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Home , Acclimatization, Cold hardening

Insect Cold-Hardiness: To Freeze or Not to Freeze

Richard E. Lee, Jr.

BioScience, Vol. 39, No. 5. (May, 1989), pp. 308-313.

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http://www.jstor.org Tue Jan 8 14:40:48 2008 Cold-hardiness: To Freeze or Not to Freeze How survive low temperatures

by Richard E. Lee, Jr.

n temperate regions, few insects process of cold-hardening re- are able to avoid exposure to low The capacity to quires at least several weeks to com- I environmental temperatures dur- plete. ing the winter. Notable exceptions cold-harden is required include the monarch butterfly, which Two strategies. Overwintering insects for overwintering mav, migrateu more than 1000-milesto appear to use two major strategies, warmer climates, and the honeybee, depending on whether they can sur- which maintains constant hive tem- vive the " of their bodv water. peratures near 35" C by behavioral Freeze-susceptible insects must avoid and physiological activities of the col- to survive long-term or short-term freezing; freeze-tolerant insects can ony (Heinrich 1981, Southwick and exposure to low temperature. This survive extracellular ice formation Heldmaier 1987). Other species avoid capacity varies as a function of the within their tissues. low-temperature extremes by vertical specific developmental stage, season FREEZE-SUSCEPTIBLEINSECTS. migration into the soil or the selection of the year, nutritional status, and the Freeze-susceptible (or freeze-intol- of protected hibernacula. However, duration of exposure. Physiological erant) species take advantage of su- for the many insects unable to avoid and biochemical processes that en- percooling. Small volumes of water, exposure to low temperatures, the hance low-temperature tolerance re- whether in or not, can of- capacity to cold-harden is required sult in cold-hardening. ten be cooled many degrees below the for overwintering survival. The study of insect cold-hardiness melting point before spontaneous nu- Although many overwintering in- is a comparatively new one. Based on cleation. For example, 5-microliter sects seek protected overwintering his publications in the 1950s and samples of tap water commonly can sites in leaf litter or beneath the bark 1960s, R. W. Salt is generally credited be cooled to -18" C or lower. The of trees, the most cold-tolerant spe- as the first to examine rigorously on capacity to supercool decreases as cies are those that overwinter in biochemical and physiological levels volume increases and as the duration exposed sites, such as the Arctic cat- the nature of low-temperature toler- of exposure to subzero temperatures erpillar (Gynaephora), which over- ance in insects (Salt 1961, 1969). In- increases. Therefore. it should not be on rocky surfaces, or fly lar- terest in this area has grown steadily, surprising that insects, being essen- vae in plant galls that extend above as evidenced by the number of publi- tially small bags of water, have the the snowline, thus exposing their in- cations listed in two recent bibliogra- capacity to supercool extensively. In- habitants to the extremes of fluctua- phies on this subject (Baust et al. sects can sometimes avoid freezing tion in ambient temperatures (Ring 1982, Lee et al. 1986). This article until their temperature has fallen and Tesar 1981). provides a brief introduction to insect many degrees below the melting point Cold-hardiness or cold tolerance cold-hardiness with emphasis on of the body fluids (Figure 1). refers to the capacity of an some recent findings, including the The supercooling point is the tem- description of an extremely rapid perature at which spontaneous nucle- cold-hardening response protective ation occurs and the ice lattice begins Richard E. Lee, Jr., is an associate profes- against short exposures to cold. to grow. Even for small insects or sor in the Department of Zoology at " Miami University, Hamilton, Ohio their eggs, this value is easily mea- 45011. His cryobiological research fo- Overwintering cold-hardiness sured by using thermocouples to de- cuses on mechanisms of freeze tolerance tect the release of the heat of crystal- and low-temperature in insects To prepare for the cold of winter, lization. The heat usuallv dissi~ates and frogs. O 1989 American Institute of insects undergo a variety of physio- within a few minutes, and their body Biological Sciences. logical and biochemical changes. The temperatures decrease to the ambient

