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Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear. Here, we report a comprehensive evaluation of the effects of five different AFPs on the activity of bacterial ice nucleators using a high-throughput ice nucleation assay. We find that bacterial INPs are inhibited by certain AFPs, while others show no effect. Thus, the ability to inhibit the activity of INPs is not an intrinsic property of AFPs, and the interactions of INPs and different AFPs proceed through protein-specific rather than universal molecular mechanisms.

Despite the fact that much progress of understanding the antifreeze proteins, AFPs, (ice-binding proteins ad recrystallization inhibitor proteins) has been made during the last four decades, many questions are still unanswered and each time one question is answered, more seems to appear.

In the first volume of the book, we present the history of the antifreeze proteins (AFPs), their physiological role in freeze-avoiding or freeze-tolerating species, as well as the evolution and grouping of the antifreeze proteins. In the present volume we delve into the functions and interactions of antifreeze proteins at the molecular level. The volume is divided in three parts (Part I, II, and III of this work) followed by a final summary of both volumes. Part I focuses on the biochemistry of the antifreeze proteins and their interaction with both ice and water. Part II describes the mode of actions for obtaining thermal hysteresis and inhibition of recrystallization and how to measure it. Part III concerns the fields in which the applications of antifreeze proteins have come the furthest, as well as the possibilities of mutating and tailoring the proteins. In this chapter we give a short summary of the chapters that constitutes this second and final volume of the book.

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

The study investigated whether evaporative water loss (EWL) in frogs stems from water diffusing through the skin or fluid secreted by mucous glands. Osmolality of cutaneous surface fluid (CSF) of Rana esculenta (Pelophylax kl. esculentus) subjected to isoproterenol or 30°C-34°C was 191±9.3 and 181±7.5 mosm/kg, respectively, as compared to lymph osmolality of, 249±10 mosm/kg. Cation concentrations of CSF were likewise independent of pre-treatment with averages of, [Na(+)]=65.5±5.1 and [K(+)]=14.9±1.6 mmol/L, and lymph concentrations of 116 mmol Na(+)/L and 5.1 mmol K(+)/L. The relatively high [K(+)] confirms that CSF is produced by submucosal glands. Since the chemical energy of water of CSF was always higher than that of body fluids, diffusion of water would be from CSF to the interstitial fluid and not in the opposite direction. It is concluded that volume and composition of CSF are regulated by subepidermal exocrine gland secretion balanced by EWL into the atmosphere and ion reuptake by the epidermal epithelium. Previously discovered regulatory mechanisms of epithelial ion absorption, hitherto not ascribed a body function, fit well with a role in regulating turnover of CSF. As a regulated external physiological compartment, CSF would be of importance for the immune defenses that amphibians employ in protecting their skin.

Carassius carassius responds to hypoxic conditions by conversion of lactate into ethanol, which is excreted over the gills. However, a closely related species, Cyprinus carpio, does not possess the ability to produce ethanol and would be expected to accumulate lactate during hypoxic exposure. While the increase in oxygen consumption in fish required following strenuous exercise or low environmental oxygen availability has been frequently considered, the primary contributing mechanism remains unknown. This study utilized the close relationship but strongly divergent physiology between C. carpio and C. carassius to examine the possible correlation between excess post-hypoxic oxygen consumption (EPHOC) and lactate accumulation. No difference in the EPHOC:O2 deficit ratio was observed between the two species after 2.5 h anoxia, with ratios of 2.0 ± 0.6 (C. carpio) and 1.3 ± 0.3 (C. carassius). As predicted, lactate accumulation dynamics did significantly differ between the species in both plasma and white muscle following anoxic exposure. Significant lactate accumulation was seen in both plasma and muscle in C. carpio, but there was no accumulation of lactate in white muscle tissue of C. carassius. These findings indicate that lactate accumulated as a consequence of 2.5 h anoxic exposure is not a major determinant of the resulting EPHOC

This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of β-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these β-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall β-helical structure with the ice-binding motifs stacked in a β-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a β-helical conformation.

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml − 1 . Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V max and K M was practically equivalent in pure buffer and in 200–250 mg BSA ml − 1 supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml − 1 partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k cat , K M and k cat / K M . The osmolytes decreased k cat / K M in the order: NaCl > Urea > TMAO/glycerol > betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein–water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k cat / K M .

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28 degrees C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean +/- SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 +/- 0.04 (10 degrees C) to 0.54 +/- 0.09 nmol O(2) egg(-1) h(-1) (28 degrees C). The mean +/- SD Q(10)-value was 2.51 +/- 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.

