Regulation of Seat-patch Water Potential in Anurans
Lee A. Lemenager1
C. Richard Tracy1
Christopher R. Tracy2
Keith A. Christian3
1
Department of Biology,
University of Nevada, Reno
Reno, Nevada 89557
USA
2
Department of Biology
California State University Fullerton
800 N. State College Blvd.
Fullerton, CA 92831-3599
USA
3
School of Environment and Life Sciences
Charles Darwin University
Darwin, Northern Territory 0909
AUSTRALIA
Correspondent: C. Richard Tracy, dtracy@unr.edu
Key words: seat patch, water exchange, water potential, water relations, frogs,
hydroregulation, aquaporins.
-1-
Lemenager et al.
pg. 2
Abstract
Decades of research on water exchange in frogs has assumed that blood osmotic potential
drives water exchange, but recent studies have suggested that the seat-patch (the primary
site of water exchange in many species) may act as a compartment with a water potential
that can be controlled to be different from that of the blood. Here, we report on
measurements of the water potential of the seat-patch and blood in six frogs (Xenopus
laevis, Rana pipiens, R. catesbeiana, Anaxyrus boreas, Pseudacris cadaverina and P.
hypochondriaca) differing in ecological relationships to environmental water. We
inferred seat-patch water potentials from water exchanges by frogs in different sucrose
solutions that ranged from 0 to1000 -kPa. In general, species that are more aquatic had
seat-patch water potentials that were less negative than those of terrestrial species, and
their seat-patch water potentials were different from the water potentials of their blood.
Terrestrial and arboreal species had seat-patch water potentials that were more negative,
and their seat-patch water potentials were similar to the water potentials of their blood.
These findings indicate a physiological mechanism to control water potential of the seatpatch. Additionally, our results suggest that frogs can adjust the hydric conductance of
their skin when they are absorbing water from very dilute solutions (with water potentials
close 0). Both the differences in water potential of seat patches among species differing
in ecological habit, and adjustments in hydric conductance might be explained by
complex (largely unexplored) mechanisms involving aquaporins.
-1-
Lemenager et al.
pg. 3
Introduction
Amphibians can be found in a diverse range of habitats ranging from fully aquatic, to
fully terrestrial or arboreal (Hillman et al. 2009). Some habitats are xeric and can
potentially cause rapid desiccation and death due to dehydration. However, some
amphibians employ physiological and behavioral mechanisms to prevent water loss and
to increase water uptake (Bentley 1971). Understanding the successes of amphibians in
environments differing in degree of terrestriality requires understanding the intricacies of
adaptations for water exchange in different hydric environments.
Nearly all anurans (frogs and toads) must maintain a moist skin to survive (Duellman and
Trueb 1994, Jorgensen 1997b) because they do not have cornified skin layers or scales to
protect underlying tissue skin layers (Drewes et al. 1977, Shoemaker and McClanahan
1975). Loss of water from a moist skin can be lethally rapid (Jorgensen 1991, 1997a,
1997b, Young et al. 2005, Hillman et al. 2009), and anurans often replace rapid water
losses by efficiently absorbing water through their skin (Sinsch 1991, Jorgensen 1994,
McClanahan and Baldwin 1968, Hillman et al. 2009). Most frogs are able to absorb water
rapidly through a highly vascularized area of skin called the “seat-patch” which is
associated with both behavioral and physiological adaptations for water uptake
(McClanahan and Baldwin 1969). The behavioral adaptations consist of body posturing,
so that the seat-patch is adpressed against a wet environmental surface from which water
can be absorbed (Heatwole et al. 1969). Physiological adaptations of the seat-patch
include control of rates of water uptake via antidiuretic hormones (reviewed in Hillman et
al. 2009), which can influence the permeability of the seat-patch largely by poorly-known
Lemenager et al.
pg. 4
mechanisms that include changes of skin conductance (Tracy 1976, 1982) and seat-patch
water potential (Tracy and Rubink 1978, Hillman et al. 2009).
