Tolerance of chronic hypercapnia by the European eel Anguilla anguilla
1 School of Biosciences, University of Birmingham, Birmingham, B15 2TT,
UK
2 Department of Pharmacological Sciences, Via Balzaretti, 9, University of
Milan, 20133 Milan, Italy
3 Marine Biological Laboratory, University of Copenhagen, Strandpromenaden
5, DK-3000 Helsingør, Denmark
* Author for correspondence at present address: CREMA-l'Houmeau, B.P. 5, 17137 l'Houmeau, France (e-mail: David.McKenzie{at}ifremer.fr)
Accepted 5 March 2003
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Summary |
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Key words: hypercapnia, European eel, Anguilla anguilla, acidbase balance, aerobic scope, hypoxia, metabolic rate, stress, swimming performance
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Introduction |
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Steffensen and Lomholt
(1990), however, measured
water PwCO2 levels exceeding 30 mmHg in
European eel farms using closed-cycle recirculating water systems. It is
unlikely that teleosts would ever experience such severe hypercapnia in
nature, and no previous investigations into the effects of chronic hypercapnia
have employed a PwCO2 of above 25 mmHg and
rarely for longer than 10 days (Heisler,
1984
,
1993
;
Dimberg, 1988
;
Larsen and Jensen, 1997
;
Fivelstad et al., 1998
,
1999
). Unusually low plasma
Cl concentrations have, however, been reported to occur
spontaneously in the European eel (Farrell
and Lutz, 1975
), which indicates that it may have an elevated
capacity to accumulate plasma HCO3 and compensate
for acidosis. Furthermore, McKenzie et al.
(2002
) found that the eel was
extremely tolerant of hypercapnic acidosis per se. That is, although
acute sequential 30 min exposures to water PCO2
levels (PwCO2) of 5, 10, 20, 40, 60 and 80 mmHg
caused a reduction in arterial pH (pHa) from 7.9 to below 7.2 and a consequent
80% decline in arterial blood total O2 content
(CaO2), this had no effect on cardiac output or
whole animal O2 uptake
(McKenzie et al., 2002
). The
selective pressures that led to the evolution of these physiological
adaptations are not known, but they do indicate that the eel may be
particularly tolerant of chronic hypercapnic acidosis and hypoxaemia.
Nonetheless, severely hypercapnic water
(Steffensen and Lomholt, 1990
)
should represent a sub-optimal aquaculture environment and, therefore, elicit
elements of a teleost stress response
(Barton and Iwama, 1991
;
Wendelaar Bonga, 1997
) in the
eels. In particular, chronic hypoxaemia consequent to Bohr and Root effects
has the potential to impair the regulation of O2 delivery, and
therefore aerobic metabolism, in response to changes in water O2
supply (hypoxia) or increased tissue O2 demand (e.g. sustained
aerobic exercise). Acute exposure to a PwCO2 of
25 mmHg caused a decline in the ability of the eel to regulate metabolic rate
in hypoxia (Cruz-Neto and Steffensen,
1997
).
In the present study, physiological adaptations to chronic severe
hypercapnia were examined by exposing eels to
PwCO2 levels of either 0.8 mmHg (ambient), 15
mmHg, 30 mmHg or 45 mmHg for 6 weeks. The extent of acidbase
compensation was assessed both with reference to control normocapnic eels, and
to the acidbase disturbances elicited by acute exposure to hypercapnia
(McKenzie et al., 2002), to
investigate the hypothesis that a capacity to tolerate low plasma
Cl concentrations would permit the accumulation of large
quantities of plasma HCO3. The hypothesis that
chronic hypercapnia would elicit a stress response in the eels was
investigated using plasma catecholamine and cortisol titres, plasma osmolality
and standard metabolic rate as indicators
(Barton and Iwama, 1991
;
Wendelaar Bonga, 1997
).
Finally, responses to progressive hypoxia and sustained aerobic exercise
performance were measured to investigate the hypothesis that hypoxaemia would
impair the eels' capacity to regulate O2 delivery and aerobic
metabolism. The results reveal that the European eel is exceptionally tolerant
of chronic hypercapnia, and only confirm the first of the three stated
hypotheses.
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Materials and methods |
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Duplicate tanks, of each size of eel, were then exposed to one of four
PwCO2 levels: 0.8±0.1 (ambient control),
15±1 mmHg, 30±1 mmHg and 45±1 mmHg.
PwCO2 levels were monitored indirectly as water
pH and regulated around each hypercapnic setpoint with the automated feedback
system described in McKenzie et al.
