Adaptations to temperature in the cellular membranes of crustacea: membrane structure and metabolism

J. therm. Biol. Vol. 15, No. 1, pp. 1-8, 1990

Printed in Great Britain. All rights reserved

0306-4565/90 $3.00 + 0.00 Copyright (~ 1990PergamonPress plc

ADAPTATIONS TO TEMPERATURE IN THE CELLULAR MEMBRANES OF CRUSTACEA: MEMBRANE STRUCTURE A N D METABOLISM NANCY L. PRUITT Department of Biology, Colgate University, Hamilton, NY 13346, U.S.A. Abstract--1. Acute changes in environmental temperature have deleterious effects on the structure and

function of cellular membranes of crustaceans, as well as other poikilothermic animals. Low temperature increases membrane order, and decreases "fluidity", in addition to influencing the activity of membranebound enzymes. 2. Crustaceans adapt to prolonged exposure to low temperatures by increasing the fluidity of membrane lipids, which serves to restore membrane function. 3. Membrane fluidity is regulated by changing the proportions of saturated and unsaturated fatty acids on membrane phosphatides at different temperatures. Unsaturation increases at low temperatures. 4. The proportion of different phospholipid head groups also changes in response to temperature, particularly among winter-active crustaceans, Ethanolamine phosphatides increase at the expense of choline phosphatides at low temperatures. 5. The experimental evidence for various metabolic mechanisms of membrane restructuring in crustaceans is discussed. Key Word Index: Crustacea; membrane; fluidity; unsaturation; phospholipids; homeoviscous adaptation

INTRODUCTION

Physiological adaptations to changes in environmental temperature play a major role in the life history of most crustacean species. Whilst many, if not all, physiological functions show some degree of compensation for the disruptive effects of thermal fluctuation, few are as widespread or as well-documented as those involving the structure and function of cellular membranes. My objectives are to summarize some of the established adaptations of membrane fluidity and function in crustaceans, and to review briefly the proposed biochemical and metabolic mechanisms that account for compensatory membrane function under different thermal regimes, Temperature and cell membranes

Many of the detrimental effects of changes in temperature on the physiology of crustaceans have been attributed to acute perturbations of the structure and function of cellular membranes. Membranes are responsible for maintaining the uniqueness of the intracellular milieu, both by providing a barrier to diffusion between the intra- and extracellular compartments, and as the functional microenvironment for enzymes, receptors, and other cell-surface markers that establish and maintain that milieu. Changes in temperature impact both the permeability of cell membranes, and the activity of integral membrane proteins, presumably via changes in the physical characteristics of the lipid bilayer. These characteristics encompass both the phase state (e.g. liquid-crystalline or gel) of the hydrophobic portion of the bilayer (or, in the case of phase separations, domains within the bilayer), and the degree of lateral diffusion, intramolecular motion, and intermolecular packing of membrane constituents within a phase. It

is generally assumed that, for a given set of membrane constituents, there is an optimal range of temperatures within which suitable molecular interactions (either lipid-lipid or lipid-protein) occur for proper membrane structure and function, and that this range is above the critical phase transition temperature of the bilayer. Nowhere is the maintenance of membrane function more critical than in excitable cells. Heat death in the crayfish Astacus is associated with a dramatic increase in the permeability of excitable membranes to cations (Bowler et al., 1973; Gladwell et al., 1975). Even within a physiological range of temperatures (10-32°C), passive membrane conductance and membrane potential is temperature dependent. Potential, for example, increases by 0.3 mV/°C in the stretch receptor neuron of Astacus (Moser et al., 1979). Both the conductance of the lipid bilayer and the stability and activity of lipoprotein complexes (Na ÷, K ÷ATPase) are implicated. Studies on artificial bilayers of homogeneous phospholipid composition indicate that cation permeabilities increase dramatically at the gel/liquid crystalline phase transition temperature (Singer, 1981). In some crustacean species taken directly from their natural habitat, the temperature of onset of membrane phase separations coincides with the lowest temperature in the ambient range (Farkas et al., 1984, 1988), implying that, in the absence of physiological adaptation, relatively minor decreases in environmental temperature could present permeability problems for these species. Adaptations in membrane fluidity

A growing body of literature suggests that poikilotherms in general and crustaceans in particular are capable of regulating membrane fluidity in the face of

