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Integrative
Organismal
Biology A Journal of the Society
for Integrative and
Comparative Biology
academic.oup.com/icbIntegrative Organismal Biology
Integrative Organismal Biology, pp. 1–11
doi:10.1093/iob/obaa043 A Journal of the Society for Integrative and Comparative Biology
RESEARCH ARTICLE
Nature or Nurture: Can Prey-Based Diets Influence
Species-Specific Physiological Performance Traits of Epidermal
Lipid Content and Cutaneous Water Loss?
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J. M. Weidler*,† William. I. Lutterschmidt1,*
*Department of Biological Sciences, Sam Houston State University, Huntsville, TX 77341, USA; †South Dakota Bureau of
Information and Telecommunications, Pierre, SD 57501, USA
1
E-mail: lutterschmidt@shsu.edu
Synopsis Epidermal lipids serve as the primary barrier to Synopsis
cutaneous water loss (CWL) and play a significant role in Spanish Resumen Los lıpidos epidermicos crean la prin-
water conservation and homeostasis. Previous studies have cipal barrera para prevenir la perdida de agua a traves de
shown the correlation between increased aridity of habitats la epidermis; esto es un factor muy importante en la ho-
and the amount of epidermal lipids among species. meostasis y en la prevenci on de la deshidrataci on. En
Generally, increased amounts of epidermal lipids lower estudios anteriores se estableci o que en algunas especies
skin permeability. Species-specific differences in CWL existe una correlaci on entre los habitats donde se incre-
and prey preferences between two sympatric snake species, menta la aridez y la cantidad de lıpidos epidermicos. En
the Northern Cottonmouth (Agkistrodon piscivorus) and general, cuando se incrementa la cantidad de lıpidos epi-
the Eastern Copperhead (Agkistrodon contortrix), moti- dermicos, se reduce la permeabilidad de la piel.
vated us to question if prey-base can result in these ob- Considerando que existen diferencias en la perdida de
served species-specific differences in CWL. We experimen- agua a traves de la piel y las preferencias de presas entre
tally controlled the diets for a captive colony of Northern dos especies simpatricas de vıboras, la serpiente mocasın
Cottonmouths (A. piscivorus) by feeding either fish de agua del norte (Agkistrodon piscivorus) y la vıbora
(Notemigonus crysoleucas) or mice (Mus musculus) to in- cobriza del este (Agkistrodon contortrix), decidimos inves-
vestigate if diet can affect the quantity and quality of epi- tigar si el tipo de dieta podrıa explicar las diferencias
dermal lipids and the rates of CWL. Snakes fed mice observadas en perdida de agua. Hicimos un experimento
gained consistently more mass, but diet treatments did donde alimentamos a una colonia en cautiverio de ser-
not affect growth rate. We found no significant differences pientes mocasın de agua del norte con dos tipos de presas,
in quantitative lipid content or rates of CWL between diet pescado (Notemigonus crysoleucas) o ratones (Mus muscu-
treatments. An analysis for qualitative lipid content using lus), para investigar si el tipo de dieta afecta la cantidad y
infrared spectrophotometry also showed no diet effect, la calidad de los lıpidos epidermicos, ademas de la tasa de
thus suggesting that lipid content and CWL are strong perdida de agua a traves de la piel. Las serpientes que se
species-specific physiological performance traits not influ- alimentaron con ratones mostraron un incremento consis-
enced by recent dietary history. While there is some evi- tente en la masa, pero las diferencias en dietas no afec-
dence that epidermal permeability may be variable under taron la tasa de crecimiento. Tampoco encontramos difer-
certain environmental conditions (e.g., humidity), our encias significativas en la cantidad de lıpidos, como
findings show that diet has no effect and that a shift in tampoco en la tasa de perdida de agua a traves de la
prey preference may not influence or enhance physiologi- piel. En un analisis cualitativo del contenido lipıdico
cal performance for decreasing CWL. usando espectrofotometrıa infrarroja se demostr o que no
hay efecto atribuido al tipo de dieta, lo cual sugiere que el
contenido lipıdico y la perdida de agua son caracterısticas
fisiol
ogicas muy arraigadas y especıficas de las especies, y
que no estan influenciadas por los habitos alimenticios
recientes. Aunque hay evidencia de que la permeabilidad
epidermica puede ser variable debido a ciertas condiciones
ß The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.
All rights reserved.
For permissions please email: journals.permissions@oup.com.2 J. M. Weidler and W. I. Lutterschmidt
ambientales (ej., humedad), nuestros resultados demues-
tran que la dieta no tiene efecto, y que alg
un cambio en
la preferencia de dietas no deberıa influenciar el
desempe~no fisiologico debido a la perdida cutanea de
agua.
Introduction Copperheads consume mainly small mammals
The physiological and behavioral conservation of wa- (Garton and Dimmick 1969; Brown 1979) which con-
ter and protecting against dehydration is an impor- tain substantially more lipid than fish and amphibians
tant performance trait for many terrestrial species, consumed mainly by cottonmouths (Clark 1949;
especially those in arid climates. Species in arid hab- Kofron 1978). Thus, we raised the question: Is the
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itats tend to have adaptations for lowering rates of lower CWL in copperheads (Moen et al. 2005;
evaporative water loss compared with those species Miller and Lutterschmidt 2014) an adaptive species-
from more mesic or aquatic habitats. Numerous specific physiological performance trait enhancing
studies have demonstrated the negative correlation their ability to use more upland mesic habitats? Or,
between evaporative water loss and habitat aridity is the lower CWL in copperheads simply the product
(e.g., Bentley and Schmidt-Nielsen 1966; Gans et al. of a lipid-rich mammalian prey-preference more read-
1968; Prange and Schmidt-Nielsen 1969; Elick and ily available in upland mesic habitats? Parkinson et al.
