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Plasticity of the water barrier in vertebrate integument
International Congress Series 1275 (2004) 283 – 290
www.ics-elsevier.com
Plasticity of the water barrier in vertebrate integument Harvey B. Lillywhite* Department of Zoology, University of Florida, Gainesville 32611, USA
Abstract. Water barrier function in vertebrates is typically relatively fixed and characteristic of species. However, both plasticity and genetic adaptation can account for covariation between environment and resistance to transepidermal water passage (R s). The capacity to adjust R s may involve phenotypic plasticity, acclimation, or developmental plasticity, and fundamental mechanisms include changes in barrier thickness, composition and physicochemical properties of cutaneous lipids, and/or geometry of the barrier within the epidermis. In amniotes, lipid barriers are structured within keratin complexes that, while important to efficacy of function, appear to constrain the possibilities and time course of plastic responses. Studies of the relative importance of plastic responses and genetic variation are few and inconclusive. D 2004 Elsevier B.V. All rights reserved.
Keywords: Vertebrate; Skin; Epidermis; Lipid; Water permeability
1. Introduction Terrestrial environments pose numerous challenges to the physiology of animals, especially larger, more active species that interact conspicuously with the physical landscape and microclimate [1]. Survival dictates that animals acquire sufficient water and energy from the environment to maintain cellular function and to support a level of metabolism sufficient for maintenance, growth and reproduction. Water is key in providing an adequate milieu for biochemical transformations and is potentially lost across body surfaces and in secretions and excreta. Evaporation of water from permeable surfaces generally constitutes the greatest source of loss, key routes of transfer being the skin and respiratory surfaces.
* Tel.: +1 352 392 1101; fax: +1 352 392 3704. E-mail address: [email protected]. 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.08.088
284
H.B. Lillywhite / International Congress Series 1275 (2004) 283–290
Clearly, features of integument that limit cutaneous water loss (CWL) are of key significance in the evolutionary radiation of vertebrates in terrestrial and especially xeric environments. Although numerous aspects of cutaneous morphology potentially influence CWL, it is well accepted that the principal barrier to transepidermal water flux consists of lipids having adaptive geometry and composition. Indeed, lipids provide a range of waterproofing mechanisms in terrestrial plants, arthropods, and vertebrates [2]. While a fair number of studies representing all three principal taxa demonstrate badaptiveQ variation in CWL in the sense that lower rates are often associated with drier environments, less is known about the stimuli and mechanisms that are causally related to this variation, either in terms of genetic adjustments or of phenotypic responses to the environment. Even less is known relating these mechanistic issues to fitness consequences of plasticity in nature and potentially related genetic constraints. This paper briefly reviews what is known concerning plasticity of water barrier function from a perspective that considers evolutionary trends and habitat demands of vertebrates. Understanding the water relations of vertebrate integument has benefited from technological advances and physicochemical studies of lipids from other systems such as arthropods and artificial membranes [3,4]. It is hoped that such advances and techniques from dermatological disciplines will find marriage with investigations that embrace comparative and evolutionary perspectives as well as application to understanding of pending environmental change [5]. 2. Water barriers and morphology of integument Although numerous attributes of skin have been proposed to influence skin resistance to evaporative water loss (R s), it is widely accepted that lipids provide the principal barrier to transepidermal water loss (TEWL). In both plants and arthropods, lipids are associated with or deposited on a multilayered cuticle. These surface lipids constitute the principal resistance to water passage across the cuticle or integument of these organisms [2]. Importantly, the cuticle of these organisms is a rigid structure and provides a stable platform for stability of the lipid barrier. Terrestrial vertebrates evolved from aquatic ancestors, and a permeability barrier was an important feature of integument that evolved in concert with increased mechanical resilience related to increasing size and terrestrial demands. Mechanical toughness of the integument was provided by the evolution of a stratum corneum comprised of multiple layers of fibrous keratins with crude correlations between degree of terrestriality and degree of keratinization. Living aquatic, semi-aquatic and terrestrial but dehydrationsensitive amphibians exhibit a stratum corneum comprised of but one or two cell layers of keratin. In contrast, reptiles, birds and mammals possess a much thicker stratum corneum having multiple layers of keratinized cells and, in sauropsids (reptiles and birds), added strength and rigidity due to presence of h-type keratins. The amphibian skin is an important site of gaseous and ionic exchange, and is highly permeable to bi-directional water flux in many species, whereas the amniotes have better developed lungs and generally do not exchange gases and ions extensively across the skin. The multilayered stratum corneum of amniotes also provides a structural template for sealing lipids that perform a water barrier function. Unlike plants and arthropods, the water barrier lipids of
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Fig. 1. Schematic models of vertebrate integument illustrating fundamental dichotomy in means of preventing dehydration of epidermis and whole animal. In the amphibious anuran (left), either externally present moisture or aqueous mucus secreted from dermal glands maintains a wet exterior of epidermis, and therefore water evaporates from the external water film rather than from the underlying epidermis. Water is replenished from the environment, either directly or (in the case of mucus discharge) by means of cutaneous absorption across the ventral seat patch, then via blood circulation to the mucous secretions within mucous glands. In the arboreal frog (left center), lipids are secreted onto the skin surfaces from dermal glands and form a barrier to water evaporation once they dry on the exterior. A lipid barrier functions similarly in the squamate reptile (center right), bird and mammal (right) except the barrier lipids are structured internally within the stratum corneum. Lipids are shown in gray shading. The large bold broken lines represent stratum germinativum.
