Water-Proofing Properties of Cuticular Lipids

SYNOPSIS. Epicuticular lipids play a critical role in allowingarthropods to thrive in terrestrial environments, by reducingtranspiration of water through the cuticle. These lipids consistof a diverse array of compounds, especiaUy long-chain hydrocarbons.Rates of water loss are correlated with hydrocarbon structuralfeatures, including chain length, unsaturation and methyl-branching.The water-proofing abilities of cuticular lipids appear to dependlargely on their physical properties. In most arthropods, ratesof water loss increase rapidly above a "transition" temperature.A widely accepted model proposes that this transition is dueto melting of the surface lipids to a fluid, permeable state.Evidence for this hypothesis has primarily been correlative,due to experimental limitations. Recent technical advances inlipid biophysics and water loss measurements have made it possibleto test the lipid melting model more directly. Experiments usingmodel cuticles, in vitro preparations and intact arthropodssupport the idea that the phase behavior of cuticular lipidsis a major factor determining cuticular permeability.     

Cuticular pheromones and water balance in the house fly,Musca domestica

Epicuticular lipids serve two major roles in insects. Their waterproofing properties are crucial to survival in terrestrial environments, and they serve as contact pheromones in a wide array of taxa. Both functions may be affected by the physical properties of the surface lipids. This provides the opportunity for natural selection on water conservation, mediated by lipid phase behavior, to interact with and perhaps conflict with sexual selection on communication and mate recognition. We used the common house fly, Musca domestica, as a model for these interacting selective forces. Male house flies preferred female models treated with a high melting-point lipid mixture, suggesting that sexual and natural selection may both act to favor longer-chain, more saturated hydrocarbons. However, higher melting points did not result in lower rates of water loss. We propose a working model in which phase separation between the unsaturated female pheromone and saturated hydrocarbons results in areas of melted, pheromone-rich lipids and regional variation in cuticular permeability.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores ('stomatal transpiration') to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid 14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.     

Evidence for water-organized structure in the cuticular water barrier of the American cockroach,Periplaneta americana

Summary Cuticle discs cut from the wings ofPeriplaneta and cuticular lipid extracts were analysed by differential scanning calorimetry. Two major endothermic events, one beginning at 7.1±0.3°C, and the other increasing with temperature above 24.7±0.7°C, were associated with extractable lipids in intact cuticle discs. Heating progressively destroyed the molecular structures responsible for the high temperature event but had no effect on the lower one. These results complement changes in cuticle permeability observed in a recent study and thought to be associated with structural change in the cuticular water barrier. The molecular structures responsible for both events depended on the presence of water in the cuticle. Cuticular lipid extracts lack the molecular organization found in intact cuticle, even when water is present.     

CUTICULAR LIPIDS OF TERRESTRIAL PLANTS AND ARTHROPODS: A COMPARISON OF THEIR STRUCTURE, COMPOSITION, AND WATERPROOFING FUNCTION

1. Lipids deposited on the surface or embedded within the cuticle of terrestrial plants and arthropods are primarily responsible for the observed low rates of water loss through the cuticle. 2. These lipids are a mixture of long-chain compounds which include hydrocarbons (saturated, unsaturated, branched), wax esters, free fatty acids, alcohols, ketones, aldehydes, and cyclic compounds. 3. The cuticle of both plants and arthropods is a continuous, non-cellular multilayered membrane which overlies the epidermal cells. 4. In arthropods, horizontal division of the cuticle into layers is clearly visible. In plants, the layers comprising the cuticle are not sharply demarcated. 5. The substance responsible for the structural integrity of the plant cuticle (cutin) is composed of cross-esterified fatty acids; structural integrity in arthropod cuticle is provided by a chitin-protein complex. 6. Cuticular lipids are probably formed near the surface in both plants and arthropods; however, specific sites of synthesis are known for only a few species and little is known about their transport from these sites to the surface. The elaborate pore canal and wax canal system of arthropod cuticle is absent from plants. 7. The physical structure and arrangement of the lipid deposits on the cuticular surface that are so important in controlling water movement depend on both quantity and chemical composition, and are, in turn, specific to each species in relation to its environment. 8. Different lipid components are not equally efficient in reducing transpiration. Maximum waterproofing effectiveness is provided by long-chain, saturated, non-polar molecules containing few methyl branches. 9. Plants and arthropods can, within genetic constraints, alter the composition of their cuticular waxes to improve impermeability when conditions require increased water conservation. 10. None of the models proposed to explain the change in arthropod cuticular permeability which occurs at a species-specific temperature (‘transition temperature’) in many species is supported by the experimental data now available.     

