Dogmas and controversies in the handling of nitrogenous wastes: Ureotely and ammonia tolerance in early life stages of the gulf toadfish, Opsanus beta
1 Division of Marine Biology and Fisheries, Rosenstiel School of Marine and
Atmospheric Science, University of Miami, Miami, FL 33149-1098, USA
2 Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1,
Canada
* Author for correspondence (e-mail: jbarimo{at}rsmas.miami.edu)
Accepted 23 February 2004
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Summary |
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Key words: ureogenesis, ureotely, Opsanus beta, toadfish, ontogeny, ammonia toxicity, ornithineurea cycle, ornithine transcarbamylase, Florida Bay, Biscayne Bay, Batrachoididae
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Introduction |
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As adults, O. beta express a full complement of OUC enzymes in the
liver, which includes carbamoyl-phosphate synthetase III (CPSase III) as
opposed to the isoform CPSase I, commonly found in terrestrial vertebrates
(Read, 1971;
Mommsen and Walsh, 1989
). The
nitrogen-donating substrate for CPSase I is ammonia directly, while CPSase III
is dependent upon glutamine as an intermediary
(Mommsen and Walsh, 1989
). In
the toadfish, glutamine synthetase (GSase) is intimately linked to the OUC
and, during facultative shifts to ureotelism, hepatic GSase activities
increase 5-fold while white muscle activities double
(Walsh et al., 1994
;
Walsh et al., 2003
).
Adult O. beta have a high tolerance to elevated external or
environmental ammonia, which may be related to ammonia detoxification
via the OUC, with GSase functioning as an ammonia trap
(Wang and Walsh, 2000). In a
96-h lethal concentration (96-h LC50) test, 50% mortality of test
subjects occurred at a total ammonia concentration of 9.75 mmol N
l1 (166 mg l1) with a calculated unionized
ammonia (NH3) fraction of 519 µmol N l1 (8.8
mg l1) (Wang and Walsh,
2000
). The toadfish LC50 values are relatively high
when compared with NH3 mean acute toxicity values of 164 and 109
µmol N l1 for 32 freshwater and 17 marine teleost
species, respectively (in Randall and
Tsui, 2002
). Ammonia tolerance in the early life stages of
toadfish remains unknown although it is reported that in rainbow trout
(Rice and Stokes, 1975
), the
spotted seatrout (Daniels et al.,
1987
) and other teleosts (see
Steele et al., 2001
) that
embryonic stages are more tolerant of environmental ammonia than their
corresponding adult stages.
The majority of urea produced by adult O. beta is excreted in
distinct pulsatile events across the gill membrane once or twice daily
(Wood et al., 1995;
Gilmore et al., 1998
).
Confined O. beta excrete 80% of their nitrogenous waste as urea with
pulses ranging from 1192 to 4334 µmol N kg1 in
concentration and 0.53 h in duration (reviewed in
Wood et al., 2003
).
Furthermore, the oyster toadfish, Opsanus tau, was classified as
`moderately' ureotelic when compared with the `fully' ureotelic O.
beta (Wang and Walsh,
2000
). O. tau was found to switch from ammonotely to
ureotely upon hatching and through the subsequent larval stage
(Stephen and Griffith, 2001
),
yet little else is known regarding the ontogeny of nitrogen
metabolism/excretion in toadfish.
The reproductive ecology of toadfish, however, has been widely studied.
O. beta are reported to actively spawn when water temperatures range
from 15 to 22°C (Breeder,
1941), which tends to occur from March to May in the shallow
coastal estuaries of southern Florida. Male toadfish establish nesting sites
under stones and in large gastropod shells
(Ryder, 1886
;
Gill, 1907
) but are noted to
prefer the `debris of civilisation' such as tin cans and broken jars
(Clapp, 1899
). Nesting males
subsequently attempt to attract mates with courtship vocalizations known as
boatwhistles (Gray and Winn,
1961
; Breeder,
1968
). Eggs are solidly adhered to the nest substrate by females,
who leave attending males to brood and guard offspring through the yolk-sac
larval stage where larvae remain connected to the substrate by a pedicel until
they become free-swimming juveniles
(Ryder, 1886
;
Gudger, 1908
).