308 BioScience Vol. 39 No. 5 temperatures. INSECT RESPONSE TO LOW TEMPERATURE In preparation for overwintering, freeze-susceptible species, such as ladybugs and springtails (Collem- bola), commonly decrease their su- BODY TEMPERATU percooling point, thereby increasing the chance of winter survival (Figure 1). For this group, supercooling points often range in winter between -15" and -3.5" C (Sarmme 1982), al- MELTING POINT though some species from the interior OF BODY FLUIDS of Alaska supercool to approximately -60" C (Miller 1982). Evacuation of the gut, resulting in the removal of potential ice-nucleating agents, is be- lieved to be one mechanism of en- hancing supercooling capacity (Cannon and Block 1988). Also, endogenous ice-nucleating agents lo- cated in the tissues may be inactivated or masked during cold-hardening (Baust and Zachariassen 1983). In other species, water balance and des- iccation appear to influence the super- cooling point (Cannon et al. 1985)...... The insects also accumulate certain ...... chemicals, called ...... FREEZE-TOLERANTINSECTS. Other insects are able to survive extensive extracellular ice formation within ...... body tissues and organs when tem- ...... peratures may fall as low as -70" C -60 (Miller 1982, Storey and Storey 1988). These freeze-tolerant insects, TIME such as the goldenrod gall fly (Eu- rosta solidaginis), commonly synthe- Figure 1. Responses of insects cooled to subzero temperatures. Insect body tempera- size ice-nucleating agents during win- tures (heavy line) in relation to the melting point, the supercooling point, and the ter cold-hardening. These unique nucleation of ice in body fluids. Although the bars on the right convey general ranges or lipoproteins serve as cata- of insect response to low temperatures, the top of the bar for the range of freeze tolerance and the bottom of the bar for freeze avoidance correspond to the supercooling lysts for the extracellular nucleation point value illustrated in the center of the figure. However, a number of freeze-tolerant of ice in body fluids at temperatures insects have supercooling points in the range of -8" to -10" C, whereas some of -5" to -10" C (Duman et al. freeze-susceptible species supercool extensively, to -60" C or below. 1985). By limiting the amount of su- percooling, these proteins apparently serve to avoid lethal intracellular decrease in the size of unfrozen chan- concentrations corresponding to 25% freezing. nels in the extracellular space (Mazur of its body weight (Salt 1961). In Freeze tolerance refers to a specific 1984). some species, combinations of two or temperature range in which tissue more of these compounds are accu- freezing is tolerated; cooling below Cryoprotectants. One of the most re- mulated during cold-hardening. In that range may result in mortality markable characteristics of overwin- freeze-intolerant species, the tradi- from excessive cellular dehydration tering insects is their capacity to pro- tional view held that these antifreezes (Figure 1). Because only water mole- duce both sim~lecom~ounds and functioned primarily in a colligative cules join the ice lattice as it grows in proteins that act as antifreeze. Insects fashion (i.e., based on the number of the extracellular space, the remaining can accumulate large amounts of solute particles in solution), causing a extracellular solute becomes more , sorbitol, trehalose, or other depression in both the hernolymph concentrated. This process results in low-molecular-weight carbohydrates freezing point and the supercooling an osmotic gradient that removes wa- or polyols. These compounds, made point (Salt 1961, Zachariassen 1985). ter from the cells. Injury due to freez- by both freeze-susceptible and freeze- One of the most significant ad- ing results from excessive concentra- tolerant s~ecies. are referred to as vances in the last few years is the tion of solutes and shrinkage, and low-molecular-weight antifreezes or realization that low-molecular-weight more recently it has been suggested cryoprotectants. One larval wasp, polyols and sugars can protect biolog- that damage may be a function of the Bracon cephi, produces 5-M glycerol ical molecules and systems via non-

May 1989 309 colligative mechanisms (Crowe et al. the freezing and the supercooling results in the rapid loss of cryopro- 1983, Crowe et al. 1987). The brine points (Duman and Horwath 1983). tectants and cold tolerance (Ring shrimp Artemia, the slime mold Dic- Zachariassen and Husby (1982) offer 1982). tyostelium, certain nematodes, and an alternative explanation: the anti- Biological clocks also play a role in some other organisms can survive for freeze proteins may function to stabi- the cold-hardening process; exposure years in a dry (anhydrobiotic) state, lize the supercooled state by adsorb- to short photoperiods induces the syn- and this survival correlates closely ing to the surface of embryonic ice thesis of thermal hysteresis proteins with