Physiological changes of sturgeon Acipenser naccarii caused by increasing environmental salinity
1 Dept Biología Animal y Ecología, Univ. de Granada, 18071
Granada, Spain
2 Dept I+D, Piscifactoría `Sierra Nevada', 18313 Riofría,
Granada, Spain
* Author for correspondence (e-mail: anasanz{at}ugr.es)
Accepted 21 August 2002
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Summary |
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Key words: Acipenser naccarii, osmoregulation, water salinity, oxidative stress, physiology
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Introduction |
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Cataldi et al. (1995)
pointed out that, if the euryhaline character of A. naccarii is
verified, new perspectives for widening culture areas to littoral zones would
become of great interest. Anadromous fish must develop complex osmoregulatory
mechanisms to survive successfully both in hypoosmotic environments (e.g.
rivers) and hyperosmotic environments (e.g. estuaries and open sea). Some
aspects of these osmoregulatory processes have been previously studied in
several sturgeon species (Natochin et al.,
1985
; McEnroe and Cech,
1985
; Krayushkina,
1998
), including A. naccarii (Cataldi et al.,
1995
,
1999
;
McKenzie et al., 1999
). Age
(maturation stage) and body size (ratio of gill surface area:body surface
area) have been postulated as determining factors of the salinity tolerance of
the fish (McEnroe and Cech,
1985
; García-Gallego et
al., 1998
).
Any environmental disturbance can be considered a potential source of
stress, as it prompts a number of responses in the animal to deal with the
physiological changes triggered by exterior changes. In theory, these
responses can be detected in fish and in other vertebrates in the form of
changes in hormonal or substrate concentrations in the plasma or alterations
in erythrocyte parameters, such as cell volume or enzyme activities
(Donaldson, 1981). If the
internal perturbation of the fish, either directly or as a result of
alterations of the environment, overwhelms the physiological mechanisms of the
animal for response and adaptation to new conditions, survival can be
threatened and death can result.
In addition, as a consequence of metabolic activity, reactive oxygen
species (ROS) are continuously produced and act as strong oxidants. As a
defence mechanism, a large repertory of antioxidant enzymes, in addition to
small antioxidant molecules, are produced by the cell. Superoxide dismutase
(SOD), which hastens the dismutation of O2.- to
H2O2, catalase (CAT), and glutathione peroxidase (GPX),
which converts H2O2 to H2O, are the most
important antioxidant enzymes found in all vertebrates. Their activities
differ among the organs and tissues of freshwater and marine fish
(Wdzieczak et al., 1982),
depending upon feeding behaviour (Radi and
Matkovics, 1988
), environmental factors and other ecological
conditions (Winston and Di Giulio,
1991
; Roche and Bogé,
1996
). When the antioxidant defences are inadequate to combat the
action of the ROS, the result is oxidative stress. The formation of ROS can be
increased in response to different variations in the internal or external
medium, whereupon oxidative alterations occur in the cellular constituents.
One of the alterations is increased lipid peroxidation as a consequence of the
oxidation of the lipid constituents of cell membranes.
One circumstance involving great metabolic changes in fish is their
acclimation and survival at different degrees of salinity. Thus, Roche and
Bogé (1996) reported
changes in antioxidant enzymes in the red blood cells of sea bass
(Dicentrarchus labrax) subjected to hipoosmotic shock. Osmoregulation
undoubtedly implies, among other circumstances, a greater energy expense,
which is caused in turn by a metabolic activation and, consequently, an
increase in ROS formation. In fish, a direct relationship has been found
between metabolic intensity and activation of oxidative enzymes
(Wilhelm Filho et al.,
1993
).
The present study examines the possible repercussions that osmoregulatory processes can induce on the classical indicators of stress (e.g. cortisol, glycaemia and haemoglobin) in A. naccarii subjected to gradually increasing environmental salinity and maintained for 20 days at a constant salt concentration of 35%. The activities of SOD, CAT and GPX, as well as lipid-peroxidation levels in blood (plasma and red blood cells), liver and heart, were studied.
