J Comp Physiol B (2001) 171: 23±32
Ó Springer-Verlag 2001
ORIGINAL PAPER
S. D. Bradshaw á K. D. Morris á F. J. Bradshaw
Water and electrolyte homeostasis and kidney function
of desert-dwelling marsupial wallabies in Western Australia
Accepted: 24 August 2000
Abstract Prolonged drought, necessitating conservation
of water, is one of the major environmental challenges
faced by many Australian marsupials. Radioactive isotopes of water and sodium were used to assess the ability
of two species of marsupial wallabies to maintain water
and electrolyte balance during periods of extreme water
deprivation in the arid Pilbara region of Western Australia. The spectacled hare-wallaby, Lagorchestes
conspicillatus, has the lowest mass-speci®c rate of water
turnover at 27.5 ml á kg)0.82 á day)1 yet reported for any
mammal and was two to three orders of magnitude
lower than that of the Rothschild's rock-wallaby, Petrogale rothschildi. Studies of renal function show that
the hare-wallaby conserves water by producing a highly
concentrated urine under the in¯uence of lysine vasopressin (LVP), the anti-diuretic hormone (ADH) in
macropodid marsupials. In contrast, rock-wallabies
show unusual renal responses to water deprivation, with
no change in LVP levels and a limited response to water
deprivation involving a reduction in renal plasma ¯ow
and glomerular ®ltration rate, with no signi®cant change
in tubular function. Both species are able to maintain
water and electrolyte homeostasis during periods of
drought, highlighting the ecacy of their di€ering
adaptive solutions to the problem of water scarcity,
Communicated by I. D. Hume
Preliminary communications of some of these data have been made
at the 13th International Congress of Comparative Endocrinology
in Yokohama in 1997 and at the Australian Mammal Society
meeting in Perth in December 1998
S. D. Bradshaw (&) á F. J. Bradshaw
Department of Zoology and Centre for Native Animal Research,
The University of Western Australia, Perth, WA 6009, Australia
e-mail: [email protected]
K. D. Morris
Science Information Division,
Department of Conservation & Land Management,
Woodvale, WA, 6026, Australia
although the hare-wallaby is superior to the rockwallaby in this respect. Rock-wallabies appear to rely
primarily on behavioural rather than physiological
responses for their survival in the Pilbara and appear to
be more vulnerable to extinction in the event of signi®cant habitat modi®cation. The secure nature of their
rock habitat, however, means that they have su€ered less
than hare-wallabies in the recent past.
Key words Marsupial á Kidney function á Wallaby á
Desert á Water
Abbreviations ADH anti-diuretic hormone á CH2 O freewater clearance á CI condition index á COSM osmolar
clearance á CPAH clearance of 3H-para-amino hippuric
acid á ECFV extracellular ¯uid volume á FF ®ltration
fraction á FRH2 O fractional reabsorption of the
®ltrate á LVP lysine vasopressin á PAH 3H-para-amino
hippuric acid á RH relative humidity á TBW total body
water content á U/POSM urine-to-plasma ratio of
osmolytes
Introduction
Seventy percent of the Australian environment is classi®ed as arid or desert and water is a major limiting
resource for animals inhabiting these regions. Measurements of rates of water turnover of free-ranging individuals, using isotopes of hydrogen (Nagy and Costa
1980; Nagy and Peterson 1988; Green 1997), provide a
means of assessing the extent to which di€erent species
are bu€ered from the e€ects of long-term drought by the
ecacy of their homeostatic regulatory systems (Bradshaw 1997). Studies of renal function also enable us to
gauge the extent to which pituitary and adrenal hormones
are responsible for maintaining ¯uid and electrolyte homeostasis of desert-dwelling marsupials that are routinely
exposed to long periods of water deprivation that may
involve stress responses de®ned as: ``the physiological
24
resultant of demands that exceed an organism's regulatory capacities'' (Bradshaw 1986, 1992).
Bakker and Bradshaw (1983) studied renal function
in the spectacled hare-wallaby, Lagorchestes conspicillatus, from Barrow Island and suggested that its extremely ecient water economy was linked to its ability
to recycle nitrogen in the form of urea to its digestive
tract. They also proposed a mechanism for urea recycling based on a signi®cant positive correlation that they
observed between urea clearance and rates of urine
production ± suggesting that urea recycling was initiated
by elevated levels of anti-diuretic hormone (ADH).
Their study was limited, however, by the lack of a
suitable assay for marsupial ADH. This was subsequently identi®ed as lysine vasopressin (LVP), rather
than arginine vasopressin in the macropodid marsupials
by Chauvet et al. (1983). The development of an heterologous radio-immunoassay for LVP paved the way for
further studies of the role of this hormone in controlling
the water economy of a variety of marsupial species,
including those from desert habitats (Bradshaw 1990,
1997; Jones et al. 1990).
The present study focused on two desert-dwelling
macropodid wallabies found living on islands o€ the
arid north-west coast of Western Australia. One, the
spectacled hare-wallaby L. conspicillatus, is now only
found in abundance in spinifex grasslands on Barrow
Island, some 1500 km north of the capital, Perth, and
its ability to maintain thermal balance in this hot, dry
habitat has been previously studied by Dawson and
Bennett (1978). Rothschild's rock-wallaby, Petrogale
rothschildi, is a species endemic to the Pilbara region of
WA and found associated with large rock piles o€ering
caves and caverns suitable for refuge during the day.
The two species thus di€er in the extent of their exposure to the aridity of their habitat ± the hare-wallaby
shelters during the day within large spinifex clumps
where temperatures rise to over 40 °C. The rock-wallaby, on the other hand, avoids the heat of the day by
sheltering in cool, humid caves where the air temperature rarely exceeds 30 °C and one would expect its
water economy to bene®t as a result of this. The two
species thus o€er an unique opportunity to compare
the water and electrolyte balance and ecacy of the
hormonal control systems of two macropodid marsupials occupying an arid habitat but possibly di€ering in
the extent to which they rely on physiological adaptations for their ultimate survival.
