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First published online November 1, 2006
Journal of Experimental Biology 209, 4475-4489 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02557
Active ammonia transport and excretory nitrogen metabolism in the climbing perch, Anabas testudineus, during 4 days of emersion or 10 minutes of forced exercise on land
1 Department Of Biological Science, National University Of Singapore, Kent
Ridge, Singapore 117543, Republic Of Singapore
2 Natural Sciences and Science Education, National Institute of Education,
Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic
of Singapore
3 Ecofisiologia CIMAR, Rua dos Bragas 289, 4050-123 Porto,
Portugal
* Author for correspondence (e-mail: dbsipyk{at}nus.edu.sg)
Accepted 21 September 2006
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Summary |
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Key words: ammonia, Anabas testudineus, emersion, nitrogen, metabolism, urea
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). It
possesses a special accessory breathing organ (ABO), situated just above the
gills in a large extension on the upper part of each gill chamber, which
facilitates the utilization of atmospheric air
(Graham, 1997). The ABO is
composed of lamellae which are covered with an extremely vascular layer of
skin and convoluted into numerous folds to maximize the surface area for
respiration (Hughes and Munshi,
1973; Munshi et al.,
1986). Because the ABO structure resembles a complicated maze, it
has been dubbed the labyrinth organ. A. testudineus periodically
gulps air from the water surface, and air is channelled to the labyrinth
organs for gaseous exchange. During droughts, A. testudineus stays in
pools associated with submerged woods and shrubs
(Sokheng et al., 1999) or
buries under the mud (Rahman,
1989). In addition, it is able to travel long distances on land
(Das, 1927;
Herre, 1935). It crawls about
with its spiky gill covers propped by its pectoral fins, and the hind part of
the fish twitches violently to propel it forward
(Liem, 1987;
Davenport and Abdul Matin,
1990). When the air is sufficiently humid, it is able to cover
several hundred meters per trip using this method of locomotion. To date,
however, no information is available on the type of energy store utilized by
the climbing perch to support locomotion on land.
Ammonia is produced in fish mainly through the catabolism of amino acids in
the liver (Ip et al., 2001b),
and is usually excreted as NH3 through the gills
(Wilkie, 2002;
Evans et al., 2005). However,
excretion of ammonia is impeded when a fish moves out of water, and ammonia
subsequently accumulates in the body. Ammonia is toxic, and therefore
amphibious air-breathing fishes have special adaptations to defend against
ammonia toxicity (for reviews, see Ip et
al., 2001b; Ip et al.,
2004a; Chew et al.,
2006). These adaptations include reduction in ammonia production
through suppression of amino acid catabolism or via partial amino
acid catabolism leading to the formation of alanine, conversion of ammonia to
less toxic compounds such as glutamine for storage or urea for subsequent
excretion, and volatilization of NH3.
Saha and Ratha (Saha and Ratha,
1989) reported that A. testudineus possessed all the
ornithine-urea cycle (OUC) enzymes, including carbamoyl synthetase I (CPS I)
which uses ammonia as a substrate, in its liver. However, they
(Saha and Ratha, 1989) should
have determined the activity of CPS III, which uses glutamine as a substrate
because fishes are known to possess CPS III instead of CPS I. These include
both marine and freshwater elasmobranchs
(Anderson, 2001;
Tam et al., 2003), a few
teleosts (Anderson and Walsh,
1995; Iwata et al.,
2000; Randall et al.,
1989), the coelacanth (Mommsen
and Walsh, 1989) and African lungfishes
(Chew et al., 2003b;
Loong et al., 2005). Saha and
Ratha (Saha and Ratha, 1987;
Saha and Ratha, 1989) also
reported the presence of hepatic CPS I in the Asian walking catfish
Clarias batrachus and the Indian air-sac catfish Heteropneustes
fossilis, but subsequently Saha et al.
(Saha et al., 1997;
Saha et al., 1999) suggested
that both CPS I and CPS III were present in the liver. However, using a more
sensitive radiometric assay, Ip et al. (Ip
et al., 2004d) were unable to detect CPS I or CPS III activities
from the liver of C. batrachus and Clarias gariepinus (for a
review, see Chew et al.,
2006). Therefore, this study was undertaken to determine whether a
full complement of OUC enzymes, including CPS I and/or III, was indeed present
in the liver of A. testudineus.
In addition, Saha and Ratha (Saha and
Ratha, 1989) proposed that A. testudineus was able to
detoxify ammonia to urea during emersion, but did not present any result to
support their proposition. Although there was an earlier study
(Ramaswamy and Reddy, 1983) on
the same species (as Anabas scandens), these researchers did not
examine nitrogenous excretion during emersion, nor did they determine the
amount of ammonia and urea in the muscle, which constituted the bulk of the
fish. Thus, another objective of this study was to examine whether urea
accumulation occurred in A. testudineus during 4 days of emersion,
and whether an increase in the rate of urea excretion would occur in fish
reimmersed in water on day 5.
This study also aimed to elucidate whether A. testudineus would adopt, (1) a reduction in ammonia production, (2) partial amino acid metabolism leading to alanine formation, and/or (3) detoxification of ammonia to glutamine, to ameliorate ammonia toxicity during emersion. During the course of the study, it was discovered unexpectedly that A. testudineus was able to excrete ammonia continuously during emersion without difficulty, resulting in high concentrations of ammonia in a small volume of external medium. Therefore, an attempt was made to determine ammonia concentrations in water samples collected from the branchial or cutaneous surfaces of fish exposed to terrestrial conditions for 24 h. Efforts were also made to determine whether A. testudineus was capable of excreting ammonia against a concentration gradient in water containing 12 mmol l-1 NH4Cl. Furthermore, because there were significant increases in tissue ammonia content and muscle total essential free amino acids in fish exposed to terrestrial conditions, experimental fish were exercised on land to explore the possibility that amino acids were utilized as substrates to fuel locomotor activities on land.
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Determination of activities of ornithine-urea cycle enzymes and glutamine synthetase from the liver
Fish in control conditions (N=4) were killed with a strong blow to
the head. The liver was excised quickly and homogenized in 5 volumes (w/v) of
ice-cold extraction buffer containing 50 mmol l-1 Hepes (pH 7.6),
50 mmol l-1 KCl, 0.5 mmol l-1 EDTA, 1 mmol
l-1 dithiothreitol and 0.5 mmol l-1 PMSF. The homogenate
was then sonicated (110 W, 20 kHz; Misonix Incorporated Farmingdale, NY, USA)
three times for 20 s each, with a 10 s break between each sonication. The
sonicated sample was centrifuged at 10 000 g and 4°C for
15 min. After centrifugation, the supernatant was passed through a Bio-Rad
P-6DG column (Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with the
extraction buffer without EDTA and PMSF. The filtrate obtained was used
directly for enzymes assay.
