2. 2. Many of these freeze-susceptible terrestrial arthropods employ low molecular weight antifreezes. such as polyols and sugars. These solutes lower the supercooling point of the insect approximately 2 × more than they lower the melting point. 3. 3. Proteins, similar to the antifreeze proteins and glycoproteins of polar marine fishes, which produce a thermal hysteresis (differences between freezing and melting points) of several degrees function as antifreezes in a number of insects, spiders and a centipede. In some species these proteins are the only identifiable antifreezes, while in other species polyols are also produced. 4. 4. Temperature and photoperiod are the most important environmental cues used to trigger the production and loss of the thermal hysteresis proteins (THPs). The physiological timing processes which control antifreeze levels involve the insects circadian system. 5. 5. Several insect THPs have now been purified and their compositions analyzed. In general, these insect THPs have more hydrophilic amino acids and a much lower alanine content than fish THPs. Perhaps the most interesting of the insect THPs is one from Tenebrio molitor , which contains 28% cysteine residues.