We studied the seat-patch water potentials of six species that differ in habitat use. The
African clawed frog, Xenopus laevis, is a fully aquatic species while the American
bullfrog, Rana catesbeiana, is a model of the semi-aquatic species. The semi-terrestrial
mesic species is the Northern leopard frog, Rana pipiens, and the semi-terrestrial xeric
species is the Western toad, Anaxyrus boreas. Finally, two scansorial species; the Pacific
chorus frog, Pseudacris hypochondriaca, which can be found both in trees and under logs,
and the California tree frog, Pseudacris cadaverina, which is often found basking in
sunlight on rocks near streams. Because six frogs are found in a wide range of hydric
environments, they should be able to regulate their water uptake in ways that work
differently in different hydric environments. Thus, highly terrestrial species would benefit
from a seat-patch water potential that is much lower than that of the environment to
facilitate rapid water uptake, but fully-aquatic frogs would benefit from a seat-patch
water potential that is closer to that of water as a way to prevent excessive water uptake.
Here, we report on experiments to measure seat-patch water potentials of several frog
species that differ in their ecological habit.
Materials and Methods
Animals and lab conditions
Specimens (mean mass±SD) of X. laevis (3.4 ±1.0g, n=17) and R. pipiens (52.1 ±4.1g,
n=20) were purchased from commercial suppliers (X. laevis from Aquatic Plant Depot,
Tampa FL; and R. pipiens from Charles D. Sullivan Co., Inc. Nashville, TN). R.
Lemenager et al.
pg. 5
catesbeiana (60.8 ±12.8g, n=15) and A. boreas (93.6 ±14.7g, n=20) were captured at
Rancho San Rafael Park (39.550186 °N, -119.828606 °W; 4672 m) in Reno, NV. P.
cadaverina (4.2 ±1.6g, n=8) were collected from Orange County, CA, and P.
hypochrondriaca (5.4 ±1.5g, n=18) were collected from Limekiln Canyon Wash, Rinaldi
Park, Northridge, Los Angeles Co., California, USA (34.27743 °N; 118.56192 °W; 329
m). Between experiments, all frogs were maintained in ten gallon aquaria in a laboratory
at the University of Nevada, Reno where air temperatures ranged from 21 to 26 °C. The
larger frogs were housed in groups of two to three individuals per aquarium, and the
smaller frogs were maintained in groups of three to five individuals per aquarium.
Water exchange with the environment
The seat-patch water potential was inferred from experiments using different
environmental water potentials to find the conditions where frogs do not exchange water
with the environment – i.e., when the water potential of the environment and seat-patch
are equal. Prior to each experiment, frogs were placed in pure water for two hours to
allow them to become fully hydrated. Then, frogs were catheterized to remove any water
in the bladder. The body mass of the frog with an empty bladder was then recorded as the
“standard body mass” (Ruibal 1962, McClanahan 1972, Tracy 1976). The frogs were
then dehydrated in a wind tunnel until they dehydrated to 90% of their standard body
mass, and then the frogs were placed in an apparatus similar to the one used in Tracy and
Rubink (1978) in which the frogs sat in contact with a sucrose solution with only the
bottom few millimeters of their body being in contact with the solution, and the rest of
their body exposed to the air in the apparatus. Subsequently, the body masses of the frogs
were measured every 10 min after the frog was blotted dry with a paper towel.
pg. 6
Lemenager et al.
Cutaneous evaporative water loss during the water uptake experiment was estimated from
experiments using frog models made from 3% agar molded into the shape of the frog.
Negative molds of each species of frogs were made by pouring dental Alginate on live
frogs that had been given MS222 (Tricaine) as an anesthetic to minimize the frog’s
movements while the mold was being made (Alginate sets in less than one minute, so no
harm is caused to the frog). Plaster of Paris was then poured into the alginate mold to
create a positive mold of the frog. By this approach, the agar frog models were made to
be the same size, shape and posture as the living frogs from which molds were made.
