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First published online January 19, 2006
Journal of Experimental Biology 209, 484-492 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02002
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Exposure to brackish water, upon feeding, leads to enhanced conservation of nitrogen and increased urea synthesis and retention in the Asian freshwater stingray Himantura signifer
1 Natural Sciences and Science Education, National Institute of Education,
Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic
of Singapore
2 Department of Biological Sciences, National University of Singapore, 10
Kent Ridge Road, Singapore 117543, Republic of Singapore
* Author for correspondence (e-mail: sfchew{at}nie.edu.sg)
Accepted 16 November 2005
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to 15 over a 10-day
period, did not lead to an increase in daily food intake. However, there were
significant reductions in daily rates of ammonia and urea excretion in H.
signifer during salinity changes, especially between day 5 (in 10
water) and day 10 (in 15 water) when compared to those of the control
kept in 1 water. Consequently, there was a significant decrease in the
percentage of nitrogen (N) from the food being excreted as nitrogenous waste
(ammonia-N+urea-N) during this period. On day 10, the tissue urea contents in
fish exposed to 15 water were significantly greater than those of fish
kept in 1 water, and the excess urea-N accumulated in the former fish
could totally account for the cumulative deficit in excretion of
urea-N+ammonia-N during the 10-day period. Thus, it can be concluded that
H. signifer is N-limited, and conserved more N from food when exposed
to brackish water. The conserved N was converted to urea, which was retained
in tissues for osmoregulation. The second objective of this study was to
elucidate whether the retention of the capacity of N conservation in H.
signifer would lead to an accumulation of urea in fish exposed to not
only 15 water, but also 1 water, upon feeding. For fish
pre-acclimated to 1 water or 15 water for 10 days and then
fasted for 48 h, the rate of ammonia excretion in fish exposed to 15
water was consistently lower than that of fish exposed to 1 water,
throughout the 36-h post-feeding period. In addition, the hourly rate of urea
excretion in the former was significantly lower than that of the latter
between hours 12 and 36. There were postprandial increases in ammonia contents
in the muscle, liver, stomach, intestine, brain and plasma of fish kept in
1 water; but postprandial increases in ammonia occurred only in the
liver and brain of fish exposed to 15 water, and the magnitudes of
increases in the latter were smaller than those in the former. Indeed,
postprandial increases in tissue urea contents occurred in both groups of
fish, but the greatest increase in urea content was observed in the muscle of
fish exposed to 15 water. Taken together, these results indicate that
H. signifer in freshwater could be confronted with postprandial
osmotic stress because of its capacity of conserving N and increasing urea
synthesis upon feeding.
Key words: ammonia, feeding, stingray, Himantura signifer, nitrogen metabolism, osmoregulation, urea
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; Anderson,
2001). Their extracellular fluids are actively regulated to have
considerably lower salt concentrations than the environment, with the osmotic
difference balanced by extracellular (as well as intracellular) nitrogenous
organic osmolytes (Yancey,
2001). Because urea is retained at high concentrations (300-600
mmol l-1) in the body fluid and tissues for osmotic water retention
(Ballantyne, 1997;
Perlman and Goldstein, 1998),
marine elasmobranchs are described as ureosmotic. Urea retention is
accomplished by a low permeability of the branchial epithelium to urea and by
re-absorption of urea in the gills (Smith
and Wright, 1999) and kidney (Morgan et al.,
2003a,b).
However, in spite of low urea permeabilities
(Fines et al., 2001), marine
elasmobranchs are ureotelic, and the majority of their waste nitrogen (N) is
excreted as urea via the gills
(Shuttleworth, 1988;
Wood, 1993;
Wood et al., 1995;
Perlman and Goldstein, 1998)
as a result of the large gradient of urea concentration between their bodies
and the ambient seawater.
Urea synthesis is energy intensive; 5 µmol of ATP are required for the
formation of every mole of urea. Because urea-N is much more costly to make
than ammonia-N, Mommsen and Walsh
(1991) postulated that marine
elasmobranchs would excrete excess nitrogen, over and above the needs of
osmoregulation, in the form of ammonia-N rather than urea-N after feeding.