Carbamoyl phosphate synthetase I and III (E.C. 2.7.2.5; CPS) activities
were determined according to published methods
(Anderson and Walsh, 1995;
Chew et al., 2003b;
Loong et al., 2005). The CPS
III activity was determined in the presence of glutamine,
N-acetylglutamate and UTP in order to eliminate the activity of CPS
II. Radioactivity was measured using a Wallac 1414 liquid scintillation
counter (Wallac Oy, Turku, Finland). Enzyme activity was expressed as µmol
[14C]urea formed min-1 g-1 wet mass.
Ornithine transcarbamoylase (E.C. 2.1.3.3) activity was determined by
combining the methods of Anderson and Walsh
(Anderson and Walsh, 1995) and
Xiong and Anderson (Xiong and Anderson,
1989). Absorbance was measured at 466 nm using a Shimadzu 160 UV
VIS recording spectrophotometer (Shimadzu Co., Kyoto, Japan). Enzyme activity
was expressed as µmol citrulline formed min-1 g-1 wet
mass. Argininosuccinate synthetase (E.C. 6.3.4.5) and lyases (E.C. 4.3.2.1)
activities were determined together assuming that both were present, by
measuring the formation of [14C]fumarate from
[14C]aspartate using the method of Cao et al.
(Cao et al., 1991), and
activity was expressed as µmol [14C]fumarate formed
min-1 g-1 wet mass. Arginase (E.C. 3.5.3.1) was assayed
as described (Felskie et al.,
1998) and activity was expressed as µmol urea formed
min-1 g-1 wet mass. Glutamine synthetase (E.C. 6.3.1.2)
was assayed as transferase activity according to the method of Shankar and
Anderson (Shankar and Anderson,
1985) and activity was expressed as µmol
-glutamylhydroxymate formed min-1 g-1 wet
mass.
Determination of effects of emersion on ammonia and urea excretion rates
After 2 days of fasting to clear the gut (0 h), control fish
(N=12) were exposed individually to 25 volumes (w/v; approximately
1.4 l) of freshwater in plastic containers (22 cmx11.5 cmx13 cm,
LxWxH). Water samples (3.6 ml) were collected every 24 h,
acidified with 0.04 ml of 2 mol l-1 HCl, and stored at 4°C.
Water was changed daily after sampling and the experiment was continued for 4
days. Ammonia and urea concentrations in water samples were determined within
1 week according to the method of Anderson and Little
(Anderson and Little, 1986) and
the method of Jow et al. (Jow et al.,
1999), respectively.
Experimental fish (N=12) were transferred individually after 2 days of fasting to similar plastic containers (22 cmx11.5 cmx13 cm, LxWxH) but with only 80 ml of freshwater, which formed a thin film at the bottom. The experimental conditions were designed to examine effects of emersion, but not desiccation. Under such experimental conditions, the fish could not maintain its normal posture and usually lay on its side at the bottom of the tank. In spite of this, the gills and the ABOs were not in direct contact with the thin film of water due to the presence of spikes on the surface of the opercula, which were puffed open to facilitate air-breathing. Very often, the fish produced jerking movements through flipping of its tail, and occasionally it would be able to right itself by leaning against the side wall of the tank. This happened especially during the first day of emersion. After 24 h, 50 ml of water was poured over the fish for brief rinsing (<1 min), and the total amount of water was determined using a measuring cylinder. An aliquot of the water sample was acidified and stored at 4°C for subsequent ammonia and urea assays. Fish were then transferred to new plastic tanks containing 80 ml of freshwater. The experiment continued for 4 days.
To evaluate the effects of recovery in water on fish after 4 days of emersion, fish (N=6) were exposed to the control conditions, as described above, for 5 days. Experimental fish were exposed to terrestrial conditions, as described above, for 4 days, and on day 5, they were re-immersed in 25 volumes of freshwater and water samples were collected 24 h later for ammonia and urea assays.
To verify that ammonia and urea excretion rates obtained were not affected by bacterial activities, several water samples (20 ml without acidification) were left at 25°C in glass beakers for 24 h. Preliminary results confirmed that the ammonia and urea concentrations in the water sample before and after the 24 h of incubation at 25°C were not statistically different. Ammonia and urea excretion rates were expressed as µmol day-1 g-1 fish.
Because results obtained indicated unexpectedly that the climbing perch could excrete a large amount of ammonia into the thin film of water during emersion, a separate set of experiments was performed following the protocol described above, except that there was no change of water in the tank and the experiment lasted only 2 days. In this set of experiments, we aimed to determine how high the concentration of ammonia could reach in the external medium after 48 h. A 2-day period was chosen because preliminary experiments revealed that bacterial activities could become an important factor if the experimental period were extended beyond 2 days.
Determination of ammonia concentrations in water collected from branchial or cutaneous surfaces in fish after 15 min or 24 h of emersion
Control fish (hour 0; N=5) were taken out of water and
anaesthetized immediately on land in a saturated atmosphere of diethylether,
which took approximately 15 min. Experimental fish (N=5) were exposed
to terrestrial conditions in plastic containers (14.7 cmx8.2
cmx9.1 cm, LxWxH) with 40 ml of freshwater for 24 h before
being anaesthetized with diethylether. Because one side of the fish was in
direct contact with the thin film of water, it was important to take note of
which side of the fish was exposed to air. The opercular opening of the
air-facing side of the anaesthetized fish was open and a small piece of
pre-weighed glass microfibre paper (2 mmx10 mm; Whatman GF/C) was
applied gently to the surface of each gill arch to absorb the water. The
volume of water absorbed was determined gravimetrically. This procedure was
repeated a second time with a new piece of fibreglass paper. The two pieces of
glass microfibre paper were transferred immediately into 0.5 ml of water
acidified with 2 µl of 1 mol l-1 HCl for subsequent ammonia
assay. Similarly, a water sample was collected from the surface of the
air-facing skin using pre-weighed glass microfibre paper (4 mmx10 mm)
and transferred to 0.5 ml of acidified water. Efforts were made to absorb
water from below the scales by pressing the fibreglass paper lightly along the
body surface. Then, the fish was flipped over carefully, and the procedure of
water collection was repeated. The whole process took approximately 10 min.
Ammonia was assayed as described above, and results were expressed as mmol
l-1.
Determination of the capacity to excrete ammonia against a concentration gradient
The normal ammonia and urea excretion rates of fish fasted for 48 h
(N=7) were determined as described above. They were then transferred
individually into Ziplock plastic bags (double layered) containing 5 volumes
(w/v) of 12 mmol l-1 NH4Cl at pH 7.0. Under such
conditions, the fish was completely immersed in the medium and maintained in
the upright position inside the bag. The opening of the bag was partially
clipped to prevent the fish from escaping. Water samples were collected as
described above every 24 h for a total period of 2 days for the estimation of
ammonia and urea excretion rates.