the presence of large amounts of crystals, thereby preventing addi- (Duman and Horwath 1983). Beck sugars (Crowe et al. 1987). Further- tional growth of the ice lattice. Later (1987) recently demonstrated that daily more, studies have demonstrated the Knight and Duman (1986) demon- cycles of temperature (thermoperiods) direct action of sugars to preserve and strated that these unique proteins in- interact with the photoperiodic cues in stabilize , phospholipid bilay- hibit recrystallization, growth of the the regulation of dormancy. The possi- ers, and liposomes during drying ice lattice in tissue that is already ble role of thermoperiodism in cold- (Crowe et al. 1987). Potential cryo- frozen. This observation raises the hardening should be examined. protectants can be evaluated by their possibility that antifreeze proteins ability to protect freeze-labile en- may also protect freeze-tolerant in- Nonfreezing injury and zymes and liposomes (Loomis et al. sects from injury due to recrystalliza- cold shock 1988). tion. Anhydrobiotic and freeze-tolerant It has been assumed freauentlv that survival are directly analogous; both Environmental regulation of winter the supercooling point represents the require the tolerance of extensive cel- cold-hardening. During the last dec- lethal temperature for freeze-suscep- lular dehydration. Even relatively low ade, several investigators have begun tible insects. Although the supercool- concentrations of trehalose, glycerol, to elucidate the mechanisms that syn- ing point represents the theoretical and other cryoprotectants may func- chronize cold-hardening with sea- lower limit of temperature tolerance tion to stabilize structure and sonal changes in the environment. for this group, recent studies have membranes during cooling andlor The process is antici- shown that chilling, in the absence of freezing. patory in that it occurs in advance of tissue freezing, may result in lethal To date, most studies examining exposure to sub-zero temperatures in injury (Knight et al. 1986, Lee and the function of endogenous nuclea- the field. The exposure of insects to Denlinger 1985).In some species, non- tors and cryoprotectants required the low temperature, particularly in the freezing injury is evident only after extraction of these compounds from range of 0" to 5" C, has been repeat- extended periods of low-temperature the insect. Baust and Rojas (1985) edly demonstrated to trigger the accu- exposure, as in the bertha armyworm, caution that this approach is limited mulation of cryoprotectants and to Mamestra configurata (Turnock et al. for understanding in vivo mecha- enhance cold-hardiness (Baust and 1983). In his recent review, Bale nisms of cold tolerance. In contrast, Lee 1981). Low temperature acts di- (1987) emphasizes the importance nuclear magnetic resonance (NMR) rectly to increase the activity of spe- that prefreezing mortality may have spectroscopy provides a specific, but cific enzymes (glycogen, phosphory- on survival in the field and urges., noninvasive, tool for studying cold- lase, hexokinase, and phospho- future studies to determine carefully hardiness in living animals. With car- fructokinase) and thereby channel the whether the supercooling point may bon-13 NMR, Buchanan and Storey flow of carbon into polyol synthesis be used reliablv as a measure of the (1983) detected cryoprotectants and (Storey and Storey 1981). Acclima- lower lethal temDerature. in freeze-tolerant larvae of the tion to high temperatures frequently In other species, short-term expo- goldenrod gall fly. More recently, Kukal et al. (1988) obtained in vivo time-lapse carbon-13 spectra of glyc- SARCOPHAGA CRASSlPALPlS DROSOPHlLA MELANOGASTER erol in freeze-tolerant lar- vae of an arctic caterpillar. In addition to accumulating low- molecular-weight cryoprotectants, a number of overwintering insects pro- duce antifreeze proteins (Duman 1977, Duman and Horwath, 1983). Sometimes these proteins are referred to as thermal hysteresis proteins, be- cause they cause a difference between the melting and freezing points of CONTROL - O" bodv fluids similar to that of anti- 10" C CI- I 0" C CONTROL -5" C 5" CI-5" C freeze peptides and glycopeptides Figure 2. Cold shock and rapid cold-hardening in the flesh fly, Sarcophaga crassipalpis in the of polar (De- (Chen et al. 1987),and in the fruit fly, (Czajka and Lee 1988). Vries 1971). In freeze-intolerant in- Control flies were held continuously at 25" C, a second group was directly transferred sects, these proteins appear to func- to subzero temperatures for 2 hours, and a third group was chilled for 2 hours at 0" or tion as an antifreeze, lowering both 5" C, respectively, before subzero exposure. At least 50 flies were tested in each group.

