PHYSIOLOGICAL STRATEGIES OF DORMANCY
OF KINOSTERNON FLAVESCENS
by
WILLIAM M. CHILIAN, B.A.
A THESIS
IN
ZOOLOGY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
Accepted
May, 1976
dt
ACKNOWLEDGMENTS
I thank Dr. F. L. Rose for advice, encouragement, and
assistance throughout this study and Drs. L. A. Roberts,
J. M. Burns, and J. S. Sevall for their review of this
manuscript.
I wish to thank those who aided in collection
of specimens and my wife, Susan, who typed the manuscript.
This research was partially supported by a Grant-in-Aid
of Research from Sigma Xi, The Scientific Research Society
of North America.
11
T A B L E OF C O N T E N T S
ACKNOWLEDGMENTS
ii
L I S T OF T A B L E S
iv
L I S T OF ILLUSTRATIONS
I.
II.
III.
IV.
v
INTRODUCTION
1
CARBOHYDRATE METABOLISM
2
Introduction
2
M a t e r i a l s and M e t h o d s
4
Results
5
Discussion
10
Summary
19
W A T E R , N I T R O G E N , AND SALT M E T A B O L I S M
20
Introduction
20
M a t e r i a l s and M e t h o d s
21
Results
25
Discussion
34
Summary
39
CONCLUSIONS
40
REFERENCES
41
111
LIST OF TABLES
Table
1.
2.
Page
Cationic and Uric Acid Content of
Precipated Urate Salts During Dormancy. Values (mT'l) or (mEq) are
Absolute Amount of Uric Acid or
Cation Content of the Precipitated
Urate Salt. Values (mEq/mM) are
Relative Concentrations of mEq of
Cation per mM of Uric Acid
31
Per cent Urea-, Urate-, or Ammonia-N
of the Total Urine and Plasma is
Represented in Table 2. Urine NPN
was Calculated Using N from Precipitated and Solvated Sources
33
IV
LIST OF ILLUSTRATIONS
Figure
1.
2.
3.
4.
Page
Shows the mean concentrations of ketone
bodies in the blood plasma with the 95%
confidence interval. 6-OHBut. is betahydroxybutyric acid. AcAc. is acetoacetic acid. Total represents the sum
of AcAc. and S-OHBut. The sample sizes
are: n=10 for hydrated animals and
animals dormant for 4 Mo; n=9 for animals dormant for 2 Mo; n=7 for animals
dormant for 6 Mo. Units are expressed
as mg solute per 100 ml of blood plasma
(mg/100 ml)
6
Shows the mean concentration of glucose,
fructose, and lactate in the blood
plasma with the 95% confidence interval.
Units are mg of solute per 100 ml of
blood plasma (mg/100 ml). The sample
sizes are: n=10 for both groups of
hydrated animals and animals dormant
for 4 months; n=9 for animals dormant
for 2 Mo; and n=7 for animals dormant
for 6 Mo
8
Shows the mean concentration of glycogen
in hepatic, skeletal muscle, and cardiac
muscle tissues with the 95% confidence
interval. Units are mg glycogen per g
of wet tissue (mg/g). The sample sizes
are: n=10 for hydrated animals and
animals dormant for 4 Mo; n=9 for animals
dormant for 2 Mo; and n=7 for animals
dormant for 6 Mo
9
Shows the mean concentration of sugars in
the urine with the 95% confidence interval.
Units are mg of sugar per 100 ml of urine.
The sample sizes are: n=10 for the hydrated animals and animals dormant for 4
Mo; n=9 for animals dormant for 2 Mo;
n=7 for animals dormant for 6 Mo
11
5.
6.
7.
Shows the mean ± S.E. of plasma and
urine electrolytes and osmolarity.
Sample sizes are: n=10 for both
groups of hydrated animals, saltloaded animals (S.L.), and 4-month
dormant animals; n=9 for 2-month dormant animals; and n=7 for 6-month dormant animals
25
Shows the mean ± S.E. of plasma and
blood nitrogenous wastes. Sar.ple
sizes are: n=10 for both groups of
hydrated animals, salt-loaded animals
(S.L.), and 4-month dormant animals;
n=9 for 2-month dormant animals; and
n=7 for 6-month dormant animals
29
A. Shows the mean ± S.E. of integumental water absorption. The sample
is 4 for both groups.
B. Shows evaporative water losses.
The sample sizes are: n=10 for hydrated and 4-month dormant animals;
n=9 for 2-month dormant animals; and
n=7 for 6-month dormant animals
35
VI
CHAPTER I
INTRODUCTION
The problems caused by the incompatible requirements
of excreting wastes and conserving v;ater for vital purposes
are compounded in animals inhabiting arid lands.
All
animals must maintain an internal environment compatible
with life by physiologically regulating a constant balance
between water and energy losses and water and energy gains.
The Yellow Mud Turtle, Kinosternon flavescens, is an
organism that inhabits ponds in arid areas; however, during
periods of drought the ponds dry and the turtles must adapt
to aridity, which they do by entering a dormant state,
characterized by inanition.
Their problems of maintaining
physiological integrity are manifold:
during activity,
they encounter osmotic flooding in their aquatic environment and during dormancy, they encounter desiccation in
their terrestrial environment.
Dormancy imposes problems
of energy and water limitations on the animals, because
they have a finite reserve of each.
The purpose of this
study is to understand how the water and energy metabolisn^
of dormant turtles differs from that of active turtles, in
otherwords, the adaptive strategy of K. flavescens to heat
and drought.
CHAPTER II
CARBOHYDRATE METABOLISM
Introduction
Water and energy stress impose several problems on any
animal with regard to maintenance of an internal environment compatible with survival.
Systems regulating energy
and water metabolism are especially important for animals
which encounter limitations of both in different stages of
their life cycle.
The Yellow Mud Turtle, Kinosternon
flavescens, has an active and dormant stage of its life
cycle.
During the active stage the animal is aquatic, and
carries out courtship, reproduction, and all feeding.