It is believed that ureotely in toadfish must provide some adaptive
significance to counterbalance the bioenergetic cost of 2.5 ATP per unit N
excreted (reviewed by Wood et al.,
2003). Several viable hypotheses exist pertaining to why toadfish
are ureotelic; however, the ultimate causal factor(s) remains unknown. It was
suggested that male toadfish are facultatively ureotelic to avoid poisoning
progeny with ammonia in confined nests with restricted water flow
(Griffith, 1991
;
Stephen and Griffith, 2001
).
In the present study, the mechanisms of ureogenesis and patterns of urea
excretion across early life history stages of O. beta were
investigated. In addition, the hypothesis that ureotelism in the toadfish
exists as a mechanism to protect offspring from toxic levels of ammonia in
confined nests was addressed. Ammonia-N and urea-N were measured in water
collected from within O. beta shelters at field sites and in
containers in the laboratory along with an ammonia 96-h LC50 test.
Enzymatic activities of eggs, larvae and juveniles were measured for the OUC
and accessory enzymes CPSase III, ornithine transcarbamylase (OTCase),
arginase and GSase from samples collected in the field and on surviving
juveniles from high ammonia exposures.
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Materials and methods |
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At each cluster of shelters, salinity was determined with a refractometer and water depth was measured with a PVC pole marked in cm increments. Shelters were retrieved by divers (AprilMay 2003) who capped the shelter's open end, thereby encapsulating nesting toadfish and `shelter' water. Once onboard the attending research vessel, shelters were opened over a basin, water samples were collected, and pH and temperature were measured with a WTW model pH 331i meter set. To allay concerns that encapsulating toadfish (<2 min) influenced nitrogen excretions, a subset of sampling was compared with shelter water collected rostral to the toadfish by syringe prior to capping. Although the capped samples were slightly higher (3.7±2.3 mmol N l1) than the syringe samples (2.7±1.7 mmol N l1), these differences are not significant (P=0.50, N=5) with the paired Wilcoxon signed rank test. The increased values for the encapsulated samples probably represent uniform mixing of the shelter water since ventilated water probably pools caudal to the toadfish at the rear of the shelter. Water-quality samples were also collected from the ambient water column near shelters to access background concentrations of ammonia and urea. Water samples were acidified to pH 2 with 12 mol l1 HCl (2 µl ml1 seawater) to inhibit bacterial degradation and prevent NH3 volatilization, stored on ice for 46 h and frozen at 20°C upon return to Miami.
Adult toadfish collected from shelters were euthanized in the field with an overdose of anaesthetic (clove oil), sexed by the internal examination of gonads, and their gastrointestinal tracts examined for the presence or absence of food items. Eggs, larvae and juveniles were also collected from shelters, blotted dry, wrapped in foil packets, immediately placed in liquid nitrogen and stored at 80°C. Samples were shipped to Guelph, Ontario on dry ice and stored at 80°C for 5 weeks until assays were run for OUC enzyme activities. In addition, four shelters including guardian male and eggs/larvae were transported to the Miami lab in 75-litre insulated coolers supplied with continually refreshed oxygenated seawater. Once a sufficient number of offspring emerged as juveniles, they were used in an ammonia 96-h LC50 test and nitrogen excretion experiments.
Based upon observations of a cohort of O. beta from a natural
spawning event in an 8000-litre mesocosm with a known date of fertilization
(J. F. Barimo, personal observations), observed developmental stages of O.
beta were consistent with previous descriptions of Opsanus spp.
