Insect antifreezes and ice-nucleating agents

CRYOBIOLOGY

19,613-627 (1982)

Insect Antifreezes

and Ice-Nucleating

Agents’

JOHN G. DUMAN Biology

Department,

University

of Notre Dame, Notre Dame, Indiana 46556

The ability of certain species of insects to survive extremely low temperatures during the winter has been the subject of considerable scientific interest, especially since the pioneering work of Salt (48-50), Somme (56), and others during the late 1950s and early 1960s. Cold-hardy insects are generally categorized as being either freeze tolerant (able to survive freezing of their body fluids) or freeze susceptible. Freezesusceptible species must adapt by producing antifreeze agents which lower their freezing and supercooling points and/or by selecting a thermally buffered overwintering site. The purpose of this paper is to review some of the more recent studies on two groups of molecules which, although both are intimately involved in the physiological adaptations of insects to subzero temperatures, have completely diametric functions. Antifreeze agents, as mentioned previously, are obviously essential to the cold tolerance of many freeze-susceptible species. In contrast, it is becoming ever more apparent that ice-nucleating agents, which initiate ice formation in the hemolymph at relatively high temperatures (thereby preventing lethal intracellular freezing), are important to the survival of many, if not most, freeze-tolerant species of insects. I. ANTIFREEZE

AGENTS

Although the unique abilities of freezetolerant insects have attracted the bulk of Received: December 11, 1981; accepted May 27, 1982. 1Presented at the Symposium “Basic Cryobiology: Antifreeze and Anti-Antifreeze Agents” at the meeting of the Society for Cryobiology in St. Louis, MO., June 1981.

the research effort concerning cold-tolerant insects, it is perhaps useful to point out that freeze-susceptible species may also become extremely cold tolerant. An example of this point is illustrated in Table 1 which lists the supercooling points of several far northern freeze-susceptible insects. Note that these insects have supercooling points which are below the temperature of homogeneous nucleation of pure water. The antifreeze agents which have been identified in freeze-susceptible insects are low-molecular-weight polyhydroxy alcohols and sugars, and high-molecularweight peptides and proteins which produce a thermal hysteresis (a difference between the freezing and melting points of an aqueous solution) of several degrees. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins found in polar marine teleost fishes (9). Research concerning the antifreeze activity of glycerol and other low-molecularweight solutes in insects has been carried out since the 1950s and the results of these studies are reasonably well known (8, 50, 56). However, more recently quite interesting data on these antifreezes and their role in the cold hardiness of freeze-susceptible insects has been obtained. By contrast, the available information on the proteinaceous antifreezes in insects is more sparse, owing to the more recent discovery of their presence in certain cold-tolerant species. A. Low-Molecular-Weight

Antifreezes

Glycerol and sorbitol are antifreezes commonly found in freeze-susceptible insects, but other compounds may serve a

613 001l-2240/82/060613-15$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

614

JOHN G. DUMAN

TABLE 1 Supercooling Points of Certain Freeze-Susceptible Insects Demonstrating their Extreme Cold Tolerance Insect Bracon cephi Pytho sp.

Willow gall insects (Diptera and Hymenoptera) Rhabdophaga sp.

similar function. Table 2 lists the polyols and sugars which have been reported as antifreezes. These compounds depress the freezing point of water in the “normal” colligative manner. The freezing point temperature of an overwintering insect’s body fluids is of some ecological consequence, as inoculative freezing across the cuticle from exterior ice can occur (51, 59). However, the temperature to which the body fluids supercool before spontaneous ice formation takes place is generally considered to be more important ecologically than the actual freezing point of the insect’s body fluids. Under normal circumstances the freezing and supercooling points are related, as an increase in solute concentration generally depresses both the freezing and supercooling points. In fact, until relatively recently it was thought, based on the work of Dorsey (11) and Lusena (32), that there was a l/l relationship between the depression of the supercooling and freezing points such that if sufficient solute was added to an

Supercooling point (“C)

Reference

-45 -46

(49) (45)

-- 60 -66

(47)

(37)

aqueous solution to lower the freezing point 1°C then the supercooling point would also be lowered 1°C (Fig. 1). More recent studies (6, 33) have demonstrated that low-molecular-weight solutes, such as the polyols and sugars found in overwintering insects, lower the supercooling point of

j I//

( DORSEY ‘4G, LUSENA ‘55 )

!iv.

,

-a5

0

TABLE 2 Low-Molecular-Weight Compounds Which Have Been Reported to Function as Antifreezes in Freeze-Susceptible Species of Insects Compound Glycerol Sorbitol Mannitol Arabitol or ribotol Trehalose Glucose Fructose

Reference (49, 56) (35) (57) (7) (7, @a (7, 60)

(60)

Note. The references given are by no means a complete list.

MELTING

,

,

- 1.0

POINT DEPRESSION PC)

FIG. 1. Relationships between the melting point depressions of aqueous solutions of varying concentrations of glucose and glycerol and the supercooling points, both homogeneous and heterogeneous, of these same solutions. Early studies by Dorsey (11) and Lusena (32) indicated a l/l relationship between melting point and supercooling point depressions of glycerol solutions. More recent work has shown, for both glucose and glycerol solutions, a 2/l ratio of supercooling (homogeneous nucleation) to melting point depression [MacKenzie (33)], and for glycerol solutionsa~~2/1 ratio between the depression of the temperature of heterogeneous nucleation and melting point depression has also been demonstrated [Block and Young (6)].