How-
ever, many of the ponds inhabited by Kinosternon are temporary and during inclement conditions (heat and drought) the
ponds dry.
The turtles must adapt to aridity, which is
done by burrowing into the ground and entering a dormant
state, characterized by inanition.
as inclement conditions persist.
Dormancy lasts as long
The length of dormancy
of K. flavescens has been documented for up to 2 years
(Rose, pers. comm.).
are obvious:
Physiological stresses of dormancy
there is a finite amount of water and energy
for the maintenance of physiological integrity.
How the
turtles regulate their carbohydrate metabolism to conserve
energy reserves is the concern of this study.
To understand how carbohydrate metabolism of dormant
turtles differs from active turtles, the following changes
were investigated:
(1) Glucose and fructose blood plasma
and urine concentrations; (2) Lactic acid blood concentrations; (3) Skeletal muscle, cardiac muscle, and hepatic
tissue glycogen; and (4) Blood plasma concentrations of
ketone bodies.
Though ketones are derived from fats, they
are important for the integration of lipid and carbohydrate
metabolism, as well as for the control of carbohydrate
metabolism.
Carbohydrate metabolism of poikliotherms has attracted
much attention in recent years (Miller, 1961; Barwick and
Bryant, 1966; Bennett, et_ a_l. , 1975; and Hutchinson and
Turney, 1975).
Most investigations dealt with anerobic
and aerobic metabolism during activity (Moberly, 1968;
Bennett and Dawson, 1975; Bennett and Licht, 1972; and
Bennett, 1975), cold torpor (Rapatz and Musacchia, 1957;
Barwich, 1966), and seasonal changes in carbohydrate metabolism (Dessuaer, 1953).
Control of carbohydrate metabolisn
of reptiles has focused primarily on hormonal regulation
(Boldyreff and Stewart, 1932; Coulson and Hernandez, 1953;
and Miller and Wurster, 1956).
Control of carbohydrate
metabolism in mammals was researched extensively, with most
studies dealing with in vitro systems (Krebs, et al., 1964;
Williamson, et al., 1967; and Exton, 1967).
There is a paucity of research dealing with the control of reptilian carbohydrate metabolism and of carbohydrate metabolism ±n_ vivo.
Little is known about the
changes in carbohydrate metabolism during heat- and
drought-induced dormancy, or estivation.
Materials and Methods
Turtles were caught in May and June, 1975 in Lubbock,
Crosby, and Lamesa counties, Texas.
They were acclimated
to the laboratory in a large aquarium with a long dry
photoperiod (16 hrs.), at 30°C for at least 1 month, and
food was provided ad^ lib.
Samples of blood, tissues, and
urine from fasted-hydrated animals were collected after 1
month of food deprivation.
Dormancy was induced by placing
the turtles in a large box of dirt where they dug beneath
the soil.
Groups were removed from dormancy at 2, 4, and
6 months.
Following decapitation, blood was collected in
a heparinized syringe, centrifuged at 3000 rpm for 3
minutes, and the plasma decanted and frozen.
The plastron
was removed and a urine sample obtained via bladder puncture.
Heart, liver, and some skeletal muscle (approxi-
mately 1 gm) was excised and frozen.
Blood lactic acid was determined by the ultraviolet
lactate dehydrogenase method (Sigma tech. bull. no. 826-UV)
Glucose was determined enzymatically using the glucose
oxidase-peroxidase method (Sigma tech. bull. no. 510). Ketone bodies, beta-hydroxybutyric acid and acetoacetic acid
were determined enzymatically using beta-hydroxybutyrate
dehydrogenase (Williamson, et al., 1962).
Glycogen was
determined by the sulfuric acid-phenol procedure (Montgomery, 1957).
Fructose was determined by the recorsinol
reaction (Roe, 1934).
Differences between means were analyzed by the StudentNeuman-Keuls multiple range test (Zar, 1974).
The cri-
terion of statistical difference was P < .05.
In the
figures, the 95% confidence interval has been plotted based
on the variance of each group.
Results
Ketones
The concentration of beta-hydroxybutyric acid was
significantly greater during dormancy than in both groups
of hydrated animals (Fig. 1). Plasma concentration of
acetoacetic acid did not vary among the experimental groups
(Fig. 1). Total ketones did not differ among both groups
of hydrated animals and animals dormant for 2 months.
The
total ketone concentration was significantly increased in
the 4- and 6-month dormant turtles.
Glucose
In hydrated animals glucose concentrations were less
FED
FASTED
HYDRATED
DORMANT
Fig. 1. Shov;s the mean concentrations of ketone
bodies in the blood plasma with the 95% confidence interval.
S-OHBut. is beta-hydroxybutyric acid. AcAc. is
acetoacetic acid. Total represents the sum of AcAc. and
3-OHBut. The sample sizes are: n=10 for hydrated animals
and animals dormant for 4 Mo; n=9 for animals dormant for
2 Mo; n=7 for animals dormant for 6 Mo. Units are expressed as mg solute per 100 m.l of blood plasma (mg/100 ml)
during fasting than during feeding (Fig. 2 ) . Plasma glucose concentration of dormant animals was less than that
of both groups of hydrated animals.
There were no dif-
ferences among the plasma glucose concentrations of any of
the experimental groups of dormant animals.
Fructose
There were no differences of plasma fructose concentrations between both groups of hydrated animals (Fig. 2).
Fructose concentrations of dormant animals were greater
than that of active (hydrated) animals and increased progressively with period of dormancy.
Lactic Acid
Concentration of plasma lactate between hydrated animals did not differ (Fig. 2). However, concentration of
plasma lactate of dormant animals was greater than that of
hydrated animals.
Hepatic Glycogen
Concentration of hepatic glycogen of hydrated animals
was significantly less after 1 month of fasting and during
dormancy than during feeding (Fig. 3). However, there was
a higher concentration of hepatic glycogen in animals
dormant for 4 and 6 months than in fasted-hydrated anir^.als.
