Temperature alters the respiratory surface area of crucian carp Carassius carassius and goldfish Carassius auratus
1 Department of Molecular Biosciences, University of Oslo, PO Box 1041,
N-0316 Oslo, Norway
2 Department of Zoophysiology, Institute of Biological Sciences, University
of Aarhus, C. F. Moellers Alle 131, DK-8000 Aarhus C, Denmark
* Author for correspondence (e-mail: jorund.sollid{at}bio.uio.no)
Accepted 20 January 2005
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Summary |
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Key words: teleost, crucian carp, goldfish, gill, morphology, temperature, Hb, haemoglobin, respiration, Q10
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Introduction |
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Being freshwater fish, crucian carp and goldfish are faced with a dilemma:
they have to cope with a continuous ion loss and water influx over the
respiratory surface area in the gills
(Evans, 1979), but still
maintain sufficient oxygen uptake. The water influx must be compensated by a
large urine production resulting in an even greater loss of ions. These ion
losses must be compensated by energetically demanding ion transport over the
gills. Thus, being able to modulate the respiratory surface area in response
to oxygen supply and demand should be of advantage.
We have previously shown that crucian carp kept in normoxia at 8°C lack
protruding lamellae, but if exposed to hypoxia, a morphological alteration is
triggered resulting in protruding lamellae and a 7.5-fold increase of
respiratory surface area (Sollid et al.,
2003). This caused a fall in the critical oxygen concentration
([O2]crit), i.e. the lowest ambient [O2]
where the fish is able to sustain its resting oxygen consumption
(
O2). The gill remodelling
is due to an induction of apoptosis and cell-cycle arrest in the mass of cells
filling up the space between adjacent lamellae, causing this interlamellar
cell mass (ILCM) to shrink. A reduction in respiratory surface area in
normoxia should lead to lower water and ion fluxes and thus reduction of
osmoregulatory costs. At the same time, the crucian carp's ability to maintain
a sufficient rate of oxygen uptake without protruding lamellae indicates a
very high oxygen affinity of its haemoglobin (Hb), which has remained to be
studied.
Fish are ectothermic organisms; hence increased temperature profoundly
raises their metabolic rates. Increased temperature also decreases the amount
of oxygen dissolved in the water. Temperature-related changes in metabolism
are met with behavioural, respiratory, cardiovascular, hematological and
biochemical adjustments (Aguiar et al.,
2002; Burggren,
1982
; Butler and Taylor,
1975
; Caldwell,
1969
; Fernandes and Rantin,
1989
; Goldspink,
1995
; Houston et al.,
1996
; Houston and Rupert,
1976
; Maricondi-Massari et
al., 1998
). The responses to increased temperature may include air
gulping, increased gill ventilation, increased lamellar perfusion, increased
cardiac output, changes in Hb function and altered expression of metabolic
enzymes. Studies related to gill morphology and temperature are scarce and
only cover acute temperature changes
(Hocutt and Tilney, 1985
;
Jacobs et al., 1981
;
Nolan et al., 2000
;
Tilney and Hocutt, 1987
),
which often reflect more pathophysiological responses that are not necessarily
adaptive.
Changes in Hb function could result from changes in the levels of
erythrocytic effectors such as organic phosphates (ATP, often supplemented by
guanosine triphosphates in fish) or changes in Hb isomorphs
(Weber, 2000). In addition to
the `standard' electrophoretically `anodic' Hb components that display
pronounced Bohr shifts, some fishes (salmonids, catfishes and eels) also have
electrophoretically `cathodic' Hbs, that have lower Bohr shifts and show
divergent phosphate sensitivities (which are insignificant in salmonids and
large in eels and catfishes). Hb composition of goldfish (that is closely
related to crucian carp) changes with temperature: electrophoresis reveals two
isoHbs in fish acclimated to 2°C, and three isoHbs in fish acclimated to
20°C and 35°C (Houston and Cyr,
1974
). This modification also occurs in isolated cells and in
hemolysates, suggesting that it is caused by altered aggregation of
pre-existing subunits rather than de novo Hb synthesis
(Houston and Rupert,
1976
).
