SUMMARY

Evolutionary transitions between different environmental media such as air and water pose special problems with respect to skin permeability because of the dramatic changes in the driving gradients and nature of water exchange processes. Also, during the transitional periods prior to complete adaptation to a new medium, the skin is exposed to two very different sets of environmental conditions. Here, we report new data for transepidermal evaporative water loss (TEWL) and cutaneous resistance to evaporative water loss (Rs) of sea snakes that are transitional in the sense of being amphibious and semi-terrestrial. We investigated three species of sea kraits (Elapidae: Laticaudinae) that are common to Orchid Island (Lanyu),Taiwan. Generally, Rs of all three species is lower than that characteristic of terrestrial/xeric species of snakes measured in other taxa. Within Laticauda, Rs is significantly greater (TEWL lower) in the more terrestrial species and lowest (TEWL highest)in the more aquatic species. Previously reported losses of water from snakes kept in seawater exhibit a reversed trend, with lower rates of loss in the more aquatic species. These data suggest selection for adaptive traits with respect to increasing exposure to the marine environment. Thus, a countergradient of traits is reflected in decreased TEWL in aerial environments and decreased net water efflux in marine environments, acting simultaneously in the three species. The pattern for TEWL correlates with ultrastructural evidence for increased lipogenesis in the stratum corneum of the more terrestrial species. The skin surfaces of all three species are hydrophobic. Species differences in this property possibly explain the pattern for water efflux when these snakes are in seawater, which remains to be investigated.

INTRODUCTION

Vertebrate integument evolved with selective compromise between the needs for mechanical protection and those of sensing the environment and regulating the exchange of materials and energy. One of the very important exchange functions of integument is the regulation of water content with an associated permeability barrier that limits excessive transepidermal evaporative water loss (TEWL) in terrestrial species(Lillywhite, 2006). Lipids comprise the principal water barrier in terrestrial plants, arthropods and vertebrates and they assume increasing importance in the water relations of organisms in arid environments (Hadley,1989; Hadley,1991; Lillywhite,2006).

The barrier to water loss in a majority of terrestrial tetrapods is conferred by the outer layers of epidermis, consisting of dead, keratin-filled cells embedded within a lipid matrix that has been likened to a`bricks-and-mortar' configuration (Elias,1983; Elias and Menon,1991). The barrier prevents dehydration, protects the body against infection and contamination from the environment, and is essential for terrestrial life. As species invaded harsher and drier environments, the skin became an important target of natural selection, producing variation in both quantitative and qualitative features of the permeability barrier and the associated efficacy of skin resistance to evaporative water loss(Rs). Generally, Rs reflects adjustments of the composition and quantity of lipids that comprise the water barrier, which in turn reflect the nature of the environmental challenge that is presented to a given species. Numerous studies have demonstrated that rates of TEWL vary inversely with habitat aridity(Dmi'el, 1998; Gans et al., 1968; Lahav and Dmi'el, 1996; Mautz, 1982a; Mautz, 1982b; Muñoz-Garcia and Williams,2005; Roberts and Lillywhite,1980).

Evolutionary transitions between different environmental media pose special problems with respect to skin permeability because of the dramatic changes in the driving gradients and nature of water exchange processes. Also, during the transitional periods prior to complete adaptation to a new medium, the skin is exposed to two very different sets of environmental conditions. Moreover,competing needs can complicate the issues; e.g. the need to conserve water in marine environments while simultaneously disposing of nitrogen and CO2 with oxygen acquisition in the reverse direction. Aerial environments impose strong gradients for evaporation, and species that occur in arid habitats exhibit the higher Rs values that have been measured in terrestrial organisms(Lillywhite, 2006).

