Metabolic responses to freezing and anoxia by the periwinkle Littorina littorea

Pergunoa

0306-&65(%)ooo22-4

J. therm. Biol. Vol. 21, No. I, pp. 57-63, 1996 Copyright 0 1996 Elsevicr Science Ltd Printed in Great Britain. All rights resewed 0306-4565/96 $15.00 + 0.00

METABOLIC RESPONSES TO FREEZING AND ANOXIA BY THE PERIWINKLE LITTORINA LITTOREA THOMAS A. CHURCHILL*

and KENNETH

B. STOREYt

Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario, Canada KlS SBS (Received I May 1995; accepted 20 May 1995)

The metabolic responses to freezing at - 8°C for up to 72 h vs 72 h anoxia exposure under

Abstract-l.

N, gas at 5°C were

compared in foot muscle of the intertidal gastropod, Littorina littorea.

2. Freezing resulted in the accumulation of o-lactate and succinate (net increases of 1.1 and 3.9 pmol/g wet weight, respectively, compared with SC-acclimated controls) with an opposite decrease in the fermentative substrate, L-aspartate, whereas, by contrast, anoxia resulted in only a small, 0.5 pmol/gww, accumulation of D-lactate. 3. Neither freezing nor anoxia exposure had a significant effect on muscle adenylate energy charge, suggesting that strong metabolic arrest mechanisms lowered energy demand to a level that could be met by fermentative metabolism alone. 4. Activation of glycogenolysis was implied by elevated glucose-6-phosphate levels in muscle under both stresses but only during freezing did glucose accumulate (a net of 1.2 pmollgww); changes in the levels of other glycolytic intermediates indicated regulatory control of glycolysis at pyruvate kinase under both stresses. 5. No carbohydrate cryoprotectants were accumulated during freezing and neither stress changed the composition or size of the free amino acid pool (approx. 100 pmol/gww total amino acids composed of 17% taurine and 12% alanine). 6. The data show that the metabolic responses to freezing and anoxia share some elements, indicating the importance of a good anaerobic capacity to freezing survival, but other aspects of metabolic response to freezing differ from anoxia and suggest factors that may be specifically important to cryoprotection. Key Word Ina’ex: Intertidal gastropods; anaerobiosis; cryobiology; energy metabolism; metabolic arrest

INTRODUCTION The intertidal changeable

zone

is one of the most

environments

inhabited

harsh

by animals.

and To

in oxygen and water availability, temperature, and salinity on both daily and seasonal time scales. Most intertidal invertebrates show a well-developed anoxia tolerance that includes the capacity to drop aerial metabolic rate to less than 10% of the resting aerobic rate in water and the ability to enhance anaerobic ATP output from fermentative reactions linked to succinate and volatile fatty acid production (de Zwaan, 1983; Storey and Storey, 1990; Storey, 1992). These species also have a high tolerance for variation in body fluid osmolality whether caused by aerial succeed there, animals must endure wide variations

*Present address: Academic Department of Surgery, Royal Free Hospital School of Medicine, London NW3 2QG, U.K. tTo whom all correspondence should be addressed.

desiccation or by the variable salinity of water in tide pools (Gilles, 1979). At high latitudes, intertidal fauna may also be exposed to freezing temperatures during winter low tides and most species living under these conditions have developed freeze tolerance. The characteristics of freeze tolerance among marine invertebrates, including the effects on survival of different subzero temperatures, duration of freezing, freezing rates, and percent body ice accumulated, have been described for several species of mussels, snails, and barnacles (for review Aarset, 1982; Murphy, 1983). One freeze tolerant intertidal species is the periwinkle Littorina littorea (Mollusca, Gastropoda). Periwinkles can endure freezing at - 8°C for at least 8 days but mortality increased sharply with lower temperatures, LDSo values at -9, - 11, and - 13°C being 7 days, 28 hours, and 4 hours, respectively (Murphy and Johnson, 1980; Murphy, 1983). Tolerance of freezing by L. littorea was greater in winter than in summer, and enhanced by acclimation to

Thomas A. Churchill and Kenneth B. Storey

58 higher salinities,

but independent

of acclimation

tem-

perature and photoperiod (Murphy, 1979). As with other freeze tolerant species, intertidal molluscs

periwinkles,

Littorina

littorea,

were

exhibit hemolymph ice nucleator proteins that act to induce ice formation in extracellular fluid spaces

artificial sea water (1000 mOsmol)

(Aunaas,

3 weeks prior to experimentation.

