Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus
1 Laboratorio de Fisioloxía Animal, Facultade de Ciencias do Mar,
Universidade de Vigo, 36310 Vigo, Spain
2 Departamento de Biología, Facultad de Ciencias del Mar y
Ambientales, Universidad de Cádiz, 11510 Puerto Real, Cádiz,
Spain
* Author for correspondence (e-mail: jsoengas{at}uvigo.es)
Accepted 26 September 2005
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Summary |
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Key words: gilthead sea bream, Sparus auratus, osmoregulation, energy metabolism
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Introduction |
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There are several studies addressing metabolic changes in euryhaline fish
during acclimation to brackish water (BW), seawater (SW) or hypersaline water
(HSW; Kelly and Woo,
1999a,b
;
Kelly et al., 1999
; Nakano et
al., 1997
,
1998
;
Sangiao-Alvarellos et al.,
2003
; Soengas et al.,
1995a
,b
;
Vijayan et al., 1996
;
Woo and Murat, 1981
). However,
few studies have assessed what metabolic changes occur in osmoregulatory and
non-osmoregulatory organs during acclimation to low salinity water (LSW; Kelly
and Woo,
1999a
,b
;
Kelly et al., 1999
;
Roche et al., 1989
;
Woo and Fung, 1981
), and very
few of those studies have assessed the time course of metabolic changes during
acclimation to LSW, BW or HSW (Kelly and Woo,
1999a
,b
;
Kelly et al., 1999
).
Gilthead sea bream Sparus auratus is an euryhaline teleost capable
of living in different environmental salinities ranging from 5 to 60 p.p.t.
(Chervinski, 1984). The
osmoregulatory system of this species has been studied previously by analysing
aspects related to changes in chloride cells, gill
Na+,K+-ATPase activity and plasma parameters in fish
adapted to different salinities or subjected to salinity transfer
(Laiz-Carrión et al.,
2005a
,b
;
Mancera et al., 1993a
),
modifications in adenohypophyseal cells after acclimation to a hypo-osmotic
environment (Mancera et al.,
1993b
,
1995
), and the role of
different hormones in the adaptation to hyperosmotic and hypo-osmotic
environments (Guzmán et al.,
2004
; Laiz-Carrión et al.,
2002
,
2003
; Mancera et al.,
1994
,
2002
; Sangiao-Alvarellos et
al., 2003
,
2005b
).
In a previous study using gilthead sea bream, we addressed modifications in
osmoregulatory and metabolic parameters after a 14-day acclimation of this
euryhaline species to brackish water (BW, 12 p.p.t.), seawater (SW, 38 p.p.t.)
or high salinity water (HSW, 55 p.p.t.;
Sangiao-Alvarellos et al.,
2003). In addition, our group has studied the time course of
changes in osmoregulatory parameters during transfer from seawater to
different environmental salinities (5, 15, 38 and 60 p.p.t.;
Laiz-Carrión et al.,
2005a
). In different euryhaline species
(Holmes and Donaldson, 1969
;
Maetz, 1974
), including
gilthead sea bream (Laiz-Carrión et
al., 2005a
; Mancera et al.,
1993a
), osmoregulatory changes occurring during acclimation to
different osmotic conditions normally exhibit two different stages: (i) an
adaptive period, with changes in osmotic parameters, and (ii) a chronic
regulatory period, where these parameters again reach homeostasis. However,
there is no information about (i) what metabolic adjustments occur during the
time course of acclimation to different salinities, and (ii) whether or not
metabolic changes display a two-stage pattern similar to that exhibited by
osmoregulatory parameters.
The purpose of the present study was therefore to assess the time course of the osmoregulatory and metabolic changes occurring during acclimation of S. auratus to hyperosmotic and hypo-osmotic environments. Thus, changes in different osmoregulatory parameters and levels of several metabolites and activities of key enzymes of the major pathways of energy metabolism (use of exogenous glucose, glycolysis, glycogenolysis, gluconeogenesis, pentose phosphate) were measured throughout a 14-day period in gills, kidney, liver and brain of S. auratus transferred to SW (38 p.p.t.), HSW (55 p.p.t.), and LSW (6 p.p.t.).