ANTIFREEZES

AND NUCLEATING

615

AGENTS IN INSECTS

water approximately twice more than they lower the freezing point (Fig. 1). Therefore, for every 1°C decrease in the freezing point there is a 2°C depression of the supercooling point. This relationship appears to hold for both homogeneous and heterogeneous nucleation. The biological consequences of this relationship should be kept in mind when interpreting data on increases in polyol and sugar concentrations in overwintering freeze-susceptible insects. The importance to supercooling point depression of small, but significant, increases in polyol or sugar concentrations in freezesusceptible insects must then be interpreted along with information on the microhabitat temperatures experienced by the insect before one can speculate upon the possible antifreeze function of the solute increase. In addition to the above-mentioned, apparently colligative effects (23) of polyols and sugars on the supercooling point, it has been suggested that they may also lower the supercooling point by “masking” heterogeneous nucleation sites found in freezesusceptible insects, (4, 5). This interesting possibility must remain speculative at this time. However, recent studies have shown that the polyols and sugars commonly found in insects do not mask the icenucleating agent found in the freezetolerant beetle Eleodes bfanchardi (29).

aqueous solution are well known for their antifreeze function in cold water marine teleost fishes (9). The best explanation for this unique behavior suggests that the repeating structure of these molecules allows them to adsorb, via hydrogen bonding, to the surface of the ice crystals at “steps” where growth of the crystal is preferred. This effectively poisons the crystal and prevents it from growing at what would be the normal freezing- melting point temperature (10, 43). The first description of this thermal hysteresis behavior came, not from fishes, but from Ramsay’s classical study of the cryptonephridial rectal complex of larvae of the beetle, Tenebrio molitor (24, 42). However, Ramsay was unable to determine a function for the proteins which he associated with the thermal hysteresis of hemolymph and perirectal space fluid. More recently overwintering larvae of another Tenebrionid beetle, Meracantha contracta, from northern Indiana were also shown to have thermal hysteresis activity in their hemolymph (12, 13) (Table 3). Note that the supercooling points, and therefore the lower lethal temperatures, of this freeze-susceptible beetle, are lowered in the winter at the same time that the freezing point is depressed to -5°C by proliferation of the thermal hysteresis proteins. Since polyols and sugars are not concentrated in B. Protein Antifreezes the winter in this species it appears that the Proteins and glycoproteins which pro- thermal hysteresis proteins (THPs) are reduce a thermal hysteresis, a difference be- sponsible for the lowered supercooling tween the freezing and melting points, in points. It must also be mentioned that TABLE 3 Freezing and Melting Points, Demonstrating Thermal Hysteresis, of the Hemolymph of Meracantha contracta in Winter and Summer (12)

Month

Melting point (“Cl

Freezing point (“C)

Thermal hysteresis (“Cl

Supercooling point (“C)

Lower lethal temperature (“0

Feb. June

-1.31 + 0.16 -0.81 ‘- 0.04

-5.02 2 1.02 -0.81 z!z0.04

3.71 2 1.12 0

-10.3 IT 1.2 - 3.8 T 0.4

-11 -4

Note. Supercooling points and lower lethal temperatures are also included. Values indicate means 5 one standard deviation.

616

JOHN

G. DUMAN

Meracantha overwinter under large decaying logs and are thereby thermally buffered. Consequently a supercooling point of -10.3”C appears to be sufficiently low to ensure their survival. Figure 2 shows seasonal changes in thermal hysteresis in Meracunthu. The THPs begin to appear in late September or early October and peak in midwinter. THPs decline somewhat in late winter and early spring, but note that final loss of the THPs does not occur until late May when the ‘likelihood of a frost is negligible. This illustrates one advantage of the THPs over low-molecular-weight antifreezes which depress the freezing and supercooling points on the basis of colligative properties. THPs can be produced at times, such as early autumn and late spring, when short periods of subzero temperatures may be interspersed with relatively warm weather. Thus the THPs can afford some protection against freezing without the drastic and disruptive increase in osmotic pressure which would result from proliferation of polyols or sugars at warmer temperatures. Low environmental temperatures induce THP production in Mercunthu. However, from an ecological perspective it may be more important that short photoperiods will also induce THPs in the autumn and are

involved in cueing their loss in the spring (13, 14). Change in daylength is a most reliable indicator of seasonal progression while temperature is notoriously poor in this regard. THP production in the fall is stimulated by either short photoperiod or low temperature. Of course under normal natural conditions a combination of these factors would be most likely to prevail. In the spring a combination of both high temperatures and long photoperiod is required before Merucunthu loses the THPs, thus providing a failsafe system which prevents THP loss (even if warm weather occurs) until the likelihood of a late frost is past. Studies in our laboratory have shown that use of photoperiod as an environmental cue is a common feature of THP production and loss in insects (19). Recently circadian rhymicity has been implicated in the timing mechanism used by some insects to control THP levels (26). The situation in larvae of the beetle Tenebrio rnolitor, the insect in which thermal hysteresis was first demonstrated by Ramsay (24, 41), is quite similar to that just described for Merucunthu. A major difference, however, is that Tenebrio larvae maintained under “summer” conditions of high temperatures and long photoperiod still have a significant amount of thermal hysteresis in the hemolymph (Table 4) (37). However, as with Merucunthu, acclimation to low temperatures and/or short photoperiod will induce a significant increase in the levels of thermal hysteresis. Coincident with the increases in thermal hysteresis the supercooling points, and therefore the lower lethal temperatures of these freezesusceptible insects, are also decreased. Like Merucunthu, Tenebrio does not accumulate polyols or sugars during acclimation to winter conditions and consequently TIME OF YEAR the lowered supercooling points appear to FIG. 2. Variations in the freezing (0) and melting be at least partially attributable to the in(0) points of the hemolymph of Meracantha larvae (demonstrating the degree of thermal hysteresis) over crease in THPs. Why Tenebrio maintains the time period from August 1975 to July 1976 (13). THPs during summer conditions remains an Values indicate means 2 standard deviation. unanswered question. The obvious impli-

ANTIFREEZES

AND

NUCLEATING

AGENTS

IN

617

INSECTS

TABLE 4 Thermal Hysteresis Activity and Supercooling Points of Tenebrio molitor Larvae Held under Various Acclimation Conditions (38) Acclimation conditions T (“‘3

Photoperiod (L/D)

