Preferred temperature of juvenile Atlantic cod Gadus morhua with different haemoglobin genotypes at normoxia and moderate hypoxia
Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingør, Denmark
* Author for correspondence (e-mail: mfpetersen{at}zi.ku.dk)
Accepted 28 October 2002
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Summary |
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Key words: haemoglobin, Atlantic cod, Gadus morhua, preferred temperature, hypoxia, swimming speed
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Introduction |
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When searching for the reason why Atlantic cod have polymorphic haemoglobin
we examined the frequency distribution of the two alleles coding for the
different haemoglobin types. The distribution is extremely heterogeneous
across the North Atlantic. The HbI2 allele reaches up to 99%
frequency in Greenland waters and is also dominant in northern Norway,
Iceland, the Faeroe Islands, Canada and in the northern part of the Baltic,
whereas the HbI1 allele is dominant in the warmer areas (Sick,
1965a,
b
;
Frydenberg et al., 1965
). The
difference between the homozygous genotypes is limited to an extra
histidine-containing peptide in the HbI-1 type, and therefore the structural
differences are only minimal (Rattazzi and
Pik, 1965
). However, differences in biochemical properties have
been described for the genotypes. The oxygen affinity of haemoglobin is higher
for HbI-2 cod at low temperatures (<10°C)
(Karpov and Novikov, 1981
;
Brix et al., 1998
;
McFarland, 1998
), and for
HbI-1 cod, at some blood pH values, at high temperatures (>14°C)
(Karpov and Novikov, 1981
;
Brix et al., 1998
). The
heterozygous haemoglobin type is generally found to have oxygen affinity
values that are intermediate to HbI-1 and HbI-2
(Karpov and Novikov, 1981
).
This information suggests that temperature could be a selective parameter for
the distribution of Atlantic cod with different haemoglobin types. The present
study examined whether this was the case by measuring the preferred
temperature of HbI-1 and HbI-2 cod.
The preferred temperature of a species is not a fixed value because it can
be influenced by other parameters. Seasonal fluctuation of the preferred
temperature is highest during summertime and lowest during winter
(Clark and Green, 1991). The
amount of food consumed by the fish also changes their preferred temperature.
Fish fed with the lowest food ration prefer the lowest temperature
(Despatie et al., 2001
). In
addition, hypoxia results in a decrease of the preferred temperature
(Schurmann et al., 1991
;
Schurmann and Steffensen,
1992
). Since hypoxia is a common phenomenon in several habitats
where the Atlantic cod is represented, especially in coastal regions such as
the Gulf of St Lawrence (Chabot and Dutil,
1999
) and the Baltic Sea
(Nielsen and Gargas, 1984
), we
analysed the effect of hypoxia on the preferred temperature for the different
haemoglobin types.
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Materials and methods |
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Determination of haemoglobin genotype
The haemoglobin genotypes were determined by agar gel electrophoresis
(Fyhn et al., 1994). 0.2ml of
blood was sampled from the caudal vein with a heparinized syringe. The blood
sample was centrifuged at 3000g in an Eppendorf tube and the
plasma discarded. 50 µl of the red blood cells (RBC) were transferred to a
new Eppendorf tube and washed twice with 60 µl of 1.17% NaCl solution. 80
µl of cold distilled water was added to haemolyse the RBC. The sample was
refrigerated for 10 min, and subsequently centrifuged for 5 min at 3000
g. To ensure proper sinking in the gel, 40 µl of haemolysed
RBC (supernatant) was mixed with 35 µl of 40% sucrose solution. A gel was
prepared using 1% agar (Difco bactoagar) dissolved by warming in Smithies
buffer (pH 8.6) diluted 1:1 with distilled water. Diluted Smithies buffer was
also used as electrophoresis buffer. All samples were electrophoresed at a 40
mA current for 35 min at 10°C, using a Pharmacia EPS600 power supply.
