4. DISCUSSION
Seat patch water potentials were related to terrestriality (including
arboreality) in the six frog species studied, with species that were
more aquatic having higher (less negative) seat patch water potentials
than those that were more terrestrial or arboreal. Similarly,
differences between water potential of the seat patch and of the blood
were related to terrestriality insofar as the aquatic species and the
terrestrial R. pipiens had different osmotic potentials of the
blood in comparison with their seat patch water potentials, but the
terrestrial toad and arboreal species had osmotic potentials of the
blood that were similar to those of their seat patches.
The basic physiology of water uptake through the ventral skin of
amphibians has been studied extensively (reviewed by Jørgensen 1997b)
working under the assumption that blood osmotic water potential drives
water exchange. Thus, the prevailing paradigm neither explains how
aquatic frogs avoid taking up too much water, nor does it explain the
disequilibrium between the seat patch water potential and blood water
potential in species that live in or near water (Fig. 3). Studies of
water uptake in amphibians have emphasized the behavioral, physiological
and ecological mechanisms that allow “thirsty” terrestrial frogs to
take up water rapidly (Tracy 1976; Jørgensen 1997b) with little emphasis
on how frogs in aquatic environments avoid taking up too much water.
Our results suggest that species that are more aquatic appear to control
their seat‑patch water potentials to be higher (less concentrated) than
the osmotic potential of their blood, but species that are more
terrestrial 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 limit the influx of water.
Unrestricted water influx into frogs that spend considerable time
exposed to water could create metabolic and electrolytic costs
associated with ridding the body of excess water.
Terrestrial species face challenges to balancing their water budgets
different from challenges faced by species that are primarily aquatic.
For example, many terrestrial anurans absorb water from moist soils, and
may be the primary source of water for some terrestrial species. The
water potentials of soils depend upon soil composition and water content
of the soil (Rose 1966; Tracy 1976). Thus, terrestrial frogs would
benefit from having a lower (more concentrated) seat-patch water
potential because that would allow absorption of water from soils with a
wider range of water potentials, including relatively dry soils.
Although circulatory changes are associated with water uptake through
the seat patch (Viborg and Hillyard 2005; Viborg et al. 2006), it seems
unlikely that circulatory adaptations alone could account for the
disequilibrium between the seat patch and blood water potentials that we
observed in the more aquatic species. The mechanisms by which frogs
could regulate their seat patch water potential may involve hormonal
control of water channel proteins called aquaporins (Preston and Agre
1991) that are located in the bladder, in the tissues of the ventral
skin, and possibly around the capillaries that connect the seat patch
with the rest of the body’s circulatory system. A number of different
types of aquaporins have been described from various amphibian tissues,
and there are differences among species with respect to aquaporin types
and expression (Suzuki et al. 2015). We suggest that the conductance of
the walls of the capillaries that connect the seat patch with the rest
of the body’s circulatory system can change in response to hormonal
signals acting on aquaporins. This change in conductance regulates water
uptake into the body’s circulatory system. Furthermore, this regulation
can, under some conditions (see below), result in a disequilibrium
between the water potentials of the seat patch and the blood.
The rates of water exchange between a frog and its environments can be
modelled with the equations in Fig. 4 (Tracy 1976; Tracy and Rubink
1978; Tracy 1982). If all properties, or system states, of EquationA (viz., A v, K sp,Ψ sp) are constant, then equation Adescribes a straight line where the slope of the line is
(A v * K sp), and the
x‑intercept is where Ψ sp =Ψ en. However, in experiments in which the
environmental (sucrose) water potential was a variable, our results (in
Fig. 1) indicate that the lines, for most species, are not uniformly
straight lines, but that they change slope relative to environmental
water potentials. Thus, K sp is a constant at more
negative environmental water potentials (closed circles in Fig. 1), but
in environments with osmotic potentials closer to pure water there was a
variable conductance of the frog (K sp) sufficient
to offset the changing environmental (sucrose) water potential, leading
to the observed leveling of the relationship between water uptake and
environmental water potential (open circles in Fig. 1). It seems likely
that this pattern is due to changes in aquaporin activity that prevents
uptake of excess water in dilute environments. This indicates that water
potential of seat patches (likely due to hormonally-controlled aquaporin
activity), and hydric conductances of seat patches are variables that
appear to act differently among different species, related to their
ecological habits.