(2002). The eels were
acclimated to hypercapnia gradually, by increasing the
PwCO2 by 5 mmHg every 23 days. Thus, 9
days were required to reach a PwCO2 of 15 mmHg,
higher levels requiring proportionally more time. The eels were then exposed
to their appropriate PwCO2 for at least 6
weeks' prior to use in any experiments, and were fed at a rate of 1% body mass
day1 throughout the exposure period.
Surgical preparation
The large eels were anaesthetised and cannulated in the dorsal aorta and
operculum, as described in McKenzie et al.
(2000), in water equilibrated
either with air (controls) or the appropriate air/CO2 mixture.
Following surgery, fish were transferred to individual 6 l opaque black
Plexiglas chambers where they recovered for 48 h in a continuous flow of water
at the appropriate PwCO2. The two cannulae were
drawn out of a small hole in the lid of the chamber so that they could be
manipulated without disturbing the fish. The dorsal aortic cannula was flushed
twice daily with heparinised (10 i.u. l1) Cortland's saline
(Wolf, 1963
).
Blood acidbase and dissolved gas status
Following 48 h recovery from surgery, a 2 ml blood sample was collected
from the dorsal aortic cannula and replaced by an equal volume of Cortland's
saline. Arterial blood pH (pHa) was measured with a Radiometer BMS2 capillary
pH electrode (Brønshøj, Denmark) thermostatted to the same water
temperature as the fish, with the signals displayed on a Radiometer PHM73
acidbase analyser. Arterial blood total CO2 content
(CaCO2) and total O2 content
(CaO2) were measured as described by Cameron
(1971) and Tucker
(1967
), respectively, with
appropriate Radiometer electrodes thermostatted to 37°C. Arterial plasma
PCO2 (PaCO2) and
arterial [HCO3] were calculated from the measured
values of pHa and arterial [CO2] using the
HendersonHasselbach equation and apparent pK and
CO2
values for trout plasma at 23°C, calculated as described in Boutilier et
al. (1984
). The remaining blood
was rapidly centrifuged and the decanted plasma stored in liquid nitrogen for
subsequent analysis of osmolality, ion concentrations and hormone titres.
Ventilation
In all cases, ventilation was measured prior to collection of the blood
sample and care was taken to minimise disturbance to the fish prior to and
during measurements. The water-filled opercular cannula was attached to a
differential pressure transducer (Validyne 45DF, Northridge, CA, USA) and gill
ventilation rate (fG, beats min1) and opercular
pressure amplitude (POP, in Pa) were displayed on a chart
recorder (Gould Windograf, Valley View, OH, USA) as described by McKenzie et
al. (2000). Following
manipulation of the opercular cannula, the eels were allowed at least 1 h for
ventilatory activity to stabilise prior to collection of data for 30 min. To
quantify ventilation, the rate was counted for three 5 min periods within each
sampling interval, and POP averaged from 10 measurements
of individual waveforms within that period. POP was used
as an index of ventilatory effort.
Tissue intracellular pH
Following collection of the blood sample, the eels were injected with 1 ml
of a 5 g l1 solution of MS-222 in saline, which caused loss
of ventilatory movements within 5 s. The animals were removed rapidly from
their chamber, decapitated, and samples of white muscle and the heart
`freeze-clamped' with aluminium tongs and frozen in liquid nitrogen within 30
s of death. Tissue intracellular pH (pHi) was measured as described by
Pörtner et al. (1990).
The tissue was ground to a fine powder while frozen under liquid nitrogen. The
powdered tissue was then defrosted in a sealed microcentrifuge (Eppendorf)
tube containing metabolic inhibitor solution and, following centrifugation,
the pH of the supernatant was measured with a Radiometer capillary electrode
thermostatted to the temperature of the fish and attached to a Radiometer PHM
73 blood-gas analyser. Care was taken to ensure that the Eppendorf tube
contained absolutely no air bubbles, as preliminary measurements revealed that
defrosting and centrifugation of the samples with access to air caused highly
variable tissue pH values, presumably as a consequence of the rapid loss of
gaseous CO2 from the defrosted sample's supernatant.
Plasma ion concentrations and osmolality
Plasma Cl concentration was measured amperometrically
with a chloride titrator (American Instruments Company, USA). Plasma cations
(Na+ and Ca2+) were analysed by atomic absorption
spectrophotometry (Phillips PYE Unicam SP9, Cambridge, UK). Plasma osmolality
was measured with an osmometer (Fiske One-Ten, Fiske Associates, Norwood, MA,
USA).