The aim of this study was to investigate if increased temperature, leading
to an increased oxygen demand, can trigger the morphological response recently
found in hypoxia-exposed crucian carp
(Sollid et al., 2003). At our
latitude the typical seasonal temperature range for the crucian carp habitat
is 0°C to 25°C. We thus acclimated crucian carp to temperatures
ranging from 10 to 25°C to examine the possible effects of changing oxygen
demand on gill morphology. In addition goldfish were acclimated at 7.5, 15 and
25°C to see if the gill remodelling seen in crucian carp also is expressed
in this closely related species when kept at low temperatures. Since goldfish
normally are kept at room temperature, an ability to remodel the gills may not
have been noticed. To identify adaptations in oxygen transport functions we
also investigated Hb multiplicity in fish acclimated to the different
temperatures, and measured the intrinsic oxygen-binding properties and
effector sensitivities of crucian carp Hbs.
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Materials and methods |
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Goldfish Carassius auratus L. (weighing 8.0-16.5 g; all adults),
bred and cultivated in Singapore, were bought from a commercial wholesaler.
They where kept in a tank (100 fish per 500 l) with aerated, ion strength
adjusted to 500 µS cm-1 (dH-Salt, NOR ZOO, Bergen, Norway) and
dechlorinated Oslo tap water (25°C) for 1 month before experiments. The
light regime and feeding were the same as for crucian carp.
Temperature acclimation
Crucian carp were transferred to new holding tanks (10 fish per 25 l)
held at 10°C, 15°C, 20°C and 25°C, respectively, and
acclimated 1 month before respirometry experiments (see below). The fish were
fed until 24 h before respirometry. Each fish was placed in the respirometer
with a continuous flow of aerated water until 12 h before commencing
measurements. To examine if gill morphology was affected by the respirometry,
four fish from each group were sampled before and after respirometry and the
left first and second gill arches were dissected out. As a control for
possible effects of the confinement in the respirometer, crucian carp kept at
15°C were placed in the respirometer for 24 h and continuously supplied
with aerated and dechlorinated Oslo tapwater. After exposures, the fish were
killed with a sharp blow to the head.
Goldfish were transferred to a new container (10 fish per 25 l) with
ion strength adjusted, aerated and dechlorinated Oslo tapwater (25°C) for
1 month, whereafter the gills of four fish, were sampled. Subsequently, the
water temperature in the container was reduced to 15°C. After 5 days at
this temperature four additional fish were sampled. The temperature was
finally reduced to 7.5°C and the gills of four fish were sampled after 5
days and 1 month at this temperature. The fish were fed during temperature
acclimation. The fish were killed for dissection of the left first and second
gill arches and treated as the crucian carp.
Respirometry
O2 during falling water
oxygen concentration was measured with closed respirometry, and the
[O2]crit was determined as described previously
(Nilsson, 1992
). The
temperature in the 1 l respirometer was the same as the acclimation
temperature. Oxygen levels in the respirometer were measured with an oxygen
electrode (Oxi340i, WTW, Weilheim, Germany) and recorded on a laptop computer
via an analog-digital converter (Powerlab 4/20, AD Instruments Ltd.,
Oxon, UK). The fish were removed from the respirometer for dissection of gills
when the recorded oxygen content became 0 mg O2 l-1.
Scanning electron microscopy (SEM)
The gill morphology of all groups was investigated as previously described
(Sollid et al., 2003). In
brief, gills were fixed in 3% glutaraldehyde in 0.1 mol l-1 sodium
cacodylate buffer before dried, AuPd coated, and examined using a JSM 6400
electron microscope (JEOL, Peabody, USA).
Hb oxygen binding
IsoHb composition was probed using PhastSystem (Amersham Biosciences,
Piscataway, NJ, USA) by isoelectrofocusing on polyacrylamide gels in the 5-8
pH range. The crucian carp had been acclimated for 1 month at 16°C or
26°C prior to blood samples.
Crucian carp Hb for oxygen-binding studies was prepared from washed red
cells as previously described (Weber et
al., 1987). The Hb was `stripped' of ionic effectors by column
chromatography on Sephadex G25 Fine gel
(Berman et al., 1971
). Major
isoHbs were separated using preparative isoelectric focusing using Pharmacia
ampholytes (0.22% pH 5-7, 0.22% pH 6-8 and 0.11% pH 6.7-7.7). Retrieved pools
were concentrated using Amicon Ultra-15 (molecular weight cut-off 10.000)
filters. All Hb samples were subsequently dialyzed for at least 24 h against
three changes of 10 mmol l-1 Hepes buffer containing 0.5 mmol
l-1 EDTA. All preparation procedures were carried out at 0-5°C.
Samples were frozen at -80°C and freshly thawed for subsequent analyses.