There was no difference between hepatic glycogen of fastedhydrated animals and animals dormant for 6 months.
8
150
^ G U cose
m ya/ i O O ml
IFruct ose
J. Lactate
100
1
1
50r>
\
rr^->..U
1
•1
^53 J
0 V > i :j!.T3aLt*w-J-
FED
6/nO.
FASTED
HYDRATED
D 0 R r/. A N T
Fig. 2. Shows the mean concentration of glucose,
fructose, and lactate in the blood plasma with the 95%
confidence interval. Units are mg of solute per 100 ml
of blood plasma (mg/100 m l ) . The sample sizes are: n=10
for both groups of hydrated animals and animals dormant
for 4 months; n=9 for anim.als dormant for 2 Mo; and n=7
for animals dormant for 6 Mo.
\
• ^
Hepatic
m s/
Cardiac
U Skeletal
f iSHO
HYDRATED
e
Muscle
2 MO
DORMANT
Via 3 Shows the mean concentration of glycogen in
hepatic!-s^;xetal - s o l e ana - - ^ - 0 - 3 = ^ ^ , , - - - % : ^ofw^f t - s ^ r ( - r / . ) ? ^ " - ; a / p ? : " f 4 a e Jn^iO .o.
fnrar/.=t%:f/Horin/n^rfo/°an.Lrs'aS..ant.or
6 Mo.
10
Cardiac Glycogen
Cardiac glycogen of fasted-hydrated and dormant animals was less than that of fed animals.
Animals dormant
for 2 months had higher levels of cardiac glycogen than
fasted-hydrated animals.
No difference in cardiac glycogen
content existed between fasted-hydrated animals and animals
dormant for 4 months; however, animals dormant for 6 months
showed less cardiac glycogen content than all groups.
Skeletal Muscle Glycogen
There was no difference among the content of skeletal
muscle glycogen among any of the experimental groups (Fig.
3).
The glycogen sample of skeletal muscle from the 2-
month dormant animals was inadvertently destroyed.
Urine Sugars
No measurable glucose was found in the urine of
hydrated animals (Fig. 4). Glucose concentrations in the
urine during dormancy increased significantly.
Urinary
fructose concentration of hydrated animals was greater
during fasting than during feeding (Fig. 4). During dormancy, fructose concentration was significantly greater
than in the hydrated animals.
Discussion
In mammals, ketone bodies increase during fasting
(Cahill, 1964), which agrees with the trend of increasing
11
0.
mg
100 ml
20
rt
10
i"
4 MO.
HYDRATED
6/.^0.
D 0 R r.: A N T
Fig. 4. Shows the mean concentration of sugars in
the urine with the 95% confidence interval. Units are mg
of sugar per 100 ml of urine. The sample sizes are: n=10
for the hydrated animals and animals dormant for 4 Mo; n=9
for animals dormant for 2 Mo; n=7 for animals dormant for
6 Mo.
12
ketones during dormancy in K. flavescens (Fig. 1). An increase in ketone bodies during dormancy might be advantageous, for if they become the main metabolic fuel, they
would cause a conservation of other fuels, e.g., glucose,
glycogen, and triglycerides.
In this respect, it is sig-
nificant that beta-hydroxybutyric acid and acetoacetic acid
are substrates which are utilized preferentially by cardiac
muscle, sparing the catabolism of other metabolic fuels
(Williamson and Krebs, 1961).
Moreover acetoacetate can
furnish 70-80% of the energy requirements of resting
skeletal muscle, with insulin not affecting its uptake
(Houghton and Ruderman, 1971).
During periods of prolonged
fasting the brain adapts to low levels of blood glucose and
metabolizes ketone bodies as the predominate fuel (Cahill,
et £l. , 1967; Owen, et a^., 1967).
The mechanism by which beta-hydroxybutyric acid increases during dormancy and acetoacetic acid remains constant is unknown; however, acetoacetate is the transitory
ketone body between the Krebs Cycle and the end point of
ketone body metabolism, beta-hydroxybutyric acid.
Since
acetoacetate has two metabolic fates, a smaller concentrating effect would be suspected than of betahydroxybutyrate, which has one metabolic fate.
Another
reason for the concentration of beta-hydroxybutyrate could
be the K
eq
of beta-hydroxybutyrate dehydrogenase.
J
J
^
^
The K
gq
13
of this enzyme, which catalyzes the dehydrogenation of
acetoacetate to beta-hydroxybutyrate, is reported to be .13
(Delafield and Doudoroff, 1969); thus, for the presence of
1 mole of acetoacetate there would be 7.7 moles of betahydroxybutyrate.
Consequently, as more ketones are pro-
duced by lipid catabolism, one would expect a greater concentration of beta-hydroxybutyrate than of acetoacetate.
Lipids may be an important source of oxidative water
during dormancy in K. flavescens (Rose, pers. comm.).
The
observed increase in ketone bodies indicates increased
lipid catabolism; thus, dormant animals may be utilizing
lipids as a water and an energy source.
Figure 2 shows increases of fructose and lactate with
a decrease of glucose during dormancy.
Ketone bodies cause
a decrease of blood glucose (Neptune, 1956; Madison, et^ al. ,
1964; Balasse, et a]^. , 1967; and Little, et al. , 1970).
This hypoglycemic mechanism is thought to be insulinmediated (Madison, 1964; Bjorntorp and Shersten, 1967; and
Little, 1970), but Balasse and Ooms (1968) reports that
ketone bodies do not affect blood insulin concentrations;
instead, liver output of glucose is decreased.
Ketone
bodies were thought to be gluconeogenic, causing an increase in glucose (Krebs, et_ a_l. , 1964; Wieland, et_ al. ,
1964; and Krebs, et al., 1965); however, these experiments
were done in vitro or in diabetic animals with no insulin.
14
Earlier researchers thought that fasting in reptiles caused
an increase in blood glucose (Bolydreff, 1932; Emerson,
1967).