(Gudger, 1908;
Breeder, 1941
;
Dovel, 1960
). Developmental
stages were categorized as: (1) egg I stage, 17 days
post-fertilization, with a distinctive amber colour and no sign of embryonic
development; (2) egg II stage, 721 days post-fertilization, with a
discernible embryo and an orange appearance due to vascularization in the
yolk; (3) yolk-sac larvae I stage, 17 days post-hatch, noted by the
absence of a chorion exposing the yolk-sac, which was adhered to the
substrate; (4) yolk-sac larvae II stage, 814 days post-hatch, with
>50% of the yolk-sac absorption and increased tail flexing and mouth
movement but still adhered to the substrate and (5) juvenile stage, where
precocious toadfish are free swimming with yolk-sac completely absorbed.
Individuals >16 mm total length (TL) are still categorically
juvenile but were not used since they appear to be less tied to nesting sites.
However, juveniles <16 mm TL were commonly found inside nesting
shelters with adult guardians present. It should be noted that the terms
`prolarvae', `cling young' and `eleutheroembryo' have been used by some
workers to describe the more common term `yolk-sac larvae'.
Experimental design
Standard laboratory prophylactic treatments routinely performed on adult
toadfish (Wood et al., 2003)
were not used on toadfish nests (shelter + eggs/larvae + guardian male) so as
to avoid potential stress to eggs/larvae. Emerging juveniles were fed freshly
hatched brine shrimp, Artemia salina (1020 per juvenile), for
1 week, after which they were fasted for 24 h prior to 96-h LC50
and nitrogen excretion flux experiments.
Ammonia and urea flux experiments were conducted with individual juvenile
toadfish in capped 28x61 mm high-density polyethylene vials with 10 ml
of static seawater (35) at 2325°C. Aeration and exhaust
(PE-90 tubing) were permitted via an access hole in the vial cap, and
aeration was adjusted to 12 small bubbles s1. Water
samples were manually sampled by pipette (3 ml) at 24 h intervals over the 72
h trial (N=7) to obtain daily waste-N flux rates. Water samples were
taken hourly from a second group of toadfish (N=7) for 26 h to
determine finer-scale patterns of nitrogen excretion. Given the small water
volume in flux chambers, samples were limited to 3 ml and water lost to hourly
sampling was replaced with fresh seawater with subsequent dilutions factored
into final calculations. Three blank vials were used to evaluate the
contribution of nitrogen waste by microbial activity in the sand-filtered
seawater utilized.
Ammonia toxicity experiments were designed after Wang and Walsh
(2000) with 125, 250, 500,
1000, 2000 and 4000 µmol N l1 treatments based on an
initial range-finding test. The corresponding fractions of unionized ammonia
(NH3) and ammonium (NH4+) were calculated in
accordance with the HendersonHasselbalch equation:
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Toadfish juveniles (N=30 per treatment) were placed in 2-litre
polyethylene containers with small PVC shelters and allowed 24 h to acclimate.
The juveniles used were <16 mm TL and ranged in wet mass from 27.9
to 48.9 mg. Treatments were spiked with variable volumes of a 3.57 mol N
l1 stock solution of NH4Cl to attain desired
concentrations. Water changes (50%, 1 litre) were conducted at 24 h intervals
to prevent ammonia degradation and otherwise maintain high water quality.
Water temperature, pH and salinity were maintained within a range of
20.022.4°C, 8.218.25 and 3536, respectively.
Water samples were collected at the onset of the LC50 test, at 24 h
intervals following water changes and at completion of the test to assure that
desired ammonia concentrations were maintained. Surviving toadfish were
sacrificed and frozen in liquid N2. Attempts to remove embryos and
larvae from the substrate and re-adhere them without subsequent physical
damage was unsuccessful and hence these stages were not utilized for ammonia
toxicity testing.