When the dogfish shark was infused with ammonia at a rate of 1500 µmol
kg-1 h-1 for 6 h
(Wood et al., 1995), both
ammonia-N and urea-N excretion increased by similar extents during infusion,
though the former more rapidly, and the entire ammonia-N load (actually 132%)
was excreted within 18 h. Based on this, Wood
(2001) concluded that the
postulate of Mommsen and Walsh
(1991) might be partially
correct, although he pointed out that NH4Cl infusion was very
different from natural feeding. At the same time, Wood
(2001) argued that marine
elasmobranchs were N-limited, and suggested that they would avoid the loss of
N after feeding by converting as much excess N as possible to urea. Indeed,
feeding via a stomach tube results in no increase in urea-N excretion, but
only a very small increase in excretion of ammonia in the Pacific spiny
dogfish (Wood et al., 2005).
So, these results (Wood et al.,
2005) are in support of the proposition made by Wood
(2001) earlier. Because there
was only a small increase in ammonia excretion after feeding, Wood et al.
(2005) concluded that their
results also supported the postulate of Mommsen and Walsh
(1991).
Since the nitrogen-limiting status of a marine elasmobranch is defined by
the salinity of the external medium, the most direct approach to test the
postulate of Mommsen and Walsh
(1991) is to acclimatize
marine elasmobranchs to diluted or full-strength seawater before feeding
experiments. However, marine elasmobranchs are rarely euryhaline and can
usually tolerate no more than 30% change in ambient salinity. The availability
of the white-edge freshwater whip ray, Himantura signifer Compagno
and Roberts 1982 (Family: Dasyatidae), which can survive well in brackish
water (Tam et al., 2003) in
South East Asia presented us with the opportunity to examine the hypotheses of
Mommsen and Walsh (1991) and
Wood (2001) using an approach
different from that of Wood et al.
(2005). H. signifer
is found in the Batang Hari Basin in Jambi of Sumatra in Indonesia. It retains
the ability to synthesize urea but reduces the capacity of retaining it in
freshwater (Tam et al., 2003).
Although this stingray can be found in Batang Hari, as far as 400 km from the
South China Sea, it may re-enter estuarine and marine environments during the
breeding season. Indeed, H. signifer possesses ureosmotic
osmoregulatory mechanisms to survive in brackish water
(Tam et al., 2003). However,
that means, in freshwater, H. signifer has to suppress both urea
production and urea retention, including active urea re-absorption
(Tam et al., 2003). Hence,
H. signifer is the most desirable species for studies on the effects
of salinity changes on the excretion and retention of food-N after feeding,
because it is ureogenic, ureosmotic and euryhaline.
In the first series of experiments, fish were divided into two groups. One
group was kept in 1 water (control) for 10 days, while the other group
was exposed to a progressive increase in salinity, reaching 15 on days
9 and 10. Food was provided every day during this 10-day period, and the
objective was to examine the effects of salinity changes on the daily food
ration of H. signifer. The daily excretion rates of ammonia and urea
were also determined in order to estimate the percentage of food-N being
excreted as ammonia-N+urea-N. On day 10, fish were sacrificed for the
collection of tissues for analyses of ammonia and urea. We aimed to answer
three important questions in this series of experiment. Would a progressive
increase in ambient salinity lead to a greater food intake in H.
signifer? Would an increase in ambient salinity result in a reduction in
nitrogenous waste excretion, and therefore an increase in retention of N,
after feeding in H. signifer? Would there be a greater rate of urea
synthesis and a greater retention of urea in specimens of H. signifer
exposed to brackish water as compared with those kept in freshwater?
Through the determination of ammonia and urea excretion rates and the
examination of the gut content of fish being sacrificed after feeding, we
obtained preliminary results which indicated that complete digestion of a meal
in H. signifer took at least 48 h, which was longer than the time
taken by some other fishes (e.g. Protopterus dolloi,
Lim et al., 2004;
Periophthalmodon schlosseri, Ip et
al., 2004). So, in order to determine the effects of a single food
ration on N excretion and retention in H. signifer, fish were kept in
freshwater (1) or exposed to a progressive increase in salinity
through a 10-day period as in the first series of experiments. On day 11, both
groups of fish were fasted for 48 h; food was then provided on day 13. Water
samples were collected during the next 36 h for the determination of hourly
ammonia and urea excretion rates. Some fish were sacrificed at various time
points for the analyses of tissue ammonia and urea contents. A period of 36 h
was chosen because preliminary experiments indicated that ammonia and urea
contents in tissues of this fish would reach the highest levels between 24 and
36 h post-feeding. Here, we aimed to answer two other questions. Would feeding
lead to an increase in urea content in tissues of H. signifer kept in
freshwater (1 water) because of its capacity of N conservation after a
meal? Would fish kept in 15 water conserve a greater percentage of the
daily food-N as urea than fish kept in 1 water?