Sealander 1972; Cohen 1975; Baeyens and Rountree (2000) investigated the phylogeography of the North
1983; Roberts and Lillywhite 1983; Dmi’el 1998; American Agkistrodon species and found that A. con-
Lillywhite 2006). Interestingly, evaporative water tortrix exhibits the ancestral condition of terrestriality,
loss seems to correlate with habitat aridity regardless with A. piscivorus exhibiting the only derived shift in
of taxonomic position (Dmi’el 1998) and is observed aquatic habitat preference. Therefore, a more informed
in other vertebrate taxa (e.g., Tieleman et al. 2003). question may be: Is the higher CWL in cottonmouths
Although many of these studies have examined a derived adaptive trait for lowering energetic cost in
interspecific comparisons of evaporative water loss maintaining epidermal lipids less needed in an aquatic
and habitat aridity among a broad range of ophidian habitat? Or, is higher CWL in cottonmouths simply
taxa, fewer studies have compared congeneric the product of lipid-poor prey (i.e., fish and amphib-
(Dunson and Freda 1985; Dmi’el 1998; Moen et al. ians) more readily available in aquatic habitats?
2005) or conspecific (Agugliaro and Reinert 2005) As epidermal lipids (Roberts and Lillywhite 1980)
taxa. Miller and Lutterschmidt (2014) compared and diets deficient in essential fatty acids influence
copperheads and cottonmouths, two closely related CWL (Menton 1970; Elias and Brown 1978;
congeneric sister species in the genus Agkistrodon Williams and Elias 1987), we compared the rates of
(Parkinson et al. 2000), to investigate if species dif- CWL and conducted both quantitative and qualita-
ferences in mesic versus aquatic habitat preferences tive analyses of epidermal lipids for Northern
correlated with their rates of cutaneous water loss Cottonmouths (A. piscivorus) fed either diets of
(CWL). The species-specific physiological ability for fish (low-lipid) or mice (high-lipid). The cotton-
limiting CWL may reflect individual adaptations that mouth served as an ideal model because this species
serve an important role in differences in habitat pref- is a diet generalist (Burkett 1966) and readily feeds
erence and resource partitioning (Miller and on either fish or mice in the laboratory. This unique
Lutterschmidt 2014). opportunity to manipulate diet experimentally
Because cutaneous (not respiratory) water loss is allowed us to investigate if skin permeability and
the primary source of evaporative water loss in squa- increased CWL in the cottonmouth are influenced
mates (Prange and Schmidt-Nielsen 1969; Cohen by prey-based lipid content. Negative results for
1975; Dmi’el 1985; Dmi’el 2001), epidermal lipids in the influence of diet would then suggest that CWL
the integument serve a major role in regulating CWL is a fixed species-specific trait reflective of selection
in reptiles (Roberts and Lillywhite 1980). Potential pressures for increased physiological performance in
species-specific differences in epidermal lipids may preferred microhabitats.
then serve as a potential mechanism allowing copper-
Materials and methods
heads (Agkistrodon contortrix) to limit CWL and use
and forage in more mesic and upland habitats, thus Experimental subjects and captive care
avoiding both direct and indirect competition with Adult northern cottonmouths (A. piscivorus) were
sympatric cottonmouths (Agkistrodon piscivorus). collected in July 2016 from Harmon Creek locatedCottonmouth skin lipids and CWL 3
in Walker County, TX (Texas Parks and Wildlife (Summer 2017) and began once the second shed
Scientific Research Permit SPR-0715-127 issued to was collected from all 24 snakes. Shed epidermis
WIL). Only female snakes (n ¼ 24) were used in samples were stored between 8 and 234 days prior
experiments to control for potential sex differences to analysis. Dunson and Freda (1985) showed no
in skin lipids (Mason et al. 1987; Ball 2000). changes in rates of water influx and efflux with snake
Beginning in August 2016, each snake was housed skins stored for two years.