In response to drought bdelloid and monogonont rotifers undergo anhydrobiosis and are assumed to synthesize protective chemicals, which are commonly sugars. In contrast to most anhydrobionts, bdelloids have earlier been shown to lack trehalose as protective chemical, and more importantly to lack trehalose synthase (tps) genes. It remains to be assessed if the absence of trehalose is a characteristic common to the entire taxon Rotifera, or if it is limited to bdelloids, or is peculiar to the two bdelloid species investigated so far. In this study, anhydrobiotic adults of a bdelloid species (Macrotrachela quadricornifera) and resting eggs of a monogonont species (Brachionus plicatilis) were analysed by thin layer chromatography and gas chromatography to detect the presence of trehalose. No trehalose was detected in the bdelloid, while the anhydrobiotic resting egg of the monogonont rotifer contained about 0.35% trehalose of its dry weight. Although very little, the presence of trehalose in B. plicatilis suggests that the trehalose synthase genes, absent in bdelloid rotifers, are present in non-bdelloid rotifers.

The eutardigrade Adorybiotus (Richtersius) coronifer survives cryptobiosis for years. During entrance into anhydrobiosis this species accumulates the disaccharide trehalose reaching a maximum content of 2.3% d.w. In the present study we examined the survival of anhydrobiotic A. (R)coronifer during exposure to alcohols of various polarity, and to high temperatures, as well as qualitative changes in protein synthesis during entrance into anhydrobiosis. Results showed that A. (R) coronifer in anhydrobiosis survived exposure to ethanol for less than 10 minutes whereas exposure to 1-butanol only decreased survival to 40% after the first 7 days and 1-hexanol did not change survival from the controls after the first 7 days. A. (R) coronifer survived temperatures up to approximately 70 °C for 60 minutes without any decrease in survival. However, survival decreased rapidly when the exposure temperature was increased to above 70 °C and no animals survived exposure to 100 °C. During the entrance into anhydrobiosis a protein with a molecular weight of approximately 71 kDa appeared on acryl amide gels showing protein bands after the animals had been incubated with 3H-Leucine. This protein may belong to the Heat-shock protein (Hsp) 70 family. The results on the survival of A. (R) coronifer during exposure to alcohols and high temperature are discussed in light of the trehalose content earlier described in this animal during anhydrobiosis.

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn.

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270μmol.

We report measurements of the supercooling point of the hemolymph of the freeze-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera; Stenopelmatidae). Dilution curve and supercooling points following ultrafiltration argue that the ice-nucleation agents present are proteinaceous. We also report dynamic light-scattering measurements leading to hydrodynamic diameters of large molecules found in the weta hemolymph. The measured diameters agree well with theoretical estimates for sizes of ice-nucleating proteins corresponding to the measured supercooling points.

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principal inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH4(+), Ca(2+), Mg(2+), F(-), SO4(2-) and PO4(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared with that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO4(3-), ×700-2800; SO4(2-), ×30-150). In contrast, in the marine species H. crispae, Na(+), Cl(-) and SO4(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq l(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.

1. 1.|The New Zealand weta, Hemideina maori, is shown to withstand freezing, below its supercooling point, for at least 5 h at −9°C and for at least 8 h at −8°C. 2. 2.|The lower lethal temperature is about −10°C. 3. 3.|Insects collected in both summer and winter are freezing tolerant. 4. 4.|Haemolymph of “winter” insects has a much higher osmotic pressure (about 700 mOsm/kg) than “summer” forms, (about 450 mOsm/kg). 5. 5.|A large contribution to this high osmolality is made by unidentified compounds, which are non-ionic, and are not carbohydrate.

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of -3 degrees C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and -1 degrees C). Lactate accumulation was only observed in the liver (4.1+/-0.8 against 16.6+/-2.4 micromol g(-1) fresh weight (FW) between 4 and -1 degrees C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4+/-0.7 microl O(2) g(-1) FW h(-1) at -4 degrees C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.

