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PubMed1991

Freeze-fracture cytochemistry: a simplified guide and update on developments.

A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.

Animals↗
PubMed1965

FREEZE-DRYING OF FOOT-AND-MOUTH DISEASE VIRUS AND STORAGE STABILITY OF THE INFECTIVITY OF DRIED VIRUS AT 4 C.

Foot-and-mouth disease virus, type A, strain 119, propagated in cultures of calf kidney cells and in the tongue epithelium of cattle was used. The process of freeze-drying was conducted in two cycles on unit volumes of 4 ml in Pyrex ampoules, averaging 150 ampoules per run, and was studied separately from the problems of storage. Ampoules containing freeze-dried virus were flame-sealed for either immediate study or storage at 4 C for later reference. Tissue-culture virus dried with various additives had a mean processing loss of 0.8 log LD(50) per ml for six different preparations. Virus freeze-dried in tissue suspension had a mean loss of 0.8 log LD(50) per ml for three different preparations. A second set of preparations was processed and specifically studied for storage quality at 4 C. The virus in 14 freeze-dried tissue-culture preparations had a mean loss of 0.75 log LD(50) per ml while stored at 4 C for 1 year. Virus in four freeze-dried tissue suspensions had a mean loss of 0.05 log LD(50) per ml held at 4 C for 1 year. None of the specific additives used for conservation of the virus during the freeze-drying process or during storage at 4 C contributed significantly to the stability of the virus preparations over and above that observed with the normal growth medium of the tissue culture or the ordinary diluents used in making suspensions of tissue virus.

Animals↗
PubMed1985

Freeze-drying and related preparation techniques for biological microprobe analysis.

An X-ray microanalytical and morphological investigation has been carried out on rapidly frozen, freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze-substitution and freeze-drying. The investigation also included an analysis of specimens infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl HM20. The morphological preservation of Lowicryl embedded tissue was adequate for the identification of different cell structures like nuclei, mitochondria, lysosomes and different types of endoplasmic reticulum. X-ray microanalytical investigation of low temperature embedded material displayed an elemental composition of cells and organelles similar to that found in freeze-dried cyosections. Compared with freeze-dried cryosections, low temperature embedded material could be sectioned for light microscopy and area of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogenous cell populations.

Animals↗
PubMed1998

Ultrastructure of human scalp hair shafts as revealed by freeze-substitution fixation.

Human scalp hair is important as a diagnostic clue to many diseases, in medical jurisprudential investigations, and also as a subject of cosmetic treatments. While many ultrastructural studies of the human hair root including the hair follicle have been reported, few studies have been done on the human hair shaft. We report here the ultrastructure of human scalp hair shafts prepared by a rapid-freezing technique followed by freeze-substitution fixation that allows the observation of fine cell structures. Healthy scalp hair shafts from Japanese females 12-13 years of age were rapid-frozen and then freeze-substituted in OsO4-acetone. In addition, this technique was applied to the study of some changes of the hair shafts (i.e., hair damaged by thioglycolic acid cold permanent waving and white hair). By this method, the hair shaft was rapid-frozen throughout without appreciable ice damage although the hair shaft was nearly 100 microm in diameter. The rapid-freezing technique resulted in excellent preservation of the ultrastructure of the hair shafts: lamellar structures in the cuticle and fine fibrous ultrastructures in the cortex were observed without chemical treatments. Thioglycolic acid treatment affected the ultrastructure of both the cuticle and the cortex. Except for the absence of melanin granules, no significant differences in the ultrastructure were observed between white hair and black hair. The rapid-freezing technique followed by freeze-substitution fixation appears to be the most reliable approach for the morphological evaluation of fully keratinized cells and tissues.

Adolescent↗
PubMed1988

Surface modifications at the periosseous region of chick osteoclast as revealed by freeze-substitution.

