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Recrystallization in a Freezing Tolerant Antarctic Nematode,Panagrolaimus davidi,and an Alpine Weta,Hemideina maori(Orthoptera; Stenopelmatidae)
33, 607–613 (1996) 0064
CRYOBIOLOGY ARTICLE NO.
Recrystallization in a Freezing Tolerant Antarctic Nematode, Panagrolaimus davidi, and an Alpine Weta, Hemideina maori (Orthoptera; Stenopelmatidae) HANS RAMLØV,* DAVID A. WHARTON,†,1
AND
PETER W. WILSON‡
*Roskilde University Center, Institute of Biology and Chemistry, P.O. Box 260, DK-4000 Roskilde, Denmark; and †Department of Zoology and ‡Department of Physiology, University of Otago, P.O. Box 56, Dunedin, New Zealand The ability of haemolymph from the freezing tolerant weta, Hemideina maori, and supernatant from homogenates of the freezing tolerant nematode Panagrolaimus davidi to inhibit the recrystallization of ice was examined using the ‘‘splat freezing’’ technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls. P. davidi survives intracellular freezing and recrystallization inhibition may be important for the survival of this stress. q 1996 Academic Press, Inc.
Recrystallization, during which there is a change in the size distribution of ice crystals in a tissue as larger crystals grow at the expense of smaller crystals, has been proposed as a possible cause of damage in freezing tolerant animals (5). The theory of the grain boundary migration which is involved in recrystallization, and of its inhibition, is given by Knight et al. (7). Thermal hysteresis proteins (THPs) are usually associated with freeze avoiding animals where they lower the freezing point without affecting the melting point by attaching to the surface of ice crystals and inhibiting their growth. THPs are also found in a number of freezing tolerant insects (2). A thermal hysteresis effect would have no obvious function in these animals since the insects promote freezing at high subzero temperatures by the production of ice nucleating agents (5). It has been suggested that recrystallization inhibition is the role of THPs in freezing tolerant insects (2, 5). Recrystallization inhibition by THPs has been demon-
Received March 15, 1996; accepted September 16, 1996. 1 To whom correspondence and reprint requests should be addressed. Fax: 064 3 479 7584.
strated using the ‘‘splat freezing’’ technique developed by metallurgists (6). Panagrolaimus davidi is a freezing tolerant Antarctic nematode which has been shown to survive intracellular freezing (13). Recrystallization inhibition could be involved in this capacity. Hemideina maori, a New Zealand alpine weta, is a large orthopteran which is freezing tolerant during all seasons, with a lower lethal temperature of about 0107C. It can survive the freezing of 82% of its body water (8, 9). This animal is likely to be exposed to diurnal cycles of freezing. Recrystallization inhibition may be of particular importance to animals which are exposed to cycles of freezing and thawing (5). As freezing is very slow in nature the grain boundaries may be more stable than they appear to be in the experimental splat freezing technique (7). Therefore Knight et al. (7) suggest that the role of THPs in freezing tolerant animals may not be to prevent recrystallization but rather to prevent the migration of stillliquid domains, present in the frozen tissue due to the freeze concentration of the body fluids, as a function of temperature gradients. The migration of such liquid domains could be potentially harmful to the tissue (7). This hypothesis could certainly be pertinent in H.
607 0011-2240/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Weta haemolymph (top row) and insect saline (bottom row) during annealing at 087C for 0 h (left column), 1 h (middle column), and 3 h (right column). The change in appearance of the weta haemolymph and insect saline indicates ice recrystallization. Scale bars, 100 mm.
maori, which are fairly large insects, having a body weight of up to 7 g. Here temperature gradients are likely to occur during thawing. In contrast, P. davidi is a very small animal and large temperature gradients are not likely to occur within a single animal. In this paper we describe our initial experiments using the splat freezing technique and cryomicroscopy to investigate recrystallization in haemolymph from H. maori and in P. davidi.
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MATERIALS AND METHODS
Adult weta, H. maori, were collected during early Autumn in the Rock and Pillar Range, New Zealand, and stored in the laboratory in plastic boxes at 47C and fed a diet of apples, carrots, and Selmisia sp. until use. Haemolymph was collected by puncturing the cuticle at the base of a leg, squeezing the animal gently, and drawing the haemolymph into capillaries. It was stored at 0207C until use. An insect Ringer was used as a control sample (12).