1982; Hayes and Loomis,

1985; Madison

et al., 1991) but they lack the high concentrations

Animal experiments

low subzero

Control seawater.

However,

although

(Storey and Storey.

colligative

1988).

cryoprotectants

are

lacking, freeze tolerance is improved by acclimation to higher salinities (Williams, 1970; Murphy. 1979. 1983) and various amino acids, anaerobic ucts, and Ca’+ appear

to be effective

end prod-

in stabilizing

membrane structure during freezing (Murphy, 1983; Loomis et al., 1988. 1989). Pre-exposure to anaerobic conditions

has also been found

to enhance

during subsequent freezing exposures due to one of several characteristics including

elevated

survival

but this may be of anaerobiosis

levels of end products

and Ca” ,

anoxia-induced metabolic arrest, and reduced potential for damage by oxygen free radicals when freezing begins

from an anoxic,

vs aerobic.

state (Murphy.

1983: Loomis, 1987; Storey and Storey, 1988). The present study investigates metabolic responses to freezing and anoxia exposures by L. littorea, comparing and contrasting the metabolic responses to these stresses in order to determine whether biochemical adaptations for anoxia tolerance also support freezing survival and whether unique metabolic responses to freezing (such as might provide cryoprotection)

occur. Extracellular

freezing

in

Marine In the aerated

at 5°C for at least

of

low molecular weight carbohydrate cryoprotectants that help terrestrial insects to endure freezing at very temperatures

collected

March by staff from the Huntsman Laboratory, St. Andrews, New Brunswick. laboratory, animals were acclimated in

has two funda-

mental effects on cells: (1) it leads to a reduction of cell volume with consequences including elevated osmolality and ionic strength of cellular fluids and potential damage due to physical distortion of membranes or denaturation of proteins, and (2) it causes ischemia. a disruption of oxygen and hemolymphborne fuel supplies to cells. a condition that is similar to the effects of anoxia. Hence the metabolic responses to freezing and anoxia should have some common features, representing metabolic adjustments that deal with the freezing-induced ischemia, whereas differences between the two stresses may highlight responses that are particularly important for the physical cryoprotection of cells during freezing. METHODS AND MATERIALS

snails were sampled directly from the 5~C For freezing exposure animals were

removed from the seawater, placed in closed plastic containers, and transferred to -8.0’ C in an incubator for freezing. In initial tests, cooling and nucleation

were monitored

All chemicals and biochemicals were purchased from Sigma Chemical Co., St. Louis, MO or Boehringer-Mannheim Corp., Montreal, PQ. Marine

the the

thermistor was attached to a YSI telethermometer with output to a linear recorder. The rate of cooling

to

- 8~C was approximately

and nucleation experimental allowed

occurred studies

- 1.0 C/min

within 45 min; therefore,

a 45 min cooling

before beginning

period

in was

to time the 12, 24 or 72 h

experimental freezing exposure times. For anoxia exposure, snails were placed in closed jars with about

1 cm of seawater in the bottom; the water had been previously bubbled with 100% nitrogen gas for at least

10min.