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Materials and methods |
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The experimental fish were subdivided into two groups: one group was left undisturbed throughout the experimental period except for salinity changes and daily measurements of water-quality parameters (`undisturbed' fish), while the other group was sampled at pre-determined intervals, after anaesthetization to allow blood and tissue samplings. The survival index in the undisturbed sturgeons was 96.4%.
Sampling
Fish were sampled at salinities of 0%, 15%, 22%, 29% and 35% and after the
period at constant 35% salinity (N=5 fish at each concentration). At
each sampling, blood, muscle, liver and heart were taken from killed fish
after anaesthetization by immersion, until sedated, in an
ethylene-glycol-mono-phenyl-ether (Merck) 1:2000 (v:v) solution. For 0%
salinity samples, only blood and muscle were taken to determine plasma
osmolality and muscle moisture, respectively. The rest of the determinations
were made at 15% salinity.
Blood was drawn from the caudal vein and collected into heparinized
syringes. Haematocrit (Hct), total haemoglobin (Hb) concentration and
red-blood-cell count (RBCC) were immediately determined. Plasma was separated
by centrifugation (650 g, 15 min), and the haemolysate
supernatant from red blood cells was obtained according to Marcon and Wilhelm
Filho (1999). Both plasma and
haemolysate were stored at -80°C until analysis.
Muscle samples were taken from the anterior dorsal area, weighed and placed in an oven to determine tissue moisture. Samples of heart and liver were submerged in liquid nitrogen and then stored at -80°C until analysis.
Analytical methods
The RBCC was performed after blood dilution (1:100) with Hendrick's reagent
(made with reagents from Sigma, Akobendas, Spain) by using a Neubauer chamber
(Afora, Sevilla, Spain) and light microscope (magnification x100). Hct
was determined by blood centrifugation (2600 g, 5 min) using a
special microfuge. The Hb concentration in the samples was determined by
colorimetry (540 nm), after mixing blood with Drabkin's solution
(Van Kampen and Zijlstra,
1961).
Plasma cortisol and glucose were assayed by immunoassay (Immulite®,
Diagnostic Products Corporation, Los Angeles, USA) and the glucose oxydase
method (Hugget and Nixon,
1957), respectively; plasma osmolality was evaluated using an
osmometer (Osmostat OM620, Daiichi Kagaku Co Ltd, Kyoto, Japan). The protein
contents of plasma, liver and heart were measured according to Bradford
(1976
). For moisture-level
measurements, muscle samples were weighed before and after drying in an oven
(Heraeus, Madrid, Spain) at 105°C until they reached constant weight.
For the determination of the parameters indicating oxidative stress (CAT, GPX, SOD and lipid-peroxidation levels), samples of plasma, haemolysate, liver and heart were used. The tissue extracts were prepared by homogenization in nine volumes of extraction buffer (100 mmol l-1 Tris HCl, 0.1 mmol l-1 EDTA, 0.1% (v/v) Triton X-100, pH 8). These homogenates were centrifuged at 27 170 g for 30 min at 4°C. The supernatants were divided into different aliquot samples and stored at -80°C until analysis.
CAT (EC 1.11.1.6) activity was determined by measuring the decrease of
hydrogen peroxide concentration at 240 nm. The decline in absorbance was
registered in 12s intervals for the first 2 min in a cuvette containing 50
mmol l-1 potassium phosphate buffer (pH 7) and 10.6 mmol
l-1 hydrogen peroxide freshly added, according to Aebi
(1984).
GPX (EC 1.11.1.9) activity was measured following the method of
Flohé and Günzler
(1984), where freshly prepared
glutathione reductase solution (2.4 U ml-1 in 0.1 mol
l-1 potassium phosphate buffer, pH 7) was added to a cuvette
containing 50 mmol l-1 potassium phosphate buffer (pH 7), 0.5 mmol
l-1 EDTA, 1 mmol l-1 sodium azide, 0.15 mmol
l-1 NADPH and 0.15 mmol l-1 H2O2.