310 BioScience Vol. 39 No. 5 sure to cold temperatures above the Table 1. Summary of characteristics associated with cold-hardening for winter and the rapid supercooling point is lethal. For ex- cold-hardening response that protects against injury due to cold shock. Adapted from Lee et al. ample, even though active pupae of 1988. the freezing-intolerant flesh fly, Sarco- Characteristics Winter cold-hardening Rapid cold-hardening phaga crassipalpis, have a supercool- Differences ing point of -23" C, this stage does Cold tolerance Long-term freeze tolerance Short-term prevention of non- not survive as little as 20 minutes of andlor supercooling freezing injury exposure to -17" C (Lee and Denlin- Stage Only in overwintering Present in more than one ger 1985). Aphids are particularly stage developmental stage (larva, pupa, adult) susceptible to chilling injury (Knight Activity Inactive (, Active feeding and reproduction et al. 1986). This type of injury is quiescence) (sometimes diapause) termed cold shock and occurs after Timing Seasonal All year rapid cooling, but in the absence of Rate of cold-hardening Slow (weeks-months) Rapid (minutes-hours) ice formation (Morris 1987). This form of cellular injury is Similarities known for a wide variety of biologi- cal systems including bacteria, fungi, Present Present algae, and higher plants, as well as Induction trigger Low temperature Low temperature mammalian spermatozoa and em- bryos. The mechanism of cold-shock stages. mental stages, even when the insects injury is thought to be the induction If adults of the flesh fly, S. crassi- are actively feeding and reproducing, of phase transitions in the mem- palpis, are exposed directly to -10" C and it may operate at any time of the brane leading to the subsequent loss for two hours, few individuals survive year. The most unexpected and re- of membrane integrity (Quinn 1985). (Figure 2). Because this stage has a markable difference is the rapidity of Alternatively, or perhaps concomi- supercooling point of -23" C, this the response: typically weeks are re- tantly, membrane failure may be due result suggests that injury is due to quired for the completion of the sea- to excessive thermoelastic (Mc- cold shock. Remarkably, however, sonal hardening process in prepara- Grath 1987). chilling at 0" C for as little as 10 tion for overwintering, whereas as Cold shock and chilling injury are of minutes enables 50% of the flies to little as 20 minutes of chilling is suf- considerable economic interest, not survive a two-hour exposure at ficient to elicit the rapid coth-hard- only with respect to plants and agri- -10" C. Two hours of chilling at 0" C ening response. cultural products, but in the develop- allows nearlv all flies to survive this ment of methods for the cryopreserva- treatment. Short-term chilling of lar- Survival value and economical im~or- tion of semen, mammalian embryos, vae and pupae before -10" C expo- tance. Under natural conditions, the and other cells and organs (Morris sure greatly enhances survival. capacity to cold-harden rapidly may 1987). Nearly all of these applications Freeze-intolerant adults of Droso- be important for survival throughout pertain to cells, tissues, or organs from phila melanogaster have a supercool- the year (Lee et al. 1987). In temper- tropical plants or from , in- ing point of -20" C, but they do not ate regions, insects may be exposed to cluding humans, which do not natu- survive even short intervals of expo- daily temperature cycles of 20" to rally experience internal low tempera- sure to -5" C (Czajka and Lee 1988). 