Plasma catecholamines and cortisol
Catecholamines were extracted from 250 µl samples of plasma added to
2.75 ml of phosphate buffer, pH 3, with octanesulphonic acid (0.02 mg
ml1) and then loaded onto a Sep-Pak C18 cartridge
(Waters, Milford, MA, USA) preactivated by washing with methanol (3 ml) and
water (6 ml). The cartridge was eluted with 5 ml of water and 5 ml of 30%
methanol, pH 3. The organic phase was collected and dried under vacuum and the
residue suspended in 125 µl of methanol. Recovery rates were approximately
90% on extracted standards (three determinations). The extracted samples were
measured by high performance liquid chromatography (HPLC) (Waters) with a
Model 510 pump (Waters) and Rheodyne 7715 injector (Rohnert Park, CA, USA). A
50 µl sample was injected onto a 5 µm Symmetry (Waters) C18
Shield column (250 mm x 4.6 mm i.d.) with precolumn. The eluent was
composed of two solutions, A and B (87:13 mixture, v/v), where A was 100 mmol
l1 NaH2PO4, 100 mg
l1 EDTA, 250 mg l1 octansulphonic acid, pH
3, and B was a 3:2 (v/v) solution of methanol:acetonitrile. Elution was
performed in isocratic mode at a flow rate of 1.3 ml min1. A
coulometric detector (Coulochem II, ESA, Chelmsford, MA, USA) was used with
the following analytical conditions: Guard cell, +250 mV; Cell 1, 50
mV; Analytical cell, 200 mV (gain range 200 nA). The data were acquired
and integrated by dedicated software (Waters Millenium 2010). Samples were
compared against curves derived from standard solutions of noradrenaline (25.5
ng l1) and adrenaline (29.5 ng l1) (Sigma,
Sigma-Aldrich Srl, Milan, Italy).
Cortisol was extracted by adding 250 µl plasma to 20 µl of glacial
acetic acid, loading the resultant solution onto Ultrafree-PF filter (5000
Mr cut-off, Millipore, Bedford, MA, USA), and filtering by
centrifuging at 100 g for 10 min. The filtrate was then
transferred to a 10 ml test tube, and extracted twice with 4 ml of diethyl
ether. The organic phases were collected, dried under vacuum, and the residue
resuspended in 70 µl of methanol. Recovery was approximately 90% when
measured on duplicate standard samples. Cortisol was measured by HPLC, as
described in Volin (1992). A
50 µl sample of extract was injected onto a Symmetry C18 250
mmx4.6 mm i.d. column with precolumn. Analyses were performed in
gradient mode using two pumps (Waters Model 510) equipped with a Rheodyne 7715
injector coupled with a Model 996 photodiode-array detector (Waters). The
eluents were (A) 30 mmol l1 NaH2PO4,
pH 3, and (B) acetonitrile, and the linear gradient was 30 min at 40:60 ratio
of A:B, followed by 35 min at 100% A; flow-rate was 1.5 ml
min1. Detection was carried out at 245 nm and the
chromatograms stored in the range 200400 nm. The data were acquired and
integrated by dedicated software (Waters Millenium 2010). Samples were
compared against a curve derived from a standard solution of cortisol (50 mg
l1) (Sigma).
Metabolic rate and tolerance of hypoxia
Metabolic rate was measured as O2 uptake. Automated intermittent
flow-through respirometry (Steffensen,
1989) was used to measure the instantaneous O2 uptake
rates (
O2, in mmol
O2 kg1 h1) of individual
non-instrumented eels once every 10 min, using a system described in detail by
McKenzie et al. (2000
). Eels
were placed in a respirometer chamber and allowed to recover overnight in
water at the appropriate PCO2.
O2 was then measured for
24 h, and routine metabolic rate (RMR) calculated as the average rate measured
over the entire 24 h period. Standard metabolic rate (SMR), the amount of
O2 required for minimum maintenance metabolism, was estimated as
the average of the six lowest measured values of
O2 for each individual
animal within the 24 h period, thus representing, a cumulative time of 1 h
(Cruz-Neto and Steffensen,
1997
).
Hypoxia tolerance in the eels was assessed as the critical water
PO2 level (PwO2)
at which the eels were no longer able to maintain SMR during progressive
hypoxia (McKenzie et al.,
2000). Eels from each experimental group were exposed to gradual
progressive hypoxia, with PwO2 reduced from
saturation (PwO2=circa 150 mmHg) to
120, 100, 80, 60, 40, 30 and 20 mmHg every 20 min (two complete 10 min
measurement cycles of the automated system), and the critical
PwO2 for maintenance of SMR then calculated as
described by McKenzie et al.
(2000
).
Sustained aerobic exercise performance
Exercise performance and associated respirometry were measured on small
eels (mean live mass approximately 100 g) with the automated Brett-type
swim-tunnel respirometer described by McKenzie et al.
