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Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes
J. Insecr Physiol. Vol. 30, No. 3, pp. 235-239, 1984 Printed
in Great
Britain.
Copyright
AH rights reserved
0022-1910/84$3.OO+O.UU IQ 1984 Pergamon Press Ltd
CHANGE IN OVERWINTERING MECHANISM OF THE CUCUJID BEETLE, CUCUJUS CLAJ4PES JOHN G. DUMAN Biology Department,
University of Notre Dame, Notre Dame, IN 46556. U.S.A.
(Received 16 June 1983: revised Seprember 1983) Abstract-Overwintering larvae of the Cucujid beetle, Cucujus clatlipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 19781979. 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 - IO%), thus preventing lethal intracellular ice formation. In contrast, C. &wipes 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. Key Word Index:
Cold tolerance. freeze tolerance, antifreeze. ice nucleators, Cucujus ciuuipes
INTRODUCTION The overwintering stages of insects have generally been classified as either freeze tolerant (those able to survive freezing of the extracellular body fluids) or freeze susceptible (those which die if frozen) (Danks,
1978; Ring, 1980; Block, 1982). Freeze-susceptible species must avoid freezing by seeking thermally buffered microhabitats and/or depressing the haemolymph freezing point and the temperature to which they can supercool before spontaneous nucleation occurs (the supercooling point) (Sbmme, 1982). Antifreeze agents which lower the freezing and supercooling points of insects include polyhydroxy alcohols (such as glycerol), sugars, thermal hysteresis proteins (Duman et al., 1982) and ethylene glycol (Gehrken, 1982). Removal and/or masking of ice nucleating agents from the gut, haemolymph, etc. in winter may also be important in extending supercooling in some species (Zachariassen, 1982; Baust and Zachariassen, 1983). The physiological and biochemical adaptations involved in insect freeze tolerance are complicated and not completely understood (Baust, 1973, 1981; Ring, 1980). Polyols, expecially glycerol and sorbitol, are generally important as cryoprotective agents to help prevent freeze damage (Zachariassen, 1979; Baust, 1973; Ring, 1980). However, high polyol levels alone do not render an insect freeze tolerant (Somme, 1964). Another adaptation found in many freeze tolerant species is the presence of haemolymph ice nucleating agents which function to initiate ice formation in the extracellular fluid at relatively high subzero temperatures, generally between - 4” to - 10°C (Zachariassen and Hammel, 1976; Zachariassen, 1982; Duman, 1982; Duman and Horwath, 1983). The ice nucleators thus prevent lethal intracellular ice formation, which can result
from extensive supercooling prior to freezing. The insect haemolymph ice nucleating agents appear to be proteinaceous (Zachariassen and Hammel, 1976; Duman and Patterson, 1978; Somme and ConradiLarsen 1979). An ice nucleating protein has been purified from the haemolymph of the freeze tolerant queens of the bold faced hornet, Vespula maculata (Duman et al., 1983). Although most freeze tolerant insect species which have been investigated have haemolymph ice nucleating agents, some species do not (Miller, 1982; Ring, 1982). Studies conducted during the late 1970’s on the overwintering larvae of the Pyrochroid beetle Dendroides canadensis from northern Indiana showed that the larvae were freeze tolerant (Duman, 1979, 1980). However, more recent work demonstrated that D. canadensis larvae from the same population had changed to a freeze-susceptible overwintering mechanism with extended supercooling (Horwath and Duman, 1984). Levels of thermal hysteresis proteins and polyols (glycerol and sorbitol) were the same in both freeze-tolerant and susceptible larvae. Freezetolerant larvae had ice nucleating proteins in their haemolymph which initiated spontaneous nucleation at - 11“C. The only identified difference between the former freeze tolerant and the more recent freezesusceptible larvae was the absence of the ice nucleators in the latter. This was the only known example of such a switch in overwintering mechanism in a cold hardy insect and consequently it might be assumed that such changes are rare. The larvae of the Cucujid beetle, Cucujus clavipes (Fabricius), overwinter under the loose bark of dead deciduous trees, in the same habitat as D. canudensis larvae. Adult C. clavipes also successfully overwinter, but are not nearly as numerous as the larvae. Previous work showed that the larvae were freeze toler235
236
JOHN G.
ant during the winter of 197881979 (Duman, 1979). The high supercooling points of these larvae ( - - I IC) indicated the presence of haemolymph ice nucleators which initiated spontaneous nucleation just below the haemolymph freezing point temperature. This manuscript demonstrates a switch in overwintering strategy in C. claoipes larvae from the freeze tolerant to a freeze-susceptible mechanism involving supercooling, the details of which are very similar to those previously documented in D. canadensis larvae. MATERIALS AND METHODS