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) at 25°C in the laboratory. Water was changed daily. No
attempt was made to separate the sexes. Fishes were acclimated to laboratory
conditions for at least 1 week before experimentation. During that period,
fish were fed with freshwater shrimp. All experiments performed in this study
were under a 12 h:12 h light:dark regime.
Feed analysis
The wet masses of samples of freshwater shrimp (approximately 1 g,
N=3) were obtained to the nearest milligram. They were then
freeze-dried and the dry masses recorded. Subsequently, the samples were
analyzed for N and carbon (C) using a Eurovector EA3011 Elemental Analyzer
(Milan, Italy) equipped with Callidus software. BBOT
(C26H26N2O2S) standard obtained
from Eurovector (Milan, Italy) was used as a standard for comparison. In
addition some samples were extracted in 70% ethanol for 24 h to remove
non-protein N-compounds before being freeze-dried for N and C analyses. The
difference obtained between samples with and without ethanol extraction gives
an indication of the combined contribution of ammonia, urea, free amino acids,
purines and pyrimidines to the N and C contents of the freshwater shrimp.
Series 1
Fish were divided into two groups. Both groups of fish were fed with live
freshwater shrimps at about 2% of their body mass. Feeding was performed
ad libitum until satiation, which took no more than 2 h, as described
previously for the giant mudskipper (Ip et
al., 2004) and the slender lungfish
(Lim et al., 2004). Excess
food was removed when the fish stopped feeding. The actual mass of the feed
consumed by the fish was calculated as the difference between the mass of
shrimps provided initially and the mass of shrimps left over. Fish were then
gently transferred individually, by hand, to new tanks (60 cmx30
cmx20 cm, lengthxwidthxheight) containing either 2 or 4 l of
1 water at 25°C, depending on the size of the fish; this point was
considered as hour 0 at the start of the experiment. H. signifer
usually stays relatively quiescent after feeding, and there would be minimal
struggling during the transfer when the fish was lifted transiently out of
water with its ventral surface supported by both palms. Water samples (3 ml),
acidified with 70 µl of 1 mol-1 HCl, were collected 24 h later
for ammonia and urea analyses. The above procedure was repeated with 1
water for the control group (N=5) for 10 days. For the experimental
group (N=5), fish were exposed to daily increases in salinity from
1 on days 1 and 2 to 5 on days 3 and 4, followed by 10
on days 5 and 6, 13 on days 7 and 8, and 15 on days 9 and 10.
A gradual increase in salinity was necessary to allow for acclimatization and
survival. Water samples were collected for ammonia and urea assays every 24 h.
Concentrations of ammonia and urea in water samples were determined according
to the methods of Anderson and Little
(1986) and Felskie et al.
(1998) as modified by Jow et
al. (1999), respectively.
Ammonia and urea excretion rates were expressed as µmol day-1
g-1 fish. Five fish in 1 water were killed, by severing the
spinal cord, for the collection of tissues on day 0. On day 10, 5 fish in
1 water and another 5 in 15 water for a second day were killed
and their tissues collected.
Series 2
Fish were kept in 1 water or taken through a progressive increase
in ambient salinity as described above. They were allowed to feed ad
libitum during this 10-day period. On day 11, fish were fasted for 48 h.
A known amount of food was provided on day 13 and the fish was allowed to feed
until satiation. They were then transferred to other tanks (60 cmx30
cmx20 cm, lengthxwidthxheight) containing either 2 or 4 l of
1 or 15 water, depending on the size of the fish. Water
samples were collected at 12 h intervals during the subsequent 36 h period
post-feeding. Fish were killed at 0 h (before the provision of food) and 12,
24 and 36 h post-feeding for tissue collection. Fish transferred to tanks
without the provision of food served as controls.
Collection of tissues and analyses of ammonia and urea
Fish were killed with a strong blow to the head, the blood samples were
collected from the severed caudal peduncle into heparinized capillary tubes.