If water uptake rates are slowed in environments with higher water
potentials (i.e., water potentials closer to that of pure water) by the
actions of aquaporins located between the seat patch and the circulatory
system (Equation B in Fig. 4), this would provide a mechanism that would
explain the data designated by open circles in Fig. 1. Five of the six
species (all except P. cadaverina ) maintained a relatively
similar water uptake rate while immersed in a range of high water
potentials. Thus, if the flow of water from the seat patch to the
circulatory system was restricted by the controlling action of
aquaporins, the seat patch water potential (Ψ sp)
would be high and similar to the environmental water potential, but the
water potential of the blood would remain low (i.e., the blood would
maintain its osmotic concentration) rather than becoming excessively
dilute. This (possibly aquaporin‑mediated) regulation would be
particularly beneficial for amphibians that spend a lot of time in
water, and is consistent with our results (Figs. 1, 2 and 3) that show
that the more aquatic species have fluids in the seat patch that are
significantly more dilute than blood. Such a disequilibrium cannot be
explained simply by the water potential gradients between the seat patch
and the blood, but it could result from an aquaporin-mediated decrease
in hydric conductance between the seat patch and blood
(K bl). As mentioned above, there is evidence that
the influx of water across the skin is independent of cutaneous blood
flow (Viborg and Hillyard 2005; Viborg et al. 2006). However, Burggren
and Viborg (2005) have raised the possibility that blood flow may play a
role in regulating water influx by changing the “functional” surface
area (i.e., Abl in Equation B in Fig. 4). Thus, it is
possible that blood flow may play a role in regulating rates of water
uptake in addition to changes in conductance (by way of aquaporins).
Word and Hillman (2005) provide evidence that water taken across the
skin of cane toads (Bufo marinus ) is taken directly into the
capillaries rather than accumulating in lymphatic spaces. As we have
shown in Fig. 3, for the boreal toad, the water potentials of the blood
and seat patch are not different. Thus, there is no barrier preventing
water being taken into the capillaries, so we would not expect an
accumulation of fluids in the lymph. Therefore, the result for cane
toads is wholly consistent with our results for boreal toads and with
the model in Fig. 4. However, our model predicts that there would be an
accumulation of dilute fluids in the ventral tissues in aquatic frogs
such as Xenopus – a hypothesis that could be tested using the
techniques of Word and Hillman (2005). An alternative hypothesis would
be that the relatively impermeable skin of Xenopus could be
explained by a lack of AVT-stimulated aquaporins in this species.
However, the lack of an AVT response does not explain what mechanism
would prevent osmotic dilution of blood over time. More importantly, in
the context of the results shown in Fig. 1, a generalized low
permeability of Xenopus skin might explain the low rate of water
uptake, but it would not explain the less negative water potential
(compared to the other species in our study) represented by the 0 uptake
point on the X-axis intercept. This less negative water potential
suggests that there is an accumulation of dilute fluid inXenopus . Thus, the aquaporin-mediated model in Fig. 4 is
consistent with both the ability of terrestrial frogs to take up water
from a relatively dry environment, and the ability of aquatic frogs to
prevent an excessive water influx from a fresh water environment.
What currently is known about the types and roles of aquaporins in frog
skin, bladders, and kidneys has been reviewed by Suzuki et al. (2007),
Suzuki & Tanaka (2009), Ogushi et al. (2010) and Suzuki et al. (2015).
These reviews, based on data from relatively few species of frogs,
present a mechanistic aquaporin model for the transport of water across
frog epidermis that involves hormonal control of three or more different
aquaporin types. Although the model describes the transport of water
from the environment across the epithelium, it does not explain the
control of water transport into the sub-epidermal capillaries that link
the seat patch fluids with the circulatory system of the body. Thus, the
aquaporin model for cutaneous water uptake is incomplete. Much like the
historical paradigm of water uptake that assumed that osmotic water
potential of blood drives water exchange, the current aquaporin model
explains how terrestrial frogs take up water, but it does not explain
how aquatic species avoid taking up too much water. To address this
fundamental question, we need to know more about aquaporin types from a
wider range of species, and more about the mechanisms that control their
levels of expression. Furthermore, to achieve a comprehensive
understanding of frog water balance, the physiological and molecular
studies of aquaporins should be designed within an ecological context by
carefully selecting the species studied.
Evaluating the mechanisms by which the biophysical variables illustrated
in Fig. 4 change to produce an osmotic disequilibrium in different parts
of a frog is beyond the scope of this report, but it represents an
exciting and logical next step in investigating the physiology of water
uptake in frogs. An integrated approach involving physiological,
molecular and ecological principles is needed to understand the
evolutionary adaptations for water exchange between frogs and their
environments.