20 20 5 5

16/8 6l18 18/6 6118

Thermal hysteresis (“Cl

Supercooling point (“C)

0.75 k 0.12 1.67 -c 0.70 1.47 2 0.26 1.35 f 0.38

-7.7 -13.6 -14.0 -14.9

k + ” 2

2.6 2.7 2.3 2.1

Note. Values are means t standard deviation.

cation is that the THPs have a secondary function in addition to that as an antifreeze. One possibility, first put forth by Ramsay (24,42) because the THPs in Tenebrio were concentrated in the perirectal space, is that the THPs may be involved in water balance. Evidence has been presented both for (38) and against (21) this possibility but since the present review concerns cold hardiness, this topic will not be pursued further. Several other insect species also have “low” levels of thermal hysteresis in their hemolymph during the summer (15). One of these is Uloma impressa, a freezesusceptible Tenebrionid beetle which over-winters as an adult under the bark of dead trees. Like Tenebrio, Uloma greatly increases (over fourfold) the levels of thermal hysteresis in the hemolymph in winter (Table 5). One important difference between Uloma and both Tenebrio and Meracantha is that during the winter Uloma proliferates glycerol as well as THPs. The glycerol greatly lowers the hemolymph melting and freezing points. The

THPs further depress the freezing point, and this combination of glycerol and THPs greatly lowers the supercooling point and lower lethal temperature of Uloma. To summarize, to this point we have seen three freeze-susceptible species which produce THPs. Meracantha produces THPs only during the winter. Tenebrio and Uloma have low levels of THPs in summer but these are significantly increased in winter. In addition the supercooling points of the insects are lowered, presumably by the increased levels of THPs alone (Meracantha and Tenebrio) or by THPs in conjunction with polyols (Uloma). Of course the advantage to freeze-susceptible species of having antifreeze proteins is obvious . However, certain freeze-tolerant insect species are also known to produce THPs (15, 17). In these cases it is not obvious why a freeze-tolerant insect, with its physiology and biochemistry designed to survive freezing, would produce an antifreeze. This is especially perplexing because some of these THP-producing freezetolerant species also produce ice nucleators

TABLE 5 Mean Values of Hemolymph Melting and Freezing Points and Thermal Hysteresis and Whole Organism Supercooling Points of Vloma impressa during Winter and Summer (15)

Month

Melting point (“Cl

Freezing point (“C)

Thermal hysteresis (“(2

Supercooling point (“Cl

Feb. June

-9.90 2 1.20 -0.88 2 0.01

-14.68 f 2.28 -2.00 f 0.36

4.76 f 2.24 1.14 k 0.36

-21.5 k 5.8 -6.2 t 0.3

618

JOHN

G. DUMAN

became freeze tolerant (during the winter of 1978-1979) in late November and for the most part remained freeze tolerant until late March. One notable exception was that during a midwinter thaw (the first of the winter) in late February the larvae became freeze susceptible for a short period after which they again became freeze tolerant. This particular winter was exceptionally cold (the coldest on record in Indiana) and free of winter thaws. Consequently, the length of time during which the Dendroides larvae were freeze tolerant may also have J J A S FMAMJJ i 0 N D J been unusual. Periodic collections during Time of Year FIG. 3. (A) Freezing (0) and melting (0) points and subsequent, less severe, winters have demonstrated that Dendroides larvae typically (B) thermal hysteresis of the hemolymph of Denlose freeze tolerance during winter thaws. droides larvae collected between June 1978 and July 1979. Values plotted represent means + standard de- During mild winters the larvae in fact may viation (17). spend considerable periods of time in a freeze-susceptible state. As a result of this which inhibit supercooling. One of these oscillation between freeze tolerance and species is the Pyrochroid bettle Den- susceptibility, the advantage to Dendroides droides canadensis which overwinters of having the THPs becomes more apparin the larval form under the bark of dead ent. The THPs would be useful as antitrees (17). Seasonal variations in THP freezes during those periods during the levels are shown in Fig. 3. As in Uloma, winter when the larvae are freeze susceptilow levels of THPs are present in Den- ble. Since the larvae also have the ice nudroides over the summer. However, the cleator proteins present at these times, the THPs increase in October, peak in winter, freezing point depressing ability of the and decline slowly through the spring. THPs may be especially critical to their As shown in Fig. 4 the Dendroides larvae survival. In addition the presence of THPs

-=m

JJASONDJFMAMJJ

FIG. 4. Lower lethal temperatures (0) and supercooling points (0) of Dendroides larvae collected between June 1978 and July 1979(17). Asterisks indicate that on that date not all the population was freeze tolerant or susceptible and the numbers in parentheses indicate the percentage of the population freeze tolerant on that date.

ANTIFREEZES

AND

NUCLEATING

in the autumn (prior to the development of freeze tolerance) and in the late spring (after the loss of freeze tolerance) would afford the freeze-susceptible larvae some protection from low temperature. In addition to the 4 species of beetles mentioned above, 12 other insects are known to produce THPs. Most of the known THP-producing insects are beetles, 13 species in all representing the families Tenebrionidae (4 species) (15, 38), Elateridae (4 species) (15), Cucujidae (1 species) (15), Pyrochroidae (2 species) (15, 19), Lampyridae (1 species) (19), and Coccinellidae (1 species) (19). Also the wood roach, Parcoblatta pennsylvanica (Orthoptera, Blatellidae) (15), the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae) (41), and the snow scorpionfly, Boreus westwoodi (Mecoptera, Boreidae) (27), are known to produce THP antifreezes as do three species of freeze-susceptible spiders and a centipede (16, 19, 27). The biochemistry of insect THPs has not been as extensively investigated as has that of fishes. However, THPs have been purified from two insect species-the milkweed bug, Oncopeltus fascia&s (41), and the larvae of the beetle, Tenebrio molitor (39, 40, 54, 61). (Tenebrio is quite unusual in that it produces several THPs which differ significantly in amino acid composition.) It may be instructive to compare what is known of these insect THPs with those of fishes. For details of the fish antifreeze proteins the reader is referred to (9). Glycoproteins with thermal- hysteresis activity have not as yet been demonstrated in insects. Consequently at this time the best studied and one of the most widely distributed fish antifreezes, the glycoproteins with their truly unique repeating structure, have no known counterpart among the insects. It is likely, however, that future work will change this. A second major type of protein antifreeze found in fishes is a non-carbohydrate-