O2 equilibrium measurements at different pH values and in the
presence of 0.1 mol l-1 KCl were carried out using a modified gas
diffusion chamber as previously detailed
(Weber, 1981
;
Wells and Weber, 1989
).
Statistics
All values are given as means ±
S.E.M. and statistically significant
differences were detected with a one-way ANOVA test with Tukey's test as post
test using GraphPad InStat (GraphPad, San Diego, CA, USA).
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Results |
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Throughout closed respirometry, the oxygen tension in the respirometer
drops, eventually to a level below the [O2]crit. Hence
the fish will experience a hypoxic environment and finally anoxia (0 mg
O2 l-1). At 8°C an increase of respiratory surface
in hypoxia takes 3 days before it is pronounced
(Sollid et al., 2003). The
present results show, that this time period is dramatically reduced at higher
temperatures. In the respirometer at 15 and 20°C the fish experienced
hypoxia and anoxia on average 6 h before sampled. Crucian carp at 15°C
(not shown) and 20°C (Fig.
1d) underwent the characteristic remodelling of their gills to
increase the respiratory surface area during these few hours in the
respirometer. This change was not due to confinement (not shown).
In our previous study, morphometric measurements indicated a 7.5-fold
increase in the lamellar area exposed to water in crucian carp kept in hypoxia
(Sollid et al., 2003
). The
gill morphological changes of crucian carp kept at 25°C, and exposed to
15°C and 20°C in the respirometer in this study appeared to be
identical in extent to those seen after hypoxia in our previous study.
However, since the gills were only examined by SEM in the present study, no
quantitative morphometrical measurements were attempted.
Goldfish at 20°C had protruding lamellae (not shown), which were indistinguishable from those seen in the 15°C group (Fig. 1f). However, in goldfish exposed to 7.5°C, a clear change in the gill filament morphology occurred. This was clearly visible after 5 days (Fig. 1e) and no further changes were apparent after 1 month (not shown). The space between adjacent lamellae was partially filled with a cell mass, as seen in crucian carp, although slightly less pronounced, as the edges of the lamellae were still visible.
Respiration
The respirometry data for the crucian carp showed, as expected, that
O2 increased with
temperature (P<0.0001, Fig.
2A). A temperature rise from 10°C to 25°C increased the
O2 more than fivefold, from
38.9±4.5 mg kg-1 h-1 to 209.5±15.1 mg
kg-1 h-1 (P<0.001,
Table 1). The increase of
O2, from 10°C to
25°C, lead to an increase of [O2]crit from
1.43±0.13 kPa to 4.02±0.27 kPa (P<0.001,
Table 1). However, there was a
strikingly small increase in [O2]crit between 20°C
and 25°C (Fig. 2B). This
corresponds well with the transformation in gill morphology that occurred
between these two temperatures (Fig.
1b,d).
|
The relationship between
O2 and
[O2]crit in crucian carp
(Fig. 2C) was similar to
literature data for goldfish (Fig.
2F). Both species show relatively low
[O2]crit at high temperatures, which indicates an
improvement of their oxygen uptake capabilities that is likely to coincide
with the remodelling of the gills. By contrast, the Atlantic cod
(Schurmann and Steffensen,
1997
) shows a steady increase in [O2]crit
with rising
O2
(Fig. 2F), indicating that this
species is incapable of any major morphological or physiological adjustments
to improve its O2 uptake capacity at high temperatures. Also,
crucian carp showed lower [O2]crit values than the
goldfish. For example at a
O2 of approximately 85 mg
kg-1 h-1, the [O2]crit values were
2.4 kPa and 3.9 kPa for crucian carp and goldfish, respectively. This
indicates an ability of crucian carp to extract more oxygen from the
surrounding water than goldfish. The Q10 was also similar between
the two species (Table 2). However, in contrast to goldfish, crucian carp exhibited a higher
Q10 between 20-25°C than between 15-20°C
(Table 2).
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Hb and oxygen binding
The thin-layer isoelectrofocusing of Hbs from fish acclimated to 14 or
26°C (Fig. 3) showed at
least three major bands. Importantly no consistent differences were seen in
the number or relative intensities of the bands between fish acclimated to the
two temperatures.
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As shown (Fig. 4A,B),
stripped crucian carp Hbs show an extremely high oxygen affinity
(P50=0.8 and 1.8 at pH 7.6 at 10 and 20°C, respectively). The
Bohr effect that approximates -0.7 at pH 7.0 decreases markedly with
increasing pH and is virtually absent at pH above 7.7 at 20°C.