Latex was then painted onto the plaster of Paris mold in several layers to make a thick
and durable negative mold that could be reused many times. A 3% agar solution was then
poured into the latex mold and allowed to set. Water absorption by the resulting agar
model was prevented by coating the venter of the model with fingernail polish
approximately to where the frog would be in contact with the sucrose solution. The agar
frog model was placed in the experimental apparatus for measuring water uptake, but the
liquid solution was not allowed to come into contact with agar frog model. Thus, the
relative humidity in the apparatus was the same as that in experiments for measuring seatpatch water potential. The change in body mass of the agar frogs was recorded every ten
minutes, while the model dehydrated, by weighing only the apparatus containing the agar
model.
The mean change in body mass in the apparatus reflects the sum of water uptake and
evaporative water loss, so the water influx into the frog was obtained by adding the
cutaneous evaporative water loss to the net water exchange. This calculated water influx
was then plotted against water potential of the environment (sucrose solutions) to obtain
Lemenager et al.
pg. 7
the x-intercept of this relationship (the point at which no liquid water is exchanged
because the water potential of the seat-patch is equal to the water potential of the sucrose
solution). Regressions of water uptake for each individual frog that had an R2 less than
0.6 were removed from calculations of the mean and standard deviation of the seat patch
water potential. A series of t-tests were used to test for differences of seat-patch water
potentials among the six species.
Osmotic Potential of Blood
Blood was collected from frogs dehydrated to 100% and 90% of their standard (fully
hydrated) body mass. Individuals of X. laevis had to be dehydrated osmotically by
placing them in a sucrose solution of -600 kPa because this species seemed to be unable
to withstand dehydration in air (perhaps they cannot supply mucus to the skin as can
more terrestrial anurans). Blood samples were collected from all frogs via the abdominal
midline vein using a 23-gauge hypodermic needle and blood osmolality was measured
immediately with a freezing point osmometer (Advanced Instruments 3MO).
The osmotic potentials of blood for each species at 100% and 90% hydration were
compared using a t–test. For each species, the water potential of the seat-patch and the
osmotic potential of blood at 90% hydration were also compared using a t-test.
Results
Water exchange with the environment
Cutaneous evaporative water losses (Table 1) ranged from 0.42 mg/min in X. laevis to 4.7
mg/min in R. catesbeiana, representing, respectively,0.88% to 0.17% of body mass lost
Lemenager et al.
pg. 8
per hour. These data were added to species water uptake rates to account for water lost
through cutaneous evaporation while in the experimental setup (Fig. 1). The water uptake
rate for the fully aquatic X. laevis ranged from 1.44 ± 0.84 mg/min at 0 kPa to -0.27 ±
0.57 mg/min at -300kPa and in R. catesbeiana it ranged from 29.77 ± 19.01 mg/min at 0
kPa to -14.94 ± 13.85 mg/min at -400kPa. For the semi-terrestrial mesic R. pipiens the
water uptake rate ranged from 43.01 ± 10.44 mg/min at 0 kPa to -1.93 ± 5.89 mg/min at 500kPa and for the semi-terrestrial xeric A. boreas it ranged from 41.89 ± 14.69 mg/min
at 0 kPa to -1.44 ± 11.97 mg/min at -600 kPa. For the arboreal species, P. cadaverina
ranged from 5.48 ± 2.59 mg/min at 0kPa to -0.57 ± 0.64 mg/min at -700kPa and P.
hypochondriaca ranged from 3.79 ± 2.42 mg/min at 0 kPa to -1.29 ± 1.32 mg/min at 1000kPa.
Seat-patch water potentials were generally related to the ecological habit of the species
(Fig. 2). The seat-patch water potential of the fully aquatic X. laevis was -283±21kPa and
the semi-aquatic R. catesbeiana was -316±76kPa. The semi-terrestrial mesic R. pipiens
had a seat-patch water potential of -487±67kPa and the semi-terrestrial xeric A. boreas
was -589±144kPa. The seat-patch water potential of the first arboreal species P.
cadaverina was -674±70kPa and second, P. hypochondriaca, was -837±114kPa.