AGENTS

IN

INSECTS

619

containing type characterized by that found in the winter flounder (10, 18). Other fish species from divergent families have antifreezes with compositions remarkably similar to that of the winter flounder shown in Table 6 (25, 44). The extremely high percentage of alanine residues is the most unusual characteristic of the flounder antifreeze. Thermal hysteresis activity is associated with the carboxyl group side chains of the aspartate and/or glutamate residues (18). Sequence work has demonstrated a primary structure in which the polar residues are grouped together in a regular fashion with long stretches of alanine residues between them (10). This regular spacing allows the hydrophilic side chains to bind to oxygen atoms in the ice lattice as described previously. The amino acid compositions of four insect THPs are shown in Table 6 for comparison. Three of these (T-l, T-2, S&T) have been purified from Tenebrio (39,54, 61) and one from the milkweed bug, Oncopeltus (41). In general, the insect THPs are composed of considerably greater percentages of hydrophilic amino acids and also lack the large percentage of alanine found in the fish antifreezes. The Oncopeltus protein is particularly interesting because of the unusually high percentage of serine (30%). It is possible that the hydroxyl group side chains of the serine are involved in binding to the ice lattice. The Tenebrio proteins likewise have high percentages of hydrophilics, especially T- 1 and S&T, although no individual amino acid is dominant. One interesting similarity between all the Tenebrio THPs listed in Table 6 is the similar proline content of each. The effect of proline on the structure-function relationships of thermal hysteresis proteins and glycoproteins has not been investigated. However, given the importance of higher-order structure to the activity of these molecules, coupled with the unique affects of proline residues on protein structure (i.e., the “kinking” effect on secondary structure, the ability to form

620

JOHN G. DUMAN TABLE 6 Amino Acid Compositions of THPs from Winter Flounder (18), Tenebrio [T-l (39), T-2 (61), S&T (54)], and Oncopeltus (41) Tenebrio THPs

Amino acid

T-l

T-2

GlY Ala CYS Val Met Ile Leu LYS Arg Tyr Phe His

11.3 11.0 14.8 15.3 5.9 7.6 9.6 7.2 3.3 3.9 4.8 1.1 1.2 1.5 1.5

7.3 6.6 7.4 8.9 5.9 8.3 14.3 11.5 4.8 7.1 6.8 2.6 2.3 3.9 1.9

13.0 9.0 9.0 11.0 6.0 9.0 7.0 3.0 7.0 3.0 5.0 7.0 5.0 3.0 3.0 3.0

7.1 2.7 30.5 12.3 20.0 6.8 3.0 1.9 3.1 7.5 2.0 1.1 2.3

13.3 10.6 3.2 1.6 61.3 5.3 2.8 1.9 -

Percentage hydrophilics

58.3

40.0

54.0

59.8

33.4

Asx Thr Ser Glx RO

S&T

Oncopeltus

Winter flounder

Note. Values are in mol%. The percentage of amino acid residues on each protein which are hydrophilic (Asp, Glu, Lys, Asn, Gln, Arg, Ser, Thr) according to the groupings of Manavalan and Ponnuswamy (34) is also included.

cis peptide bonds, the low-energy barrier between the cis and trans configurations of peptide bonds containing a proline residue), the presence of proline residues in certain of the THPs may be of considerable importance. Other THPs, interesting because of their high cysteine content, have also been purified from Tenebrio (40). These are shown in Table 7. TC-2, with its 28% cysteine content, is particularly unusual. A fish THP with 7.6% cysteine has been purified from the sea raven, Hemitripterus americanus (55). The composition of the Hemitripterus protein is also shown in Table 6. One obvious major difference between the Tenebrio and Hemitripterus THPs is the substantially greater cysteine content of the Tenebrio proteins. At this time the function of the cysteine residues is unknown. However, both the fish (55) and

the Tenebrio (54) THPs are inactivated by dithiothreitol, indicating that at least some of the cysteine residues are involved in disulfide bridges and that these are critical for thermal hysteresis activity. Like the other insect THPs, TC-1 and TC-2 also contain high percentages of hydrophilic residues (46.7 and 46.5%, respectively). The nearly identical percentage of hydrophilics in the two THPs is interesting, especially since the actual percentages of the individual hydrophilic residues vary considerably. Tenebrio is the only animal known to produce such structurally divergent THPs. The reason for this is unknown at this time. However, there is evidence that these various THPs may have a synergistic effect on thermal hysteresis activity (54). The specific thermal hysteresis activities of the fish and insect THPs are similar; however, differences do exist (Fig. 5). Note

ANTIFREEZES

AND

NUCLEATING

AGENTS

IN

621

INSECTS

TABLE 7 Amino Acid Compositions of Two Tenebrio THPs with High Cysteine Contents, TC-1 and TC-2 (40) Amino acid

TC-1

TC-2

GUY Ala CYS Val Met Be Leu LYS Arg Tyr Phe His Trp

7.9 4.0 15.8 13.0 15.5 7.9 15.4 3.8 2.5 4.7 6.0 3.1 -

5.3 2.3 11.1 12.4 11.4 5.0 28.0 2.3 1.0 2.2 15.4 3.1 -

10.7 7.9 8.2 9.1 6.7 8.1 14.4 7.6 1.2 5.4 1.7 6.2 2.1 2.3 2.0 2.0 2.5 2.8

Percentage Hydrophilics

46.7

46.5

40.3

Asx

Thr Ser Glx PI-O

Hemitripterus

6

PROTEIN CONCENTRATION (mg/mll

5. Comparison of the freezing point depressing activities of various THPs. 1 = winter flounder antifreeze (18); 2 = mixture of glycoprotein antifreezes 3-5 from the Antarctic fish Trematomous borchgrevinki (DeVries, personal communication); 3 = glycoprotein antifreezes 7 and 8 from the Antarctic fish Trematomous borchgrevinki (30); 4 = Tenebrio, T-2 (61); 5 = Tenebrio TC-2 (40), 6 = approximate melting point depressions of all the THPs. FIG.