2

NANCY L. PRUITT

changing environmental temperatures. This phenomenon, first described in detail in E. coli, has been termed "homeoviscous adaptation" (Sinensky, 1974). The physical properties of crustacean membrane lipids have been measured using a variety of techniques including fluorescence polarization (Georgescauld et al., 1979; Farkas et al., 1988) and electron spin resonance spectroscopy (Farkas et al., 1984), which indirectly measure the extent of lateral diffusion within the bilayer, and intramolecular mobility, respectively, and differential scanning calorimetry (Sanina et al., 1987), a technique that identifies the temperatures and breadth of phase transitions based on their endothermic characteristics. Van't Hoff's curves relating temperature to the fluorescence polarization parameter, P, in phospholipids isolated from copepods demonstrate that membrane components from animals collected in temperate seas have more lateral mobility than those from tropical seas at all temperatures of measurement. Lateral mobility is greatest when lipids are isolated from copepods collected in the spring. Breaks in the P vs T curves that convey the onset of phase separation, i.e. the formation of rigid "domains" of phospholipids in an otherwise liquid-crystalline bilayer, occur at higher temperatures in tropical copepods (Farkas et al., 1988). In addition, phase separation temperatures corresponded quite closely to the temperatures at which the copepods were collected. The capacity for adjusting membrane fluidity to prevailing thermal conditions is most evident in crustacean species that remain active at low temperatures and, in fact, the inability to do so may limit an animal's thermal range of activity. Sanina et al. (1987) measured the thermotropic properties of phosphatidylcholine (PC) from eight species of marine invertebrates collected in winter. The area of thermograms corresponding to temperatures above the crystalline phase directly correlated to the level of physiological activity of the species in question. More active species had a larger proportion of liquidcrystalline PC, and less active species had larger zones of crystalline PC. Planktonic crustaceans include both winter-active copepods and sympatric, winterdormant cladocerans. The molecular order of phospholipids from both subclasses, measured via electron spin resonance parameter (S), is very similar when animals are grown at 2 0 (Farkas et aL, 1984), and breaks in the S vs T curves, corresponding to the onset of phase separations, are nearly identical. Following a shift in growth temperature to 10'C, the winter-active copepods increase the overall fluidity of phospholipids, and lower the temperatures of both the onset and completion of phase separation by an amount exactly corresponding to the magnitude of the temperature shift. The winter-passive cladocerans do neither. Adaptations in membrane function The functional correlates of homeoviscous adaptation are well documented in poikilothermic species, particularly (but not exclusively) in excitable cells (Lagerspetz, 1980). Indeed, crustaceans have frequently been used as the model system in studies of thermal adaptation of neuromuscular function. In the crayfish, Astacus. acclimated to either 12 or 25°C,

resting membrane potential (Vm) of the closer muscles of walking legs is linearly correlated with temperature (Harri and Florey, 1979). Breaks in the Vmvs temperature curve occur at 17 and 24°C in coldand warm-acclimated crayfish, respectively, indicating that compensatory shifts in functional transition temperatures occur with thermal acclimation. In addition, the optimal temperature ranges of both excitatory junction potentials and facilitation at the neuromuscular junction are shifted toward the temperature of acclimation, as is the temperature at which maximal tension can be developed in the muscle itself. These authors suggest that homeoviscous adaptation of membrane phospholipids may account for acclimation of neuromuscular electrical parameters. Adaptations to temperature in membrane phenomena of excitable cells may be due to the compensatory changes in permeability properties of the phospholipids, or in interactions between lipids and integral membrane proteins, although recent data support the latter. The thermal stability of the Ca 2+-ATPase from crayfish sarcoplasmic reticulum is highly dependent upon the presence of membrane phospholipids (Volmer and Veltel, 1985), and reconstitution of delipidated ATPase with saturated phosphatidylcholine yields a lipoprotein with higher thermostability than does reconstitution with a more fluid, unsaturated PC. Thermal stability of membrane lipoproteins, and consequently membrane function, is clearly affected by the conformation of the lipids in the bilayer microenvironment, and adaptations in fluidity result in functional conservation. A decreased resistance to high lethal temperatures in cold-acclimated Austropotamobius at 18h photoperiod is accompanied by an increase in the proportion of unsaturated fatty acids in muscle phospholipids (Cossins, 1976), a nearly ubiquitous adaptation to low temperature that accounts, in large part, for homeoviscous adaptation (refer to "Mechanisms of homeoviscous adaptation--fatty acids", next section). Acclimation to a short-day photoperiod increases the unsaturation of lipids from coldacclimated crayfish to an even greater extent, but has no effect on resistance adaptation, implying that fatty acid composition and resistance adaptation are not directly related. Cossins (1976) postulates that resistance adaptation involves the thermal sensitivity of a membrane-bound protein factor and not the integrity of the bulk lipid bilayer. Viscotropic adaptation of membrane function to temperature is well documented in non-excitable cells, as well, particularly in the membrane-bound enzymes of energy metabolism and in Na ÷, K ÷ATPase, but most of these studies have focused on either non-crustacean poikilotherms, or unicellular organisms (for review, see Hazel, 1973). Findings among crustaceans are consistent with the notion that membrane function is conserved via adjustments in membrane composition, and thus, fluidity. In crayfish, for example, the activity of cytochrome c oxidase is dependent upon the presence of the phospholipids in its inner-mitochondrial membrane milieu for activity, and the composition of mitochondrial lipids depends upon the thermal history of the crayfish. Reconstitution of the delipidated enzyme