The collected blood was centrifuged at 5000 g at 4°C for 1
min to obtain the plasma. 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. The resulting supernatant was kept at -25°C until
analysed. The muscle, liver, stomach, intestine and brain were quickly
excised. The excised tissues and organs were immediately freeze-clamped with
aluminium tongs pre-cooled in liquid N2. Frozen samples were kept
at -80°C until analysis. The frozen tissue samples were weighed, ground to
a powder in liquid nitrogen and homogenized three times in 5 volumes (w/v) of
6% HClO4 at 24 000 revs min-1 for 20 s, using an
Ultra-Turrax homogenizer, with intervals of 10 s between each homogenization.
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. Ammonia was determined according to the method of Bergmeyer
and Beutler (1985) and urea
determined as described above. Results were expressed as µmol
g-1 wet mass tissue or µmol ml-1 plasma.
Statistical analyses
Results are presented as means ± standard error of the mean
(s.e.m.). Data in all the figures were analysed using repeated-measures
analysis of variance (ANOVA) followed by least-square means (LS-MEANS) to
evaluate differences between means. Data in all the tables were assessed using
one-way analysis of variance followed by Bonferroni's multiple range test to
evaluate differences between means. Differences where P<0.05 were
regarded as statistically significant.
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Series 1
The amounts of food consumed by the fish daily remained relatively constant
throughout the 10-day period of exposure to a progressive increase in
salinity, and were comparable to those of fish kept in 1 water
(Fig. 1). Using the values in
Fig. 1, the total food intakes
during the 10-day period for fish kept in 1 and 15 water were
calculated to be 0.142 and 0.150 g g-1 fish, respectively.
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water
excreted a total of 88.5 µmol N g-1 fish of ammonia and 73.4
µmol N g-1 fish of urea (calculated from
Fig. 2). For fish exposed to a
progressive increase in salinity, the total amounts of ammonia and urea
excreted during the 10-day period were 56.8 and 52.4 µmol N g-1
fish, respectively. The daily ammonia excretion rate of H. signifer
exposed to a progressive increase in salinity was significantly lower than
that of fish kept in 1 water during the period of day 3 to day 10
(Fig. 2A). The theoretical
total amount of ammonia retained in fish exposed to salinity changes can be
estimated from Fig. 2A as
(3.2+2.5+3.1+3.5+4.9+5.1+4.2+4.1)=30.6 µmol N g-1 fish. In
addition, the daily urea excretion rate of the experimental fish exposed to
increased salinity was significantly lower than that of the control kept in
1 water from day 4 onwards (Fig.
2B). So, the theoretical total amount of urea-N retained can be
estimated from Fig. 2B as
(1.1+1.9+1.7+1.5+2.1+1.9+1.2)x2= 22.8 µmol N g-1 fish.
Overall, there was a significant decrease in the percentage of food-N being
excreted as ammonia-N+urea-N (total-N) by the fish exposed to a progressive
increase in salinity on day 5 (10 water) and thereafter.
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Ammonia contents in the muscle, liver, stomach, intestine, brain and plasma
of control fish in 1 water on day 0, fish kept in 1 water for
10 days and fish exposed to a progressive increase in salinity through a
10-day period were not significantly different from each other
(Table 1). Urea contents in the
muscle, liver, stomach, intestine, brain and plasma of fish in 1 water
on day 0 and those kept in 1 water for 10 days were comparable. By
contrast, there were significant increases in urea contents in all these
tissues and organs of fish exposed to a progressive increase in salinity
(Table 1).
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Because contents of ammonia and urea in fish kept in 1 water for 10
days were not significantly different from those of the day 0 fish in
1 water, the rate of urea excretion must be balanced with the rate of
urea synthesis. Therefore, the averaged daily rate of urea synthesis during
this 10-day period can be estimated as (2.4+2.2+3.0+4.1+4.2+4.1+4.4+4.3+4.0+
4.2) µmol 10 days-1 g-1 fish/10=3.7 µmol
day-1 g-1 fish.