separately in plastic cages (38 26 22 cm) with
aspen bedding (Harlan Teklad, Madison, WI) and Measures of CWL
water provided ad libitum. Snakes were kept in a We used the in vitro technique (Agugliaro and
laboratory and acclimatized to temperature Reinert 2005; Miller and Lutterschmidt 2014) to
(25 6 2 C), relative humidity (50 6 3%), and photo- measure CWL of intact epidermis (Dunson and
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period (12 L : 12 D cycle) with the photophase cen- Robinson 1976; Zucker and Maderson 1980; Stokes
tered on 1200 h. Snakes were randomly selected for and Dunson 1982). Only the mid-dorsal region of
one of two diet treatments; a low-lipid fish diet the shed epidermis was used to control for potential
(n ¼ 12) or high-lipid mice diet (n ¼ 12). Generally, differences in CWL along the body’s dorsal surface
fish (i.e., golden shiners) have a mean body fat 9% (Miller and Lutterschmidt 2014). Three mid-dorsal
of Mb (Lochmann and Phillips 2012) while mice samples (ca. 2 2 cm) were cut from each snake’s
have more than double the mean body fat 25% second shed and inspected microscopically for the
of Mb (Reed et al. 2007). Beginning September presence of holes or tears in the integument. These
2016, each of the two diet groups were fed weekly shed samples (n ¼ 72), with the mucosal surface fac-
and offered either fish or mice equal to 20% of their ing outward, were then stretched over the opening
Mb (Lutterschmidt and Rayburn 1993; Byars et al. (0.58 cm2) of a culture tube (10 75 mm) contain-
2010; Sparkman et al. 2010). We measured each ing 1 mL of deionized water. We then secured the
snake’s initial snout–vent–length (SVL) to the near- shed to the opening of the culture tube and created a
R
est 0.1 cm (mean ¼ 51.07, SE ¼ 1.167, n ¼ 24) and tight seal using waxed tread and ParafilmV (Pechiney
body mass (Mb) to the nearest gram (mean ¼ 193.9, Plastic Packaging, Menasha, WI). The culture tube
SE ¼ 11.98, n ¼ 24) prior to experimentation and was then inverted and suspended inside a 30-mL
R
Mb was measured monthly (September 2016 to July specimen bottle containing 5 g of t.h.e.V desiccant
2017) for growth. Food amounts were adjusted to (EMD Chemicals, Inc., Gibbstown, NJ). Water is
ensure food per unit Mb remained constant. Fish then drawn from the culture tube, through the
(golden shiners, Notemigonus crysoleucas) were pur- shed epidermis under simulated natural physiological
chased from Oakhurst Bait Co. (Oakhurst, TX) and conditions (Burken et al. 1985a; Agugliaro and
R
CD-1V IGS Laboratory mice (Mus musculus) were Reinert 2005). The initial and final mass of each
supplied by the Sam Houston State University culture tube was measured at 120 h. We calculated
Science Annex. the rate of CWL for each sample from the difference
of the initial and final mass of the culture tube, di-
Shed epidermis collection vided by the 120 h. The three shed samples from
Snakes were maintained in captivity until all snakes each snake were then averaged for a total of 24 in-
produced a second shed epidermis for collection and dependent mean values of CWL rate.
study. The first shed was discarded and not used for
experimentation as these shed integuments are af- Epidermis lipid extraction and quantitative analyses
fected by each snake’s natural diet regime and other The quantitative lipid content of a shed (mg lipid/g
differences such as surface abrasion from traversing of shed) was determined by the initial and final
habitat structures under field conditions. Thus, the masses of a shed after lipid extraction. We separated
second shed was used because all snakes experienced both the dorsal and ventral surfaces of each shed
identical acclimatization and captive care regimes en- allowing for comparison of lipid content between
suring valid between-treatment comparisons of diet. shed surfaces (dorsal versus ventral) and the sum
Cages were inspected daily for the presence of of these surfaces provided total lipid content for
fresh sheds. Collected sheds were immediately dried, each shed. Standard techniques for lipid extraction
sealed in plastic bags, and frozen (20 C) to pre- from shed epidermis were used (Roberts and
serve the integument (Burken et al. 1985a; Agugliaro Lillywhite 1980; Stokes and Dunson 1982; Burken
and Reinert 2005). All experiments on individual et al. 1985b; Agugliaro and Reinert 2005). The dorsal
sheds were performed over the same time period and ventral sheds were first placed in 240-mL4 J. M. Weidler and W. I. Lutterschmidt
R
specimen jars with 100 g of t.h.e.V desiccant for 24 h, Table 1 Molecular geometries of each peak and wavenumber
removed and immediately massed to the nearest (cm1) identified by IR spectrometry
0.0001 g using a Denver Instruments A-250 analytical Peak Wavenumber Assigned molecular
balance (Denver Instruments Company, Bohemia, number (cm21) geometry
NY). We then placed each shed surface in a 400- 1 3268 OH
mL jar for 24 h containing a 120-mL, 2:1 chloroform 2 2927 CH2
to methanol solution (Folch et al. 1957) to extract 3 1630 CO, amide I
total lipids. After lipid extraction, sheds were re-
4 1526 CO and dNH, amide II
moved from the chloroform–methanol solution and
5 1450 dCH2
washed once in fresh chloroform–methanol solution
6 1397 dCH3
and rinsed three times in distilled water. We then
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placed the sheds in jars containing fresh desiccant for 7 1236 CN, amide III
24 h prior to measuring final dry mass. Quantitative 8 1066 CC
lipid content (mg) was determined from the differ- 9 486 SS
ence in initial and final dry masses (to the nearest
0.0001 mg) of each shed surface. The lipid content
per shed epidermis mass (mg/g) was standardized for Molecular geometries of functional groups and the
analysis by dividing lipid content by the initial mass chemical composition within sheds were identified
of the dorsal and ventral surfaces. (Barry et al. 1993; Ripamonti et al. 2009).