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before proceeding. The temperature was slowly raised to 087C and held at this temperature for up to 5 h. The specimen was photographed between crossed polaroids immediately after transfer to the cold stage at 0207C, when the temperature reached 087C, after a further 30 min at 087C, after 1 h, and then at hourly intervals. The change in crystal size during annealing was determined by measuring the maximum diameter of the 10 largest crystals on the negative using a X7 measuring magnifier (Peak scale lupe). Specimens consisted of weta haemolymph, insect saline, nematode supernatant, ATW, and nematode supernatant heated at 657C for 1 h. FIG. 2. Change in crystal diameter of weta haemolymph (l) and insect saline (s) at 0207C and during annealing at 087C. Vertical lines represent the standard error of the mean (N Å 10).
P. davidi, an Antarctic nematode, was cultured on a soya flour medium (16) at 157C. The nematodes were separated from the medium by the Baermann funnel technique (4). They were washed in tap water and transferred to an artificial tap water (ATW) (3). The nematodes were homogenized and stored at 0207C. The sample was thawed before use and centrifuged at 10,000g for 10 min and a sample of supernatant was taken. Ten-microliter samples were frozen using a ‘‘splat freezing’’ technique (6). This involved dropping droplets from a height onto the polished surface of an aluminum block, which was cooled to 0787C by surrounding it with dry ice. The block was enclosed within a deep polystyrene container which was filled with carbon dioxide by evaporation from the dry ice restricting frosting. Portions of the ice were transferred rapidly, using forceps cooled in dry ice, onto a microscope cold stage at 0207C mounted on a Zeiss Axiophot Photomicroscope. The cold stage was surrounded by a collar which was flushed with nitrogen gas to prevent condensation and frosting. The design of the cold stage was similar to that described previously (14). The ice splat specimen was checked to see if it was free of frost
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RESULTS
Weta Haemolymph The changes in appearance of splat-frozen weta haemolymph and in insect Ringer controls are shown in Fig. 1. In both samples there is a change in appearance during annealing at 087C from many small ice crystals to fewer larger crystals. This is reflected in a change in crystal diameter measured from the negatives (Fig. 2). There was also an increase in crystal size during warming from 0207C to 087C. Ice recrystallization occurs in both samples. Nematode Supernatant Splat-frozen nematode supernatant showed little change in appearance during annealing at 087C for 3 h (Fig. 3). ATW and heat-treated nematode supernatant, however, did show ice recrystallization during annealing (Fig. 3). This is reflected in changes in crystal diameter measured from the negatives (Fig. 4). There was an increase in crystal diameter during warming from 0207C to 087C in ATW and heat-treated supernatant but not in untreated nematode supernatant (Fig. 4). The presence of recrystallization inhibition in the samples tested is summarized in Table 1. There was a significant increase in crystal size in all samples (ANOVA: P õ 0.05), but in the nematode supernatant the increase only occurred during the first hour of annealing and
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RECRYSTALLIZATION IN A WETA AND A NEMATODE TABLE 1 Recrystallisation Inhibition in the Samples Tested Crystal size after 3 hours annealing (mm)
Sample Weta haemolymph Insect saline Nematode supernatant Heated supernatant ATW a
126 140 42 152 132
{ { { { {
3a 7 2 12 9
Percentage of change during annealing at 087C
Recrystallization inhibition
182 233 175 214 275
Absent Absent Present Absent Absent
Mean { se.