C. clavipes and D. canadensis larvae were collected from under the bark of dead deciduous trees in Potato Creek State Park in northern Indiana. Individuals were maintained at 4°C upon return to the laboratory and were sampled within 2 h. Haemolymph was taken by puncturing the cuticle in the dorsal midline with a 2%gauge needle and collecting the haemolymph in a 10~1 glass capillary tube. Lower lethal temperatures were determined by placing groups of individuals (15-20) into a freezing chamber, consisting of a small flask in a Brinkman model 1496 or a Lauda K 4R refrigerated bath, and slowly lowering the temperature (0.2” C/min) to various set point temperatures. The groups were kept at these temperatures for 24 h then removed and kept at 4’ C. An individual was considered to have survived a given temperature when it was mobile 24 h after removal from the freezing chamber. The temperature at which 50% of the individuals died was taken as the lower lethal temperature. The supercooling points of the larvae were measured by attaching a thermistor, using Vaseline, onto the cuticle of an individual, placing the specimen into a freezing chamber and lowering the temperature at a rate of 0.5” C/min. The thermistor was connected to a YSI model 42 SL telethermometer and a recorder (Fisher 5000). The temperature at which the release of the heat of fusion marked the initiation of freezing was recorded as the supercooling point (Salt, 1966). A modification of the technique of Ramsay and Brown (1955) was used to measure the freezing and melting points of the haemolymph (Duman, 1977). The haemolymph was placed in a sealed capillary tube, a small ( - 0.25 mm) seed crystal was sprayfrozen (Cryokwik) in the sample and the capillary was placed into a refrigerated alcohol bath which was equipped with a viewing chamber through which the crystal could be observed with a microscope. The bath temperature was controlled to f O.Ol”C. The temperature was slowly raised (O.O2”C/5min) until the crystal disappeared. This temperature was taken as the melting point of the sample. Another crystal was spray-frozen in the sample, the capillary again placed in the chamber and the bath temperature lowered (0.1 C/2.5 min) until the crystal grew. The temperature at which the seed crystal began to grow was taken as the haemolymph hysteretic freezing point. Aqueous solutions which do not contain thermal hysteresis antifreeze proteins exhibit identical freezing and melting points. However, in the presence of these proteins the freezing point is often depressed several degrees below the melting point. This
DUMAN difference between the melting and freezing points is termed thermal hysteresis. The magnitude of the thermal hysteresis exhibited by a sample is dependent upon the concentration of the antifreeze protein present (Patterson and Duman. 1982; Tomchaney et al., 1982). The level of undercooling exhibited by C. ckacipes larvae was calculated by taking the difference between the haemolymph freezing points (mean) and whole body supercooling points (mean) for a group. An undercooling of only a few degrees is indicative of the presence of ice nucleating factors (Duman. 1980). In the absence of potent ice nucleating agents an aqueous solution, or an insect. will typically supercool l5-2o’C below the freezing point. Antifreeze agents, such as glycerol, extend the amount of undercooling by approximately twice the amount that they depress the freezing point (MacKenzie, 1977; Block and Young. 1979). The effect of the absence of ice nucleating agents on the lack of freeze tolerance in cold-tolerant. winter-collected (February 1983) larvae of the beetles Dendroides canadensis and Cucuj~(s rlacipes was checked in the following manner. A group of larvae (20-25 individuals) was placed in a freezing chamber at 0°C and lowered slowly (0.2 Cimin) to - 10 C. This temperature was Y 3 C below the mean hysteretic freezing point of the larvae and well above the supercooling point temperature ( - 25 to - 30°C). Prior to being placed in the freezing chamber each larva was pricked in the dorsal midline with a 28.gauge needle. This caused a drop of haemolymph to well-up around the wound. The wounding caused most of the larvae to freeze at or above - IVC as ice crystals on the surface of the larvae (formed as water condensed and froze on the surface of the cuticle as the temperature was lowered) seeded the haemolymph across the wound. Those individuals which had frozen were obvious because of the opaque nature of the droplet of haemolmph around the wound. Unfrozen individuals were seeded by touching the haemolymph droplet with the frozen tip of a Pasteur pipette which had been filled with water and placed in a freezer at -2o’C. A control group (25 individuals) was wounded and placed in a freezing chamber at 0 C and lowered slowly to -4 ‘C. Since this temperature was above the freezing point of the haemolymph, these larvae did not freeze. A second control (25 individuals) consisted of unwounded larvae which were placed in the freezing chamber at 0 ‘C and slowly lowered to - 10 C. Because the larvae, in the absence of wounding. would supercool to - -3OXZ, these larvae did not freeze. The groups were held in the freezing chamber at the stated temperature for 24 h. The groups were then removed and held at 4°C. Those individuals which were mobile 24 h later were considered to have survived the treatment. RESULTS
As demonstrated in Table I. the supercooling points of C. cluvipes larvae collected in February 1983 ( - 30°C) were much lower than those of larvae collected in February, 1979 ( - IOC). However, the 24 h lower lethal temperatures of the 2 groups were
Overwintering strategy change
231
Table I, Comparisons of the 24 h lower lethal temperatures. supercooling points, haemolymph hysteretic freezing points and the amount of undercooling (freezing point minus organismal supercooling point) of C. cluuipes larvae collected in early February of 1979 with those collected in early February of 1983 Lower lethal temperature (“C)
Date February 2. 1979 February 16. 1983
Freezing point (“C)
Supercooling point (“C)
- 26 - 30
- 10.7 * 1.7 (10) - 30.1 k 2.3 (10)
Undercooling ( ‘C)
- 6.93 f 1.18 (7) - 5.71 f 1.50 (10)
3.8 24.4
Values indicate means k standard deviation, except for the lower lethal temperatures and undercooling. Values in parentheses indicate sample size.
Table 2. Comparisons of the haemolymph melting points, hysteretic freezing points and thermal hysteresis (melting point minus freezing point) of C. clavipes larvae collected in early February of 1979 with those collected in early February of 1983 Date February 2, 1979 (7) February 16, 1983 (10) Values indicate mean sample size.