For fish exposed to a progressive increase in salinity, the averaged daily
urea synthesis rate during the 10-day period is equivalent to the summation of
the averaged daily rate of urea excretion and the excess urea accumulated in
the fish on day 10 divided by 10 days. The averaged rate of urea excretion is
estimated as (2.6+3.0+2.5+2.9+2.3+2.4+2.8+2.3+2.1+ 3.0) µmol 10
day-1 g-1 fish/10=2.6 µmol day-1
g-1 fish. There were 50 g muscle, 3 g liver, 2 g stomach, 3 g
intestine, 1 g brain and 2 ml plasma in a 100 g H. signifer
(Tam et al., 2003). So, the
excess amount of urea that would accumulate in the body of a hypothetical 100
g fish exposed to 15 water on day 10 can be estimated (from
Table 1) as [(126-72) µmol
g-1x50 g]+[(118-60) µmol g-1x3
g]+[(117-57) µmol g-1x2 g]+[(93-63) µmol
g-1x3 g]+[(105-67) µmol g-1x1
g]+[(153-74) µmol ml-1x2 ml]=3280 µmol (equivalent to
6560 µmol N). This is equivalent to 32.8 µmol urea g-1 fish,
accumulated during a 10-day period. Hence, the averaged daily rate of urea
accumulation amounts to 3.3 µmol day-1 g-1 fish. This
means that the rate of urea synthesis in fish exposed to a progressive
increase in salinity was 2.6+3.3= 5.9 µmol day-1 g-1
fish.
Series 2
The ammonia excretion rate of fish fasted for 48 h in 15 water was
significantly lower than that of the control fasted for 48 h in 1
water throughout the 36-h period (0-12 h, 12-24 h and 24-36 h) post-feeding
(Fig. 3A). In addition, the
urea excretion rate of fish in 15 water was significantly lower than
that of the control in 1 water during the 12-24 h and 24-36 h
post-feeding (Fig. 3B).
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water, the liver ammonia content
increased significantly at 12, 24 and 36 h post-feeding
(Table 2). In addition, there
were significant increases in ammonia content in the intestine, brain and
plasma at 24 and 36 h (Table
2). For the muscle and the stomach, significant increases in
ammonia contents occurred only at 36 h
(Table 2). Twenty-four hours
and 36 h after feeding, there were significant increases in urea content in
the muscle, liver, stomach, intestine, brain and plasma of fish kept in
1 water (Table 2).
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For H. signifer kept in 15 water, there was no significant
change in ammonia contents in the muscle, stomach, intestine and plasma during
the 36-h post-feeding period (Table
3). However, the ammonia content in the liver increased
significantly at 24 and 36 h, and that in the brain also increased
significantly at 36 h, post-feeding (Table
3). Twenty-four hours and 36 h after feeding, there was a
significant increase in urea content in the muscle of fish kept in 15
water (Table 3). In addition,
there were significant increases in urea content in the liver, stomach,
intestine and brain at 36 h post-feeding
(Table 3). However, no
significant change in plasma urea concentration was observed throughout the
36-h period.
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The excess urea that would accumulate 36 h post-feeding in a hypothetical
100 g fish in 1 water can be estimated (from
Table 2) to be [(23 µmol
g-1x50 g)+(29 µmol g-1x3 g)+ (26 µmol
g-1x2 g)+(25 µmol g-1x3 g)+(34 µmol
g-1x1 g)+ (70 µmol ml-1x2 ml)]=1538
µmol (equivalent to 3076 µmol N). A similar calculation reveals that the
excess ammonia-N accumulated in these tissues and organs would be only 140
µmol N.
At 36 h, the excess urea that would accumulate in a 100 g fish in
15 water after feeding can be estimated (from
Table 3) to be [(51 µmol
g-1x50 g)+(31 µmol g-1x3 g)+ (21 µmol
g-1x2 g)+(25 µmol g-1x3 g)+(25 µmol
g-1x1 g)]= 2485 µmol (equivalent to 4970 µmol N), which
is 1.6-fold greater than that (1538 µmol) of fish kept in 1
water.
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). That could
be the reason why it retains the capacity to synthesize and accumulate urea
for osmoregulation and can survive well in brackish water. H.
signifer suppresses urea synthesis and reduces urea retention to survive
in freshwater, but in brackish water, it up-regulates urea synthesis and
retains urea in its tissues (Tam et al.,
2003). It is important to point out that Tam et al.
(2003) performed their studies
with fish fasted for 48 days before experiments and withheld food throughout
the experimental period. Thus, there is a dearth of knowledge on relationships
between feeding and osmoregulatory needs in H. signifer at present.
H. signifer exposed to brackish water has to increase the internal
urea content for osmoregulation, and so when food is available, there would
theoretically be a greater nitrogenous intake through, and/or a greater
conservation of N from, food consumption.