Here, we report new data for TEWL and Rs of sea snakes that are transitional in the sense of being amphibious and semi-terrestrial. We investigated three species of sea kraits (Elapidae: Laticaudinae) that are common to Orchid Island (Lanyu), Taiwan. Laticauda colubrina(Schneider) is the more terrestrial of the three species and spends considerable time ashore, where individuals seclude themselves in caves or among rocks, sometimes moving considerably inland and spending as much as half their time out of the sea. Laticauda semifasciata (Reinhardt) is the more fully marine species and comes ashore seldom, for comparatively short distances and primarily to oviposit eggs. The third species, Laticauda laticaudata (Linnaeus), is intermediate with respect to terrestrial habits, spending limited sojourns ashore and usually in a narrow intertidal zone without penetrating inland (Brischoux and Bonnet, 2009). The terrestrial environments of all three species can be considered mesic insofar as they consist of moist and secluded places generally within or very near to the intertidal zone, with possible exceptions for L. colubrina.

The purposes of our investigation were to measure rates of TEWL and to compare values of permeability and Rs among these three species. We compare these features of skin with rates of water loss that were determined previously from snakes kept in seawater. We also compare the ultrastructural features of the permeability barrier among these species.

MATERIALS AND METHODS

Animals

We investigated TEWL and Rs in each of the three species of sea kraits. All animals were collected at coastal sites on the island of Lanyu (Orchid Island), Taiwan(Lillywhite et al., 2008a) and returned to the laboratory at the National Taiwan Normal University in Taipei.

Measurements of evaporative water loss and skin resistance

We measured TEWL, skin temperatures and calculated Rsin 15 Laticauda colubrina, nine L. laticaudata and 12 L. semifasciata. We measured rates of TEWL by means of a Delfin VapoMeter® (Delfin Technologies Ltd, Kuopio, Finland) applied to the dorsolateral skin at mid-body of live snakes while they rested within mesh bags. The bag was opened just enough to allow access to a patch of skin that was measured, allowing minimum disturbance to a snake. In most cases, the snake remained quiescent while the body at the site of measurement reflexly pushed against the open port of the VapoMeter. Thus, the reflexive behavior of the snakes ensured a tight seal during measurements, each taking 14–34 s. If a snake moved during the measurement, the datum was discarded and the measurement repeated. Each individual was measured repeatedly 5–10 times, and these data were averaged to produce a single mean measurement for each individual snake. A 40-gauge thermocouple was applied to the skin at the site of measurement to determine the skin surface temperature, using a TES 1314 30-gauge thermocouple thermometer (TES Electrical Electronic Corporation,Taipei, Taiwan). During measurements, ambient air temperature and humidity varied from 24.5 to 25.6°C and from 46.2 to 56.8%, respectively, among different trials but remained virtually stable during individual trials.

The skin resistance to evaporative water flux was calculated as Rs=RtRb,where Rt is the total resistance and Rb is the boundary layer resistance. The total resistance was calculated as follows:
\[R_{\mathrm{t}}=[\mathrm{WVD}_{\mathrm{s}}-(\mathrm{RH}{\times}\mathrm{WVD}_{\mathrm{a}})]{\ }\mathrm{TEWL}^{-1},\]
where WVDs is the saturated water vapor density at skin temperature, WVDa is the saturated water vapor density at ambient chamber temperature, and RH is the relative humidity of ambient air(Spotila and Berman, 1976). The boundary layer resistance was determined as above, utilizing measurements of TEWL from patches of saturated tissue paper that evaporated as a free water surface. Values of Rb were less than 5% of Rt.

Measurements of water efflux in seawater

For comparisons of aquatic water fluxes with evaporative losses of water in air, we used data that were published (for `control' animals) previously(Lillywhite et al., 2008a). In brief, snakes of each species were kept in full seawater for a period of 37 days. They were removed from water momentarily every third day, dried by gentle blotting with a towel, weighed and returned to seawater. These snakes do not drink seawater and were not fed during the dehydration trials.