f
Melting point (“C)
Freezing point (“C)
Thermal hysteresis (“C)
- 3.04 * 0.89 - 2.58 + 0.57
-6.93* 1.18 - 5.71 + 1.50
3.89 + 1.52 3.13 + 1.12
standard
deviation. Numbers in parentheses
approximately the same ( - 26°C in 1979 and - 30°C in 1983). It is obvious that C. cluvipes larvae were freeze tolerant during February of 1979 (The lower
lethal temperature was well below the supercooling point I, but the larvae were not freeze tolerant during February of 1983. (The lower lethal temperature and the supercooling points were nearly identical.) The minor amount of undercooling (3.8”C) below the haemolymph freezing point in 1979 indicates the presence of ice nucleating agents in these freeze tolerant larvae. In contrast, the extensive undercooling (24&C) in 1983 larvae shows the absence of the ice nucleator, a point of major importance in freeze-susceptible larvae in which the ability to supercool is critical. Table 2 illustrates that the melting and freezing points of the haemolymph, and also the levels of thermal hysteresis (indicative of the concentration of thermal hysteresis antifreeze proteins), were not significantly different in larvae collected in February of the 2 years. Table 3 compares the concentrations of glycerol and sorbitol in the haemolymph of larvae during the 2 years. Glycerol levels were nearly twice as high in February 1979 than in February 1983. Likewise, the sorbitol concentrations were significantly higher in 1979. The major difference measured between the freezetolerant larvae collected in 1979 and the freezesusceptible C. culvipes studied in 1983 was the absence of the ice nucleating agent in the latter. In an
indicate
attempt to determine whether the absence of the ice nucleator was the critical factor in the lack of freeze tolerance in C. &viper in 1983, larvae were artificially seeded across a wound in the cuticle (See Materials and Methods section) at a temperature close to the supercooling point which had been naturally induced by the ice nucleator in the 1979 larvae (- 1OC). As shown in Table 4, these artificially seeded larvae did not survive freezing. In contrast, unfrozen control groups which were (1) wounded and held at -4°C or (2) held supercooled at -10°C both had high percentages of survival. Similar experiments with freeze-susceptible Den droides cnnadensis larvae collected in February 1983 yielded nearly identical results. It should be stated that D. CQnadenSiSlarvae had haemolymph freezing points (= - 6°C) and supercooling points (_ - 30°C) similar to those of C. chvipes. These data indicate that the primary cause of the lack of freeze tolerance in the larvae of both C. clupives and D. canadensis either was not the absence of an ice nucleator or that the absence of the ice nucleator was not the only critical factor. DISCUSSION Previous studies (Horwath and Duman, 1984) demonstrated that Dendroides canadensis larvae had
switched from a freeze-tolerant anism (involving haemolymph
Table 3. Comparison of haemolymph polyol (glycerol and sorbitol) concentrations of C. clavipes larvae collected in early February 1979 with those collected in early February 1983 Date February 2, 1979 (7) February 16, 1983 (9)
Glycerol (M)
Sorbitol (M)
0.46 f 0.23 0.25 + 0.14
0.07 _+0.02 0.04 + 0.01
Values indicate means + standard deviation. Numbers in parentheses indicate sample size.
overwintering mechice nucleators) to a
238
JOHN
G. DUMAN
Table 4. The effect on survival of (1) artificially nucleating freeze susceptible larvae across a puncture wound in the cuticle at a temperature of - 10°C (the approximate temperature at which the haemolymph nucleators caused natural nucleation in 1979 larvae which were freeze tolerant); (2) holding wounded, but unfrozen larvae at -4-C (above the hysteretic freezing point): and (3) holding unwounded, supercooled larvae at - 10 C (well above the supercooling point) Species
Treatment
~~
C. chipes
( I ) Wounded
L?. cunadensis
artificial nucleation (2) Wounded (3) None ( 1) Wounded artificial cucleation (2) Wounded (3) None
Temperature (“C) ..~ -10
7” Survival 0
-4 - 10 -10
96 100 0
-4 -10
92 100
See Materials and Methods for details. All larvae were collected in early February, 1983.
supercooling strategy in which the haemolymph ice nucleators were no longer present and in which the
larvae died if frozen. This study documents a nearly identical occurrence in the overwintering larvae of Cucujus chipes. It is evident from these studies that it can no longer be taken for granted that an insect species will be freeze tolerant during a given year based on previous determinations of freeze tolerance in former years. This underscores the need for longterm monitoring of other insect populations to determine whether these swiitches are common. In both D. canadensis and C. clauipes a critical unanswered question concerns whether these species will revert to a freeze-tolerant strategy in the future. In addition, the factors (environmental cues etc.) which may have triggered the change are unknown. The winter of 1982-1983 was much less severe than that of 1978-1979 and it is possible that this difference was responsible for the observed physiological changes, however this is unlikely. The temperatures throughout the preceding autumns during the 2 years were quite similar and consequently the temperatures, and of course photoperiods, experienced during the seasonal acclimatizations should have been much the same. In past years C. clauipes, as well as other freeze-tolerant species which we have studied from this area, became freeze tolerant in late November or early December (Duman. 1979). A somewhat related situation occurs between northern (New York) and southern (Texas) populations of the goldenrod gall fly, Eurosta solidagensis, where the development of freeze tolerance in response to temperature cues varies (Baust et al., 1979). However, the change in overwintering mechanisms described for both C. clauipes and D. catladensis has taken place within a population. An additional unknown factor concerns the advantage of one strategy over the other. Recall that in both species the lower lethal temperatures were approximately identical during both freeze tolerant and freeze susceptible years. Consequently there does not appear to be any advantage in either system in regard to short term tolerance of a given minimal temperature. Of course other possible advantages are likely to pertain, but these remain obscure. The haemolymph concentrations of polyols (both glycerol and sorbitol) were significantly greater when the C. clavipes larvae were freeze tolerant in February 1979 than they were in the freeze-susceptible larvae in
February 1983. As the cryoprotective effect of polyols, especially glycerol, is fairly well established (Baust, 1973; Zachariassen, 1979; Ring, 1980) it is possible that the lower polyol levels in 1983 contributed to the loss of freeze tolerance. However, this is unlikely. Early in the winter of 1978-1979 C. clavipes larvae were collected (December 8, 1978) which were freeze tolerant but lacked high glycerol concentrations. (Glycerol was not detectable with the same chromatographic technique used in this study.) Sorbitol (0.03M) was the only polyol detected (Duman, 1979). Consequently, although it is probable that polyols were important contributing factors to the freeze tolerance of the larvae in the winter of 1978-1979, it is not likely that the lowered polyol levels in 1983 larvae were responsible for the lack of freeze tolerance. Also, the function of the polyols in the 1983 freeze-susceptible larvae is not obvious. The antifreeze function of polyols in depressing the freezing and supercooling points of freeze-susceptible insects is well documented (Sijmme, 1982; Duman et al., 1982). When multimolar concentrations of poly01s are found in a freeze-susceptible insect the contribution of the polyols to the freezing and supercooling point depression can easily be calculated and appreciated. For example, the 5M glycerol concentration in larvae of overwintering Bracon cephi larvae obviously is a major factor in lowering the freezing point of the larvae to - 15°C and the supercooling point to -45°C (Salt, 1959). The polyol levels of C. cfavipes larvae in February 1983 are obviously elevated above those of summer insects, in which polyols are not detectable with the chromatographic technique. However, based on the molar freezing point constant for water (1.86”Q the 0.25M concentration of glycerol in February 1983 larvae would only lower the freezing point of the haemolymph by - 0.47”C. The effect of glycerol on the supercooling point of water is to depress the supercooling point approximately twice as much as it depresses the freezing point (Block and Young, 1979, MacKenzie. 1979). Consequently a 0.25M glycerol concentration should lower the supercooling point by only w 0.94% In fact, the combined contribution of both glycerol and sorbitol to the supercooling point depression would only be slightly more than l”C, hardly an impressive amount. Consequently, a nonantifreeze function for the elevated polyols may be their major contribution to the cold tolerance of the
Overwintering
1983 larvae. The stabilizing effects of glycerol on proteins at low temperatures is one possibility (Gekko and Timashiff, 1981 a,b). As in the earlier studies on D. canadensis, a major physiological variable shown to change in C. clavipes larvae was the presence of haemolymph ice nucleators during 1978-1979 when the larvae were freeze tolerant and the absence of ice nucleators from the haemolymph when freeze tolerance was lost. Consequently, the lack of an ice nucleator and subsequent lethal intracellular ice formation following extensive supercooling might have explained the loss of freeze tolerance in 1983 larvae. However, the experiments in which freeze susceptible larvae collected in February 1983 were artificially seeded, at temperatures approximating those at which they would have frozen had the natural haemolymph ice nucleators been present, demonstrated that in both D. canadensis and C. clavipes the absence of ice nucleators is not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance. It may be that if this other critical unknown factor(s) could be identified and replaced, then the absence of the ice nucleator might prove to be critical. This raises the possibility that these insect species which demonstrate the ability to change overwintering mechanisms could be excellent model systems in which to investigate and hopefully identify these critical unknown factors which are involved in insect freeze tolerance. Acknowledgemenfs-This work was supported Science Foundation Grant PCM 8109708.