Determination of effects of emersion on content of tissue ammonia, urea and free amino acids
A total of 24 fish were kept in the control condition as described above.
Fish [(N=6) for each time point] were killed with a strong blow to
the head at the end of days 1, 2, 4 and 5, for tissue sampling. Blood was
collected from the severed caudal artery into sodium heparin-coated capillary
tubes. The collected blood was centrifuged at 4000 g at
4°C for 10 min to obtain the plasma. The plasma was deproteinized in an
equal volume (v/v) of ice-cold 6% trichloroacetic acid (TCA) and centrifuged
at 10 000 g at 4°C for 15 min. The resulting supernatant
was kept at -80°C for analysis of ammonia, urea and free amino acids
(FAAs). The muscle, liver and brain tissues were excised and immediately
freeze-clamped with tongs pre-cooled in liquid nitrogen. Frozen samples were
stored at -80°C until analysis.
Experimental fish (N=24) were killed at the end of days 1, 2 and 4 of emersion (N=6 each) for tissue sampling as described above. After 4 days of emersion, the remaining six fish were immersed in 25 volumes (w/v) of freshwater, and were killed for tissue sampling after 24 h of immersion.
To determine ammonia, urea and FAA content, the frozen liver, muscle and
brain samples were weighed and ground to a powder in liquid nitrogen. Five
volumes of ice-cold 6% TCA were added and the mixture was homogenized three
times for 20 s each (with intervals of 10 s) with an Ultra-Turrax (Staufen,
Germany) homogenizer at 24 000 revs min-1. The samples were then
centrifuged for 15 min at 10 000 g at 4°C and the
supernatant were stored at -80°C for subsequent analysis. Ammonia and urea
assays as well as FAA analysis were performed within 3 weeks. For analysis of
ammonia, the pH of the deproteinized sample was adjusted to 6.0-7.0 with 2 mol
l-1 KHCO3. Ammonia and urea content in the muscle, liver
and plasma samples were determined as described by Bergmeyer and Beutler
(Bergmeyer and Beutler, 1985)
and Jow et al. (Jow et al.,
1999), respectively. Results were expressed as µmol
g-1 wet mass tissue or µmol ml-1 plasma. For FAA
analysis (N=4), the supernatant of the deproteinized muscle and liver
samples were diluted with an equal volume of 0.2 mol l-1 lithium
citrate buffer (pH 2.2) and adjusted to pH 2.2 with 4 mol l-1 LiOH.
The filtered samples were then analyzed for FAAs using a Shimadzu LC-6A Amino
Acid Analysis System with a Shim-pack ISC-07/S1504 Li-type column (Kyoto,
Japan). FAAs, total FAA (TFAA) and total essential FAA (TEFAA) were expressed
as µmol g-1 wet mass tissue.
Determination of effects of 10 min of exercise on A. testudineus during emersion
Fish (N=10) were transferred individually after 2 days of fasting
to plastic aquaria containing 10 ml of freshwater, which formed a thin film at
the bottom. Control fish (N=5) for this set of experiment were left
inside the container without any disturbance. The control fish naturally
exhibited some jerky movements, albeit much less frequently than the
experimental fish. After 10 min, 50 ml of water was added to briefly rinse the
fish. Water samples were collected for ammonia and urea assays as described
above. Fish were killed with a strong blow to the head for the collection of
muscle samples. Experimental fish (N=5) were stimulated mechanically
and continuously, which resulted in frequent jerky movements, within the
plastic aquaria. After 10 min, water samples were collected and the fish was
killed for muscle collection.
Ammonia, urea and FAA were determined in the muscle samples as described
above. Despite analysing all the FAAs, only content of alanine, glutamate,
lysine, TEFAA and TFAA are presented. Glucose and glycogen content were
determined according to the method of Lim and Ip
(Lim and Ip, 1987). Lactate
and succinate content were determined by the method of Gutmann and Wahlefeld
(Gutmann and Wahlefeld, 1974)
and Beutler (Beutler, 1985),
respectively. ATP, ADP and AMP were determined spectrophotometrically
following the method of Scheibel et al.
(Scheibel et al., 1968).
Determination of O2 consumption rate
In this set of experiments, the volume of the fish was determined by water
displacement. Control fish (N=4) were transferred to air-tight
plastic boxes (13.5 cmx7 cmx8.5 cm, LxWxH) containing
400 ml of water with continuous aeration 24 h before the experiment. For the
determination of O2 consumption rate, the box, containing 400 ml of
water, 120 ml of air and the fish was sealed. The air space was reduced to 120
ml by the inclusion of a piece of polystyrene (Styrofoam) which had a
thickness of 1.85 cm and a volume of 100 ml. The polystyrene was positioned in
the box so that the fish was restricted to respire through an air space
closest to the O2 sensor. Water in the box was stirred slowly with
a magnetic bar. Changes in aerial and aquatic PO2 were
monitored using an Ocean Optics FOXY Fiber Optics O2 sensing system
S2000 with two FOXY-R O2 electrodes (Ocean Optics Inc., Dunedin,
FL, USA) inserted separately into the air and water compartments. For the
control fish, two measurements were made for 30 min each, with a 60 min
interval during which the water was aerated. As for the experimental fish
(N=4), they were transferred directly from water into air tight
plastic boxes (13.5 cmx7 cmx4.2 cm, LxWxH), which
contained 210 ml of air and 3 ml of water. Two pieces of polystyrene which
each had a thickness of 1.85 cm and a volume of 100 ml were put inside the box
to reduce the air volume. The O2 consumption rate in air was
monitored with a FOXY-R O2 electrode. Another set of experimental
fish (N=4) was exposed to terrestrial conditions for 24 h before the
determination of the O2 consumption rate.
To calibrate the FOXY-R O2 electrode for measurements of water PO2, it was immersed in sodium sulphite solution (2 g l-1; 0%) or air-saturated water (100%) until stable readings were recorded. For air PO2 measurements, the electrode was calibrated in a partially closed tube supplied continuously with humidified N2 gas (0%) or air (100%).
Statistical analyses
Results are presented as means ± standard errors (s.e.m.). Results
shown in Fig. 1 were analyzed
using repeatedmeasures analysis of variance (ANOVA) followed by leastsquare
means (LSMEANS) to evaluate differences between means. Results in Tables
1 and
2 were assessed using two-way
ANOVA followed by Bonferroni's multiple range test to evaluate differences
between means, and those in Fig.