Ultrastructure and histochemistry

Skin samples from two or three individuals of each species were fixed in Karnovski's fixative for 24 h, washed in sodium cacodylate buffer, osmicated in 1% osmium tetroxide (OsO4), dehydrated through a graded series of alcohol, and routinely embedded in a low viscosity mixture of epon-epoxide(McNutt and Crain, 1981). To demonstrate the barrier lipid structures, skin samples were post-fixed with 0.5% ruthenium tetroxide (RuO4; instead of OSO4) for 1 h and then processed as above. With respect to routine histology, semi-thick sections (0.5–1 mm) of OSO4-fixed samples were stained with Toluidine Blue for light microscopy, while silver-gray sections were double-stained with uranyl acetate and lead citrate, then visualized under a Zeiss EM 12 electron microscope. Silver-gray sections from RuO4-fixed samples were observed with and without double staining for evaluating the lipid structural organization.

Statistics

Data are reported as means ± s.e.m. To evaluate variation in measured variables related to TEWL, we performed an ANOVA and then employed post-hoc tests to examine differences between species. In other circumstances, we employed paired t-tests as described elsewhere in the text. All analyses were performed using SAS StatView© 5.0.1 for Windows (Cary, NC, USA).

RESULTS

Evaporative water loss and skin resistance

The measured mean TEWL of semi-terrestrial L. colubrina is 0.189±0.03 mg cm–2 h–1, that of the relatively more aquatic L. laticaudata is 0.294±0.05 mg cm–2 h–1 and that of the most fully aquatic L. semifasciata is 0.469±0.05 mg cm–2h–1 (ANOVA, P<0.0001). Corresponding Rs values are 401±103, 176±33 and 93±12.9 s cm–1, respectively. Thus, Rs is significantly greater in the more terrestrial species and lowest in the more aquatic species; accordingly, there is a reverse ranking of skin permeability (ANOVA, P<0.0001)(Fig. 1).

Losses of water from snakes kept in seawater exhibit a reversed trend, with lower rates of loss in the more aquatic species (P<0.0001)(Lillywhite et al., 2008a). The mean % body mass lost per day is 0.68±0.05 in L. colubrina, 0.50±0.04 in L. laticaudata and 0.33±0.02 in L. semifasciata. Taking metabolic losses into account (Lillywhite et al.,2008a), roughly 75% of the total mass loss is attributed to losses of body water (Fig. 2).

The Rs measured in Laticauda spp. are below the known measurements of most other snakes for which data are available(Lillywhite, 2006), with the exception of Rs reported for the marine file snake, Acrochordus granulatus(Lillywhite and SanMartino,1993), which is similar to that of the highly marine L. semifasciata (Fig. 3). There are no published data for other species of elapid snakes, so we took the opportunity while in Taiwan to measure Rs in a terrestrial krait (Bungarus multicinctus Blyth) and a terrestrial cobra (Naja atra Cantor). The Rs measured in B. multicinctus, a secretive, mesic elapid, was similar to that of L. semifasciata, whereas that of N. atra was somewhat greater than that of the semi-terrestrial L. colubrina(Fig. 3).

Ultrastructure of the epidermis

All three species of Laticauda had common features typical of ophidian epidermal organization, such as the vertical differentiation into an outermost beta layer, followed by a mesos layer (5–8 cell layers thick)that separates it from the alpha keratinizing layer, nucleated layers of immature alpha cells (one cell thick) and, below that, a single germinative layer (Fig. 4). The beta layer is essentially similar in all three species, being composed of what resembles spongy, `air-filled' cells, the boundaries of which are well defined(Fig. 5), unlike in terrestrial snakes. The spongy appearance of beta cells is reminiscent of the air-filled keratinous rachis of avian feathers. Due to the very different mechanical properties of these layers, it is almost impossible to obtain thin sections where the layers are not separated from each other, especially when exposed to the electron beam under the microscope.

Two features distinguish the sea snake epidermis from terrestrial snake epidermis: (1) the absence of a syncytial appearance of the beta layer(Fig. 4 and Fig. 5) and (2) a pronounced lipogenesis in the alpha keratinizing layer. The lipogenesis was most pronounced in the more terrestrial L. colubrina(Fig. 4 and Fig. 6), followed by L. laticaudata (Fig. 7), and was least obvious in the more fully aquatic L. semifasciata(Fig. 8).