amniotes are contained within, not upon, the integument. In either case, however, the barrier is located superficially and distal to the vascularized tissues of the inner skin. Among vertebrates, there is a fundamental dichotomy reflecting structure of integument and associated strategies for water balance. Either the epidermis is protected from excessive water loss by a lipid or keratin–lipid water barrier (amniotes and some amphibians), or the epidermis is maintained in a hydrated state by an overlying aqueous film or external moisture from an external source (many amphibians and some reptiles). In either strategy, the location of the lipid or aqueous film reflects the requirement for maintaining water balance of the skin itself as well as that of the entire organism (Fig. 1). Thus, when bullfrogs are basking in dehydrating environments, discharge of mucus onto the skin surfaces increases with increasing body temperature—a response interpreted to maintain a hydrated integument as well as evaporative cooling in the interest of thermoregulation [6]. These frogs do not bask or risk dehydration if water is unavailable to replenish body stores that contribute to the mucus secretions and TEWL. On the other hand, various arboreal frogs inhabiting yet drier environments secrete lipids onto the skin and wipe them over the body surfaces to form a protective water barrier that restricts losses of body water and protects the hydration status of underlying epidermis. In terrestrial amniotes, the skin is protected by a water barrier of variable effectiveness in the lipid– keratin complex of the stratum corneum (Fig. 1). 3. Mechanisms for renewal and regulation of the water barrier There are numerous potential means by which water barriers associated with vertebrate integument can be adjusted to meet environmental demands. Fundamentally, these can be reduced to the categories of response that follow in the subsequent subsections.
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3.1. Presence or absence of barriers Whether or not an effective water barrier is present in skin is perhaps most relevant to evolutionary radiation of animals and genetically determined adaptations of species. Barrier function in amniotes appears to be relatively fixed, heritable, and characteristic of species (e.g. Ref. [7]). There are few known examples of vertebrates that can produce a water barrier anew—facultatively—in response to environmental demands, while otherwise not possessing one. With respect to both genetic and plastic responses, species of amphibians provide well-studied examples. Many amphibians, and perhaps a majority, evaporate water across the skin at rates similar to that from a free water surface, and there are no known barriers to water passage in these species. In contrast, various species of anurans inhabiting arid or semi-arid environments exhibit features of integument that pose well-known barriers to TEWL [8]. The more efficacious examples include cocoons that form in burrowing anurans during aestivation and epicutaneous lipids that are secreted from dermal glands and wiped over the body surfaces in elaborate behaviors thought to stimulate secretion and spreading of lipids over the entire body surface [9]. Such behaviors are observed prior to animals retiring from activity and assuming a water-conserving posture, or when frogs are subjected to gradual dehydration and disturbance by handling [9,10]. The stimulus that elicits the behavior is not clear, however, and it is not known how bcleanQ the skin becomes between bouts of wiping when R s might potentially decrease as lipids wear away. 3.2. Barrier thickness The amphibian examples also are relevant to mechanisms for increasing thickness of the water barrier. Characteristic cocoons consist of multiple single cell-thick layers of shed stratum corneum resulting from multiple shedding of the skin layers during periods of dormancy in drying soils. Such cocoons may consist of 40–60 layers of cornified cells with secreted lipids and proteinaceous materials sandwiched between them [11]. These structures impose considerable resistance to water movement, and the barrier efficacy presumably increases with the numbers of layers or thickness of the structure. In frogs that wipe lipids over the body surface, tactile stimulation of the wiping elicits reflexive release of lipids from dermal glands, so the thickness of the final lipid layer could presumably be controlled to some extent by the duration and nature of the wiping [9]. The externally wiped layer of lipid was estimated to be about 0.2 Am in phyllomedusine frogs [12], but nothing is known about its variability either among or within species of anurans. In amniotes, the barrier region of the stratum corneum provides a tough and resilient framework for intercellular lamellar lipids, and the laminated feature of the entire lipid– keratin structure is a means by which thickness of the barrier might be altered. Hypothetical proliferation of additive layers would enhance R s to water permeation if the requirement was increased in response to increased drying power of the ambient atmosphere. Is there evidence this mechanism is utilized? Recently, it was demonstrated that R s essentially doubles following the first postnatal ecdysis in California king snakes (Lampropeltis getula), and the increased resistance correlates nicely with a doubling in thickness and number of strata of the specialized
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mesos layer of epidermis that constitutes the water barrier in this and other species of squamates [13]. This response is interpreted as an adaptive adjustment of R s during the natal transition from aqueous environment of the embryo to atmospheric air that surrounds the newborn. Some lizards also exhibit upregulation of the cutaneous water barrier as a response to changes in environmental conditions, and changes in cutaneous lipids are known to be involved [14]. However, it remains unclear as to whether such changes in R s might be correlated with changes in thickness of the water barrier. Chicks of the Japanese Quail (Coturnix c. japonica) exhibit a 43% decrease in skin water permeability and approach adult values within 13 days post-hatching. Histological data suggest that increased barrier efficacy is attributable to epidermal thickening, as in snakes [15]. In other studies, reptilian skin responds to trauma such as tape stripping with a bthickeningQ response involving hyperplasia of the a-keratin keratinocytes [16]. Human disorders of cornification in various disease states can result in different densities or thickness of stratum corneum and intercellular bilayer structures [3]. However, comparative studies of possible adaptive modifications of such structures in relation to natural environments have not been undertaken. 3.3. Composition of barrier lipids While a few generalizations are appropriate regarding composition of lipid classes in various water barriers of vertebrates, the subject is one that has amazing complexity. In every case examined, permeability barriers contain a complex mixture of lipid molecules. Longer chain-length hydrocarbons tend to comprise a dominant category of lipids in most barriers examined. These will tend to melt at higher temperatures and will result in reduced water permeation, whereas short chain length molecules reduce the intensity of van der Waals interactions between hydrocarbon molecules and create a more fluid structure that is more readily penetrated by water. Relative saturation of hydrocarbons also contributes to a tighter water barrier, whereas unsaturation and methyl branching tends to introduce kinks in molecules and disrupts packing. In some systems, however, chain elongation and unsaturation may offset one another, such that chain length alone is not necessarily a reliable indicator of water permeation [17]. Hydrocarbons and wax esters are both very nonpolar, which also assists in repelling water. Polar phospholipids and other classes of lipids having intermediate polarity, in addition to branching, might be important in structuring the geometry of a water barrier and providing a degree or specific orientation of fluidity important with respect to potential mechanical distortion or disruption of the barrier structure. Detailed information on cutaneous lipids of amphibians can be found in relatively few published studies. Analyses of the epicutaneous lipids secreted during wiping behaviors of phyllomedusine frogs demonstrate a mixture of hydrocarbons, triacylglycerols, cholesterol, cholesterol esters, free fatty acids and wax esters [12]. Wax esters are dominant and average about 46 carbons in length. Other studies demonstrate a cocktail of lipids that can be extracted from skin of frogs that do not exhibit wiping behaviors, and there is generally no correlation between these lipid properties and rates of evaporative water loss [18]. However, lipid mixtures extracted from whole skin undoubtedly include elements of membrane lipids not associated with a water barrier as well as precursor molecules that might be converted to other components when a barrier is present. There is no information