there were no significant changes in crystal size after this (least significant difference posthoc test, LSD: P ú 0.05). Ice crystal size was significantly smaller in the nematode supernatant after 3 h annealing than in all the other samples tested (LSD: P õ 0.05). DISCUSSION
Recrystallization inhibition as well as thermal hysteresis activity (Wilson and Ramløv, unpublished) is absent in the haemolymph of H. maori. However, thermal hysteresis activity has been described in a few freezing tolerant animals (1, 5, 10, 11) in some of which recrystallization inhibition has also been shown. Recrystallization inhibition has been suggested as the major role of THPs in freezing tolerant animals (1, 5). Tursman et al. (11) speculate that the recrystallization inhibition shown in the centipede Lithobius fortificatus may not be of great importance to this animal because of a requirement for inoculative freezing (above 027C) and a high lower lethal temperature (above 067C), but presumably the size and shape of the ice crystals are still im-
FIG. 3. Nematode supernatant (top row), heated supernatant (bottom row), and ATW controls (middle row) during annealing at 087C for 0 h (left column), 1 h (middle column), and 3 h (right column). There is little change in appearance in the nematode supernatant, suggesting recrystallization inhibition. Ice recrystallization is, however, apparent in heated supernatant and in ATW controls. Scale bar, 100 mm.
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portant (11). The alpine weta H. maori also has a relatively high lower lethal temperature (c. 0107C (9)) and has ice nucleating agents in the haemolymph (nucleating at c. 087C (15)). Recrystallization inhibition may not therefore be important for the survival of this species in the frozen state in nature, although it experiences several freeze/thaw cycles in the course of the year and even daily during some periods (Ramløv, unpublished results). However, we only used haemolymph collected at one time of the year (early autumn). In the centipede L. fortificatus recrystallization inhibition was
FIG. 4. Change in crystal diameter of nematode supernatant (l), heated nematode supernatant (s), and ATW (h) at 0207C and during annealing at 087C. Vertical lines represent the standard error of the mean (N Å 10).
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present in winter-collected but absent in summer-collected haemolymph (11). Recrystallization inhibition was, however, present in the supernatant from homogenates of the freezing tolerant nematode P. davidi. Recrystallization inhibition was indicated by the small size of ice crystals in comparison with other samples. Also, there was no increase in crystal size during warming of the sample from 020 to 087C. Recrystallization was not, however, completely inhibited. An increase in crystal size occurred during the first hour of annealing but there was no further increase in size during the subsequent 3 h of annealing. We have been unable to detect thermal hysteresis activity in P. davidi using Differential Scanning Calorimetry (Wharton and Block, unpublished results). However, Knight and Duman (5) report that recrystallization is inhibited in the haemolymph from Dendroides canadensis when diluted up to 104 times. THPs are thus extremely potent in inhibiting recrystallization and recrystallization inhibition may still occur where thermal hysteresis activity is low or absent. THPs are present in the freezing tolerant intertidal mollusc Mytilus edulis, although the levels of thermal hysteresis are low (10). In M. edulis the THPs may therefore be of importance in inhibiting recrystallization (5). The inhibition of recrystallization may be important for the ability of P. davidi to survive intracellular freezing. The redistribution of ice during recrystallization might be expected to be particularly damaging when ice is located intracellularly, although the nematode can survive the stresses associated with the initial freezing of the cells. The recrystallization inhibition was destroyed in heated supernatants of the homogenates, suggesting that a protein is responsible. Substances other than THPs show recrystallization inhibition activity. Particles, for example, may inhibit grain boundary migration (7). Some synthetic peptides synthesized as variants of an antifreeze peptide from winter flounder may show no THP activity or modification of ice crystal growth and yet still show
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recrystallization inhibition activity. Other peptides and some polyaminoacids and polyolefins also showed recrystallization inhibition activity (7). A large proportion of the water is frozen in H. maori, even at moderate subzero temperatures (9). However, we did not find any recrystallization inhibition in the haemolymph from this species. Knight et al. (7) hypothesized that THPs in freezing tolerant animals have a function as inhibitors of migration of still-liquid domains. This does not seem to be important in H. maori. In contrast we found recrystallization inhibition in relatively dilute homogenates of the freezing tolerant nematode P. davidi. This species survives the formation of intracellular ice (13) and recrystallization inhibition may be a special adaptation for the survival of this stress. From the results presented in this paper we suggest that recrystallisation inhibition is important in animals which can survive intracellular freezing. In contrast, in freezing tolerant animals which cannot survive intracellular freezing recrystallization inhibition is not essential. The presence of recrystallization inhibition in some freezing tolerant animals which do not survive intracellular freezing may indicate that these organisms are especially vulnerable to recrystallization or to movements of salty still-liquid domains in the extracellular spaces. Clearly more work on the role of recrystallization inhibition is needed to elucidate these problems. ACKNOWLEDGMENT We thank the Danish Research Council for financial support. REFERENCES 1. Duman, J. G., Wu, D. W., Xu, L., Tursman, D., and Olsen, T. M. Adaptations of insects to subzero temperatures. Quart. Rev. Biol. 66, 387–410 (1991). 2. Duman, J. G., Xu, L., Neven, L. G., Tursman, D., and Wu, D. W. Hemolymph proteins involved in insect subzero-temperature tolerance: ice nucleators and antifreeze proteins. In ‘‘Insects at Low Temperatures’’ (R. E. Lee and D. L. Denlinger,
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3.