Improved preservation of osteoclast fine structure can be achieved by quick freezing, freeze-substitution, or detergent extraction. With such techniques the ruffled border mainly contains a disorganized, interconnected meshwork of microfilaments (5-7 nm in diameter), whereas in the clear zone a few ordered arrays of intermediate-type filaments (10-12 nm in diameter) are detectable among the network of microfilaments. In well-frozen samples, well-preserved matrix may have occluded the cytoskeleton; detergent extraction permits visualization of the cytoskeletal components. In fresh-frozen cells an extracellular fuzzy coat overlays the ruffled border. At the site of attachment of the clear zone to the bone surface, extracellular cementing material is detected only after quick freezing. The superiority of quick freezing to preserve ultrastructure is shown in various cytoplasmic organelles. Most vesicles and vacuoles found close to the ruffled border seemed not to make contact with the extracellular matrix. Anhydrous procedures using quick freezing and freeze-substitution stabilize bone mineral in some vacuoles and in the channels of the ruffled border.

Animals↗
PubMed2001

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states.

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.

Adenosine Triphosphatases↗
PubMed2005

Neural mechanisms of freezing and passive aversive behaviors.

Cues that predict aversive outcomes often produce marked inhibitions of behavior known as freezing, but it is unknown exactly what neural pathways cause this inhibition. The amygdala and bed nucleus of the stria terminalis, along with their projections to the periaqueductal gray, are strongly implicated in freezing, but it is not known how these structures inhibit motor output. The median raphe nucleus (MRN), which contains a major population of serotonin neurons, has also been implicated in freezing, but the serotonin neurons themselves do not seem to be involved, leaving it uncertain which neurons in this area promote freezing. Our recent work suggests that GABAergic neurons just lateral to the MRN, but not within the MRN, regulate freezing via projections to midbrain dopamine neurons. Because freezing pathways may control a variety of other passive aversive behaviors, their elucidation may help understand the mechanisms of addictions and compulsions, which involve a failure of aversive outcomes to inhibit behavior.

Amygdala↗
PubMed1995

Changes in histological structure and physiochemical properties of raw, cured, smoked pork loins resulting from freezing near cryoscopic temperature.

The aim of the study was to determine the effects of unintended freezing during cold storage (deep chilling conditions) and low freezing temperatures (-10 degrees C) of raw pork loins, cured and smoked, on their histological structure and some physicochemical characteristics. The experimental products after freezing at -10 degrees C, were stored at the same temperature for 0, 2, 4 and 6 days, thawed thereafter and immediately deep chilled at near cryoscopic temperature (-3 degrees C) for 0, 2, 4 or 6 days. These conditions were changing the histological structures of pork loin, the water holding capacity (WHC) was reduced by frozen storage but improved by deep chilling; the maximum shear force and energy were increased. The more the freezing at -10 degrees C was prolonged, the more unfavourable were the changes in structure, WHC and tenderness. Deep chilling at -3 degrees C after thawing of the experimental material on the other hand favourably affected the parameters measured. Freezing at -10 degrees C and deep chilling at -3 degrees C did not affect the electrophoretic picture of pork loin proteins nor the water activity. Freezing itself had no influence on WHC. Changes were due to storage.

Animals↗
PubMed2001

Control of pore structure and size in freeze-dried collagen sponges.

Because of many suitable properties, collagen sponges are used as an acellular implant or a biomaterial in the field of tissue engineering. Generally, the inner three-dimensional structure of the sponges influences the behavior of cells. To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable, and homogeneous pore structure. Collagen sponges can be produced by freeze-drying of collagen suspensions. The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing. In industrial production, the collagen suspensions are solidified under time- and space-dependent freezing conditions, resulting in an inhomogeneous pore structure. In this investigation, unidirectional solidification was applied during the freezing process to produce collagen sponges with a homogeneous pore structure. Using this technique the entire sample can be solidified under thermally constant freezing conditions. The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension. Collagen sponges with a very uniform and defined pore structure can be produced. Furthermore, the pore size can be adjusted between 20-40 microm. The thickness of the sponges prepared during this research was 10 mm.

Biocompatible Materials↗
PubMed2001

Differential scanning calorimetry studies of intraembryonic freezing and cryoprotectant penetration in zebrafish (Danio rerio) embryos.