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that sheds light on how composition of barrier lipids are altered in relation to facultative waterproofing of skin by wiping or formation of a cocoon. Studies of squamates demonstrate a complex mixture of lipids including hydrocarbons, wax and sterol esters, di- and triacylglycerols, free fatty acids, alcohols, cholesterol, ceramides, and a variety of phospholipids present in skin, with concentration shown histochemically in the mesos layer of epidermis [19–21]. Analyses of lipids extracted from the skin of snakes are complicated potentially by presence of lipid classes important as sex attractant pheromones, in addition to barrier lipids that might be present. Again, there is no information regarding possible regulation of compositional features of lipids in relation to plasticity of barrier function. The cutaneous lipids of mammals have been studied extensively, but largely in humans and laboratory rodents and in contexts of disease and cosmetic interests in skin. Lamellar bodies provide precursor lipids consisting mainly of glycosphingolipids, free sterols and phospholipids that are delivered to the extracellular spaces in developing stratum corneum. Subsequently, these are enzymatically converted to nonpolar products and assembled into lamellar structures surrounding the corneocytes. The changes in lipid composition result in a barrier structure consisting largely of ceramides, free fatty acids, and cholesterol [22]. It is known in mammals that barrier structure and function is genetically determined and is rapidly restored following trauma. Furthermore, transformation of the precursor lipids to nonpolar-enriched barrier lipids of the stratum corneum is susceptible to disturbance by a number of factors, including essential fatty acid deficiency, genetic defects and enzyme deficiencies, environmental factors and hydration status [3]. These and various disease states are known to cause dysfunction of the water barrier and abnormally elevated TEWL. Barrier defects are indeed reflected in the lipid composition of the stratum corneum. Experimental studies of mammalian epidermis demonstrate that mechanical or chemical disruption of normal barrier morphology influences cellular kinetics, protein and lipid synthesis within 15–20 h that restore the barrier efficacy (reviewed in Ref. [23]). However, covariation of lipids and TEWL in relation to variation of natural stressors in the environment of mammalian species has not been investigated. Recent investigations have demonstrated facultative waterproofing in the skin of birds. When subjected to conditions of dehydration, adult zebra finches are capable of rapid upregulation of the cutaneous water barrier. Within 16 h of water deprivation, TEWL decreases by 50%, and the skin barrier efficacy continues to improve until mammal-like values are achieved [24]. Similarly, hoopoe larks in extremely arid regions of the Arabian Peninsula are characterized by TEWL rates about 30% lower than that of larks from mesic environments, and these rates decrease significantly when birds are acclimated to high temperatures [25]. In these desert birds, adjustments of lipid ratios have been shown to favor ceramides over free fatty acids and sterols, and these changes correlate with reductions of TEWL [26]. The higher ratios of ceramides evidently allow the lipids of the permeability barrier to exist in a more highly ordered crystalline phase, which creates a tighter barrier to water vapor diffusion. Acclimation of water loss rates does not appear to occur in skylarks and woodlarks from mesic environments in Europe, nor in Dunn’s larks from the Arabian desert [25]. Thus, capacity for plasticity of the permeability barrier appears to vary among species and, in the case of larks, is characteristic only of species inhabiting more extreme desert regions.
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3.4. Physical properties and geometry There is a great deal of information relating to physicochemical properties of lipids and lipid membranes, and it is well accepted that permeability can differ quantitatively because of the physical nature of the water movement pathway. It is important to note that in models of vertebrate barriers, lipids form a continuous barrier in the horizontal dimension parallel with the skin surface (Fig. 1). Either immobility of the animal (surface lipids of amphibians) or a tough, resilient lattice of keratin (stratum corneum–lipid complex of amniotes) appears to be important for optimal integrity of barrier function. It is not known to what degree physical disruption of the barrier structure necessitates repair or renewal during normal activity of animals that might bend or jar the skin. Recent studies that employ molecular models suggest that resistance to water permeation of mammalian stratum corneum is related, in part, to tight, gel-like packing of hydrocarbon chains and changes in lipid phase behavior related to component ratios and molecular arrangement of cholesterol [27,28]. Additionally, fluid and crystalline phases of the lipid lattice alternate vertically in repetition with stacked lamellae [29]. The crystalline phase enhances barrier efficacy, while the presence and localization of fluid domains facilitate elasticity in relation to geometry of the barrier and lipid interactions with keratin components. Models further suggest that diffusion of water is limited in directions both parallel and perpendicular to the plane of the lipid bilayers due to their organization [28]. In avian skin, the efficacy of facultative waterproofing resides in a capacity to modulate the organization of secreted lipids, viz. non-bilayer, electron-lucent lipids under basal conditions, but lamellar lipid structures in response to xeric stress, leading to significantly decreased TEWL [24]. Both compositional features of cutaneous lipids and the hydration status of the skin will influence the phase properties of the barrier, which becomes disrupted as the skin becomes progressively hydrated. Unlike terrestrial mammals, the stratum corneum of marine mammals retains appreciable amounts of glycolipids [30]. 4. Conclusion In spite of extensive literature on membrane physiology and the structure and function of mammalian skin in relation to disease and cosmetics, there is comparatively little understanding of adaptive adjustments of permeability barriers in contexts of evolution, phylogeny and environment. Comparative and experimental approaches with goals of understanding mechanisms of plasticity in relation to adaptation will hopefully remedy this deficit of understanding in the future. Such investigations should include, or interface with, genetics, morphology, ecology and evolutionary disciplines, as well as physiology. References [1] W.P. Porter, et al., Calculating climate effects on birds and mammals: impacts on biodiversity, conservation, population parameters, and global community structure, Am. Zool. 40 (2000) 597 – 630. [2] N.F. Hadley, Integumental lipids of plants and animals: comparative function and biochemistry, Adv. Lipid Res. 24 (1991) 303 – 320. [3] G. Menon, R. Ghadially, Morphology of lipid alterations in the epidermis: a review, Microsc. Res. Tech. 37 (1997) 180 – 192. [4] A.G. Gibbs, Water-proofing properties of cuticular lipids, Am. Zool. 38 (1998) 471 – 482.