Materials and methods
Study areas
Field studies were carried out on Enderby Island (Latitude:
20°36¢33¢¢S, Longitude: 116°31¢12¢¢E)and Barrow Island (Latitude:
20°47¢57¢¢S Longitude: 115°24¢18¢¢E) in the arid Pilbara region of
Western Australia, some 1500 km north of Perth. Field trips to
Barrow Island were carried out in November 1990, April 1991 and
April 1992; trips to Enderby Island were in March and November
1986, December 1987 and February 1989. Barrow Island is approximately 80 km from the coast whereas Enderby Island forms
part of the Dampier Archipelago and lies 5 km o€ the coast. The
islands are exposed to a summer rainfall reÂgime, derived from periodic cyclones, which deposit extremely variable amounts of rain
from year to year. The ocial ``average'' rainfall on Barrow Island
is, for example, 330 mm (1968±1988) but may vary from 122 mm
(in 1990) to 750 mm in 1974 following a large cyclone which deposited over 250 mm in 24 h. Monthly mean rainfall data for
Barrow Island are shown in Fig. 1.
Microclimatic data
Temperature and humidity data collected during each of the ®eld
trips to Barrow and Enderby Islands are summarised in Tables 1
and 2, along with the amount of rainfall that had fallen in the
1 month prior to each trip. Temperature and relative humidity
were recorded continuously with Thiess hygrothermographs that
were calibrated with a Schlutheis mercury thermometer and a sling
psychrometer before use.
Animals and turnover measurements
Rock-wallabies (P. rothschildi) were collected on Enderby Island in
Bromilow traps (Kinnear et al. 1988), baited with apple. Traps
were set at dusk and cleared throughout the night at 3-h intervals.
Wallabies were taken to a ®eld laboratory set up on the Island
where blood (ca. 10 ml) for hormone measurements was immedi-
Fig. 1 Average monthly rainfall for the period 1968±1998 on Barrow
Island o€ the arid Pilbara coast of Western Australia
Table 1 Microclimatic data for Barrow Island ®eld trips 1990±1992. (NS not signi®cant, RH relative humidity)
Trip no.
and date
No. of
records
Rainfall 1 month
prior to trip (mm)
Mean max
temp (°C)
Mean min
temp (°C)
Mean max
RH (%)
Mean min
RH (%)
1 Nov 1990
2 April 1991
4 April 1992
Statistical
signi®cance
17
12
6
0
0
112
34.8 ‹ 1.3
35.8 ‹ 0.6
30.2 ‹ 0.8*
F2,32 = 4.26
P = 0.02
21.2 ‹ 0.4
24.3 ‹ 0.8*
20.2 ‹ 1.2
F2,32 = 9.88
P = 0.0005
96.9 ‹ 1.0*
89.4 ‹ 3.3
88.7 ‹ 3.6
F2,32 = 3.95
P = 0.02
41.2 ‹ 3.9
39.8 ‹ 2.7
47.8 ‹ 7.2
F2,32 = 0.64
NS
* Indicates which trips are the source of the signi®cant di€erences
25
Table 2 Microclimatic data for Enderby Island ®eld trips 1986±1989
Trip no.
and date
No. of
records
Rainfall 1 month
prior to trip (mm)
Mean max
temp (°C)
Mean min
temp (°C)
Mean max
RH (%)
Mean min
RH (%)
6 Mar 1986
7 Nov 1986
8 Dec 1987
10 Feb 1989
Statistical
signi®cance
9
9
6
7
118
0
0
211
31.8 ‹ 0.6
31.0 ‹ 0.5
31.9 ‹ 0.3
33.2 ‹ 1.2
F3,27 = 1.61
NS
28.0 ‹ 0.3*
25.3 ‹ 0.3
25.7 ‹ 0.8
28.3 ‹ 0.8*
F3,27 = 8.65
P = 0.0003
92.7 ‹ 2.1*
73.7 ‹ 1.5
19.2 ‹ 1.7
91.0 ‹ 3.3*
F3,27 = 18.25
P = 0.0001
66.7 ‹ 2.9*
44.4 ‹ 2.2
56.2 ‹ 0.4
68.3 ‹ 5.4*
F3,27 = 12.70
P = 0.0001
* Indicates which trips are the source of the signi®cant di€erences
ately taken by cardiac puncture, with approval from the Animal
Ethics Committee of the University of Western Australia. The
blood sample was centrifuged at 3000 rpm, the plasma separated
and then frozen in liquid nitrogen for subsequent analysis. The
wallabies were then weighed (to 0.01 kg, Salter), measured with
vernier calipers (short-leg and pes length in mm used to calculate
the Condition Index ± see Bakker and Main 1980), marked with ear
tags and injected intramuscularly with 1.0 ml solution containing
tritium (3HHO, 16.6MBq/ml, 450 lCi) and sodium-22 (22NaCl,
0.19 MBq/ml, 5 lCi). The animals were then placed in individual
metabolism cages and held for 12 h in the dark for the collection of
voided faeces and urine. The animals were not provided with water
and ambient temperature varied between 15 °C and 28 °C over the
12-h period. Twelve hours was chosen as the most suitable time
from which a 24-h rate of urine production could be estimated and
dilution of the tritium isotope by metabolic eater production during
this period was calculated to be minimal. On removal from the
metabolism cages, the rock-wallabies were bled a second time from
the lateral tail vein (ca. 1 ml) for an equilibration sample from
which the volume of distribution of the injected isotopes was calculated. The rock-wallabies were released, at their site of capture,
late in the afternoon on the next day, once ambient temperatures
had fallen below 30 °C. The rock-wallabies were left undisturbed
for 7±10 days before attempting any recaptures where the procedure was much simpler ± a small blood sample (ca. 1 ml, the
recapture sample) was taken from the tail vein contra-lateral to that
bled initially and the body weight of the animal was recorded. The
wallabies were then released at their site of capture. The procedure
used for processing spectacled hare-wallabies on Barrow Island
di€ered only in that the wallabies were captured by hand from a
moving vehicle using long-handled nets, rather than being trapped
(Bakker and Bradshaw 1989). Hormone blood samples were taken
within 5 min of capture of the hare-wallaby and they were then
transported to the ®eld laboratory (some 5 km distant) for processing as described for rock-wallabies. Metabolism cages were
maintained in an air-conditioned room with an average overnight
temperature of 23 °C.