Infrared spectroscopy and qualitative lipid analyses Statistical analyses
R R
Prior to total lipid extraction from sheds, a fourth We used SigmaPlotV 11.0 and SPSS StatisticsV 22.0
mid-dorsal and a single mid-ventral shed sample (ca. for all statistical analyses, for testing assumptions of
1 1 cm) were cut from each shed for qualitative normality (Shapiro–Wilks) and equal variance
lipid analyses. We used infrared (IR) spectrometry (Levene’s), and for graphing. Linear regression anal-
(Bruker Optics Alpha Fourier Transform IR) to ex- ysis (Zar 2010) was used to confirm that storage
amine and quantify the qualitative composition of times between 8 and 234 days did not affect CWL
lipids within the shed epidermis. Spectroscopy (Li or lipid content of shed epidermis samples. A
et al. 2014; Ismail et al. 1999; Zarini et al. 2019) is Student’s t-test was used to test for differences in
the study of how radiated energy and matter inter- CWL between diets. To investigate differences in to-
act. Different chemical bond types respond to radi- tal shed lipid content, we used a multivariate analysis
ation differently allowing one to identify various of variance (MANOVA) with diet (fish and mice)
functional groups and distinguish differences in representing treatment groups and shed surface
chemical composition between samples. IR spectros- (dorsal and ventral) as dependent variables within
copy specifically uses infrared radiation to excite the treatments. This multivariate analysis appropriately
molecules of a compound generating an infrared tests the null hypothesis that snakes with different
spectrum of energy absorbed by molecules as a func- diets have the same dorsal and ventral lipid content
tion of frequency or wavelength. We used IR spec- because dorsal and ventral shed samples are obtained
trometry, the application of spectroscopy, to from the same shed of an individual and inter-
examine the absorbance wavenumbers that corre- correlated (Zar 2010). As shed surfaces were sampled
spond to molecular geometries of organic molecules from the same shed and individual, paired t-tests
within the shed epidermis. The qualitative composi- were used to investigate differences between shed
tion of lipids was examined quantitatively by record- surfaces from each within each diet treatment.
ing the absorbance values of threshold wavenumber Principal components analysis (PCA) with the
peaks in each sample. Each shed sample was dry, non-orthogonal oblique rotation method (Oblimin
allowed to reach ambient temperature (23 C), cen- with Kaiser normalization), assuming non-
tered within the instrument, and measured once. We independence among IR peaks, was used to investi-
examined nine standardized wavenumber positions gate possible differences in molecular geometries and
(corresponding to chemical bonds and molecular ge- the variation in qualitative lipids between diets and
ometries, Table 1) to investigate qualitative differen- shed surfaces. Group-mean PC scores of the first two
ces in lipid content between sheds from the fish principal components are illustrated with 95% con-
(low-lipid) and mice (high-lipid) diets. fidence intervals and were statistically analyzed for
Additionally, we compared dorsal and ventral sheds. separation using one-way analysis of variance ofCottonmouth skin lipids and CWL 5
component scores followed by Tukey’s a posteriori
tests. Differences between treatments (diet) and var-
iables (shed surface) were considered significant at
P < 0.05. The corresponding author may be con-
tacted for data availability.
Results
Linear regression analysis confirmed that storage
time of shed epidermis did not affect CWL or quan-
titative lipid content in our samples. We found no
time-effect on CWL for both the fish (F ¼ 3.16; df ¼
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1, 10; P ¼ 0.106) and mouse (F ¼ 3.13; df ¼ 1, 10;
P ¼ 0.107) diets. Additionally, we found no effects of
storage time on quantitative lipid content (fish diet,
F ¼ 0.37; df ¼ 1, 10; P ¼ 0.554 and mouse diet, Fig. 1 Mean (695% CI) rates of CWL for the fish (0.649, SE ¼
0.0798, n ¼ 12) and mouse (0.702, SE ¼ 0.0828, n ¼ 12) diet
F ¼ 0.01; df ¼ 1, 10; P ¼ 0.920). These results con-
treatments are show in black. Gray box plots show the median
firm the stability of shed epidermal tissue for storage (central gray line within box), the 25th and 75th percentiles
and the later testing of CWL and lipids (Dunson and (bottom and top lines of box), and the 10th and 90th percentiles
Freda 1985; Miller and Lutterschmidt 2014). (gray error bars).
We found no difference in the rates of CWL (t ¼
0.456; df ¼ 22; P ¼ 0.653) between the fish and
mouse diet treatments (Fig. 1). Using an
MANOVA, we also found no difference in quantita-
tive lipid content between the fish and mouse diet
treatments when considered jointly on the variables
dorsal and ventral shed surfaces (Wilk’s K ¼ 0.983;
F ¼ 0.181; df ¼ 2, 21; P ¼ 0.835; partial g2 ¼ 0.017).
The MANOVA between-subject effects for each de-
pendent variable indicated that there were no signif-
icant difference between fish and mouse diet
treatments for dorsal sheds (F ¼ 0.180; df ¼ 1, 22;
P ¼ 0.676; partial g2 ¼ 0.008) or ventral sheds
(F ¼ 0.380; df ¼ 1, 22; P ¼ 0.544; partial g2 ¼
0.017). Following the MANOVA, paired t-tests
were used to investigate differences between shed Fig. 2 Quantitative lipid content of shed epidermis for both
surfaces within each diet treatment. We found that dorsal (84.15, SE ¼ 5.515, n ¼ 12) and ventral (59.91, SE ¼
lipid content between dorsal and ventral shed sam- 6.445, n ¼ 12) shed surfaces in the fish diet treatment are shown
ples differed significantly within both the fish in the first pair of box plots. The second pair of box plots show
both mean (695% CI) for dorsal (80.06, SE ¼ 7.899, n ¼ 12) and
(t ¼ 4.911; df ¼ 11; P < 0.001) and mouse
ventral (54.12, SE ¼ 6.840, n ¼ 12) shed surfaces in the mouse
(t ¼ 4.975; df ¼ 11; P < 0.001) diet treatments diet treatment. Group means (black circles) and 695% CI (black
(Fig. 2). error bars) are shown with gray box plots showing the median
Using IR spectroscopy, we found nine predomi- (central gray line within box), the 25th and 75th percentiles
nant peaks in both the dorsal and mid-ventral shed (bottom and top lines of box), and the 10th and 90th percentiles
surfaces (Fig. 3), corresponding to nine molecular (gray error bars). Paired t-tests indicated statistically significant
geometries (Table 1). Of the 48 shed samples, differences between the dorsal and ventral shed surfaces within
both diets.
