The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays
1 Department of Biological Science, National University of Singapore, Kent
Ridge, Singapore 117543, Republic of Singapore
2 Natural Sciences, National Institute of Education, Nanyang Technological
University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
3 Department of Zoology, University of Guelph, Guelph, Ontario, Canada, NIG
2W1
* Author for correspondence (e-mail: dbsipyk{at}nus.edu.sg)
Accepted 19 May 2003
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Summary |
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Key words: ammonia, amino acid, elasmobranch, freshwater stingray, Himantura signifer, ornithine-urea cycle, osmoregulation, Potamotrygon motoro, stingray, Taeniura lymma, urea, urea transporter
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Introduction |
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In tropical waters in Southeast Asia (Thailand, Indonesia and Papua New
Guinea) and South America (Amazon River basin), a number of elasmobranch
species occur in low salinity waters. For the Amazon freshwater stingrays, a
reduction in tissue urea concentration occurs as the result of reduced
synthesis (Forster and Goldstein,
1976) and/or a higher renal clearance rate
(Goldstein and Forster, 1971
).
Potamotrygon spp. is a stenohaline Amazonian stingray permanently
adapted to freshwater. Although it has low levels of some of the enzymes
related to urea synthesis (Anderson,
1980
), it retains virtually no urea or trimethylamine oxide (TMAO)
in situ, cannot accumulate urea in laboratory salinity stress
(Thorson et al., 1967
;
Gerst and Thorson, 1977
) and,
like other teleosts, is primarily ammonotelic
(Goldstein and Forster, 1971
;
Barcellos et al., 1997
). In
comparison, there is a dearth of knowledge about the freshwater elasmobranchs
in Southeast Asia.
The river Batang Hari originates from the Barisan Range, flows eastwards
through the whole of Jambi, Indonesia and drains into the South China Sea. The
white-edge freshwater whip ray Himantura signifier
(Compagno and Roberts, 1982;
Family: Dasyatidae) is a stingray found in the Batang Hari basin in Jambi,
Sumatra. It is believed to occur only in freshwater. In the laboratory, H.
signifier can survive in freshwater (0.7
) indefinitely or in
brackish water (20
) for at least two weeks. Unlike
Potamotrygon spp., which have been isolated from the sea for millions
of years, H. signifer can travel freely along the river and may
encounter brackish water during certain periods of the year. Hence, H.
signifer represents an ideal species to study the effects of salinity
changes on the capacity for urea synthesis and retention in a primarily
freshwater elasmobranch with euryhaline capability.
This study was undertaken to test the hypothesis that H. signifer
has retained the capability to synthesize urea de novo but has a
lower capability to retain urea compared with its marine counterparts.
Specimens of H. signifer were exposed progressively from 0.7
water to 20
water through an 8-day period. The rates of ammonia and
urea excretion and the concentrations of ammonia, urea and free amino acids
(FAAs) in various tissues and organs were measured. At the same time,
activities of various enzymes involved in the OUC were determined. For
comparison, the rate of urea excretion and the activities of OUC enzymes in
the liver of the marine blue-spotted fantail ray Taeniura lymma,
which is found in Indonesian waters, were determined. Efforts were also made
to study the contents of ammonia and urea in various tissues of
Potamotrygon motoro, a stenohaline freshwater stingray from the
Amazon Basin River for direct comparison. P. motoro was exposed
progressively from 0.7
water to only 13
water, beyond which it
would not survive.
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Materials and methods |
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Exposure of specimens to experimental conditions and collection of
water samples and tissues
Specimens were submerged individually in plastic aquaria tanks (60 cm
length x 30 cm width x 20 cm height) containing 10 volumes (w/v)
of aerated water at 25°C. For H. signifer, control specimens were
exposed to freshwater at pH 7 for 8 days. Experimental specimens were exposed
to daily increases in salinity from 0.7 (day 1) to 5
(day 2)
to 10
(day 3) to 15
(day 4) to 20
(day 5), remaining
at 20
on days 6-8. For P. motoro, control specimens were
exposed to freshwater for 4 days. The experimental specimens were exposed to
daily salinity changes from 0.7
(day 1) to 4
(day 2) to
7
(day 3) to 13
(day 4). Gradual ascent in salinity was
necessary to allow for acclimatization and survival. The salinity was capped
at 20
and 13
for H. signifer and P. motoro,
respectively, because a preliminary study revealed that, beyond these
salinities, mortality could be high. Water samples (3 ml) were collected
daily, acidified with 70 µl of 1 mol l-1 HCl and kept at 4°C
until analysed. Concentrations of ammonia and urea were determined according
to the methods of Bergmeyer and Beutler
(1985
) and Felskie et al.
