The Role of Lipid Physical Properties in Lipid Barriers1

AMER. ZOOL., 38:268-279 (1998)

The Role of Lipid Physical Properties in Lipid Barriers1

ALLEN G. GIBBS2

Evolutionary and Comparative Physiology Group, Department of Ecology and Evolutionary Biology,University of California, Irvine, California 92697-2525

SYNOPSIS. The hydrophobic nature of lipids means that they provide good bar-riers to the movement of charged and polar molecules. Barrier function appearsto depend on the physical state of the lipids. Two well-investigated examples in-clude cell membranes and epicuticular lipids of arthropods. Ecologically relevantchanges in temperature significantly affect lipid properties, and both evolutionaryand acclimatory differences in lipid composition appear to preserve the physicalproperties of lipids under different environmental conditions. These differences aregenerally believed to be beneficial to the organism, but rigorous examination oftheir adaptive significance is rare. Important issues are how lipid properties areregulated; which properties are physiologically relevant, how are these propertiessensed, and what biochemical and molecular mechanisms regulate lipid properties?Progress has recently been made in understanding how membrane lipid propertiesare regulated, but regulatory mechanisms for cuticular lipids and other lipid sys-tems remain completely unknown.

INTRODUCTION

Lipids comprise those compounds thatare insoluble in aqueous media and solublein non-polar solvents. They are uniqueamong biological molecules in that they aredefined by their physical properties ratherthan by their chemical structure. Unlikeproteins and nucleic acids, which are sim-ply polymers of a limited set of smallermolecules, lipids span a range of chemical-ly distinct forms (Hadley, 1985; Stanley-Samuelson and Nelson, 1993). This diver-sity contributes to the wide range of bio-logical functions in which lipids are in-volved, from major structural elements ofcells to chemical signals within and amongindividuals.

In many cases, physiological functions oflipids appear to depend upon their physicalproperties. The most well-documented ex-amples concern lipids that serve as barriers{e.g., cell membranes, cuticular lipids of ar-thropods). Ecologically relevant changes inenvironmental conditions such as tempera-ture have substantial effects on the physical

1 From the Symposium The Biology of Lipids: In-tegration of Structure and Function presented at theAnnual Meeting of the Society for Integrative andComparative Biology, 26-30 December 1996, Albu-querque, New Mexico.

: E-mail: [email protected]

properties of lipids. Membranes and cutic-ular lipids share the fact that their compo-sition and properties appear to be closelyregulated. The primary focus of this reviewis on the maintenance of lipid-based phys-iological processes in the face of environ-mental stress, although I emphasize at theoutset that much of the evidence for the im-portance of lipid properties is only correl-ative. This situation is beginning to change,as new techniques from molecular and evo-lutionary biology are applied to these sys-tems.

PHYSICAL PROPERTIES OF CELLULARMEMBRANES

Cell membranes are by far the most thor-oughly studied lipid systems. In the pres-ence of water, phospholipids spontaneouslyassemble into ordered bilayer membranesthat are held together by hydrophobic in-teractions between acyl chains (Cevc,1991). The insolubility of lipids in polarsolvents carries the converse implicationthat polar and charged compounds will beinsoluble in lipids. Thus, membrane bilay-ers form excellent barriers to the movementof water and ions into and out of the cell.The flow of molecules and informationacross the cell membrane is regulated bymembrane proteins. An important issue in

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PHYSICAL PROPERTIES OF LIPID BARRIERS 269

membrane biology is the extent to whichchanges in the membrane lipid environmentaffect the function of transport and receptorproteins (Cossins, 1994; Gibbs, 1995). In afew cases, proteins require specific lipids inorder to function (e.g., Robinson and Ca-paldi, 1977). Such specificity is rare, andmost proteins will function to some extentin a wide range of membrane environments.However, it is clear that not all membranesare equal, and that membrane proteins areaffected by the types of lipids around them.