(2001). Eels were placed in
the respirometer and trained to swim at a water speed equivalent to 0.5 body
lengths per second (BL s1) for at least 12 h
(overnight). The following day, the eels were exposed to progressive
increments in swimming speed, of 0.25 BL s1 every
40 min, until exhaustion. Measurements of
O2 were collected at each
swimming speed, and used to calculate the theoretical rate of O2
uptake of a stationary resting fish (Brett,
1958
). This notional value, termed `immobile metabolic rate'
(IMR), was used to derive aerobic scope and the net aerobic metabolic cost of
swimming at each speed (Beamish,
1978
), as described in detail by McKenzie et al.
(2001
). Maximum sustainable
(critical) swimming speed (Ucrit) was calculated (in
BL s1) as described by Brett
(1964
).
Statistical analyses
Comparisons amongst groups for any given variable were made by one-way
analysis of variance (ANOVA) with Bonferroni post-hoc tests to
establish significant differences amongst groups. Statistical significance was
attributed at a 95% limit of confidence (P<0.05).
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Results |
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Extracellular and intracellular acidbase compensation
All of the hypercapnic groups exhibited a marked increase in
a[CO2], progressively from 13 mmol l1 in control
animals to almost 75 mmol l1 in animals exposed to 45 mmHg
PwCO2 (Table
1). This was associated with a progressive decline in pHa
(Table 1). Calculation of
PaCO2 revealed that it was equilibrated at
approximately 2 mmHg above PwCO2 in each group,
while plasma bicarbonate concentrations were elevated in all hypercapnic
groups, to above 70 mmol l1 in the group at a
PwCO2 of 45 mmHg
(Table 1). As can be seen in
the pH/bicarbonate (Davenport) diagram
(Fig. 1), the decline in pHa
during hypercapnia did not parallel the non-bicarbonate buffer line calculated
by Hyde et al. (1987) for
plasma of the closely related American eel Anguilla rostrata, but
deviated significantly above it. Fig.
1 also carries data reported by McKenzie et al.
(2002
), describing the effects
on acidbase status of acute exposure to hypercapnia, consisting of
sequential exposure at 30 min intervals to
PwCO2 levels of 5, 10, 20, 40, 60 and 80.
Comparison with the data from the present study reveals that the animals in
chronic hypercapnia exhibited a profound accumulation of plasma bicarbonate
and a consequent compensation of pHa for any given
PaCO2. The compensation of pHa was, however,
only partial and animals at a PwCO2 of 30 and
45 mmHg were suffering from chronic extracellular acidosis
(Table 1). All of the
hypercapnic groups, however, regulated the intracellular pH of their white
muscle and heart to levels unchanged from those of normocapnic animals
(Table 1).
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|
Plasma ion concentrations
As shown in Fig. 2, there
was a highly significant negative linear relationship between plasma
bicarbonate and plasma chloride (P<0.0001), with an almost
equimolar loss of plasma chloride for each bicarbonate ion accumulated.
However, plasma Cl concentrations were only significantly
lower than the controls at a PwCO2 of 45 mmHg,
due to wide individual variations (Farrell
and Lutz, 1975) within all groups. There were no significant
effects of chronic hypercapnia on plasma Na+ or Ca2+
concentrations (Table 2).
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|
Blood O2 content and ventilatory responses
The increased PaCO2 in the hypercapnic
groups, and the accompanying extracellular acidosis, was linked to a
progressive reduction in CaO2, presumably as a
result of Root and Bohr effects on haemoglobin-O2 binding. The
hypoxaemia was significant in animals at 30 and 45 mmHg
PwCO2 (Table
3). Fig. 3 compares
the percentage reduction in CaO2 in eels from
the present study with data replotted from McKenzie et al.
(2002), describing the effects
of acute hypercapnia. The comparison reveals that during chronic exposure the
eels had a less severe hypoxaemia at any given
PaCO2. However,
Fig. 3 also shows that the
relationship between pHa and CaO2 was the same
in eels exposed to chronic hypercapnia (this study) and in those exposed to
acute hypercapnia as reported by McKenzie et al.
(2002
). Thus, the increased
CaO2 at any given
PwCO2 in chronic versus acute exposure
was only a result of compensation of pHa, not modifications of
haemoglobin-O2 affinity. Despite the chronic hypoxaemia at a
PwCO2 of 30 or 45 mmHg, however, there were no
significant effects on either ventilatory frequency or effort, where this
latter was measured as POP
(Table 3).