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Materials and methods |
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Experimental protocol
In a first experiment (May 2004), we assessed changes due to hyperosmotic
acclimation. Water in three tanks (N=32) was progressively replaced,
within 1 h, by hypersaline water (HSW, 55 p.p.t., 1354 mOsm
kg1 H2O), while water in the other three tanks
(N=32) was replaced by SW. Salinity in the group denoted as HSW was
obtained by mixing full SW with natural marine salt (Unionsal, Cádiz,
Spain).
In a second experiment (June 2004), we assessed changes due to hypo-osmotic acclimation. Water in three tanks (N=32) was progressively replaced, within 1 h, by low salinity water (LSW, 6 p.p.t., 160 mOsm kg1 H2O), while water in the other three tanks (N=32) was replaced by SW. Salinity in the group denoted as LSW was obtained by mixing full SW with dechlorinated tapwater.
In both experiments fish remained in their specific salinity conditions for 2 weeks, during which the common water quality criteria were assessed with no major changes being observed. Average values for those parameters were 5 mg l1 for oxygen, 0.3 mg l1 for nitrite, 0.4 mg l1 for nitrate, 0.4 mg l1 for ammonia, and less than 0.1 mg l1 for chlorine, calcium and hydrogen sulphide. The water salinity was checked every day and corrected as necessary. No mortality was observed during the experiments.
Sampling
For both experiments, on day 0, eight fish from one tank containing SW were
sampled (time 0). In experiment 1, on days 1, 3, 7 and 14 after transfer,
eight fish from each group (SW and HSW) were dipnetted from six tanks (three
replicates per group), anaesthetized with 2-phenoxyethanol (1 ml
l1 water), weighed and sampled. A similar procedure was used
in experiment 2 to obtain samples from fish in SW and LSW. Blood was obtained
in ammonium-heparinized syringes from the caudal peduncle. Plasma samples were
obtained after centrifugation of blood (30 s at 13 000 g;
Eppendorf 5415R, Eppendorf AG, Hamburg, Germany), and divided into two
portions. One portion was immediately frozen on liquid nitrogen for assessment
of plasma osmolality, cortisol and protein levels, whereas the other portion,
for assessment of plasma metabolites, was deproteinized immediately (using 6%
perchloric acid) and neutralized (using 1 mol l1 potassium
bicarbonate) before freezing on liquid nitrogen and storage at 80°C
until further assay. In order to assess gill Na+,
K+-ATPase activity, 35 filaments coming from the second
branchial arch were cut just above the septum with fine-point scissors and
placed in 100 µl of ice-cold SEI buffer (150 mmol l1
sucrose, 10 mmol l1 EDTA, 50 mmol l1
imidazole, pH 7.3) and frozen at 80°C. Brain, liver, kidney, and
the remaining branchial arches were removed within a few seconds from each
fish, freeze-clamped in liquid nitrogen, and stored at 80°C until
assay.
Analytical techniques
Plasma samples were centrifuged and supernatants used to assess
metabolites. Glucose and lactate concentrations were measured using commercial
kits from Spinreact (Barcelona, Spain) adapted to microplates. Plasma protein
was measured using the bicinchoninic acid method with the BCA protein kit
(Pierce, Rockford, IL, USA) for microplates, and bovine serum albumin as
standard. Plasma triglyceride levels were determined enzymatically using a
commercial kit from Spinreact (Spain) in microplates. Those assays were run on
a Bio Kinetics EL-340i Automated Microplate Reader (Bio-Tek Instruments,
Winooski, VT, USA) using DeltaSoft3 software for Macintosh (BioMetallics, Inc.