The NADPH-consumption rate was monitored for 2 min. After the addition of 1
mmol l-1 GSH (reduced glutathione), the decrease in absorption at
340 nm at intervals of 12s for the first 2 min was measured.
SOD (EC 1.15.1.1) activity was measured spectrophotochemically by the
ferricytochrome c method using xanthine/xanthine oxidase as the
source of superoxide radicals. One unit of activity was defined as the amount
of enzyme necessary to produce a 50% inhibition of the ferricytochrome
c reduction rate (McCord and
Fridovich, 1969).
Lipid-peroxidation levels were determined by quantifying the concentration
of thiobarbituric-acid-reacting substances (TBARS), expessed as the
malondialdehyde (MDA) concentration, according to Buege and Aust
(1978).
Statistical analysis
The differences in parameters were tested for significance using a one-way
analysis of variance (ANOVA) and the LSD (least-significant difference) test
(P<0.05).
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Results |
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The results presented in Table
1 show an increase in plasma cortisol level with salinity,
although the difference was not significant. No significant changes were found
in glycemia values. The total plasma proteins showed a statistically
significant decline with increasing salinity (from 15 to 29
),
remaining stable from 29
to 35
salinity. Hepatic protein
increased significantly during acclimation to salinity, while the protein
level in the heart did not significantly change during this process.
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Table 2 shows that Hct, Hb
concentration and RBCC showed similar responses to increasing salinity. Values
during the first phase (0-29) increased as salinity rose, only to
decline when the salinity level reached 29
and then increase again
during the 20 days at a constant salinity of 35
.
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The water content of the muscle fell significantly as the animals
acclimated to the saline water, but values returned to normal at the end of
the period of constant 35 salinity
(Fig. 2).
|
Enzyme activities and lipid-peroxidation levels in plasma and red blood
cells are shown in Fig. 3 and
Fig. 4, respectively. High
levels of SOD activity were found in red blood cells, compared with levels
found in plasma; SOD activity in plasma increased significantly when the
salinity level reached 22, remaining high at higher salinities. SOD
activity in red blood cells increased with increasing salinity, but this trend
was only significant after 20 days at 35
salinity. The CAT activity in
red blood cells was higher than in plasma and increased significantly with
increasing salinity in both samples (red blood cells and plasma). No
appreciable responses in GPX activity were found in plasma at higher salinity,
although red blood cells showed an increase in GPX as salinity increased.
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Major changes in MDA concentration were found in red blood cells at
differing salinities; levels were high at 15 salinity but fell when
salinity reached 22
. Subsequent increases in salinity (22-35
)
did not alter these low values but, after 20 days at 35
salinity, a
significant increase was found, although values did not reach those previously
recorded at 15
salinity. In addition, although lipid-peroxidation
levels in plasma were lower than those found in the erythrocytes, they rose in
relation to increased salinity.
Table 3 shows the parameters of oxidative stress in the liver and heart. First, it should be emphasized that the CAT and GPX activities in the liver were far greater than those detected in the heart. Levels of CAT and SOD in the liver significantly declined as salinity increased, while no significant change was detected in the heart with increasing salinity. Lipid-peroxidation levels showed no significant change in the two tissues with increasing salinity.
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Discussion
The effect of cortisol on osmoregulatory parameters has been amply studied
in teleosts (Maetz, 1969;
Epstein et al., 1971
;
Kamiya, 1972
;
Scheer and Langford, 1976
;
Assem and Hanke, 1979
;
Hegab and Henque, 1984
). Its
primary action seems to be the stimulation of
Na+/K+-ATPase activity. In the present study, the trend
of increasing levels of cortisol at higher salinity in A. naccarii
(Table 1) indicates that, for
this chondrostean, the role of cortisol must be similar to that in teleosts.