Analytical procedures
Water samples were analyzed for total ammonia-N and urea-N within 48 h of
collection. Field samples were adjusted to pH 8 with 10 mol
l1 NaOH to neutralize the HCl preservative. Urea-N was
measured using a standard colorimetric diacetylmonoxime assay
(Price and Harrison, 1987).
Ammonia-N was determined by a modification to the spectrophotometric
indophenol blue method of Ivancic and Deggobis
(1984
). The indophenol blue
method measures total ammonia-N, i.e. ammonia (NH3) and ammonium
(NH4+), and total ammonia-N is abbreviated to ammonia-N
in subsequent text. Spectrophotometric measures for the ammonia and urea
assays were performed using a Molecular Devices Thermo Max microplate reader
(Menlo Park, CA, USA).
Assays for the enzymes
GSase, CPSase II and III, OTCase and arginase were performed in accordance
with Steele et al. (2001) with
the following modifications. For enzyme assays, tissue samples from
48 individuals were pooled depending on developmental stage with
later stage samples composed of fewer individuals. Samples were homogenized in
1786 volumes (depending on developmental stage) of ice-cold extraction
buffer (0.05 mol l1 Hepes buffer, pH 7.5, 0.05 mol
l1 KCl, 0.5 mmol l1 EDTA, 1 mmol
l1 DL-dithiothreitol) and sonicated. Supernatants
of the centrifuged tissue homogenates (10 min at 14 000 g,
4°C) were passed through Sephadex (G25) columns to removed endogenous
substrates and modifiers (Felskie et al.,
1998
). To account for protein dilution during column purification,
a dilution factor was calculated as the pre-column protein concentration
divided by the post-column protein concentration. Protein content was assayed
by a dye-binding method using a reagent kit from Bio-Rad Laboratories
(Hercules, CA, USA). The standards used were 00.05 µmole bovine
serum albumin. Glutamine was the only substrate provided for the CPSase assay,
which measured [14C] carbamoyl phosphate as described by Anderson
et al. (1970
), since the
maximum activity with ammonia as a substrate (data not shown) was minimal and
comparable with that found in adults
(Anderson and Walsh, 1995
).
Enzyme reactions in assays were all conducted at 26°C, and reaction times
for GSase, CPSase, OTCase and arginase were 10, 30, 10 and 10 min,
respectively. All end products of enzyme and protein assays were measured
spectrophotometrically using a Perkin Elmer model Lambda 2 UV/VIS
spectrophotometer (Norwalk, CT, USA) except for the CPSase assay, which
utilized a Beckman Coulter LS 6500 multi-purpose scintillation counter
(Fullerton, CA, USA). Toadfish were considered ureotelic if they excreted most
nitrogen as urea (>50%) and expressed significant levels of OUC enzymes (as
in Anderson, 2001
). The term
percent ureotelic (as in Wood et al.,
2003
) is defined as the percent of waste-N occurring as urea-N
where:
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Data analysis
The ammonia 96-h LC50 curve was calculated by nonlinear logistic
curve regression analysis using Sigma Plot software. SPSS software was used
for Student's t-test and one-way analysis of variance (ANOVA) with
the StudentNewmanKeuls post-hoc test to examine
differences in OUC enzyme activities among developmental stages and ammonia
treatment. All data were tested for normality with the Levene Test for
Homogeneity of Variances. Any parameters that were not normally distributed
were log transformed and retested for normality before parametric analysis.
Statistical analyses performed on proportions were arcsine transformed. All
statistical procedures followed recommendations of Zar
(1996) and
=0.05.
Results are presented as means ±
S.E.M.