30" C. The cavacitvA , to cold-harden ture as a normal part of their life cycle. Similarly, short-term chilling protects rapidly may allow even nonoverwin- The study of insect model systems that these fruit flies against injury due to tering life stages to almost instanta- are naturally able to protect them- cold shock (Figure 1). neously enhance cold tolerance as selves against injury due to cold shock Clearly, the phenomenon of over- they track diurnal changes in temper- may ultimately lead to methods of wintering cold-hardiness differs dis- ature. This capacity may be of partic- preventing chilling injury in the stor- tinctly from the rapid cold-hardening ular importance in the spring and age and transport of plant products response protecting against cold- autumn, when ambient temperatures and to more successful methods for shock injury (Table 1).Although both may vary rapidly and without warn- the of human tissues processes protect against forms of in- and organs. jury caused by exposure to low tem- perature, winter cold-hardening fre- Mechanism: glycerol and heat-shock Rapid cold-hardening response quently includes the acquisition of proteins. What is the underlying freeze-tolerance or the capacity to mechanism that results in the rapid Historically, studies of insect cold- survive exposure to temperatures cold-hardening response? One line of hardening have examined seasonal near the supercooling point for a spe- evidence suggests that a cryoprotec- processes related to overwintering. In cific overwintering stage. In contrast, tive agent, glycerol, may be involved. contrast, we have identified an ex- the rapid cold-hardening response Within two hours of transfer to 0" C, tremely rapid cold-hardening re- protects against low-temperature in- glycerol titers of larvae and pharate sponse that protects insects from in- jury that occurs as much as 15" C adults of the flesh fly are elevated jury due to cold shock (Chen et al. above the supercooling point. Fur- two- to three-fold (Chen et al. 1987). 1987, Lee et al. 1987). This response thermore, the rapid cold-hardening This interpretation is consistent with is found even in nonoverwintering response occurs in various develop- glycogen phosphorylase being di-

May 1989 311 rectly activated by exposure to cold in complexity is further compounded by Burton, V., H. K. Mitchell, P. Young, and N. S. phylogenetically diverse insects (Lee the great diversity of insects and the Petersen. 1988. Heat shock protection against cold stress of Drosophila melano- et al. 1987). This catalyzes varying environments they inhabit. gaster. Mol. Cell. Biol. 8: 3550-3552. the breakdown of glycogen in the Furture studies must not only con- Cannon, R. J. C., and W. Block. 1988. Cold insect fat body, resulting in the pro- sider strategies of freeze-tolerance tolerance of microarthropods. Biol. Rev. 63: duction of glycerol. Quinn (1985)has versus intolerance, but also cold 23-77. shock and other forms of injury that Cannon, R. J. C., W. Block., and G. D. Collett. suggested that cryoprotective agents 1985. Loss of supercooling ability in Cryp- such as glycerol may stabilize rela- occur at low temperature in the ab- topygus antarcticus (Collembola: Isotomi- tionships between bilayer- and non- sence of tissue freezing. In the last dae) associated with water uptake. Cryo bilayer-forming lipids during rapid decade, in-depth research efforts have Lett. 6: 73-80. cooling. Thus, low-molecular-weight begun to focus on a few select insect Chen, C.-P., D. L. Denlinger, and R. E. Lee. 1987. Cold-shock injury and rapid cold- polyols may also play a cryoprotec- species and to search for mechanistic, hardening in the flesh fly Sarcophaga crassi- tive role in preventing injury due to as opposed to correlative, explana- palpis. Physiol. Zool. 60: 297-304. cold shock. tions of cold-hardiness at biochemical Crowe, J. H., L. M. Crowe, J. F. Carpenter, Another hypothesis suggests that and physiological levels. Future ad- and C. A. Wistrom. 