Membrane adaptations in crustacea 10

T ~

8 0

.]E

[]

o

0

Lipid

donor

o°,°°o°o4 • o,,° ,o44 ....

.X.','~

5 ° acclimated 20 ° acclimated

,°,..°%d

:+:':'1

6

L*

~g

,.-.,,.. • ,%% .-, , *,,.° • °.,,°.%° • .°.,. *, %%'.%,, • ....,

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o

',*.'°'.'2 ,.,°° ,.,,, .... °.*°,

> 0

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I NO LIPID

TPL

'.'.'.'.'.'

I

CL

Fig. I. Activity of cytochrome c oxidase from hepatopancreas of the crayfish, Cambarus bartoni. Velocities are means + SEM of n = 4 animals. Activities are measured in lipid-depleted preparations of the enzyme (acetone extracts; NO LIPID) and in enzyme preparations reconstituted with lipid fractions: TPL = enzyme reconstituted with the total phospholipid extract from donor crayfish; CL = enzyme reconstituted with the cardiolipin fraction from donor crayfish. The enzyme was isolated from crayfish acclimated to 5"C. Asterisks indicate a statistically significant difference (P ~<0.05, t-test) from the lipid-depleted enzyme. with phospholipids (especially cardiolipin) isolated from the mitochondria of a cold-acclimated conspecific results in higher levels of cytochrome c oxidase activity than when the source of lipid is a warm-acclimated donor, regardless of the source of the enzyme protein (Fig. 1; Hodge and Neas, 1985; Neas and Hodge, 1985). Changes in lipid composition influence the rates of lipid-associated enzymes in an adaptive manner. Functional adaptations in crustacean membranes are reflected in gross physiological processes as well. The permeability of gill membranes to water in Gammarus duebeni is perfectly conserved following cold-acclimation, and is accompanied by an increase in the proportions of monoenoic and polyunsaturated fatty acids at the expense of saturates in gill phospholipids; the phospholipids of non-gill tissues in this species do not change upon acclimation (Dawson et al., 1984). Increased cold-tolerance in specimens of the barnacle Balanus balanoides collected in winter is accompanied by increased iodine number of whole body lipids (Cook and Gabbot, 1972). Indeed, the ability to maintain any level of activity with the onset of winter is greatest in species that can modify the composition of cellular membrane lipids in an adaptive manner, a phenomenon documented both in crayfish (Pruitt, 1988) and planktonic crustaceans (Farkas, 1979; Farkas et al., 1984). Mechanisms o f homeoviscous adaptation--fatty acids

The major determinant of phase state and fluidity of the hydrophobic portion of cellular membranes at a given temperature is the fatty acid composition of the phospholipids comprising the bilayer. Although the capillary melting points of phospholipids are