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[5] P.M. Kareiva, J.G. Kingsolver, R.B. Huey (Eds.), Biotic Interactions and Global Change, Sunderland, Sinauer Associates, Massachusetts, 1993. [6] H. Lillywhite, Thermal modulation of cutaneous mucus discharge as a determinant of evaporative water loss in the frog, Rana catesbeiana, Z. Vgl. Physiol. 73 (1971) 84 – 104. [7] F. Furuyama, K. Ohara, Genetic development of an inbred rat strain with increased resistance adaptation to a hot environment, Am. J. Physiol. 265 (Part 2) (1993) R957 – R962. [8] R.C. Toledo, C. Jared, Cutaneous adaptations to water balance in amphibians, Comp. Biochem. Physiol. 105A (1993) 593 – 608. [9] H.B. Lillywhite, et al., Wiping behavior and its ecophysiological significance in the Indian tree frog Polypedates maculatus, Copeia 1997 (1997) 88 – 100. [10] L.A. Blaylock, R. Ruibal, K. Plat-Aloia, Skin structure and wiping behaviour of phyllomedusine frogs, Copeia 1976 (1976) 283 – 295. [11] R. Ruibal, S.S. Hillman, Cocoon structure and function in the burrowing hylid frog, Pternohyla fodiens, J. Herpetol. 15 (1981) 403 – 408. [12] L.L. McClanahan, J.N. Stinner, V.H. Shoemaker, Skin lipids, water loss, and energy metabolism in a South American tree frog (Phyllomedusa sauvagei), Physiol. Zool. 51 (1978) 179 – 187. [13] M.C. Tu, et al., Postnatal ecdysis establishes the permeability barrier in snake skin: new insights into lipid barrier structures, J. Exp. Biol. 205 (2002) 3019 – 3030. [14] G.H. Kattan, H.B. Lillywhite, Humidity acclimation and skin permeability in the lizard Anolis carolinensis, Physiol. Zool. 62 (1989) 593 – 606. [15] F.M.A. McNabb, R.A. McNabb, Skin and plumage changes during the development of thermoregulatory ability in Japanese quail chicks, Comp. Biochem. Physiol. 58A (1977) 163 – 166. [16] P.F.A. Maderson, A.H. Zucker, S.I. Roth, Epidermal regeneration and percutaneous water loss following cellophane stripping of reptile epidermis, J. Exp. Zool. 204 (1978) 11 – 32. [17] A.G. Gibbs, A.K. Louie, J.A. Ayala, Effects of temperature on cuticular lipids and water balance in desert Drosophila: is thermal acclimation beneficial? J. Exp. Biol. 201 (1998) 71 – 80. [18] P.C. Withers, S.S. Hillman, R.C. Drewes, Evaporative water loss and skin lipids of anuran amphibians, J. Exp. Zool. 232 (1984) 11 – 17. [19] J.B. Roberts, H.B. Lillywhite, Lipid barrier to water exchange in reptile epidermis, Science 207 (1980) 1077 – 1079. [20] J.B. Roberts, H.B. Lillywhite, Lipids and the permeability of epidermis from snakes, J. Exp. Zool. 228 (1983) 1 – 9. [21] R.R. Burken, P.W. Wertz, D.T. Downing, A survey of polar and nonpolar lipids extracted from snake skin, Comp. Biochem. Physiol. 81 (1985) 315 – 318. [22] J.A. Bouwstra, et al., Structure of the skin barrier and its modulation by vesicular formulations, Prog. Lipid Res. 42 (2003) 1 – 36. [23] E. Proksch, et al., Barrier function regulates epidermal lipid and DNA synthesis, Br. J. Dermatol. 193 (1993) 473 – 482. [24] G.K. Menon, et al., Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing, J. Morphol. 227 (1996) 1 – 13. [25] B.I. Tieleman, J.B. Williams, Cutaneous and respiratory water loss in larks from arid and mesic environments, Physiol. Biochem. Zool. 75 (2002) 590 – 599. [26] M.J. Haugen, et al., Lipids of the stratum corneum vary with cutaneous water loss along a temperature– moisture gradient, Physiol. Biochem. Zool. (2003) 907 – 917. [27] R.O. Potts, M.L. Francoeur, Lipid biophysics of water loss through the skin, Proc. Natl. Acad. Sci. 87 (1990) 3871 – 3873. [28] T.J. McIntosh, Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol, Biophys. J. 85 (2003) 1675 – 1681. [29] J.A. Bouwstra, et al., The lipid organization in the skin barrier, Acta Derm.-Venereol. Supp 208 (2000) 23 – 30. [30] P.M. Elias, et al., Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical and functional considerations, Am. J. Anat. 180 (1987) 161 – 177.