The fully aquatic X. laevis, and the semi-aquatic R. catesbeiana had statistically similar
seat-patch water potentials (t19 = 1.11, P > 0.2), and these species were statistically higher
(less negative) than all other species (P < 0.001, See Appendix A for statistical
comparisons). The seat-patch of the semi-terrestrial mesic species, R. pipiens, was
Lemenager et al.
pg. 9
significantly higher than A. boreas, the semi-terrestrial xeric species (t32 = 2.59, P <
0.02), and higher than both of the arboreal species (R. pipiens vs. P. cadaverina t21 =
6.08, P < 0.001, R. pipiens vs. P. hypochondriaca t28 = 10.41, P < 0.001). The seat-patch
water potential of A. boreas was similar to the chorus frog, P. cadaverina (t23 = 1.48, p >
0.1), but it was statistically higher than the other arboreal species, P. hypochondriaca (t30
= 5.28, p < 0.001). P. cadaverina had a seat-patch water potential statistically higher than
P. hypochondriaca (t19 = 3.45, p < 0.01) which had the lowest (most negative) seat-patch
water potential.
Osmotic Potential of Blood Compared to Seat-patch Water Potential
The osmotic potentials of blood (Table 2) were lower (more negative) at 90% hydration
than at 100% hydration (i.e. they had a greater concentration of osmolytes). Among the
more aquatic species, X. laevis and R. catesbeiana, and the semi-terrestrial species, R.
pipiens (Figure 3), seat-patch water potentials were significantly higher than (i.e. less
negative, and less concentrated) the blood osmotic potential (X. laevis: t12 = 29.71, P <
0.001; R. catesbeiana:t18 = 8.33, P < 0.001, R. pipiens: t21 = 5.54 P < 0.001). Among the
more terrestrial, and arboreal species, seat-patch water potentials were not significantly
different from the blood osmotic potential (A. boreas: t24 = 1.86, P > 0.05; P. cadaverina:
t8 = 0.78, P>0.40 ; and P. hypochondriaca:t21 = 0.08, P>0.90).
Discussion
Seat-patch water potentials were related to terrestriality (including arboreality) in the six
frog species studied, with species that were more aquatic generally having higher (less
Lemenager et al.
pg. 10
negative) seat-patch water potentials than those that were more terrestrial. Similarly,
differences between water potential of the seat-patch and blood were related to
terrestriality insofar as aquatic and the semi-terrestrial mesic species had different
osmotic potentials of the blood compared with the seat-patch, but the semi-terrestrial
xeric and arboreal species had osmotic potentials of the blood that were similar to those
of the seat-patch.
Our results suggest that more aquatic species are able to control their seat-patch water
potentials to be higher (less negative) than the osmotic potential of their blood, but more
terrestrial species appear unable to do so. This ability to control the water potential of the
seat-patch is potentially beneficial to aquatic species, as it would allow them to slow
influx of water, thus reducing the requirement to urinate excessively, which could
negatively affect the body’s ion balance. The opposite is true for species that are more
terrestrial; having a high seat-patch water potential would allow these species to absorb
water more rapidly when these frogs are dehydrated.
The mechanisms by which frogs could regulate their seat-patch water potential may
involve control of aquaporins (Preston and Agre 1991) located in the tissues of the seatpatch. One amphibian-specific type of aquaporin, AQP-h3 is found in the ventral skin of
all anurans regardless of the frog’s ecological habit (aquatic, semi-terrestrial, terrestrial
and arboreal); another amphibian-specific aquaporin, AQP-h2 is found in the urinary
bladder of species across all habitat types as well as the ventral skin of some species
(Suzuki et al. 2007, Suzuki and Tanaka 2009, Ogushi et al. 2010). Specifically, the pelvic
skin of aquatic and semi-terrestrial species lack AQP-h2, but it is found in the ventral
Lemenager et al.
pg. 11
skin of species that are more terrestrial or arboreal species (Suzuki et al. 2007, Ogushi et
al. 2010). Thus, species that are more terrestrial appear to have two types of aquaporins in
the seat-patch while those that are more aquatic appear to have only one type. We use the
word “appear” here because to date, very few anuran species have been assayed for
aquaporin type in the ventral skin. Nevertheless, the patterns among aquaporins in the
work by Suzuki et al. (2007), Suzuki and Tanaka (2009) and Ogushi et al. (2010) are
consistent with our observations of control of seat-patch water potential in relation to
ecological habit of the frogs.