4.
5.
6.
7.
8.
9.
Eds.), pp. 94–127. Chapman and Hall, New York/ London, 1991. Greenaway, P. Sodium regulation in the freshwater mollusc Limnea stagnalis (L) (Gastropoda, Pulmonata). J. Exp. Biol. 53, 147–163 (1970). Hooper, D. J. Extraction of free-living stages from soil. In ‘‘Laboratory Methods for Work with Plant and Soil Nematodes’’ (J. F. Southey, Ed.), pp. 5– 30. HMSO, London, 1986. Knight, C. A., and Duman, J. G. Inhibition of recrystallization of ice by insect thermal hysteresis proteins: A possible cryoprotective role. Cryobiology 23, 256–262 (1986). Knight, C. A., Hallett, J., and DeVries, A. L. Solute effects on ice recrystallization: An assessment technique. Cryobiology 25, 55–60 (1988). Knight, C. A., Wen, D., and Laursen, R. A. Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology 32, 23–34 (1995). Ramløv, H., Bedford, J., and Leader, J. Freezing tolerance of the New Zealand alpine weta, Hemideina maori Hutton (Orthoptera; Stenopelmatidae). J. Therm. Biol. 17, 51–54 (1992). Ramløv, H., and Westh, P. Ice formation in the freeze tolerant alpine weta Hemideina maori Hutton (Orthoptera; Stenopelmatidae). Cryo-lett. 14, 169– 176 (1993).
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10. Theede, H., Schneppenheim, R., and Bevess, L. Frostschutz-Glycoproteine bei Mytilus edulis? Marine Biol. 36, 183–189 (1976). 11. Tursman, D., Duman, J. G., and Knight, C. A. Freeze tolerance adaptations in the centipede, Lithobius fortificatus. J. Exp. Zool. 268, 347–353 (1994). 12. Tyrrell, C., Wharton, D. A., Ramløv, H., and Moller, H. Cold tolerance of an endoparasitic nematode within a freezing tolerant orthopteran host. Parasitology 109, 367–372 (1994). 13. Wharton, D. A., and Ferns, D. J. Survival of intracellular freezing by the Antarctic nematode Panagrolaimus davidi. J. Exp. Biol. 198, 1381–1387 (1995). 14. Wharton, D. A., and Rowland, J. J. A thermoelectric microscope stage for the measurement of the supercooling points of microscopic organisms. J. Microsc. 134, 299–305 (1984). 15. Wilson, P. W., and Ramløv, H. Hemolymph ice nucleating proteins from the New Zealand alpine weta Hemideina maori (Orthoptera: Stenopelmatidae). Comp. Biochem. Physiol. 112B, 535–542 (1995). 16. Wouts, W. M. Mass production of the entomogenous nematode Heterorhabditis heliothidis (Nematoda: Heterorhabditidae) on artificial media. J. Nematol. 13, 467–469 (1981).
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CRYOBIOLOGY ARTICLE NO.
Recrystallization in a Freezing Tolerant Antarctic Nematode, Panagrolaimus davidi, and an Alpine Weta, Hemideina maori (Orthoptera; Stenopelmatidae) HANS RAMLØV,* DAVID A. WHARTON,†,1
AND
PETER W. WILSON‡
*Roskilde University Center, Institute of Biology and Chemistry, P.O. Box 260, DK-4000 Roskilde, Denmark; and †Department of Zoology and ‡Department of Physiology, University of Otago, P.O. Box 56, Dunedin, New Zealand The ability of haemolymph from the freezing tolerant weta, Hemideina maori, and supernatant from homogenates of the freezing tolerant nematode Panagrolaimus davidi to inhibit the recrystallization of ice was examined using the ‘‘splat freezing’’ technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls. P. davidi survives intracellular freezing and recrystallization inhibition may be important for the survival of this stress. q 1996 Academic Press, Inc.