15-20 min maintained

Bubbling

with N? was continued

and then the jars were at 5’C for 24 h or 72 h.

sealed

for and

Tissue sampling and extruction Foot

muscle

from

individual

snails

was rapidly

dissected out (within 15 set), frozen in liquid nitrogen, and transferred to - 80°C for storage. Perchloric acid extracts

of frozen

foot

muscle

samples

were

prepared and spectrofluorometric coupled enzyme assays for metabolites were conducted as described by Churchill and Storey (1989). Amino acids were analyzed in aliquots perchloric precolumn

of the

acid extracts using a Waters HPLC after derivatization with orthophthalaldehyde.

L-alanine and taurine co-eluted from the column but because taurine predominated in the mixture, peak areas were quantified

relative

to taurine

standards

and then values for alanine (determined enzymatic assays) were subtracted.

from

Data and statistics Data are reported

Animals and chemicnls

for a few snails by placing

a thermistor inside the shell in contact with mantle (secured with tape around the shell);

as means rt_SEM, N = 8 in most

cases and N = 5 for 24 h anoxic snails. Data were analyzed using one-way analysis of variance and when significant differences were found among groups, the Dunnett’s test (2-tailed) was applied.

Metabolic responses to freezing vs anoxia RESULTS

59

Table 1. Levels of the major amino acids in foot muscle of the periwinkle Lirtorina lirtorea

During sampling of snails after -8°C exposure, we observed the presence of large amounts of extracellular ice surrounding all internal body organs, thereby indicating that snails were frozen and not simply supercooled. Recovery after freezing and anoxia stresses was also tested. To assess freezing survival, some snails were removed from the -8.O”C incubator at each sampling time, and were placed at 0°C in air for 1 h followed by returning them to the 5°C seawater. Survival was assessed 6 days later by the reappearance of normal movements (e.g., snails climbed the walls of aquaria) and by contraction of the foot in response to probing. Based on these criteria, recovery after freezing was 8/9 snails at 12 h, S/l0 at 24 h, and lo/IO at 72 h. Recovery after anoxia exposure was 100% at both timepoints. The effects of freezing or anoxia exposure on the levels of L-aspartate, a fermentative substrate, and the accumulation of anaerobic endproducts by foot muscle are shown in Fig. 1. During freezing the foot accumulated both b-lactate and succinate. Levels of both compounds had increased significantly after 12 h at - 8°C and after 72 h had risen by a net M-fold for o-lactate and 2.1-fold for succinate. L-Alanine content did not change significantly during

D-Lactate

L-Alonine

Succinate

L-Asportate

Fig. 1. Effects of freezing and anoxia exposures on the levels of (A) o-lactate and L-alanine and (B) succinate and L-aspartate in foot muscle of Littorina littorea. Bars are: 0, control; N, frozen 12 h; n , frozen 24 h; q3,frozen 72 h; E, anoxic 24 h; qO,anoxic 72 h. Data are means * SEM, N = 8 except for N = 5 for 24 h anoxic. u,bSignificantly different from the corresponding control value using one-way analysis of variance followed by the Dunnett’s test, P < 0.01; P < 0.05.

Amino acid Aspartate Glutamate Serine Glutamine Histidine Glycine Alanine Taurine Gamma-aminobutyric Valine Lysine Total amino acids

nmol/g wet weight

acid

3980+419 125 & 24 119+27 131 + 22 305+71 3235 5 663 11283 + 547 73700 k 8844 131 f 15 1337 + 125 509 + 77 95543 + 9557

Data are means f SEM, N = 8; no changes in free amino acid levels were detected between experimental groups so pooled values combine N = 3 control, N = 3, 72 h frozen, and N = 2, 72 h anoxic samples. Asparagine and methionine were not detected in any samples; other amino acids were present in amounts less than 1OOnmol/gww and values for these are not shown.