The decline in the speci®c activity of the injected isotopes was
measured by liquid scintillation spectrometry (Packard Tri-Carb
300CD) in 100 ll plasma samples diluted in 5 ml Pico-Fluor 15
scintillant (Packard). All samples were counted to less than 1%
error and quenching was estimated and corrected for by automatic
external standardisation. Rates of turnover of water were calculated using the equations of Nagy and Costa (1980) and sodium
turnover was calculated after measuring the sodium-23 concentrations of the equilibration and recapture samples in a Varian
(Model 475) atomic absorption (AA) spectrophotomer.
Renal parameters
Renal parameters were measured on selected wallabies after their
removal from the metabolism cages, and prior to their release in the
late afternoon. Glomerular ®ltration rate (GFR) and renal plasma
¯ow were measured using a clearance technique based on that of
Sapirstein et al. (1955) and Reid (1969) and modi®ed for use with
the hare-wallaby by Bakker and Bradshaw (1983). The wallabies
were held in a Hessian bag during the procedure with their tail
exteriorised and remained very quiet throughout the whole procedure. A lateral tail vein was cannulated with a Bardicath which was
extended up the vein until it entered the vena cava and then taped
securely in place. A bolus injection of 1 ml sterile saline containing
14
C-inulin (0.18 MBq) and 3H-Para-amino hippuric acid (PAH;
3.7 MBq) was given by the cannula and washed in rapidly with
5 ml 2% Heparin in sterile saline. Successive 0.5-ml blood samples
at 5, 10, 20, 40, 60, 80 and 120 min were then taken via the cannula,
centrifuged, and the plasma processed for 3H/14C double isotope
counting. The decline in activity of the two isotopes over time was
plotted and curve ®tting, as shown in Fig. 2, enabled the calculation of the clearances of both inulin and PAH. The extracellular
¯uid volume (ECFV) of each wallaby was estimated from the calculated dilution volume at time zero of the injected 14C-inulin ±
estimated by linear regression from the last four points on the
disappearance curve (40, 60, 80 and 120 min) according to the
procedure of Sapirstein et al. (1955) which is detailed in Bakker
and Bradshaw (1983). Clearances of electrolytes and osmolytes
were calculated knowing their respective concentrations in plasma
(P) and urine (U) samples and the rate of urine production (V)
from: Cx ˆ Ux/Px á V, where x is the osmolyte concerned. This was
estimated from the urine voided over the 12-h period that the
Electrolyte concentrations and osmolality
Plasma sodium and potassium concentrations in the hormone
sample and urine samples were measured in 5-ll aliquots by AA.
Chloride concentrations were measured by amperometric titration
in a Buchler-Cotlove chloridometer on 10-ll aliquots. The osmotic
pressure of plasma and urine samples was measured on 10-ll samples in a Wescor model 5100B vapor pressure osmometer, with urine
samples being appropriately diluted with triple distilled water.
Fig. 2 Representative disappearance curves for intravenously injected
14
C-inluin and 3H-para-amino hippuric acid (PAH) used to estimate
simultaneously renal plasma ¯ow and glomerular ®ltration rate
(GFR) in wallabies
26
wallabies had been held in the metabolism cages. The metabolism
cages were calibrated individually for losses by adding known
volumes of distilled water onto the silicone-treated, stainless steel
collecting funnels (0.5±50 ml) and then measuring the volume
collected. A linear regression of y ˆ mx + c was calculated where
y ˆ volume of ¯uid collected and x ˆ volume of ¯uid added.
Separate equations were derived for the di€erent metabolism cages
used for hare-wallabies and rock-wallabies and the equations
were y ˆ 1.0262x + 0.1322 (r2 ˆ 0.98 ) and y ˆ 1.93x + 0.796
(r2 ˆ 0.99) respectively. All urine samples were corrected using
these equations and then converted to ml (kg á day))1 to calculate
rates of V. Free-water clearance (CH2O) was calculated from
V ˆ CH2O + COSM.
Assay of LVP
Lys8-vasopressin (LVP) was assayed by RIA using the method
originally developed for arginine vasotocin by Rice (1982) and
modi®ed for the measurement of LVP in marsupial plasma by Jones
et al. (1990). Synthetic [Lys8]-Vasopressin (Sigma V-6879) was
iodinated (125I) using the chloramine-T oxidation method
of Greenwood et al. (1963) and a high-speci®c activity fraction
isolated on a Sephadex G25 column. The antibody used (courtesy
of Dr G.E. Rice) was originally raised to Arg8-vasotocin (AVT) but
also showed high cross-reactivities with Arg8-vasopressin (AVP),
LVP and phenypressin (Phe3-Arg8-vasopressin). LVP in 1-ml
plasma samples was extracted by absorption of plasma proteins on
octadecasilyl silica followed by subsequent elution with acetonitrile
(Bennett et al. 1977) using C-18 Sep-Paks (Waters No. 51910). Free
and protein-bound LVP were separated by precipitation with an
anti-rabbit globulin (IDS Tyne and Wear, UK) and all samples
were counted to less than 1% error in a Packard Prias PGD
autogamma scintillation counter. Intra- and inter-assay variabilities were 9.8% and 6.4%, respectively.