Nucleation temperatures of intraembryonic water and cryoprotectant penetration in zebrafish embryos were studied using differential scanning calorimetry. The effects of embryo developmental stage, dechorionation, partial removal of yolk, cooling rate, and cryoprotectant treatment on the temperatures of intraembryonic freezing were investigated. Embryo stages were found to have a significant effect on the nucleation temperatures of intact embryos. Freeze onset temperatures of -11.9 +/- 1.5, -15.6 +/- 0.3, and -20.5 +/- 0.1 degrees C were obtained for intact embryos at 6-somite, prim-6, and high-pec stages, respectively. After dechorionation, the freeze onset temperatures of intraembryonic water shifted to significantly lower temperatures, being -23.5 +/- 0.8, -18.7 +/- 0.7, -24.9 +/- 0.8 degrees C for 6-somite, prim-6, and high-pec stages, respectively. Yolk-reduced high-pec stage embryos showed significantly lower nucleation temperatures with an average onset at -27.9 +/- 0.4 degrees C. The effect of cryoprotectant treatment on the nucleation temperatures of intraembryonic water varies among different embryo stages and different cryoprotectants. Thirty-minute treatment with 2 M methanol significantly decreased the nucleation temperatures of dechorionated 6-somite embryos whilst no temperature decrease was observed for prim-6 or yolk-reduced high-pec embryos. Thirty-minute exposure to 1 M propylene glycol did not significantly affect the nucleation temperatures of dechorionated 6-somite, prim-6, or yolk-reduced high-pec embryos. In order to increase the permeability of embryos to cryoprotectants, the yolk sacs of dechorionated embryos at 6-somite or prim-6 embryos were punctured with a sharp micro-needle before exposure to cryoprotectants. The punctured prim-6 embryos showed significantly lower temperatures of intraembryonic freezing after 30 min of exposure to 2 M methanol following the multi-punctures. The nucleation temperatures of punctured 6-somite or prim-6 embryos were also decreased significantly after exposure to 1 M propylene glycol for 30 min. These results suggested that in intact embryos, intraembryonic freezing appeared to be seeded by the external ice in the perivitelline fluid and that in dechorionated embryos (in the absence of external water) intraembryonic freezing was more likely a consequence of heterogeneous nucleation. Methanol was demonstrated to show a limited degree of penetration into prim-6 stage embryos, but it did not penetrate later-stage embryos such as prim-6 and yolk-reduced high-pec. No propylene glycol permeation was observed for embryos at all stages. However, multi-punctures of yolk resulted in the permeation of both cryoprotectants into prim-6 embryos and propylene glycol permeation into 6-somite embryos. These findings may have important implications in overcoming the problem associated with the low membrane permeability of zebrafish embryos to cryoprotectants.

Adaptation, Physiological↗
PubMed1992

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.

Adaptation, Physiological↗
PubMed2002

Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems.

Previous studies have established that the selective precipitation of a less soluble buffer component during freezing can induce a significant pH shift in the freeze concentrate. During freezing of sodium phosphate solutions, crystallization of the disodium salt can produce a pH decrease as great as 3 pH units which can dramatically affect protein stability. The objective of our study was to determine how the presence of glycine (0-500 mM), a commonly used bulking agent in pharmaceutical protein formulations, affects the pH changes normally observed during freezing in sodium phosphate buffer solutions and to determine whether these pH changes contribute to instability of model proteins in glycine/phosphate formulations. During freezing in sodium phosphate buffers, the presence of glycine significantly influenced the pH. Glycine at the lower concentrations (< or = 50 mM) suppressed the pH decrease normally observed during freezing in 10 and 100 mM sodium phosphate buffer, possibly by reducing the nucleation rate of salt and thereby decreasing the extent of buffer salt crystallization. The presence of glycine at higher concentration (> 100 mM) in the sodium phosphate buffer resulted in a more complete crystallization of the disodium salt as indicated by the frozen pH values closer to the equilibrium value (pH 3.6). Although high concentrations of glycine can facilitate more buffer salt crystallization and these pH shifts may prove to be potentially damaging to the protein, glycine, in its amorphous state, can also act to stabilize a protein via the preferential exclusion mechanism.

Ammonium Sulfate↗
PubMed2002

Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability.