Shuttle box technique
To determine the preferred temperature, cod of body mass 90-200 g were
allowed to thermoregulate in an electronic shuttle box. The shuttle box
consisted of a warm and a cold water chamber connected by a tube. The fish was
able to thermoregulate by shuttling between the two chambers. The temperature
difference between the two chambers was 2°C and was kept constant by the
use of two pumps (Eheim 1046). When the fish occupied the cold chamber the
entire system was cooled down (4°Ch-1), whereas the system
started heating up when the fish entered the warm chamber
(4°Ch-1). The position of the fish was registered by a CCD
camera connected to a PC video frame-grabber (Visionetics VFG-512 BC),
digitising at 25 frames s-1, at a resolution of 256x256
pixels. Three 40 W red light bulbs illuminated the shuttle box from below and
made the fish look like a black silhouette on a light background. The contrast
between the fish and its surroundings was used by a custom-designed software
program to detect the geometric centre of the silhouette of the fish. The
x,y coordinates of the position of the fish were transmitted
via an RS-232 port to a second computer. The second computer was
equipped with Labtech Notebook programmed to switching on either cooling or
heating, depending on the position of the fish (for further details, see
Schurmann et al., 1991;
Schurmann and Steffensen,
1992
,
1994
).
The oxygen saturation was measured by a microprocessor oximeter (WTW oxy 96) in the warm part of the shuttle box. Test measurements showed that the difference in oxygen saturation between the two chambers never exceeded 2%. The oxygen saturation could be regulated via a computer system equipped with Labtech Notebook, which controlled solenoid valves. When the oxygen saturation was above the selected set point (35% oxygen saturation) compressed nitrogen was added to the system.
Experimental protocol
The cod was introduced to the shuttle box 24 h prior to the experiment at a
water temperature of 10°C. The experiments were carried out during
daytime, where the highest activity and the most precise thermoregulation of
the cod were previously observed. The data collection period consisted of 4 h
of normoxia (>80% oxygen saturation) followed by a 2 h reduction in oxygen
saturation, again followed by 3 h of hypoxia (35% oxygen saturation).
Calculations and analysis
To estimate the body temperature of a fish in a shuttle box system Newton's
Law of Cooling may be used:
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Analyses of variance (ANOVA) tested the effects of the independent factors (HbI-genotype and oxygen saturation) and their interactions on the preferred temperature or swimming speed. The Student NewmanKeul method was used in cases where the two-way ANOVA showed a significant difference (P<0.05).
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Results |
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Discussion |
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Periods of hypoxia occur in coastal areas, and in the late summer months
the oxygen saturation can be as low as 20-40% in Danish waters
(Nielsen and Gargas, 1984). In
the present study an oxygen saturation of 35% resulted in a significant
decrease in the preferred temperature for cod with HbI-1 genotype from
15.4±1.1 to 9.8±1.8°C. There are several physiological
advantages in lowering the body temperature during hypoxia, including lower
metabolic rate, higher oxygen affinity for haemoglobin and higher oxygen
solubility in the water (Jobling,
1994
). The disadvantages of preferring lower water temperatures
during hypoxia are a reduction in swimming speed as well as a reduction in
food intake and digestion rate, which results in decreased growth
(Brett, 1971
). Changes in
enzyme conformation, membrane structure and acidbase regulation are
also consequences of a sudden decrease in temperature
(Reynolds and Casterlin, 1980
;
Jobling, 1994
). A decrease in
the preferred temperature as a consequence of hypoxia has both advantages and
disadvantages for the fish. Hypoxia, however, seems to be nothing but a
disadvantage for the fish. Chabot and Dutil
(1999
) found that the growth
rate of Atlantic cod at 10°C decreased significantly when the oxygen fell
below 56% saturation. In the same study it was observed that spontaneous
activity also decreases during hypoxia.
The observation that the preferred temperature of HbI-2 cod did not
decrease as a consequence of hypoxia (8.2±1.5-8.0±2.9°C)
indicates that the energy saving advantages of an even lower temperature was
not necessary for the cod to survive. This conclusion makes sense, since the
preferred temperature of HbI-1 cod never went below this temperature during
hypoxia. Schurmann and Steffensen
(1992) found that the lowest
preferred temperature correlated with the lowest oxygen saturation (15%). If
the HbI-2 genotypes had been exposed to the same low level of hypoxia,
possibly the preferred temperature would have been significantly lower.