Note. A cysteine containing THP from the fish Hemitripterus americanus (55) is included for com-

which have 5°C of thermal hysteresis in the hemolymph in winter (in some individual cases considerably more is present), may have higher specific activities than those of Tenebrio. It is clear that much work needs to be done on this aspect of the that the activity of the fish antifreezes (1 - 3) insect THPs. An even more critical area for future is higher at the low protein concentrations, but that one of the Tenebrio THPs (T-2) has work concerns the effects of THPs on higher activity than the fish THPs at higher supercooling. The correlation between the concentrations. It is unclear from Fig. 5, proliferation of THPs by certain insects in because the thermal hysteresis activity of winter and the decreased supercooling points which occur at the same time prothe Tenebrio THPs plateaus at 1. l- 1.X, how some insects can have thermal hys- vides indirect support for the supposition teresis levels in their hemolymph of several that the THPs not only lower the freezing degrees. (See Tables 3, 5 and Figs. 2, 3.) point of water, but also lower the superThe synergistic effect of different THPs, cooling point. Preliminary unpublished data mentioned previously, is one possible ex- from our laboratory indicate that this is the planation. Other possibilities are that fac- case. The THPs tested appear to lower the tors (such as pH, ion concentrations, etc.) temperature of heterogeneous nucleation which have not been controlled in determi- approximately two times more than they nations of specific THP activity may be lower the freezing point of water. The procritical. Another explanation is that the posed mechanism to explain the thermal THPs of other insect species, such as Den- hysteresis activity of THPs can quite logiparison. Values are in mol%. The percentage of amino acid residues of each protein which are hydrophilic (Asp, Glu, Lys, Asn, Gln, Arg, Ser, Thr) according to the groupings of Manavalan and Ponnuswamy (34) is also included.

droides

622

JOHN

G. DUMAN

tally be extended to explain how THPs could lower supercooling point temperatures. If the THPs would bind to embryo crystals forming around a heterogeneous nucleation site, thus inhibiting them from reaching the critical size, then nucleation would be delayed and the supercooling point lowered. It is perhaps more feasible that THPs could bind directly to heterogeneous motes, thereby inhibiting the formation of embryo crystals around these motes. II.

ICE-NUCLEATING

AGENTS

One of the most interesting recent developments in the study of insect freeze tolerance concerns the discovery, first shown by Zachariassen and Hammel (63), that many, if not most, freeze-tolerant insects possess nucleating agents in their hemolymph. These nucleating agents function to inhibit supercooling and thereby ensure that ice formation occurs in the extracellular fluid at fairly high temperatures. Extensive supercooling can result in lethal intracellular ice formation even when ice forms initially in the extracellular fluid (22). Normally as extracellular ice forms solute is excluded from the ice crystal lattice, thus increasing the osmotic pressure of the unfrozen portion of the extracellular fluid. Consequently, water is drawn osmotically from the intracellular to the extracellular pool, thereby dehydrating the cells. The cell membrane normally prevents seeding of the intracellular fluid by the ice present in the extracellular fluid. As the cells are dehydrated, the freezing point of the intracellular fluid is depressed, thus decreasing the likelihood of intracellular ice formation. However, if the rate of ice crystal growth in the extracellular fluid is high, as would occur if the insect was extensively supercooled prior to freezing, the osmotic flux of water out of the cells may not be sufficient to lower the freezing point of the intracellular fluid to that of the extracellular fluid and consequently lethal intracellular ice may form. Some freeze-tolerant insect species have

adapted to this apparent problem by producing extracellular ice-nucleating agents. Zachariassen and Hammel (63) described the presence of heat-sensitive ice nucleators in the hemolymph of three species of freeze-tolerant Tenebrionid beetles. Addition of a small volume of hemolymph (5% of final volume) containing the nucleators to aqueous solutions with varying concentrations of glycerol or NaCl caused the supercooling points of the solutions to increase to a temperature only 5.3”C below the freezing point of the solution, regardless of the osmolality. With the hindsight provided by this initial work, when one reviews prior studies (pre-1976) which concerned the supercooling points of freezetolerant insects, it becomes fairly obvious that most freeze-tolerant insects have these ice nucleators. The supercooling points of these insects are generally high, above - lO”C, even though high polyol concentrations are the general rule. Likewise, several more recent studies have reaffirmed Zachariassen and Hammel’s work (2,3, 15, 17, 20, 36, 46, 47, 58, 62). It has been suggested that the ice nucleators could have adaptive significance in addition to preventing lethal intracellular ice formation (3). Because most physiological and biochemical processes are slowed by the presence of ice, early freezing at a high temperature should conserve energy reserves in the overwintering insect. For example, an insect held at -8°C in the frozen state would use less energy reserves than the same insect held at -8°C in the supercooled state. Studies from our laboratory on two freeze-tolerant species of insects will be used to further illustrate the nature of the nucleators. One of these insects is the white-faced hornet, Vespula maculata (20). The large papery nests of V. maculata generally contain up to 200 individuals by late summer. The colony consists of an egglaying queen and female workers throughout the early summer with males and future