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|
Stress indicators
Exposure to hypercapnia had no effect on plasma noradrenaline, adrenaline
or cortisol levels, which were low in all groups
(Table 4). There were no
differences in plasma osmolality amongst the four experimental groups, the
evidence of increased osmolality at a PwCO2 of
30 mmHg and 45 mmHg was not significant
(Table 4). Furthermore, as can
be seen in Table 4, there were
no significant effects of hypercapnia on RMR or SMR.
|
Regulation of aerobic metabolism during hypoxia and sustained aerobic
exercise
In all groups, exposure to hypoxia was associated with a decline towards
and beyond SMR as hypoxia deepened (data not shown), but there was no effect
of hypercapnia on the critical water PO2 for
maintenance of SMR (Table 5),
indicating that all groups were equally tolerant of hypoxia. There were also
no significant effects of hypercapnia on any aspect of exercise metabolism or
performance. The eels from all groups showed an exponential increase in
O2 consumption with increased swimming speed,
Fig. 4 shows this exponential
relationship between swimming speed and total O2 uptake in the
control group and the group exposed to the highest
PCO2, at 45 mmHg, and also the power
relationship between swimming speed and net cost of swimming in these same two
groups. These exponential and power relationships were essentially
indistinguishable amongst all the four groups, and maximum
O2, aerobic scope and
Ucrit were not statistically different
(Table 5).
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Discussion |
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The accumulation of plasma HCO3 to levels
above 70 mmol l1 by the eels exposed to a
PwCO2 of 45 mmHg is the highest yet reported in
a teleost (Börjeson, 1977;
Jensen and Weber, 1982
;
Dimberg, 1988
;
Larsen and Jensen, 1997
) and
was linked to the ability of the European eel to tolerate unusually low plasma
Cl levels (Farrell and
Lutz, 1975
), as demonstrated by the inverse approximately 1:1
relationship between plasma [HCO3] and plasma
[Cl]. A similar equimolar relationship has been described in
rainbow trout exposed to hypercapnia
(Larsen and Jensen, 1997
). The
HCO3 accumulation allowed the eels to compensate
for the acute acidosis that occurs upon initial exposure to hypercapnia
(McKenzie et al., 2002
). It
seems unusual, however, that the eels did not completely compensate for the
chronic extracellular acidosis at a PwCO2 of 15
or 30 mmHg if they were capable of the profound accumulation of
HCO3 observed at a
PwCO2 of 45 mmHg. That is, the eels tolerated
some degree of extracellular acidosis at all levels of hypercapnia. One
possible explanation for the incomplete regulation of pHa may be that the eels
regulated extracellular pH only as far as was required to ensure regulation of
pHi. Although it is well established that teleosts regulate pHi in hypercapnia
(Heisler, 1984
,
1993
), the present study may
have been the most extreme conditions for which such regulation has been
demonstrated.
Given the ability of the eel to regulate pHi during hypercapnia, the
primary physiological imbalances appear to have been the chronic extracellular
acidosis, exceptionally low plasma chloride levels and hypoxaemia. McKenzie et
al. (2002) found that an 80%
reduction in CaO2 elicited by acute hypercapnia
exposure had no effect on routine metabolic rate in the eel, so the reduction
in CaO2 levels of approximately 50% observed at
a PwCO2 of 45 mmHg in the present study would
not, therefore, be expected to limit the ability of the eels to meet their
routine O2 requirements. Indeed, there were no significant effects
of chronic hypercapnia on RMR or SMR, and this would explain the absence of
any hyperventilation in the hypercapnic groups. However, the
PaCO2, pHa and
CaO2 levels measured in the hypercapnic groups
in the present study elicited a significant hyperventilation when they were
created by acute exposure (McKenzie et
al., 2002
), revealing compensation of the ventilatory response
during chronic exposure (Larsen and
Jensen, 1997
).
The circulating levels of the catecholamines adrenaline and noradrenaline
increase in teleost fishes immediately in response to a variety of physical
and environmental stresses that require enhanced O2 transport, such
as exhaustive exercise, hypoxia or hypercapnia
(Randall and Perry, 1992). It
is perhaps somewhat surprising, therefore, that the hypercapnic eels did not
show a chronic elevation of plasma catecholamines, despite their extracellular
acidosis and hypoxaemia. Catecholamines are, however, an indicator of acute
rather than chronic stress in fish
(Randall and Perry, 1992
), and
members of the genus Anguilla do not show a pronounced catecholamine
release when compared with salmonids
(Gilmour, 1998
). An elevation
of plasma cortisol is the most widely used primary indicator of chronic
sublethal physiological stress in fish
(Barton and Iwama, 1991
;
Wendelaar Bonga, 1997
). The
absence of any increase in plasma cortisol is an indication, therefore, that
hypercapnia at the levels tested was not stressful to the eel. In many
freshwater teleosts, reductions in plasma osmolality and increases in
metabolic rate can occur as a secondary consequence of an underlying endocrine
response (Wendelaar Bonga,
1997
). The absence of any variations in osmolality or SMR in the
eels is, therefore, consistent with the absence of an endocrine response, and
is further evidence that chronic hypercapnia did not present a sublethal
stress. The fact that hypercapnia did not cause a decline in SMR indicates
that there was no anaesthetic effect of CO2
(Bernier and Randall, 1998
).