www.ics-elsevier.com
Plasticity of the water barrier in vertebrate integument Harvey B. Lillywhite* Department of Zoology, University of Florida, Gainesville 32611, USA
Abstract. Water barrier function in vertebrates is typically relatively fixed and characteristic of species. However, both plasticity and genetic adaptation can account for covariation between environment and resistance to transepidermal water passage (R s). The capacity to adjust R s may involve phenotypic plasticity, acclimation, or developmental plasticity, and fundamental mechanisms include changes in barrier thickness, composition and physicochemical properties of cutaneous lipids, and/or geometry of the barrier within the epidermis. In amniotes, lipid barriers are structured within keratin complexes that, while important to efficacy of function, appear to constrain the possibilities and time course of plastic responses. Studies of the relative importance of plastic responses and genetic variation are few and inconclusive. D 2004 Elsevier B.V. All rights reserved.
Keywords: Vertebrate; Skin; Epidermis; Lipid; Water permeability
1. Introduction Terrestrial environments pose numerous challenges to the physiology of animals, especially larger, more active species that interact conspicuously with the physical landscape and microclimate [1]. Survival dictates that animals acquire sufficient water and energy from the environment to maintain cellular function and to support a level of metabolism sufficient for maintenance, growth and reproduction. Water is key in providing an adequate milieu for biochemical transformations and is potentially lost across body surfaces and in secretions and excreta. Evaporation of water from permeable surfaces generally constitutes the greatest source of loss, key routes of transfer being the skin and respiratory surfaces.
* Tel.: +1 352 392 1101; fax: +1 352 392 3704. E-mail address: [email protected]. 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.08.088
284
H.B. Lillywhite / International Congress Series 1275 (2004) 283–290
Clearly, features of integument that limit cutaneous water loss (CWL) are of key significance in the evolutionary radiation of vertebrates in terrestrial and especially xeric environments. Although numerous aspects of cutaneous morphology potentially influence CWL, it is well accepted that the principal barrier to transepidermal water flux consists of lipids having adaptive geometry and composition. Indeed, lipids provide a range of waterproofing mechanisms in terrestrial plants, arthropods, and vertebrates [2]. While a fair number of studies representing all three principal taxa demonstrate badaptiveQ variation in CWL in the sense that lower rates are often associated with drier environments, less is known about the stimuli and mechanisms that are causally related to this variation, either in terms of genetic adjustments or of phenotypic responses to the environment. Even less is known relating these mechanistic issues to fitness consequences of plasticity in nature and potentially related genetic constraints. This paper briefly reviews what is known concerning plasticity of water barrier function from a perspective that considers evolutionary trends and habitat demands of vertebrates. Understanding the water relations of vertebrate integument has benefited from technological advances and physicochemical studies of lipids from other systems such as arthropods and artificial membranes [3,4]. It is hoped that such advances and techniques from dermatological disciplines will find marriage with investigations that embrace comparative and evolutionary perspectives as well as application to understanding of pending environmental change [5]. 2. Water barriers and morphology of integument Although numerous attributes of skin have been proposed to influence skin resistance to evaporative water loss (R s), it is widely accepted that lipids provide the principal barrier to transepidermal water loss (TEWL). In both plants and arthropods, lipids are associated with or deposited on a multilayered cuticle. These surface lipids constitute the principal resistance to water passage across the cuticle or integument of these organisms [2]. Importantly, the cuticle of these organisms is a rigid structure and provides a stable platform for stability of the lipid barrier. Terrestrial vertebrates evolved from aquatic ancestors, and a permeability barrier was an important feature of integument that evolved in concert with increased mechanical resilience related to increasing size and terrestrial demands. Mechanical toughness of the integument was provided by the evolution of a stratum corneum comprised of multiple layers of fibrous keratins with crude correlations between degree of terrestriality and degree of keratinization. Living aquatic, semi-aquatic and terrestrial but dehydrationsensitive amphibians exhibit a stratum corneum comprised of but one or two cell layers of keratin. In contrast, reptiles, birds and mammals possess a much thicker stratum corneum having multiple layers of keratinized cells and, in sauropsids (reptiles and birds), added strength and rigidity due to presence of h-type keratins. The amphibian skin is an important site of gaseous and ionic exchange, and is highly permeable to bi-directional water flux in many species, whereas the amniotes have better developed lungs and generally do not exchange gases and ions extensively across the skin. The multilayered stratum corneum of amniotes also provides a structural template for sealing lipids that perform a water barrier function. Unlike plants and arthropods, the water barrier lipids of
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Fig. 1. Schematic models of vertebrate integument illustrating fundamental dichotomy in means of preventing dehydration of epidermis and whole animal. In the amphibious anuran (left), either externally present moisture or aqueous mucus secreted from dermal glands maintains a wet exterior of epidermis, and therefore water evaporates from the external water film rather than from the underlying epidermis. Water is replenished from the environment, either directly or (in the case of mucus discharge) by means of cutaneous absorption across the ventral seat patch, then via blood circulation to the mucous secretions within mucous glands. In the arboreal frog (left center), lipids are secreted onto the skin surfaces from dermal glands and form a barrier to water evaporation once they dry on the exterior. A lipid barrier functions similarly in the squamate reptile (center right), bird and mammal (right) except the barrier lipids are structured internally within the stratum corneum. Lipids are shown in gray shading. The large bold broken lines represent stratum germinativum.