2 and Tables 3,
4,
5 and
6 were analyzed by Student's
t-test. Differences with P<0.05 were regarded as
statistically significant.
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Effects of emersion on rates of ammonia and urea excretion
With daily change of water, the ammonia concentration in the thin film of
water (80 ml) reached approximately 4-5 mmol l-1 after 24 h
throughout the 4-day period of emersion. This indicates that the rate of
ammonia excretion in A. testudineus during emersion was high. Indeed,
the daily ammonia excretion rates (N=12) in A. testudineus
on days 1, 3 and 4 of emersion were not significantly different from those of
the control immersed in water (Fig.
1A). Surprisingly, on day 2, the daily ammonia excretion rate of
the fish exposed to terrestrial conditions was significantly greater than that
of the control. When summed together over a 2-day period, however, the
excretion rate of 11.5±2.1 µmol 2 days-1 g-1
was not significantly different from the control value of 13.9± 0.7
µmol 2 days-1 g-1. Similarly, the total rate of
ammonia excretion in the experimental (24.1±4.6 µmol 4
days-1 g-1) and control (32.2±1.9 µmol 4
days-1 g-1) fish over a 4-day period were comparable.
Upon re-immersion on day 5, the daily ammonia excretion rate (N=6) of
the experimental fish was not significantly different from that of the
immersed control (Fig. 1A).
By contrast, in spite of significant increases, the urea concentration in the thin film of water remained relatively low throughout the 4-day emersion period. Emersion led to a significant decrease in the daily urea excretion rate on day 1, but had no significant effect on urea excretion thereafter (Fig. 1B). There was no significant change in urea excretion in fish during re-immersion on day 5 as compared with day 1 or day 5 controls (Fig. 1B).
In a separate set of experiments in which daily change of water was omitted for a 2-day period, the ammonia concentration (N=5) in the thin film of water reached 6.68±1.11 and 13.2±2.1 mmol l-1 at the end of day 1 and day 2, respectively. The respective daily ammonia excretion rates were 9.98±1.92 and 8.64±1.85 µmol day-1 g-1 fish, which were not significantly different from the corresponding values (7.45±1.32 and 7.54±1.21 µmol day-1 g-1 fish, respectively) of the control fish (N=5) immersed in water.
Ammonia concentrations in water samples collected from branchial or cutaneous surfaces of fish after 15 min or 24 h of emersion
The ammonia concentration in water samples collected from the branchial
surface of the air-facing side of the fish after 24 h of emersion
(N=5) was not significantly different from those of the control
exposed to terrestrial conditions for 15 min during anaesthesia (N=5;
Fig. 2). However, for the
water-facing side of the experimental fish, the ammonia concentration in the
branchial water increased to 21.5±2.4 mmol l-1, which was
significantly greater than that of the control fish
(Fig. 2). In water samples
collected from the air-facing cutaneous surface of the experimental fish, the
ammonia concentration varied greatly and was not significantly different from
the control value (Fig. 2). By
contrast, the ammonia concentration (20.8±3.5 mmol l-1) in
water samples collected from the waterfacing cutaneous surface was
significantly greater than that of the control. The concentration of ammonia
in the thin film of water at the bottom (80 ml) was 5.32±0.87 mmol
l-1.
Effects of environmental ammonia (12 mmol l-1) on rates of ammonia and urea excretion
The ammonia excretion rate of A. testudineus (N=7) in
normal freshwater without NH4Cl was 7.12±1.04 µmol
day-1 g-1 fish. At the beginning of the experiment, the
ambient ammonia concentration without any fish was 12.3 mmol l-1.
With an initial pH of 7.0, the NH4+ and NH3
concentrations were calculated to be 12.19 and 0.11 mmol l-1,
respectively. In comparison, the concentrations of NH4+
and NH3 in the plasma were 0.188 and 0.012 mmol l-1,
respectively, taking the plasma ammonia concentration to be 0.2 mmol
l-1 (from Table 1)
and the blood pH to be 7.6 (Y.K.I., unpublished results). So, both the
NH4+ and NH3 gradients were directed inwards;
but, surprisingly, the experimental fish could excrete ammonia against such a
large ammonia gradient. As a result, the concentration of ammonia in the
external medium increased to 13.1±0.2 mmol l-1 at the end of
day 1. A simple calculation reveals that the ammonia excretion rate decreased
significantly to 4.31±0.81 µmol day-1 g-1 fish
(N=7) during this 24 h period. On day 2, the ambient ammonia
concentration increased further to 14.7±0.3 mmol l-1, and
the ammonia excretion rate (8.32±1.44 µmol day-1
g-1 fish; N=7) returned back to a level comparable with
the initial control value.
Effects of emersion on the contents of ammonia, urea and FAAs
Ammonia in the muscle and liver of A. testudineus (N=6)
increased significantly during emersion and peaked at 4.06 µmol
g-1 on day 2 (4.5-fold of the control value) and 10.7 µmol
g-1 on day 1 (5.5-fold of the control value), respectively
(Table 1). The ammonia
concentration in the plasma of A. testudineus increased significantly
during the first 2 days of emersion, but returned to the control level
thereafter (Table 1). Although
urea also increased significantly in the muscle and liver of A.
testudineus during emersion, the peak levels (0.79 µmol g-1
and 0.81 µmol g-1 on day 2, respectively) attained were much
lower than those of ammonia (Table
2).
Emersion led to significant increases in isoleucine, leucine, phenylalanine, tyrosine and valine in the muscle of A. testudineus (N=4) (Table 3). By contrast, the aspartate and glutamine content of muscle in fish emersed for 2 or 4 days were significantly lower than the corresponding control value. Although emersion had no significant effect on the muscle TFAA, there was a significant increase in the muscle TEFAA in fish exposed to 2 days of emersion. Two days of emersion led to a significant increase (8.8-fold) in the lysine content of the liver of A. testudineus (Table 4). However, 4 days of emersion resulted in significant increases in arginine and phenylalanine in the liver. In addition, there was a significant decrease in the glutamate in the liver of fish after 2 or 4 days of emersion. After 2 days of emersion, the brain glutamine content of A. testudineus increased significantly by 2.5-fold (Table 5). There were also significant decreases in alanine, aspartate and glutamate in the brain of fish during the first 2 days of emersion. However, the TFAA and TEFAA content in the liver and brain of A. testudineus were unaffected by emersion.