freezing. Levels of aspartate, however, showed a progressive decrease throughout the freezing timecourse, dropping by 40% after 72 h. The response to anoxia was different. Only D-lactate accumulated and amounts were much lower after 72 h anoxia than after 72 h freezing; the maximal increase was 2.6-fold. Alanine and aspartate levels did not change and succinate levels actually showed a significant decrease in foot of anoxic animals. HPLC analysis of free amino acids in L. littorea foot revealed no changes in the levels of other amino acids as the result of either freezing or anoxia exposures; because of this, data were pooled and means for all samples are shown in Table 1. The major free amino acid present in foot was taurine; the mean concentration of 73.7 pmol/g wet weight represented 77% of the total amino acid pool. Other major amino acids were alanine, aspartate, glycine and vahne representing 1184.2, 3.4, and 1.4% of the total pool size, respectively. A search for putative carbohydrate cryoprotectants was made. Glycerol levels in foot muscle samples ranged from 0.20 to 2.0 pmol/gww, showing no consistent pattern between experimental groups and failing to accumulate in amounts that could be relevant for colligative cryoprotection. Assays for sorbitol, mannose, and fructose showed that levels of each were below 0.08 pmol/gww in all cases. The effect of freezing and anoxia exposures on glucose levels in foot muscle is shown in Table 2. Glucose increased by 2.3-fold after 72 h of freezing but levels were unaffected by anoxia exposure.

Thomas A. Churchill and Kenneth B. Storey

60

Table 2. Effects of freezing and anoxia exposures on the levels of glycolytic intermediate metabolites in foot muscle of the periwinkle Lirrorinalittorea Freezing-exposed Control Aerobic Glucose G6P F6P F1,6Pz PEP Pyruvate

940 + 160 97 _t 9 16+ 3 20 + 4 90& 15 136 k I5

Anoxic

~.12h 750 * 90 76+ 10 6 f 3b II f 1 17+3* 129+6

24 h

72 h

nmol/g wet weight 137Ok 120 218Ok310” 131 * 10s 137 f 19s lo* I 18&4 18&4 17&4 25 _+6” 70 * 5 136 i 12 226 + 24”

24 h

72h

1150_+ 180 134 + 17b 9+1 14+5 31 F9” 87 + 22

570* 110 76k4 9_+2 15_+3 35 & 9” 92 k 8

Data are means k SEM. N = 8 except for N = 5 for the 24 h anoxic group. “,bSignificantly different from the corresponding control value using one-way analysis of variance followed by the Dunnett’s test 2-tailed. P < 0.01, P < 0.05. Changes in the levels of glycolytic intermediates in foot muscle as the result of freezing exposure are also

shown in Table

2. Levels

of glucose-6-phosphate

(G6P) were unaffected after 12 h freezing but rose by 3540% with longer periods of freezing, in line with

the increase in glucose. F6P content showed the opposite response, decreasing significantly in 12 h frozen muscle but then reverting to control levels with longer periods of freezing. Levels of fructose-l ,6bisphosphate were constant during freezing. PEP content of foot muscle was strongly reduced (to 19% of control values) after 12 h of freezing exposure but then increased again with longer periods of freezing. Pyruvate responded differently. remaining constant over the shorter freezing times but increasing by 66% in 72 h frozen snails. Table 2 also shows the effect of anoxia on glycolytic intermediates in foot muscle. After 24 h of anoxia exposure, G6P content had risen by 38% but decreased again after 72 h. PEP content dropped to about one-third of control values in both 24 and 72 h anoxic muscle. However, levels of other metabolites were unaffected by anoxia exposure. The effects of freezing and anoxia exposures on energetics in foot muscle of L. littorea are shown in Fig. 2. Neither stress caused a major disruption of energy levels. Although ADP content of muscle rose by 2-3 fold in all experimental animals, neither ATP and AMP levels showed significant changes during either freezing or anoxia. Total adenylates remained constant with an overall mean of 1.47 f 0.25 pmol/gww for all samples and energy charge which was 0.93 + 0.02 in control foot muscle remained constant at 0.86-0.88 under experimental conditions.