sufficiently high to be of. little or no physiological consequence (in the range of 200-300°C), the melting points of many fatty acids that are abundant in phospholipids fall within the range of environmental temperatures normally encountered by poikilothermic organisms. Membranes have been observed to undergo a distinct change in phase at temperatures corresponding to melting (or freezing) of the hydrophobic core at physiological temperatures. The temperature at which membranes change from liquid-crystal to hydrated gel upon cooling is termed the transition temperature (T~) to distinguish it from the molecular melting point of phospholipids. In artificial bilayers of homogeneous fatty acid composition, T¢ is a discrete temperature characteristic of the molecular composition; in biological membranes, T~ reflects the heterogeneous nature of membrane composition, and is usually characterized by a broad endothermic range limited by temperatures that represent the onset and completion of phase transitions of the individual fatty acid components. Membranes comprising large proportions of unsaturated acids have lower Tcs than those with saturated fatty acids, due to the lower melting points of unsaturated acids. The presence of cis carbon-to-carbon double bonds in unsaturated fatty acids introduces kinks in the linear structure of the fatty acid, and consequently disrupts the molecular packing of phospholipid molecules, increases the area of the phospholipid bilayer, decreases the membrane thickness, increases the lateral mobility of membrane constituents, and lowers the T~ (Brenner, 1984). One widespread strategy among poikilotherms for achieving homeoviscous adaptation is the substitution of more unsaturated fatty acy| moieties for saturated ones in membrane phosphatides at low temperatures, and crustaceans do it extensively. Antarctic (Clarke, 1977; Meyer-Rochow and Pyle, 1980) species are enriched in polyunsaturated fatty acids relative to temperate (Holz, 1974; Gastaud, 1977) and tropical species (Farkas et al., 1988), and among temperate zone species, the lipids of animals collected in winter are generally more unsaturated than those from warmer waters of summer (Saether et al., 1986; Tooke et al., 1985b; Guary et al., 1975; Morris, 1971; Herodek, 1969). Even in the Antarctic, the onset of winter increases the degree of unsaturation of both phospholipids and triglycerides in the prawn (Clarke, 1977). Some of these adaptations in situ may reflect differences in the fatty acid content of the diet; i.e. passive accumulations of fatty acids of appropriate physical properties for the prevailing conditions from phytoplanktonic food organisms who themselves undergo an accumulation of unsaturates in the cold (Saether et al., 1986). Indeed, it is postulated that the cladoceran, Moina, is entirely dependent upon diet for its source of fatty acids; and Moina grows at a higher rate at low temperatures when the food includes unsaturated fatty acids (D'Abramo, 1979). The fatty acid content of the diet can influence both the triglyceride and phospholipid composition of crustaceans (Saether et al., 1986). However, laboratory acclimation studies of various species indicate that low temperature alone can increase the degree of membrane unsaturation in crayfish (Cossins, 1976; Farkas and Nevenzel, 1981;

NANCY L. PRU1TT Table 1. Fatty acid composition (weight percent) of phospholipids from thermally acclimated crustaceans Saturates Monoene PUFA* U/S§ Acc. temp. Animal Tissue* cold/warm CA5" WA CA WA CA WA CA WA Source Austropotamobius m 4,/25 18.4 22.6 27.3 26.1 51.3 47.6 4.4 3.4 Cossins, 1976 Procambarus h 4/23 20.7 25.5 17.1 16.0 42,5 37.7 3.0 2.1 Farkasand Nevenzel, 1981 Cambarus (winter-active) h 5/20 18.7 21.6 28.4 29.0 41.3 37.9 3.7 3.1 Pruitt, 1988 Oreonectes (winter-quiescent) h 5/20 18.9 24.4 20.4 30.2 49.6 42.1 3.7 3.0 Pruitt, 1988 Carcinus m 7/27 18.2 20.8 26.9 38.6 53.6 40.3 4.4 3,8 Chapelle, 1978 Careinus g 7/27 20.0 27.4 31.9 36.8 48.9 36,2 4.0 2.7 Chapelle,1978 Gammarus g 5/15 18.3 22.6 30.1 44.3 51.4 31,4 4.5 3.4 Dawsonet al., 1984 Gammarus wb 5/15 28.9 23.8 33.1 27.6 37.9 48.5 2.5 3.2 Dawsonet al., 1984 *m = muscle; h = hepatopancreas; g = gill; wb = whole body (excluding gills). tCA = cold-acclimated; WA = warm-acclimated. ~PUFA = polyunsaturated fatty acids. §U/S = weight percent of monoenes + polyunsaturated fatty acids/weight percent of saturates. Pruitt, 1988), crabs (Chapelle, 1978), barnacles (Cook and Gabott, 1972), and gill tissue of amphipods (Dawson et al., 1984), among others, when different acclimation groups are either starved or fed identical diets. The results of some of these studies are summarized in Table 1. In all species and tissues that undergo an increase in U/S (weight percent of monoenes + polyunsaturated fatty acids/weights percent of saturates), cold acclimation increases the proportions of polyunsaturated fatty acids at the apparent expense of saturates. Monoenes may decrease in abundance at low temperatures, or remain unchanged, depending upon the species and tissue in question, but, unlike some fish (the green sunfish, for example; Cossins et al. 1980), an accumulation of monoenes does not occur at low temperatures in crustacean phospholipids (with the exception of the winterdormant Daphnia; Farkas et al., 1981). In north Atlantic krill, species of both Euphausia and Thysanoessa (Saether et al., 1986), that cannot, or do not, accumulate polyunsaturates at extremely low sea temperatures, compensate by increasing the proportions of monounsaturated fatty acids in wax esters. Kriil that accumulate neither polyunsaturated phosphatides nor monoenoic wax esters, Meganyctiphanes norvegica, do not spawn in the same cold Norwegian fjords as Thysanoessa (Saether et al., 1986). Some evidence indicates that the phospholipid fatty acids of crayfish (Cossins, 1976) and marine mysids (Morris, 1971) are sensitive to changes in photoperiod in the absence of thermal fluctuation. Mysids begin accumulating polyenoic acids in late summer and early fall, before the temperature of the water begins to drop. Cossins (1976) reports an enrichment of unsaturates in crayfish phospholipids exposed to 8 h days at 4 ' C over that of 4°C-acclimated crayfish exposed to 18h days.