Statistical analyses
The distribution of all data was assessed for normality by constructing probability plots (Gnanadesikan 1977) and, where
appropriate, variables were logarithmically transformed prior
to statistical analysis. Patterns of variation in the data set were
explored initially through analysis of variance (ANOVA) coupled
with either a Student-Neuman Keuls Test (SNK), a Bonferroni
Test or Tukey HSD post hoc multiple comparisons. The signi®cance of di€erences between selected group means was also
assessed, where appropriate, by Student's t-test.
Results
Environmental conditions and microclimatic data
Both Barrow and Enderby Islands are located in the arid
Pilbara region of Western Australia and subjected to the
same summer rainfall reÂgime. Maximum ambient temperatures ranged from 30.2 °C to 35.8 °C on Barrow
Island, being signi®cantly lower in April 1992 than in
April 1991, along with the mean minimum temperature
of 20.2 °C (see Table 1). Levels of dew formation are
always high on Barrow Island and this is re¯ected in the
elevated measurements for relative humidity (RH) on all
the trips. Mean maximum temperatures did not di€er
signi®cantly over the four ®eld trips to Enderby Island
(see Table 2) but trips 6 and 10 had signi®cantly higher
mean minimum temperatures. Mean maximum humidity levels were high in March 1986 and February 1989,
corresponding with the wet season, and this was also
re¯ected in mean minimum values for RH.
Water and electrolyte homeostasis
Data are available for three successive ®eld trips to
Barrow Island for the hare-wallaby and four to Enderby
Island for the rock-wallaby. The trips were timed to
compare the condition of the rock-wallabies during the
driest part of the year (November±December) with that
seen following cyclonic rain in late summer and early
autumn (February±April). Turnover data and information on plasma composition for hare-wallabies are
shown in Table 3 and compared with similar information for rock-wallabies in Table 4.
Trip 1 in November 1990 coincided with the driest
year yet recorded on Barrow Island with a total of only
122 mm of rain recorded (see Fig. 1 for average precipitation data). Despite this drought, the condition of the
hare-wallabies appeared excellent, with condition indices
(CI) ranging from 4.3 to 4.9. ANOVA shows that the CI
was just signi®cantly higher in trip 4 (after rain) than on
trip 2 (dry) with F2,101 ˆ 3.29 and P ˆ 0.04. Body mass
showed a similar trend, with trip 4 animals being signi®cantly heavier than those collected on dry trips, but
the total body water content (TBW) did not vary signi®cantly. Changes in water in¯ux and e‚ux between
trips were however dramatic, increasing signi®cantly
from 27.5 ml (kg0.82 á day))1 on trip 1 to 139.1 ml á
(kg0.82 á day))1 on trip 4. Paired t-tests of in¯ux versus
out¯ux show that the wallabies were in hygric balance on
trips 2 and 4 but water e‚ux was signi®cantly greater
than in¯ux on trip 1 with paired t13 ˆ 6.93 and
P < 0.001. Given that the rock-wallabies collected on
the next trip in April 1991 showed no decrease in either
body mass or condition, it would appear that this imbalance was only transitory and perhaps re¯ected the
additional stress brought about by capture and handling.
Sodium in¯ux and e‚ux also varied between trips,
falling signi®cantly after rain, presumably as a result of
the extremely high water intake and reduced sodium
content of the vegetation. The sodium pool was
low at 42.6 ‹ 1.0 mmol á kg)1 on trip 1 but increased
signi®cantly to 73.3 ‹ 1.5 mmol á kg)1 and 72.6 ‹
1.2 á mmol á kg)1 on trips 2 and 4 respectively (F2,88 ˆ
231.1, P < 0.001).
Plasma sodium concentrations varied slightly between the three trips, ranging from 155.3 ‹
1.1 mmol l)1 in the dry seasons to 146.7 ‹ 0.7 mmol l)1
after rain (F2,101 ˆ 17.85 P < 0.001). Plasma potassium
concentrations remained constant however, as did the
plasma osmolality, indicating that the dry season animals were not su€ering from any dehydration which
would elevate their plasma solute concentrations.