Recrystallization, during which there is a change in the size distribution of ice crystals in a tissue as larger crystals grow at the expense of smaller crystals, has been proposed as a possible cause of damage in freezing tolerant animals (5). The theory of the grain boundary migration which is involved in recrystallization, and of its inhibition, is given by Knight et al. (7). Thermal hysteresis proteins (THPs) are usually associated with freeze avoiding animals where they lower the freezing point without affecting the melting point by attaching to the surface of ice crystals and inhibiting their growth. THPs are also found in a number of freezing tolerant insects (2). A thermal hysteresis effect would have no obvious function in these animals since the insects promote freezing at high subzero temperatures by the production of ice nucleating agents (5). It has been suggested that recrystallization inhibition is the role of THPs in freezing tolerant insects (2, 5). Recrystallization inhibition by THPs has been demon-
Received March 15, 1996; accepted September 16, 1996. 1 To whom correspondence and reprint requests should be addressed. Fax: 064 3 479 7584.
strated using the ‘‘splat freezing’’ technique developed by metallurgists (6). Panagrolaimus davidi is a freezing tolerant Antarctic nematode which has been shown to survive intracellular freezing (13). Recrystallization inhibition could be involved in this capacity. Hemideina maori, a New Zealand alpine weta, is a large orthopteran which is freezing tolerant during all seasons, with a lower lethal temperature of about 0107C. It can survive the freezing of 82% of its body water (8, 9). This animal is likely to be exposed to diurnal cycles of freezing. Recrystallization inhibition may be of particular importance to animals which are exposed to cycles of freezing and thawing (5). As freezing is very slow in nature the grain boundaries may be more stable than they appear to be in the experimental splat freezing technique (7). Therefore Knight et al. (7) suggest that the role of THPs in freezing tolerant animals may not be to prevent recrystallization but rather to prevent the migration of stillliquid domains, present in the frozen tissue due to the freeze concentration of the body fluids, as a function of temperature gradients. The migration of such liquid domains could be potentially harmful to the tissue (7). This hypothesis could certainly be pertinent in H.
607 0011-2240/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Weta haemolymph (top row) and insect saline (bottom row) during annealing at 087C for 0 h (left column), 1 h (middle column), and 3 h (right column). The change in appearance of the weta haemolymph and insect saline indicates ice recrystallization. Scale bars, 100 mm.
maori, which are fairly large insects, having a body weight of up to 7 g. Here temperature gradients are likely to occur during thawing. In contrast, P. davidi is a very small animal and large temperature gradients are not likely to occur within a single animal. In this paper we describe our initial experiments using the splat freezing technique and cryomicroscopy to investigate recrystallization in haemolymph from H. maori and in P. davidi.
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MATERIALS AND METHODS
Adult weta, H. maori, were collected during early Autumn in the Rock and Pillar Range, New Zealand, and stored in the laboratory in plastic boxes at 47C and fed a diet of apples, carrots, and Selmisia sp. until use. Haemolymph was collected by puncturing the cuticle at the base of a leg, squeezing the animal gently, and drawing the haemolymph into capillaries. It was stored at 0207C until use. An insect Ringer was used as a control sample (12).
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before proceeding. The temperature was slowly raised to 087C and held at this temperature for up to 5 h. The specimen was photographed between crossed polaroids immediately after transfer to the cold stage at 0207C, when the temperature reached 087C, after a further 30 min at 087C, after 1 h, and then at hourly intervals. The change in crystal size during annealing was determined by measuring the maximum diameter of the 10 largest crystals on the negative using a X7 measuring magnifier (Peak scale lupe). Specimens consisted of weta haemolymph, insect saline, nematode supernatant, ATW, and nematode supernatant heated at 657C for 1 h. FIG. 2. Change in crystal diameter of weta haemolymph (l) and insect saline (s) at 0207C and during annealing at 087C. Vertical lines represent the standard error of the mean (N Å 10).