The mesos layers consist of approximately 5–8 cell layers, which is quite similar to the mesos layer reported for terrestrial snakes(Tu et al., 2002). Some of the cells show slightly electron-lucent core areas. Ruthenium tetroxide penetration into these layers was poor, possibly owing to the nature of the beta layer and resulting in patchy staining. However, stained areas of mesos layers showed multiple lamellae surrounding the mesos layer cells, some of which were etched away by reactive RuO4. In such areas, the lamellar lipids were well stained and visualized(Fig. 9).

The alpha layer also showed some interesting features. Several areas of a section show electron-lucent domains within individual alpha cells, similar to what is seen in the soft scales of avian feet(Menon and Aggarwal, 1982). Subjacent to these areas, the immature alpha cells appear highly lipogenic,containing several free lipid droplets (see Fig. 4, Fig. 6 and Fig. 7). Sometimes these highly lipogenic cells are separated by one or more cells that show no lipid inclusions at all.

In all three species, the cell-to-cell junction between the alpha layer and the immature alpha cells consistently showed interesting junctional specializations. These are invaginations into the nucleated layer, almost regularly placed between two adjacent desmosomes(Fig. 10) and they appear to be housing nerve endings (Fig. 11). Furthermore, tonofilaments that anchor on to the desmosomes at this interface extend well down into the nucleated cells, suggesting a specialized mechanism that couples the fully keratinized alpha cells with the nucleated cells below (Fig. 8and Fig. 10).

DISCUSSION

The water relations of sea snakes

The skin of reptiles is an important route of osmotic and evaporative water exchange, as is true in many other vertebrates(Lillywhite and Maderson,1982; Mautz,1982b; Minnich,1979; Lillywhite,2006). The resistance of skin to water passage varies considerably with environmental demands, and variation in Rs can be physiologically labile as a consequence of acclimation(Kattan and Lillywhite, 1989; Kobayashi et al., 1983) [for a review of mammalian data, see also Lillywhite(Lillywhite, 2007)]. Thus,skin resistance might vary intraspecifically as well as interspecifically with season and habitat, at least in some species(Dunson and Freda, 1985). Snakes have provided useful animals for studies of skin permeability, largely because of their whole-body synchronized ecdysis – characteristic of many squamates – and the variability exhibited among species that occupy a broad range of habitats. The variation of skin resistance among snake species correlates closely with the evaporative stress that is associated with different environments (Dmi'el,1998; Lahav and Dmi'el,1996; Lillywhite and SanMartino, 1993; Prange and Schmidt-Nielsen, 1969; Roberts and Lillywhite, 1983). Thus, heterogeneity of skin properties is related, in part, to the hydric stresses of the environment (see also Lillywhite, 2006).

Evolutionary transitions between different media (air/water) involve profound changes in the hydric environment, with maximum gradients of water potential driving water exchanges in terrestrial settings. Although less steep, gradients of water potential promoting losses of water from skin also exist in marine environments. Our data for water loss from semi-terrestrial sea kraits in the two environments are interesting and suggest there is dual adaptation to the different media. Generally, Rs of all three species is lower than that characteristic of terrestrial/xeric species of snakes measured in other taxa(Lillywhite, 2006). Moreover, Rs of the more aquatic L. semifasciata is similar to that reported for the marine snake A. granulatus(Lillywhite and SanMartino,1993) and our unpublished data for a secretive and mesic elapid, B. multicinctus (Fig. 3). Skin resistance of the more terrestrial L. colubrinafalls more closely to values we have measured in a terrestrial elapid N. atra, which is less secretive and occupies more exposed situations than does B. multicinctus (Fig. 3). This being said, values of Rs measured in three species of sea kraits vary positively with terrestrial tendencies(Fig. 1).