Besides, the increase of serum cortisol levels is considered to be a primary
indicator of stress response (McDonald and
Milligan, 1992
; Cataldi et al.,
1998
). In a previous test with animals at the same salt
concentrations but at a higher environmental temperature (21°C;
Martínez-Álvarez et al., manuscript submitted), the values for
plasma cortisol were higher than in the present study, and, in this case, the
increase at higher salinity proved significant. The cortisol levels found in
our tests were comparable with those reported by Cataldi et al.
(1998
) for the same fish
species. These also show that, after acclimation to low temperatures, the
cortisol concentration diminished significantly. According to McKenzie et al.
(1999
), although these
sturgeons showed no fluctuations in plasma cortisol levels at different
salinities (0
, 11
and 23
), poor growth and high
mortality following disturbances in animals in water at 20
salinity
indicate only a partial tolerance to brackish water (20
salinity).
These circumstances, among others, induced these authors to conclude that a
salinity of 20
represents the upper tolerance limit in A.
naccarii (Cataldi et al.,
1995
,
1999
). Our animals, on the
other hand, remained for 20 days at a salinity of 35
, with only 4%
mortality. In addition, some specimens were transferred afterwards to an
exhibition marine aquarium in northwestern Spain, where they survived for
several months, although monitoring was no longer possible. In addition, the
results presented in the present and other studies of different experiments
involving acclimation to rising salinity indicate a compensatory response that
enables survival at an environmental salinity of 35
.
Thus, during a number of saltwater-acclimation experiments with sturgeons
of >2 years of age (mean initial mass 1500 g), we have found an increase in
chloride cells (Carmona et al., manuscript submitted) and branchial
Na+/K+-ATPase activity
(Morales et al., 2001),
changes in fatty-acid composition of branchial-cell membranes (R. M.
Martínez-Álvarez, unpublished data) and rising levels of plasma
urea (Martínez-Álvarez et al., manuscript submitted; in this
study we also used fish of
1 year and a mean initial mass of 750 g). All
these responses encourage osmolality (Fig.
1) and the electrolyte levels in blood
(Martínez-Álvarez et al., manuscript submitted) to stabilize in
a hyperosmotic medium. The different age/mass of the sturgeons used in our
studies (14 months, 932 g) and in those of Cataldi et al.
(1995
) (20 months, 1900 g),
Cataldi et al. (1999
) (0-150
days, 0.015-35.9 g) and McKenzie et al.
(1999
) (5 months, 56 g) and/or
the differences in acclimation times and rhythms could explain the differences
in the results found in these studies compared with the present study.
The trend for the cortisol level to rise in response to growing
environmental salinity should, like a hyperglycemia-causing hormone, raise the
plasma glucose level. However, we found no such rise
(Table 1). Previous studies of
this issue are contradictory, showing both a rise
(Assem and Hanke, 1979;
Bashamohideen and Parvatheswararao,
1972
) and a fall (Krumschnabel
and Lackner, 1993
; Soengas et
al., 1991
) in glucose during seawater adaptation. There appears to
be a high glucose demand in order to supply the energy by osmoregulatory
mechanisms (Krumschnabel and Lackner,
1993
; Plaut,
1998
), whereupon glyconeogenesis even increases
(Jürss and Bittorf,
1990
). The greater use of glucose could mask the plasma glucose
increase prompted by the cortisol.
The decline found in total plasma proteins
(Table 1) during increasing
salinity could also be accounted for by the high osmoregulatory energy demand.
This, together with a reduced appetite of the animals at higher salinity
(Usher et al., 1991;
Plaut, 1998
), would account
for this reduction. The latter would also contribute to the failure to find a
rise in blood glucose.
During the first stage of acclimation, the Hct, Hb concentration and RBCC
increased (Table 2); they
subsequently decreased as the salinity rose further and returned to initial
values when the fish had remained for 20 days at a constant salinity of
35. These changes can be attributed to changes in the water content in
the blood, caused by the change in environmental salinity
(Plaut, 1998
). Thus, at the
beginning of exposure to a hyperosmotic environment, the fish would lose water
passively, and thereby undergo increases in the concentrations of blood-cell
elements. Afterwards, the compensatory increase in water ingestion would
provide a transitory dilution of the blood parameters. Finally, these would
return to initial values as a result of the rest of the osmoregulatory
mechanisms, which act to re-establish the extracellular volume. This
hypothesis is reinforced by the results found for the muscle water content
(Fig. 2), which, on the one
hand, reflect a certain dehydration of the muscle during acclimation to the
saline water and, on the other hand, reflect the return to normal values after
20 days at 35
.