amniotes are contained within, not upon, the integument. In either case, however, the barrier is located superficially and distal to the vascularized tissues of the inner skin. Among vertebrates, there is a fundamental dichotomy reflecting structure of integument and associated strategies for water balance. Either the epidermis is protected from excessive water loss by a lipid or keratin–lipid water barrier (amniotes and some amphibians), or the epidermis is maintained in a hydrated state by an overlying aqueous film or external moisture from an external source (many amphibians and some reptiles). In either strategy, the location of the lipid or aqueous film reflects the requirement for maintaining water balance of the skin itself as well as that of the entire organism (Fig. 1). Thus, when bullfrogs are basking in dehydrating environments, discharge of mucus onto the skin surfaces increases with increasing body temperature—a response interpreted to maintain a hydrated integument as well as evaporative cooling in the interest of thermoregulation [6]. These frogs do not bask or risk dehydration if water is unavailable to replenish body stores that contribute to the mucus secretions and TEWL. On the other hand, various arboreal frogs inhabiting yet drier environments secrete lipids onto the skin and wipe them over the body surfaces to form a protective water barrier that restricts losses of body water and protects the hydration status of underlying epidermis. In terrestrial amniotes, the skin is protected by a water barrier of variable effectiveness in the lipid– keratin complex of the stratum corneum (Fig. 1). 3. Mechanisms for renewal and regulation of the water barrier There are numerous potential means by which water barriers associated with vertebrate integument can be adjusted to meet environmental demands. Fundamentally, these can be reduced to the categories of response that follow in the subsequent subsections.
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3.1. Presence or absence of barriers Whether or not an effective water barrier is present in skin is perhaps most relevant to evolutionary radiation of animals and genetically determined adaptations of species. Barrier function in amniotes appears to be relatively fixed, heritable, and characteristic of species (e.g. Ref. [7]). There are few known examples of vertebrates that can produce a water barrier anew—facultatively—in response to environmental demands, while otherwise not possessing one. With respect to both genetic and plastic responses, species of amphibians provide well-studied examples. Many amphibians, and perhaps a majority, evaporate water across the skin at rates similar to that from a free water surface, and there are no known barriers to water passage in these species. In contrast, various species of anurans inhabiting arid or semi-arid environments exhibit features of integument that pose well-known barriers to TEWL [8]. The more efficacious examples include cocoons that form in burrowing anurans during aestivation and epicutaneous lipids that are secreted from dermal glands and wiped over the body surfaces in elaborate behaviors thought to stimulate secretion and spreading of lipids over the entire body surface [9]. Such behaviors are observed prior to animals retiring from activity and assuming a water-conserving posture, or when frogs are subjected to gradual dehydration and disturbance by handling [9,10]. The stimulus that elicits the behavior is not clear, however, and it is not known how bcleanQ the skin becomes between bouts of wiping when R s might potentially decrease as lipids wear away. 3.2. Barrier thickness The amphibian examples also are relevant to mechanisms for increasing thickness of the water barrier. Characteristic cocoons consist of multiple single cell-thick layers of shed stratum corneum resulting from multiple shedding of the skin layers during periods of dormancy in drying soils. Such cocoons may consist of 40–60 layers of cornified cells with secreted lipids and proteinaceous materials sandwiched between them [11]. These structures impose considerable resistance to water movement, and the barrier efficacy presumably increases with the numbers of layers or thickness of the structure. In frogs that wipe lipids over the body surface, tactile stimulation of the wiping elicits reflexive release of lipids from dermal glands, so the thickness of the final lipid layer could presumably be controlled to some extent by the duration and nature of the wiping [9]. The externally wiped layer of lipid was estimated to be about 0.2 Am in phyllomedusine frogs [12], but nothing is known about its variability either among or within species of anurans. In amniotes, the barrier region of the stratum corneum provides a tough and resilient framework for intercellular lamellar lipids, and the laminated feature of the entire lipid– keratin structure is a means by which thickness of the barrier might be altered. Hypothetical proliferation of additive layers would enhance R s to water permeation if the requirement was increased in response to increased drying power of the ambient atmosphere. Is there evidence this mechanism is utilized? Recently, it was demonstrated that R s essentially doubles following the first postnatal ecdysis in California king snakes (Lampropeltis getula), and the increased resistance correlates nicely with a doubling in thickness and number of strata of the specialized
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mesos layer of epidermis that constitutes the water barrier in this and other species of squamates [13]. This response is interpreted as an adaptive adjustment of R s during the natal transition from aqueous environment of the embryo to atmospheric air that surrounds the newborn. Some lizards also exhibit upregulation of the cutaneous water barrier as a response to changes in environmental conditions, and changes in cutaneous lipids are known to be involved [14]. However, it remains unclear as to whether such changes in R s might be correlated with changes in thickness of the water barrier. Chicks of the Japanese Quail (Coturnix c. japonica) exhibit a 43% decrease in skin water permeability and approach adult values within 13 days post-hatching. Histological data suggest that increased barrier efficacy is attributable to epidermal thickening, as in snakes [15]. In other studies, reptilian skin responds to trauma such as tape stripping with a bthickeningQ response involving hyperplasia of the a-keratin keratinocytes [16]. Human disorders of cornification in various disease states can result in different densities or thickness of stratum corneum and intercellular bilayer structures [3]. However, comparative studies of possible adaptive modifications of such structures in relation to natural environments have not been undertaken. 3.3. Composition of barrier lipids While a few generalizations are appropriate regarding composition of lipid classes in various water barriers of vertebrates, the subject is one that has amazing complexity. In every case examined, permeability barriers contain a complex mixture of lipid molecules. Longer chain-length hydrocarbons tend to comprise a dominant category of lipids in most barriers examined. These will tend to melt at higher temperatures and will result in reduced water permeation, whereas short chain length molecules reduce the intensity of van der Waals interactions between hydrocarbon molecules and create a more fluid structure that is more readily penetrated by water. Relative saturation of hydrocarbons also contributes to a tighter water barrier, whereas unsaturation and methyl branching tends to introduce kinks in molecules and disrupts packing. In some systems, however, chain elongation and unsaturation may offset one another, such that chain length alone is not necessarily a reliable indicator of water permeation [17]. Hydrocarbons and wax esters are both very nonpolar, which also assists in repelling water. Polar phospholipids and other classes of lipids having intermediate polarity, in addition to branching, might be important in structuring the geometry of a water barrier and providing a degree or specific orientation of fluidity important with respect to potential mechanical distortion or disruption of the barrier structure. Detailed information on cutaneous lipids of amphibians can be found in relatively few published studies. Analyses of the epicutaneous lipids secreted during wiping behaviors of phyllomedusine frogs demonstrate a mixture of hydrocarbons, triacylglycerols, cholesterol, cholesterol esters, free fatty acids and wax esters [12]. Wax esters are dominant and average about 46 carbons in length. Other studies demonstrate a cocktail of lipids that can be extracted from skin of frogs that do not exhibit wiping behaviors, and there is generally no correlation between these lipid properties and rates of evaporative water loss [18]. However, lipid mixtures extracted from whole skin undoubtedly include elements of membrane lipids not associated with a water barrier as well as precursor molecules that might be converted to other components when a barrier is present. There is no information