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Results |
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Environmental parameters, i.e. salinity, depth, pH and temperature, were relatively consistent between sampling dates except for mean water temperature, which rose 2.8°C (Table 1). Within inhabited shelters, ammonia-N concentration was 8.1±1.0 µmol N l1 on 24 April 2003 and 9.7±1.5 µmol N l1 on 15 May 2003 while urea-N was measured at 17.1±2.6 µmol N l1 and 11.1±1.8 µmol N l1, respectively (Table 1). The mean value for waste-N concentration (ammonia-N + urea-N) was 23.0± 2.1 mmol N l1, and the maximum waste-N value was 76.2 mmol N l1, occurring in a nest containing one male, one female and freshly laid eggs. Ammonia and urea measured within shelters originated from resident toadfish, with background ammonia levels in the adjacent water column being undetectable in all but two cases (both <2 µmol N l1) and background urea never being detected. Resident toadfish excreted 9.6100% of waste-N as urea-N; however, the majority of these toadfish were classified as functionally ureotelic (35 of 51). Overall, toadfish with offspring present excreted 58.3±2.8% of waste-N as urea-N (N=46) while toadfish without offspring excreted 59.1±12.0% urea-N (N=5) and there was no statistical difference between groups (t=0.2080, P=0.83, d.f.=51).
The stomachs and intestines of O. beta were generally devoid of food, but mud and shell hash was occasionally present, indicating sediment excavation. Only two guardian males had food items present, one with five toadfish eggs (possibly culled) and the other with a 7 cm TL toadfish (presumably an unwelcome visitor).
Daily nitrogen flux experiment
Juvenile O. beta were predominantly ureotelic over the 72 h trial,
excreting 81.9±4.0% (N=7) of their waste-N as urea-N. In
addition, ammonia and urea excretion rates increased slightly after the first
24 h interval (Fig. 2). The
mean urea-N daily flux rate for juvenile toadfish over the 72 h test was
1.018±0.084 µmol N g1 h1 whereas
ammonia-N was 0.235±0.095 µmol N g1
h1.
|
Hourly nitrogen flux experiment
Juveniles sampled hourly were also determined to be predominately
ureotelic, excreting 65.7±7.4% of waste-N as urea-N over the 26 h
experiment. The mean urea-N daily flux rate was 1.275±0.327 µmol N
g1 h1 while ammonia-N was
0.726±0.212 µmol N g1 h1.
Background urea or ammonia was not detected in the three blank control vials.
All juveniles tested were noted to excrete urea in pulsatile events as do
adults; however, urea also appeared to be excreted continually at a lower
basal rate similar to that of ammonia excretion
(Fig. 3). Urea pulses accounted
for 62.0±5.9% (N=7 fish) of cumulative urea excreted, and
these pulses are defined as short duration increases (>2-fold) in the basal
excretion rate.
|
Ammonia toxicity
The total ammonia 96-h LC50 value for juvenile O. beta
was calculated as 875 µmol N l1 with 13.3% and 60.0%
mortality for 500 µmol N l1 and 1000 µmol N
l1 treatments, respectively
(Fig. 4). At pH 8.23, the
corresponding NH3 concentration was calculated as 38 µmol N
l1. Prior to death, gill opercula tended to flare open,
exposing unusually red gill filaments accompanied by a loss of balance and a
subsequent loss of buoyancy.
|
OUC enzyme activities and developmental stages
All developmental stages of O. beta expressed significant levels
of OUC enzymes. There was an overall trend of increased enzyme activities with
development stage and statistically significant differences were noted for
each enzyme assayed: GSase (F=25.9108, P<0.0001,
d.f.=23), CPSase (F=59.2644, P<0.0001, d.f.=23), OTCase
(F=12.2383, P=0.0001, d.f.=23) and arginase
(F=68.5322, P<0.0001, d.f.=23)
(Fig. 5). The majority of total
CPSase existed as CPSase III, which is the first enzyme in the teleost OUC.
However, CPSase II accounted for 19.1% of total CPSase activity in the egg II
stage and gradually declined to 7.7% in the juvenile stage
(Fig. 6). These changes in
CPSase II and CPSase III activities were statistically significant across
developmental stages (F=16.8173, P<0.0001, d.f.=23).