Despite the presence of AQP-h3 in the seat-patch, the permeability of the seat patch of
the fully aquatic species, X. laevis, is unaffected by antidiuretic hormones such as AVT
(Bentley 1969, Bentley and Main 1972, Cree 1988). Interestingly, compared to other
species the sequence of AQP-h3 in X. laevis has an additional eleven amino acid terminal
segment, called a CT tail (Ogushi et al. 2010). This unique aquaporin, named AQP-x3,
has lost its functionality as an aquaporin, and it does not affect membrane permeability
while the CT tail is present. When AQP-x3 has the CT tail experimentally removed, it
regains its functionality as an aquaporin (Ogushi et al. 2010).
The water potential of the substrate from which terrestrial frogs obtain water plays a large
role in frog water balance. The water potential of soil changes depending on the
composition of the soil, and how much water is in the soil (Rose 1966, Tracy 1976).
Terrestrial frogs would benefit from having a lower (more negative) seat-patch water
potential because this would allow absorption of water from soils with a wider range of
water potentials. Semi-terrestrial mesic species would benefit from having intermediate
pg. 12
Lemenager et al.
seat-patch water potentials as they spend a majority of their time sitting on moist
substrates. Semi-terrestrial mesic species would still have a range of soil water potentials
from which to take up water while on short forays away from water. It appears that
terrestrial species possess both AQP-h3 and AQP-h2 types, but a broader survey of the
molecular diversity of aquaporins present in the seat-patch of terrestrial species is
certainly warranted.
An unexplained observation in our research reported here was the ability of five species,
all except P. cadaverina, to maintain a relatively similar water uptake rate while
immersed in different water potentials (see data designated with unfilled circles in Fig.
1). The rates of water exchange between a frog and its environments are described with
the equation (Tracy 1976),
•
mw = AvKfrog (Ψfrog – Ψe )
where:
•
mw
Is the rate of water exchange
Av
Is the area of skin through which water is transported
Kfrog
Is the hydric conductance of the frog skin
(Ψfrog – Ψe )
Is the water potential difference between the seat patch and the environment
If all properties of the frog (viz., Av, Kfrog, Ψfrog) are constant, then the equation describes
a straight line where the slope of the line is (Av * Kfrog), and the x-intercept is where Ψfrog
= Ψe.
However, our results indicate that the lines, for most species, are not uniformly
straight lines, but that they change slope at environmental water potentials between 0 and
Lemenager et al.
pg. 13
-200 kPa. That indicates that Kfrog is a constant at more negative environmental water
potentials (<-200 kPa), but in environments with osmotic potentials closer to pure water
(0 to -200kPa) there was a variable conductance of the frog (Kfrog). This pattern may be
due to changes in aquaporin activity that prevents uptake of excess water in dilute
environments, but this pattern could also indicate a change in effective surface area
through which water is absorbed (Av). This indicates that water potential of seat patches
(potentially due to hormonally-controlled aquaporin activity), hydric conductances of
frogs, and even surface area through which water is absorbed are variables that appear to
act somewhat independently in different environments, and certainly these variables can
portray important biophysical mechanisms governing water exchange by frogs in
different environments. Evaluating the physiological (and maybe behavioral)
mechanisms by which these biophysical variables change is beyond the scope of this
report, yet it represents an exciting and logical next step in investigating the physiology
of water uptake in frogs.
Acknowledgements
We thank Nichole Maloney for her help in getting the research started. We also thank the
members of the Tracy Lab for their invaluable input, and especially thank John Gray for
his countless hours of providing animal care. This research was conducted under IACUC
approval number 2008-0386 and 2008-00108.