8 were not used due to poor condition. Thus, sample
sizes were 0.427) indicated non- (eigenvalue ¼ 8.349) with peak numbers 4 and 7
independence among IR peaks. The first two correlating strongest with this axis (r ¼ 0.982 and6 J. M. Weidler and W. I. Lutterschmidt
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Fig. 3 An example output of the IR spectroscopy showing the
nine predominant absorbance peaks of a shed sample. The x-axis
(wavenumber) represents the vibration frequency of molecular
bonds within the sample. Stronger bonds and lighter atoms vi-
brate at higher frequencies, so their location along the x-axis
corresponds to different functional groups. Peaks indicate the IR
absorbance units (y-axis) specific to those wave numbers or
frequencies characteristic of molecular geometries.
Fig. 5 PCA of IR absorbance units for the nine predominant
peaks and molecular geometries within lipids with 95% confi-
dence intervals of mean component scores for each group il-
lustrated on both axes. Extensive overlap of confidence intervals
of the combined dorsal and ventral shed surfaces within the fish
and mouse diet treatments (A) indicate that diet had no effect on
qualitative lipid content. Separation of the 95% confidence
intervals for both principal components of the combined diet
treatments within each shed surface (B) indicate that the dorsal
and ventral shed surfaces differed in qualitative lipid content.
and PC2 (F ¼ 0.629; df ¼ 1, 38; P ¼ 0.433) scores
indicated no significant differences between diet
Fig. 4 PCA of IR absorbance units for the nine predominant
peaks and molecular geometries within lipids of both the dorsal treatments (Fig. 5A). However, group mean differ-
and ventral shed surfaces and within each diet treatment. This ences between dorsal and ventral shed surfaces
illustrates that the primary source of variation is associated with (Fig. 5B) differed significantly for PC1 (F ¼ 67.085;
differences between dorsal and ventral epidermal samples and df ¼ 1, 38; P < 0.001; Tukey’s test, q ¼ 11.583,
not diet. P < 0.001) but not PC2 (F ¼ 1.105; df ¼ 1, 38;
P ¼ 0.300) scores.
0.981, df ¼ 38). The second principal component (y- As would be expected with captive feeding, there
axis) explained 3.8% of the variation (eigenvalue ¼ were significant relationships between cumulative Mb
0.340) with peaks 2 and 8 correlating strongest with gain and time in captivity for both the fish
this axis (r ¼ 0.350 and 0.273, df ¼ 38). The 95% (F ¼ 127.87; df ¼ 1, 10; P < 0.001; r2 ¼ 0.93) and
confidence intervals of mean principal component mouse (F ¼ 272.15; df ¼ 1, 10; P < 0.001; r2 ¼ 0.96)
scores for both PC1 and PC2 overlapped heavily be- diets. Differences in lipid content between the prey-
tween diet treatments (Fig. 5A) but showed signifi- based diet treatments, where mice contain more than
cant separation between the dorsal and ventral shed double the mean body fat (Reed et al. 2007) of fish,
surfaces for PC1 (Fig. 5B). One-way analyses of var- also resulted in significant differences in growth
iance for both PC1 (F ¼ 0.639; df ¼ 1, 38; P ¼ 0.429) (Fig. 6). Comparison of regression coefficients andCottonmouth skin lipids and CWL 7
lipids (Miller and Lutterschmidt 2014). Epidermal
permeability does not seem to correlate with phylog-
eny (Dmi’el 1998; Tieleman et al. 2003), making the
pronounced differences in CWL between these
closely related sympatric species a result of evolu-
tionary adaptation, and not phenotypic plasticity.
Miller and Lutterschmidt (2014) found species-
specific differences in CWL and epidermal lipid con-
tent (under identical and controlled mouse diets for
both species) and suggest that these performance
traits may potentially serve as a physiological mech-
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anism for the ecological partitioning of two closely
related sympatric species. These authors add that
such theoretical concepts in physiological ecology
are well illustrated when traits for physiological per-
Fig. 6 Mean (6SE) cumulative gain of body mass (Mb) for snakes formance and tolerance correlate with species natural
in both the fish (n ¼ 12) and mouse (n ¼ 12) diet treatments. The
history and ecology. Here, we questioned if cotton-
gray square represents the initial gain of Mb equal to zero and
resulting regression lines for both diets include zero. mouths (by consuming increased lipids in a prey-
based diet) could decrease skin permeability and
slopes (Zar 2010) indicated that the rate of growth CWL thus creating potential overlap in niche space
did not differ between fish and mouse diets (t ¼ and competitive interactions with copperheads.
0.499; df ¼ 18, P ¼ 0.624). However, snakes in While these two taxa do partition by diet and mi-
the mouse-diet treatment gained consistently more crohabitat, diet was not likely the driving force in the
Mb as indicated by the highly significant difference divergence of these two taxa. Our results suggest that
in regression elevations (t ¼ 5.594, df ¼ 19, CWL and epidermal lipids are not influenced by diet
P < 0.001). The final accumulative gain in Mb for and may represent more relatively fixed species-
mouse-fed snakes (mean ¼ 234.4, SE ¼ 35.13, specific traits (Lillywhite 2004) reflective of evolu-
tionary selection for physiological performance.
n ¼ 12) averaged 37.0 g (SE ¼ 42.24, n ¼ 12) greater
Roth (2005) found the spatial distributions of cot-
than fish-fed snakes (mean ¼ 197.4, SE ¼ 25.87,
tonmouths to be mostly riparian with 83% of snake
n ¼ 12) at the end of the 11-month captivity period.