Electron paramagnetic resonance spectroscopy of spin-labelled cuticle of Centruroides sculpturatus (Scorpiones: Buthidae): Correlation with thermal effects on cuticular permeability

Cuticular phase transitions and molecular dynamics have been studied in the buthid scorpion Centruroides sculpturatus using the techniques of thermogravimetric analysis (TGA) and electron paramagnetic resonance (EPR). TGA studies of the cuticular permeability reveal discontinuous changes in thermal dependence of transcuticular water loss rates (transitions). The first transition occurs between 30–40 C and results in a small increase in water loss rate, whereas the second transition begins near 55 C and is accompanied by a large increase in water loss rate. EPR spectra of spin-labelled cuticle indicate that the epicuticular lipids are very mobile at ambient temperature, with the translational diffusion coefficient being about 5 × 10^?6 cm² sec at 22 C. and that the low-temperature transition is associated with an increase in mobility of the hydrocarbon chains of the epicuticular lipids. The high-temperature transition probably results from melting of the epicuticular lipids. The results of this study are discussed with reference to current models of the structure of the arthropod cuticle.     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Water permeability of plant cuticular membranes: the effects of humidity and temperature on the permeability of non-isolated cuticles of onion bulb scales

Abstract. Water permeability of cuticular membranes (CM) from the inner bulb scales of Allium cepa has been investigated. CM have a thickness ranging from 0.6 to 1.3 μm. They are composed of a thin (120–200 nm) lamellated cuticle proper and a thicker (300–900 nm) cuticular layer. Permeability coefficients for diffusion of water across these thin membranes are very low (4 × lO⁻¹⁰ms⁻¹⁰). There was no difference in permeability of CM from successive scales of the same onion. Extraction of soluble cuticular lipids (SCL) with chloroform increased permeability by a factor of 1350 to 2050. Preliminary data indicate that only 1 μg cm⁻¹⁰ of SCL are removed by this treatment, hydrocarbons being the main (75%) consistuent. Permeability coefficients of cuticular transpiration were little affected by relative humidity, showing that transport is limited by a hydrophobic barrier that lacks dipoles. However, following extraction, permeability of the membranes depended strongly on humidity due to the presence of polar functional groups in the polymer matrix. Soluble cuticular lipids undergo a phase transition around 47°C. Temperatures higher than that irreversibly increased water permeability.     

Physical properties of insect cuticular hydrocarbons: Model mixtures and lipid interactions

Cuticular lipids include a diverse array of hydrophobic molecules that play an important role in the water economy of terrestrial arthropods. Their waterproofing abilities are believed to depend largely on their physical properties, but little is known about interactions between different surface lipids to determine the phase behavior of the total lipid mixture. I examined the biophysical properties of binary hydrocarbon mixtures, as a model for interactions between different epicuticular lipids of insects. The midpoint of the solid/liquid phase transition (T_m) for mixtures of n-alkanes differing in chain length equaled the weighted average of the T_ms of the component lipids. This was also true for n-alkane-methylalkane mixtures. However, alkane-alkene mixtures melted at temperatures up to 17°C above the temperature predicted from the weighted average of component lipid T_m values. Hydrocarbon mixtures did not exhibit biphasic melting transitions indicative of independent phase behavior of the component lipids. Instead, melting occurred continuously, over a broader temperature range than pure hydrocarbons.     

Surface lipids and plant defenses

The major function of the plant epidermis is to form the cuticle, a functional permeability barrier of the cell wall which prevents excessive water loss and the entry of harmful substances and pathogens into the host. This type of cell wall modification is mainly composed of a polyester matrix, cutin, and soluble waxes embedded in the matrix and deposited on the external surface. Cuticle-associated proteins may also be important. Recent observations are starting to reveal complex inter-relationships between cuticular lipids and immunity. This suggests that the cuticle is not simply a physical barrier, but a dynamic host defense with signaling circuits and effector molecules. Furthermore, these studies have also demonstrated that cuticular lipids and immunity may intersect in common pathways, although the significance of this is not fully understood. In this review, we examine the functions of the plant cuticle in host–pathogen interactions, and discuss the possibilities of integrating the membrane and cuticular glycerolipid biosynthesis.     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.     

Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Biochemical analysis of elastic and rigid cuticles of Cirsium horridulum

The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.     

Thermodynamics of cuticular transpiration

Water conservation is a significant physiological problem for many insects, particularly as temperature increases. Early experimental work supported the concept of a transition temperature, above which water-loss rates increase rapidly as temperature increases. The transition phenomenon was hypothesized to result from melting of epicuticular lipids, the main barrier to cuticular transpiration. This explanation has been challenged on theoretical grounds, leading to thermodynamic analyses of cuticular transpiration based on reaction rate theory. These studies have not directly addressed the mechanistic basis of the transition temperature. Models developed in the context of cell membrane transport provide potential explanations that can be tested experimentally. These models include changes in the activation entropy for diffusion through the cuticular lipids, increased solubility of water in melted lipids, and lateral heterogeneity of the cuticle.     