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Elevated ammonia and OUC enzyme activities
The effect of environmental ammonia exposure on OUC enzyme activity was
studied in the juvenile survivors of the ammonia 96-h LC50 test.
Only the control, 500 µmol N l1 and 1000 µmol N
l1 treatments had sufficient biomass for analysis. There
were no statistical differences between treatments in GSase, CPSase III or
arginase activities; however, there was a 3-fold rise in OTCase activity in
the 1000 µmol N l1 treatment over the control group,
which was significantly different from the control and 500 µmol N
l1 treatments (F=5.38, P=0.0138, d.f.=17)
(Fig. 7). There was also no
significant difference in the activity of either CPSase II or CPSase III
(F=2.1420, P=0.1734, d.f.=11) among treatment groups, with
the proportion of CPSase III comprising 89.892.5% of total CPSase
activity.
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Discussion |
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Environmental parameters measured in Florida Bay were consistent between
sampling dates and reflected seasonal weather trends. Water temperatures
recorded at spawning sites in the present study were 6°C higher than
previously reported for O. beta at a site 200 km NW of our FB-1
(Breeder, 1941
). Success of
spawning may be less dependent upon actual water temperature than anticipated
and thus less susceptible to inter-annual temperature fluctuations unless each
population represents a distinct ecotype.
Ureotely in early life history stages
OUC enzyme activities measured in embryonic, larval and juvenile O.
beta demonstrate that the metabolic pathway for ureogenesis is present
during the entire life history of this species. The nitrogen excretion data
indicate that juvenile O. beta are ureotelic and that urea excretion
was primarily pulsatile when juvenile fish were kept in relatively confined
conditions. It is likely that juveniles possess the same mechanism for urea
excretion as adults, including expression of tUT, a facilitated diffusion urea
transporter in the gill (Walsh et al.,
2000). Further experiments are necessary to validate this
hypothesis.
In the present study, mass-specific OUC enzyme activities were found to increase steadily from the embryonic to the juvenile stage. However, the increased activities probably reflect an initial dilution effect caused by yolk (presumably lacking OUC enzymes), which is gradually consumed during subsequent development. It is likely that OUC enzyme activities are at sufficient levels to synthesize urea de novo during each developmental stage. For example, in juveniles, CPSase III activity is 0.028 µmol min1 g1 while urea excretion is 0.008 µmol min1 g1 (Table 2), demonstrating an excess capacity for this apparent rate-limiting enzyme relative to excretion rates. However, the functionality of the OUC remains unknown for embryonic and larval stages of O. beta. Additionally, CPSase II accounted for 19.1% of total CPSase activity in the egg II stage and gradually declined to 7.7% by the juvenile stage. This reduction in CPSase II probably represents an ontogenic shift in pyrimidine biosynthesis while toadfish develop through early life stages.
|
In a study of the congener O. tau, yolk-sac larvae and juveniles
were found to be ureotelic while embryos were ammoniotelic
(Stephen and Griffith, 2001).
However, urea may accumulate within the embryo and yolk, which may explain the
>5-fold post-hatch increase in urea excretion reported by Stephen and
Griffith (2001
). In embryonic
rainbow trout, Pilley and Wright
(2000
) suggest that the
yolk-sac membrane is relatively impermeable to urea and demonstrate that urea
excretion is dependent, in part, on a phloretin-sensitive facilitated urea
transporter. It is unknown if such a urea transport mechanism is functional in
the yolk-sac membrane of O. tau. Additionally, it should be noted
from a comparative aspect that juvenile O. beta were on average 82%
ureotelic during their first week as juveniles while O. tau were
60% ureotelic. These findings are consistent with studies of adults where
O. tau were classified as `moderately' ureotelic but where O.
beta were considered `highly' ureotelic
(Wang and Walsh, 2000
).