by National
REFERENCES Baust J. G. (1973) Mechanisms of cryoprotection in freeze tolerant animals. Cryobio/ogy 10, 197-205. Baust J. G. (1981) Biochemical correlates to cold hardening in insects. Cryobiology 18, 186198. Baust J. G., Grandee R., Condon G. and Morrissey R. E. (1979) Physiol. Zool. 52, 572-580. Baust J. G. and Zachariassen K. E. (1983) Seasonally active cell matrix associated ice nucleators in an insect. CryoLetters 4, 65-71. Block W. (1982) Cold hardiness in invertebrate poikilotherms. Comp. biochem. Physiol. 73A, 581-593. Block W. and Young S. R. (1979) Measurement of supercooling in small arthropods and water droplets. CryoLetters 1, 85-91. Duman J. G. (1977)The role of macromolecular antifreeze in the darkling beetle, Meracantha confracta. J. romp. Physiol. 115, 279-286. Duman J. G. (1979) Thermal hysteresis factors in overwintering insects. J. Insect Physiol. 25, 805-810. Duman J. G. (1980) Factors involved in the overwintering survival of the freeze tolerant beetle, Dendroides cunadensis. J. romp. Physiol. 136, 53-59. Duman J. G. (1982) Insect antifreezes and ice nucleating agents. Cryobiology 19, 613-627. Duman J. G., Horwath K. L., Tomchaney A. and Patterson J. L. (1982) Antifreeze agents of terrestrial arthropods. Comp. biochem. Physiol. 73A, 545-555.
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change
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Duman J. G. and Horwath K. L. (1983) The role of hemolymph proteins in the cold tolerance of insects. A. Reu. Physio. 45, 261-270. Duman J. G., Morris J. P. and Castellino F. J. (1983) Purification and composition of an ice nucleating protein from queens of the hornet, Vespula maculata. J. camp. Physiol. In press. Duman J. G. and Patterson J. L. (1978) The role of ice nucleators in the frost tolerance of overwintering queens of the bald faced hornet, Vespula maculata. Camp. hiothem. Physiol. 59A, 69-72. Danks H. V. (1978) Modes of seasonal adaotation in the insects. I Winter survival. Can. Em. 110. i 167-1205. Gehrken U. (1982) Role of ethylene glycol in the coldhardening of the adult bark beetle. Ips acuminufus. CryoLeflers 3, 307. Gekko K. and Timasheff S. N. (1981a) Mechanism of protein stabilization by glycerol: Preferential hydration in glycerol-water mixtures. Biochemistry 20, 4667-4676. Gekko K. and Timasheff S. N. (1981 b) Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry 20. 46774686. Horwath K. L. and Duman J. G. (1984) Yearly variations in the overwintering mechanism of the cold hardy beetle Dendroides canadensis. Phpsiol. 2001. In press. MacKenzie A. P. (1977) Non-equilibrium freezing behavior of aqueous systems. Phil. Trans. R. Sot. 278, 1677189. Miller K. (1982) Cold-hardiness strategies of some adult and immature insects overwintering in interior Alaska. C’omp. biochem. Physiol. 73A. 595-604. Patterson J. L. and Duman J. G. (1982) Purification and composition of protein antifreezes with high cysteine contents from larvae of the beetle, Tenebrir) moliror. J. exp. Zool. 219, 381-384. Tomchaney A. P., Morris J. P., Kang S. H. and Duman J. G. (1982) Purification, composition and physical properties of a thermal hysteresis antifreeze protein from larvae of the beetle, Tenebrio moliror. Biochemistry 21, 716-721. Ramsay J. A. and Brown R. H. (1955) Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid. J. scient. Inswum. 32. 372-375. Ring R. A. (1980) Insects and their cells. In Low Temp&ature Preservation in Medicine and Biology (Ed. by Ashwood-Smith M. J. and Farrant J. ) on. 187.-217. Univ. Park Press, Baltimore. Ring R. A. (1982) Freezing tolerant insects with low supercooling points. Comp. biochem. Physiol. 73A, 605-612. Salt R. W. (1959) Role of glycerol in the cold hardening of Bracon cephi (Gahan). &n. J. 2001. 37, 59-69. Salt R. W. (1966) Factors influencine nucleation in sunercooled insects.‘Can. J. Zool. 44. 117-133. Samme L. (1964) Effects of glycerol on cold-hardiness in insects. Can. J. Zool. 42. 87-101. Siimme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. biochrm. PIIj:riol. 73A. 519-543. S(imme L. and Conrad&Larsen E. M. (1979) Nucleating agents in the hemolymph of third instar larvae of Eurosta solidugensis (Fitch) (Diptera, Tephritidae) Norm. J. Enf. 25. 187-188. Zachariassen K. E. (1979) The mechanism of the cryoprotective effect of glycerol in beetles tolerant to freezing. J. Insect Phvsiol. 25, 29-32. Zachariassen K. E. (1982) Nucleating agents in cold-hardy insects. Comp. biochem. Physiol. 73Ar 557-563. _ Zachariassen K. E. and Hammel H. T. (1976) Nucleating agents in the heamolymph of insects tolerant to freezing. Nature 262, 285-287. .I
in Great
Britain.