locations occurring within 10 m of water. This well-
Discussion documented aquatic habitat preference by cotton-
mouths (Gloyd and Conant 1990); Dixon and
Our results indicate that prey-based diets of either Werler 2005) may have other associated adaptations
fish or mice do not affect CWL nor the quantity or for decreased epidermal lipids and increased CWL.
quality of epidermal lipids in the northern cotton- Behavioral aggregation and increased social interac-
mouth (A. piscivorus). Thus, significantly greater tion in cottonmouths (Roth 2005; Roth and
rates of CWL in cottonmouths compared with cop- Lutterschmidt 2011) may be adaptive for limiting
perheads (Moen et al. 2005; Miller and evaporative water loss by reducing the surface area
Lutterschmidt 2014) result most likely from differ- of exposed integument (Graves et al. 1986; Tu et al.
ences in species-specific physiological performance 2002; Agugliaro and Reinert 2005). Additionally, in-
traits that are not influenced by diet. However, fac- creased skin permeability may aid also in water ab-
tors such as habitat and microhabitat acclimatization sorption (Cohen 1975), evaporative cooling for
(e.g., Kattan and Lillywhite 1989), phenotypic plas- thermoregulation, and cutaneous gas exchange
ticity (e.g., Haugen et al. 2003; Lillywhite 2004), and (Standaert and Johansen 1974; Heatwole and
ontogenetic changes (e.g., Agugliaro and Reinert Seymour 1978) for increased respiration in an
2005; Mu~ noz-Garcia and Williams 2008) may influ- aquatic environment (Winne et al. 2001).
ence water barrier function that is typically and rel- There is a well-established aridity gradient for skin
atively characteristic of a species (Lillywhite 2004). permeability (Cohen 1975; Roberts and Lillywhite
As copperheads are more terrestrial, the derived shift 1980; Stokes and Dunson 1982; Baeyens and
from terrestrial to aquatic microhabitat preference by Rountree 1983; Roberts and Lillywhite 1983; Dmi’el
cottonmouths (Parkinson et al. 2000) may reflect an 1998; Lillywhite 2006) which correlates with the
adaptive trait for reducing epidermal lipids and the amount and quality of lipids in the epidermis
energetic costs associated with maintaining these (Lillywhite and Maderson 1982; Burken et al.8 J. M. Weidler and W. I. Lutterschmidt
1985b; Lillywhite 2006; Miller and Lutterschmidt fish and mouse diet treatments (Fig. 2). Miller and
2014). However, it seems that increased consump- Lutterschmidt (2014) also found similar results for
tion of dietary lipids from prey does not necessarily both copperheads and cottonmouths. This greater
result in increased epidermal lipids. Ingested lipids amount of lipid in the dorsal integument most likely
are not simply deposited into the epidermis (e.g., aids in reducing water loss from the dorsal surface.
Sheridan 1994; Price 2017). Dietary triglycerides are Here, we demonstrate that diet does not influence
hydrolyzed in the intestine by pancreatic lipase pro- this difference in either the quantity (Fig. 2) or qual-
ducing free fatty acids and monoacylglycerol (Patton ity (Fig. 5A) of lipids in the dorsal or ventral shed
1975; Kammoun et al. 2008) where these compo- surfaces. Additionally, the ventral integument is 50%
nents are then absorbed and re-esterified by enter- thicker than the dorsal integument (Jayne 1988). The
ocytes to produce triglycerides for transport increased amount of keratin likely protects against
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throughout the body (Price 2017). ground abrasion during locomotion. The thinner
The anabolism of epidermal lipids results from dorsal surface likely has proportionally more lipids
sequestered acetate, the main carbon source for lipid as scale thickness does not prevent water loss across
synthesis (Wertz 1996). Multiple lipid bilayers the integument (Lillywhite and Maderson 1982).
obstructing the intercellular space of the outermost The main motivating factor for this study was to
layer (i.e., stratum corneum) of the epidermis serve further investigate the significant difference in dorsal
as the permeability barrier preventing trans CWL CWL between copperheads and cottonmouths
(Landmann 1988). Generally, the lipids in reptile (Miller and Lutterschmidt 2014) and a potential cor-
epidermis consist of cholesterol, free fatty acids, relate with lipid content in a prey-based diet. In
and ceramides (Roberts and Lillywhite 1983; addition to Miller and Lutterschmidt (2014) finding
Burken et al. 1985b; Landmann 1988; Elias and no significant species difference in the mean amount
Menon 1991; Ball 2004; Torri et al. 2014). A high of dorsal lipid, we found no significant difference in
concentration of ceramides may decrease permeabil- the mean amount of lipid in the dorsal and ventral
ity by allowing lipid lamellae to form tight, highly shed surfaces between prey-based diets. This suggests
ordered crystalline phases (Velkova and Lafleur 2002; that the observed differences in CWL between cop-
Bouwstra et al. 2003). A high concentration of cho- perheads and cottonmouths are related to species-
lesterol more tightly packs the lipid fatty acid chains specific differences in the qualitative properties of
together, creating a more impermeable barrier dorsal lipids. Further investigation for qualitative dif-
(Hadley 1989; Raffy and Teissie 1999). Thus, despite ferences in epidermal lipids between copperheads
the majority of the epidermis being composed of and cottonmouths would help inform the observed
keratin, lipids in the mesos layer provide the barrier species-specific differences in CWL (Miller and
to water loss (Roberts and Lillywhite 1980). Lutterschmidt 2014).