1987. Stabilization of vances in understanding are expected dry phospholipid bilayers and proteins by rapid cold-hardening is related to an sugars. Biochem. 1. 242: 1-10. apparently universal biological re- from tissue culture and molecular Crowe, J. H., L. M. Crowe, and R. Mouradian. sponse, known as heat shock. The technology, as well as from NMR, 1983. Stabilization of biological membranes heat-shock response is a cellular re- differential scanning calorimetry, and at low water activities. 20: sponse to environmental stress, char- cryomicroscopy. 346-356. Czajka, M. C., and R. E. Lee. 1988. Cold acterized by the synthesis of a specific shock and rapid cold-hardening in Droso- set of proteins, the heat-shock pro- Acknowledgments phila melanogaster. Cryobiology 25: 546. teins (Burdon 1986, Petersen and DeVries, A. L. 1971. Glycoproteins as biologi- Mitchell 1985). Although the specific This paper is dedicated to Andrew A. cal antifreeze agents in Antarctic . Sci- mechanism is unknown, heat-shock Weaver, College of Wooster, for in- ence 172: 1152-1155. Duman, J., and K. Horwath. 1983. The role of proteins are correlated with enhanced troducing me to the joys of research. I hemolymph proteins in the cold tolerance of thermotolerance. This response is in- thank Dennis L. Claussen and David insects. Ann. Rev. Physiol. 45: 261-270. duced not only by exposure to sub- L. Denlinger for their helpful com- Duman, J. G. 1977. The role of macromolecu- lethal heat treatments, but also by ments on an earlier draft of this lar antifreeze in the darkling beetle, Mera- manuscript. Support from the Na- cantha contracta. J. Comp. Physiol. 115: anoxia, transition-series metals, etha- 279-286. nol, teratogens, and some other tional Science Foundation (grant Duman, J. G., L. G. Neven, J. M. Beals, K. R. chemicals. Due to the wide range of DCB-8811317) and the National In- Olson, and F. J. Castellino. 1985. Freeze- environmental inducers, heat-shock stitutes of Health (grant SAT 1 R15 tolerance , including hae- proteins are sometimes referred to as HL40535-01) is gratefully acknowl- molymph protein and lipoprotein nucleators, in the larvae of the cranefly Tipula trivittata. stress proteins. edged. 1.Insect Physiol. 3: 1-8. Striking similarities exist between Heinrich, B. 1981. Energetics of honeybee the heat-shock response and the rapid References cited swarm thermoregulation. Science 212: 565- cold-hardening response against cold- 566. shock injury. Both occur in less than Bale, J. S. 1987. Insect cold hardiness: freezing Knight, C. A,, and J. G. Duman. 1986. Inhibi- and supercooling-an ecophysiological per- tion of recrystallization of ice by insect ther- an hour. Each provides protection spectlve. ]. lnsect Physiol. 33: 899-908. mal hysteresis proteins: a possible cryopro- against a form of temperature stress. Baust, J. G., and R. E. Lee. 1981. Divergent tective role. Cryobiology 23: 256-262. Both responses are induced by expo- mechanisms of frost-hardiness in two popu- Knight, J. D., J. S. Bale, F. Franks, S. F. Ma- sure to specific temperature triggers, lations of the gall fly, Eurosta solidaginis. I. thias, and J. G. Baust. 1986. Insect cold slightly below the upper lethal tem- lnsect Physiol. 27: 485-490. hardiness: supercooling points and pre- Baust, J. G., R. E. Lee, and R. A. Ring. 1982. freeze mortality. Cryo Lett. 7: 194-203. perature for heat shock or slightly The and of low Kukal, O., A. S. Serianni, and J. G. Duman. above the lower lethal temperature temperature tolerance in insects and other 1988. Glycerol metabolism in a freeze- for rapid cold-hardening. The strong- terrestrial arthropods: a bibliography. Cryo tolerant arctic insect: an in vivo 13 C NMR est tie is that protection against heat Lett. 3: 191-212. study. I. Comp. Physiol. 