In the present study swimming speeds for HbI-1 and HbI-2 cod at normoxia
were 0.38 and 0.29 BL s-1, respectively. These swimming
speeds correspond with values obtained in previous studies
(Schurmann and Steffensen,
1994). Whether the haemoglobin type actually affects the swimming
speed is not known, but since the preferred temperature is thought to be the
zone where physiological processes are optimised or maximized
(Jobling, 1994
), the question
is relevant.
Hypoxia (35% oxygen saturation) significantly lowered the swimming speed
for both haemoglobin types (0.17 and 0.16 BL s-1). The
preferred temperature also decreased for HbI-1 exposed to hypoxia. Since
Schurmann and Steffensen
(1994) found a tendency of a
higher swimming speed at 10°C compared to 15°C, the decrease in
swimming speed for HbI-1 cod in the present study was probably caused by
hypoxia and not by the temperature. The levels of swimming speed at 35% oxygen
saturation correspond to previous measurement for Atlantic cod; 0.18
BL s-1 was the average swimming speed at 30% oxygen
saturation measured at 5°C and 10°C by Schurmann and Steffensen
(1994
). The reduction in
swimming speed during hypoxia will not only decrease the oxygen requirement of
the Atlantic cod, but also reduce the chance of reaching a more favourable
environment. Another strategy for fish during hypoxia is to increase the
activity level, which enhances the probability of encountering better oxygen
conditions. The latter strategy is observed for the skipjack tuna
(Katsuwonus pelamis) (Dizon,
1977
) and the red hake (Urophycis chuss)
(Bejda et al., 1987
).
The consequence of the difference in the preferred temperature of HbI-1 and
HbI-2 cod should be a differentiation of physiological processes
(Jobling, 1994). So far only a
limited number of studies have distinguished between the haemoglobin types
when studying physiology and behaviour of Atlantic cod. An exeption was
McFarland (1998
) who
differentiated between the haemoglobin types when measuring the metabolism,
and found that HbI-2 cod had a significantly lower standard metabolic rate at
4°C compared with HbI-1 cod. Feeding behaviour has also been examined and
the result showed that the competitive performance was highest for HbI-2 cod
measured at 6°C (Salvanes and Hart,
2000
). The advantageous feeding behaviour for HbI-2 cod at low
water temperatures is also reflected in growth-related parameters; HbI-2 cod
from mid to northerly Norwegian coastal waters has a higher growth rate and
earlier age of first spawning than HbI-1 cod
(Jørstad and Nævdal,
1994
; Mork et al.,
1983
,
1984
). It seems that the
theory of optimised physiological performances at the preferred temperature
fits HbI-2 cod perfectly, whereas the advantages of HbI-1 cod at high water
temperatures are more blurred and less well known. We predict that there are
more physiological and behavioural differentiations between the haemoglobin
types due to their different preferred temperatures. These results suggest
that for physiological and behavioural studies as well as for aquacultural
use, Atlantic cod should be subdivided according to its haemoglobin type.
The coupling between temperature and the occurrence of cod with different haemoglobin genotypes is of interest when discussing the biological consequences of increased water temperatures due to global warming. The results from the present study indicate that increasing water temperatures will result in an increased frequency of HbI-1 cod, because this haemoglobin type prefers a higher temperature. Furthermore, Atlantic cod stocks will move north if the seawater temperature exceeds the preferred temperature of the HbI-1 genotype. If a combination of increased water temperature and hypoxia should occur, the predicted superiority of HbI-1 cod as a consequence of increased water temperatures, and the fact that HbI-1 cod prefers a lower temperature during hypoxia, will cause an unfavourable situation for the HbI-1 cod. This situation is especially relevant in coastal regions where hypoxia is common, and could cause extensive damage to the Baltic cod stock, for example.
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Acknowledgments |
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References |
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