NJ, USA). Plasma osmolality was measured using a vapour pressure osmometer
(Fiske One-Ten Osmometer, Fiske, Norwood, VT, USA).
Gill and kidney Na+,K+-ATPase activity was determined
using the micro assay method of McCormick
(1993) adapted to S.
auratus (Mancera et al.,
2002
), as previously described
(Laiz-Carrión et al.,
2003
; Sangiao-Alvarellos et
al., 2003
).
Plasma cortisol levels were measured (in duplicate) by indirect enzyme
immunoassay (ELISA) validated for gilthead sea bream
(Tintos et al., 2005). The
ELISA satisfied the criteria of specificity (testing cross-reactivity with
other steroids), reproducibility (interassay coefficient of variation <6%),
precision (intrassay coefficient of variation <4%) and accuracy (average
recovery >98%).
Frozen brain, liver, gill and kidney samples were quickly minced on a
chilled Petri dish to very small pieces that were mixed and (still frozen)
divided into two homogeneous portions to assess enzyme activities and
metabolite levels, respectively. The tissue to be used for assessment of
metabolite levels was homogenized immediately by ultrasonic disruption in the
cold with 7.5 vol. of ice-cooled 6% perchloric acid, and neutralized (using 1
mol l1 potassium bicarbonate). The homogenate was
centrifuged (2 min at 13 000 g, Eppendorf 5415R), and the
supernatant used for assays. Tissue lactate levels were determined
spectrophotometrically using a commercial kit (Spinreact, Spain). Tissue
glycogen levels were assessed using the method of Keppler and Decker
(1974). Glucose obtained after
glycogen breakdown (after subtracting free glucose levels) was determined
enzymatically using a commercial kit (Biomérieux, Spain).
The tissue used for assessment of enzyme activities was homogenized by ultrasonic disruption in the cold with 10 vol. of ice-cold stopping-buffer containing: 50 mmol l1 imidazole-HCl (pH 7.5), 15 mmol l1 2-mercaptoethanol, 100 mmol l1 KF, 5 mmol l1 EDTA, 5 mmol l1 EGTA, and a protease inhibitor cocktail (Sigma, P-2714). The homogenate was centrifuged (2 min at 13 000 g, Eppendorf 5415R) and the supernatant used for assays. In those cases where non-cytosolic enzymes were assessed, appropriate centrifugations were carried out to obtain samples.
The activities of several enzymes representative of major pathways of
carbohydrate metabolism (glycogen phosphorylase, GPase; 6-phosphofructo
1-kinase, PFK; pyruvate kinase, PK; fructose 1,6-bisphosphatase, FBPase;
glutamate dehydrogenase, GDH; hexokinase, HK; glucose 6-phosphatase, G6Pase;
glucose 6-phosphate dehydrogenase, G6PDH) were assessed. Reaction rates of
enzymes were determined using a Unicam UV-2 spectrophotometer (Thermo Unicam,
Waltham, MA, USA). Reaction rates of enzymes were determined by the increase
or decrease in absorbance of NAD(P)H at 340 nm. The reactions were started by
the addition of homogenates (0.05 ml), at a pre-established protein
concentration, omitting the substrate in control cuvettes (final volume 1.35
ml), and allowing the reactions to proceed at 15°C for pre-established
times. No changes were found in tissue protein levels in any of the groups
studied, and therefore enzyme activities are expressed in terms of
mg1 protein. Homogenate protein was assayed in triplicate as
detailed by Bradford (1976),
using bovine serum albumin (Sigma, St Louis, MO, USA) as standard. Enzyme
analyses were all carried out to achieve maximum rates in each tissue, as
defined in preliminary tests. The specific conditions for enzyme assays were
described previously (Laiz-Carrión et al.,
2002
,
2003
; Sangiao-Alvarellos et
al., 2003
,
2005b
).