Previously unpublished data from our laboratory, however, show no influence of photoperiod on phospholipid fatty acid composition of 20°C-acclimated crayfish, regardless of whether the species in question spends the winter in an active state or a quiescent one (Table 2). Photoperiod effects on fatty acid composition appear to be somewhat contradictory, and may be quite species-specific, or only apparent at low temperatures. Membrane phospholipids comprise about six major classes that differ in the nature of their hydrophilic head groups. The distribution of fatty acids among the various phospholipid classes is not random, and the extent to which the fatty acid composition of different classes changes in response to temperature varies with class. The most abundant classes, choline phosphoglycerides (PC) and ethanolamine phosphoglycerides (PE), tend to be the most highly unsaturated classes, and PE is generally more unsaturated than PC in most crustacean tissues (Table 3). These phosphatides also tend to undergo the greatest degree of fatty acid substitution with temperature, a point illustrated by the higher magnitude of differences in the U/S ratios between warmand cold-acclimated conspecifics. Of studies summarized in Table 3, the average increase in U/S of PE with cold-acclimation is 2.2; in PC, 1.5; in sphingomyelin, 1.2; and in serine and inositol phosphoglycerides, 0.4. Very few generalizations about crustaceans can be made from the manner in which these reductions in U/S are accomplished within a lipid class. For example, in two species of crayfish, Austropotamobius pallipes and Orconectes propinquus, ethanolamine phosphoglycerides at low temperatures are greatly depleted of saturated fatty acids (Cossins, 1976; Pruitt, 1988), whereas in another species, Cambarus bartoni, the level of saturates in PE

Table 2. The effects of photoperiod on the fatty acid composition of crayfish Orconectes propinquus Cambarus bartoni (winter-quiescent) (winter-active) 8L:I6D 16L:8D 8L:I6D 16L:8D Saturates 28.06 (3.26) 26.14 (2.28) 22.61 (0.40) 26.32 (I. 14) Monoenes 31.13 (1.44) 29.74 (3.36) 32.60 (1.03) 29.08 (2.44) n-6 26.93(0.73) 28.00(1.31) 25.14(2.54) 25.50(2.23) n-3 6.71 (0.22) 8.97(1.25) 7.92(0.42) 8.99(I.51) U/S 2.41 (0.28) 2.63 (0.31) 2.90 (0.27) 2.44 (0.21) n 4 4 4 4 Values are mean (SEM) weight percent. Crayfish were acclimated to 20'C