Lemenager et al.
pg. 14
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Lemenager et al.
Tables
TABLE 1. Mean (±SD) cutaneous evaporative water loss (CEWL) of six anuran species in
our experimental apparatus. Data were collected using agar frog models (3%
agar). Comparisons were made between species by looking at both the percent
body mass lost per hour, and the CEWL in mg/min.
Species
Mean CEWL % body mass Body mass lost Sample
(mg/min)
lost per hour
(mg/min/g)
size
Xenopus laevis
0.42± 0.02
0.88
0.1174
4
Rana catesbeiana
4.7 ± 0.93
0.17
0.0735
3
Rana pipiens
2.8 ± 0.42
0.32
0.0535
4
Anaxyrus boreas
2.5 ± 0.16
0.18
0.0270
3
Pseudacris cadaverina
0.54 ± 0.15
0.58
0.1321
4
Pseudacris hypochondriaca 0.53 ± 0.06
0.63
0.0985
4
pg. 18
Lemenager et al.
TABLE 2. Osmotic potentials of blood (mean ±SD) in six anuran species while at
100% and 90% standard body mass (the mass of a fully hydrated frog with
an empty bladder). The osmotic potential of blood was measured using a
freezing point osmometer. Sample sizes are given in parentheses.
Osmotic Potential (-kPa)
Species
Blood (100%)
Blood (90%)
Xenopus laevis
581 ± 23 (6)
700 ± 29 (7) *
Rana catesbeiana
480 ± 32 (12)
580 ± 13 (6) *
Rana pipiens
593 ± 22 (7)
697 ± 31 (7) *
Anaxyrus boreas
590 ± 7 (6)
685 ± 11 (8) *
Pseudacris cadaverina
635 ± 35 (4)
707 ± 13 (3) *
Pseudacris hypochondriaca
710 ± 44 (6)
840 ± 41 (9) *
* denotes a statistical difference between 90 and 100% hydration at p = 0.05
pg. 19
Lemenager et al.
Appendix A
TABLE A-1. Statistical comparisons between the seat-patch water potentials of Rana
catesbeiana and Xenopus laevis and other anuran species used in the
experiment using a t-test.
Species vs. Species
Student-t
df
P
R. catesbeiana vs. P. hypochondriaca
14.23
26
< 0.001
R. catesbeianavs. P. cadaverina
10.43
19
< 0.001
R. catesbeianavs. A. boreas
6.42
30
< 0.001
R. catesbeianavs. R. pipiens
6.55
28
< 0.001
X. laevisvs. P. hypochondriaca
12.59
19
< 0.001
X. laevisvs. P. cadaverina
14.16
12
< 0.001
X. laevisvs. A. boreas
5.53
23
< 0.001
X. laevisvs. R. pipiens
7.80
21
< 0.001
pg. 20
Lemenager et al.
Figure Legends
Fig. 1. The rate of water exchange, corrected for cutaneous evaporative water
loss, for six anuran species (mean ± SD) exposed to different
environmental water potentials (sucrose solutions). The x-intercept of the
slope is taken to be the point at which no net water exchange occurs and
is therefore where the seat-patch water potential is equal to the
environmental water potential. Filled circles are mean water uptake rates
that follow the linear equation for the rate of water exchange between a
frog and its environment. Mean and standard deviations of standard body
masses are given under the names of species.
Fig. 2. The seat-patch water potential (mean ± SD) of six anuran species dehydrated to
90% standard body mass. Sample sizes are given in parentheses. Species have
been arranged in order of increasing terrestriality from left to right.
Fig. 3. Comparisons between the seat patch water potentials and the blood osmotic
potentials of six anuran species. Data are means ± SD; * denotes statistical
significance at p < 0.05.
Lemenager et al.
Figure 1
pg. 32
Lemenager et al.
Figure 2
pg. 33
Lemenager et al.
Figure 3
pg. 34
Lemenager et al.
pg. 35