With respect to terrestrial exposure, variation of Rsamong the three species of sea kraits is graded in a direction that suggests these traits are adaptive (Fig. 1 and Fig. 3). While our data do not permit estimation of Rs for snakes subjected to dehydration in seawater, rates of water loss among the three species suggest there is selection for adaptive traits related to increasing exposure to the marine environment (Fig. 3). Thus, a countergradient of traits is reflected in the relative water loss patterns acting simultaneously in the three species: decreased TEWL(in air) but higher rates of water loss in seawater in L. colubrina,which spends much time in aerial environments, and an increased TEWL (in air)but lower rates of water loss in seawater in L. semifasciata, which is highly aquatic and spends most time in seawater. The rates of TEWL and water loss in seawater in L. laticaudata are intermediate to the other two species (Fig. 3). Using these data and a regression analysis for mass and surface area in another marine snake (Lillywhite and SanMartino, 1993), we estimated the rates of surface-specific water losses in seawater and found that expressing the water losses this way does not change the pattern relating species and the magnitude of such losses depicted in Fig. 3. How can we explain the differential behavior of the skin in air and in seawater?

There is increasing evidence to suggest that responses of skin that result in adjustments of the water permeability are stimulated by external humidity in terrestrial vertebrates (reviewed by Lillywhite, 2007). Exposure to dry air increases the rate of barrier recovery following tape stripping of mammalian epidermis, and exposure to low humidity increases DNA synthesis in normal epidermis and amplifies the DNA synthetic response to barrier disruption (Denda et al.,1998a; Denda et al.,1998b). Prolonged exposure to dry air results in a thicker, more competent stratum corneum and is regarded as a homeostatic adjustment of the barrier to environmental humidity. The precise signals that mediate these responses remain to be defined, but studies in which Vaseline® or treatment with a humectant prevents these changes suggest that humidity is a key variable and acts locally as an external stimulus(Denda et al., 1998b). Environmental humidity, stratum corneum water content, water flux through the stratum corneum, calcium gradients, altered osmotic pressure and induction of cytokines are likely all involved in complex, interactive ways(Lillywhite, 2007). Recently,it was shown that maturation of postnatal skin and development of the permeability barrier are more rapid in preterm infants that are nursed in environments of low relative humidity(Ågren et al., 2006). Studies by Kattan and Lillywhite (Kattan and Lillywhite, 1989) and Tu et al.(Tu et al., 2002) suggest that external humidity might act similarly on the integument of squamate reptiles. Therefore, we assume as a working hypothesis that adjustments of the ophidian permeability barrier occur in response to water potential gradients acting between skin and the environment.

The evolution of Rs among marine elapids can be inferred by the data from sea kraits, which exhibit higher Rs in the more terrestrial species and a reduction of Rs with progressively more marine habits. The more terrestrial L. colubrina possibly represent a transitional,amphibious stage in which elapids ancestral to sea snakes spent much time in terrestrial near-shore environments. Selection for high skin resistance to water loss was reduced in comparison with more terrestrial and xeric-habitat elapids, with further reduction as animals spent increasing time in the sea. We will assume further this sets the fundamental structural properties of the permeability barrier, which is comprised of mesos-layer lipids within the epidermis (Fig. 6, Fig. 7 and Fig. 9). This barrier cannot be changed rapidly, or reversed frequently, when amphibious animals enter or leave the sea. Therefore, another explanation must be invoked to explain the reversal of water loss properties when the snakes are in seawater. The countergradient properties of skin in seawater could possibly be explained by variation in the surface hydrophobic properties of the skin, which are currently the subject of ongoing investigations. The external skin surfaces of all three species of Laticauda we studied are hydrophobic, suggesting that the skin interacts with a microfilm of air at the surface of the stratum corneum (Fig. 12). We are exploring the hypothesis that variation in the nature of this air film is correlated with species differences in the efflux of water when these snakes are aquatic.