Copyright
AH rights reserved
0022-1910/84$3.OO+O.UU IQ 1984 Pergamon Press Ltd
CHANGE IN OVERWINTERING MECHANISM OF THE CUCUJID BEETLE, CUCUJUS CLAJ4PES JOHN G. DUMAN Biology Department,
University of Notre Dame, Notre Dame, IN 46556. U.S.A.
(Received 16 June 1983: revised Seprember 1983) Abstract-Overwintering larvae of the Cucujid beetle, Cucujus clatlipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 19781979. 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 - IO%), thus preventing lethal intracellular ice formation. In contrast, C. &wipes 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. Key Word Index:
Cold tolerance. freeze tolerance, antifreeze. ice nucleators, Cucujus ciuuipes
INTRODUCTION The overwintering stages of insects have generally been classified as either freeze tolerant (those able to survive freezing of the extracellular body fluids) or freeze susceptible (those which die if frozen) (Danks,
1978; Ring, 1980; Block, 1982). Freeze-susceptible species must avoid freezing by seeking thermally buffered microhabitats and/or depressing the haemolymph freezing point and the temperature to which they can supercool before spontaneous nucleation occurs (the supercooling point) (Sbmme, 1982). Antifreeze agents which lower the freezing and supercooling points of insects include polyhydroxy alcohols (such as glycerol), sugars, thermal hysteresis proteins (Duman et al., 1982) and ethylene glycol (Gehrken, 1982). Removal and/or masking of ice nucleating agents from the gut, haemolymph, etc. in winter may also be important in extending supercooling in some species (Zachariassen, 1982; Baust and Zachariassen, 1983). The physiological and biochemical adaptations involved in insect freeze tolerance are complicated and not completely understood (Baust, 1973, 1981; Ring, 1980). Polyols, expecially glycerol and sorbitol, are generally important as cryoprotective agents to help prevent freeze damage (Zachariassen, 1979; Baust, 1973; Ring, 1980). However, high polyol levels alone do not render an insect freeze tolerant (Somme, 1964). Another adaptation found in many freeze tolerant species is the presence of haemolymph ice nucleating agents which function to initiate ice formation in the extracellular fluid at relatively high subzero temperatures, generally between - 4” to - 10°C (Zachariassen and Hammel, 1976; Zachariassen, 1982; Duman, 1982; Duman and Horwath, 1983). The ice nucleators thus prevent lethal intracellular ice formation, which can result
from extensive supercooling prior to freezing. The insect haemolymph ice nucleating agents appear to be proteinaceous (Zachariassen and Hammel, 1976; Duman and Patterson, 1978; Somme and ConradiLarsen 1979). An ice nucleating protein has been purified from the haemolymph of the freeze tolerant queens of the bold faced hornet, Vespula maculata (Duman et al., 1983). Although most freeze tolerant insect species which have been investigated have haemolymph ice nucleating agents, some species do not (Miller, 1982; Ring, 1982). Studies conducted during the late 1970’s on the overwintering larvae of the Pyrochroid beetle Dendroides canadensis from northern Indiana showed that the larvae were freeze tolerant (Duman, 1979, 1980). However, more recent work demonstrated that D. canadensis larvae from the same population had changed to a freeze-susceptible overwintering mechanism with extended supercooling (Horwath and Duman, 1984). Levels of thermal hysteresis proteins and polyols (glycerol and sorbitol) were the same in both freeze-tolerant and susceptible larvae. Freezetolerant larvae had ice nucleating proteins in their haemolymph which initiated spontaneous nucleation at - 11“C. The only identified difference between the former freeze tolerant and the more recent freezesusceptible larvae was the absence of the ice nucleators in the latter. This was the only known example of such a switch in overwintering mechanism in a cold hardy insect and consequently it might be assumed that such changes are rare. The larvae of the Cucujid beetle, Cucujus clavipes (Fabricius), overwinter under the loose bark of dead deciduous trees, in the same habitat as D. canudensis larvae. Adult C. clavipes also successfully overwinter, but are not nearly as numerous as the larvae. Previous work showed that the larvae were freeze toler235
236
JOHN G.