Membrane fluidity and membrane proteinfunction

Physiologically relevant decreases intemperature or increases in pressure makecell membranes less fluid (more viscous;Hazel and Williams, 1990; Cossins, 1994).Studies using purified Na/K-adenosine tri-phosphatase (Na/K-ATPase) in defined lipidenvironments have shown that enzyme ac-tivity is highly correlated with membranefluidity (Chong et al, 1985; Harris, 1985).Similar results have been obtained usingother proteins (Squier et al, 1988). Thus,the effects of temperature on fluidity mayaffect ion transport and other cellular pro-cesses mediated by membrane proteins.

The direct effects of temperature onmembrane fluidity can be offset by chang-ing the composition of the membrane lipids.In most membrane systems studied to date,acclimation to lower temperatures results inincreased fluidity, due primarily to in-creased unsaturation of fatty-acyl chains ofphospholipids (Hazel and Williams, 1990).This response has been termed homeovis-cous adaptation, to emphasize the apparentimportance of maintaining constant mem-brane microviscosity (Sinensky, 1974). Theterm "adaptation" is used here in a broadsense, including both genetic adaptationamong species and phenotypic responses tothe environment within an individual's life-span (Bennett, 1997). The important ques-tion, of course, is whether homeoviscousresponses are in fact adaptive (i.e., benefi-cial to the organism).

Homeoviscous adaptation is the mostwidespread cellular response to temperatureknown, and has been demonstrated in alltaxa studied to date. Even the polycyclic

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Measurement temperature (°C)

FIG. 1. Effects of temperature and thermal acclima-tion on the activity of cytochrome c oxidase from carp.Cytochrome oxidase activity of mitochondrial mem-branes was assayed at 10 and 30°C. Fish were accli-mated to either 10°C (open bars) or 30°C (filled bars).Data from Table 1 in Wodtke (1981).

membrane-spanning ether lipids of ther-mophilic archaebacteria exhibit acclimatoryresponses consistent with homeoviscous ad-aptation (Kaneshiro and Clark, 1995). Ac-cording to homeoviscous theory, increasesin membrane fluidity at low temperatureshould result in increased enzyme catalyticrates and help to compensate for the inhib-itory effects of reduced temperature. How-ever, few studies have tested this hypothesisdirectly, because of difficulties in distin-guishing between changes in the number ofenzyme molecules and the per-moleculeturnover rate. In one such study, acclima-tion to low temperature resulted in in-creased unsaturation and fluidity of mito-chondrial inner membranes from carp(Wodtke, 1981). These changes were cor-related with increased specific activity of aninner membrane enzyme, cytochrome c ox-idase, such that enzyme turnover rates were30-35% higher in individuals acclimated tolow temperature (Fig. 1). These results sup-port the idea that homeoviscous adaptationaffects cytochrome oxidase activity, butother factors (e.g., post-translational modi-fications of the protein) could also havebeen involved (Poly, 1997).

Manipulation of the membrane environ-ment provides a more direct test of the in-fluence of membrane fluidity on enzymeproperties. Trout acclimated to low temper-ature exhibited increase turnover rates oferythrocyte Na/K-ATPase and increasedmembrane fluidity (Raynard and Cossins,

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0 10 20 30 40 50 60 70

Pressure (MPa)

FIG. 2. Effects of pressure on gill Na/K-ATPase ac-tivity in marine fishes. Pressure increases by approxi-mately 1 MPa for each 100 m increase in depth. Theaverage depth of the ocean is -3800 m (Gibbs, 1997),so pressures >10 MPa are typical of deep-sea envi-ronments. Species (common name, habitat depths,temperature range) were: ( • ) Coryphaenoides armatus(rattail, 1,900-4,800 m, 2-4°C), (O) C. acrolepis (rat-tail, 700-1,820 m, 4-8°C), (T) Pohchthys notatus(midshipman, 0-300 m, 6-15°C) and (V) Sphyraenahelleri (barracuda, 0-20 m, 24°C). Data from Gibbsand Somero (1989).

1991). Cholesterol supplementation re-duced both fluidity and Na/K-ATPase activ-ity to an extent similar to thermal accli-mation, suggesting that acclimation ofmembrane fluidity helps to maintain iontransport rates under varying temperatures.