Effects of 10 min of forced exercise on land
Forced exercise for 10 min on land led to significant increases in
excretion of ammonia (0.103±0.009 µmol 10 min-1
g-1 fish) and urea (0.015±0.002 µmol 10 min-1
g-1 fish) as compared with the control on land but without exercise
(0.047±0.014 µmol g-1 fish, and 0.003±0.001
µmol g-1 fish, respectively). As a result, the concentration of
ammonia in the thin film of water in the container with the exercised fish
reached 1.79±0.05 mmol l-1 which was significantly higher
than that of the control (0.86±0.20 mmol l-1). In addition,
there were significant increases in ammonia, alanine, lysine and TEFAA in the
muscle of fish after forced exercise on land as compared with the control fish
exposed to terrestrial conditions for 10 min without disturbance
(Table 6). However, forced
exercise on land did not have any significant effect on ATP, ADP, AMP,
glucose, glycogen, lactate and succinate in the muscle of A.
testudineus (Table 6).
Rate of O2 consumption
For the control fish in freshwater (N=4), the O2
consumption rates in water and air were 1.38±0.13 and 3.10± 0.54
µmol h-1 g-1 fish, respectively. Taken together, the
total O2 consumption rate was 4.47±0.48 µmol
h-1 g-1 fish. For fish exposed to terrestrial conditions
(N=4 for each group) for 1, 24 or 48 h, the O2 consumption
rates in air were 5.25±0.61, 4.67±0.22, and 6.43±0.53
µmol h-1 g-1 fish, respectively, and the value
obtained from those exposed to terrestrial conditions for 48 h of emersion was
significantly greater than that of the immersed control.
Construction of a balance sheet of ammonia and urea-N excretion and ammonia and urea-N retention in a 50 g fish
A 50 g fish contained approximately 30 g of muscle and 1 g of liver. Based
on our results, a balance sheet was constructed for changes in nitrogen
excretion and changes in ammonia-N and urea-N content in a 50 g fish after 2
or 4 days of immersion or emersion (Table
7). Although there was no significant change in the overall
ammonia excretion rate during 4 days of emersion, we took into consideration
the small changes involved and presented them in
Table 7. After considering the
amount of ammonia-N and urea-N stored in the muscle and liver, it becomes
apparent that there was an increase in nitrogen production in A.
testudineus after 2 or 4 days of emersion.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) adopted in this study. Thus, our results contradict those of
Saha and Ratha (Saha and Ratha,
1989), and the discrepancy could be a result of differences in
methodology. Saha and Ratha assayed for CPS I activity in the liver extract of
A. testudineus using a colorimetric method that involved a coupled
enzyme system, the accuracy of which depends heavily on the purity of the
coupled enzymes obtained commercially. The radiometric assay adopted in this
study is less prone to interference and more sensitive than the colorimetric
method. Because of its high sensitivity, the radiometric assay is more
specific, being able to differentiate CPS II and CPS III in the presence of
UTP, and is therefore more reliable. Incidentally, using the same radiometric
assay, Ip et al. (Ip et al.,
2004d) were unable to detect CPS I or CPS III in the livers of
C. gariepinus and C. batrachus, although their presence has
been reported previously in the latter species based on a colorimetric method
(Saha and Ratha, 1989;
Saha et al., 1999). Similarly,
activities of argininosuccinate synthetase and argininosuccinate lyase
reported by Saha and Ratha (Saha and
Ratha, 1989) for A. testudineus (0.32 µmol
h-1 g-1 tissue and 0.21 µmol h-1
g-1 tissue, respectively) using two separate colorimetric methods
were much higher than the combined activity of argininosuccinate synthetase +
argininosuccinate lyase (0.006 µmol h-1 g-1 tissue)
reported herein, based on a more sensitive and specific radiometric assay. Unlike other ureogenic fishes which have CPS III in the liver, the hepatic glutamine synthetase activity of A. testudineus was very low. Thus, together with the absence of detectable CPS I or III activities (detection limit=0.001 µmol min-1 g-1 tissue), it can be concluded that A. testudineus is non-ureogenic and hence highly unlikely to be ureotelic. Indeed, A. testudineus is ammonotelic in water, excreting 94% of its nitrogenous wastes (ammonia-N + urea-N) as ammonia-N.
Contrary to the belief that there is a tendency towards predominance of
ureotelism in amphibious air-breathing teleosts
(Walsh, 1997;
Saha and Ratha, 1998;
Wright and Land, 1998;
Sayer, 2005), most adult
tropical amphibious teleosts studied so far are predominantly ammonotelic in
water (Graham, 1997;
Ip et al., 2004a). More
importantly, they do not detoxify ammonia through enhanced ureogensis when
exposed to terrestrial conditions (for reviews, see
Ip et al., 2001b;
Ip et al., 2004a;
Ip et al., 2004b;
Chew et al., 2006). In a
recent review on tropical fishes (Sayer,
2005), results on increased urea excretion in a few species of
mudskippers upon return to water after a period of emersion were cited as
support for a physiological role of urea in ammonia detoxification in these
fishes during emersion. However, the fact remains that these mudskippers are
nonureogenic and they do not possess a full complement of OUC enzymes in their
livers (Gregory, 1977;
Lim et al., 2001). Although a
complete OUC with very low CPS activity is present in the liver of the giant
mudskipper, Periophthalmodon schlosseri
(Lim et al., 2001), increased
urea synthesis has a very minor role in ammonia detoxification in fish exposed
to terrestrial conditions (Ip et al.,
1993; Ip et al.,
2001c), alkaline pH (Chew et
al., 2003a) or high concentrations of environmental ammonia
(Peng et al., 1998;
Randall et al., 1999). In
another recent review (Eddy,
2005), it was reiterated that urea was produced by the African
catfish C. gariepinus (as C. mossambicus) and the Indian
air-sac catfish H. fossilis during air exposure. However, C.
gariepinus is non-ureogenic and does not possess a functional OUC
(Ip et al., 2004d); it does
not accumulate urea during emersion either
(Ip et al., 2005). As for
H. fossilis, it is debatable if increased urea synthesis plays a
major role in ammonia detoxification during emersion based on results reported
in the literature (for a review, see Chew
et al., 2006). More importantly, we were unable to detect CPS I or
III activity from the liver of H. fossilis using the same radiometric
assay as in this study (Y.K.I. and S.F.C., unpublished results).
Likewise, contrary to a previous proposition
(Saha and Ratha, 1989), our
results confirm that increased urea synthesis and excretion was not adopted by
A. testudineus to detoxify ammonia during 4 days of emersion. In
fact, the urea-N excreted by A. testudineus during emersion accounted
for <4% of the total waste-N (ammonia-N + urea-N), and there was no
significant increase in urea excretion when the fish was reimmersed in water
on day 5. In addition, there were only minor increases in urea content in
tissues of A. testudineus after 4 days of emersion despite large
increases in tissue ammonia. Because a functional OUC is absent from the liver
of A. testudineus, it is logical to deduce that the urea accumulated
in the tissues of A. testudineus during emersion originated from
argininolysis and/or purine catabolism followed with uricolysis.