Impact of feeding,molting and relative humidity on cuticular wax deposition and water loss in the lone star tick,Amblyomma americanum

To determine whether there is a correlation between the amount of lipids on the surface of ticks and their permeability to water, we quantified cuticular surface wax and measured water-loss rates in the ixodid tick Amblyomma americanum from nonfed nymph to egg-laying female. Ticks deposited no extra cuticular lipids during feeding, permitting maximum transpiratory water loss that presumably helps to concentrate the bloodmeal; and ticks deposited additional cuticular wax after apolysis that reduced integumental water loss, which likely prepares ticks for off-host existence. A remarkable three-fold boost in surface wax deposition and extreme water retention were noted after host drop-off following feeding. This wax is likely host-derived. Fed nymphs could discriminate between low and high relative humidity, enabling pharate adults to conserve lipid that would otherwise be lost with the exuvia and feces. This conservation strategy likely adds to the lipid pool needed by the tick to survive in a dry environment and complements the tick's behavioral abilities for seeking out optimum conditions for water conservation and host location.     

Water composition and loss by body color and form mutants of the German cockroach (Dictyoptera: Blattellidae)

Body water composition and rates of water loss were determined for adult males of five body color or form mutants of the German cockroach, Blattella germanica (L.). Body water content ranged from 68.4% for a yellow body strain to 74.7% for the balloon wing strain. Dark body strains (black and black×orange) contained less water than lighter color strains (orange and yellow). There were no differences in whole-body lipid content among the strains. Cuticular permeability at 30°C ranged from 18.44 to 24.8 μg cm^-2 h^-1 mmHg^-1 for black body and balloon wing strains, respectively; dark body strains had lower cuticular permeabilities than light body strains. Brief, whole-body extraction with hexane increased cuticular permeability from 5.6-fold for the balloon strain to 8.3-fold for the orange body strain. Cuticular permeabilities of hexane-extracted dark body strains again had lower cuticular permeabilities than those of light body strains, indicating that differences in permeability were due to the cuticle itself and not differences in the amount or composition of epicuticular lipids. Measurements of cuticle water content indicated that light body strains contained significantly more water than dark body strains. Rate of percent original mass and percentage of total body water lost increased linearly with time. Implications of cuticular water content to cuticular permeability are discussed.     

Characteristic properties of proteins from pre-ecdysial cuticle of larvae and pupae of the mealworm Tenebrio molitor

Proteins extracted from the cuticle of pharate larvae and pupae of the mealworm Tenebrio molitor are more soluble at low temperatures than at higher temperatures, a behaviour characteristic of hydrophobic proteins. When the temperature of an unfractionated cuticular extract is raised from 4 to 25 degrees C the solution becomes turbid, droplets of a heavy, protein-rich phase are formed, which gradually settles, leaving an upper protein-poor phase, indicating that the aggregation process is a coacervation. The aggregation of the dissolved cuticular proteins is influenced by changes in temperature, pH, and ionic strength. The process has been studied by measuring development of turbidity in unfractionated cuticular extracts and in solutions of three purified proteins from Tenebrio pharate larvae and pupae (TmLPCP-A1a, TmLPCP-E1a, and TmLPCP-G1a), while temperature, pH or ionic strength of the solutions were varied. Protein aggregation was also studied by determination of changes in fluorescence intensity, when the hydrophobicity probe, 8-anilinonaphthalenesulfonic acid (ANS) was added to solutions of the cuticular proteins. Only when the protein solutions had developed a measurable turbidity was an increase in ANS-fluorescence observed, indicating formation of tightly packed clusters of hydrophobic amino acid residues during aggregation. The temperature range for aggregation depends upon protein concentration: the higher the concentration the lower and more narrow is the temperature range within which aggregation occurs. The tendency for the individual cuticular proteins to aggregate is most pronounced near their isoelectric points, and most of the cuticular proteins have alkaline isoelectric points. The influence of salts on the tendency of the proteins to aggregate varies among the proteins and depends upon how close they are to their isoelectric point. A solution containing both protein TmLPCP-A1a and TmLPCP-E1a becomes more turbid and develops a more intense ANS-fluorescence when warmed from 10 to 30 degrees C than corresponding to the sum of measurements performed on separate solutions of the two proteins, indicating that the two proteins interact during aggregation. The Tenebrio larval/pupal cuticular proteins are characterized by an abundance of hydrophobic amino acid residues, and especially their contents of alanine and proline are high. The behaviour of the cuticular proteins in solution resembles that of another hydrophobic protein, tropoelastin, and it seems reasonable to suggest that similar interactions govern the folding and aggregation of the peptide chains in the two types of proteins. The proline and alanine rich chain segments in the pharate cuticular proteins are suggested to form a series of beta-turns and to fold into a relatively open structure at low temperatures, giving water access to the hydrophobic residues and making the proteins water soluble. At increased temperatures the structure of the ordered water layer surrounding the hydrophobic groups breaks down, and the peptide chains tend to collapse into a more closed structure and to interact more tightly with hydrophobic regions in neighbouring molecules. In dilute solutions in the test tube this results in aggregation and precipitation of the proteins; in intact, pharate cuticle at ambient temperatures the proteins will preferably be in an aggregated, easily dissociated state. Accordingly, small changes in intercuticular pH and ionic strength can produce pronounced changes in the mechanical properties of unsclerotized solid cuticle by interference with protein interactions, in agreement with reports that some cuticles undergo plasticization during and/or immediately after ecdysis.