Interestingly, cooperativity increased with decreasing pH over the entire
range investigated (8.4-6.4, Fig.
4A), whereas n50 values at low pH fall to unity and
lower (reflecting anticooperativity) in fish Hbs that express Root effects
(Brittain, 1987). The oxygen
affinities decrease with increasing temperature (in agreement with the
exothermic nature of haem oxygenation). As expressed by the heats of
oxygenation (
H=58 and 49 kJ mol-1 at pH 7.6 and 7.0,
respectively) the temperature sensitivity of P50 decreases with pH.
This correlates with the parallel increase in the Bohr effect and, thus, in
the endothermic dissociation of the Bohr protons. By contrast, the ATP
sensitivity of the Hb decreases with increasing pH
(Fig. 4A) in accordance with
the associated decrease in positive charge of the phosphate binding sites.
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Crucian carp red cells contain at least three major isoHbs (II, III and IV)
and two minor ones (I and V). The elution profile
(Fig. 4C) indicates relative
abundance of 6% HbI, 27% HbII, 62% HbIII+IV and 5% HbV, and that Hbs I, II,
III and IV are isoelectric at pH values of 6.7, 6.4, 6.9 and 5.8,
respectively. All components exhibit similar, high oxygen affinities and
similar Bohr effects (P50 of 1.5-1.8 mmHg at pH 7.6 and 20°C,
and -0.30). These properties correspond with those of stripped
hemolysates, indicating the absence of functionally significant interaction
between the isolated components.
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Discussion |
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The increase of respiratory surface area of crucian carp kept at 25°C
coincided with a relatively low [O2]crit at this
temperature, indicating an increase in the capacity for oxygen uptake
(Fig. 2B,C). This was clearly
reflected in the high
O2:[O2]crit
ratio of crucian carp kept at 25°C
(Table 1). The relationship
between temperature,
O2 and
[O2]crit of crucian carp observed in the present study
resemble that found in a study on goldfish
(Fig. 2A-C) in more than half a
century ago (Fry and Hart,
1948
), which also showed an unexpectedly low
[O2]crit at higher temperatures. These results can now
be explained by the present finding that goldfish have protruding lamellae at
high, but not low, temperatures. The reason why this transformation of gill
morphology has not been observed in goldfish earlier is most likely that
goldfish are traditionally kept at rather high temperatures, usually at room
temperature.
By contrast, Atlantic cod, a species that presumably does not have the
ability to adjust the respiratory surface area to its oxygen needs, shows a
linear relationship between [O2]crit and
O2
(Fig. 2C; see also
Schurmann and Steffensen,
1997
).
The results suggest that the oxygen demand of crucian carp does not trigger
a remodelling of the gills unless the water temperature reaches 25°C,
which is near the highest temperature that crucian carp normally experiences
in its habitat for short periods during the summer months (J. S., G. E. N.,
unpublished observations from the Oslo area). This indicates that gills with
non-protruding lamellae are able to supply the crucian carp with sufficient
oxygen to sustain aerobic metabolism at 20°C where its
O2 is around 120 mg
kg-1 h-1 (Table
1). The capacity to sustain a high
O2 with a small respiratory
surface area could rely on a high O2 affinity of the Hb. We
measured oxygen affinity in the presence of 0.1 mol l-1 KCl, which
decreases the oxygen affinity, mimicking the intracellular condition. Our data
show that the high oxygen affinity (P50=1.8 mmHg at pH 7.7 and
20°C) increases markedly with falling temperature (P50=0.7 mmHg
at 10°C) due to the pronounced temperature sensitivity at high in
vivo pH (7.7), where the phosphate sensitivity is low
(Fig. 4A,B). These properties
that appear to characterise all major isoHbs
(Fig. 4D) witness a high blood
oxygen affinity as previously recorded in goldfish (P50=2.6 mmHg at
pH 7.56 and 26°C; Burggren,
1982
).
The remodelling of the gills appears to be rapid, since we did not observe
any intermediate stages in crucian carp kept at 15°C or 20°C. Thus, it
appears to be an `on/off' response that is triggered either by hypoxia or high
temperature, or maybe by their common denominator: an increased demand for
oxygen uptake. When we reduced the acclimation temperature for goldfish to
7.5°C, they remodelled their gills to a state with almost no protruding
lamellae. Since no intermediate stages were seen in goldfish gills during the
25°C to 15°C transfer, it seems, like in crucian carp, that this is an
`on/off' response that is triggered by either temperature or
O2.