In contrast to the values for the hare-wallabies, data
from the four rock-wallaby trips shown in Table 4 were
much more variable. Much of this variation in the various parameters recorded is due to trip 6 in March 1986
F2,38 = 8.46
P = 0.014
2.21 ‹ 0.23
(13)
3.47 ‹ 0.32
(12)
F2,38 = 12.95
P < 0.001
2.18 ‹ 0.34
(13)
3.52 ‹ 0.22
(12)
3.07 ‹ 0.31
(14)
Body mass
(kg)
5.28 ‹ 0.16
(32)
6.77 ‹ 0.14
(39)
6.59 ‹ 0.19
(36)
6.33 ‹ 0.14
(31)
F4,138 = 12.54
P < 0.001
Condition
Index (CI)
81.3 ‹ 1.46
(12)
73.0 ‹ 1.42
(26)
70.3 ‹ 1.69
(30)
78.2 ‹ 1.69
(24)
F4,92 = 6.57
P < 0.001
Total body
water (%)
160.1 ‹ 15.6
(4)
60.0 ‹ 3.3
(18)
65.6 ‹ 6.8
(13)
68.9 ‹ 5.2
(14)
F4,48 = 20.5
P 0.001
157.9 ‹ 14.6
(4)
63.9 ‹ 4.2
(18)
77.2* ‹ 6.8
(13)
81.1* ‹ 5.0
(14)
F4,48 = 16.3
P < 0.001
4.91 ‹ 1.35
(4)
2.27 ‹ 0.38
(17)
5.74 ‹ 0.61
(13)
2.88 ‹ 0.37
(14)
F4,47 = 7.75
P < 0.001
4.79 ‹ 1.30
(4)
2.57 ‹ 0.36
(17)
6.31 ‹ 0.59
(13)
3.49 ‹ 0.32
(14)
F4,47 = 9.1
P < 0.001
5.6 ‹ 0.1
(42)
5.2 ‹ 0.1
(23)
5.6 ‹ 0.2
(36)
[K+]p
mmol á l)1
285.8 ‹ 3.3
(41)
280.1 ‹ 2.2
(23)
287.3 ‹ 2.1
(22)
[OP]p
mosmol á kg)1
146.9 ‹ 1.13
(15)
134.8 ‹ 4.0
(33)
137.6 ‹ 1.12
(32)
145.9 ‹ 0.86
(28)
F4,108 = 3.8
P = 0.006
5.17 ‹ 0.20
(15)
4.6 ‹ 0.15
(32)
3.8 ‹ 0.12
(32)
3.9 ‹ 0.12
(28)
F4,107 = 11.98
P < 0.001
[K+]p
mmol á l)1
283.9 ‹ 2.57
(15)
293.7 ‹ 2.7
(33)
275.8 ‹ 2.8
(32)
291.3 ‹ 2.5
(28)
F4,109 = 7.5
P < 0.001
[OP]p
mosmol á kg)1
F2,101 = 17.85 F2,100 = 1.35 F2,99 = 1.61
P < 0.001
NS
NS
146.7 ‹ 0.7
(42)
155.3 ‹ 1.3
(24)
151.9 ‹ 1.1
(36)
Water in¯ux
Water E‚ux
Na In¯ux
Na E‚ux
[Na+]p
ml á (kg0.82 day))1 ml á (kg0.82 day))1 mM á (kg day))1 mM á (kg day))1 mmol á l)1
* In¯ux and out¯ux di€er signi®cantly with paired t13 = 4.47 and t12 = 6.3 for trips 8 and 10 respectively, P < 0.001
6 Mar 1986 2.53 ‹ 0.11
(32)
7 Nov 1986 3.11 ‹ 0.13
(39)
8 Dec 1987 3.07 ‹ 0.14
(36)
10 Feb 1989 3.32 ‹ 0.12
(32)
Signi®cance F4,139 = 5.13
P = 0.001
Trip no.
and date
Table 4 Water and electrolyte turnover and homeostasis of rock-wallabies on Enderby Island (Mean ‹ SE with n in parentheses)
* In¯ux and out¯ux di€er signi®cantly with paired t13 = 6.93 and P < 0.001
Signi®cance F2,102 = 22.6 F2,101 = 3.18 F2,87 = 2.1 F2,38 = 273.1
P < 0.001
P = 0.04
NS
P 0.001
F2,38 = 246.9
P 0.001
138.8 ‹ 5.6
(13)
4 April 1992 2.8 ‹ 0.04
(43)
4.92 ‹ 0.14 79.7 ‹ 0.74 139.1 ‹ 5.9
(42)
(29)
(13)
35.9 ‹ 2.3
(12)
2.66 ‹ 0.29
(14)
Total body Water in¯ux
Water E‚ux
Na In¯ux
Na E‚ux
[Na+]p
water (%) ml á (kg0.82 á day))1 ml á (kg0.82 á day))1 mM á (kg á day))1 mM á (kg á day))1 mmol á l)1
2 April 1991 2.31 ‹ 0.07 4.29 ‹ 0.19 79.5 ‹ 1.20 35.3 ‹ 2.0
(24)
(24)
(23)
(12)
Condition
Index (CI)
36.3* ‹ 2.5
(14)
Body mass
(kg)
1 Nov 1990 2.31 ‹ 0.06 4.56 ‹ 0.18 76.4 ‹ 1.60 27.5 ‹ 2.0
(36)
(36)
(36)
(14)
Trip no.
and date
Table 3 Water and electrolyte turnover and homeostasis of spectacled hare-wallabies on Barrow Island (Mean ‹ SE with n in parentheses)
27
3.7 ‹ 0.4
3.4 ‹ 0.3
NS
12.2 ‹ 0.5
18.8 ‹ 1.1
P < 0.001
5.80 ‹ 0.1
7.94 ‹ 0.46
P = 0.004
99.4 ‹ 0.12
99.2 ‹ 0.09
NS
6.7 ‹ 1.4
14.3 ‹ 5.5
P = 0.01
0.82 ‹ 0.05
1.28 ‹ 0.12
P = 0.005
8, Dec 1987 dry
10, Feb 1989 wet
Signi®cance
14.4 ‹ 0.96
16.9 ‹ 2.43
NS
)29.4 ‹ 3.3
)42.5 ‹ 4.4
NS
37.4 ‹ 4.3
59.2 ‹ 6.8
NS
U/POSM
FRH2O
ml á (kg day))1
COSM
CH2O
ml á (kg day))1 ml á (kg day))1
CIN
V
FF (%)
ml á (kg min))1 ml á (kg day))1
Trip no.
date season
Table 6 Renal parameters in rock-wallabies on Enderby Island compared in dry and wet seasons (Mean ‹ SE, n = 7)
5.4 ‹ 0.4
4.2 ‹ 0.5
NS
CPAH
ECFV (%)
ml á (kg min))1
LVP
pg á ml)1
16.0 ‹ 2.6
8.8 ‹ 0.9
P = 0.02
18.3 ‹ 0.8
18.2 ‹ 1.4
NS
6.05 ‹ 0.43
9.04 ‹ 1.32
NS
99.6 ‹ 0.06
97.8 ‹ 0.3
P = 0.002
6.9 ‹ 0.6
50.2 ‹ 4.6
P < 0.001
1.45 ‹ 0.09
2.69 ‹ 0.23
P = 0.001
1, Nov 1990 dry
4, April 1992 wet
Signi®cance
24.3 ‹ 0.4
31.3 ‹ 2.5
P = 0.01
)51.7 ‹ 5.2
)26.6 ‹ 9.3
P = 0.02
58.6 ‹ 5.7
107.0 ‹ 13.3
P = 0.003
8.0 ‹ 0.4
1.26 ‹ 0.2
P < 0.001
LVP
pg á ml)1
CPAH
ECFV
ml á (kg min))1 (%)
U/POSM
FRH2O
ml á (kg day))1
COSM
CH2O
ml á (kg day))1 ml á (kg day))1
CIN
V
FF (%)
ml á (kg min))1 ml á (kg day))1
Trip no.