Conversely, the absence of any increase in SMR, which represents the minimum
costs of organismal maintenance, may indicate that energetic costs for
acidbase and ion regulation are low in the eel, as they appear to be in
other teleosts (Morgan and Iwama,
1999
).
It was unexpected that the ability of eels exposed to hypercapnia to
regulate aerobic metabolism in hypoxia was equal to the normocapnic controls,
despite significant hypoxaemia in animals at
PwCO2 levels of 30 mmHg and 45 mmHg. A previous
study found that acute hypercapnia exposure decreased tolerance of hypoxia in
the eel (Cruz-Neto and Steffensen,
1997), and this difference from the results of the present study
may be linked to the severe effects of acute exposure on blood O2
content (McKenzie et al.,
2002
) that were ameliorated in chronic hypercapnia by the
compensation of acidbase status. The differences in hypoxia tolerance
may also have been a consequence of the effects of water chemistry on the
regulation of acidbase balance
(Larsen and Jensen, 1997
).
Cruz-Neto and Steffensen
(1997
) studied eels in water
with a hardness of 150 mg l1 as CaCO3, compared
with the hardness of 240 mg l1 as CaCO3 in the
present study. Nonetheless, the ability of the eels in the present study to
regulate their aerobic metabolism in hypoxia, despite quite profound
hypoxaemia, demonstrates that their cardiovascular and ventilatory systems
possess an exceptional capacity to meet the O2 demands of their
tissues (McKenzie et al.,
2002
).
In salmonids, sustained aerobic swimming performance is impaired by
elevated water CO2 levels
(Dahlberg et al., 1968), and
their maximum performance appears to be closely matched to the capacity of
their cardiovascular system for O2 convection, as reductions in
CaO2 cause reductions in aerobic scope, AMR and
Ucrit (Jones, 1971
;
Brauner et al., 1993
;
Gallaugher et al., 1995
,
2001
). Contrary to
expectations, chronic hypercapnia did not have any negative impact on these
performance traits in the eel and, despite a 50% reduction in
CaO2, the animals adapted to a
PwCO2 of 45 mmHg exhibited the same fivefold
increase in
O2 and
achieved the same AMR as observed in normocapnia. The fact that eels in
normocapnia did not perform better than those adapted to a
PwCO2 of 45 mmHg, despite having twice the
CaO2, leads to the conclusion that the capacity
for O2 convection in the eel is not closely matched to maximum
tissue O2 demands during exercise, but can exceed them. Although it
must be presumed that sufficiently extreme reductions in
CaO2 will eventually limit O2
convection and exercise performance, the present results may indicate that,
unlike in salmonids, aerobic scope and AMR in the eel can be determined by the
maximum capacity of the respiring tissues to use O2 and perform
work. Compensation of heart pHi may have been important in preserving cardiac
performance and O2 convection during exercise, although the eel
heart possesses an exceptional intrinsic tolerance of both hypercapnic
acidosis (McKenzie et al.,
2002
) and hypoxia (Davie et
al., 1992
). Compensation of pHi in the working muscles may have
ensured their maximum performance and oxygen consumption, and would explain
the absence of any effects of hypercapnia on the net aerobic metabolic costs,
the energetic efficiency (Beamish,
1978
), of swimming. Sustained aerobic exercise performance has
long been considered a valid means of revealing underlying sublethal stresses
in fish (Brett, 1958
;
Randall and Brauner, 1991
), so
these results are yet further evidence that the eel was not stressed by such
severe chronic hypercapnia.
In conclusion, the results indicate that the eel's exceptional tolerance of
chronic hypercapnia is due to an ability to withstand chronic extracellular
acidosis, hypoxaemia and extremely low Cl levels, an ability
to regulate pHi independently of extracellular acidbase status, and a
remarkable capacity to meet the O2 demands of routine and active
metabolism, despite hypoxaemia. The life history of the eel must have provided
the selective pressures that led to the evolution of these particular
physiological adaptations. One ecological trait that may be particularly
relevant is the habit of eels to make excursions into air, reportedly to
escape poor water quality or to migrate through damp vegetation to colonise
new water bodies (Tesch,
1977). Such air-exposure inhibits O2 uptake and
CO2 excretion, causing acidosis and hypoxaemia
(Hyde et al., 1987
), which can
be expected to be exacerbated if the animals were also exercising. This, then,
might select for the observed ability to tolerate acidosis, and also for an
ability to meet the O2 demands of routine and active metabolism
when O2 supply is limited a profound reserve capacity for
O2 convection by the cardiovascular system. The habit of the eel to
live in confined spaces when in water
(Tesch, 1977
), where
O2 supply may be limited, may also have selected for a reserve
capacity for O2 convection. It is less easy to speculate about
which selective pressures might have led to the eel's ability to withstand
extremely low plasma Cl levels
(Farrell and Lutz, 1975
), and
the unusual physiological adaptations that allow the eel to tolerate chronic
severe hypercapnia are all interesting topics for further study.