Changes in the trehalose level in the tardigrade Adorybiotus coronifer were ob- served during induction of, and arousal from, anhydrobiosis. A trehalose accumulation surpassing 1.6% dry weight (d.w.) in anhydrobiotic animals collected dry on their biotope was rapidly reduced on rehydration, reaching the level of active animals (0.1-0.2% d.w.) after 6 hours. Tardigrades dried while embedded in sand in the laboratory accumulated trehalose from 0.1% to 2.3% d.w. within 5-7 hours. Induction of aerobic acidosis in arousing tardigrades by CO, perfusion reversibly arrested re- activation for at least 36 hours and induced a reduction in anabolic and catabolic activities, mea- sured as a significant reduction in trehalose degradation, and a sevenfold reduction in the rate of protein synthesis. These data support the hypotheis that trehalose generally serves a protective role in desiccation- tolerant Metazoa, but indicate that tardigrades require only a moderate level for efficient pro- tection. Trehalose accumulation A positive correlation between the survival of the cryptobiotic state and the accumulation of the disaccharide trehalose has been demonstrated for a number of anhydrobiotic organisms, and con- centrations as high as 13-18% dry weight have been reported in anhydrobiotic cysts of the crusta- cean Artemia salina' (Clegg, '64; '65) and in the nematode Aphelenchus avenae (Madin and Crowe, '75; Crowe et al., '77). Moreover, trehalose reduces damage, induced by freeze-drying, to iso- lated liposomes (Crowe et al., '861, microsomes from sarcoplasmatic reticulum (Crowe et al., '83), and the enzyme phosphofructokinase (Carpenter et al., '87; '88). Other data such as the physical properties of nearly dry Artemia salina cysts (Clegg et al., '82; Clegg, '86) and computerized modeling of molecular interactions between tre- halose and a phospholipid bilayer (Gaber et al., '86) indicate that trehalose is a likely candidate for the polyhydroxy compound that, according to the water replacement hypothesis (Webb, '65; Crowe, '711, replaces the structural water of the cellular compounds during desiccation. Anhydrobiosis is widespread in the phylum Tardigrada, but quantification of trehalose accu- mulation during the induction of anhydrobiosis has not yet been made in this phylum. Crowe ('75) reported that paper chromatography analysis of ethanol extracts of the tardigrade Macrobiotus

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor , and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP H4 , was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.

Water vapour absorption (WVA) is an important mechanism for water gain in several xeric insects. Theoretical calculations indicate that the energetic cost of WVA should be small (5–10% of standard metabolic rate) assuming realistic efficiencies. In this study we explored the relationship between WVA, metabolic heat flux (HFmet.) and CO2 release in larvae of Tenebrio molitor using microcalorimetry. By comparing metabolic heat flux with the catabolic rate estimated from V CO 2 , we were able to differentiate anabolic and catabolic rates prior to and during WVA, while simultaneously monitoring water exchange. Three to four hours before the onset of WVA, larvae showed clear increases in HFmet. and catabolic flux, and a simultaneous decrease in anabolic flux. Following the onset of WVA, HFmet. decreased again until indistinguishable from control (non-absorbing) values. Possible factors contributing to the “preparatory phase” are discussed, including mobilization of Malpighian tubule transporters and muscular activity in the rectum. Absorbing larvae reduced the water activity of the calorimetric cell to 0.906, agreeing with gravimetric estimates of the critical equilibrium activity. Periods of movement during WVA coincided with decreased uptake fluxes, consistent with the animal's hydrostatic skeleton and the need to close the anus to generate pressure increases in the haemocoel.

The eelpout Zoarces viviparus is a common inhabitant in the shallow waters along the Danish coastline. Specimens were caught in the brackish (12-16 per thousand) Roskilde fjord where water temperatures range from20 degrees C during summer to subzero in winter. The serum melting points found in Z. viviparus varied between -0.76 (September) to -0.94 degrees C (January). Eighty to 97% of the serum melting points could be attributed to sodium, chloride and potassium. Hysteresis freezing points showed seasonal variation varying from -0.83 (September) to -2.08 degrees C (February). Serum antifreeze activity showed a seasonal variation with high levels (1.2 degrees C) in winter and low levels (0.1 degrees C) during summer and autumn. Antifreeze proteins are responsible for this antifreeze activity. Antifreeze activity was also found in Z. viviparus during their embryological development in the female ovary. Embryo thermal hysteresis reached the maximum level (approx. 0.6 degrees C) during December and maintained this level until parturition in January. Antifreeze activity seems unaffected by diminishing ice crystal fractions at ice fractions below 0.1 whereas ice fractions above 0.1 caused a decline in antifreeze activity.

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.

The present study revealed that hibernating freeze-avoiding Rhagium inquisitor beetles have thermal hysteresis antifreeze agents in the intracellular fluid as well as in the intestinal fluid and the haemolymph. The antifreeze activity in all three compartments increased with diminishing size of the seeding ice crystal, suggesting that all three compartments are well protected against spontaneous ice nucleation at low sub-zero temperatures.