(1998
), respectively. The
rates of ammonia and urea excretion were expressed as µmol day-1
g-1 fish.
Specimens of H. signifer were killed on day 8 and those of P. motoro on day 4 for tissue collection. The caudal peduncle of the experimental specimen was severed, and blood exuding from the caudal vessels was collected in heparinized capillary tubes. The blood sample was centrifuged at 4000 g at 4°C for 10 min to obtain the plasma. A portion of the plasma was used for analyses of osmolality and concentrations of Na+ and Cl-. Another portion was deproteinized in 2 volumes (v/v) of ice-cold 6% HClO4 and centrifuged at 10 000 g at 4°C for 15 min. The resulting supernatant fluid was kept at -80°C until analysed. The brain, liver, stomach, intestine and muscle were quickly excised, with the stomach and intestine flushed with ice-cold saline solution (0.9% NaCl). The excised tissues and organs were immediately freeze-clamped with tongs pre-cooled in liquid nitrogen. Samples were kept at -80°C until analysed.
Analyses of plasma osmolality and concentrations of Na+
and Cl-
Plasma osmolality was analysed using a Wescor 5500 vapour pressure
osmometer. Na+ and Cl- concentrations were determined by
a Corning 410 flame photometer and Corning 925 chloride analyzer, respectively
(Corning Ltd, Halstead, Essex, UK.
Analysis of free amino acids (FAAs)
The frozen muscle and liver samples were weighed, ground to a powder in
liquid nitrogen and homogenized three times in 5 volumes (w/v) of 6%
trichloroacetic acid using an Ultra-Turrax homogenizer at 24 000 revs
min-1 for 20 s each with 10-s off intervals. The homogenate was
centrifuged at 10 000 g at 4°C for 15 min to obtain the
supernatant for FAA analyses. The plasma was deproteinized in 2 volumes (v/v)
of ice-cold 6% trichloroacetic acid and centrifuged at 10 000
g at 4°C for 15 min.
For the analysis of FAA, the supernatant fluid obtained was adjusted to pH 2.2 with 4 mol l-1 lithium hydroxide and diluted appropriately with 0.2 mol l-1 lithium citrate buffer (pH 2.2). FAAs were analyzed using a Shimadzu LC-6A amino acid analysis system (Kyoto, Japan) with a Shim-pack ISC-07/S1504 Li-type column. Results are expressed as µmol g-1 wet mass tissue or mmol l-1 plasma.
Analyses of ammonia and urea
Samples were homogenized as stated above except in 5 volumes of 6%
HClO4. After centrifugation at 10 000 g for 15 min,
the supernatant was decanted and the pH adjusted to 5.5-6.0 with 2 mol
l-1 KHCO3. The ammonia and urea concentrations were
determined as stated above. Results were expressed as µmol g-1
wet mass tissue or mmol l-1 plasma.
Determination of TMAO
TMAO was assayed by the iron sulphate method of Wekell and Barnett
(1991), which has been
extensively modified for the analysis of small samples by Raymond
(1998
). The difference in
absorbance obtained from the sample in the presence and absence of Fe-EDTA and
heat treatment (reduction step of TMAO to TMA) was used for the estimation of
TMAO in the sample. TMAO obtained from Sigma Chemical Co. (St Louis, MO, USA)
was used as a standard for comparison.