Membrane adaptations in crustacea

5

Table 3. Unsaturation/saturation*ratios of fatty acids from individualphospholipidclasses of laboratory acclimatedcrustaceans PC + PE Serine/lnosotol¶/ SM Acc. temp. Animal Tissue:~ cold/warm CAt WA CA WA CA WA CA WA Source Austropotamobius m 4/25 3.3 2.6 15.6 7.8 2.5 1.7 --Cossins,1976 Cambarus (winter-active) h 5/20 5.7 3.4 5.9 5.0 1.9 1.9 3.7 2.4 Pruitt,1988 Orconectes (winter-quiescent) h 5/20 5.7 2.3 5.0 3.3 2.0 1.7 2.5 1.5 Pruitt,1988 Carcinus m 7/27 3.0 2.4 5.0 4.7 . . . . Chapelle,1978 Carcinus g 7/27 3.7 3.1 2.9 2.6 . . . . Chapelle,1978 *U/S = weight percent of monoenes+ polyunsaturatedfatty acids/weightpercent of saturates. tCA = cold-acclimated;WA = warm-acclimated. :~m= muscle; h = hepatopancreas; g = gill. §PC = phosphotidylcholine;PE = phosphatidylethanolamine;SM = sphingomyelin. ¶ISerine/Inositolis summeddata for serineand inositolphosphoglycerides. is independent of thermal history. PC from coldacclimated crayfish becomes consistently more unsaturated, but shows only minor changes with cold-acclimation in the crab (Cossins, 1976; Pruitt, 1988; Chapelle, 1978). Tooke and Holland (1985a, b) examined seasonal changes in fatty acid content of different phospholipid classes from two species of barnacles that differ in their ability to acquire coldtolerance with the onset of winter in an attempt to relate cold-tolerance to membrane composition. The fatty acid composition of most major phosphatides was similar between the two species, and differed very little between specimens caught in summer and winter. The one major exception was sphingomyelin (SM), a phosphatide largely restricted in its intracellular distribution to the plasma membrane. The degree of unsaturation of this lipid was slightly greater in winter (vs summer) in the cold-tolerant species, Balanus balanoides, but significantly more saturated in winter (vs summer) in the cold-susceptible species, Elminius modestus. These authors postulate that this single phosphatide may play a major role in cold-tolerance in barnacles. Species with different overwintering strategies clearly have different capacities for retailoring the fatty acid composition of membrane phosphatides. Crustaceans fall into three main categories: (A) species that are active only in summer and spend the winter either quiescent or in resting stages, (B) species that are active only in winter and spend the summer in resting stages, and (CA species that remain active year-round. Among the planktonic crustaceans, Farkas (1979) found that species in category B have significant accumulations of long chain polyunsaturated fatty acids, predominantly of the n-3 family, in the total lipid fraction relative to species in category C. Daphnia magna, a passively-overwintering cladoceran (category A), shows no increase in overall unsaturation at low temperature (Farkas et al., 1984), and is incapable of incorporating very long chain acids (22:6n-3) into phospholipids. Crayfish in categories A and C are both capable of increasing total membrane unsaturation, but the former group do so to a greater extent than the latter (Pruitt, 1988). Several metabolic mechanisms could account for the accumulation of unsaturates in phospholipids at low temperatures but, among the crustaceans, most studies have focused on the biosynthetic step of fatty acid metabolism. Cold exposure reduces the rate of l-[~4C]acetate incorporation into fatty acids by a factor of 2 in the crayfish, Procambarus (Farkas et al., 1981), and by a factor of 4 in Daphnia and Cyclops

(Farkas et al., 1981), but all three species synthesize more unsaturated than saturated acids at low temperatures. Both intact animals and hepatopancreas slices of Procambarus incorporate more label from acetate into polyunsaturates at 5°C (27-38%) than at 23°C (12-14%). At 23°C, 73-80% of label from acetate is found in saturates, whereas only 51-73% is found in polyunsaturates. Daphnia, which is apparently incapable of synthesizing long chain polyenoic acids, preferentially incorporates labelled acetate into monoenes (18:1) following a shift in temperature from 25 to 5°C. Cyclops, exposed to the same thermal regime, directs radiolabel into both monoenes and docosahexeonic acid (22: 6n-3). In non-crustacean poikilotherms, the retailoring of existing membrane phospholipids via a deacylation-reacylation pathway has been implicated as a possible mechanism for fatty acid substitution. Either step of the cycle may be influenced by temperature in an adaptive manner: Low temperature favours the removal of more saturated fatty acids via phospholipases (Neas and Hazel, 1984) and the insertion of more unsaturated acids via acyltransferases (Hazel et al., 1987). Circumstantial evidence indicates that this pathway may function in crustaceans as well. Chapelle et al. (1977) have reported an increase in the synthesis of lysophosphatidylcholine relative to PC at low ambient temperatures. No data on the substrate specificity of either crustacean phospholipases or acyltransferases are reported. Bottino et al. (1980) observed a lag in time of 2 months between a decrease in the environmental water temperature and the corresponding change in the degree of unsaturation of fatty acids in three species of shrimp from the Gulf of Mexico. They interpreted this to mean that changes effected through the food chain of the shrimp predominate over endogenous adjustments in metabolism, but their studies did not distinguish between membrane phospholipids, where homeoviscous adaptation would be most critical, and depot lipids. Mechanisms o f homeoviscous adaptation--phospholipids The physical properties of a phospholipid bilayer are dependent upon the proportions of its constituent molecules and their geometries, and phospholipid geometry is a function of both the acyl chain composition, and the nature of the polar headgroup (for review, see Hazel, 1988). Phospholipids with large, extensively hydrated headgroups, such as phosphatidylcholine, assume cylindrical shapes, and are