The closest parallels to our study are those studies Reptiles have mainly cholesterol, free fatty acids,
that fed mice diets deficient in essential fatty acids, and ceramides in the mesos layer of the epidermis
thus increasing rates of epidermal water loss (Roberts and Lillywhite 1983; Burken et al. 1985b;
(Menton 1970; Elias and Brown 1978; Williams Landmann 1988; Elias and Menon 1991; Lillywhite
and Elias 1987). The lamellar bodies of these mice 2006; Torri et al. 2014). An increase in polar ceram-
were void of lipids (Elias and Brown 1978), suggest- ides is associated with lower permeability in birds
ing that such diet deficiencies completely disrupt the and bats (Haugen et al. 2003; Mu~ noz-Garcia and
lipid barriers to water loss. Although our study for Williams 2007; Mu~ noz-Garcia et al. 2012) and a
increasing lipid consumption in a prey-based diet high amount of cholesterol in lipid bilayers also low-
(mouse versus fish) failed to show differences in epi- ers permeability (Hadley 1989; Raffy and Teissie
dermal lipids and skin permeability, we did observe 1999). Our qualitative lipid analysis for the presence
significant growth differences between diet treat- of particular molecular geometries indicated no ef-
ments (Fig. 6). This observed difference in growth fect of diet (Fig. 5A). It is important to note that
is most likely due to the greater mean body fat in the differences in molecular geometries may not be
mouse diet (25% of Mb) compared with the fish completely associated with lipid composition, as
diet (9% of Mb). Similar differences in growth other molecular compounds are within epidermis.
were observed in fish (e.g., Vergara et al. 1999) However, the dorsal and ventral surfaces were found
with diets of higher lipid content. to be significantly different (Fig. 5B) with the dorsal
We found significant differences in quantitative surface often lacking a discernible peak 6 (dCH3).
lipid content between the dorsal and ventral shed The significance for the absence of this molecular
surface of the northern cottonmouth for both the geometry in the dorsal integument is unknown.Cottonmouth skin lipids and CWL 9
Finally, we comment on the observed treatment Use Committee (IACUC) approval No. 16-10-27-
effects of diet on the Mb of snakes. Although the 1003-3-01. J.M.W. thanks Renae Weidler for her
growth rate between snakes in the fish and mouse endless encouragement and support during his grad-
diet treatments did not differ, snakes fed mice gained uate studies and research.
consistently more Mb each month (Fig. 6) and
resulted in a significantly higher regression elevation Conflict of interest
than snakes fed fish. At the end of the 11-month The authors of this manuscript declare no conflicts
captivity period, mouse-fed snakes gained an average of interest related to the data and/or analyses pre-
Mb 37.0 g greater than fish-fed snakes. Although sented in this original research report.
there are numerous abiotic and biotic factors that
influence growth in natural populations, it might References
Downloaded from https://academic.oup.com/iob/article/3/1/obaa043/6126408 by guest on 19 September 2021
be of interest to question if diet composition as a
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Swedish Abstrakt Epidermala fetter anv€ands som prim€ara Russian ff,cnhfrn Kbgblß gblehvbcf cke;fn
barri€arer för kutan vattenförlust i skinnet och spelar en jcyjdyßv ,fhehjv ghjnbd gjnehb djlß xeheÅ
stor roll i vattenkonservering och homeostas. Tidigare rj;yßq gjrhjd b buhfn df;ye hjk d cj[hfyeybb
undersökningar har visat kopplingar mellan ökad torka i djlyjuj ,fkfycf ;bdjnyß[. Ghelßleøbe
miljön och epidermala fetter bland arter av vilda djur. bcckeljdfybz gjrfÅfkb ceøecndjdfybe rjhhekzçbb
Vanligtvis så ger en ökad m€angd epidermala fetter en l€agre ve;le edekbxeybev Åfcełkbdjcnb chelß j,bnfybz
permeabilitet i skinnet. Artspecifika skillnader av kutan b rjkbxecndjv kbgbljd e hfÅkbxyß; dbljd. Rfr
vatterförlust i skinnet och skillnaden i preferenserna av ghfdbkj, edekbxeybe cjleh;fybz kbgbljd d
föda mellan de två sympatriska ormarterna Northern gblehvbce cyb;fen djljghjybçfevjcn rj;yjuj
Cottonmouth (Agkistrodon piscivorus) och Eastern gjrhjdf. fflbljdje hfÅkbxbe d gjnehe djlß xeheÅ
Copperhead (Agkistrodon contortrix), fick oss att ifrågas€atta
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rj;yßq gjrhjd b cjcnfde lbenß ve;le ldevz
om rovdjurens föda i sin tur kan leda till denna artspeci- cbvgfnhbxecrbvb dblfvb Åveq, djlzyßv
fika skillnad av kutan vattenförlust. Vi genomförde ett ex- øbnjvjhlybrjv (Agkistrodon piscivorus) b
periment d€ar vi kontrollerade kosten för en testkoloni av velyjujkjdßv øbnjvjhlybrjv (Agkistrodon contor-