Emersion does not impede ammonia excretion in A. testudineus - a novel observation
In water, fishes excrete ammonia as NH3 though their gills
(Wilkie, 2002;
Evans et al., 2005). Because
no water current is available to flush the excreted ammonia away from the
gills, the partial pressure of NH3 (PNH3)
increases quickly in the boundary layer, leading to a reduction in the blood
to boundary water 
PNH3 during emersion. In
addition, most fish gills collapse in air, and so branchial ammonia excretion
via diffusion of NH3 is repressed. Indeed, for the
tropical air-breathing teleosts Oxyeleotris marmoratus
(Jow et al., 1999),
Boleophthalmus boddarti (Lim et
al., 2001), P. schlosseri
(Lim et al., 2001;
Ip et al., 1993;
Ip et al., 2001c),
Misgurnus anguillicaudatus (Chew
et al., 2001); Bostrichyths sinensis
(Ip et al., 2001a), Channa
asiatica (Chew et al.,
2003a), Monopterus albus
(Tay et al., 2003) and C.
gariepinus (Ip et al.,
2005) exposed to terrestrial conditions, ammonia excretion rates
decreased to 10-25% of that of the corresponding immersed control fish.
Therefore, the unique observation made in this study that A.
testudineus was able to increase ammonia excretion on day 2 and sustain
the normal (immersed) daily rate of ammonia excretion on days 1, 3 and 4 of
emersion is unexpected. It would imply that A. testudineus was able
to effectively excrete ammonia despite a lack of water to irrigate its
branchial epithelial surfaces and a 50% reduction in the cutaneous surface
being in direct contact with the external medium.
Results obtained in a separate experiment in which daily change of water was omitted during a 2-day period indicate that A. testudineus was able to continuously excrete ammonia into the small volume of external medium and increase the ammonia concentration therein to 13.2 mmol l-1. Therefore, it can be deduced that A. testudineus was able to excrete ammonia against a concentration gradient during emersion.
High ammonia concentrations in water samples collected from brachial or cutaneous surfaces of fish exposed to terrestrial conditions
Although the contribution of the kidney to ammonia excretion is less than
that of the gills in water-breathing fishes, it might have an important role
in ammonia excretion for amphibious fishes. However, during emersion, water
conservation is an important issue in A. testudineus and consequently
urine flow rate could not be high. Therefore, it is unlikely that increased
ammonia excretion occurred through the kidney in fish exposed to terrestrial
conditions.
Because the ammonia concentration in the water collected from the branchial
surface of the water-facing side of the fish after 24 h of emersion increased
to 21.5±2.4 mmol l-1, it can be concluded that A.
testudineus was able to actively excrete ammonia against a concentration
gradient across its gills. However, the ammonia concentration in the branchial
water collected from the air-facing side remained relatively unchanged
compared with the control. It is probable that water in the buccal cavity was
drained naturally from the air-facing side towards the water-facing side as
the fish lay flat in the bottom of the container, facilitating the continual
excretion of ammonia through the gills over that side of the body. Thus, it
can be deduced that the presence of water in the buccal cavity is a
prerequisite to active branchial ammonia transport. By contrast, the giant
mudskipper, P. schlosseri, has a torpedoshaped body and maintains an
upright posture on land. Because of this, water would be drained away from the
gills towards the ventral side of the buccal cavity; therefore, it had to
develop gills with intrafilamentous interlamellar fusions
(Low et al., 1988) to trap
water in order to facilitate the continual and active excretion of ammonia
during emersion. Unlike P. schlosseri, the skin of A.
testudineus might also be involved in active ammonia excretion because
the ammonia concentration (20.8 mmol l-1) in the water samples
collected from the waterfacing cutaneous surface was greater than that (5.32
mmol l-1) in the small volume of ambient water.
A. testudineus can excrete ammonia against 12 mmol-1 NH4Cl
Indeed, A. testudineus was able to excrete ammonia continuously
when immersed in water containing 12 mmol l-1 NH4Cl,
although there was an initial decrease in the rate of ammonia excretion on day
1. Hence, A. testudineus is one of a few fish species that are
capable of excreting ammonia against a steep ammonia gradient; the others are
the giant mudskipper P. schlosseri
(Randall et al., 1999;
Ip et al., 2004c) and the
African catfish C. gariepinus (Ip
et al., 2004d). P. schlosseri, which inhabits a brackish
environment, is able to actively excrete NH4+
via a basolateral Na+, K+-ATPase and an apical
Na+/H+ exchanger of the branchial epithelium
(Randall et al., 1999;
Wilson et al., 2000). However,
Na+/NH4+ exchange may not occur across the
gills of A. testudineus because apical electroneutral
Na+/H+ exchange is likely to be absent from gills of
freshwater fishes (Wilkie,
2002). Therefore, some novel mechanisms may be involved in active
ammonia excretion in A. testudineus during emersion or ammonia
exposure. Taken together, our results confirm that the ability to excrete
ammonia against an ammonia gradient facilitates the survival of A.
testudineus during emersion because ammonia can continue to be excreted
into an extremely small volume of external medium without being impeded.
Emersion also leads to accumulation of ammonia in tissues of A. testudineus
The steady state content of ammonia in the tissue is maintained by a
balance between ammonia production and ammonia excretion/detoxification.
Theoretically, the ammonia content in the experimental fish should remain
relatively unchanged after 4 days of emersion, because they were able to
excrete ammonia on land at a rate comparable to that of fish immersed in
water. Surprisingly, results obtained proved otherwise, and emersion led to
significant increases in ammonia content in the muscle, liver and brain. Thus,
the only logical explanation is that an increase in ammonia production had
occurred. Indeed, a balance sheet (Table
7) on changes in ammonia and urea-N excretion, and in the content
of ammonia, urea and FAAs in various tissues revealed that an increase in
ammonia production could have occurred in A. testudineus during 4
days of emersion. This is the first report of such a phenomenon because
previous studies in various air-breathing fishes (B. boddarti, B.
sinensis, C. asiatica, C. gariepinus, M. albus, M. anguillicaudatusm,
O. marmoratus, P. schlosseri and the slender African lungfish
Protopterus dolloi) revealed that the summation of debits in
nitrogenous excretion and credits in nitrogenous accumulation in fish exposed
to terrestrial conditions would lead to negative values, indicating the
occurrence of a decrease in ammonia production to ameliorate ammonia toxicity
(Jow et al., 1999;
Lim et al., 2001;
Tay et al., 2003;
Chew et al., 2001;
Chew et al., 2003a;
Chew et al., 2003b;
Ip et al., 2001a;
Ip et al., 2005).
