1921
The Journal of Experimental Biology 212, 1921-1929
Published by The Company of Biologists 2009
doi:10.1242/jeb.028704
Water exchange and permeability properties of the skin in three species of
amphibious sea snakes (Laticauda spp.)
H. B. Lillywhite1,*, J. G. Menon2, G. K. Menon3, C. M. Sheehy 3rd4 and M. C. Tu5
1
Department of Zoology, University of Florida, Gainesville, FL 32611, USA, 2Department of Biology, William Paterson University of
New Jersey, Wayne, NJ 07470, USA, 3Department of Ornithology and Mammalogy, California Academy of Sciences, Golden Gate
Park, San Francisco, CA 94118, USA, 4Department of Biology, Amphibian and Reptile Diversity Research Center, University of
Texas at Arlington, Arlington, TX 76019, USA and 5Department of Biology, National Taiwan Normal University, Taipei, Taiwan 116,
Republic of China
*Author for correspondence (e-mail: [email protected])
Accepted 24 March 2009
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.
Key words: snake, sea snake, skin, ecophysiology, evaporative water loss, skin resistance, lipid, epidermis, permeability barrier, mesos layer,
alpha keratin, marine, seawater.
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
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1922 H. B. Lillywhite and others
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 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 postfixed 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
Measurements of evaporative water loss and skin resistance
We measured TEWL, skin temperatures and calculated Rs in 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=Rt–Rb, where Rt is the total resistance and Rb is the boundary
layer resistance. The total resistance was calculated as follows:
Rt = [WVDs – (RH ⫻ WVDa)] 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.
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–2 h–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,
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Transepidermal water loss in sea snakes
1900
TEWL
Xeric/terrestrial
snake species
700
=
X Naja atra
550
500
Resistance (s cm–1)
Permeability (104⫻cm s–1)
TEWL (µg cm–2 h–1)
550
500
450
400
350
300
250
200
150
100
140
1923
Permeability
120
100
450
400
350
Elapidae
300
250
200
Acrochordus
granulatus
X
150
100
80
X Bungarus
multicinctus
50
60
co
lub
rin
a
40
lat
ica
ud
ata
se
mi
fas
cia
ta
20
Resistance (s cm–1)
550
500
450
400
350
300
250
200
150
100
50
Fig. 3. Skin resistances (Rs) of sea kraits (means ± s.e.m.), shown with
comparisons to other species. The range indicated in the upper left of the
figure illustrates the range of values published for other snakes, which are
mostly terrestrial and xeric-habitat species (see Lillywhite, 2006). All are
non-elapids. In the lower right of the figure is a single data point for a
marine species of file snake [Acrochordus granulatus (Lillywhite and Ellis,
1994)]. The data points at the extreme right of the graph for a terrestrial
cobra (Naja atra) and a mesic, terrestrial krait (Bungarus multicinctus) were
measured from Taiwanese specimens during the present study. Both are
elapids and span the values of Rs measured in the three species of sea
kraits.
Rs
colubrina laticaudata semifasciata
Fig. 1. Comparisons of values for rates of cutaneous evaporative water loss
(TEWL), skin permeability and skin resistance (Rs) measured in three
species of sea kraits. Values shown are means ± s.e.m.
whereas that of N. atra was somewhat greater than that of the semiterrestrial 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
Percent mass loss per day
0.8
Estimated water loss
in seawater
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
colubrina
laticaudata
semifasciata
Fig. 2. Estimated rates of total water loss for sea kraits kept in seawater.
Data are means ± s.e.m., calculated from data in Lillywhite et al. (Lillywhite
et al., 2008a) after correcting total mass losses for metabolic losses. The
species differences are significantly different (ANOVA, P<0.005).
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
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1924 H. B. Lillywhite and others
Fig. 4. Low-magnification electron micrograph of the skin
of Laticauda colubrina, showing the basal cell layer,
immature alpha (ai) and mature alpha cells (a), as well
as the mesos layer (m) and beta layer (b) above the
mesos layer. The separation of the different layers is an
artifact of sectioning, as well as the drifting of plastic
(embedding medium) under the electron beam (the clear,
white region marked with an asterisk). Note the presence
of lipid droplets in two of the immature alpha cells, as
well as in the mature alpha cells (arrows).
(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. 8 and 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.