A potential problem with interpretingthermal effects on membrane proteins isthat temperature has large effects on nearlyall biochemical processes. Thus, it can bedifficult to distinguish the direct effects oftemperature on membrane proteins from in-direct effects mediated by membrane lipids.Hydrostatic pressure provides one means ofaltering membrane fluidity without chang-ing temperature. The direct effects of hy-drostatic pressure on membrane propertiesare very similar to those of low tempera-ture: reduced fluidity, increased thicknessetc. Deep-sea organisms tend to have higherlevels of unsaturated lipids that offset themembrane-ordering effects of high pres-sures (Gibbs, 1997). Because phospholipidbilayers are more sensitive to pressure {i.e.,more compressible) than proteins, the ef-fects of pressure on lipid-protein systemsshould be due largely to effects on the lipidcomponent.

Pressure strongly inhibits Na/K-ATPase

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FIG. 3. Effects of lipid substitution on Na/K-ATPasefrom the deep-sea fish, Coryphaenoides armatus. Thesubstitution procedure of Warren et al. (1974) wasused to modify the lipid environment of Na/K-ATPase.Filled circles indicate the effects of pressure on ATP-ase activity in the enzyme's native lipid environment.Native lipids were replaced with: (O) phospholipids(PLs) isolated from C. armatus; (A) PLs from a cold,shallow-living fish, Anoplopoma fimbria; ( 0 ) PLsfrom a warm-living fish, Sphyraena barracuda; (V)chicken egg phosphatidylcholine; and (•) a 1:1 mix-ture of cholesterol and A. fimbria PLs. Data fromGibbs and Somero (1990).

activity (Gibbs and Somero, 1989). Differ-ences among species in pressure responsesare correlated with presumed differences inmembrane fluidity; sodium pump activity isleast affected by pressure in deep-sea spe-cies, and most inhibited in species fromwarm, shallow habitats (Fig. 2). These ef-fects can be altered by changing the mem-brane environment around the protein, us-ing a lipid substitution procedure (Gibbsand Somero, 1990). Sodium pump activityis most inhibited by high pressure when theenzyme is placed in a relatively viscous,more saturated membrane environment, andleast inhibited by pressure in the presenceof relatively fluid lipids isolated from deep-sea and cold-water fishes (Fig. 3). These re-sults demonstrate that the catalytic proper-ties of membrane enzymes can be directlyaffected by the membrane lipid environ-ment, and therefore support the idea thathomeoviscous adaptation is physiologicallyimportant.

Although fluidity has received the mostattention from physiologists, many otherphysical properties of membranes are alsoaffected by the environment. These includegel <-> fluid and bilayer <-» nonbilayer phase

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transitions, membrane thickness, and mo-lecular cross-sectional area of phospholip-ids. Phospholipids in a fluid membranehave a greater molecular surface area dueto increased molecular mobility, and themembrane as a whole will be thinner andwill tend to freeze at lower temperatures.These correlations make distinguishingwhich properties of membranes are impor-tant a difficult experimental problem. Ad-ditionally, membrane properties may differsubstantially within a given membrane,with consequent effects on membrane pro-teins (Post and Klodos, 1996; Williams,1997). At even finer scales, membraneproperties differ according to the depth inthe bilayer, with sections closer to the mid-dle of the bilayer being more fluid and hav-ing reduced lateral pressures (Scarlata,1990; Cantor, 1997). Several recent authorshave challenged the significance of mem-brane viscosity (e.g., Lee, 1991; Zakim etal., 1992; Hazel, 1995), and this remains anactive area of research.

Evolutionary and acclimatory changes inphysical properties of membranes

Organisms adapting over evolutionarytime scales face differences in temperaturesimilar to those experienced by eurythermalorganisms, which need to acclimate to tem-perature within an individual's life span.Broadly speaking, differences in membranecomposition between species from warmand cold habitats are similar to those ofthermally-acclimated members of a givenspecies. Animals from colder environmentshave relatively greater levels of lipid unsat-uration in both cases, and their membranesare more fluid (Hazel and Williams, 1990).