However, these and other studies (Coulson and Her-
nandez, 1953; Miller and Wurster, 1956; and Algauhari,
1967) were done using a reducing sugar test, which would
not reveal fluctuations of separate reducing sugars; i.e.,
glucose and fructose.
The observed increased in plasma lactate during
dormancy (Fig. 2) may be due to ketones since, ketone
bodies increase production of lactate (Williamson and
Krebs, 1961).
An increase in lactic acid may be due also
to anerobic respiration caused by hypoxia or unequal distribution of blood flow (Huckabee, 1969/70).
The increase
in blood lactate in K. flavescens was probably a combination of the above factors, since torpid turtles have reduced peripheral blood flow (Stitt, et^ al. , 1970) .
Fructose metabolism during dormancy may be advantageous because it may be easier to metabolize than glucose
during ketoacidosis (Mayes and Felts, 1967; Shreeve, 1974).
Fructose enhances production of glycerol-phosphate, which
leads to esterification of free fatty acids, so more ketone
bodies are not produced (Mayes and Felts, 1967; Shreeve,
1974).
An increase in fructose can occur in the presence
of insulin (Cahill, 1964).
Insulin halts by 50% the con-
version of fructose to glucose and increases the incorpor-
15
ation of fructose into triglycerides (Cahill, 1964).
One
likely mechanism for an increase in blood fructose would be
a reduction in blood volume, thus concentrating solutes in
the plasma, which would explain also the increase in blood
lactate and beta-hydroxybutyrate.
Kinosternon flavescens has a higher lipid index than
emy other known reptile (Rose, pers, comm.).
Fructose is
a lipogenic sugar and its metabolism leads to the production of glycerol-phosphate, which esterifies with free
fatty acids into lipids (Mayes and Felts, 1967; Shreeve,
1974).
A high fructose metabolism may possibly be advan-
tageous for the production and storage of lipids.
Hepatic glycogen deposition is known to be glucoseand/or insulin-dependent (Shreeve, 1974; Huijing, 1975).
If ketone bodies stimulate insulin release (Madison, 1964),
glycogen content would rise.
There was a greater concen-
tration of hepatic glycogen of dormant animals (2 and 4
months) than in fasted-hydrated animals (Fig. 3), which
corresponded with the increase in ketone bodies.
In Fig.
3 fed-hydrated animals had a greater concentration of
hepatic glycogen than in the other experimental groups.
The reason for this may be that the function of hepatic
glycogen is to maintain blood glucose levels (Hers, et al.,
1970).
As hepatic glycogen is depleted, blood glucose de-
creases and the liver catabolizes fats, producing ketones.
16
Tissues adjust to the increased levels of ketone bodies and
metabolize them preferentially, even in the presence of
glucose; thus, there is no need to anabolize hepatic
glycogen during the months of inanition.
In K. flavescens,
ketones may be important for the reduction in hepatic
glycogen catabolism during dormancy.
An animal dormant
for 4 months had a greater hepatic glycogen concentration
than an animal fasted for 1 month, which indicates a reduction of catabolism during dormancy.
Cardiac glycogen content of animals dormant for 2
months is greater than that of fasted-hydrated animals
(Fig. 3). The content of cardiac glycogen.of dormant animals and fasted-hydrated animals was less than fed-hydrated
animals (Fig. 3). Cardiac glycogen was reported to increase during fasting (Shreeve, 1974).
However, in
Kinosternon fasting was not a matter of a few days, but a
minimum of one month.
With such a time difference it is
difficult to compare the decrease of cardiac glycogen during fasting and dormancy with the increase of cardiac
glycogen during fasting reported by other workers (Adrouny,
1969; Neely and Morgan, 1974; and Shreeve, 1974).
The
higher concentration of cardiac glycogen of animals dormant for 2 months over that of fasted-hydrated animals
parallels the rise in ketone bodies in dormant animals.
Ketones have a sparing effect on cardiac glycogen catab-
17
olism, which may be due to the heart preferentially
metabolizing them (Williamson and Krebs, 1961).
Through
ketone-stimulated insulin release (Madison, 1964) cardiac
glycogenesis is enhanced (Shreeve, 1974; Huijing, 1975).
These observations help explain the higher level of cardiac
glycogen in animals dormant for 2 months from that of
fasted-hydrated animals.
Cardiac glycogen concentration
of animals dormant for 2 months is greater than cardiac
glycogen content of animals dormant for 4 and 6 months
(Fig. 3), even though the plasma ketones increased during
dormancy (Fig. 1). The degradation of cardiac glycogen in
animals dormant for 4 and 6 months could indicate hypoxia,
which caused the catabolism of cardiac glycogen (Shreeve,
1974), since hypoxia interferes with cardiac lipid and
ketone body metabolism (Neely and Morgan, 1974).
During
dormancy, the ventilation rate of K. flavescens was observed to drop too low to record effectively (Rose, pers.
comm.); such a reduction in ventilation could possibly
cause hypoxia.
Skeletal muscle glycogen remained constant in all
experimental groups (Fig. 3). The primary function of
skeletal muscle glycogen is an energy source during
anerobic activity (Hers, 1970; Shreeve, 1974).
This ob-
servation indicates that during dormancy, skeletal muscle
glycogen should remain constant.
Since resting muscle uses
18
ketone bodies as 70-80% of its energy requirements (Houghton and Ruderman, 1971), muscle glycogen probably plays a
minor role in the substrates of skeletal muscle during
dormancy; thus, it remained constant.
Urine sugar is an indication of the degree of ketoacidosis (Shreeve, 1974).
Ketoacidosis interferes with
glucose reabsorption (Shreeve, 1974).
Most fructose en-
tering the kidney is converted into glucose and actively
reabsorbed, but this conversion appears to be incomplete
under acidotic conditions or when the kidney encounters
more fructose than it can convert.
In normal hydrated
animals no glucose excretion was expected nor was any
found.
Fructose reabsorption in mammalian kidneys is sus-
pected of being an active process (Cohen and Barac-Niets,
1973), but whether or not reptiles have such a mechanism
is unknown.