Would a progressive increase in ambient salinity lead to a greater food intake in H. signifer?
Our results reveal that H. signifer was able to acclimate to a
progressive increase in salinity from 1 to 15 through a 10-day
period without increasing the daily food consumption
(Fig. 1). The averaged food
rations for fish kept in 1 water and fish exposed to a progressive
increased in salinity during the 10-day period were 0.014 and 0.015 g
g-1 fish, respectively. So, increased conservation of nitrogen from
food intake appears to be the main adaptation exhibited by H.
signifer to survive in brackish water.
Would an increase in ambient salinity result in an increase in retention of N after feeding in H. signifer?
The rates of ammonia and urea excretion in H. signifer kept in
1 water are comparable to those of the little skate, Raja
erinacea in 100% or 75% seawater
(Steele et al., 2005).
However, when H. signifer was exposed to a progressive increase in
salinity, there was a progressive decrease in the rate of ammonia excretion.
Yet, despite a reduction in ammonia excretion, there was no significant change
in ammonia contents in various tissues of fish exposed to 15 water on
day 10. Tissue urea contents, in contrast, were significantly higher in these
experimental fish than those in fish kept in 1 water for 10 days. So,
fish exposed to brackish water indeed conserved a greater portion of the N
from the relatively constant amount of food intake, and incorporated the N
into urea for osmoregulatory purposes. In addition, there was a progressive
decrease in the rate of urea excretion in fish exposed to salinity changes,
which indicates that H. signifer also retained more urea in response
to the increasing ambient salinity. Thus, our results are in support of the
postulate of Wood (2001) that
ureosmotic elasmobranchs are nitrogen-limited and would avoid the loss of
nitrogen after feeding by converting as much excess N as possible to urea.
Because preliminary results indicate that it took at least 48 h for H.
signifer to completely clear its gut of food, it would be inappropriate
to calculate the daily percentage food-N being excreted by fish in this series
of experiments. However, it would be meaningful to perform the calculation
over the 10-day period. Since the total food intake for fish kept in 1
water was 0.142 g g-1 fish and since each gram of feed contained
1.5 mmol N, the total N intake was 213 µmol N g-1 fish. During
this 10-day period, the total waste-N (ammonia-N+urea-N) excreted was
88.5+73.4=161.9 µmol N g-1 fish. Thus, (161.9x100)/213 or
76% of the food-N was excreted by H. signifer in 1 water.
Alternately, this means only 24% of the food-N was conserved, which is lower
than those for the non-ureosmotic fish species such as the African lungfish
Protopterus dolloi (Lim et al.,
2004) and the giant mudskipper Periophthalmodon
schlosseri (Ip et al.,
2004). This could be a result of H. signifer maintaining
a relatively high level of urea in its plasma, and simultaneously reducing the
capacity to retain urea in order to survive in freshwater
(Tam et al., 2003;
Ip et al., 2004).
Because the total food intake for fish exposed to a progressive increase in
salinity was 0.15 g g-1 fish, the total N intake was 225 µmol N
g-1 fish. During this 10-day period, the total N excreted was
56.8+52.4=109.2 µmol N g-1 fish. Thus, (109.2x100)/225 or
49% of the food-N was excreted by H. signifer exposed to a
progressive increase in salinity, which means 51% of the food-N was conserved.
From these calculations, it is confirmed that a 2.1-fold (=51%/24%) increase
in conservation of N occurred in H. signifer exposed to brackish
water as compared with those kept in 1 water.
Would there be a greater rate of conversion of the retained N to urea in H. signifer exposed to brackish water?
There was a significant decrease in the rate of ammonia excretion in H.
signifer exposed to a progressive increase in salinity from day 3 to day
10. The total amount of ammonia retained was 30.6 µmol N g-1. In
addition, there was a significant decrease in the rate of urea excretion in
these experimental fish between day 4 and day 10, amounting to 22.8 µmol N
g-1. Together the sum of N conserved during the 10-day period is
30.6+22.8=53.4 µmol N g-1. So, for a 100 g H. signifer,
a total of 5340 µmol N would have been conserved. On day 10, the excess
amount of urea accumulated in the tissues and organs of a 100 g fish exposed
to 15 water was estimated to be 6560 µmol N, which is even greater
than the estimated amount of 5340 µmol N conserved. Hence, it can be
concluded that the conserved N was completely converted into urea for
osmoregulation.