Determination of activities of OUC enzymes
Preliminary studies revealed that a full complement of OUC enzymes was
present in the liver and stomach of both H. signifer and T.
lymma. However, no carbamoylphosphate synthetase III (CPS III) activities
were detectable in the muscle, intestine or brain. Subsequently, the liver and
the stomach were minced and suspended in 10 volumes (w/v) of ice-cold
extraction buffer (285 mmol l-1 sucrose, 3 mmol l-1 EDTA
and 3 mmol l-1 Tris-HCl, pH 7.2), homogenized using an Ultra-Turrax
homogenizer at 24 000 revs min-1 and sonicated three times for 20 s
with a 10 s break between each sonication. The homogenate was centrifuged at
10 000 g at 4°C for 15 min to obtain the supernatant,
which was subsequently passed through a 10 ml Bio-Rad P-6DG column (Bio-Rad
Laboratories, Hercules, CA, USA) equilibrated with cold suspension buffer. The
collected filtrates were used for the subsequent enzyme analyses.
CPS III (E.C. 2.7.2.5) activity was determined as described by Anderson and
Walsh (1995). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter (Wallac Oy, Turku,
Finland). The CPS activity was expressed as µmol [14C]urea
formed min-1 g-1 wet mass.
Ornithine transcarbamoylase (OTC; E.C. 2.1.3.3) activity was determined by
combining the methods of Anderson and Walsh
(1995) and Xiong and Anderson
(1989
). Absorbance was
measured at 466 nm using a Shimadzu 160 UV VIS recording spectrophotometer.
The OTC activity was expressed as µmol citrulline formed min-1
g-1 wet mass.
Argininosuccinate synthetase (E.C. 6.3.4.5) and lyase (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.
(1991). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter. Argininosuccinate
synthetase and lyase 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 by Felskie et al.
(1998). Urea was determined as
described above. Arginase activity was expressed as µmol urea formed
min-1 g-1 wet mass.
Glutamine synthetase (GS; E.C. 6.3.1.2) activity was measured according to
the method of Shankar and Anderson
(1985). The formation of
-glutamylhydroxymate was determined at 500 nm using a Shimadzu 160 UV
VIS recording spectrophotometer. The GS activity was expressed as µmol
-glutamylhydroxymate formed min-1 g-1 wet
mass.
Statistical analyses
Results are presented as means ± S.E.M. Student's
t-test or analysis of variance (ANOVA) followed by multiple
comparisons of means by Duncan's procedure was used to evaluate differences
between means in groups where appropriate. Differences where
P<0.05 were regarded as statistically significant.
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Results |
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The ammonia level in the liver of H. signifer in 20 water
was half that of the control specimens, while plasma ammonia was almost 7-fold
higher (Table 2). Urea
concentration increased significantly in the muscle, brain and plasma but not
in the liver of these specimens (Table
2). Unlike H. signifer, there was no increase in blood
plasma osmolality or concentrations of Na+ or Cl- when
P. motoro was exposed to increasing salinity from 0.7
to
13
(Table 3). In
addition, there was a decrease in the ammonia level in the muscle of P.
motoro exposed to increasing salinity
(Table 4). Although urea levels
in the muscle, liver, brain and plasma increased significantly in P.
motoro exposed to 13
water, these values were very much lower
than those in the corresponding tissues of H. signifer
(Table 4).
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A full complement of OUC enzymes was detected in the liver of H.
signifer (Table 5). The
CPS activity could be enhanced by the addition of
N-acetyl-L-glutamate (AGA) but was refractory to uridine
triphosphate (UTP) inhibition (Table
5). The activities of GS, CPS, OTC and arginase were significantly
higher in the liver of H. signifer exposed to 20 water
compared with those in 0.7
and 10
, whereas the activities of
OUC enzymes in 10
water were intermediate between those in 0.7
and 20
water, except for arginosuccinate synthetase + lyase (ASS+L)
(Table 5). A full complement of
OUC enzymes was also found in the stomach of H. signifer
(Table 6). At higher
salinities, there was no increment in the enzymatic activities, with the
exception of CPS in specimens exposed to 20
water
(Table 6). The OUC enzymes were
absent in the muscle, brain and intestine and hence these data are not
presented.
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For the marine stingray T. lymma, a full complement of OUC enzymes was present in the liver (Table 7). The activities of all the enzymes were higher than that of H. signifer in freshwater. A full complement of OUC enzymes was found in the stomach of T. lymma as well (Table 7).