158: 175-183. stress confers protection against chill- Baust, J. G., and R. R. Rojas. 1985. Review- Lee, R. E., C.-P. Chen, and D. L. Denlinger. Insect cold hardiness: facts and fancy. ]. 1987. A rapid cold-hardening process in ing injury and vice versa. In the flesh lnsect Physiol. 3 1 : 755-759. insects. Science 238: 1414-1417. fly, S. crassipalpis, transfer from 25" Baust, J. G., K. E. Zachariassen. 1983. Season- Lee, R. E., and D. L. Denlinger. 1985. Cold to 36" C for a short period increased ally active cell matrix associated ice nuclea- tolerance in diapausing and non-diapausing survival at -10" C (Chen et al. 1987). tors in an insect. Cryo Lett. 4: 65-71. stages of the flesh fly, Sarcophaga crassi- Conversely, the production of heat- Beck, S. D. 1987. Thermoperiod-photoperiod palpis. Physiol. Ent. 10: 309-315. interactions in the determination of diapause Lee, R. E., D. L. Denlinger, and C.-P. Chen. shock proteins has been linked to in Ostrinia nubilalis. 1. Insect Physiol. 33: 1988. Insect cold-hardiness and diapause: recovery from chilling in Drosophila 707-71 2. regulatory relationships. Pages 243-262 in (Burton et al. 1988). Buchanan, G. W., and K. B. Storey. 1983. In F. Sehnal, A. Zabza, and D. L. Denlinger. vivo detection of cryoprotectants and lipids Endocrinological Frontiers in Physiological in overwintering larvae using carbon-13 nu- lnsect Ecology. Wroclaw Technical Univer- Conclusions clear magnetic resonance spectroscopy. Can. sity Press, Wroclaw, Poland. 1.Biochem. Cell Biol. 61: 1260-1264. Lee, R. E., R. A. Ring, and J. G. Baust. 1986. The nature of insect low-temperature Burdon, R. H. 1986. Heat shock and the heat Low temperature tolerance in insects and tolerance is clearly complex, and this shock proteins. Biochem. 1.240: 313-324. other terrestrial arthropods: bibliography 11.

312 BioScience Vol. 39 No. 5 Cryo Lett. 7: 113-126. Loomis, S. H., J. F. Carpenter, and J. H. Savage Heat Wmes,Waer Shortap9 Crowe. 1988. Identification of strombine and taurine as cryoprotectants in the inter- tidal bivalve Mytilus edulis. Biochim. ParchedFarms Biophys. Acta 943: 113-1 18. Mazur, P. 1984. Freezing of living cells: mech- anisms and implications. Am. I. Physiol. 247 Year After Year After Year C125-C142 (Cell Physiol. 16). McGrath, J. J. 1987. Cold shock: thermoelastic The Greenhouse Effect May Take Hold of the Earth stress in chilled biological membranes. Pages 57-66 in K. R. Diller, ed. Network Thermo- In Your Lifetime dynamics, Heat and Mass Transfer in Bio- technology. American Society of Mechanical The National Arbor Day Foundation destroying the world's forests. And Engineers Bed vol. 5, Heat Transfer Division Urges You to Plant Ihes to Fight plant trees. vol. 90. United Engineering Center, New the Greenhouse Effect You can make a difference. York. Trees you plant may be our best line Miller, K. 1982. Cold-hardiness strategies of he Earth is heating up. of defense. Trees can shade your some adult and immature insects overwinter- "The Greenhouse Effect," home in summer, and slow winter ing in interior Alaska. Comp. Biochem. says James E. Hansen, Direc- wind. While your trees absorb COP, Physiol. 73A: 595-604. T tor of NASA's Institute for Space they also reduce the amount of fossil Morris, G. J. 1987. Direct chilling injury. Pages Studies, "is here.. .it's time to stop 120-146 in B. W. W. Grout and G. J. Mor- fuel burned for cooling and heating. ris, eds. The Effects of Low Temperature on waffling." As a result, your trees can be as Biological Systems. Edward Arnold, Lon- Scientists have tracked the warm- effective as 15 forest trees in fighting don. ing of the Earth for decades. It is the Greenhouse Effect. Petersen, N. S., and H. K. Mitchell. 