ant during the winter of 197881979 (Duman, 1979). The high supercooling points of these larvae ( - - I IC) indicated the presence of haemolymph ice nucleators which initiated spontaneous nucleation just below the haemolymph freezing point temperature. This manuscript demonstrates a switch in overwintering strategy in C. claoipes larvae from the freeze tolerant to a freeze-susceptible mechanism involving supercooling, the details of which are very similar to those previously documented in D. canadensis larvae. MATERIALS AND METHODS
C. clavipes and D. canadensis larvae were collected from under the bark of dead deciduous trees in Potato Creek State Park in northern Indiana. Individuals were maintained at 4°C upon return to the laboratory and were sampled within 2 h. Haemolymph was taken by puncturing the cuticle in the dorsal midline with a 2%gauge needle and collecting the haemolymph in a 10~1 glass capillary tube. Lower lethal temperatures were determined by placing groups of individuals (15-20) into a freezing chamber, consisting of a small flask in a Brinkman model 1496 or a Lauda K 4R refrigerated bath, and slowly lowering the temperature (0.2” C/min) to various set point temperatures. The groups were kept at these temperatures for 24 h then removed and kept at 4’ C. An individual was considered to have survived a given temperature when it was mobile 24 h after removal from the freezing chamber. The temperature at which 50% of the individuals died was taken as the lower lethal temperature. The supercooling points of the larvae were measured by attaching a thermistor, using Vaseline, onto the cuticle of an individual, placing the specimen into a freezing chamber and lowering the temperature at a rate of 0.5” C/min. The thermistor was connected to a YSI model 42 SL telethermometer and a recorder (Fisher 5000). The temperature at which the release of the heat of fusion marked the initiation of freezing was recorded as the supercooling point (Salt, 1966). A modification of the technique of Ramsay and Brown (1955) was used to measure the freezing and melting points of the haemolymph (Duman, 1977). The haemolymph was placed in a sealed capillary tube, a small ( - 0.25 mm) seed crystal was sprayfrozen (Cryokwik) in the sample and the capillary was placed into a refrigerated alcohol bath which was equipped with a viewing chamber through which the crystal could be observed with a microscope. The bath temperature was controlled to f O.Ol”C. The temperature was slowly raised (O.O2”C/5min) until the crystal disappeared. This temperature was taken as the melting point of the sample. Another crystal was spray-frozen in the sample, the capillary again placed in the chamber and the bath temperature lowered (0.1 C/2.5 min) until the crystal grew. The temperature at which the seed crystal began to grow was taken as the haemolymph hysteretic freezing point. Aqueous solutions which do not contain thermal hysteresis antifreeze proteins exhibit identical freezing and melting points. However, in the presence of these proteins the freezing point is often depressed several degrees below the melting point. This
DUMAN difference between the melting and freezing points is termed thermal hysteresis. The magnitude of the thermal hysteresis exhibited by a sample is dependent upon the concentration of the antifreeze protein present (Patterson and Duman. 1982; Tomchaney et al., 1982). The level of undercooling exhibited by C. ckacipes larvae was calculated by taking the difference between the haemolymph freezing points (mean) and whole body supercooling points (mean) for a group. An undercooling of only a few degrees is indicative of the presence of ice nucleating factors (Duman. 1980). In the absence of potent ice nucleating agents an aqueous solution, or an insect. will typically supercool l5-2o’C below the freezing point. Antifreeze agents, such as glycerol, extend the amount of undercooling by approximately twice the amount that they depress the freezing point (MacKenzie, 1977; Block and Young. 1979). The effect of the absence of ice nucleating agents on the lack of freeze tolerance in cold-tolerant. winter-collected (February 1983) larvae of the beetles Dendroides canadensis and Cucuj~(s rlacipes was checked in the following manner. A group of larvae (20-25 individuals) was placed in a freezing chamber at 0°C and lowered slowly (0.2 Cimin) to - 10 C. This temperature was Y 3 C below the mean hysteretic freezing point of the larvae and well above the supercooling point temperature ( - 25 to - 30°C). Prior to being placed in the freezing chamber each larva was pricked in the dorsal midline with a 28.gauge needle. This caused a drop of haemolymph to well-up around the wound. The wounding caused most of the larvae to freeze at or above - IVC as ice crystals on the surface of the larvae (formed as water condensed and froze on the surface of the cuticle as the temperature was lowered) seeded the haemolymph across the wound. Those individuals which had frozen were obvious because of the opaque nature of the droplet of haemolmph around the wound. Unfrozen individuals were seeded by touching the haemolymph droplet with the frozen tip of a Pasteur pipette which had been filled with water and placed in a freezer at -2o’C. A control group (25 individuals) was wounded and placed in a freezing chamber at 0 C and lowered slowly to -4 ‘C. Since this temperature was above the freezing point of the haemolymph, these larvae did not freeze. A second control (25 individuals) consisted of unwounded larvae which were placed in the freezing chamber at 0 ‘C and slowly lowered to - 10 C. Because the larvae, in the absence of wounding. would supercool to - -3OXZ, these larvae did not freeze. The groups were held in the freezing chamber at the stated temperature for 24 h. The groups were then removed and held at 4°C. Those individuals which were mobile 24 h later were considered to have survived the treatment. RESULTS
As demonstrated in Table I. the supercooling points of C. cluvipes larvae collected in February 1983 ( - 30°C) were much lower than those of larvae collected in February, 1979 ( - IOC). However, the 24 h lower lethal temperatures of the 2 groups were
Overwintering strategy change
231
Table I, Comparisons of the 24 h lower lethal temperatures. supercooling points, haemolymph hysteretic freezing points and the amount of undercooling (freezing point minus organismal supercooling point) of C. cluuipes larvae collected in early February of 1979 with those collected in early February of 1983 Lower lethal temperature (“C)
Date February 2. 1979 February 16. 1983
Freezing point (“C)
Supercooling point (“C)
- 26 - 30
- 10.7 * 1.7 (10) - 30.1 k 2.3 (10)
Undercooling ( ‘C)
- 6.93 f 1.18 (7) - 5.71 f 1.50 (10)
3.8 24.4
Values indicate means k standard deviation, except for the lower lethal temperatures and undercooling. Values in parentheses indicate sample size.
Table 2. Comparisons of the haemolymph melting points, hysteretic freezing points and thermal hysteresis (melting point minus freezing point) of C. clavipes larvae collected in early February of 1979 with those collected in early February of 1983 Date February 2, 1979 (7) February 16, 1983 (10) Values indicate mean sample size.