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that sheds light on how composition of barrier lipids are altered in relation to facultative waterproofing of skin by wiping or formation of a cocoon. Studies of squamates demonstrate a complex mixture of lipids including hydrocarbons, wax and sterol esters, di- and triacylglycerols, free fatty acids, alcohols, cholesterol, ceramides, and a variety of phospholipids present in skin, with concentration shown histochemically in the mesos layer of epidermis [19–21]. Analyses of lipids extracted from the skin of snakes are complicated potentially by presence of lipid classes important as sex attractant pheromones, in addition to barrier lipids that might be present. Again, there is no information regarding possible regulation of compositional features of lipids in relation to plasticity of barrier function. The cutaneous lipids of mammals have been studied extensively, but largely in humans and laboratory rodents and in contexts of disease and cosmetic interests in skin. Lamellar bodies provide precursor lipids consisting mainly of glycosphingolipids, free sterols and phospholipids that are delivered to the extracellular spaces in developing stratum corneum. Subsequently, these are enzymatically converted to nonpolar products and assembled into lamellar structures surrounding the corneocytes. The changes in lipid composition result in a barrier structure consisting largely of ceramides, free fatty acids, and cholesterol [22]. It is known in mammals that barrier structure and function is genetically determined and is rapidly restored following trauma. Furthermore, transformation of the precursor lipids to nonpolar-enriched barrier lipids of the stratum corneum is susceptible to disturbance by a number of factors, including essential fatty acid deficiency, genetic defects and enzyme deficiencies, environmental factors and hydration status [3]. These and various disease states are known to cause dysfunction of the water barrier and abnormally elevated TEWL. Barrier defects are indeed reflected in the lipid composition of the stratum corneum. Experimental studies of mammalian epidermis demonstrate that mechanical or chemical disruption of normal barrier morphology influences cellular kinetics, protein and lipid synthesis within 15–20 h that restore the barrier efficacy (reviewed in Ref. [23]). However, covariation of lipids and TEWL in relation to variation of natural stressors in the environment of mammalian species has not been investigated. Recent investigations have demonstrated facultative waterproofing in the skin of birds. When subjected to conditions of dehydration, adult zebra finches are capable of rapid upregulation of the cutaneous water barrier. Within 16 h of water deprivation, TEWL decreases by 50%, and the skin barrier efficacy continues to improve until mammal-like values are achieved [24]. Similarly, hoopoe larks in extremely arid regions of the Arabian Peninsula are characterized by TEWL rates about 30% lower than that of larks from mesic environments, and these rates decrease significantly when birds are acclimated to high temperatures [25]. In these desert birds, adjustments of lipid ratios have been shown to favor ceramides over free fatty acids and sterols, and these changes correlate with reductions of TEWL [26]. The higher ratios of ceramides evidently allow the lipids of the permeability barrier to exist in a more highly ordered crystalline phase, which creates a tighter barrier to water vapor diffusion. Acclimation of water loss rates does not appear to occur in skylarks and woodlarks from mesic environments in Europe, nor in Dunn’s larks from the Arabian desert [25]. Thus, capacity for plasticity of the permeability barrier appears to vary among species and, in the case of larks, is characteristic only of species inhabiting more extreme desert regions.
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3.4. Physical properties and geometry There is a great deal of information relating to physicochemical properties of lipids and lipid membranes, and it is well accepted that permeability can differ quantitatively because of the physical nature of the water movement pathway. It is important to note that in models of vertebrate barriers, lipids form a continuous barrier in the horizontal dimension parallel with the skin surface (Fig. 1). Either immobility of the animal (surface lipids of amphibians) or a tough, resilient lattice of keratin (stratum corneum–lipid complex of amniotes) appears to be important for optimal integrity of barrier function. It is not known to what degree physical disruption of the barrier structure necessitates repair or renewal during normal activity of animals that might bend or jar the skin. Recent studies that employ molecular models suggest that resistance to water permeation of mammalian stratum corneum is related, in part, to tight, gel-like packing of hydrocarbon chains and changes in lipid phase behavior related to component ratios and molecular arrangement of cholesterol [27,28]. Additionally, fluid and crystalline phases of the lipid lattice alternate vertically in repetition with stacked lamellae [29]. The crystalline phase enhances barrier efficacy, while the presence and localization of fluid domains facilitate elasticity in relation to geometry of the barrier and lipid interactions with keratin components. Models further suggest that diffusion of water is limited in directions both parallel and perpendicular to the plane of the lipid bilayers due to their organization [28]. In avian skin, the efficacy of facultative waterproofing resides in a capacity to modulate the organization of secreted lipids, viz. non-bilayer, electron-lucent lipids under basal conditions, but lamellar lipid structures in response to xeric stress, leading to significantly decreased TEWL [24]. Both compositional features of cutaneous lipids and the hydration status of the skin will influence the phase properties of the barrier, which becomes disrupted as the skin becomes progressively hydrated. Unlike terrestrial mammals, the stratum corneum of marine mammals retains appreciable amounts of glycolipids [30]. 4. Conclusion In spite of extensive literature on membrane physiology and the structure and function of mammalian skin in relation to disease and cosmetics, there is comparatively little understanding of adaptive adjustments of permeability barriers in contexts of evolution, phylogeny and environment. Comparative and experimental approaches with goals of understanding mechanisms of plasticity in relation to adaptation will hopefully remedy this deficit of understanding in the future. Such investigations should include, or interface with, genetics, morphology, ecology and evolutionary disciplines, as well as physiology. References [1] W.P. Porter, et al., Calculating climate effects on birds and mammals: impacts on biodiversity, conservation, population parameters, and global community structure, Am. Zool. 40 (2000) 597 – 630. [2] N.F. Hadley, Integumental lipids of plants and animals: comparative function and biochemistry, Adv. Lipid Res. 24 (1991) 303 – 320. [3] G. Menon, R. Ghadially, Morphology of lipid alterations in the epidermis: a review, Microsc. Res. Tech. 37 (1997) 180 – 192. [4] A.G. Gibbs, Water-proofing properties of cuticular lipids, Am. Zool. 38 (1998) 471 – 482.