An interesting contrast between evolu-tionary and acclimatory changes in mem-brane fluidity involves the relative "suc-cess" of homeoviscous adaptation in main-taining the same membrane fluidity at dif-ferent temperatures. Comparison ofvertebrate species from a wide range ofbody temperatures (Tb) indicates that thefluidity of synaptosomal membranes is thesame when measured at each species' Tb(Behan-Martin et al., 1993). Thus, the "ef-ficacy" of homeoviscous adaptation is 1,i.e., evolutionary compensation for the ef-

fects of temperature on membrane fluidityhas been complete. Acclimatory responsesare generally incomplete (i.e., the efficacyof homeoviscous adaptation is less than 1).However, these responses are highly vari-able, even among membrane fractions fromthe same cell. Some membranes exhibit noacclimation of fluidity at all (Lee and Cos-sins, 1990; Crockett and Hazel, 1995).

Biochemical mechanisms of homeovis-cous adaptation within species have beenexamined for numerous organisms (Hazeland Williams, 1990). Changes in composi-tion of phospholipid headgroups are in-volved in rapid acclimatory responses totemperature (Williams and Hazel, 1994).Changes in lipid saturation are slower, butoccur rapidly enough to respond to diurnalvariations in temperature (Carey and Hazel,1989). In ectothermic vertebrates, a key en-zyme in membrane acclimation is A9-desat-urase, which catalyzes the insertion of adouble bond in the 9-10 position of fatty-acyl chains. Tiku et al. (1996), using carp,showed that changes in saturation duringcold acclimation were associated with bothincreased synthesis of A9-desaturase and ac-tivation of existing desaturase molecules. Inother taxa, activities of additional desatu-rases have been shown to increase duringexposure to cold (Macartney et al., 1994).

An unexplored issue is whether similarbiochemical mechanisms are involved inevolutionary differences among species andin acclimatory changes. Differences inmembrane saturation are clearly implicatedin both genetic and phenotypic variation,but is increased unsaturation in fishes livingat cold temperatures due to higher basallevels of A9-desaturase, or are there addi-tional biochemical differences? No consis-tent difference in headgroup compositionhas been demonstrated in inter-specificcomparisons, which suggests evolutionarydifferences in membrane fluidity are notdue to headgroup variation.

Regulation of membrane propertiesDespite extensive investigation of the

process and the results of homeoviscous ad-aptation, regulation of membrane propertiesis very poorly understood. Homeoviscousadaptation can be considered in terms of the

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feedback loops involved in many physio-logical processes. A regulated variable(which may or may not be membrane flu-idity) must be sensed by some mechanism.The signal must be integrated and inter-preted somehow, and the organism must re-spond by changing the amounts or activitiesof enzymes involved in membrane lipidsynthesis. Detailed knowledge of any ofthese regulatory steps is lacking.

One way to identify the regulated vari-able in a feedback system is to manipulatesuspected variables directly. Vigh et al.(1993) used a catalytic hydrogenation pro-cedure to increase the saturation of mem-brane phospholipids in a strain of the cya-nobacterium Synechocystis, which reducedmembrane fluidity without changing tem-perature. Thus temperature-related changesin membrane properties and other cellularprocesses could be decoupled from changesin fluidity. Cells responded to reduced flu-idity by increasing the expression of thedesA gene product, a Al2-desaturase. How-ever, other membrane properties could alsohave been affected by the hydrogenationtreatment. This study demonstrates that lip-id saturation affects membrane biosynthe-sis, but does not distinguish among modelsfor regulation of membrane properties.

How are membrane properties sensed?As noted above, it is unclear which mem-brane properties are essential for membranefunction. Because various properties ofmembranes are correlated, the membraneproperty sensed by the cell may not be themembrane property which is physiological-ly most important. Integration of informa-tion regarding cell membrane properties isalso almost completely unknown. Two linesof evidence indicate that integration is asub-cellular phenomenom. First, isolatedcells and cell cultures exhibit responses totemperature which are similar to those oftissues in intact organisms (Dey et al.,1993; Williams and Hazel, 1994). Second,different membrane systems within a cellmay exhibit different acclimatory responses(Lee and Cossins, 1990; Crockett and Ha-zel, 1995), arguing against a general exter-nal signal regulating membrane synthesis.