fermentative end products, the metabolic activity during anoxia was apparently extremely low. The only end product that accumulated in foot muscle was D-lactate with a net accumulation of only about 0.5 pmol/gww. This suggests an extremely low metabolic rate in anoxic periwinkles, undoubtedly the result of a combination of low temperature effects on metabolism and anoxia-induced metabolic depression. Indeed, even at higher temperatures, the accumulation of anaerobic products by L. littorea is low by comparison with various anoxia-tolerant bivalves (Wieser, 1980; Storey et al., 1982; Klutymans et al., 1983; de Zwaan et al., 1991). The net accumulation of alanine and succinate in L. littorea foot muscle after 24 h anoxia exposure at 12°C was only

2.0

L

-I

ATP

The metabolic responses to freezing and anoxia by of

L. littorea differed. Based on the accumulation

AMP

0.75

E.C. 0.50

Total

DISCUSSION

ADP

Admylatee

Ensrgy

Charge

Fig. 2. Effects of freezing and anoxia exposures on levels of (A) ATP, ADP, AMP and (B) total adenylates and energy charge, [ATP + ADP/Z]/[ATP + ADP + AMP], in foot muscle of Littorinalitrorea.Other details as in Fig. 1.

Metabolic responses to freezing vs anoxia 3 and l.rlpmol/gww, respectively (Storey et al., 1982). By contrast with the minimal metabolic response to anoxia at SC, freezing at -8°C had a greater effect on metabolism. Freezing exposure resulted in the accumulation of both lactate and succinate, the net accumulation being 1.1 and 3.9 pmol/gww, respectively. On the other hand, aspartate, a well known fermentative substrate for anaerobic metabolism in marine molluscs (de Zwaan, 1983), dropped by 1.9 pmol/gww, accounting for about one-half of the increase in succinate. This greater effect of freezing than of anoxia on the accumulation of fermentative end products is further surprising in light of the fact that the body temperature of the freezing-exposed snails was 13°C lower than that of the anoxic snails, and hence, a substantially lower metabolic rate at - 8°C should have been expected. Several factors may contribute to the differing responses to freezing and anoxia. Firstly, the greater end product synthesis during freezing may reflect a specific synthesis for cryoprotective purposes. This point will be further discussed later. Secondly, the greater accumulation of products in frozen muscle may be because these products are trapped within the frozen tissue whereas intertissue transport or excretion could minimize the net accumulation of these compounds in anoxic foot. This is true for succinate in some marine molluscs; some species excrete succinate whereas others further catabolize succinate to propionic acid, a volatile fatty acid, that is then excreted (de Zwaan, 1983). Arguing against this occurrence for L. littorea, however, is the observation that aspartate levels did not change in anoxic foot. The typical pattern of anaerobic metabolism in molluscs has two phases. Early in anoxia glycogen catabolism to a pyruvate derivative (lactate, alanine, or an imino acid) is coupled to aspartate conversion to succinate, whereas later (once aspartate reserves are used up) glycogen is directly converted to succinate/propionate (de Zwaan, 1983; de Zwaan et al., 1991). Because aspartate is intimately involved in anaerobic metabolism, the lack of significant change in aspartate levels during anoxia at 5°C is evidence for a very low rate of anaerobic metabolism, rather than the loss of succinate product either by excretion or conversion to propionate. The third factor that may contribute to the difference in responses to freezing versus anoxia is metabolic arrest. Anoxia-induced metabolic rate depression is a common and rapid response to oxygen lack by intertidal molluscs; anoxic metabolic rates for different species are 2-20% of the corresponding aerobic resting rate at the same temperature (Klutymans et al., 1983; de Zwaan et al., 1991;