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In H. signifer exposed to 20 water, ß-alanine and
proline accumulated in the muscle (Table
8). The total FAA (TFAA) content was higher in the muscle of
specimens exposed to 20
water as compared with that of the freshwater
control (Table 8). Higher
levels of glutamine and proline were found in the liver
(Table 8), while
ß-alanine, glutamate, glutamine and glycine accumulated in the brain (Y.
K. Ip and S. F. Chew, unpublished results). No major changes were observed in
concentrations of FAAs in the plasma of the experimental or the control fish
(Table 8). No TMAO was detected
in the muscle or liver of H. signifier kept in freshwater or exposed
to 20
water using the method adopted in this study. For T.
lymma, 30.6±8.69 µmol g-1 and 1.54±0.34
µmol g-1 of TMAO were detected in the muscle and liver,
respectively, of specimens kept in seawater (30
).
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Discussion |
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In freshwater, H. signifer was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea (Fig. 2). Similar to marine elasmobranchs, it had a functional OUC with CPS III in the liver (Table 5). CPS III characteristically uses glutamine (preferential to NH4+) as a nitrogen donor, is activated allosterically by AGA, is present in mitochondria and is not inhibited by UTP. Although significantly lower than that of the marine T. lymma in seawater (0.48 µmol min-1 g-1; Table 7), the activity of CPS III in the liver of H. signifer in freshwater (0.13 µmol min-1 g-1; Table 5) was remarkably high. Hence, it can be concluded that the capacity (maximal activity) to synthesize urea had been downregulated in H. signifier in a freshwater environment. However, the remarkable capacity to synthesize urea in freshwater implies that H. signifer must excrete urea constantly in order not to create any osmotic problem.
In order to maintain the concentration of urea at a steady state under a certain environmental condition, the rate of urea excretion must be balanced with the rate of urea production. In freshwater, the rate of urea excretion, and hence the rate of urea synthesis, in H. signifier was 2.4 µmol day-1 g-1 (Fig. 2). In a 100 g specimen, there was approximately 50 g muscle, 3 g liver, 0.8 g brain and 2 ml plasma (Y. K. Ip and S. F. Chew, unpublished results). Therefore, for a 100 g fish, the rate of urea excretion was 2.4x100=240 µmol day-1 or 0.17 µmol min-1. Hence, to sustain the steady-state level of urea in the body of H. signifer in freshwater, the liver must be synthesizing urea at a rate of 0.057 µmol min-1 g-1 (liver). This is much lower than the maximal activity (based on CPS III activity) present in a 3 g liver (0.13 µmol min-1 g-1 x 3 g=0.39 µmol min-1; Table 5) of a 100 g fish. As for T. lymma, the urea excretion rate (or the rate of urea synthesis) in seawater was 4.24±1.34 µmol day-1 g-1. In a 100 g fish, this would translate into a urea synthesis rate of 0.098 µmol min-1 g-1 in the liver, which is approximately 2-fold higher than that in H. signifer in freshwater. Hence, besides having a lower maximal capacity of urea synthesis as determined by OUC enzyme activities in vitro, freshwater H. signifier also had a lower rate of urea synthesis in situ.
The above analysis assumes that the liver is the main site of urea
formation. Recently, it has been reported that urea synthesis can also take
place in the muscle of certain teleost fishes
(Anderson, 2001). Here, we
report for the first time the presence of a complete OUC in the stomach of
elasmobranchs, i.e. both H. signifer
(Table 6) and T. lymma
(Table 7), although the
activity of CPS III in the stomach of T. lymma was relatively low.
Going by the activities of CPS III in H. signifer, gram by gram, the
capacity of OUC in the stomach was approximately 70% that in the liver. It has
been reported previously that in mammalian species the only tissue besides
liver that has both CPS I and OTC activities is the intestinal mucosa
(Jones et al., 1961
;
Raijman, 1974
). However, ASS+L
is not present in the intestinal mucosa, and hence a complete OUC is lacking
(Meijer et al., 1990
). The use
of CPS III by most piscine systems requires that GS be intimately involved
with the OUC, co-localized to the mitochondria with CPS III
(Anderson and Casey, 1984
;
Walsh and Mommsen, 2001
).