Early work with the protozoan Tetrahy-tnena pyriformis suggested that lipid desat-

urases might be activated directly by mem-brane fluidity, so that membranes would beessentially self-regulating (Thompson andNozawa, 1977). More recently, increasedbiosynthesis of desaturases at low temper-atures has been found in several organisms(Macartney et al., 1994). In fishes, post-translational modifications may also affectdesaturase activity (Tiku et al., 1996). Mo-lecular analyses of homeoviscous adapta-tion are still in the early stages, but resultsto date suggest that multiple regulatorymechanisms are operating.

Is homeoviscous adaptation adaptive?The discussion above provides a minimal

introduction into the large and diverse lit-erature on homeoviscous adaptation. Eco-logically relevant differences in temperatureand other environmental variables signifi-cantly affect the physical properties of cellmembranes, and these changes affect func-tional properties of membrane proteins. Ac-climatory responses to changes in environ-mental temperature occur rapidly and in-volve differences in gene expression andpossibly post-translational mechanisms,suggesting that membrane composition isclosely regulated. The basic biochemistryof phenotypic acclimation and genetic ad-aptation to temperature is similar in a va-riety of organisms, suggesting a universalneed to regulate membrane properties.

However, the organismal consequencesof homeoviscous adaptation are very poorlyunderstood. One could well ask whetherhomeoviscous adaptation really does bene-fit the organism. Would an organism's fit-ness or performance be compromised ifhomeoviscous adaptation did not occur?Because this appears to be a universal re-sponse to temperature, one can not inves-tigate species which lack homeoviscous re-sponses and compare them to those whichdo acclimate. A well-chosen taxon, includ-ing stenothermal and eurythermal specieswith a wide range of body temperatures,may provide an excellent system for thestudy of membrane adaptation and accli-mation. All inter-specific studies to datehave been performed without considerationof the phylogenetic relationships of the spe-cies involved. For example, Behan-Martin

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Temperature (°C)

FIG. 4. Effects of temperature on rates of water lossfrom an individual grasshopper {Melanoplus sangui-nipes). Water loss was measured by continuous weigh-ing in a temperature-controlled microbalance. Cuticu-lar lipids of M. sanguinipes melt at 38—50°C (Gibbs etal., 1991; Gibbs and Mousseau, 1994).

et al. (1993) included species ranging frompasserine birds to Antarctic fishes.

A second approach to testing the physi-ological significance of homeoviscous ad-aptation would be to manipulate membranecomposition and fluidity. This can be ac-complished by direct chemical modification(Vigh et al., 1993) or by genetic techniques.For example, a mutant deficient in synthesisof desaturases should exhibit reduced ther-mal tolerance or other deficiencies at ex-treme temperatures. Animal models such aszebrafish and fruit flies have received verylittle attention from membrane physiolo-gists, but could become useful study sys-tems.

CUTICULAR WATERPROOFING INTERRESTRIAL ARTHROPODS

Epicuticular lipids of terrestrial arthro-pods provide a second example for whichthe physical properties of lipids are thoughtto play an important physiological role. Allterrestrial organisms face the problem ofwater loss through exposed surfaces, and allstudied so far have evolved lipid layerswhich serve as the major barrier to evapo-rative water loss (Hadley, 1985, 1994). In-sects and other small organisms are partic-ularly vulnerable to dehydration, due totheir relatively high surface area:volume ra-tio. Cuticular lipids in terrestrial arthropodshave therefore been studied extensively

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Temperature (°C)

FIG. 5. Melting curves for cuticular lipids from fourinsect species. The frequency of -CH2- symmetricstretching vibrations increases as lipids undergo thesolid-fluid phase transition. From left to right, specieswere: Drosophila melanogaster, Blatella germanica,Melanoplus sanguinipes and Rhodnius prolixus.

(Blomquist et al., 1987; de Renobales et al.,1991). Most arthropods contain primarilylong-chain hydrocarbons, but wax estersand other oxygenated compounds may pre-dominate in some species (Buckner, 1993).