The mass-specific urea flux rate for juvenile O. beta (0.008
µmol min1 g1) is an order of magnitude
greater than for adults (0.0008 µmol min1
g1), which was anticipated considering mass-specific
metabolic rates are likely to be much higher in the actively growing juveniles
if toadfish conform to normal metabolic scaling patterns
(Schmidt-Nielsen, 1997). A
comparison of mass-specific OUC enzyme activities revealed that GSase, CPSase
and OTCase activities in adult livers
(Anderson and Walsh, 1995
) are
double those found in whole juveniles while the reverse is true of arginase
(Table 2). For adult toadfish,
GSase appeared to be the limiting OUC enzyme in terms of activity when GSase
transferase activities were converted to biosynthetic rates
(Anderson and Walsh, 1995
). In
juveniles, the biosynthetic rate for GSase is higher than that for CPSase III
(Table 2); however, the
potential capacity for de novo urea synthesis as demonstrated by
these activity measurements can account for observed rates of urea
excretion.
The adult OUC enzyme activities were measured in liver, the organ
traditionally associated with the OUC
(Mommsen and Walsh, 1991),
even though hepatic tissue comprises only 2.25% of total wet biomass
(Hopkins et al., 1997
). When
comparing adult liver with white muscle (see
Table 2), mass-specific CPSase
III, OTCase and GSase activities were approximately 100, 78 and 24 times
higher, respectively (Anderson and Walsh,
1995
; Julsrud et al.,
1998
; Walsh et al.,
2003
). However, it is believed that OUC enzyme activities have
been underestimated in adults, as recently demonstrated with GSase where liver
only accounted for 40% of `whole body' GSase activity while muscle comprised
28% of activity when using tissue mass to calculate glutamine synthetic
potential (Walsh et al.,
2003
). Approximate (but probably overestimated) enzymatic
activities are calculated in a simple model for GSase, CPSase and OTCase in
adult toadfish based on the assumption that liver comprises 2% of toadfish
biomass with the remaining 98% being muscle tissue
(Table 2). Muscle tissue
actually represents 70% of wet biomass
(Kennedy et al., 1989
);
however, activities of OUC enzymes in remaining tissue groups are unknown for
toadfish but are likely to have low enzymatic activities more similar to
muscle than liver, as was largely the case for GSase (see
Walsh et al., 2003
).
The adult whole body calculations in
Table 2 show that the
mass-specific enzymatic activities for GSase and OTCase are approximately an
order of magnitude greater in juveniles than adults, which is consistent with
the 10-fold difference in mass-specific urea excretion rates. However,
juvenile CPSase III activities are only double those of adults. In adults,
CPSase III activity is regulated by N-acetyl-L-glutamate
as a positive allosteric cofactor, UTP as an inhibitor and
phosphoribosylpyrophosphate for some activation
(Anderson and Walsh, 1995;
Julsrud et al., 1998
). In
juveniles, the in vivo regulatory conditions exerted on CPSase III
are unknown and these may account for the differences seen between adults and
juveniles for the in vitro CPSase III activities.
Nest fouling hypothesis
Juvenile O. beta are less tolerant to ammonia than adults when
comparing the 96-h LC50 value of 0.875 mmol N l1
in this study with 9.75 mmol N l1 reported in adults
(Wang and Walsh, 2000). The
functional role of the 3-fold increase in OTCase activity when exposed to 1000
µmol N l1 ammonia is puzzling and it appears that OTCase
may be more plastic in juveniles than adults. In vertebrates, no other
function for OTCase, other than its association with the OUC, has yet been
discovered. One possible explanation is that an excess of yolk-derived
arginine enters the OUC, is converted to ornithine by arginase and a
subsequent build up of ornithine could trigger the upregulation of OTCase
during ammonia stress. It is likely that juveniles were subsisting on
yolk-derived nutrients in this experiment since active feeding by juveniles on
Artemia was never observed. Active feeding was unlikely since
juveniles failed to survive past 1 month as juveniles except in a fallow
500-litre tank containing a wide assemblage of potential prey items (J. F.