Incidentally, the GS activity in the stomach of H. signifer was high,
reaching 124% that in the liver (Table
6). This observation is in agreement with a recent report that the
stomach and intestine of the four-eyed sleeper Bostrichthys sinensis
(teleost) exhibit high GS activity
(Anderson et al., 2002
).
Together, this information indicates that the piscine stomach is an important
organ involved in nitrogen metabolism, presumably after feeding rather than
simply as a digestive organ (see below). Elasmobranchs are carnivorous and
require high protein diets for urea synthesis; an OUC in the stomach would
represent the first line of `defence' against the release of ammonia through
protein and amino acid degradation along the digestive tract and to produce a
useful osmolyte, i.e. urea, upon feeding.
Urea synthesis is energy expensive; 5 moles of ATP is needed to synthesize
one mole of urea de novo in fish (except lungfishes, which possess
CPS I). Why would H. signifer synthesize urea and excrete 47% of its
nitrogenous wastes in this form instead of ammonia in freshwater? Unlike
potamotrygonid rays living in the Amazon River, H. signifer might
have invaded the freshwater environment only recently. It is unclear at this
moment if H. signifer would return to brackish water to reproduce,
like the bull shark Carcharhinus leucas of Lake Nicarrgua
(Thorson, 1976). Although
H. signifer can be found in Batang Hari as far as 400 km from the
South China Sea, there is still the possibility that it may re-enter estuarine
and marine environments. Therefore, it would be essential for H.
signifer to retain the ureosmotic osmoregulatory mechanisms to survive in
higher salinities.
Increase in the rate of urea synthesis during exposure to higher
salinities
In 20, the plasma osmolality in H. signifier increased
significantly from 416 mosmol kg-1 (in freshwater) to 571 mosmol
kg-1 (Table 1). The
increase in plasma Na+ and Cl- concentrations could only
account for 77% of the difference (155 mosmol kg-1) involved. The
rest was apparently made up with urea, the plasma concentration of which
increased by 38.8 mmol l-1. These results indicate that H.
signifier is ureosmotic, albeit with limited capacity in either urea
synthesis or urea retention or both (see below).
When H. signifer was exposed to a progressive increase in salinity
(0.7 to 5
to 10
to 15
to 20
) through
an 8-day period, there was a continuous decrease in the rate of ammonia
excretion (Fig. 1). By day 8,
i.e. 4 days after exposure to 20
water, the ammonia excretion rate was
only 20% that of the freshwater control. Yet, there was no change in the
ammonia contents in the muscle and brain, and the ammonia content in the liver
decreased instead (Table 2).
This suggests that ammonia was used as a substrate for the synthesis of urea,
which was essential for osmoregulation at higher salinities. Indeed, in
20
water, urea levels in the muscle, brain and plasma increased
significantly (Table 2).
In a 100 g specimen, the reduction in the ammonia excretion rate during the
8-day period can be calculated as [(5.5-5.39) + (5.77-4.32) + (5.27-3.84) +
(6.23-3.74) + (5.36-1.91) + (5.16-2.32) + (6.36-1.24) + (6.36-1.45)] x
100= 2252 µmoles (calculated from Fig.
1). The increase in ammonia-equivalents stored as urea in the
tissue at the end of day 8 in 20 water was
{[(107-70.9)x50]+[(91.6-59.3)x0.8]+
[(82.6-43.8)x2]}x2=3817 µmoles (calculated from
Table 2, without liver data
which showed no significant changes). Of this, it becomes obvious that 59%
could be accounted for simply by the reduction in ammonia excretion, with the
rest contributed by a reduction in urea excretion.
These results indirectly indicate that an increase in the rate of urea
synthesis in situ must have occurred in H. signifer during
the first 4 days of progressive exposure from freshwater to 20 water
because unexcreted ammonia was being converted to urea. An evaluation of the
activities of OUC enzymes at the end of day 8 (in 20
) supports this
proposition (Table 5). There
were significant increases in the total activities (units g-1
tissue) of CPS III, OTC, arginase and GS in specimens exposed to 20
water. In 10
water (day 3), specimens had an OUC capacity intermediate
between those of freshwater and 20
water. Hence, it can be concluded
that there was a gradual upregulation of activities of OUC enzymes in response
to increasing salinity.