Northern Cottonmouth (A. piscivorus) d€ar vi gav dem trix), gjcke;bkb njkxrjv lkz bcckeljdfybz djghjcf
antingen fisk (Notomigeonus crysoleucas) eller möss (Mus dkbzybz lbenß yf cnegey gjnehb djlß xeheÅ
musculus) för att ta reda på om kosten kan €andra rj;yßq gjrhjd. ß ghjdekb rcgehbveyn, d nexeybe
m€angden och kvaliteten av det epidermala fettet och rjnjhjuj rjkjyb djlzyß[ øbnjvjhlybrjd (A. pis-
m€angden av kutan vattenförlust. Ormarna som matades civorus) rjhvbkb bkb hß,jq (Notemigonus crysoleucas),
med möss hade konsekvent en högre viktökning, men bkb vßłfvb (Mus musculus) c çek bcckeljdfybz
skillnaden i kost verkade inte påverka tillv€axten. Vi fann dkbzybz lbenß yf rfxecndj b rjkbxecndj kbgbljd
ingen större skillnad för m€angden av epidermala fetter gblehvbcf b cnegey gjnehb djlß xeheÅ rj;yßq
eller kutan vattenförlust beroende på kosten. En analys gjrhjd. þveb, rjnjhß[ rjhvbkb vßłfvb, yf,hfkb
av kvalitativt fettinnehåll från en infraröd spektrofotometri ,jkłe vfccß, d nj dhevz rfr hfÅkbxbe d lbene ye
visade inte heller några skillander mellan de olika kosterna, gjdkbzkj yf b[ crjhjcn hjcnf. ß ye j,yfhe;bkb
vilket visar att det epidermala fettet och kutana vattenför- Åyfxbnekyjuj hfÅkbxbz yb d rjkbxecndeyyjv
lusten inte påverkades av den prövade kosten vad det cjcnfde, yb d crjhjcnb gjnehe djlß xeheÅ rj;yßq
g€aller artspecifika fysiologiska drag. Aven€ om det finns gjrhjd ve;le ldevz rcgeheveynfkyßvb
resultat som visar att den epidermala permeabiliteten kan uheggfvb. BcgjkÅjdfybe byahf-rhfcyjq
variera under vissa miljöförhållanden (t.ex. luftfuktighet), cgernhjvenhbb lkz rfxecndeyyjuj fyfkbÅf kbgbljd
så visar våra resultat att kosten inte påverkar och att en nfr ;e ye dßzdbkj dkbzybz lbenß, gjÅdjkzz
skillnad i preferenserna av föda inte nödv€andigtvis har ett ghelgjkfufn, xnj cjleh;fybe kbgbljd b ehjdey
inflytande eller förb€attrar de fysiologiska funktionerna för djljghjybçfevjcnb zdkzncz dblj-
minskad kutan vattenförlust. cgeçbabxecrbvbv abÅbjkjubxecrbvb xehnfvb, yf
rjnhße yelfdybe bÅveyeybz d lbene ye jrfÅßdfn
Åyfxbnekyjuj dkbzybz. ffl nj dhevz rfr ecn
cdblenekcndf njve, xnj ghjybçfevjcn gblehvbcf
dfhbheen gjl dkbzybev jghelekyyß[ eckjdbq
jrhe;føeq chelß (yfghbveh, ehjdyz dkf;yjcnb),
yfłb bcckeljdfybz levjycnhbhen, xnj bÅveyeybe
d lbene, jcyjdfyyje yf geherkxeybb yf lheujq
rjhv, dehjznyj, ye jrfÅßdfen dkbzybz yf
abÅbjkjubxecrbe gjrfÅfnekb cdzÅfyyße cj
cyb;eybev rj;yjq djljghjybçfevjcnb.
Dutch Epidermische lipiden dienen als de primaire bar-
rière tegen het cutane verlies van water en spelen een
belangrijke rol in het conserveren van water en homeo-
stase. Eerdere studies toonden onder species de correlatie
tussen verhoogde onvruchtbaarheid van habitatten en de
hoeveelheid epidermische lipiden. In het algemeen verlaagt
een verhoogde hoeveelheid epidermische lipiden de perme-
abiliteit van de huid. Soort-specifieke verschillen in het
verlies van water via de huid en de prooivoorkeur tussen
twee sympatrische slangensoorten, de Watermoccasinslang
(Agkistrodon piscivorus) en de Koperkop (AgkistrodonCottonmouth skin lipids and CWL 13
contortrix), heeft ons gemotiveerd ons af te vragen of de
prooibasis kan resulteren in deze waargenomen soort-spec-
ifieke verschillen in cutane waterverlies. Om te onder-
zoeken of het dieet de hoeveelheid en kwaliteit van de
epidermische lipiden en de mate van waterverlies via de
huid kan beı̈nvloeden, hebben we het dieet van een in
gevangenschap genomen kolonie Watermoccasinslang
(Agkistrodon piscivorus) experimenteel gecontroleerd door
het of vis (Notemigonus crysoleucas) o
f muizen (Mus mus-
culus) te voeren. De slangen die met muizen gevoed wer-
den kregen consequent meer massa, maar de dieet behand-
eling had geen effect op de mate van groei. Gedurende de
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voedingskuren hebben we geen significante verschillen
kunnen vinden in de kwantitatieve lipide inhoud of de
mate van waterverlies. Een analyse voor kwalitatieve lipide
inhoud met gebruik van IR spectrofotometrie toonde geen
effect door de voeding, wat dus suggereert dat lipide
inhoud en cutane waterverlies zeer species-specifieke fysio-
logische functionering eigenschappen zijn, die niet
beı̈nvloed worden door recente dieet geschiedenis.
Terwijl er sommige bewijs is dat epidermische permeabi-
liteit variabel kan zijn onder bepaalde natuurlijke omstan-
digheden (b.v. vochtigheid), laten onze bevindingen zien
dat dieet er geen effect op heeft en dat een verschuiving in
prooivoorkeur niet de fysiologische functionering hoeft te
beı̈nvloeden of te verhogen voor afnemende cutane
waterverlies.You can also read