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Acknowledgments |
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References |
---|
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Barton, B. A. and Iwama, G. K. (1991). Physiological changes in fish from stress in aquaculture, with emphasis on the responses and effect of corticosteroids. Ann. Rev. Fish Dis. 1,3 -26.[CrossRef]
Beamish, F. W. H. (1978). Swimming Capacity. In Fish Physiology, vol. 7 (ed. W. S. Hoar and D. J. Randall), pp. 101-187. New York: Academic Press.
Bernier, N. J. and Randall, D. J. (1998). Carbon dioxide anaesthesia in rainbow trout: effects of hypercapnic level and stress on induction and recovery from anaesthetic treatment. J. Fish Biol. 52,621 -637.[CrossRef]
Börjeson, H. (1977). Effects of hypercapnia on the buffer capacity and haematological values in Salmo salar (L.). J. Fish Biol. 11,133 -142.
Boutilier, R. G., Heming, T. A. and Iwama, G. K. (1984). Physicochemical parameters for use in fish respiratory physiology. In Fish Physiology vol.10A (ed. W. S. Hoar and D. J. Randall), pp.403 -430. New York: Academic Press.
Brauner, C. J., Val, A. L. and Randall, D. J.
(1993). The effect of graded methemoglobin levels on the swimming
performance of chinook salmon (Oncorhyncus tshawytscha).
J. Exp. Biol. 185,121
-135.
Brett, J. R. (1958). Implications and assessment of environmental stress. In The Investigation of Fish-Power Problems (ed. P. A. Larkin), pp.69 -93. Vancouver: University of BC, Institute of Fisheries.
Brett, J. R. (1964). The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Bd Canada 21,1183 -1226.
Cameron, J. N. (1971). Rapid method for
determination of total carbon dioxide in small blood samples. J.
Appl. Physiol. 31,632
-634.
Cameron, J. N. and Randall, D. J. (1972). The effect of increased ambient CO2 on arterial CO2 tension, CO2 content and pH in rainbow trout. J. Exp. Biol. 57,673 -680.[Medline]
Cruz-Neto, A. P. and Steffensen, J. F. (1997). The effects of acute hypoxia and hypercapnia on oxygen consumption of the freshwater European eel. J. Fish Biol. 50,759 -769.[CrossRef]
Dahlberg, M. L., Shumway D. L. and Doudoroff, P. (1968). Influence of dissolved oxygen and carbon dioxide on swimming performance of largemouth bass and coho salmon. J. Fish. Res. Bd Canada 25,49 -70.
Davie, P. S., Farrell, A. P. and Franklin, C. F. (1992). Cardiac performance of an isolated eel heart: Effects of hypoxia and responses to coronary artery perfusion. J. Exp. Zool. 262,113 -121.[Medline]
Dimberg, K. (1988). High blood CO2 levels in rainbow trout exposed to hypercapnia in bicarbonate-rich hard freshwater methodological verification. J. Exp. Biol. 134,463 -466.[Medline]
Farrell, A. P. and Lutz, P. L. (1975). Apparent anion imbalance in the fresh water adapted eel. J. Comp. Physiol. 102,159 -166.
Fivelstad, S., Berit Olsen, A., Kloften, H., Ski, H. and Stefanson, S. (1999). Effects of carbon dioxide on Atlantic salmon (Salmo salar) smolts at constant pH in bicarbonate rich freshwater. Aquaculture 178,171 -187.[CrossRef]
Fivelstad, S., Haavik, H., Lovik, G. and Berit Olsen, A. (1998). Sublethal effects and safe levels of carbon dioxide in seawater for Atlantic salmon postsmolts (Salmo salar): ion regulation and growth. Aquaculture 160,305 -316.[CrossRef]
Gallaugher, P. E., Thorarensen, H. and Farrell, A. P. (1995). Hematocrit in oxygen transport and swimming in rainbow trout (Oncorhynchus mykiss). Resp. Physiol. 102,279 -292.[CrossRef][Medline]
Gallaugher, P. E., Thorarensen, H., Keissling, A. and Farrell, A. P. (2001). Effects of high intensity exercise training on cardiovascular function, oxygen uptake, internal oxygen transport and osmotic balance in chinook salmon (Oncorhynchus tshawytscha) during critical speed swimming. J. Exp. Biol. 204,2861 -2872.[Medline]
Gilmour, K. M. (1998). Gas exchange. In The Physiology of Fishes, 2nd edn (ed. D. H. Evans), pp. 101-127. Boca Raton: CRC Press.