ANTIFREEZES

AND NUCLEATING

queens being produced late in the season. The workers and males die in the autumn. However, the inseminated future queens (as many as 100 from a single nest) hibernate individually, generally in or under partially decomposed logs. These queens are freeze tolerant and in spite of high levels of glycerol in the hemolymph (2 = 3.8 g%) the supercooling points of the queens (.? = -4.6”C) are generally less than 2°C below the equilibrium freezing points (X = -2.YC) of the hemolymph, thus indicating that an ice nucleator is present. The Vespulu ice nucleator is heat sensitive (100°C for 5 min), has a molecular weight greater than 3500 (nondialyzable), and is inactivated by a bacterial proteolytic enzyme (Table 8). This indicates that the Vespula ice nucleator is probably proteinaceous. The freeze-tolerant larvae of the Pyrochroid beetle, Dendroides canadensis, were discussed previously in regard to their production of THPs. Recall that Fig. 4 shows their supercooling and lower lethal temperatures over the course of a year. In spite of the production of THPs and also polyols (glycerol and sorbitol) the supercooling points of the larvae in winter are fairly high, thus indicating the presence of ice-nucleating agents. The presence of nucleators is quite clear when one looks at the degree of undercooling of the larvae over the course of a year (Fig. 6). Undercooling is here defined as the hemolymph freezing point minus the supercooling point of the insect and thus indicates how much the larTABLE 8 Effects of Various Treatments on the Supercooling Points of Water Containing a 1% Solution of Vespula Hemolymph (20) Supercoolingpoint Sample

(A) Distilled water (B) Winter Vespufa hemolymph pool (1%)in water (C) B + heat (lOO”C,5 min) (D) B + dialysis (E) B + proteolytic enzyme

(“C)

-17.4

f 2.4

-3.8 k 0.2

-16.7 c 2.8 -4.5 f 0.3

-15.8 C 3.4

AGENTS IN INSECTS

623

6

JJASONOJFMAMJJ Time

of year

6. Degree of undercooling (hemolymph freezing point minus organismal supercooling point) of Dendroides larvae collected between June 1978 and July 1979(17). Values plotted are means 2 standard deviation. FIG.

vae supercool below their freezing point. In interpreting Fig. 6 it should be remembered that the insects were brought in from the field and their supercooling points determined the same day. Consequently, these supercooling points reflect the presence of food in the gut of the insects during warmer periods of the year. Gut contents are known to be the site of nucleation in feeding insects (52, 53). The degree of undercooling decreased through the autumn and remained at approximately 1°C or less from November through February thereby indicating the presence of an extremely efficient nucleating factor. In December and January the larvae exhibited essentially no undercooling as the supercooling and freezing points showed no statistical difference. In early March there was a large and sudden increase in the degree of undercooling indicative of the loss of the nucleating factor. The degree of undercooling again decreased in late March and early April after the larvae had lost freeze tolerance. However, this decrease was almost certainly due to the resumption of feeding by the larvae. Like the hornet nucleator, the Dendroides nucleating agent is nondialyzable and heat sensitive (loss of activity above 80°C) and is inactivated by the bacterial protease, thus once again indicating that the nucleator may be a protein. In fact, we have now made considerable progress in purify-

624

JOHN

G. DUMAN

ing this nucleator and it does seem to be proteinaceous. However, the ice nucleator in an Afro-alpine plant, Lobelia telekii, appears to be a high-molecular-weight carbohydrate polymer (28). In addition, frost injury of various sensitive plants (corn, beans, etc.) has been associated with epiphytic bacteria which act as efficient ice nuclei between -2 and -5°C (31). This latter point leads to the interesting possibility that certain cell types in an insect, such as special hemocytes, might act as nucleation sites in insects. Clearly, once insect nucleating agents are purified, the study of the relationships between the structure of the nucleators and their unique activity should prove quite interesting. The factors which control nucleator levels in freeze-tolerant insects also deserve attention. It would seem that production of ice nucleators should be closely tied to the development or loss of freeze tolerance as the presence of an ice nucleator in a freeze-susceptible insect would appear to be a distinct liability. Yet some freezetolerant species appear to have nucleators in the summer as well as the winter (20, 62, 63). It is obvious that insect ice-nucleating agents will be intriguing subjects for investigation for many years. At this point the study of these unique factors has barely begun. They are a reminder that although the understanding of insect cold tolerance has progressed significantly, there is still much to be discovered. SUMMARY

Cold-tolerant, freeze-susceptible insects (those which die if frozen) survive subzero temperatures by proliferating antifreeze solutes which lower the freezing and supercooling points of their body fluids. These antifreezes are of two basic types. Lowmolecular-weight polyhydroxy alcohols and sugars depress the freezing point of water on a colligative basis, although at higher concentrations these solutes may

deviate from linearity. Recent studies have shown that these solutes lower the supercooling point of aqueous solutions approximately two times more than they depress the freezing point. Consequently, if a freeze-susceptible insect accumulates sufficient glycerol to lower the freezing point by 5”C, then the glycerol should depress the insect’s supercooling point by 10°C. Some cold-tolerant, freeze-susceptible insects produce proteins which produce a thermal hysteresis (a difference between the freezing and melting point) of several degrees in the body fluids. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins of polar marine teleost fishes. The THPs lower the freezing, and presumably the supercooling, point by a noncolligative mechanism. Consequently, the insect can build up these antifreezes, and thereby gain protection from freezing, without the disruptive increases in osmotic pressure which accompany the accumulation of polyols or sugars. Therefore the THPs can be more easily accumulated and maintained during warm periods in anticipation of subzero temperatures. It is not surprising then that photoperiod, as well as temperature, is a critical environmental cue in the control of THP levels in insects. Some species of freeze-tolerant insects also produce THPs. This appears somewhat odd, since most freeze-tolerant insects produce ice nucleators which function to inhibit supercooling and it is therefore not clear why such an insect would produce antifreeze proteins. It is possible that the THPs have an alternate function in these species. However, it also appears that the THPs function as antifreezes during those periods of the year when these insects are not freeze tolerant (i.e., early autumn and spring) but when subzero temperatures could occur. In addition, at least one freeze-tolerant insect which produces THPs, Dendroides canadensis, typically

ANTIFREEZES

AND NUCLEATING

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 mofitor 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 icenucleating 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