The effects of ketoacidosis on fructose re-
absorption are unknown.
The results indicate that fruc-
tose excretion follows its blood concentration; that is,
the higher the concentration of blood fructose, the greater
the amount of fructose excreted.
The question of why fruc-
tose rises in the plasma but plateaus in the urine during
dormancy, is unanswered.
More research is needed to more
fully understand kidney function and metabolism of reptiles
during dormancy.
19
Summary
1.
Heat- and drought-induced dormancy of Kinosternon
flavescens was experimentally stimulated.
2.
During dormancy the concentration of fructose,
lactate, and beta-hydroxybutyrate increased, the concentration of glucose decreased, and acetoacetate remained
constant.
3.
The concentrations of both sugars in the urine
increased during dormancy.
4.
Cardiac and hepatic glycogen decreased during
dormancy, while skeletal muscle glycogen remained constant.
However, dormant animals appeared to have a reduction in
glycogen catabolism from hydrated animals fasting 1 month.
5.
An increase in plasma ketone bodies may be impor-
tant for survival during dormancy, because of the sparing
of other substrates.
CHAPTER III
WATER, NITROGEN, AND SALT METABOLISM
Introduction
Physiological adaptations of reptiles to arid areas
have attracted much attention (Bentley and Schmidt-Nielsen,
1970; Bustard, 1967; and Campbell, 1970).
Many investi-
gators centered on adaptations during active stages of a
reptile's life cycle (Bustard, 1967; Bentley and SchmidtNielsen, 1970; and Chew, 1961).
Osmoregulatory studies of
aquatic reptiles focused mainly upon water loss and gain
through the integument; however, controversy remains on
the subject of integumental water absorption (Bentley and
Schmidt-Nielsen, 1965; Cloudsley-Thompson, 1968) . Electrolyte metabolism was researched widely in hibernating reptiles (Mahyew, 1965), hypothermic reptiles (Khamis, 1975),
and aquatic reptiles (Dunson and Weymouth, 1965).
Extra-
renal excretion was studied extensively in marine and
terrestrial reptiles by Dunson (1968) and Templeton (1965).
Although much research was done on nitrogen metabolism of
active reptiles from mesic, xeric, and hydric habitats
(Campbell, 1970; Moyle, 1949), little attention has been
directed towards water, nitrogen, and electrolyte metabolis:
20
21
during heat- and drought-induced dormancy.
Organisms which inhabit temporary ponds in arid climates may encounter a severe problem of dehydration.
Water
stress imposes several problems on any animal with regard
to maintenance of an internal environment compatible with
survival.
Systems regulating the losses of water, integu-
mentary and renal, are especially important because of
their necessary physiological adjustment from one of
"osmotic flooding" in the aquatic environment to dehydration during drought.
The Yellow Mud Turtle, Kinosternon
flavescens, is a hydrophilic animal which inhabits temporal
ponds in arid lands.
During drought the ponds dry, and the
turtles must adapt to aridity by entering a dormant state
characterized by inanition.
Dormancy lasts as long as the
unfavorable conditions persist, sometimes for as long as
2 years (Rose, pers. comm.).
The purpose of this study
was to understand how physiological systems controlling
salt, water, and nitrogen metabolism adjust to dehydration
during dormancy.
Materials and Methods
Turtles were collected in May-June, 1975.
They were
acclimated to the laboratory in a water-filled aquarium
with food ad lib. under a long day (16:8) photoperiod for
1 month.
hydration.
Some turtles were sampled during feeding and
Samples from hydrated-fasted turtles were col-
lected after 1 month of fasting.
Prior to the induction
22
of dormancy, turtles were removed from the aquarium,
weighed, and marked.
Then, to induce dormancy, they were
placed in a box of dirt where they dug immediately below
the surface.
Individuals were removed at 2, 4, and 6
months and blood and urine samples were obtained.
Salt-loading was carried out by drillin7 a hole in the
carapace, injecting 1 ml of 5 M NaCl or KCl/lOOg/24 hrs for
4 days.
The head of each animal was washed daily with 10
ml of deionized water and the wash was analyzed for salts
(Dunson, 1974).
Blood plasma from all experimental groups was obtained in the following manner:
(1) Individuals were de-
capitated; (2) Blood was drawn into a heparinized syringe
and was centrifuged at 3000 rpm for 3 minutes; (3) Plasma
was decanted and frozen.
Urine samples of dormant animals
were collected by removing the plastron, excising the
bladder, and centrifuging the bladder contents.
The
supernatent was decanted and the precipitate solubilized
in 10 ml of .5% Li^CO-..
Samples from hydrated and salt-
loaded animals V7ere obtained by holding the animal over a
50 ml beaker and pressing the hind legs into the body
cavity, inducing urination.
Evaporative water loss of active turtles was determined
after withholding food for 5-7 days so the RQ would approximate .7 (Minnich, 1972).
The animals were placed in
23
a container in a controlled environmental chamber at 50%
R.H.
Weight loss was assumed to be evaporative water loss,
provided the animal did not urinate or defecate.
Evapor-
ative water loss of dormant animals was determined by removing the animals from the soil and following the same
procedure as for hydrated animals.
Evaporative water loss
was expressed as mgH^O/g body wt./hr.
Integumental absorption of water was determined by
weighing each turtle, suturing its cloaca shut, and applying petroleum jelly over the sutures (to repel water).
The
animals were secured in a tritiated water medium with only
the posterior submerged.
was obtained.
After 16 hrs. a plasma sample
Plasma tritium was determined by liquid
scintillation (Beckman LS lOOC), the posterior skin surface
area was estimated, and the absorption of water was expressed as ml absorbed/cc skin/hr.
Solutes and precipitates were determined as follows:
(1) Urea and ammonia—the Obrink modification of the Conway
microdiffusion technique (Obrink, 1955); (2) Bicarbonate—
the Conway technique (Conway, 1955); (3) Chloride—titrimetrically (Sigma tech. bull. no. 830); (4) Sodium and
potassium—flame photometrically (Beckm.an Kline flame) ;
(5) Uric acid—enzymatically (Sigma tech. bull. no. 292UV); (6) Osmolarity—freezing point depression (Advanced
Instruments Osmometer Model 3L).