61

Storey, 1992). Presumably, the freezing of extracellular body fluids would cut off oxygen supply to tissues to create an anoxic state and, indeed, all freeze tolerant species have been shown to accumulate anaerobic end products while frozen (Storey and Storey, 1988). Therefore, freezing-induced ischemia should presumably trigger the same metabolic arrest response as anoxia. However, one key difference between the two stresses is that whereas anoxia was imposed almost instantly (by placing snails in a nitrogen gas environment), freezing is a more prolonged process that would cause a gradual transition through hypoxia to anoxia as more extracellular fluids froze and remaining tissue oxygen was used up. Anoxia-tolerant animals generally show two phases of response to hypoxia. In the first phase during mild hypoxia, a compensation strategy is used and fermentative pathways are recruited to supplement the ATP output of oxidative phosphorylation. However, when a critical minimum p0, is reached, the second phase is entered and a conservation strategy is adopted; metabolic arrest is initiated and metabolic rate is dropped to a level that can be maintained over the long term by fermentative ATP output alone. Thus, the differences in metabolic end product accumulation between anoxia and freezing might represent the metabolic consequences of a sudden versus a gradual decline in oxygen tension, resulting in a net accumulation of anaerobic end products that was much lower under the nitrogen gas atmosphere than during freezing. Low molecular weight cryoprotectants are an important feature of freeze tolerance among terrestrial insects and frogs (Storey and Storey, 1988). Amounts ranging up to 2 M or more are accumulated, the amount of cryoprotectant produced by different species being greater the lower the freezing temperature that is experienced naturally. The purpose of colhgative cryoprotectants is to prevent cell volume from dropping below a critical minimum during the cell shrinkage that is associated with the growth of ice in extracellular spaces. Previous studies have failed to indicate the accumulation of cryoprotectants in marine molluscs in amounts great enough to be of use in the colligative defense of cell volume during freezing (Loomis, 1987; Storey and Storey, 1988). The present study also showed a lack of cryoprotectant accumulation as a direct response to freezing; glucose levels increased slightly (Table 2) but compounds such as glycerol and sorbitol were not produced and the total amino acid pool of foot muscle remained constant during freezing (Table 1). However, the osmolality of the body fluids of marine molluscs is high (isosmotic with seawater at about lOOOmOsmol), and the concentration of the free

62 amino

Thomas A. Churchill and Kenneth B. Storey acid pool

in L. littorea is about

IOOmM.

Hence, the normal osmolality of cells may be sufficiently high to maintain the critical minimum cell volume during typically amounts

freezing

encountered of colligative

not accumulated

at the subzero

temperatures

in nature. Whereas large cryoprotectants are obviously

by marine molluscs during freezing,

other cryoprotective actions may be accomplished by lower levels of various metabolites including amino acids and anaerobic endproducts. olites such as trehalose and proline stabilizing freezing

the bilayer structure whereas

other

Certain metabare important in

of membranes

metabolites

may

during stabilize

protein structure at low water activities (Rudolph and Crowe, 1985; Crowe et a/.. 1987). Loomis et al. (1988), studying

the marine

mussel

Mytilus edulis.

showed that taurine was the major component

of the

hemolymph fractions that were able to counteract the freezing-induced fusion of liposomes. Thus, this compound,

at 74 mM in L. Iittorea foot

which is present

muscle (Table I), could have a natural cryoprotectant role with no need to elevate its concentration during freezing

exposure.

Further

studies

that an-

Changes in the levels of glycolytic foot muscle also indicate a difference

intermediates in in the metabolic

response to freezing vs anoxia by L. littorea (Table 2). An activation of glycogenolysis was indicated during both stresses

by the elevation

24 h. However, reversed

under

of G6P levels seen at

anoxic

by 72 h (consistent

and was never accompanied

conditions

this was

with metabolic by a significant

arrest) increase

in glucose levels whereas in frozen muscle G6P remained elevated at 72 h and glucose accumulated. There was little effect of freezing

or anoxia

on the

levels of F6P and F1.6P1, the substrate and product of phosphofructokinase, suggesting that the enzyme is not a major

regulatory

locus for the control

of

glycolysis under these situations. Both stresses stimulated changes in PEP and pyruvate, however, implicating

regulatory

control

at the

pyruvate

kinase

locus. PEP levels had dropped, with no change in pyruvate, by 12 h of freezing and remained reduced at 24 h; similarly

PEP levels were depressed

24 and 72 h anoxic snails. This observation

in both suggests

alanine, lactate, also prevent the

an activation of glycolytic flux, with regulatory control at pyruvate kinase, and is consistent with the

vesicles during freezing or

accumulation of D-lactate as an end product. The regulatory role of pyruvate kinase in glycolytic control during anoxia in marine molluscs has been well

aerobic end products such as alanopine, and propionate could fusion of small unilamellar

showed

(Storey et al..