date season
Table 5 Renal parameters in spectacled hare-wallabies on Barrow Island compared in dry and wet seasons (Mean ‹ SE, n = 6). (CH2 O free-water clearance, CIN clearance of inulin,
COSM osmolar clearance, CPAH clearance of para-amino hippuric acid, ECFV extracellular ¯uid volume, FF ®ltration fraction, FRH2 O fractional reabsorption of ®ltrate, LVP lysine
vasopressin, U/POSM urine to plasma ratio of osmolytes, V = rate of urine production)
28
which followed a cyclone and heavy rainfall on Enderby
Island, with 69 mm of rain recorded in the month previous to the ®eld trip. The rock-wallabies were in poor
condition, with a mean body mass of 2.5 ‹ 0.1 kg and
CI of 5.3 ‹ 0.2 ± both variables being signi®cantly
lower than on the other three trips (F4,139 ˆ 5.13,
P < 0.001). The TBW of trip 6 animals was also signi®cantly elevated which also re¯ects their poor body
condition with a replacement of solids in the body by
water. The rates of water in¯ux and e‚ux were over
twice as high in animals collected on trip 6 than those
measured during the three other trips, averaging
160.1 ‹ 15.6 ml (kg0.82 day))1 and 157.9 ‹ 14.6 ml
(kg0.82 day))1 respectively. Trip 6 animals were in hygric
balance, however, (i.e. in¯ux ˆ e‚ux) in contrast to the
rock-wallabies on trips 8 and 10 when e‚uxes were
signi®cantly greater than in¯uxes over the period of
measurement (Paired t12 ˆ 4.467 P < 0.001 and paired
t13 ˆ 6.30 P < 0.001 for trips 8 and 10, respectively).
The sodium pool of trip 6 animals, at 79.3 ‹
13.3 mmol kg)1, was very signi®cantly higher than
measured on the other three trips, which ranged from
46.5 mmol kg)1 to 49.7 mmol kg)1, and plasma sodium
concentrations were also higher in trips 6 and 10 than in
trips 7 and 8 (see Table 4). Plasma potassium concentrations showed less variation but were signi®cantly
elevated in trip 6 animals at 5.2 ‹ 0.20 mmol l)1 compared with means closer to 4 mmol l)1 on the other
three trips. Plasma osmolality was signi®cantly reduced
on trip 8 at 275.8 ‹ 2.8 mosmol kg)1, falling otherwise
within the normal mammalian range of 285±295
mosmol kg)1.
Renal function
Detailed analysis of renal function in both hare-wallabies and rock-wallabies was not possible on all trips, due
to logistic limitations and the occasional intervention of
cyclones which necessitated speedy evacuation from the
®eld, but data from the two species in both wet and dry
seasons are compared in Tables 5 and 6. Clearance data
are only available from six or seven individuals on each
trip but more extensive data on other related renal parameters from more individuals are reported in the text
for comparison.
Rates of urine production were low at 6.9 ‹
0.63 ml kg)1 day)1 in hare-wallabies on the very dry
trip 1 in November 1990 and the urine was highly concentrated with a urine-to-plasma ratio of osmolytes
(U/Posm) of 8.0 ‹ 0.4 and a mean osmolality of
2357 ‹ 109 mosmol kg)1. The GFR, measured by the
clearance of inulin, was low at 1.45 ‹ 0.09 ml
kg)1 min)1 and urine plasma ¯ow, measured as the
clearance of PAH, was also quite low at 6.05 ‹
0.43 ml kg)1 min)1, giving a ®ltration fraction (FF) of
24.3 ‹ 0.42%. Fractional reabsorption of the ®ltrate
(FRH2 O ) was thus exceptionally high at 99.58 ‹ 0.06%.
Osmolar clearance (CH2 O ) was also low at 58.6 ml kg)1
29
)1
day
and CH2 O highly negative at )51.7 ‹ 5.2 ml
kg)1 day)1 as would be anticipated from the elaboration
of highly concentrated urine. Plasma LVP levels were
elevated at 16.0 ‹ 2.6 pg ml)1 and the mean value of a
more extensive sample of hare-wallabies from trip 1
which were not part of the kidney function study was
even higher at 39.8 ‹ 8.8 pg ml)1 (n ˆ 18).
Trip 4 in April 1992 followed a cyclone that deposited considerable rain on Barrow Island and renal
parameters from the six hare-wallabies studied show
signi®cant changes when compared with the animals
collected in the drought year of 1990. Renal plasma ¯ow
was not signi®cantly higher, but the GFR had increased
to 2.69 ‹ 0.23 ml kg)1 min)1 as a result of a signi®cant
increase in the FF to 31.3 ‹ 2.5%. Rates of urine
production were dramatically increased almost nine-fold
to a mean of 50.2 ‹ 4.6 ml kg)1 day)1 and the urinary
concentration, as measured by the U/Posm ratio had
fallen signi®cantly from 8.0 to 1.26 ‹ 0.18, with a signi®cant decrease in FRH2 O from 99.6% to 97.8 ‹ 0.3%
(P ˆ 0.0001). Cosm was signi®cantly higher, as would be
anticipated from the increased urinary volumes, and
CH2 O was less negative at )26.6 ‹ 9.3 ml kg)1 day)1.
There was no change in ECFV between the two trips,
remaining at approximately 18%, but plasma LVP
levels fell signi®cantly to 8.8 ‹ 0.9 pg ml)1. As
with trip 1, LVP levels were measured in other individuals not destined for the study of kidney function,
and the overall mean plasma concentration was 9.3 ‹
1.2 pg ml)1 (n ˆ 39).