This work investigates the use of spray freeze-drying (SFD) to produce protein loaded particles suitable for epidermal delivery. In the first part of the study, the effects of formulation and process conditions on particle properties are examined. Aqueous solutions of trehalose produce SFD particles in the size range 20-80 microm, with a smooth, textured surface, but having high internal porosity. The latter was visualized using SEM and a novel particle embedding and sectioning technique. Use of an annealing step during the freeze-drying cycle caused the particles to shrink, reducing hereby porosity and also the measured rate of moisture uptake into these amorphous particles. SFD pure mannitol was approximately 40% amorphous, but not hygroscopic. Incorporation of dextran 37,500 into a combined amorphous trehalose/mannitol formulation led to increased particle shrinkage and lower particle porosity on annealing. The model protein trypsinogen lost approximately 15% activity during SFD of solutions containing 50 mg/mL protein, but was only marginally aggregated (1.4%). It is suggested that trypsinogen forms an irreversible partially unfolded state or molten globule on SFD/rehydration. The pure protein was also partially inactivated without aggregation during atomization into air. Surprisingly, neither activity loss nor aggregation were detected on atomization of the protein solution into liquid nitrogen. Quench-freezing of small droplets may reverse the partial unfolding of trypsinogen occurring on atomization into air. The origin of the trypsinogen inactivation during SFD must therefore be the subsequent freeze-drying step of this multistep process. Isolated freeze drying of trypsinogen produces strong aggregation and equivalent inactivation. This result suggests that trypsinogen behaves differently during freeze drying from frozen droplets and from bulk solution in a vial. In the former case the protein forms an irreversible partially unfolded state, whereas in the latter case aggregates are formed. Trypsinogen inactivation during SFD could be completely prevented by the presence of trehalose in the formulation. Electron Spectroscopy for Chemical Analysis (ESCA) showed a high surface excess of the protein in the SFD particles, which was reduced on inclusion of Polysorbate 80, but not trehalose. Taken together, these results help to elucidate the complex destabilization behavior of trypsinogen during SFD.

Chromatography, Gel↗
PubMed2001

Annealing to optimize the primary drying rate, reduce freezing-induced drying rate heterogeneity, and determine T(g)' in pharmaceutical lyophilization.

In a companion paper we show that the freezing of samples in vials by shelf-ramp freezing results in significant primary drying rate heterogeneity because of a dependence of the ice crystal size on the nucleation temperature during freezing.1 The purpose of this study was to test the hypothesis that post-freezing annealing, in which the product is held at a predetermined temperature for a specified duration, can reduce freezing-induced heterogeneity in sublimation rates. In addition, we test the impact of annealing on primary drying rates. Finally, we use the kinetics of relaxations during annealing to provide a simple measurement of T(g)', the glass transition temperature of the maximally freeze-concentrated amorphous phase, under conditions and time scales most appropriate for industrial lyophilization cycles. Aqueous solutions of hydroxyethyl starch (HES), sucrose, and HES:sucrose were either frozen by placement on a shelf while the temperature was reduced ("shelf-ramp frozen") or by immersion into liquid nitrogen. Samples were then annealed for various durations over a range of temperatures and partially lyophilized to determine the primary drying rate. The morphology of fully dried liquid nitrogen-frozen samples was examined using scanning electron microscopy. Annealing reduced primary drying rate heterogeneity for shelf-ramp frozen samples, and resulted in up to 3.5-fold increases in the primary drying rate. These effects were due to increased ice crystal sizes, simplified amorphous structures, and larger and more numerous holes on the cake surface of annealed samples. Annealed HES samples dissolved slightly faster than their unannealed counterparts. Annealing below T(g)' did not result in increased drying rates. We present a simple new annealing-lyophilization method of T(g)' determination that exploits this phenomenon. It can be carried out with a balance and a freeze-dryer, and has the additional advantage that a large number of candidate formulations can be evaluated simultaneously.

Crystallization↗
PubMed2004

Effect of collapse on the stability of freeze-dried recombinant factor VIII and alpha-amylase.

Recombinant Factor VIII (rFVIII) and alpha-amylase were used as model proteins to examine the effect of freeze-drying process conditions on the long-term stability of these proteins as freeze-dried solids. The same sucrose/glycine formulation was used for all treatments. Three freeze-drying protocols were used-an "aggressive" and a "conservative" cycle that both produced pharmaceutically acceptable product, and a protocol that produced a collapsed matrix. For rFVIII, there was no difference in the biological activity versus the time profile for product freeze-dried under the three different conditions when stored at 5 or 25 degrees C. At 40 degrees C, however, the stability of the collapsed product appeared to be better than that of product freeze-dried with no collapse. Also, the level of residual moisture in the collapsed product was higher than that of the product with no collapse. For alpha-amylase, there was no significant difference in the stability profile at any of the temperatures over the time course of the study. The results support the conclusion that collapse is not necessarily detrimental to the long-term stability of freeze-dried proteins.