In freshwater, the rate of urea excretion in a 100 g H. signifer
was 0.17 µmol min-1 (see above), which was one-third of the
total activities of CPS III in a 3 g liver (0.39 µmol min-1)
determined in vitro in the presence of saturating concentrations of
substrates and AGA. Assuming the amount of ammonia retained in the body on day
3 was all turned into urea, there must be an increase of
(5.27-3.84)x100/(24x60)= 0.10 µmol min-1 in the rate
of urea synthesis (Fig. 1),
bringing up the total urea synthesis rate to 0.27 µmol min-1.
This is actually well within the capacity of the CPS III present in the liver
(0.39 µmol min-1). Hence, theoretically it would be unnecessary
to induce CPS III activity through increasing the concentration of this
enzyme. Moreover, by the time the new steady-state concentration of urea was
reached in specimens exposed to 20 water on day 5, the rate of urea
synthesis returned back to the control value, which could be adequately
sustained by the OUC capacity in the liver of the specimen in freshwater.
Why then did the activities of CPS III in the liver increase 2.4-fold in
specimens exposed to 20 water
(Table 5)? This could be due to
the fact that CPS III activity depends on the presence of AGA, the
concentrations of which in the liver and stomach of H. signifer are
unknown at present. Results obtained with H. signifer indicate that
hepatic AGA might not reach the level required to derive maximal capacity of
CPS III during salinity adaptation, leading to the need to produce greater
concentrations of CPS III. Alternatively, CPS III may not function at
Vmax in vivo due to the lower than saturation
concentrations of glutamine or ATP. Exposure to higher salinity also led to an
increase in the activities of CPS III in the stomach of H. signifer
(Table 6), which supports the
above proposition that the stomach could be involved in producing urea for
osmoregulation in addition to its digestive function.
H. signifer has limited capacity to retain urea in brackish water
of high salinity
Working with the marine little skate Raja erinacea, Goldstein and
Forster (1971) reported that
the plasma urea concentration decreased from 390 mmol l-1 to 240
mmol l-1 when the skate was adapted progressively from 100% to 50%
seawater. By contrast, the urea concentration in the plasma of H.
signifier in 20
(67%) seawater was only 82.6 mmol l-1.
Despite the much lower concentration of urea in the plasma, the rate of urea
excretion in H. signifer (2.4±0.42 µmol day-1
g-1; Fig. 2) in
freshwater was obviously high, although not as high as that of the marine
T. lymma (4.24±0.80 µmol day-1 g-1),
which has a much higher plasma urea concentration (383±11.1 mmol
l-1), in seawater. This suggests that H. signifer reduced
its capability to retain urea, in addition to reducing its capability to
synthesize urea, in order to survive in a freshwater environment. There was a
significant decrease in the rate of urea excretion in H. signifer
during passage through 5
, 10
and 15
water
(Fig. 2). However, the rate of
urea excretion increased back to the control value (3.2 µmol
day-1 g-1) when the specimens reached 20
water
on day 5, presumably resulting from the steeper urea gradient built up between
the blood plasma (83 mmol l-1)
(Table 2) and the external
medium (0 mmol l-1).
The rate of urea loss from the body can be calculated according to the
formula R=[urea]plasmaxk, where R
is the rate of urea excretion in µmol day-1 g-1,
[urea]plasma is plasma urea concentration in mmol l-1,
and k is the rate constant in ml g-1 day-1.
Assuming the urea concentration in the external medium to be zero, the value
for k in specimens exposed to freshwater can be calculated as
2.4/(44-0)=0.055 (Table 2;
Fig. 2). This value is 5-fold
greater than that of T. lymma, which had a plasma urea concentration
of 380 mmol l-1 and a k value of 4.24/(380-0)=0.011. This
could mean that either the permeability of the branchial epithelial and skin
surfaces to urea was increased or the uptake of urea through the gills and
kidney was reduced in H. signifer in freshwater in comparison with
the marine T. lymma in seawater. H. signifer could
apparently alter the k value for urea when exposed to higher
salinities. On day 5 (first day in 20 water), the k value for
H. signifer decreased by 29% to 3.2/(83-0)=0.039
(Table 2; Fig. 2), which is still 3-fold
higher than the value of 0.011 for T. lymma. Since H.
signifer could upregulate its urea synthetic capacity (as indicated by
the activity of CPS III) to that of T. lymma, the reason for its
inability to survive well in waters of >20
appears to be its
limited capacity to retain urea as a consequence of adapting to live in a
freshwater environment.