The ability of cuticular lipids to preventwater loss is thought to depend on theirphase state. Rates of water loss from insectsincrease rapidly above a species-specific"critical" temperature (Fig. 4; Wiggles-worth, 1945; Loveridge, 1968; Hadley,1994). Early investigators suggested that in-creased transpiration is caused by the melt-ing of the cuticular lipids. A number of bio-physical studies, using various techniques,have found evidence consistent with thishypothesis (Beament, 1945; Holdgate andSeal, 1956; Toolson et al., 1979). A limi-tation of many experiments has been thatsurface lipids occur in very small quantitiesand have broad phase transitions. Thus,some biophysical techniques used in othersystems may be inappropriate for these lip-ids. In some cases, only one species wasinvestigated, so correlations between lipidmelting and cuticular permeability mayhave been mere coincidence.

Recent work using infrared spectroscopyrevealed extensive variation in lipid prop-erties. Surface lipids melt at temperaturesas low as 25°C, although mealworm (Te-nebrio molitor) lipids remain solid up to80°C (Fig. 5; Gibbs and Crowe, 1991).Thus, lipid melting occurs at physiological-ly relevant temperatures in some, but notall, species. Variation in melting point (Tm)

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FIG. 6. Effects of temperature on water flux througha model membrane. A Gore-Tex membrane was coatedwith n-tricosane (Tm = 42-44°C), and water fluxthrough the membrane was measured using a flow-through system.

exists within species as well. Grasshoppers(Melanoplus sanguinipes) exhibit geo-graphic variation correlated with latitude,and Tm is higher in individuals acclimatedto warmer temperatures (Gibbs et al, 1991;Gibbs and Mousseau, 1994). This variationcan be used to examine the questions ofwhether the properties of surface lipids re-ally do affect rates of water loss, and whichdifferences in lipid composition are likelyto affect water balance.

I emphasize that the relationship betweenlipid melting and cuticular permeability stillneeds to be clearly resolved. In general, theevidence is favorable; organismal measure-ments, made using a variety of techniques(Figure 4; Beament, 1945; Toolson et al,1979), and model cuticles (Fig. 6) exhibitsimilar transitions in water flux at the melt-ing temperature of the lipid barrier. Anotherimportant issue is whether arthropods en-counter environmental temperatures highenough to affect lipid physical properties.In M. sanguinipes, field body temperaturescan exceed 40°C, which would be highenough to melt cuticular lipids partially(Chappell, 1983; Gibbs et al, 1991). To-gether, these results suggest that increasedrates of water loss associated with lipidmelting can occur under ecologically rele-vant conditions.

Genetic and acclimatory variation incuticular lipid properties

The cuticular lipids of arthropods exhibitsuch great diversity that broad comparisons

across disparate taxa are largely uninform-ative. Only a few sets of closely related spe-cies have been used in comparative studiesof the relationship between lipid composi-tion and rates of water loss. Species withlonger-chain hydrocarbons generally losewater less rapidly (Hadley, 1978; Hadleyand Schultz, 1987). Unfortunately, the phy-logenetically-based approaches developedin recent years have not yet been applied toinsect water balance, but future work willcertainly need to take evolutionary relation-ships into consideration.

Recent studies have demonstrated geo-graphic variation in surface lipid composi-tion (Ferveur et al, 1996). These differ-ences have been shown to have a geneticbasis, but the effects of this variation onwater balance have not been investigated.In M. sanguinipes, populations from south-ern California tend to have lipids whichmelt at higher temperatures than those fromnorthern populations (Gibbs et al, 1991).Recent evidence indicates that individualsfrom southern populations lose water lessrapidly at the same measurement tempera-ture (Rourke, personal communication).This pattern is consistent with local adap-tation to higher environmental temperaturesand lower humidities at lower latitudes, al-though other, non-adaptive explanations cannot be excluded (Endler, 1986).