24
Precipitated urate salts were analyzed for:
(1) Total
uric acid; (2) Total amount of cation bound to urates
(mEq); and (3) Relative amount of cation bound (mEq of
cation/mM uric acid).
The differences between means of each multiple comparison, i.e., a comparison of the significance of one mean
from all other means was tested by the Student-::eumanKeuls multiple range test.
Standard errors are calculated
from the variance of each group.
For the comparison of
significance of 2 means, a Students t-Test was used.
P <
.05 was used as the criterion of significant difference.
Results
Urine Sodium
Sodium concentrations in the urine of hydrated animals
were not significantly different (Fig. 5). Urinary sodium
concentrations of all groups of dormant turtles were significantly greater than those of hydrated animals, with an
increase of sodium during dormancy.
Plasma Sodium
Plasma sodium concentrations of hydrated animals were
not different, but plasma sodium concentrations of dormant
animals were greater than those of hydrated animals (Fig.
5).
Plasma sodium concentrations of dormant animals in-
creased significantly with time.
25
URINE
PLASMA
HCO3
mEq/l
1000
400
mOsm/l
[^
500
200
ri^
r^
r^
^i^
100
CI"
mEq/l
50
J f] n
0
lOO
rh
r^-i
mEq/l
50
f*-i
100
fj^
•
No'
mEq/l
rli
rfi
^
50-
*
r^ rh
x>
w
HYD.
^_
0
0
0
0
5
5
2
<M
^j-
>r
DORMANT
to
U.
HYD
li-
0
0
5
5
3-
-•
cvj
V
»X)
t^
DORMANT
Fig. 5. Shows the mean ± S.E. of plasma and urine
electrolytes and osmolarity. Sample sizes are: n=10 for
both groups of hydrated animals, salt-loaded animals
(S.L.), and 4-month dormant animals; n=9 for 2-month dormant animals; and n=7 for 6-month dormant animals.
26
Urine Potassium
Urinary potassium concentrations of hydrated animals
were not significantly different, but were considerably
less than urinary potassium of dormant animals (Fig. 5).
Urinary potassium of turtles dormant for 6 months were
significantly greater than those of animals dormant for
2 and 4 months.
Plasma Potassium
Plasma potassium between hydrated animals was not
different (Fig. 5). Plasma potassium of dormant animals
was greater than that of hydrated animals, with an increase
of plasma potassium during dormancy.
Urine Chloride
Urinary chloride concentrations of animals dormant
for 2 months and both groups of hydrated animals were not
significantly different.
Chloride concentrations in urine
of animals dormant for 4 and 6 months were greater than
were the other 3 experimental groups.
Plasma Chloride
Plasma chloride concentrations of hydrated animals
were not different, but these 2 groups had plasma chloride
concentrations significantly less than that of the dormant
animals.
Plasma concentration of chloride of animals dor-
mant for 2 months was greatly less than that of animals
27
dormcint for 4 and 6 months.
Urine Bicarbonate
Urinary bicarbonate concentration decreased during
dormancy (Fig. 5). There was no difference in bicarbonate
concentrations among hydrated animals (both groups) and
animals dormant for 2 months.
However, bicarbonate con-
centrations decreased in animals dormant for 4 and 6
months.
Urine Osmolarity
Urine osmolarity of the hydrated animals was not significantly different, but the osmolarity of these 2 groups
was less than that of dormant and salt-loaded animals
(Fig. 5). There was an increase in urine osmolarity during dormancy.
Urine osmolarity of salt-loaded animals was
not significantly different from dormant animals.
Plasma Osmolarity
Plasma osmolarity was greater in dormant and saltloaded animals than in hydrated animals (Fig. 5). Plasma
osmotic pressure increased during dormancy, but was highest
in salt-loaded animals.
Urine Urea
There was no difference in urinary urea concentration
of hydrated animals, but the concentration of these two
28
groups was significantly less than urinary urea concentrations of dormant animals (Fig. 6). The concentration of
urinary urea was greatest in animals dormant for 2 months.
Plasma Urea
Plasma urea concentrations of hydrated animals were
not significantly different, but these 2 groups had a lower
concentration of plasma urea than dormant animals (Fig. 6).
Urea plasma concentrations were highest in animals dormant
for 2 and 4 months.
Salt-loaded animals had increased
plasma urea levels, though below that of dormant animals.
Urine Uric Acid
Urinary uric acid concentrations (only uric acid in
solution) between hydrated animals were not statistically
different; however, the concentrations were less than those
of dormant animals.
Urinary uric acid concentrations of
animals dormant for 2 and 6 months were not statistically
different, nor was any difference found between the concentration of turtles dormant for 4 and 6 months; however,
urate concentration of animals dormant 4 months was statistically greater than concentration in 2-months dormant
animals.
Plasma Uric Acid
Plasma uric acid concentrations of hydrated animals
were not different, and these 2 groups had a lower concen-
29
URINE
PLASMA
_
<
o
24
F]
mWI
2 0 --
>
<
mq/dl
ri El
rlr
12
r^
r.. ["1
5
1 0
0
0
0
0
50-
rh
4
9 mW/1
mg/dl
25
o
2-
rin
3
'*•< n
J±L
200
40
mg/dl
<
mM/1
100
LU
^
20
rJi
n.
x>
«/1
o
Lu
HYD
•o
J!La.
o
o
s
5
^
CJ
d
o
5
_j
u?
l/^
DORMANT
HYD
6
5
o
5
o
2
to
DORMANT
Fig. 6. Shows the mean ± S.E. of plasma and blood
nitrogenous wastes. Sample sizes are: n=10 for both
groups of hydrated animals, salt-loaded animals (S.L.),
and 4-month dormant animals; n=9 for 2-month dormant ani'
mals; and n=7 for 6-month dormant animals.