Since ammonia is produced mainly through amino acid catabolism, it can be
deduced that amino acid catabolism increased in A. testudineus during
emersion and the resulting carboxylic acid was fuelled into the tricarboxylic
acid cycle for energy production. Glutamate holds an important position in
amino acid metabolism because many amino acids can be channelled into
glutamate for deamination through the reaction catalyzed by glutamate
dehydrogenase. Glutamate could be catabolized to ammonia (or alanine through
transamination) and 
-ketoglutarate, a tricarboxylic acid cycle
intermediate for energy production. Support for increased amino acid
catabolism can be inferred from the decrease in glutamate content, which
indicates that glutamate degradation exceeded its synthesis, in both the
muscle and liver of fish during 4 days of emersion.
The brain is often the organ undergoing the largest increase in glutamine
concentration in fish exposed to ammonia because it is the most vulnerable to
ammonia toxicity (Mommsen and Walsh,
1992). The significant increase in the glutamine content in the
brain of fish after 2 days of emersion is in agreement with the conclusion
that an increase in ammonia production had occurred in extracranial tissues,
and the brain was transiently confronted with ammonia toxicity. Glutamate is
not only a substrate for glutamine synthesis catalyzed by glutamine
synthetase, but also acts as a substrate for the formation of alanine or
aspartate through transamination reactions. The decreases in alanine and
aspartate content in the brain of A. testudineus exposed to
terrestrial conditions for 2 days suggested that glutamate was selectively
channelled into glutamine synthesis, detoxifying ammonia derived from the
blood to protect the brain cells.
Accumulation of certain FAAs (essential and non-essential) in the muscles during emersion and its implication
In this study, food was withdrawn before and during the experiment.
Therefore, the rate of protein degradation should be higher than the rate of
protein synthesis, leading to a net proteolysis. Because there was an apparent
increase in amino acid catabolism, it can be predicted that decreases in
tissue FAA, and consequently TFAA, would theoretically occur if the rate of
proteolysis remained unaffected during the emersion period. However, there
were significant increases in several essential amino acids (isoleucine,
leucine, lysine, valine and phenylalanine) and TEFAA in the muscle of A.
testudineus after 2 days of emersion as compared with the immersed
control. Thus, it can be confirmed that an increase in proteolysis had indeed
occurred. Furthermore, the decreases in aspartate, glutamine and glutamate in
the muscle suggested that certain amino acids were preferentially catabolized
during emersion. The significant increase in muscle alanine content indicate
that there might also be an increase in partial amino acid catabolism, which
released carboxylic acids for energy production without producing ammonia
(Ip et al., 2001c;
Chew et al., 2003a). Hence, it
can be concluded that emersion led to simultaneous increases in rates of
proteolysis and amino acid catabolism in A. testudineus.
Most tropical air-breathing teleosts studied so far, with the exception of
the small snakehead C. asiatica
(Chew et al., 2003a), reduce
the rate of amino acid catabolism/proteolysis to slow down the accumulation of
endogenous ammonia during emersion (for reviews, see
Ip et al., 2001b;
Ip et al., 2004a;
Ip et al., 2004b;
Chew et al., 2006). However,
such an adaptation naturally prevents the use of amino acids as energy sources
when the fish is out of water. A. testudineus is unique with respect
to nitrogen metabolism and excretion on land, because there was an apparent
increase in ammonia production during 4 days of emersion
(Table 7). This could be
related to the fact that the fish frequently struggled with jerky movements
under such conditions.
Effects of air-exposure on the rate of oxygen consumption
Carbon chains released after the deamination of amino acids are usually
shuttled into the tricarboxylic acid cycle, which produces NADH in the
mitochondria. The balance of mitochondrial redox involves the electron
transport chain which utilizes oxygen as the terminal electron acceptor.
Therefore, it is highly unlikely that A. testudineus would reduce
O2 consumption or undergo anaerobic energy metabolism during
emersion. Many amphibious air-breathing fishes can sustain aerial
O2 consumption rate at or near the same level or higher than in
water (Graham, 1997). However,
it had been reported previously that A. testudineus had a reduced
O2 consumption rate in air (by 10-24%)
(Hughes and Singh, 1970;
Natarajan, 1978), in spite of it being a triphasic breather with a
demonstrated amphibious behaviour (Graham,
1997). By contrast, the non-amphibious anabantoid,
Trichogaster trichopterus, is known to have the same O2
consumption rate in air as in water
(Burggren and Haswell,
1979).
Contrary to previous reports, our results, while comparable with those in
the literature (Hughes and Singh,
1970; Natarajan, 1978), reveal for the first time that the
O2 consumption rate of A. testudineus exposed to
terrestrial conditions for 2 days was significantly higher than that of the
control. What leads to this discrepancy is uncertain at present, but based on
our result, it can be concluded that the ABOs of A. testudineus
functioned effectively to absorb O2 from air and this could have
facilitated A. testudineus to utilize amino acids as sources of
energy for locomotor activities during emersion.
Effects of forced exercise on land
Lipid is the dominant fuel (35-68%) in resting non-fed fish; carbohydrate
is the second most important fuel, and its contribution increases to a much
greater extent during starvation (Lauff
and Wood, 1996a; Lauff and
Wood, 1996b). In comparison, the contribution of protein oxidation
(14-30%) to the overall metabolic rate is low (for a review, see
Wood 2001). When fish are made
to swim at sustainable and submaximal velocities, the contribution of protein
oxidation to overall fuel use stays the same or decreases with increasing
velocity. Many tropical air-breathing fishes can actively move on land; and,
out of these, the Boddart goggled-eye mudskipper, B. boddarti
(Ip et al., 2001c), and the
small snakehead, C. asiatica
(Chew et al., 2003c), utilize
glycogen to support bursts of locomotor activities during emersion. However,
the giant mudskipper, P. schlosseri, which can actively excrete
NH4+, utilizes amino acids to fuel locomotor activities
on land (Ip et al.,
2001c).
Similar to P. schlosseri, 10 min of forced exercise on land did not result in decreases in glycogen and glucose in the muscle of A. testudineus. In addition, there were no significant changes in lactate or succinate content in muscle. Taken together, these results indicate that carbohydrate was not the major fuel for locomotor activity in A. testudineus exposed to terrestrial conditions. Because 10 min of exercise resulted in no changes in ATP, ADP and AMP in the muscle, other energy stores must have been utilized. Forced exercise for 10 min on land led to both increased ammonia excretion and increased ammonia accumulation in A. testudineus. The excess ammonia produced during exercise was unlikely to be released from AMP deamination because there was no change in the muscle total adenylate content. Therefore, it can be concluded that the excess ammonia was released through increased amino acid catabolism in the exercised fish. In addition, there could be an increase in partial amino acid catabolism because there was a significant increase in the alanine content of the muscle. The simultaneous increases in lysine and TEFAA in the muscle confirms that there was an increase in proteolytic rate during the 10 min of exercise on land.