Thermal acclimation of cuticular hydro-carbons has also been correlated with ratesof water loss (Toolson and Hadley, 1979;Toolson, 1982). Despite the lack of bio-physical information in these studies, theyare also consistent with the idea that accli-mation of lipid properties affects water bal-ance. Lipids from warm-acclimated individ-uals tend to have longer chain lengths.These differences should result in highermelting temperatures of the surface lipidsand reduced rates of water loss. This maynot be the case; acclimation to high tem-perature in the desert fruit fly, D. mojaven-sis, results in longer-chain length hydrocar-bons (Markow and Toolson, 1990), butthese changes in lipid composition are notassociated with changes in Tm or rates ofwater loss (Gibbs et al, 1998). In M. san-guinipes, acclimation to warm temperaturesactually results in relatively greater synthe-

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sis of shorter-length, unbranched hydrocar-bons (Gibbs and Mousseau, 1994). Becausestraight-chain lipids melt at higher temper-atures than branched lipids (Gibbs and Po-monis, 1995), overall Tm values are greaterin warm-acclimated individuals (Gibbs etal, 1991; Gibbs and Mousseau, 1994).

Total lipid quantities also usually in-crease at higher temperatures, so it is oftenunclear whether higher- Tm lipids provide abetter water-proofing barrier or whether thethickness of the barrier is the main factordetermining cuticular permeability. Similarconcerns apply in inter-specific compari-sons. Both lipid amounts and lipid proper-ties probably affect cuticular permeability.

Regulation of Cuticular Lipid PropertiesThe mechanisms whereby insects sense

and maintain the properties of the cuticularlipids are completely unknown. It wouldseem beneficial for an insect to be able todetect and assess the physical properties ofits surface lipids, but no such sensorymechanism has been described. In the caseof mealworms and some other species, lip-ids melt at such high temperatures (>80°C;Gibbs and Crowe, 1991) that no regulationwould seem necessary. The surface lipidsremain in a completely solid state at allnon-lethal temperatures. In other species,such as M. sanguinipes and some Drosoph-ila, melting temperatures may be in the en-vironmental range (Chappell, 1983; Gibbset al., 1997). One may then pose the ques-tion: Why don't all arthropods producehigh-rm lipids? Low-7m lipids would not bea problem for insects with ready access towater, but M. sanguinipes in California canbe found in very hot, dry habitats, wherefree water is not available.

A potential benefit of low-T^ surface lip-ids involves their dispersal over the cuticle.Cuticular lipids are deposited on the surfacethrough pores, and in some species formvisible wax blooms (Hadley, 1994). In oth-ers, the lipids appear to form a uniform lay-er, whose formation may depend on theability of lipids to disperse by flowingacross the surface. Measurements of surfacelipid viscosity are needed to test this hy-pothesis.

Low-rm lipids might also affect chemical

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FIG. 7. Time course of thermal acclimation of cutic-ular lipids in the grasshopper, Melanoplus sanguinipes.Seven-day old nymphs were transferred from 27°C to29°C or 34°C (open and filled symbols respectively),and shed skins were collected for determination of lip-id melting temperatures. Each series of points indicatesTm values for successive instars from a single individ-ual. From Gibbs and Mousseau (1994).

communication. Many insects use tactilepheromones in mate and colony recognition(Howard, 1993). The ability of a conspe-cific to detect a signal possibly could de-pend on whether the pheromone is embed-ded in a solid or fluid lipid matrix. The neu-rological basis for detection of volatilepheromones has been investigated in nu-merous insects, but mechanisms for detect-ing contact pheromones have been investi-gated in only a few cases (Stadler et al.,1994).

Finally, perhaps the physical propertiesof cuticular lipids are not under active reg-ulation. Acclimatory changes can be soslow that their utility in responding to en-vironmental variation is questionable. Forexample, surface lipids of juvenile M. san-guinipes acclimate over a period of 2—3weeks, spanning 4 instars (Fig. 7). Otherarthropods are certainly capable of chang-ing their surface lipids much more rapidlythan this. Surface lipids of houseflies con-sist primarily of methyl-branched alkanes(and very few alkenes) immediately afterecdysis, but alkenes are the dominant com-ponents a few days later. These sweepingchanges include the production and depo-sition of lipid pheromones, a process that isunder hormonal control (Blomquist et al.,1993). Changes in surface compositionhave also been observed in social insects(Howard, 1993). The causes and mecha-

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nisms whereby these latter changes occurare unknown, and may involve biosynthesisof new lipids or acquisition of them fromother colony members.