30
tration of plasma uric acid than those of dormant animals
(Fig. 6). Concentrations of plasma uric acid increased
during dormancy with the plasma uric acid concentration of
turtles dormant for 6 months significantly greater than all
other experimental groups.
Urine Ammonia
Urinary ammonia concentrations (only ammonia in solution) among all experimental groups were different (Fig.
6).
The results were too variable to show any trend.
Plasma Ammonia
No measurable plasma concentrations of ammonia were
found, except in salt-loaded animals.
Urate Salts
Total precipitated uric acid was significantly different in all groups, with an increase during dormancy as
in Table 1 (hydrated animals had no precipitate).
The
cimount of total cation bound was significantly different
for each cation among experimental groups.
Relative amounts
of sodium and potassium in precipitated urate salts did not
change during dormancy; however, the relative amounts of
bound ammonia increased.
Urea-N
The percentages of molar urea-N of non-proteinnitrogen (NPN) in blood plasma of hydrated fasting animals,
31
TABLE 1
CATIONIC AND URIC ACID CONTErTT OF PRECIPATED URATE
SALTS DURING DORMANCY. VALUES (mM) OR (mEq) ARE
ABSOLUTE AMOUNT OF URIC ACID OR CATION C0:;TE::T
OF THE PRECIPITATED URATE SALT. VALUES
(mEq/mM) ARE RELATIVE CONCENTRATIONS
OF mEq OF CATION PER m:'i OF
URIC ACID
6 Months
2 Months
4 Months
.159
.022
.447
.028
1.230
.060
Na"*"
X
(mEq) S.E.
.055
.001
.142
.001
.525
.003
Na"*"
X
(mEq/mM) S.E.
.346
.050
.318
.019
.427
.021
K*
X
(mEq) S.E.
.117
.002
.337
.004
.998
.001
.734
.084
.754
.013
.811
.017
.027
.627
.001
.134
.002
.168
.300
.510
.027
.134
.031
Uric X
Acid S.E.
(mM)
K"*"
X
(mEq/mM) S.E.
NH4
X
(mEq) S.E.
NH^
X
fmEa/mM) S.E.
n
10
.003
32
and animals dormant for 2 and 4 months were not different
(Table 2).
These 3 groups had a statistically higher per-
centage of plasma urea-N than hydrated-fed animals and animals dormant for 6 months.
Urine percentage urea-N was
significantly different among all groups, with a lower percentage during dormancy.
Uric Acid-N
The percentage of plasma uric acid-N of total plasma
NPN between hydrated-fed animals and animals dormant for
6 months was not significantly different (Table 2).
These
2 groups had a significantly higher percentage of plasma
uric acid-N than any of the other 3 groups.
The percentage
of total uric acid-N (precipitated and solvated uric acid)
of urine NPN was different among all groups, with a greater
percentage occurring during dormancy.
Ammonia-N
The percentage of urinary ammonia-N of the urine NPN
was statistically equivalent in animals dormant for 4 and
6 months and in fasted-hydrated animals (Table 2).
The
percentage urinary ammonia-N was significantly greater in
hydrated-fed animals than in animals dormant for 2 months.
These 2 groups had a significantly greater percentage of
ammonia-N than the other 3 groups.
Water Relations
Evaporative water loss of hydrated animals was sig-
33
TABLE 2
PER CENT UREA-, URATE-, OR A:-LM0NIA-N OF THE TOTAL
URINE AND PLASI-IA IS REPRESENTED IN TABLE 2.
URINE NPN WAS CALCULATED USING N FROM
PRECIPITATED AND SOLVATED SOURCES
Hydrat ed
Fasted
Dormant
Fed
2 Months
4 Months
6 Months
94.5
94.3
86.1
(4.7)
Plasma
Urea-N
X(S.E.)
Urate-N
X(S.E. )
90.9
83.8
(12.5)
(20.5)
(7.4)
(4.0)
9.1
(1.9)
17.2
5.7
(1.1)
5.5
(0.3)
(0.1)
13.9
(0.8)
84.0
52.3
41.8
22.6
10.7
(2.3)
(4.7)
(3.9)
(9.1)
(1.8)
5.1
(0.8)
14.4
39.9
66.7
76.2
(1.9)
(7.3)
(3.2)
(3.8)
10.9
33.3
18.3
10.7
13.1
(0.6)
(1.7)
(1.1)
(1.6)
(0.5)
Urine
Urea-N
X(S.E.)
Urate-N
X(S.E.)
NH^-N
X(S.E.)
n
10
10
10
34
nificantly greater than that of dormant animals (Fig. 7).
All groups of dormant animals demonstrated no differences
in evaporative water loss.
Dormant animals absorbed sig-
nificantly less water across their integument than active
cuiimals.
Salt-Loading
The fluid collected from rinsing the head of saltloaded animals indicated only a trace of sodium and potassium (less than 1 mEq/l), nor were any salt crusts observed on the head of Kinosternon flavescens.
Discussion
Many reptiles and amphibians are able to withstand
large changes in the osmotic pressure of their body fluids
(Gordon, 1962; Bentley, 1959; and Goldstein, 1972).
The
observed increase of plasma osmolarity in K. flavescens was
approximately 250 mOsm after 6 months of dormancy (Fig. 5).
Evaporative water losses would reduce volume of body water,
thus solutes would be concentrated, which would cause the
observed increase in osmolarity.
Kinosternon flavescens
had no extrarenal excretion of salts, so its main adaptation to osmotic stress during dormancy was its ability to
withstand large changes in the osmotic pressure in its body
fluids.
Urinary bicarbonate secretion in the turtle's bladder
35
20
ml/cc/hr
0
1
0
HYD.
DORMANT
B.
i
1
e
-3
IIO.OXIO
i
I
mg HpO/g/hr
_^
1.5X10
;i
-3
L25 X 10
-3
.0 XIO
HYD.