Proteins and amino acids are major fuels in anorexic salmon during the
spawning migration (Mommsen et al.,
1980), although lipid is the first substrate to be used
(Wood, 2001). At present,
whether lipid acts as a source of energy during exercise in amphibious fishes
is uncertain, but using amino acids to fuel activity on land is not a common
phenomenon among amphibious fishes (Ip et
al., 2001c; Chew et al.,
2003c). This is because provisions must be made to ameliorate
ammonia toxicity during exercise. Results obtained from A.
testudineus and P. schlosseri
(Ip et al., 2001c) suggest
that active ammonia excretion is an important prerequisite to the use of amino
acid to support locomotor activities on land.
Why is A. testudineus so unique? Its body shape, air-breathing capacity and ability to migrate on land during drought
Because of its laterally compressed body, A. testudineus usually
lies on one side during emersion. Very often, it flips the body and the tail
fin to produce jerky movements which constitutes, in effect, vaulting actions
supported by the spiny edges of the gill plates
(Davenport and Abdul Martin,
1990). Through such vaulting actions, fuelled by amino acid
catabolism, it can `migrate' long distances on land. Using amino acids as
substrates for energy metabolism on land implies that it must respire in air
effectively to remain aerobic. Indeed, like many other air-breathing fishes,
A. testudineus has labyrinth organs (ABOs) which facilitate
air-breathing during immersion or emersion. In general, amphibious fishes
(e.g. catfishes, mudskippers, sleepers, snakeheads and swamp eels) develop
torpedo-shaped bodies to maintain an upright posture on land, but A.
testudineus possesses a laterally compressed body. Why would A.
testudineus retain such a body shape during evolution? Our results
suggest at least one advantage in doing so. Increased amino acid catabolism
naturally leads to increased ammonia production, and excess ammonia must be
excreted even in the absence of water to flush the gills. Therefore, it is
imperative for A. testudineus to possess active ammonia transport
mechanisms, which are apparently present in the branchial and cutaneous
surfaces. Lying on one side of the body during emersion renders half of the
cutaneous surface to be in direct contact with the external medium and water
in the buccal cavity to drain towards the gills over that side. This would
facilitate the continual and active excretion of ammonia through 50% of the
total branchial and cutaneous surfaces. Although both A. testudineus
and P. schlosseri can excrete ammonia actively on land, they
apparently took different evolutionary paths with respect to branchial
development. By developing intrafilamentous interlamellar fusions to
facilitate active ammonia excretion during emersion, the gills of P.
schlosseri can no longer function effectively as a respiratory and/or
osmoregulatory organ in water. By contrast, by maintaining a laterally
compressed body, A. testudineus is able to achieve the same feat as
P. schlosseri, but with unspecialized gills that may function
effectively for osmoregulation and/or acid-base balance during immersion.
Since A. testudineus is known to inhabit brackish water, efforts
should be made in the future to determine the effects of increased salinity on
its branchial functions and air-breathing capacity.
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References |
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Anderson, P. M. (2001). Urea and glutamine synthesis: environmental influences on nitrogen excretion. In Fish Physiology: Nitrogen Excretion. Vol. 20 (ed. P. A. Wright and P. M. Anderson), pp. 239-277. New York: Academic Press.[CrossRef]
Anderson, P. M. and Little, R. M. (1986). Kinetic properties of cyanase. Biochemistry 25,1621 -1626.[CrossRef][Medline]
Anderson, P. M. and Walsh, P. J. (1995). Subcellular localization and biochemical properties of the enzyme carbamoyl phosphate synthetase and urea synthesis in the batrachoidid fishes, Opsanus beta, Opsanus tau and Porichthys notatus. J. Exp. Biol. 198,755 -766.
Bergmeyer, H. U. and Beutler, H. O. (1985). Ammonia. In Methods of Enzymatic Analysis. Vol.VIII (ed. H. U. Bergmeyer, J. Bergmeyer and M. Graßl), pp. 454-461. Weinheim: Verlar Chemie.
Beutler, H. O. (1985). Succinate. In Methods of Enzymatic Analysis. Vol.VIII (ed. H. U. Bergmeyer, J. Bergmeyer and M. Graßl), pp. 25-33. Weinheim: Verlar Chemie.
Burggren, W. and Haswell, S. (1979). Aerial
CO2 excertion in the obligate air breathing fish, Trichogaster
trichopterus, a role for carbonic anhydrase. J. Exp.
Biol. 82,215
-225.
Cao, X. Y., Kemp, J. R. and Anderson, P. M. (1991). Subcellular localization of two glutamine-dependent carbamoyl-phosphate synthetases and related enzymes in liver of Micropterus salmoides (largemouth bass) and properties of isolated liver mitochondria: comparative relationships with elasmobranchs. J. Exp. Zool. 258,24 -33.[CrossRef]
Chew, S. F., Jin, Y. and Ip, Y. K. (2001). The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure. Physiol. Biochem. Zool. 74,226 -237.[CrossRef][Medline]
Chew, S. F., Hong, L. N., Wilson, J. M., Randall, D. J. and Ip, Y. K. (2003a). Alkaline environmental pH has no effect on the excretion of ammonia in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti. Physiol. Biochem. Zool. 76,204 -214.[CrossRef][Medline]
Chew, S. F., Ong, T. F., Ho, L., Tam, W. L., Loong, A. M.,
Hiong, K. C., Wong, W. P. and Ip, Y. K. (2003b). Urea
synthesis in the African lungfish Protopterus dolloi - hepatic
carbamoyl phosphate synthetase III and glutamine synthetase can be
up-regulated by 6 days of aerial exposure. J. Exp.
Biol. 206,3615
-3624.
Chew, S. F., Wong, M. Y., Tam, W. L. and Ip, Y. K.
(2003c). The snakehead Channa asiatica accumulates
alanine during aerial exposure, but is incapable of sustaining locomotory
activities on land through partial amino acid catabolism. J. Exp.
Biol. 206,693
-704.
Chew, S. F., Wilson, J. M., Ip, Y. K. and Randall, D. J. (2006). Nitrogen excretion and defence against ammonia toxicity. In Fish Physiology: The Physiology of Tropical Fishes. Vol. 21 (ed. A. Val, V. Almedia-Val and D. J. Randall), pp. 307-395. New York: Academic Press.
Das, B. K. (1927). The bionomics of certain air-breating fishes of India, together with an account of the development of their air-breathing organs. Philos. Trans. R. Soc. Lond. 216,183 -219.
Davenport,
Significantly different from the corresponding
day 1, terrestrial condition.