Renal function in the Enderby Island rock-wallabies
contrasts signi®cantly with the picture seen in harewallabies. During the dry season rates of urine production were as low as in hare-wallabies, at 6.7 ‹
1.4 ml kg)1 day)1 but the concentration of the urine, as
measured by the U/Posm ratio was lower at 5.4 ‹ 0.4.
Renal plasma ¯ow (CPAH) was similar at 5.8 ‹
0.1 ml kg)1 min)1 but the FF of the rock-wallabies was
some 10% lower at 14.4 ‹ 1.0, giving a substantially
reduced GFR of 0.82 ‹ 0.05 ml kg)1 min)1. The
FRH2 O was high at 99.4 ‹ 0.1%, slightly lower than
that of the hare-wallabies in the dry season. Circulating
levels of LVP were quite low, however, at 3.7 ‹
0.4 pg ml)1 and the ECFV was also low at 12.2 ‹
0.5%. LVP concentrations from a larger sample of
rock-wallabies collected on trip 8 averaged 3.49 ‹
0.37 pg ml)1 con®rming the low levels measured in the
seven renal animals.
Following cyclonic rain in February 1989, renal
parameters were little changed in the rock-wallabies.
Rates of urine production doubled to a mean of 14.3 ‹
5.5 ml kg)1 day)1 but the U/Posm of the urine produced
did not change signi®cantly and neither did the Cosm nor
the CH2 O . There was a signi®cant increase in CPAH to
7.94 ‹ 0.46 ml kg)1 min)1 which appeared primarily
responsible for the small but signi®cant increase in the
GFR to 1.28 ‹ 0.12 ml kg)1 min)1, as the FF remained
unchanged. ECFV increased after rain to a more usual
mean of 18.8 ‹ 1.1% but plasma LVP levels at
)1
3.4 ‹ 0.3 pg ml were not di€erent from those measured during the dry season.
Discussion
Maintenance of water and electrolyte homeostasis
The widespread use of tritium to estimate rates of water
turnover of free-ranging vertebrates (Green 1989, 1997;
Nagy and Costa 1980; Nagy 1982, 1987b; Hume 1999),
as well as the use of doubly labelled water to measure
®eld metabolic rates (Nagy 1987a; Nagy and Peterson
1988; Nagy 1994) facilitates comparisons between species as a function of habitat. A priori, one expects desertdwelling animals to display an enhanced water economy
when compared with related species occupying more
mesic or humid habitats, with lower rates of water
turnover and utilisation.
Rates of water turnover of the spectacled hare-wallaby on Barrow Island were ®rst measured by Bakker
and Bradshaw (1989), who reported a value of
43.5 ml kg)0.82 day)1 for the dry part of the year. This
contrasts with the mean value of 27.5 ‹ 2.0 ml
kg)0.82 day)1 reported here for November 1990 but this
was the driest year ever recorded on Barrow Island and
one can anticipate an extremely low value. Nagy and
Bradshaw (2000) again measured rates of turnover of
hare-wallabies on Barrow Island in December 1993 and
reported a mean value of 29.4 ml kg)0.82 day)1 con®rming that this species of wallaby indeed has the lowest
rate of water turnover of any mammal yet studied
world-wide. Nagy and Bradshaw (2000) also reported
the Water Economy Index (WEI) of this species, calculated as the ratio of the water in¯ux to daily energy
consumption (ml water per kJ FMR), as 0.104 which is
also low and re¯ects on this species' level of adaptation
to its arid habitat.
Nagy and Bradshaw (2000) further compared allometric relations of rates of water turnover of arid versus
non-arid marsupials and derived two signi®cantly
di€erent regressions. That for non-arid marsupials was
1.87g0.64 ml day)1 (r2 ˆ 0.939) compared with 0.777g0.69
ml day)1 (r2 ˆ 0.848) for nine arid-living species. This
second equation predicts a daily water in¯ux of 81.0 ml
(kg0.82 day))1 for a 3 kg wallaby which falls within the
range reported here for both hare- and rock-wallabies.
As may be seen, the water turnover measured with
tritium is not an invariant feature of the species, but
depends very much on the actual weather conditions
during the period of measurement. Following rain, for
example, rates of water in¯ux in the spectacled harewallaby rose to 139 ml kg)0.82 day)1, and turnover was
thus markedly increased over that measured during
periods of chronic water deprivation.
The water economy of a number of other arid
marsupial species occurring on Barrow Island was also
studied by Nagy and Bradshaw (2000) in December
1993, including the burrowing bettong, Bettongia
30
Table 7 Body composition
homeostasis in desert wallabies
± maximum percentage
variation (%) in parameters
recorded over all ®eld trips
Species
Body
mass
Condition
index
Total body
water content
Plasma
Na+
Plasma
K+
Plasma
osmolality
Hare-wallaby
Rock-wallaby
21.2
31.2
14.7
28.2
4.3
15.7
5.9
9.0
7.7
36.9
2.9
5.6
lesueur (85.7 ml kg)0.82 day)1), the black-footed
rock-wallaby,
Petrogale
lateralis
lateralis
(70.6 ml kg)0.82 day)1), the golden bandicoot Isoodon
auratus barrowensis (127 ml kg)0.82 day)1), the northern
brushtail possum, Trichosurus vulpecula arnhemensis
(164.3 ml kg)0.82 day)1), and the dwarf Barrow Island
euro kangaroo, Macropus robustus isabellinus
(67.1 ml kg)0.82 day)1), and all are clearly low when
compared with other values in the literature.
A primary determinant of water economy in mammals is the renal-concentrating capacity, which is in turn
determined to a large extent by the renal anatomy, and
the relative dimensions of the renal medulla. Sperber's
(1944) index of medullary thickness has been calculated
for a number of species of marsupials and the harewallaby is high at 8.7 (Purohit 1974). The mean osmolality of voided urine of hare-wallabies from Barrow
Island was signi®cantly higher than that of Enderby
Island rock-wallabies (1397 ‹ 106 versus 1207 ‹
72 mosmol kg)1 respectively, P < 0.001), although the
maximum recorded values for each species were similar
at 3,600 mosmol kg)1.