Animals↗
PubMed1998

Overexpression of dnaK/dnaJ and groEL confers freeze tolerance to Escherichia coli.

Heat shock proteins not only can protect host cells against heat stress, they can also enable freeze tolerance as well. With respect to this unexpected feature, we are able to show that, at least in Escherichia coli, the heat shock proteins DnaK/DnaJ and GroEL play a very significant role. We found that the recovery rate of E. coli cultures that had been stored at -80 degreesC in the absence of any cryoprotectant was related to the abundance of these heat shock proteins accumulated before the freeze treatment. Before freezing, the DnaK in the bacterial cells was induced to accumulate to a level comparable to that produced in response to heat shock. After the freezing treatment, the recovery rate of the induced culture was very similar to that of the heat-shocked culture. Over production of GroEL was also protective but less effective. While freezing inevitably leads to protein denaturation, we propose that advance synthesis of DnaK/DnaJ and GroEL can accordingly prevent irreversible denaturation by chaperoning the unfolded polypeptides during freezing.

Adaptation, Physiological↗
PubMed1997

Freeze drying of cardiac valves in preparation for cellular repopulation.

When freeze-dried cardiac valves have been implanted they remained acellular. This study is the initial step in the development of a method designed to repopulate the substance of the freeze-dried valve with fibroblasts and the lumenal surface with endothelial cells. In this scheme, the freeze-drying process performs three functions; it provides a porous matrix, it kills the donor cells, and it preserves the collagen structure and hence the mechanical strength of the valve. This paper describes the production of appropriate porosity in freeze-dried porcine pulmonary valve leaflets. We found that Tg' for this material is -83 degrees C, which made it impracticable to freeze-dry exclusively from the glassy state. Uncontrolled freeze-drying produced a variable structure with most of the pores considerably smaller than the desired size and a dense layer, apparently devoid of perforations, on the surface. Compacted layers also occurred within the substance of the leaflets. These appearances suggested that extensive collapse had occurred during the drying process. Variation of the cooling rate, the primary drying temperature, and the warming rate during secondary drying enabled us to identify the following conditions that provided satisfactory internal porosity: cooling at 5 degrees C/min, vacuum drying for 6 h at -20 degrees C, and secondary drying for 10 h during rewarming at 0.06-0.08 degrees C/min. The internal cavities measured 100-350 microns2 by ca. 400 microns2, which is adequate to provide access for the fibroblasts (cross-sectional area ca. 150-200 microns2 when rounded but fusiform when attached. However, the internal porous structure rarely communicated with the surface and mechanical perforation was required to provide continuity between the surface and the internal sponge. The resulting method provides a basis for studies of cell colonization.

Animals↗
PubMed1998

Respiratory responses to chilling and freezing in two sub-antarctic insects.

The effects of chilling (to temperatures above the supercooling point, SCP) and freezing on respiration of adults and larvae of two coleopterans living on sub-Antarctic South Georgia (54 degrees S, 37 degrees W), Hydromedion sparsutum and Perimylops antarcticus (Coleoptera, Perimylopidae), were quantified. Respiration rates of individual insects (live weights, 11-21 mg) were measured at 10 degrees C prior to chilling (-4 degrees C) or freezing (SCP range -3.8 to -5.3 degrees C) and posttreatment. The species possess a small amount of freeze tolerance in both adults and larvae. Chilling had no significant effects on respiration rates of P. antarcticus and H. sparsutum, although mean levels were depressed by 6-15%. Freezing produced considerable enhancement of respiratory activity. Mean values increased postfreezing in larvae (+34%) of H. sparsutum and in both larvae (+44%) (P < 0.01) and adults (77%) (P < 0.05) of P. antarcticus. Chilling and freezing had different effects on respiration rates and P. antarcticus showed the greatest metabolic response to freezing.

Adaptation, Physiological↗