P. davidi, an Antarctic nematode, was cultured on a soya flour medium (16) at 157C. The nematodes were separated from the medium by the Baermann funnel technique (4). They were washed in tap water and transferred to an artificial tap water (ATW) (3). The nematodes were homogenized and stored at 0207C. The sample was thawed before use and centrifuged at 10,000g for 10 min and a sample of supernatant was taken. Ten-microliter samples were frozen using a ‘‘splat freezing’’ technique (6). This involved dropping droplets from a height onto the polished surface of an aluminum block, which was cooled to 0787C by surrounding it with dry ice. The block was enclosed within a deep polystyrene container which was filled with carbon dioxide by evaporation from the dry ice restricting frosting. Portions of the ice were transferred rapidly, using forceps cooled in dry ice, onto a microscope cold stage at 0207C mounted on a Zeiss Axiophot Photomicroscope. The cold stage was surrounded by a collar which was flushed with nitrogen gas to prevent condensation and frosting. The design of the cold stage was similar to that described previously (14). The ice splat specimen was checked to see if it was free of frost
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RESULTS
Weta Haemolymph The changes in appearance of splat-frozen weta haemolymph and in insect Ringer controls are shown in Fig. 1. In both samples there is a change in appearance during annealing at 087C from many small ice crystals to fewer larger crystals. This is reflected in a change in crystal diameter measured from the negatives (Fig. 2). There was also an increase in crystal size during warming from 0207C to 087C. Ice recrystallization occurs in both samples. Nematode Supernatant Splat-frozen nematode supernatant showed little change in appearance during annealing at 087C for 3 h (Fig. 3). ATW and heat-treated nematode supernatant, however, did show ice recrystallization during annealing (Fig. 3). This is reflected in changes in crystal diameter measured from the negatives (Fig. 4). There was an increase in crystal diameter during warming from 0207C to 087C in ATW and heat-treated supernatant but not in untreated nematode supernatant (Fig. 4). The presence of recrystallization inhibition in the samples tested is summarized in Table 1. There was a significant increase in crystal size in all samples (ANOVA: P õ 0.05), but in the nematode supernatant the increase only occurred during the first hour of annealing and
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RECRYSTALLIZATION IN A WETA AND A NEMATODE TABLE 1 Recrystallisation Inhibition in the Samples Tested Crystal size after 3 hours annealing (mm)
Sample Weta haemolymph Insect saline Nematode supernatant Heated supernatant ATW a
126 140 42 152 132
{ { { { {
3a 7 2 12 9
Percentage of change during annealing at 087C
Recrystallization inhibition
182 233 175 214 275
Absent Absent Present Absent Absent
Mean { se.
there were no significant changes in crystal size after this (least significant difference posthoc test, LSD: P ú 0.05). Ice crystal size was significantly smaller in the nematode supernatant after 3 h annealing than in all the other samples tested (LSD: P õ 0.05). DISCUSSION
Recrystallization inhibition as well as thermal hysteresis activity (Wilson and Ramløv, unpublished) is absent in the haemolymph of H. maori. However, thermal hysteresis activity has been described in a few freezing tolerant animals (1, 5, 10, 11) in some of which recrystallization inhibition has also been shown. Recrystallization inhibition has been suggested as the major role of THPs in freezing tolerant animals (1, 5). Tursman et al. (11) speculate that the recrystallization inhibition shown in the centipede Lithobius fortificatus may not be of great importance to this animal because of a requirement for inoculative freezing (above 027C) and a high lower lethal temperature (above 067C), but presumably the size and shape of the ice crystals are still im-
FIG. 3. Nematode supernatant (top row), heated supernatant (bottom row), and ATW controls (middle row) during annealing at 087C for 0 h (left column), 1 h (middle column), and 3 h (right column). There is little change in appearance in the nematode supernatant, suggesting recrystallization inhibition. Ice recrystallization is, however, apparent in heated supernatant and in ATW controls. Scale bar, 100 mm.