The data presented in this study enable one to assess
the extent to which the two desert wallabies are able to
maintain water and electrolyte homeostasis in the face
of chronic water deprivation. Clearly, the hare-wallaby ±
despite its greater environmental exposure ± is more
e€ective at regulating its milieu inteÂrieur. The percent
change in body composition variables listed in Tables 3
and 4 for the two species over all trips is listed in Table 7
Fig. 3 Temperature records taken by datalogger on Barrow Island in
November/December 1993. The solid line records from an underground warren, built and inhabited by burrowing bettongs (Bettongia
lesueur), the dotted line records from the middle of a spinifex clump
used as a refuge by hare-wallabies and the dashed line is the ambient
temperature
and shows clearly that the rock-wallaby experiences
variations in TBW as well as plasma electrolyte concentrations and osmolality that are two-to-three-times
greater than those experienced by hare-wallabies. These
data highlight the extent to which the hare-wallaby is
able to minimise the impact of daily high thermal loads
on its overall water economy. Figure 3 shows temperature records from a data logger recording environmental
temperature and humidity records on Barrow Island in
November±December 1993. The temperature within a
large spinifex tussock (Triodia angusta), used as a daily
refuge site by hare-wallabies, is only marginally lower
than the ambient air temperature and reaches as high as
45 °C on some days. The very much lower and more
stable temperature recorded over the same period from a
burrow of the burrowing bettong, is similar to records
taken from cave sites used by rock-wallabies on Enderby
Island and shows the extent to which these privileged
sites enable this species to conserve body water. Taken
together, the data on body composition homeostasis and
perceived environmental heat loads for the two species
(Dawson and Bennett 1978), suggest that the harewallaby is far better adapted physiologically than the
rock-wallaby to cope with extended periods of water
deprivation.
Comparisons of renal function between
the two species
Although the renal data presented in Table 6 give the
impression that the rock-wallabies are unable to vary
Fig. 4 Variation in urinary-concentrating activity of the kidneys of
hare-wallabies and rock-wallabies (U/Posm) as a function of rate of
urine production. The power curve is ®tted to the data for harewallabies but does not di€er signi®cantly for that for rock-wallabies
31
Fig. 5 Variation in circulating levels of lysine vasopressin (LVP) in
hare-wallabies and rock-wallabies as a function of the concentrating
activity of the kidney (U/Posm)
signi®cantly the concentration of their urine, Fig. 4
suggests that this is not the case by plotting U/Posm ratios versus rate of urine production (V) for the complete
data set. Rates of urine production in rock-wallabies do
not exceed 55 ml kg)1 day)1 ± whereas they almost
reach 100 ml kg)1 day)1 in hare-wallabies ± but U/Posm
ratios reach levels of 10 in some individuals of both
species. The power curve ®tted to the data is for harewallabies only, but data for the rock-wallaby do not
di€er signi®cantly from this line.
What is dramatically di€erent between the two
species is the relationship between urinary concentration
(U/Posm) and plasma levels of LVP ) or the lack of any
such relationship in rock-wallabies as seen in Fig. 5. LVP
levels increase with increasing concentration of the urine
in hare-wallabies, as would be expected for a mammal
where LVP is the physiological antidiuretic hormone, but
remain essentially unchanged in rock-wallabies. The renal response to water deprivation in the rock-wallaby
thus does not appear to be hormone-mediated, resulting
instead from a reduction of renal plasma ¯ow that is
translated into a lower GFR with the production of
smaller volumes of, essentially, similar urine.
One needs to raise the possibility of whether the two
species might not di€er in the identity of their physiological anti-diuretic hormone ) LVP in the hare-wallaby
but a hormone other than LVP, or phenypressin, in the
rock-wallaby? Neither species has yet been subjected to
detailed investigation as to the precise nature of their
neurohypophysial peptides, but macropodid marsupials
generally are known to store LVP, mesotocin and
phenypressin in their pars nervosa (Chauvet et al. 1980,
1983, 1987). Mesotocin has not been shown to have any
e€ect on renal function in marsupials and, to date,
phenypressin has not been investigated as a putative
anti-diuretic hormone. Lack of binding activity in the
plasma of the rock-wallaby would suggest that phenypressin is not the ADH in this species. Plasma levels of
LVP have been assayed in only a few species of marsu-
pial. The Rottnest Island quokka (Setonix brachyurus)
inhabiting areas where brackish water was available
during mid-summer had circulating levels of LVP of
35.6 ‹ 15.8 pg ml)1. This compared with a mean of
89.2 ‹ 19.6 pg ml)1 measured in animals with no
access to free water (Jones et al. 1990). Plasma levels of
LVP in the Tammar wallaby (Macropus eugenii)
reported by Wilkes and Jannsens) 1986 averaged
21.5 ‹ 4.4 pg ml)1, con®rming earlier measurements of
circulating levels of ADH in this species reported by
Bakker et al. (1982) using a toad bioassay (Bakker and
Bradshaw 1978). Wilkes and Jannsens (1986) also
demonstrated that LVP functions as an e€ective ADH in
the developing tammar wallaby.
Given the unusual nature of the renal responses to
water deprivation in the rock-wallaby, it may well be
that this species lacks an hormonally mediated response.
Acknowledgements Wallabies on Barrow and Enderby Islands
were collected under permit from the Department of Conservation
and Land Management (CALM) and all experimental procedures
were approved by the Animal Ethics Committee (then the Animal
Welfare Committee) of The University of Western Australia.
Grateful acknowledgement is made to Brian Clay, Darren Murphy,
Chris Dickman, Phil Withers and Bob McNeice for assistance in
the ®eld, and to Zorica Kostadinovic for help with laboratory
analyses. The work was funded by the Australian Research Council
(ARC) and was greatly assisted by material support from WA
Petroleum (WAPET) which provided transport to and accommodation on Barrow Island.
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