2 Mo.
4 Mo.
6 Mo.
DORMANT
Fig. 7A. Shows the mean ± S.E. of integumental water
absorption. The sample size is 4 for both groups. Fig.
7B. Shows evaporative water losses. The sample sizes
are: n=10 for hydrated and 4-month dormant animals; n=9
for 2-month dormant animals; and n=7 for 6-month dormant
animals.
36
is energy-dependent.
The bicarbonate secretion is coupled
with chloride absorption and is dependent on metabolic
energy derived from oxidation (Oliver, et al^. , 1975).
A
buried, dormant turtle may be hypoxic, which could decrease
bladder bicarbonate secretion.
Bicarbonate concentrations
were less in dormant animals (4 and 6 months) and varied
inversely with urinary chloride (Fig. 5), suggesting that
during dormancy, oxidative energy was limited.
Desert tortoises are known to be either ureo- or
uricotelic, depending on their state of hydration (Khalil
and Haggag, 1955; Cloudsley-Thompson, 1971).
Most aquatic
poikliotherms are either ammono- or ureotelic, depending
on their state of hydration.
K. flavescens was ureo- or
uricotelic, and ammonia was never the primary excretory
product (Fig. 6). Since the animals were dehydrating during dormancy, it would be advantageous to be uricotelic,
because a greater amount of oxidative water from proteins
is produced during uricotelism than during ureotelism.
Uricotelism is also advantageous because of the formation
of insoluble urate salts, which precipitate in the bladder
allowing water to be reabsorbed (Schmidt-Nielsen, 1964;
Minnich, 1970; and Minnich and Shoemaker, 1972).
However,
urea-N was the major source of NPN in the blood plasma of
all experimental groups (Table 2). High plasma uric acid
would be deleterious because urates could precipitate and
37
interfere with circulation and kidney function.
Urinary
molar percentage of NPN showed urea as the primary source
of NPN of hydrated animals, as an equimolar NPN source (to
uric acid) in animals dormant for 2 months, and as a lesser
source than uric acid in animals dormant for 4 and 6 months
(Table 2). These results indicated that during dehydration,
uric acid was the main end-product of N metabolism, urea
was the main nitrogenous waste during hydration, and
cimmonia was only a minor source of waste N.
Precipitation of urate salts were important for survival of K. flavescens during dormancy.
Most uric acid of
reptiles is precipitated as a monobasic salt (Minnich,
1970); however, results in Table 1 showed the relative
amount of bound cation greater than 1, suggesting that some
urate was precipitated as a dibasic salt.
The percentage
of dibasic salt increased during dormancy, therefore more
solutes were bound, allowing more water to be reabsorbed.
Results indicated that Kinosternon flavescens had no
extrarenal excretion of salt.
The reason for this obser-
vation could be that the precipitation of mono- and dibasic
urate salts in the urine eliminates potential osmotic
pressure from salts, which in effect "removes" the salts.
The solubility of monobasic urate salts are:
potassium
urate > sodium urate > ammonium urate (McNabb, 1974).
These observations paralleled the increase of the relative
38
amount of ammonium urate during dormancy, while relative
amounts of the other urate salts remain relatively constant
(Table 1). Minnich (1972) reported that fasted snakes had
increased amounts of ammonium urate because of the increased protein catabolism during fasting leading to the
production of ammonia and uric acid.
During dormancy,
fasting may have caused an increased ammonia production
from protein catabolism, which may have caused the relative
amounts of ammonium urate to increase.
The concentration of plasma ammonia of salt-loaded K.
flavescens (16.1 mg/dl) suggested the possibility of a
renal sodium-ammonia exchange; however, if the rise in
plasma osmolarity was great enough to cause anuria, ammonia
from the kidney would not be excreted, but returned to the
circulation, which could possibly explain the plasma
ammonia.
Dormant K. flavescens lost less evaporative water than
did active animals (Fig. 7). The integument of dormant
animals appeared to be keratinized during dormancy, which
would retard water loss.
Desert-dwelling amphibians were
reported to retard water loss during estivation by the
formation of a keratinized layer over their skin (Lee and
Mercer, 1967; McClanahan, et_ a]^. , 1976; and Mayhew, 1965).
Keratinization of the skin also retards water absorption
through the integument during dormancy (Fig. 7).
39
Summary
1.
Heat- and drought-induced dormancy of Kinosternon
flavescens was experimentally simulated in the laboratory.
2.
Comparisons of blood and urine electrolytes and
nitrogenous wastes between active and dormant animals were
made.
3.
Electrolyte concentrations of blood and urine were
greater during dormancy than during hydration, except
urinary bicarbonate, which decreased.
4.
Osmotic pressure of urine and plasma was greater
in dormant animals than in hydrated animals.
5.
Plasma concentrations of urea and uric acid were
higher in dormant animals than in hydrated animals, and
all urinary nitrogenous wastes were greater during dormancy.
6.
No extrarenal salt excretion was found.
7.
Evaporative water loss and integumental water ab-
sorption of dormant animals was less than that of hydrated
animals, which aids in water conservation.
8.
K. flavescens adapts to dehydration by changing
from ureotelism during hydration to uricotelism during
dormancy.
Uric acid excretion is advantageous because
urate salts precipitate in the bladder, which removes some
of the osmotic potential of cations and uric acid.
CHAPTER IV
CONCLUSIONS
1.
To escape heat and drought, Kinosternon flavescens
enters a dormant state.
2.
During dormancy, carbohydrate metabolism may be
reduced due to increased production of ketone bodies.
3.
The animals store large amounts of lipids (for
energy and metabolic water), which may be related to a
high fructose metabolism.
4.
During dormancy, the animals switch from ureo-
telism to uricotelism which increases production of
metabolic water.
Uricotelism is also advantageous because
insoluble urate salts precipitate in the bladder, allowing
bladder water to be reabsorbed.
5.
K. flavescens has no extrarenal excretion of
salts and the main adaptation to increased levels of plasma
electrolytes is a tolerance to the high levels of salt and
osmolarity.
40
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