Our results indicate that, in A. naccarii, blood (plasma and red
blood cells) antioxidant defences (SOD, CAT and GPX activities; Figs
3,
4) have the ability to
strengthen under increasing environmental salinity. The increase of these
enzyme activities in red blood cells could explain the decline in
lipid-peroxidation values and the stability of these values despite changing
salinity. Nevertheless, it seems that the activity of these enzymes in the
plasma is not sufficient to avoid a certain degree of lipid oxidation at
35 salinity (Fig. 3).
However, this fact did not apply to red blood cells, where lipid oxidation
remained low at all times (Fig.
4).
It should be emphasized that the CAT and GPX activities in the liver
(Table 3) were far greater than
those detected in the heart, a circumstance that has also been noted by
Wilhelm Filho et al. (1993) in
other fish species. In addition, SOD and CAT decreased significantly in the
liver as environmental salinity rose. This trend, when not accompanied by
lipid peroxidation, would indicate a certain alteration of the cell metabolism
rather than a state of oxidative stress. It should be borne in mind that
enzymatic activities are expressed as a function of the protein content; the
relative protein content of the liver increased with rising environmental
salinity. This increase may result from a decrease in fatty content in
response to a deficit in energy intake, as discussed above, or to reduced
body-water content. Thus, the increase in hepatic protein is a possible reason
for the depressed SOD and CAT activities. In the heart, the invariability in
the protein content would promote constancy in enzyme activities during
acclimation to environmental salinity.
It could be stated that there were no alterations in oxidative state during acclimation to salinity in the sturgeon, either in the liver or in the heart, during the osmoregulatory process, with lipid-peroxidation levels remaining low at all times. The fact that we did note alterations in the antioxidant enzymatic activities in the blood could indicate that the blood was more affected by being more directly involved in osmoregulatory activities.
There are studies demonstrating that tissues differ in their responses to
oxidative stress induced by the same circumstance. However, we have not found
any studies that determine the parameters of oxidative stress under conditions
of hyperosmotic stress; only one study refers to the changes in enzyme
activities (SOD, CAT and peroxidase) in D. labrax during replacement
of seawater (37) with freshwater (5
), reporting a stimulation
of SOD and CAT activities by hypoosmotic shock
(Roche and Bogé,
1996
).
In conclusion, the sturgeon A. naccarii, when subjected to growing
environmental salinity up to 35, revealed a number of physiological
responses, such as disturbance in body fluid (detected by increased plasma
osmolality, altered number of red blood cells and decreased levels of muscle
hydration), activation of osmoregulatory mechanisms (increased cortisol
levels) and antioxidant defences (augmented antioxidant enzyme activities in
the blood), and alteration of energetic metabolites (changes in protein
concentration in the plasma and liver), indicating that the acclimation of
sturgeons to increased salinities involves osmotic stress counteracted by
osmoregulation. In fact, after the period of acclimation to growing salinity
(up to 35
) and after the fish remained for 20 days at this salinity,
the plasma osmolality values, the concentration of blood parameters, and the
water content of the muscle all returned to the values obtained for water with
low salinity, showing that osmoregulatory processes have achieved their
objective.
By contrast, levels of cortisol, antioxidant enzymes in the blood (plasma and red blood cells), lipid peroxidation in plasma, and hepatic proteins did not return to initial values. This indicates that osmoregulatory processes cause major physiological changes in the fish.
Further study on osmoregulatory physiology is needed to determine the point at which maintenance of an environmental salinity level enables growth and utilization of food compatible with the possibility of raising this fish species in brackish or seawater.
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Acknowledgments |
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References |
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