The estimated rate of urea synthesis in a fish in 1 water during
the 10-day period was 3.7 µmol day-1 g-1 fish. For
fish exposed to a progressive increase in salinity, the average urea synthesis
rate during a similar period is estimated to be 5.9 µmol day-1
g-1 fish. This means that the rate of urea synthesis in fish
exposed to a progressive increase in salinity was up-regulated 1.6-fold
(5.9/3.7).
Would feeding lead to an increase in urea contents in tissues of H. signifer kept in 1 water?
Mommsen and Walsh (1991)
postulated that marine elasmobranchs might excrete excess nitrogen intake,
over and above the needs of osmoregulation, in the form of ammonia rather than
urea after feeding. Although results obtained from our series 1 experiment
confirmed that H. signifer was N-limited, these results could not
verify whether the postulate made by Mommsen and Walsh
(1991) was correct; so, the
second series of experiments was performed.
In 1 water, H. signifer cannot be N-limited, and therefore
should excrete a major portion of the food-N as ammonia according to the
hypothesis of Mommsen and Walsh
(1991). However, our results
proved it otherwise. After a single intake of food, the percentage of total
waste-N (ammonia-N+urea-N) excreted as ammonia in H. signifer fasted
for 48 h and kept in 1 water during the 0-12, 12-24 and 24-36 h
periods was relatively constant. More importantly, besides increases in
ammonia contents, there were postprandial increases in urea contents in all
the tissues studied in H. signifer. The excess urea-N accumulated at
36 h in a 100 g fish is estimated to be 3076 µmol N. Because the excess
ammonia-N accumulated in these tissues and organs was only 140 µmol, that
means a major portion of the food-N was converted into urea in spite of H.
signifer not being nitrogen-limited in 1 water. There can be two
reasons for this as suggested by Lim et al.
(2004) and Ip et al.
(2004). Firstly, urea
synthesis, being an energy-intensive process, is not a major issue when food-N
is concerned, because food intake prescribes the availability of energy
resources. Secondly, the majority of excess amino acids from food intake are
catabolized by glutamate dehydrogenase, which is present in the matrix of
liver mitochondria. Also present in the mitochondrial matrix are glutamine
synthetase and CPS III, which convert ammonia to carbamoyl phosphate for
subsequent urea synthesis. So, naturally, ammonia produced through catabolism
of excess amino acid, unlike that infused/injected into the blood or
peritoneal cavity, is readily available for urea synthesis in the liver.
Because H. signifer retains the capacity of increased urea synthesis
and retention, our results indicate that it would be confronted with osmotic
stress transiently in 1 water after feeding.
Would fish keep in 15 water conserve a greater percentage of the food-N than fish kept in 1 water?
For H. signifer kept in 15 water and fasted for 48 h,
there was indeed a greater conservation of N after feeding. The postprandial
rate of ammonia excretion in fish exposed to 15 water was
significantly lower than that in fish kept in 1 water during the 0-12,
12-24 and 24-36 h periods. Simultaneously, there was a significantly lower
rate of urea excretion in the former as compared to the latter, indicating a
greater retention of urea in fish kept in 15 water.
In contrast to fish kept in 1 water, there was no increase in
ammonia contents in various tissues and organs, except the liver and the
brain, of fish exposed to 15 water in spite of the greater
conservation of food-N. Once again, this is in support of the conclusion that
the conserved food-N was completely converted to urea for osmoregulatory
purposes. At 36 h, the excess urea-N accumulated in a 100 g fish in 15
water is estimated to be 4970 µmol N, which is 1.6-fold greater than that
of fish kept in 1 water (3076 µmol N). Thus, these results confirm
once again that fish kept in 15 water converted a greater percentage
of the food-N to urea than fish kept in 1 water. They are in support
of the proposition that urea synthesis in elasmobranchs is adapted more for
osmoregulation than ammonia detoxification as suggested by Ip et al.
(2005), although the
ornithine-urea cycle of invertebrates may function mainly to detoxify ammonia
(Hiong et al., 2005).
Conclusion
H. signifer is N-limited, and conserves more N from food when
exposed to brackish water. The conserved N is converted to urea, which is
retained in tissues for osmoregulation. However, because of its capacity to
conserve N and increase urea synthesis upon feeding, H. signifer can
be confronted with postprandial osmotic stress in freshwater.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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