1985. Heat accelerating at an ominous pace. Free Booklet. The National shock proteins. Pages 347-366 in G. A. A sharp increase in atmospheric Arbor Day Foundation has published Kerkut and L. I. Gilbert, eds. Comprehensive carbon dloxide (COP) is a major a guidebook titled Conservatron Insect Physiology, Biochemistry and Phar- cause. 'lbo thlngs contribute - the Tkes whlch has colorful photos and macology. vol. 10. Biochemistry. Pergamon burning of fossil fuels and the illustrations and easy-to-understand Press, Elmsford, NY. Quinn, P. J. 1985. .2 lipid-phase separation destruction of forests. descriptions that will show you.. . model of low-temperature damage to biolog- How to use shade trees and wind- ical membranes. Cryobiology 22: 128-146. breaks to save energy In your Ring, R. A. 1982. Freezing-tolerant insects home with low supercooling points. Comp. Bio- How to save trees during con- chem. Physiol. 73A: 605-612. struction Ring, R. A., and D. Tesar. 1981. Adaptations The rlght way to plant trees to cold in Canadian arctic insects. Cryobiol- The right way to prune trees THE GREENH CT Increased ogy 18: 199-211. I'm pleased to offer this informa- Salt, R. W. 1961. Principles of insect cold- carbon d~ox~de hardiness. Ann. Rev. Ent. 6: 55-74. tive booklet to you free of charge. . 1969. The survival of insects at low Just return the coupon below and temperatures. Symp. Soc. Exp. Biol. 23: I'll send your Conservatzon Dees 331-350. booklet by return mail. Ssmme, L. 1982. Supercooling and winter sur- If you believe, as I do, that fighting vival in terrestrial arthropods. Comp. Bio- the Greenhouse Effect is vltally chem. Physiol. 73A: 519-543. important to our future, send for your Southwick, E. E., and G. Heldmaier. 1987. free Conservation Dees booklet Temperature control in honey bee colonies. today. BioScience 37: 395-399. Storey, K. B., and J. M. Storey. 1981. Biochem- ical strategies of overwintering in the gall fly Carbon dioxlde is a one-way filter larvae, Eurosta solidaginis: effect of low tem- It lets the sun's energy pass through @& perature acclimation on the activities of en- but traps the heat rising from the John Rosenow, Executive Director zymes of intermediary metabolism. ]. Comp. Earth - what scientists call the National Arbor Day Foundatlon Physiol. 144: 191-199. "Greenhouse Effect ." ------. 1988. Freeze tolerance in animals. Trees remove COP from the atmo- 1 Physiol. Rev. 68: 27-84. sphere, but mankind has destroyed a I YM, I want to I Turnock, W. J., R. J. Lamb, and R. P. Bod- thlrd of the world's forests. help fight the I naryk. 1983. Effects of cold stress during I GreenhouseSend my free Effect. I pupal diapause on the survival of Mamestra COP in the atmosphere has 1 configurata (Lepidoptera: Noctuidae). Oeco- increased by one-fourth since the I Conservat~onTkes I logia 56: 185-192. industrial age began, and scientists Zachariassen, K. E. 1985. Physiology of cold estimate that it could double in the I booklet. I tolerance in insects. Phvsiol. Rev. 65: 799- I I 832. nextIf thatcentury. happens, the huge polar Zachariassen, K. E., and J. A. Husby. 1982. ice caps may melt, causing I'4 Name I Antifreeze effect of thermal hysteresis agents oceans to flood coastal cities. protects highly supercooled insects. Nature I I Drought will plague America's I Addresq 298: 865-867. breadbasket. Rivers that supply I water to cities will dry up. Heat I clt~ I waves will be commonplace. I I Much can be done to slow the I State ZIP I Greenhouse Effect. We must ~Ma~ItoConservat~onTreesNat~onalArborDay Fwndal~on.Nebraska Clty NE 68410 I decrease our use of fossil fuels. Stop L -,,------,-I May 1989