AGENTS IN INSECTS

625

structure-function relationships of these unique molecules should be done. ACKNOWLEDGMENTS This work was supported by National Science Foundation Grants #PCM 77-03475 and #PCM8109708. REFERENCES 1. Baust, J. G. Biochemical correlates to cold hardening in insects. Cryobiology 18, 186-198 (1981). 2. Baust, J. G., and Edwards, J. S. Mechanisms of freezing tolerance in an Antarctic midge. Belgica antarctica. Physiol. Entomol. 4, l-5 (1979). 3. Baust, J. G., Grandee, R., Condon, G., and Morrissey, R. E. The diversity of overwintering strategies used by separate populations of gall insects. Physiol. Zool. 52, 1-5 (1979). 4. Baust, J. G., and Morrisey, R. E. Supercooling phenomenon and water content independence in the overwintering beetle, Coleomegilla maculata. J. Insect Physiol. 21, 1751-1754 (1975). 5. Baust, J. G., and Morrissey, R. E. Strategies of low temperature adaptation. Proc. Znt. Cong. Entomol. 15, 173-184 (1977). 6. Block, W., and Young, S. R. Measurement of supercooling in small arthropods and water droplets. Cryo-Letters 1, 85-91 (1979). 7. Block, W., and Zettel, J. Cold hardiness of some alpine collembola. Ecol. Entomol. 5, l-9 (1980). 8. Danks, H. V. Modes of seasonal adaptation in the insects. I. Winter survival. Canad. Entomol. 110, 1167-1205 (1978). 9. DeVries, A. L. Biological antifreezes and survival in freezing environments. In “Animals and Environmental Fitness” (R. Gilles, Ed.), pp. 583-607. Pergamon, Elmsford, N.Y., 1980. 10. DeVries, A. L., and Lin, Y. Structure of a peptide antifreeze and mechanism of adsorption to ice. Biochim. Biophys. Actn 495, 388-392 (1977). Il. Dorsey, N. E. The freezing of supercooled water. Trans. Amer. Phil. Sot. 38, 246-326 (1948). 12. Duman, J. G. The role of macromolecular antifreeze in the darkling beetle, Meracantha contracta. J Comp. Physiol. 115, 279-286 (1977). 13. Duman, J. G. Variations in macromolecular antifreeze levels in larvae of the darkling beetle, Meracantha contracta. J. Exp. Zool. 201, 85-92 (1977).

14. Duman, J. G. Environmental effects on antifreeze levels in larvae of the darkling beetle,

JOHN G. DUMAN

626

contracta. J. Exp. 2001. 201, 333-337 (1977). 1.5. Duman, J. G. Thermal hysteresis factors in over25, wintering insects. J. Insect Physiol. 805-810 (1979). 16. Duman, .I. G. Subzero temperature tolerance in spiders: The role of thermal hysteresis factors. J. Comp. Physiol. 131, 347-352 (1979). 17. Duman, J. G. Factors involved in the overwintering survival of the freeze tolerant beetle, Dendroides canadensis. J. Comp. Physiol. 136, 53-59 (1980). 18. Duman, J. G., and DeVries, A. L. Isolation characterization and physical properties of protein antifreezes from the winter flounder, Meracantha

29.

30.

31.

Pseudopleuronectes americanus. Comp. Biochem. Physiol. B 54, 375-380 (1976).

19. Duman, J. G., Horwath, K. L., Tomchaney, A., and Patterson, J. L. Antifreeze agents of terrestrial arthropods. Comp. Biochem. Physiol., in press (1982). 20. Duman, .I. G., and Patterson, J. L. The role of ice nucleators in the frost tolerance of overwintering queens of the bald faced hornet. Comp. Biochem. Physiol. A 59, 69-72 (1978). 21. Duman, J. G., Patterson, J. L., Kozak, J. J, and DeVries, A. L. Isopiestic determination of water binding by fish antifreeze glycoproteins. Biochim. Biophys. Acta 626, 332-336 (1980). 22. Farrant, J. General observations on cell preservation. In “Low Temperature Preservation in Biology and Medicine” (M. J. Ashwood-Smith and J. Farrant, Eds.), pp. 1- 18. Univ. Park Press, Baltimore, 1980. 23. Franks, F. The nucleation of ice in undercooled aqueous solutions. Cryo-Letters 2, 27-31 (1981). 24. Grimstone, A. V., Mulliger, A. M., and Ramsay, J. A. Further studies on the rectal complex of the mealworm, Tenebrio molitor (Coleoptera, Tenebrionidae). Phil. Trans. R. Sot. London Ser. B 253, 343-382 (1968). 25. Hew, C. L., Fletcher, G. L., and Ananthanarayanan, V. S. Antifreeze proteins from the scorpius: shorthorn sculpin, Myoxocephalus Isolation and characterization. Canad. J. Biochem. 58, 377-383 (1980). 26. Horwath, K. L., and Duman, J. G. Involvement of the circadian system in photoperiodic regulation of insect antifreeze proteins. J. Exp. Zool., in press. 27. Husby, J. A., and Zachariassen, K. E. Antifreeze agents in the body fluid of winter active insects and spiders. Experientia 36, 963-964 (1980). 28. Krog, J. O., Zachariassen, K. E., Larsen, B., and Smidsrod, 0. Thermal buffering in Afro-alpine

32.

33. 34.

35.