PFK activity was determined at low (0.1 mmol l1) and high (5 mmol l1) fructose 6-phosphate concentrations (omitted for controls). An activity ratio was calculated as the activity at low [fructose 6P]:high [fructose 6P]. Similarly, a fructose 2,6-bisphosphate (F 2,6-P2) activation ratio was determined using 5 µmol l1 fructose 2,6-bisphosphate, and 0.1 mmol l1 fructose 6-phosphate concentrations.
PK activity was determined at low (0.05 mmol l1 for kidney, and 0.1 mmol l1 for gills) and high (2.8 mmol l1) phosphoenolpyruvate (PEP) concentrations (omitted for controls). An activity ratio was calculated as the activity at low [PEP]:high [PEP]. Similarly, a fructose 1,6-bisphosphate (F 1,6-P2) activation ratio was determined using 0.1 mmol l1 fructose 1,6-bisphosphate, and 0.1 mmol l1 PEP concentrations.
GPase a activity was measured with 2.5 mmol l1
AMP and 10 mmol l1 caffeine present, and total GPase
activities were estimated in the presence of 2.5 mmol l1 AMP
but without caffeine. The ratio of GPase activities with and without caffeine
x100 represents the percentage of total GPase (a+b) in
the active form (%GPase a). Recent studies in mammalian skeletal
muscle (Rush and Spriet, 2001)
report that caffeine inhibits GPase a. Therefore, we cannot discard
an understimation of GPase a activity in the present study, which may
also alter the %GPase a, and therefore the results provided regarding
GPase activity must be interpreted with caution.
Statistics
Data were statistically analysed by a two-way analysis of variance (ANOVA)
test in which treatment (SW and HSW transference in experiment 1; SW and LSW
transference in experiment 2) and time (0, 1, 3, 7 and 14 days) were the main
factors. Logarithmic transformations of the data were made when necessary to
fulfil the conditions of the analysis of variance but data are shown in their
decimal values for clarity. Post-hoc comparisons were made using a
Tukey test, with the differences considered to be statistically significant at
P<0.05.
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Results |
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Plasma osmolality in fish transferred from SW to HSW and LSW significantly increased (up to 20%) and decreased (up to 15%), respectively (Fig. 1). Changes were especially apparent on the first days after transfer, followed by recovery later. Gill Na+,K+-ATPase activity increased significantly with time in HSW- and LSW-transferred groups, the increase being higher in fish in HSW (80%) than in LSW (60%). Kidney Na+,K+-ATPase activity was 70% higher in HSW- with respect to SW-transferred fish at day 1, but returned to initial values at day 3. In fish transferred to LSW this activity decreased by 20% after 7 days of transfer.
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Liver GPase activity decreased threefold in fish transferred from SW to HSW on the first day, with activity recovered at day 7 (Fig. 4). In fish transferred to LSW a lower increase (up to 30%) was observed from day 7 onwards. Also, a decrease (up to 50%) was noticed for the %GPase in the active form (data not shown) on days 1 and 3 after transfer. PFK activity displayed no significant changes in fish transferred from SW to HSW whereas a small decline (20%) was noticed after 1 day in fish transferred to LSW. The activity ratio of PFK revealed a 50% increase in fish transferred to HSW on day 3 whereas a low decline (25%) was noticed on day 14 for fish transferred to LSW. The cofactor activation ratio of PFK displayed a 50% increase on day 3 after transfer from SW to HSW whereas no changes were noticed for fish transferred to LSW (not shown). G6PDH activity continuously decreased (up to 30%) in fish transferred from SW to LSW whereas no changes were noticed in fish transferred to HSW. G6Pase activity displayed a 100% increase on the first day after transfer to HSW and a subsequent 50% increase was also observed after 14 days; in contrast, a threefold decrease was observed in activity of fish transferred to LSW from day 3 onwards. FBPase activity did not display significant changes (data not shown). Finally, GDH activity decreased approx 20% after 7 days of transfer to LSW but not presented changes in fish transferred to HSW (data not shown).