20%) of the net alanine accumulation 1982).

protect enzymes in dilute solution during freeze-thaw (Loomis et al., 1989). Membrane effects were optimal at 2@40mM levels of these compounds whereas stabilization of enzyme activity required much higher concentrations. Succinate was ineffective in mem-

established

brane stabilization

suggests that cytosolic routes for pyruvate disposition (D-lactate, alanopine) had become limiting and may

but relatively

good at protecting

enzyme activity. These compounds cryoprotective role during freezing the levels accumulated

naturally

much lower than the amounts protection of macromolecules Loomis explain

may also have a in rliz>o,although

by L. littorea were needed for effective in vitro. However,

et al. (1989) suggested that this effect could the observed enhancement of freezing sur-

vival by pre-exposure of animals to anoxic conditions (Murphy, 1983). Imino acids including alanopine, and to lesser extents octopine and strombine, were among the anaerobic end products that showed cryoprotective effects (Loomis et a/., 1989) but none of these were quantified in the present study. Only one of these has potential importance for L. littoreu since foot muscle has only D-lactate dehydrogenase and

(reviewed

by Storey,

1992). With longer

term freezing (72 h), however, PEP increased and, in addition, pyruvate levels increased.

promote

the alternative

again This

route of PEP catabolism,

via

PEP carboxykinase, that feeds PEP into the pathway of succinate synthesis. Figure stresses muscle. occurred

2 shows

that neither

freezing

nor anoxia

had major effects on the energetics A small increase in muscle ADP

of foot content

under both stresses but this was not enough

to significantly reduce energy charge. By contrast, during anoxia at 12’C, foot muscle ATP content decreased by 50% after 24 h (Storey et al.. 1982). It is obvious, then, that at low temperatures, metabolic rates in this species are so low and/or metabolic arrest is so powerful, that cellular energetics can be maintained at a high and constant value over at least 3

alanopine dehydrogenase (Plaxton and Storey, 1982). However, it is unlikely that alanopine could accumulate in frozen muscle in any significant amount for

days of freezing

two reasons: (I) levels of t,-alanine, the co-substrate (with pyruvate) of alanopine formation, did not change during freezing in viva (Fig. l), and (2)

Acknowledgemenrs-Thanks to J. M. Storey for critical commentary on the manuscript. Supported by operating grants from the National Institutes of Health (GM 43796). U.S.A. and the Natural Sciences and Engineering Research Council of Canada to K.B.S. and an N.S.E.R.C. postgraduate scholarship to T.A.C.

alanopine accumulation, when it occurred anoxia in L. littorea, was only a fraction

during (about

or anoxia.

Metabolic responses to freezing vs anoxia REFERENCES

Aarset A. V. (1982) Freezing tolerance in intertidal invertebrates (a review). Comp. Biochem. Physiol. A 73, 571-580.

Aunaas T. (1982) Nucleating agents in the haemolymph of intertidal invertebrates tolerant to freezing. Experientia 38, 1456-1457.

Churchill T. A. and Storey K. B. (1989) Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect, Epiblema scudderiana. J. Comp. Physiol. B 159, 461472. Crowe J. H., Crowe L. M., Carpenter J. F. and Wistrom C. A. (1987) Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242, l-10. Gilles R. (1979) Mechanisms of Osmoregulation in Animals. Wiley-Interscience, New York. Hayes D. R. and Loomis S. H. (1985) Evidence for a proteinaceous ice nucleator in the hemolymph of the pulmonate gastropod, Melampus bidentatus. Cryo-Lett. 6, 418421.