36.

plants due to nucleating agent induced water freezing. Nature (London) 282,300-301(1979). Lee, R. E., Zachariassen, K. E., and Baust, J. G. Effects of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions. Cryobiology 18, 511-514 (1981). Lin, Y., Duman, J. G., and DeVries, A. L. Studies on the structure and activity of low molecular weight glycoproteins from an Antarctic fish. Biochem. Biophys. Res. Commun. 46, 87-92 (1972). Lindow, S. E., Amy, D. C., Upper, C. D., and Barchet, W. R. The role of bacterial ice nuclei in frost injury to sensitive plants. In “Plant Cold Hardiness and Freezing Stress” (P. H. Li and A. Sakai, Eds.), pp. 249-263. Academic Press, New York, 1978. Lusena, C. V. Ice propagation in systems of biological interest. III. Effects of solutes on nucleation and growth of ice crystals. Arch. Biochem. Biophys. 57, 277-284 (1955). MacKenzie, A. P. Non-equilibrium freezing behavior of aqueous systems. Phil. Trans. R. Sot. London Ser. B 278, 167- 189 (1977). Manavalan, P., and Ponnuswamy, P. K. Hydrophobic character of amino acid residues in globular proteins. Nafure (London) 275, 673-674. Mansingh, A., and Smahman, B. J. Variation in polyhydric alcohol in relation to diapause and cold hardiness in larvae of Zsia isabella. J. Znsect Physiol. 18, 1565-1571 (1972). Miller, L. K. Physical and chemical changes associated with seasonal alterations in freezing tolerance in the adult northern Tenebrionid, Upis

37.

38. 39.

40.

41.

ceramboides.

J. Insect

Physiol.

24,

791-796 (1978). Miller, L. K., and Werner, R. Supercooling to -60°C: An extreme example of freezing avoidance in northern willow gall insects. Cryobiology 17, 621-622 (1980). Patterson, J. L., and Duman, J. G. The role of the thermal hysteresis factor in Tenebrio molitor larvae. J. Exp. Biol. 74, 37-45 (1978). Patterson, J. L., and Duman, J. G. Composition of a protein antifreeze from larvae of the beetle, Tenebrio molitor. J. Exp. Zool. 210, 361-367 (1979). Patterson, J. L., and Duman, J. G. Purification and composition of protein antifreezes with high cysteine contents from larvae of the beetle, Tenebrio molitor. J. Exp. Zool., in press. (1982). Patterson, J. L., Kelly, T. J., and Duman, J. G. Purification and composition of a thermal hysteresis producing protein from the milkweed

ANTIFREEZES

42.

43.

44. 45.

46.

47.

48.

49. 50. 51. 52.

AND

NUCLEATING

bug, Oncopeltus fasciatus. J. Comp. Physiol. 142, 539-542 (1981). Ramsay, .I. A. The rectal complex of the mealworm, Tenebrio molitor L. (Coleoptera, Tenebrionidae). Phil. Trans. R. Sot. London Ser. B 248, 279-314 (1964). Raymond, J. A., and DeVries, A. L. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc. Nat. Acad. Sci. USA 74, 2589-2593 (1977). Raymond, J. A., Lin, Y., and DeVries, A. L. Glycoprotein and protein anti-freezes in two Alaskan fishes. J. Exp. Zool. 193, 125- 130 (1975). Ring, R. A. Insects and their cells. In “Low Temperature Preservation in Medicine and Biology” (M. J. Ashwood-Smith, J. Farrant, Eds.), pp. 187-217. Univ. Park Press, Baltimore, 1980. Ring, R. A., and Tesar, D. Cold hardiness of the arctic beetle, Pytho americanus Kirby. Coleoptera, Pythidae (Salpingidae). J. Insect Physiol. 26, 763-774 (1980). Ring, R. A, and Tesar, D. Adaptations to cold in 18, Canadian Arctic insects. Cryobiology 199-211 (1981). Salt, R. W. Natural occurrence of glycerol in insects and its relationship to their ability to survive freezing. Canad. Entomol. 89, 491-494 (1957). Salt, R. W. Role of glycerol in the cold hardening of Bracon cephi (Gahan). Canad. J. Zool. 37, 59-69 (1959). Salt, R. W. Principles of insect cold hardiness. Annu. Rev. Entomol. 6, 55-74 (1961). Salt, R. W. Delayed inoculative freezing of insects. Canad. Entomol. 95, 1190- 1202 (1963). Salt, R. W. Factors influencing nucleation in supercooled insects. Canad. J. Zool. 44, 117-133 (1966).

AGENTS

IN INSECTS

627

53. Salt, R. W. Location and quantitative aspects of ice nucleation in insects. Canad. J. Zool. 16, 329-333 (1967). 54. Schneppenheim, R., and Theede, H. Isolation and characterization of freezing point depressing peptides from larvae of Tenebrio molitor. Comp. Biochem. Physiol. B 67,561-568 (1980). 55. Slaughter, D., Fletcher, G. L., Ananthanarayanan, V. S., and Hew, C. L. Antifreeze proteins from the sea raven, Hemitripterus americanus.

56. 57. 58.

59. 60. 61.

62. 63.

J. Biol.

Chem. 256, 2022-2026

(1981). Somme, L. Effects of glycerol on cold hardiness in insects. Canad. J. Zool. 42, 87-101 (1964). Somme, L. Mannitol and glycerol in overwintering aphid eggs. Norsk. Entomol. Tiddsskr. 16, 107-111 (1969). Somme, L. Nucleating agents in the hemolymph of third instar larvae of Eurosta solidagensis (Fitch) (Dipt. Tephritidae). Norw. J. Entomol. 25, 187-188 (1978). Somme, L. Cold tolerance of alpine, Arctic and Antarctic collembola and mites. Cryobiology 18, 212-220 (1981). Tanno, K. High sugar levels in the solitary bee, Ceratina. Low Temp. Sci. Ser. B 22, 51-57 (1964). Tomchaney, A. P., Morris, J. P., Kang, S. H., and Duman, J. G. Purification, composition and physical properties of a thermal hysteresis “antifreeze” protein from larvae of the beetle, Tenebrio molitor. Biochemistry, in press (1982). Zachariassen, K. E. The roles of polyols and nucleating agents in cold hardy beetles. J. Comp. Physiol. 140, 227-234 (1980). Zachariassen, K. E., and Hammel, H. T. Nucleating agents in the hemolymph of insects tolerant to freezing. Nature (London) 262, 285-287 (1976).