f
Melting point (“C)
Freezing point (“C)
Thermal hysteresis (“C)
- 3.04 * 0.89 - 2.58 + 0.57
-6.93* 1.18 - 5.71 + 1.50
3.89 + 1.52 3.13 + 1.12
standard
deviation. Numbers in parentheses
approximately the same ( - 26°C in 1979 and - 30°C in 1983). It is obvious that C. cluvipes larvae were freeze tolerant during February of 1979 (The lower
lethal temperature was well below the supercooling point I, but the larvae were not freeze tolerant during February of 1983. (The lower lethal temperature and the supercooling points were nearly identical.) The minor amount of undercooling (3.8”C) below the haemolymph freezing point in 1979 indicates the presence of ice nucleating agents in these freeze tolerant larvae. In contrast, the extensive undercooling (24&C) in 1983 larvae shows the absence of the ice nucleator, a point of major importance in freeze-susceptible larvae in which the ability to supercool is critical. Table 2 illustrates that the melting and freezing points of the haemolymph, and also the levels of thermal hysteresis (indicative of the concentration of thermal hysteresis antifreeze proteins), were not significantly different in larvae collected in February of the 2 years. Table 3 compares the concentrations of glycerol and sorbitol in the haemolymph of larvae during the 2 years. Glycerol levels were nearly twice as high in February 1979 than in February 1983. Likewise, the sorbitol concentrations were significantly higher in 1979. The major difference measured between the freezetolerant larvae collected in 1979 and the freezesusceptible C. culvipes studied in 1983 was the absence of the ice nucleating agent in the latter. In an
indicate
attempt to determine whether the absence of the ice nucleator was the critical factor in the lack of freeze tolerance in C. &viper in 1983, larvae were artificially seeded across a wound in the cuticle (See Materials and Methods section) at a temperature close to the supercooling point which had been naturally induced by the ice nucleator in the 1979 larvae (- 1OC). As shown in Table 4, these artificially seeded larvae did not survive freezing. In contrast, unfrozen control groups which were (1) wounded and held at -4°C or (2) held supercooled at -10°C both had high percentages of survival. Similar experiments with freeze-susceptible Den droides cnnadensis larvae collected in February 1983 yielded nearly identical results. It should be stated that D. CQnadenSiSlarvae had haemolymph freezing points (= - 6°C) and supercooling points (_ - 30°C) similar to those of C. chvipes. These data indicate that the primary cause of the lack of freeze tolerance in the larvae of both C. clupives and D. canadensis either was not the absence of an ice nucleator or that the absence of the ice nucleator was not the only critical factor. DISCUSSION Previous studies (Horwath and Duman, 1984) demonstrated that Dendroides canadensis larvae had
switched from a freeze-tolerant anism (involving haemolymph
Table 3. Comparison of haemolymph polyol (glycerol and sorbitol) concentrations of C. clavipes larvae collected in early February 1979 with those collected in early February 1983 Date February 2, 1979 (7) February 16, 1983 (9)
Glycerol (M)
Sorbitol (M)
0.46 f 0.23 0.25 + 0.14
0.07 _+0.02 0.04 + 0.01
Values indicate means + standard deviation. Numbers in parentheses indicate sample size.
overwintering mechice nucleators) to a
238
JOHN
G. DUMAN
Table 4. The effect on survival of (1) artificially nucleating freeze susceptible larvae across a puncture wound in the cuticle at a temperature of - 10°C (the approximate temperature at which the haemolymph nucleators caused natural nucleation in 1979 larvae which were freeze tolerant); (2) holding wounded, but unfrozen larvae at -4-C (above the hysteretic freezing point): and (3) holding unwounded, supercooled larvae at - 10 C (well above the supercooling point) Species
Treatment
~~
C. chipes
( I ) Wounded
L?. cunadensis
artificial nucleation (2) Wounded (3) None ( 1) Wounded artificial cucleation (2) Wounded (3) None
Temperature (“C) ..~ -10
7” Survival 0
-4 - 10 -10
96 100 0
-4 -10
92 100
See Materials and Methods for details. All larvae were collected in early February, 1983.
supercooling strategy in which the haemolymph ice nucleators were no longer present and in which the
larvae died if frozen. This study documents a nearly identical occurrence in the overwintering larvae of Cucujus chipes. It is evident from these studies that it can no longer be taken for granted that an insect species will be freeze tolerant during a given year based on previous determinations of freeze tolerance in former years. This underscores the need for longterm monitoring of other insect populations to determine whether these swiitches are common. In both D. canadensis and C. clauipes a critical unanswered question concerns whether these species will revert to a freeze-tolerant strategy in the future. In addition, the factors (environmental cues etc.) which may have triggered the change are unknown. The winter of 1982-1983 was much less severe than that of 1978-1979 and it is possible that this difference was responsible for the observed physiological changes, however this is unlikely. The temperatures throughout the preceding autumns during the 2 years were quite similar and consequently the temperatures, and of course photoperiods, experienced during the seasonal acclimatizations should have been much the same. In past years C. clauipes, as well as other freeze-tolerant species which we have studied from this area, became freeze tolerant in late November or early December (Duman. 1979). A somewhat related situation occurs between northern (New York) and southern (Texas) populations of the goldenrod gall fly, Eurosta solidagensis, where the development of freeze tolerance in response to temperature cues varies (Baust et al., 1979). However, the change in overwintering mechanisms described for both C. clauipes and D. catladensis has taken place within a population. An additional unknown factor concerns the advantage of one strategy over the other. Recall that in both species the lower lethal temperatures were approximately identical during both freeze tolerant and freeze susceptible years. Consequently there does not appear to be any advantage in either system in regard to short term tolerance of a given minimal temperature. Of course other possible advantages are likely to pertain, but these remain obscure. The haemolymph concentrations of polyols (both glycerol and sorbitol) were significantly greater when the C. clavipes larvae were freeze tolerant in February 1979 than they were in the freeze-susceptible larvae in