Kanwisher J. W. (1959) Histology and metabolism of frozen intertidal animals. Biol. Bull. 116, 258-264. Klutymans J. H., De Bont A. M. T., Kruitwagen E. C. J., Ravestein H. J. L. and Veenhof P. R. (1983) Anaerobic capacities and anaerobic energy production of some Mediterranean bivalves. Comp. Biochem. Physiol. B 75, 171-179.

Loomis S. H. (1987) Freezing in intertidal invertebrates: an update. Cryo-Lett. 8, 186-195. Loomis S. H., Carpenter J. F. and Crowe J. H. (1988) Identification of strombine and taurine as cryoprotectants in the intertidal bivalve Mytilus edulis. Biochim. Biophys. Acta 943, 113-118. Loomis S. H., Carpenter J. F., Anchordoguy T. J., Crowe J. H. and Branchini B. (1989) Cryoprotective capacity of endproducts of anaerobic metabolism. J. Exp. Zool. 252, 9-15. Madison D. L., Scrofano M. M., Ireland R. C. and Loomis S. H. (1991) Purification and partial characterization of an ice nucleator protein from the intertidal gastropod, Melampus biakntatus. Cryobiology 28, 483490.

Murphy D. J. and Johnson L. C. (1980) Physical and temporal factors influencing the freezing tolerance of the marine snail, Littorina littorea (L.). Biol. Bull. 158, 22G232.

63

Murphy D. 1. and Pierce S. K. (1975) The physiological basis for changes in the freezing tolerance of intertidal molluscs. I. Responses to subfreezing temperatures and the influence of salinity and temperature acclimation. J. Exp. Zool. 193, 313-322. Murphy D. J. (1977) Metabolic and tissue solute changes associated with changes in the freezing tolerance of the bivalve mollusc, Modiolus a’emissus. J. Exp. Biol. 69, 1-12. Murphy D. J. (1979) A comparative study of the freezing tolerances of the marine snails Littorina littorea (L.) and Nassarius obsoletus (Say). Physiol. Zool. 52, 219-230. Murphy D. J. (1983) Freezing resistance in intertidal invertebrates. Ann. Rev. Physiol. 45, 289-299. Plaxton W. C. and Storey K. B. (1982) Alanopine dehydrogenase: purification and characterization of the enzyme from Littorina littorea foot muscle. J. Comp. Physiol. 149, 5764.

Rudolph A. S. and Crowe J. H. (1985) Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline. Cryobiology 2, 367-377. Storey K. B. (1992) Molecular mechanisms of metabolic arrest in mollusks. In Surviving Hypoxia (Edited by Hochachka P. W., Lutz P. L., Sick T., Rosenthal M. and van den Thillart G.), pp. 253-269. CRC Press, Boca Raton. Storey K. B. and Storey J. M. (1988) Freeze tolerance in animals. Physiol. Rev. 68, 27-84. Storey K. B. and Storey J. M. (1990) Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation. Quart. Reu. Biol. 65, 145-174. Storey K. B., Miller D. C., Plaxton W. C. and Storey J. M. (1982) Gas-Liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates. Analyt. Biochem. 125, 5&58. Wieser W. (1980) Metabolic end products in three species of marine gastropods. J. Mar. Biol. Assoc. U.K. 60, 175-180. Williams R. J. (1970) Freezing tolerance in Mytilus edulis. Comp. Biochem. Physiol. 35, 145-161.

de Zwaan A. (1983) Carbohydrate metabolism in bivalves. In The Mollusca (Edited by Wilbur K. M.), Vol. 1, pp. 137-175. Academic Press, New York. de Zwaan A., Cortesi P., van den Thillart G., Roos J. and Storey K. B. (1991) Differential sensitivities to hypoxia by two anoxia-tolerant marine molluscs: a biochemical analysis. Mar. Biol. 111, 343-351.