2 METHODS
2.1 Animals and lab conditions
Specimens (mean mass ± SD) of X. laevis (3.4 ± 1.0 g) andR. pipiens (52.1 ± 4.1 g) were purchased from commercial
suppliers (X. laevis from Aquatic Plant Depot, Tampa, Florida,
USA; and R. pipiens from Charles D. Sullivan Co., Inc. Nashville,
Tennessee, USA). Rana catesbeiana (60.8 ± 12.8 g) and B.
boreas (93.6 ± 14.7 g) were captured at Rancho San Rafael Park
(39.550186° N, -119.828606° W; 4672 m) in Reno, Nevada, USA.Pseudacris cadaverina (4.2 ± 1.6 g) were collected from Orange
County, California, USA, and P. regilla (5.4 ± 1.5 g) 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 38 L 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.
2.2 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 (Tracy and Rubink
1978). 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), and then the frogs
were dehydrated in a wind tunnel to 90% of their standard body mass
which took from one to several hours depending on species and activity
of the frogs. Once they reached this target level of dehydration, the
frogs were placed individually into an apparatus similar to the one used
in Tracy and Rubink (1978) consisting of a plastic container containing
a sucrose solution so that only the ventral surface of the frog’s body
was in contact with the solution, and the rest of its body was exposed
to the air. For the following hour, the frogs’ body masses were measured
every 10 min after the frog was blotted dry with a paper towel. Each
frog was tested in the various sucrose solutions, with at least 7 days
between experiments. The order of testing began with pure water and
progressed towards more negative water potentials.
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, which evaporates as a free water surface of
the given size and shape (Spotila and Berman 1976). Negative molds of
each species of frogs were made by pouring dental alginate on live frogs
that had been given MS222 (Tricaine methanesulfate) 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. 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. 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. Water absorption by the resulting agar model was
prevented by coating the venter of the model with fingernail polish
approximately 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 seat
patch water potential. The body mass of the agar models was measured
every ten minutes.
The mean change in frog 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 evaporative water loss (estimated from
the agar models as described above) to the water exchange (measured in
the apparatus described above). This calculated water influx was then
plotted against water potential of the environment (sucrose solutions)
to obtain the x-intercept of this relationship, which is the point at
which no liquid water was exchanged because the water potential of the
seat patch was equal to the water potential of the sucrose solution.
Data were discarded in cases when excessive frog activity resulted in
questionable results. The bladders of the frogs were voided prior to
being dehydrated to 90% standard body mass to decrease the likelihood
of urination, but the data were discarded when either urination or
defecation occurred during the experiments. Inspection of plots of water
uptake as a function of sucrose water potential showed two patterns for
each species except P. cadaverina : a sloping linear relationship
at lower water potentials, and a horizontal relationship at high water
potentials. That is, for dilute solutions (near 0 kPa), water uptake
rates were similar despite the frogs being subjected to different water
potentials, but at more negative water potentials there was a sloping
linear relationship between water potential and water uptake. Thus,
because it was clear that different mechanisms governed the points at
different water potentials for these species, only the points along the
slope were used in regression analyses to determine the seat patch water
potentials (intercept of the X-axis). In P. cadaverina , the
X-intercept was not different if the point for the highest water
potential was used in the regression or not. We used an ANOVA followed
by a Tukey HSD to test for differences of seat patch water potentials
among the six species.
2.3 Osmotic Potential of Blood
Several months after the experiments described above were completed, the
osmotic potential of the blood was measured for each frog at 100% and
90% of their standard (fully hydrated) body mass, with a one week
interval between the two measurements. Although most species were
allowed to dehydrate in air, X. laevis had to be dehydrated
osmotically by placing them in a sucrose solution of -600 kPa because
this species showed signs of distress in air (perhaps they cannot supply
mucus to the skin as can more terrestrial anurans). Blood samples
(approximately 25 µL) were collected from all frogs via the abdominal
midline vein using a 23-gauge hypodermic needle, and whole blood
osmolality was measured immediately with a freezing point osmometer
(Advanced Instruments 3MO).
Water Potential is expressed in the SI units of kPa, but the osmometer
output was in units of mOsm. Blood osmotic potentials were converted to
units of kPa using the van’t Hoff Law (Salisbury and Ross 1969).
The osmotic potentials of blood for each species at 100% and 90%
hydration were compared using a paired 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 paired t-test.