FAAs in tissues remain relatively unchanged during salinity
adaptation
Exposure to 20 water did not have much effect on the concentrations
of FAAs in the tissues of H. signifer and it can be concluded that
FAAs were not involved in cell volume regulation in brackish water. At high
concentration, urea alters many macromolecular structures and functions; for
example, assembly of collagen and microtubules and inhibition of enzymes
(Yancey, 2001
). Some
osmolytes, especially the methylamines, stabilize macromolecular structure and
have a counteracting effect to that of urea. TMAO is usually a better
stabilizer than other known osmolytes, including betaine
(Yancey, 2001
), perhaps
explaining why it is the dominant non-urea osmolyte in most ureosmotic fishes.
However, no TMAO was detected in any of the tissues and organs of specimens
exposed to freshwater or 20
water. On the other hand, glycine,
sarcosine and ß-alanine can also act as counteracting osmolytes
(Yancey and Somero, 1980
). The
presence of two of these amino acids, glycine and ß-alanine, in
significantly higher levels in the muscle and liver of H. signifer
exposed to 20
water suggests that they might have a counteracting
function when urea built up in these tissues
(Table 8). A 1.5-fold build up
of urea in the muscle and liver (Table
2) was counter-balanced by at least a 3-fold increment in
ß-alanine and a 1.5-fold increment in glycine. Taurine is usually found
in moderate levels in tissues of marine elasmobranchs (skates;
King and Goldstein, 1983
). By
contrast, the levels of taurine in the muscle and liver of H.
signifier were high, reaching >20 µmol g-1 tissue. It is
possible that taurine can also act as a counteracting osmolyte despite the
fact that its concentration remained unchanged during salinity adaptation.
The rate of nitrogen metabolism was higher in freshwater than in
20 water
In freshwater, the rate of nitrogenous waste (ammonia + urea) excretion in
H. signifer was 11.2 µmol N day-1 g-1
(calculated from Figs 1 and
2), which was higher than that
in 20 water on day 5 (8.50 µmol N day-1 g-1).
In fact, from day 2 onwards, the total nitrogen excreted was consistently
higher in H. signifer exposed to freshwater than those exposed to
increased salinity. It is logical to deduce that a higher rate of ammonia
production, and hence amino acid catabolism, occurred in specimens exposed to
freshwater. Since the specimens were not fed during the experiment, presumably
the carbon chains released from amino acid catabolism were channelled into ATP
production. Hence, these results suggest that the energy demand in specimens
surviving in freshwater was higher than that in specimens surviving in
brackish water (20
). H. signifer maintained its blood
hyperosmotic to the external medium, with the difference in osmolality between
the blood and the external medium being much greater in freshwater (416-39=377
mosmol kg-1; Table
1) than in 20
(571-543=28 mosmol kg-1;
Table 1). As a result, the
energy demand with respect to osmoregulation alone was higher in 0.7
than in 20
, because energy was required to drive the various ionic
transporters to minimize the loss and/or to increase the uptake of
Na+ and Cl-. The energetic regulation of putative urea
transporters might be involved as well.
Conclusion
Unlike P. motoro, H. signifer is ureogenic and ureotelic in
freshwater. The hyperosmoticity of its blood is greater than other freshwater
teleost fishes because of the presence of urea (>40 mmol l-1).
Hence, it faces higher energetic demands of osmoregulation due to the steep
ionic and urea gradients between the blood and the environment. To survive in
freshwater, it reduces the OUC capacity and decreases urea retention as
compared with the marine ray T. lymma. In 20 water, H.
signifer increases the rate of urea synthesis by upregulating the OUC
enzymes, which is accompanied by a reduction in the ammonia excretion rate.
However, its capacity to retain urea at higher salinity is limited and
consequently it cannot survive well in 20
water or beyond.
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