Barimo, unpublished data).
GSase activity was unchanged in juveniles with 1000 µmol N
l1 ammonia exposure, but adult GSase activity in muscle
doubled with high ammonia exposure (Wang
and Walsh, 2000), and other factors also upregulate GSase activity
in liver (reviewed by Wood et al.,
2003
). The upregulation of GSase in association with a shift to
ureotelism and the relative position of GSase upstream from CPSase III are
thought to provide a critical control for the OUC in adult toadfish
(Wood et al., 2003
). However,
these initial results could suggest that either the GSaseCPSase axis
does not function as a control point in juveniles, that the OUC is already
functioning at maximum potential or that juvenile toadfish are obligately
ureotelic.
The level of ammonia measured within nesting shelters appeared to be below
the 96-h LC50 value for juveniles. In addition, the mean
concentration for waste-N (urea-N + ammonia-N) measured in shelters at FB-1
was 23.0±2.1 µmol N l1 (N=51) while the
maximum concentration was 76.2 µmol N l1. Thus, if all
excreted waste-N were expressed as ammonia it would still be far below the
LC50 value for juveniles. However, if the NH3 fraction
is calculated for the previous waste-N values based on field data (pH,
salinity and temperature), the mean waste-N value is 3.3±0.3 µmol N
l1 with a maximum concentration of 9.6 µmol N
l1. Although these values still fall below the calculated
NH3 96-h LC50 of 38 µmol N l1, the
safety margin for the prevention of nest fouling is reduced. Although the
ammonia 96-h LC50 test was not conducted with embryonic or larval
stages of development, their LC50 value is not expected to deviate
greatly from that of juveniles. In O. mykiss, an opposite trend was
noted where 85-day-old yolk-sac fry were actually less tolerant to ammonia
than both fertilized eggs and alevins by a factor of 20
(Rice and Stokes, 1975
).
Therefore, it appears unlikely that male toadfish are facultatively ureotelic
in order to avoid poisoning offspring with ammonia excretions in confined
nests with restricted water flow, especially since no statistically
significant difference was observed in percentage ureotely between males with
or without offspring present. This hypothesis also fails to explain why female
toadfish are also facultatively ureotelic
(Walsh et al., 1990
), since
they do not brood or guard nests, or that male toadfish are generally fasting
while rearing offspring, as determined by examination of gut contents.
However, the nest-fouling hypothesis cannot be confidently rejected without
ammonia 96-h LC50 values for embryos and larvae in addition to data
on the chronic effects of ammonia exposure to growth and survivorship.
Furthermore, the variability of environmental parameters challenging the
toadfish in the field, i.e. pH, temperature and salinity, needs careful
consideration.
The data in the present study may, however, lend support to the hypothesis
that toadfish produce urea to conserve nitrogen since guardian male toadfish
appeared to be fasting. If male toadfish are fasting for long durations, i.e.
4 or more weeks, then such a strategy may help conserve or recycle nitrogen
if, for example, mutualistic gut bacteria were harnessing energy by ureolysis
(Mommsen and Walsh, 1989;
Wood et al., 2003
). However,
this hypothesis again fails to explain why ureotelism is a characteristic
trait of both sexes (Walsh et al.,
1990
) or even why ureotely occurs across the entire life history
of the species, yet it remains a hypothesis worthy of future investigation.
Perhaps the most viable remaining hypotheses regarding the adaptive
significance of ureotelism in toadfish are: (1) predator avoidance by chemical
crypsis since pulsatile urea excretion would enable sessile epibenthic
toadfish to avoid emitting a continual chemosensory cue to predators, as would
be the case with continual ammonia excretion; (2) chemical communication or
pheromones between conspecifics; and (3) a means of coping with high levels of
ambient ammonia resulting from the decomposition of vegetation in highly
productive seagrass beds, which is the likely habitat of non-nesting
(sub-adult) toadfish.
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