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[5] P.M. Kareiva, J.G. Kingsolver, R.B. Huey (Eds.), Biotic Interactions and Global Change, Sunderland, Sinauer Associates, Massachusetts, 1993. [6] H. Lillywhite, Thermal modulation of cutaneous mucus discharge as a determinant of evaporative water loss in the frog, Rana catesbeiana, Z. Vgl. Physiol. 73 (1971) 84 – 104. [7] F. Furuyama, K. Ohara, Genetic development of an inbred rat strain with increased resistance adaptation to a hot environment, Am. J. Physiol. 265 (Part 2) (1993) R957 – R962. [8] R.C. Toledo, C. Jared, Cutaneous adaptations to water balance in amphibians, Comp. Biochem. Physiol. 105A (1993) 593 – 608. [9] H.B. Lillywhite, et al., Wiping behavior and its ecophysiological significance in the Indian tree frog Polypedates maculatus, Copeia 1997 (1997) 88 – 100. [10] L.A. Blaylock, R. Ruibal, K. Plat-Aloia, Skin structure and wiping behaviour of phyllomedusine frogs, Copeia 1976 (1976) 283 – 295. [11] R. Ruibal, S.S. Hillman, Cocoon structure and function in the burrowing hylid frog, Pternohyla fodiens, J. Herpetol. 15 (1981) 403 – 408. [12] L.L. McClanahan, J.N. Stinner, V.H. Shoemaker, Skin lipids, water loss, and energy metabolism in a South American tree frog (Phyllomedusa sauvagei), Physiol. Zool. 51 (1978) 179 – 187. [13] M.C. Tu, et al., Postnatal ecdysis establishes the permeability barrier in snake skin: new insights into lipid barrier structures, J. Exp. Biol. 205 (2002) 3019 – 3030. [14] G.H. Kattan, H.B. Lillywhite, Humidity acclimation and skin permeability in the lizard Anolis carolinensis, Physiol. Zool. 62 (1989) 593 – 606. [15] F.M.A. McNabb, R.A. McNabb, Skin and plumage changes during the development of thermoregulatory ability in Japanese quail chicks, Comp. Biochem. Physiol. 58A (1977) 163 – 166. [16] P.F.A. Maderson, A.H. Zucker, S.I. Roth, Epidermal regeneration and percutaneous water loss following cellophane stripping of reptile epidermis, J. Exp. Zool. 204 (1978) 11 – 32. [17] A.G. Gibbs, A.K. Louie, J.A. Ayala, Effects of temperature on cuticular lipids and water balance in desert Drosophila: is thermal acclimation beneficial? J. Exp. Biol. 201 (1998) 71 – 80. [18] P.C. Withers, S.S. Hillman, R.C. Drewes, Evaporative water loss and skin lipids of anuran amphibians, J. Exp. Zool. 232 (1984) 11 – 17. [19] J.B. Roberts, H.B. Lillywhite, Lipid barrier to water exchange in reptile epidermis, Science 207 (1980) 1077 – 1079. [20] J.B. Roberts, H.B. Lillywhite, Lipids and the permeability of epidermis from snakes, J. Exp. Zool. 228 (1983) 1 – 9. [21] R.R. Burken, P.W. Wertz, D.T. Downing, A survey of polar and nonpolar lipids extracted from snake skin, Comp. Biochem. Physiol. 81 (1985) 315 – 318. [22] J.A. Bouwstra, et al., Structure of the skin barrier and its modulation by vesicular formulations, Prog. Lipid Res. 42 (2003) 1 – 36. [23] E. Proksch, et al., Barrier function regulates epidermal lipid and DNA synthesis, Br. J. Dermatol. 193 (1993) 473 – 482. [24] G.K. Menon, et al., Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing, J. Morphol. 227 (1996) 1 – 13. [25] B.I. Tieleman, J.B. Williams, Cutaneous and respiratory water loss in larks from arid and mesic environments, Physiol. Biochem. Zool. 75 (2002) 590 – 599. [26] M.J. Haugen, et al., Lipids of the stratum corneum vary with cutaneous water loss along a temperature– moisture gradient, Physiol. Biochem. Zool. (2003) 907 – 917. [27] R.O. Potts, M.L. Francoeur, Lipid biophysics of water loss through the skin, Proc. Natl. Acad. Sci. 87 (1990) 3871 – 3873. [28] T.J. McIntosh, Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol, Biophys. J. 85 (2003) 1675 – 1681. [29] J.A. Bouwstra, et al., The lipid organization in the skin barrier, Acta Derm.-Venereol. Supp 208 (2000) 23 – 30. [30] P.M. Elias, et al., Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical and functional considerations, Am. J. Anat. 180 (1987) 161 – 177.