Ultrastructure of integument

From the ultrastructural features, the first line of defense/adaptation to aquatic life seems to be the beta layer, with its `spongy' structure and lack of syncytium, which possibly enhance the non-wetting properties of the skin surface. Generally, the tough outer beta layer of ophidian skin affords physical protection to the mesos layer, with its `bricks-and-mortar'organization of corneocytes and lipids that are the basis of the permeability barrier in amniote vertebrates(Lillywhite, 2006). However,this structure is known to be disrupted by hydration(Bouwstra et al., 2003; Warner et al., 1988), and marine cetaceans have adapted their stratum cornuem to hydration viaretention of more polar lipids such as the glycosphingolipids, which are compatible with higher hydration levels(Elias et al., 1987). Amphibious marine snakes are known to shed their skin with relative frequency,possibly due to hydration damage to their barrier, which seems inevitable over time. However, a non-wettable surface layer that includes a modified beta layer with `air-filled' cells could provide a degree of protection from hydration damage to the crucial permeability barrier that is housed within the subjacent mesos layer. Moreover, frequent shedding could be important with respect to maintaining effective hydrophobic properties of the skin surfaces.

The high lipogenic activity in the alpha layer, especially in the immature alpha layer, could be the basis of a facultative response to dehydration stress, thereby fortifying the mesos layer barrier by adding a second component of what appears to be triglyceride droplets. Neutral lipids are often used in facultative waterproofing strategies of amphibians(Lillywhite, 2006) and are present as the normal barrier lipids of avian and mammalian stratum corneum(Menon and Menon, 2000; Gu et al., 2008; Haugen et al., 2003; Lillywhite, 2007; Muñoz-Garcia and Williams,2008; Muñoz-Garcia et al., 2008). The observation that alpha layer lipogenesis is expressed to a greater extent in the more terrestrial Laticauda(compared with the more marine species) correlates with lower rates of TEWL and possibly reflects the evolution of permeability barrier plasticity in the amphibious species of sea snakes.

Other specializations in the epidermis of sea kraits are suggested by the ultrastructural observations reported here. The anchoring and arrangement of tonofilaments at the junctional region of alpha and immature alpha layers of epidermis suggest that these features possibly accommodate to mechanical stresses related to movement in the marine environment. The second junctional feature, shown in Fig. 11,might possibly be related to the presence of neuronal processes, almost certainly important for detecting vibrations in the marine environment. Whether structures that we believe might be nerve terminal housings are indeed present will need to be ascertained by further investigation using immuno-electron-microscopy to demonstrate the presence of neuropeptides.

Conclusion

In conclusion, three species of sea kraits exhibit physiological and ultrastructural features of integument that rank in order corresponding with presumptive adaption to the environment and ecology of the respective species. The characters we studied also reflect a progression of responses to stages of the terrestrial-to-marine transition represented by the three species of sea kraits (Lillywhite et al.,2008b). Insofar as fresh drinking water is a requirement for water balance in these marine snakes (Lillywhite et al., 2008a), properties of skin that conserve water are expected to be an important feature in the suite of characters that assist the organisms in maintaining water balance in potentially very stressful environments. The significance of these features is further emphasized by the fact that sea snakes can become severely dehydrated in nature(Lillywhite et al., 2008a). Further research must be undertaken to more fully understand the features contributing to skin permeability appropriate for a fully marine existence.

LIST OF ABBREVIATIONS

     
  • RH

    relative humidity, as decimal fraction

  •  
  • Rb

    boundary layer resistance to evaporative water loss

  •  
  • Rs

    skin resistance to evaporative water loss

  •  
  • Rt

    total resistance to evaporative water loss

  •  
  • TEWL

    transepidermal evaporative water loss

  •  
  • WVDa

    saturated water vapor density at ambient chamber temperature

  •  
  • WVDs

    saturated water vapor density at skin temperature

FOOTNOTES

We express much appreciation to Debra Crumrine (Dermatology Research, VA Medical Center, SF) for her help with electron microscopy. We are also grateful to Leslie Babonis, Hui-Min Chu and Chun-Jui Chang who assisted in aspects of logistics and field work. Special thanks are extended to the administrative office at Orchid Island for permission to remove snakes that were used in these studies. The care and experimental use of animals were within institutional guidelines and were approved by the institutional animal care and use committee. This research was supported by a grant from the National Geographic Society(#8058-06 to H.B.L.). We are also grateful to Delfin Technologies, Ltd for the use of the VapoMeter.

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