Thus, cuticular lipids involved in chem-ical communication can be modified over aperiod of days, whereas the time course ofthermal acclimation indicates a muchslower restructuring. Perhaps temperaturesimply has different effects on the activitiesof biosynthetic enzymes, such that lipidcomposition and properties change as a re-sult of altered channeling of precursors intodifferent lipid classes. As long as the cuticleremains sufficiently impermeable, details ofsurface lipid composition and physicalproperties may not affect organismal waterbalance significantly. In this scenario, slowchanges in surface lipid composition maysimply reflect accumulated, non-adaptivechanges due to the effects of temperatureon biosynthetic enzymes. This non-adaptivemodel is consistent with data for M. san-guinipes and the desert fruit fly, D. moja-vensis. Areas in which more work clearlyis include the rates of turnover of cuticularlipids, and the effects of temperature onspecific steps of lipid biosynthesis.

Future questions in cuticular lipidsCuticular lipids are essential to the sur-

vival of terrestrial arthropods. However,most studies of the water-proofing abilitiesof these lipids have been correlative in na-ture, relying on comparative approaches oracclimatory variation. Ideally, one wouldlike to manipulate surface lipid compositiondirectly and examine the effects on waterbalance. As is rapidly becoming the casefor membranes, the use of molecular tech-niques, in carefully chosen species, will be-come more common.

Many mutants for surface lipid biosyn-thesis have been discovered in plants (Post-Beittenmiller, 1996), but genetic analysis oflipid synthesis and regulation in insects hasonly recently begun. This work has mainlyfocused on cuticular pheromones in Dro-sophila (Coyne et al., 1994; Scott, 1994;Ferveur et al., 1997), but physiologists canalso use genetic approaches to study theconsequences of lipid variation for waterbalance.

CONCLUDING COMMENTS

Lipids are defined by one of their phys-ical properties (hydrophobicity). This prop-erty makes lipids an excellent barrier to themovement of water and other moleculesinto and out of cells and the whole organ-ism, but other lipid properties can be func-tionally important. Despite considerable ef-fort, it is not always apparent which lipidproperties are crucial to physiological func-tion. For example, intensive research intocell membranes has left us with several bio-physical models from which to choose. Afundamental experimental problem is thecomplexity of lipid mixtures, and the factthat they are biosynthetically related. Thus,environmental factors that affect the pro-duction of one lipid class will affect the lev-els of other lipids.

Additional complexity arises from themultiple functions exhibited by many lip-ids. For example, pheromone componentsmay make up the majority of cuticular lip-ids (Scott, 1994). In the case of cell mem-branes, unsaturated fatty acids are central tohomeoviscous adaptation, are required tomaintain the fluidity of depot fats in hiber-nating mammals (Frank, 1991, 1994), andare precursors for eicosanoids and hor-mones (Stanley and Howard, 1997). Thus,environmental effects or natural selectionon one aspect of lipid biology will likelyaffect many other processes.

In spite of these limitations, the physicalproperties of lipids are clearly important intheir biological functions. The biochemicalprocesses responsible for changing mem-brane and cuticular lipid properties arelargely known, but the regulation of theseprocesses is very poorly understood. Thetechniques of molecular genetics have beenused to identify loci involved in lipid bio-synthesis, but these studies on their own areinsufficient. Future advances will requirethe integration of biochemical, biophysicaland molecular approaches, as well as in-vestigations of the organismal effects of lip-id variation.

ACKNOWLEDGMENTS

I thank Lisa Crockett for co-organizingthe symposium, Jeannine Larabee for com-

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ments on the manuscript, Bryan Rourke foraccess to unpublished data, and the Societyfor Integrative and Comparative Biologyfor helping to fund the symposium. Fund-ing for research and manuscript preparationwas provided by NSF grant IBN-9317471,with travel support from the Ubu Endow-ment.

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