Heisler, N. (1984). Acidbase regulation in fishes. In Fish Physiology Vol.10A (ed. W. S. Hoar and D. J. Randall), pp.315 -401. New York: Academic Press.
Heisler, N. (1993). Acidbase regulation. In The Physiology of Fishes (ed. D. H. Evans), pp.343 -378. Boca Raton: CRC Press.
Howell, B. J. (1970). Acidbase balance in the transition from water breathing to air breathing. Fed. Proc. 29,1130 -1134.[Medline]
Hyde, D. A., Moon, T. W. and Perry, S. F. (1987). Physiological consequences of prolonged aerial exposure in the American eel, Anguilla rostrata: blood respiratory and acidbase status. J. Comp. Physiol. B 157,635 -642.
Jensen, F. B. and Weber, R. E. (1982). Respiratory properties of tench blood and hemoglobin. Adaptation to hypoxic-hypercapnic water. Mol. Physiol. 2, 235-250.
Jones, D. R. (1971). The effect of hypoxia and anaemia on the swimming performance of rainbow trout (Salmo gairdneri). J. Exp. Biol. 55,541 -551.[Medline]
Larsen, B. K. and Jensen, F. B. (1997). Influence of ionic composition on acid-base regulation in rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia. Fish Physiol. Biochem. 16,157 -170.
McKenzie, D. J., Cataldi, E., Owen, S., Taylor, E. W. and Bronzi, P. (2001). Effects of acclimation to brackish water on the growth, respiratory metabolism and exercise performance of Adriatic sturgeon (Acipenser naccarii). Can. J. Fish. Aquat. Sci. 58,1104 -1112.[CrossRef]
McKenzie, D. J., Piraccini, G., Piccolella, M., Steffensen, J. F., Bolis, C. L. and Taylor, E. W. (2000). Effects of dietary fatty acid composition on metabolic rate and responses to hypoxia in the European eel, Anguilla anguilla. Fish. Physiol. Biochem. 22,281 -296.[CrossRef]
McKenzie, D. J., Taylor, E. W., Dalla Valle, A. Z. and Steffensen, J. F. (2002). Tolerance of acute hypercapnic acidosis by the European eel (Anguilla anguilla). J. Comp. Physiol. B 172,339 -346.[CrossRef][Medline]
Morgan J. D. and Iwama, G. K. (1999). Energy cost of NaCl transport in isolated gills of cutthroat trout. Am. J. Physiol. 277,R631 -R639.[Medline]
Pörtner, H. O., Boutilier, R. G., Tang, Y. and Toews, D. P. (1990). Determination of intacellular pH and PCO2 after metabolic inhibition by fluoride and nitrilotriacetic acid. Respir. Physiol. 81,255 -274.[CrossRef][Medline]
Randall, D. J. and Brauner, C. L. (1991). Effects of environmental factors on exercise in fish. J. Exp. Biol. 120,113 -126.
Randall, D. J. and Cameron, J. N. (1973).
Respiratory control of arterial pH as temperature changes in rainbow trout,
Salmo gairdneri. Am. J. Physiol.
225,997
-1002.
Randall, D. J. and Perry, S. F. (1992). Catecholamines. In Fish Physiology vol.12b (ed. W. S. Hoar, D. J. Randall and A. P. Farrell), pp. 255-300. San Diego: Academic Press.
Steffensen, J. F. (1989). Some errors in the respirometry of water breathers: how to avoid and correct for them. Fish. Physiol. Biochem. 6, 49-59.
Steffensen, J. F. and Lomholt, J. P. (1990). Accumulation of carbon dioxide in fish farms with recirculating water. In Fish Physiology, Fish Toxicology and Fisheries Management (ed. R. C. Ryans), pp.157 -161. Athens, Georgia: Environmental Research Laboratories US Environmental Protection Agency, EPA/600/9-90/011.
Tesch, F. W. (1977). The Eel. London: Chapman and Hall.
Tucker, V. A. (1967). Method for oxygen content
and dissociation curves on microliter blood samples. J. Appl.
Physiol. 23,410
-414.
Van Waarde, A., Thillart, van den G. and Kesbeke, F. (1983). Anaerobic energy metabolism of the European eel, Anguilla anguilla L. J. Comp. Physiol. B 149,469 -475.
Volin, P. (1992). Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection. J. Chromatography 584,147 -155.[Medline]
Wendelaar Bonga, S. E. (1997). The stress
response in fish. Physiol. Rev.
77,591
-625.
Wolf, K. (1963). Physiological salines for freshwater teleosts. Progr. Fish. Cult. 25,135 -140.