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portant (11). The alpine weta H. maori also has a relatively high lower lethal temperature (c. 0107C (9)) and has ice nucleating agents in the haemolymph (nucleating at c. 087C (15)). Recrystallization inhibition may not therefore be important for the survival of this species in the frozen state in nature, although it experiences several freeze/thaw cycles in the course of the year and even daily during some periods (Ramløv, unpublished results). However, we only used haemolymph collected at one time of the year (early autumn). In the centipede L. fortificatus recrystallization inhibition was
FIG. 4. Change in crystal diameter of nematode supernatant (l), heated nematode supernatant (s), and ATW (h) at 0207C and during annealing at 087C. Vertical lines represent the standard error of the mean (N Å 10).
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present in winter-collected but absent in summer-collected haemolymph (11). Recrystallization inhibition was, however, present in the supernatant from homogenates of the freezing tolerant nematode P. davidi. Recrystallization inhibition was indicated by the small size of ice crystals in comparison with other samples. Also, there was no increase in crystal size during warming of the sample from 020 to 087C. Recrystallization was not, however, completely inhibited. An increase in crystal size occurred during the first hour of annealing but there was no further increase in size during the subsequent 3 h of annealing. We have been unable to detect thermal hysteresis activity in P. davidi using Differential Scanning Calorimetry (Wharton and Block, unpublished results). However, Knight and Duman (5) report that recrystallization is inhibited in the haemolymph from Dendroides canadensis when diluted up to 104 times. THPs are thus extremely potent in inhibiting recrystallization and recrystallization inhibition may still occur where thermal hysteresis activity is low or absent. THPs are present in the freezing tolerant intertidal mollusc Mytilus edulis, although the levels of thermal hysteresis are low (10). In M. edulis the THPs may therefore be of importance in inhibiting recrystallization (5). The inhibition of recrystallization may be important for the ability of P. davidi to survive intracellular freezing. The redistribution of ice during recrystallization might be expected to be particularly damaging when ice is located intracellularly, although the nematode can survive the stresses associated with the initial freezing of the cells. The recrystallization inhibition was destroyed in heated supernatants of the homogenates, suggesting that a protein is responsible. Substances other than THPs show recrystallization inhibition activity. Particles, for example, may inhibit grain boundary migration (7). Some synthetic peptides synthesized as variants of an antifreeze peptide from winter flounder may show no THP activity or modification of ice crystal growth and yet still show
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recrystallization inhibition activity. Other peptides and some polyaminoacids and polyolefins also showed recrystallization inhibition activity (7). A large proportion of the water is frozen in H. maori, even at moderate subzero temperatures (9). However, we did not find any recrystallization inhibition in the haemolymph from this species. Knight et al. (7) hypothesized that THPs in freezing tolerant animals have a function as inhibitors of migration of still-liquid domains. This does not seem to be important in H. maori. In contrast we found recrystallization inhibition in relatively dilute homogenates of the freezing tolerant nematode P. davidi. This species survives the formation of intracellular ice (13) and recrystallization inhibition may be a special adaptation for the survival of this stress. From the results presented in this paper we suggest that recrystallisation inhibition is important in animals which can survive intracellular freezing. In contrast, in freezing tolerant animals which cannot survive intracellular freezing recrystallization inhibition is not essential. The presence of recrystallization inhibition in some freezing tolerant animals which do not survive intracellular freezing may indicate that these organisms are especially vulnerable to recrystallization or to movements of salty still-liquid domains in the extracellular spaces. Clearly more work on the role of recrystallization inhibition is needed to elucidate these problems. ACKNOWLEDGMENT We thank the Danish Research Council for financial support. REFERENCES 1. Duman, J. G., Wu, D. W., Xu, L., Tursman, D., and Olsen, T. M. Adaptations of insects to subzero temperatures. Quart. Rev. Biol. 66, 387–410 (1991). 2. Duman, J. G., Xu, L., Neven, L. G., Tursman, D., and Wu, D. W. Hemolymph proteins involved in insect subzero-temperature tolerance: ice nucleators and antifreeze proteins. In ‘‘Insects at Low Temperatures’’ (R. E. Lee and D. L. Denlinger,
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