6

NANCYL. PRUIT'I" Table 4. Phospholipid class composition of tissues from thermally acclimated crustaceans PC#

Animal

Tissue*

Carcinus g (mito) Carcinus m Carcinus h Carcinus hl Cyclops (winter-active) wb Daphnia (winter-dormant) wb Austropotamobius m Cambarus (winter-active) h Orconectes (winter-quiescent) h

PE

PC/PE

SM

Acc. temp. cold/warm

CA+

WA

CA

WA

CA

WA

CA

WA

Source

13/23 7:27 7/27 13/23 10/20 10/20 4/25 5/20 5/20

44.1 56,6 82.8 74.8 27.7 43.4 54.1 21.8 24.9

48.2 63.5 76.8 77.3 37.5 52.6 55.0 43.6 25.9

25.8 22.8 12.2 14.7 54.8 40.6 26.2 44.6 42.7

21.9 17.6 13.2 12.3 44.9 31.9 22.9 35.2 40.6

1.7 2.5 6.8 5.1 0.5 1.1 2.1 0.5 0.6

2.2 3,6 5.8 6.3 0.8 1.6 2.4 1.2 0.6

9.3 5.3 2.2 7.2 1.2 3.9 4.8 19.5 7.9

10.1 5.2 4.1 7.0 3.1 5.2 6.7 9.3 8.0

Chapelle et al., 1981 Chapelle et al., 1977 Chapelle et al., 1977 Brichon et al., 1980 Farkas et al., 1984 Farkas et al., 1984 Cossins, 1976 Pruitt, 1988 Pruitt, 1988

Values are mol percent of the total phospholipids. *g = gill; m = muscle; h = hepatopanereas; hi = hemolymph; wb = whole body. tPC ~ phosphatidylcholine; PE = phosphatidylethanolamine; SM ~ sphingomyelin. ~CA = cold-acclimated; WA = warm-acclimated.

packed efficiently in the biiayer. The small size of the ethanolamine headgroup relative to the large area occupied by the acyl chains causes ethanolamine phosphatides to assume a conical shape, particularly if the acyl moieties are highly unsaturated and disordered, as is often the case in crustaceans (Table 3). Consequently, bilayers of pure or highly enriched ethanolamine phosphoglycerides are unstable and, in the absence of other phosphatides or polar molecules that stabilize the bilayer geometry, can revert to the configuration of an inverted micelle, the hexagonal lI (Hlj) phase. In heterogeneous bilayers, such as biological membranes, the presence of conical, unsaturated PE has a disordering effect on the bilayer, and may contribute to homeoviscous adaptation. In addition, at physiological pH, PE is anionic and PC is a Zwitterion. Chapelle et al. (1977) suggest that increased proportions of PE relative to PC would increase the intramolecular repulsion, and hence reduce the packing efficiency of the bilayer, rendering tt more fluid. The permeability of a bilayer increases in proportion to the ratio of PE:PC phosphatides (Fast, 1967). Thus, it is expected that modification of phospholipid headgroup composition in response to cold temperatures would involve an increase in PE relative to PC. In some crustaceans this is clearly the case. The relative percent of some of the major phospholipid classes from thermally acclimated crustaceans are listed in Table 4. In general, coldacclimation lowers the PC/PE ratio in tissues of winter-active species, with the exception of hepatopancreas of the crab, Carcinus (Chapelle et al., 1977). In winter-dormant or quiescent species, such as the copepod, Cyclops, or the crayfish, Orconectes, headgroup modification is minimal or absent (Pruitt, 1988; Farkas et al., 1984). Crustaceans synthesize phosphatides using pathways similar to those established in vertebrate systems (Shieh, 1969; Chapelle, 1977). Glycerol-3phosphate is twice acylated via acyltransferase enzymes to phosphatidic acid (PA), the precursor to other phospholipids. PA can either be subsequently dephosphorylated to 1,2-diacylglycerol, which can then be converted either to PC (via CDP-choline phosphotransferase) or to PE (via ethanolamine phosphotransferase), or it can react with CTP to yield CDP-diglyceride from whence it is converted to either serine-, glycerol-, or inositol-phosphoglycerides via CDP-diglyceride transferase in the presence of the appropriate hydrophilic acceptors (for review, see