Intriguingly, Isaia (1972)
showed that the water flux across the goldfish gills increased more than five
times from 5 to 25°C, which is much greater than would be expected from a
diffusion process. It is tempting to suggest that at least part of this
increased water flux was caused by an increase in the respiratory surface
area. Indeed, Isaia (1972
)
suggested that the `results must indicate either an important change in the
branchial permeability during adaptation or the functioning of a greater
respiratory surface at an increased temperature'. Moreover, it has been found
that the common carp Cyprinus carpio, exposed to chronic hypoxia, is
able to extract a higher percentage of the available oxygen than normoxic carp
(Lomholt and Johansen, 1979
).
This could imply that the common carp has the ability to alter its respiratory
surface area, possibly in a manner similar to that found in its cyprinid
cousins: crucian carp and goldfish. Moreover, a capacity for gill remodelling
to increase or decrease oxygen uptake and water fluxes may not be limited to
cyprinids. A gill morphology characterised by thickened lamellae with
epithelial cells being cuboidal or columnar instead of squamous has been seen
in juvenile largemouth bass kept at over-wintering temperatures close to
4°C (Leino and McCormick,
1993
).
The present data showed that the change from non-protruding to protruding lamellae occurs between 20 and 25°C in crucian carp, and between 7.5 and 15°C in goldfish. This may reflect species or population differences. Each year, the crucian carp we studied face a severely hypoxic and anoxic environment during the long winter period. Hence, they are more dependent on their glycogen stores than goldfish for survival. Thus, saving energy is likely to be a more critical feature for crucian carp. A small respiratory surface area over a large temperature interval will reduce osmoregulatory costs and, thereby, save energy that can be stored for surviving the long winter. There was also an apparent difference in the ability of these two species to handle soft water. The crucian carp population is well adapted to soft water, and do well in Oslo tapwater (20-50 µS cm-1), whereas goldfish did not do well (did not feed and were lethargic) in Oslo tapwater. Upon recommendation from the importer, we increased the conductivity in the goldfish water to 500 µS cm-1, which had a striking positive effect of the welfare of the goldfish. It is possible that these differences in water conductivity could be related to the difference seen in the temperature where gill remodelling takes place between the two species. However, at present we can only speculate.
It has been found previously that crucian carp acclimated to hypoxia has
higher O2 than normoxic
crucian carp (Johnston and Bernard,
1984
). This increase of
O2 could be due to
increased ventilation rates and/or elevated osmoregulatory costs for having a
larger respiratory surface area. Similarly, in the present study, the crucian
carp displayed a larger difference in
O2 between 20°C and
25°C (Q10=2.9) than between 15 and 20°C
(Q10=1.9) (Table 2),
which could be explained by the presence of protruding lamellae in the
25°C group, causing elevated osmoregulatory costs. By contrast,
ectothermic animals generally show Q10 values that fall with
increasing temperature (Prosser,
1986
; Withers,
1992
). Interestingly, between 10-15°C and 15-20°C, the
Q10 values in goldfish (Fry and
Hart, 1948
) (Table
2) decrease less than in the crucian carp, which may be explained
by the goldfish remodelling its gills at a lower temperature than the crucian
carp.
To conclude, the present study shows that both crucian carp and goldfish
have the ability to remodel their gills by changing the size of the ILCM
between the lamellae. Moreover, the response, which has previously shown to be
triggered by hypoxia, can also be triggered by temperature. Thus, at high
temperatures both goldfish and crucian carp display gills with clearly
protruding lamellae. The remodelling of the gills to gain protruding lamellae
is caused by increased apoptosis and cell-cycle arrest in the ILCM
(Sollid et al., 2003). In the
light of the present results, it is possible that the signals that trigger
this change could include both hypoxia and high temperature, or their common
denominator: the need for extracting more oxygen from the water. The ability
to match the respiratory surface area to oxygen needs may provide a means of
reducing water and ion fluxes and, thereby, the osmoregulatory costs. However,
our observations suggest that this is a sharp `on/off' response rather than a
graded change, since no intermediate stages are seen except during the short
transition from one state to the other. While this transition took several
days in hypoxia at 8°C (Sollid et al.,
2003
), the present study showed that, at 20°C, it could be
completed during the few hours that the fish were exposed to hypoxia in the
respirometer.
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Acknowledgments |
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