|
Gill glycogen levels displayed a 100% increase on the first day in fish transferred from SW to HSW but returned to similar values as in SW fish at day 3; a higher increase (up to 250%) was observed in levels of fish transferred to LSW from day 7 of transfer onwards (Fig. 5). Free glucose levels increased tenfold in gills of fish transferred from SW to HSW on the first day after transfer, with levels being restored on day 14; in contrast the only significant change noticed in fish transferred to LSW was a 50% decline on the first day after transfer. Lactate levels displayed a pattern similar to that of glucose, thus in HSW-transferred fish a threefold increase was observed after 7 days that recovered on day 14 whereas in LSW-transferred fish a 50% decrease was observed between 1 and 7 days after transfer.
|
Gill GPase activity did not present any significant changes (data not shown) whereas the percentage of enzyme in the active form (%GPase a) increased 50% on the first day after transfer from SW to HSW in contrast to a threefold increase in fish transferred to LSW on day 14 (Fig. 6). PK activity, either total activity or the activity ratio of the enzyme only, increased in fish transferred from SW to LSW after 14 days (a similar increase in the cofactor activation ratio of the enzyme was also observed in fish transferred to LSW after 14 days, data not shown). HK activity increased all along the transfer period in fish transferred from SW to HSW, reaching a 40% increase; in contrast, no changes were noticed in LSW. Finally, G6PDH activity was stimulated approx. 20% in the time period between 1 and 7 days of transfer from SW to HSW, whereas a higher increase (up to 100%) was observed after 7 and 14 days of transfer to LSW.
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Finally, brain GPase activity did not show any changes (data not shown) whereas the percentage of the enzyme in the active form displayed a 15% increase after 14 days of transfer from SW to LSW (Fig. 10). PFK activity increased approx. 20% on days 1 and 3 after transfer to HSW whereas no changes were noticed in fish in LSW. The activity ratio of PFK did not display any major changes (data not shown) whereas the cofactor activation ratio of PFK increased by up to 40% on days 1 and 3 after transfer to HSW. HK activity increased in fish transferred to HSW from day 7 of transfer onwards, reaching a 15% increase after 14 days; a similar increase was also observed in fish transferred to LSW but only after 14 days. Finally, G6PDH activity increased (up to 40%) in fish transferred to HSW from day 1 of transfer onwards whereas no changes were observed in fish transferred to LSW.
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Discussion |
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Gill Na+,K+-ATPase activity is related to the
capacity of this osmoregulatory organ for extrusion of excess ions in a
hyperosmotic environment (Marshall,
2002; McCormick,
1995
,
2001
) and for ion intake in a
hypo-osmotic environment (Deane and Woo,
2004
; Jensen et al.,
1998
; Lin et al.,
2004
). In the present study, the time course of changes in gill
Na+,K+-ATPase activity after hyperosmotic transfer
agrees with that previously reported for this species
(Laiz-Carrión et al.,
2005a
) and other euryhaline species
(Deane and Woo, 2004
;
Jensen et al., 1998
) as well as
with the physiological role of this ion pump in euryhaline teleosts
(Jensen et al., 1998
;
Marshall, 2002
; McCormick,
1995
,
2001
). In addition, these
results reinforce the U-shaped model for the relationship between gill
Na+,K+-ATPase activity and environmental salinity
observed for several several euryhaline teleosts
(Deane and Woo, 2004
;
Jensen et al., 1998
), including
gilthead sea bream (Guzmán et al.,
2004
; Laiz-Carrión et
al., 2005a
). Our results agree with this model and also suggest a
role for this enzyme in ion intake in hypo-osmotic environments
(Marshall, 2002
;
McCormick, 2001
). Since we
have no indication whether or not sea bream is able to actively absorb ions in
hypo-osmotic environments, it is entirely feasible that this species maintains
ion balance for 14 days by decreasing ion efflux. However, specimens of
gilthead sea bream maintained for 3 months in LSW also showed high gill
Na+,K+-ATPase activity compared to fish maintained in
brackish water (Laiz-Carrión et
al., 2005b
), suggesting that sea bream actively absorb ions in
hypo-osmotic environments.