van den Bosch, 1974). In addition, a conversion of PE to PC can take place via the transfer of methyl groups from S-adenosyl-e-methionine. Much of the work with crustaceans addressing the effects of temperature on phosphatide metabolism has been done by Chapelle and colleagues using the crab, Carcinus meanus. In general, phospholipid synthesis is more rapid at higher temperatures, but that the individual classes are metabolized independently is apparent from the differential effects of temperature on the synthesis and turnover of different lipid classes. Synthesis and turnover of PS, CL, SM and PC is generally increased at warm temperatures relative to cold temperatures, whereas PA turnover is decreased. The turnover of other lipids, i.e. PE and PI, is not as temperature-dependent (Chapelle et al., 1977; Chapelle et al., 1979; Chapelle et al., 1981). In particular, the activity of choline phosphotransferase is far more temperature-sensitive than ethanolamine phosphotransferase (Chapelle et al., 1982). Thus the accumulation of PE at low temperature may reflect the temperature-dependence of the synthesis and turnover rate relative to PC and other lipids. In hepatopancreas tissue, the rate of methylation of PE via S-adenosyl-L-methionine methyltransferase to yield PC is positively correlated with temperature, which may also partially account for the depletion of PE and accumulation of PC (Brichon et al., 1980; Chapelle et al., 1982). Finally, Chapelle et al. (1982) have shown that environmental temperature strongly influences the distribution of PC between haemolymph and tissues in the crab. Thus the rate and extent of blood-tissue exchange of phospholipids may be an important point in the regulation of tissue phospholipid composition. Mechanisms o f homeoviscous adaptation--sterols

Cholesterol is the predominant steroid in the cellular membranes of crustaceans, and can account for between 5 and 12% of the total membrane lipid (Krzynowek et al., 1982; Madiera and AntunesMadiera, 1977; Dawson et al., 1984). Its effect on phospholipid membranes is complex and dependent upon the phase state of the bilayer. In the gel phase the conical geometry of cholesterol, a consequence of the relatively small area occupied by its hydrophilic 3-/3 hydroxyl group, disrupts the packing of phospholipids and broadens the temperature range of the phase transition (Yeagle, 1985). Above the T¢, cholesterol tends to restrict the rotational freedom of acyl

Membrane adaptations in crustacea chains in the fluid hydrophobic domain, and thus further orders the membrane and increases its thickness (Yeagle, 1985). It would be expected, therefore, that long-term exposure to warm temperatures would increase the cholesterol content of membranes but, in the crustaceans studied, that does not appear to be the case. Steroid concentrations differ by < 1% in the lipids of planktonic crustaceans from arctic and temperate waters (Gastaud, 1977). In the gills of the amphipod, Gammarus, acclimated to 5 and 15°C, steroid composition increases from 9 to 12% of the total lipid, respectively, but in the bodies (minus gills) steroids account for 7 and 8%, respectively (Dawson et al. 1984). Cossins (1976) found no significant difference in the phospholipid/cholesterol molar ratio of crayfish acclimated to 4 and 25°C. Bulk membrane fluidity in crustaceans at different temperatures does not appear to be regulated to any large degree by steroids. CONCLUSIONS Complex and diverse combinations of lipids comprise the cellular membranes of organisms, which are highly dynamic and highly regulated structures. Our understanding of the influences of environment on the structure and function of membranes is growing, but yet incomplete. Temperature is among the most pervasive aspects of the physical environment that can influence membrane function, and the study of crustaceans has yielded a wealth of information on the ability of organisms to adjust and accommodate environmental challenges that are natural and recurring such as temperature. Crustacean membrane fluidity and function are compensated in the face of changing temperature, by adjusting the composition of membrane components. The mechanisms for these adjustments are diverse, and include thermally-mediated changes in the synthesis and incorporation of differentially saturated fatty acids, and in the metabolism of phospholipid classes. Mechanisms of homeoviscous adaptation that have been documented in other poikilothermic species, such as alterations in plasmologen levels or in the composition of phospholipid molecular species, are as yet unsubstantiated in crustaceans, leaving the study of crustacean membrane metabolism a fertile field of endeavor. REFERENCES

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