February 1983. As the cryoprotective effect of polyols, especially glycerol, is fairly well established (Baust, 1973; Zachariassen, 1979; Ring, 1980) it is possible that the lower polyol levels in 1983 contributed to the loss of freeze tolerance. However, this is unlikely. Early in the winter of 1978-1979 C. clavipes larvae were collected (December 8, 1978) which were freeze tolerant but lacked high glycerol concentrations. (Glycerol was not detectable with the same chromatographic technique used in this study.) Sorbitol (0.03M) was the only polyol detected (Duman, 1979). Consequently, although it is probable that polyols were important contributing factors to the freeze tolerance of the larvae in the winter of 1978-1979, it is not likely that the lowered polyol levels in 1983 larvae were responsible for the lack of freeze tolerance. Also, the function of the polyols in the 1983 freeze-susceptible larvae is not obvious. The antifreeze function of polyols in depressing the freezing and supercooling points of freeze-susceptible insects is well documented (Sijmme, 1982; Duman et al., 1982). When multimolar concentrations of poly01s are found in a freeze-susceptible insect the contribution of the polyols to the freezing and supercooling point depression can easily be calculated and appreciated. For example, the 5M glycerol concentration in larvae of overwintering Bracon cephi larvae obviously is a major factor in lowering the freezing point of the larvae to - 15°C and the supercooling point to -45°C (Salt, 1959). The polyol levels of C. cfavipes larvae in February 1983 are obviously elevated above those of summer insects, in which polyols are not detectable with the chromatographic technique. However, based on the molar freezing point constant for water (1.86”Q the 0.25M concentration of glycerol in February 1983 larvae would only lower the freezing point of the haemolymph by - 0.47”C. The effect of glycerol on the supercooling point of water is to depress the supercooling point approximately twice as much as it depresses the freezing point (Block and Young, 1979, MacKenzie. 1979). Consequently a 0.25M glycerol concentration should lower the supercooling point by only w 0.94% In fact, the combined contribution of both glycerol and sorbitol to the supercooling point depression would only be slightly more than l”C, hardly an impressive amount. Consequently, a nonantifreeze function for the elevated polyols may be their major contribution to the cold tolerance of the
Overwintering
1983 larvae. The stabilizing effects of glycerol on proteins at low temperatures is one possibility (Gekko and Timashiff, 1981 a,b). As in the earlier studies on D. canadensis, a major physiological variable shown to change in C. clavipes larvae was the presence of haemolymph ice nucleators during 1978-1979 when the larvae were freeze tolerant and the absence of ice nucleators from the haemolymph when freeze tolerance was lost. Consequently, the lack of an ice nucleator and subsequent lethal intracellular ice formation following extensive supercooling might have explained the loss of freeze tolerance in 1983 larvae. However, the experiments in which freeze susceptible larvae collected in February 1983 were artificially seeded, at temperatures approximating those at which they would have frozen had the natural haemolymph ice nucleators been present, demonstrated that in both D. canadensis and C. clavipes the absence of ice nucleators is not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance. It may be that if this other critical unknown factor(s) could be identified and replaced, then the absence of the ice nucleator might prove to be critical. This raises the possibility that these insect species which demonstrate the ability to change overwintering mechanisms could be excellent model systems in which to investigate and hopefully identify these critical unknown factors which are involved in insect freeze tolerance. Acknowledgemenfs-This work was supported Science Foundation Grant PCM 8109708.
by National
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Duman J. G. and Horwath K. L. (1983) The role of hemolymph proteins in the cold tolerance of insects. A. Reu. Physio. 45, 261-270. Duman J. G., Morris J. P. and Castellino F. J. (1983) Purification and composition of an ice nucleating protein from queens of the hornet, Vespula maculata. J. camp. Physiol. In press. Duman J. G. and Patterson J. L. (1978) The role of ice nucleators in the frost tolerance of overwintering queens of the bald faced hornet, Vespula maculata. Camp. hiothem. Physiol. 59A, 69-72. Danks H. V. (1978) Modes of seasonal adaotation in the insects. I Winter survival. Can. Em. 110. i 167-1205. Gehrken U. (1982) Role of ethylene glycol in the coldhardening of the adult bark beetle. Ips acuminufus. CryoLeflers 3, 307. Gekko K. and Timasheff S. N. (1981a) Mechanism of protein stabilization by glycerol: Preferential hydration in glycerol-water mixtures. Biochemistry 20, 4667-4676. Gekko K. and Timasheff S. N. (1981 b) Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry 20. 46774686. Horwath K. L. and Duman J. G. (1984) Yearly variations in the overwintering mechanism of the cold hardy beetle Dendroides canadensis. Phpsiol. 2001. In press. MacKenzie A. P. (1977) Non-equilibrium freezing behavior of aqueous systems. Phil. Trans. R. Sot. 278, 1677189. Miller K. (1982) Cold-hardiness strategies of some adult and immature insects overwintering in interior Alaska. C’omp. biochem. Physiol. 73A. 595-604. Patterson J. L. and Duman J. G. (1982) Purification and composition of protein antifreezes with high cysteine contents from larvae of the beetle, Tenebrir) moliror. J. exp. Zool. 219, 381-384. Tomchaney A. P., Morris J. P., Kang S. H. and Duman J. G. (1982) Purification, composition and physical properties of a thermal hysteresis antifreeze protein from larvae of the beetle, Tenebrio moliror. Biochemistry 21, 716-721. Ramsay J. A. and Brown R. H. (1955) Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid. J. scient. Inswum. 32. 372-375. Ring R. A. (1980) Insects and their cells. In Low Temp&ature Preservation in Medicine and Biology (Ed. by Ashwood-Smith M. J. and Farrant J. ) on. 187.-217. Univ. Park Press, Baltimore. Ring R. A. (1982) Freezing tolerant insects with low supercooling points. Comp. biochem. Physiol. 73A, 605-612. Salt R. W. (1959) Role of glycerol in the cold hardening of Bracon cephi (Gahan). &n. J. 2001. 37, 59-69. Salt R. W. (1966) Factors influencine nucleation in sunercooled insects.‘Can. J. Zool. 44. 117-133. Samme L. (1964) Effects of glycerol on cold-hardiness in insects. Can. J. Zool. 42. 87-101. Siimme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. biochrm. PIIj:riol. 73A. 519-543. S(imme L. and Conrad&Larsen E. M. (1979) Nucleating agents in the hemolymph of third instar larvae of Eurosta solidugensis (Fitch) (Diptera, Tephritidae) Norm. J. Enf. 25. 187-188. Zachariassen K. E. (1979) The mechanism of the cryoprotective effect of glycerol in beetles tolerant to freezing. J. Insect Phvsiol. 25, 29-32. Zachariassen K. E. (1982) Nucleating agents in cold-hardy insects. Comp. biochem. Physiol. 73Ar 557-563. _ Zachariassen K. E. and Hammel H. T. (1976) Nucleating agents in the heamolymph of insects tolerant to freezing. Nature 262, 285-287. .I