Fig. 5. High-magnification electron micrograph showing
the beta layer in Laticauda semifasciata with clear cell
boundaries, unlike the syncytial beta layer observed in
terrestrial snakes. Also note the ‘spongy’ or ‘air-filled’
appearance of individual beta cells, reminiscent of what
is seen in avian feathers. The dark triangles at the
upper corners are due to the copper grid bars
supporting the section.
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Transepidermal water loss in sea snakes
1925
Fig. 6. Electron micrograph illustrating a higher
magnification of part of the immature (ai) and mature (a)
alpha layers in Laticauda colubrina, as well as one
separated part of the mesos layer (m). Note the
lipogenic nature (arrows) of the immature alpha cells
and small amount of lipids trapped in the mature alpha
cells (arrow).
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. colubrina falls 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 Rs among 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?
Fig. 7. Electron micrograph illustrating the beta (b),
mesos (m) and alpha layers (a) in semi-aquatic
Laticauda laticaudata. The profile of lipogenesis in the
various cell layers is similar to that seen in L. colubrina
(Fig. 6). Note that even the mesos layer contains cellular
inclusions of small amounts of lipid.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1926 H. B. Lillywhite and others
Fig. 8. Electron micrograph illustrating the alpha (a) and
mesos (m) layers in the highly aquatic Laticauda
semifasciata. There is hardly any evidence of
lipogenesis in the alpha layers. What appear as minute
electron-lucent areas are actually intercellular spaces,
not intracellular inclusions. The interface of mature and
immature alpha layers in L. semifasciata shows deep
interdigitations between the two cell layers (arrows).
Tonofilaments radiating from the desmosomal plaques
criss-cross the entire breadth of the cytoplasm, and
anchor on to the desmosomes of the proximal cell
membranes, which in turn show radiating tonofilaments
into the next cell layer. Thus, a cytoskeletal system
providing ‘tensegrity’ to the skin is very much in
evidence.
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
Fig. 9. Electron micrograph showing the mesos layers in
Laticauda semifasciata, stained with RuO4. The image
shows individual lipid lamellae (L) surrounding individual
mesos cells. Note that the highly reactive RuO4 etches
away the keratin-containing cells while staining the
lipids exceptionally well. The poor penetration of RuO4,
especially in the snake epidermis, coupled with the high
degree of cellular disruption on the edges of the mesos
layer, makes it challenging to visualize and document
the lipids and intact mesos cells in the same frame.
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Transepidermal water loss in sea snakes
1927
Fig. 10. Electron micrograph of epidermis from Laticauda
semifasciata at higher magnification. The tonofilaments
(t) circumscribing a nucleus as well as interdigitations
between the mature (a) and immature (ai) alpha layers
are clearly discernible.
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
Fig. 11. High-magnification electron micrograph of
epidermis from Laticauda semifasciata, highlighting
unusual morphology of the interface between the
mature (a) and immature (ai) alpha layers. The apical
cell membrane of the outermost immature alpha layer
cells show modifications that appear to be numerous
specialized invaginations housing nerve-ending-like
structures. These invaginations are regularly placed
between adjacent desmosomes (d). These possibly
represent ‘wrinkles’ in the plasma membrane that are
invaginated into the cell by physical constraints of being
trapped between adjacent desmosomes, as the
desmosomes are anchored to less flexible, fully
keratinized mature alpha cells.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1928 H. B. Lillywhite and others
Fig. 12. Photos illustrating the hydrophobic nature of the
skin of sea kraits. (A) Water droplets (white arrow) and
curved water–air interface (black arrow) on the surface
of Laticauda colubrina, photographed while moving at
the edge of the sea (photo by Leslie Babonis).
(B) Droplets of seawater (arrows) illustrate hydrophobic
beading on the outer scale surfaces of Laticauda
semifasciata.
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 via
retention 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; MuñozGarcia 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-electronmicroscopy 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-tomarine transition represented by the three species of sea kraits
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Transepidermal water loss in sea snakes
(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
Rb
Rs
Rt
TEWL
WVDa
WVDs
relative humidity, as decimal fraction
boundary layer resistance to evaporative water loss
skin resistance to evaporative water loss
total resistance to evaporative water loss
transepidermal evaporative water loss
saturated water vapor density at ambient chamber temperature
saturated water vapor density at skin temperature
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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