In euryhaline fish, kidney Na+,K+-ATPase activity
also presents changes in response to variation in environmental salinity
(Kelly and Woo,
1999a,b
;
Kelly et al., 1999
;
Lasserre, 1971
;
Venturini et al., 1992
). To
our knowledge, this is the first report on the time course of kidney
Na+,K+-ATPase activity in gilthead sea bream after
transfer from SW to HSW or LSW. The increase in activity seen on the first day
of acclimation to HSW could be attributed to a reduction in urine production
and/or to increased ion transport in the kidney. Since plasma osmolality also
increased during acclimation to HSW, fish needed to eliminate the excess ions.
However, no changes occurred in gill Na+,K+-ATPase
activity during the adaptive period, so it is possible that the excess ions
could be driven by rapid activation of kidney
Na+,K+-ATPase activity, which returned to basal activity
once the activity in gills was stimulated during the regulatory period. So, as
shown in Fig. 1, at day 1,
plasma osmolality and kidney Na+,K+-ATPase activity are
high whereas gill Na+,K+-ATPase activity was normal.
However, at day 3 an increase was noticed in gill
Na+,K+-ATPase whereas renal
Na+,K+-ATPase activity and plasma osmolality had almost
recovered, suggesting that the heightened level of gill
Na+,K+-ATPase activity may be responsible for correcting
the osmotic disturbance.
Plasma cortisol and metabolites
Cortisol is considered to be an important hormone for acclimation to
hyperosmotic and also hypo-osmotic environments (McCormick,
1995,
2001
). In gilthead sea bream,
a role for cortisol in osmotic acclimation
(Laiz-Carrión et al.,
2005a
; Mancera et al.,
1993a
,b
,
2002
) and regulation of energy
metabolism (Laiz-Carrión et al.,
2002
,
2003
) has been proposed. The
changes in plasma cortisol observed in the present study (i.e. increase in the
first days of exposure to changed salinities followed by a subsequent return
to basal levels) agree with similar cortisol pulses observed previously in
several fish species, including gilthead sea bream
(Laiz-Carrión et al.,
2005a
), after transfer to HSW
(Marshall et al., 1999
;
Morgan et al., 1997
; Richards
et al., 2004; Scott et al.,
2004
) or LSW (Roche et al.,
1989
). Thus, it seems that the transient increase in plasma
cortisol levels during the adaptive period of acclimation would be necessary
for the enhancement of gill Na+,K+-ATPase activity and
adaptation to the new salinity (McCormick,
1995
,
2001
). Since cortisol presents
an important metabolic role in teleosts
(Mommsen et al., 1999
),
including gilthead sea bream (Laiz-Carrión et al.,
2002
,
2003
), this hormone could also
assist to the osmoregulatory process by providing energy substrates for ion
regulation. The higher increase in plasma cortisol levels in fish transferred
to HSW than LSW also suggests more metabolic changes in HSW than in LSW (see
below).
Plasma glucose levels in fish acclimated to HSW increased 1 day after
salinity exposure followed by a slow decline over the following days, which is
different from those reports in the literature following acclimation from
freshwater (FW) to SW in rainbow trout
(Soengas et al., 1993), from
FW to brackish water in carp (De Boeck et
al., 2000
) and from SW to HSW in black sea bream
(Kelly et al., 1999
). In
contrast, tilapia transferred from FW to SW showed a two-stage behaviour
comparable to that described in the present study
(Nakano et al., 1998
). On the
other hand, fish transferred to LSW displayed a peak of glucose on day 1
followed by elevated levels up to the end of experiment. This results agree
with those previously reported for S. auratus under similar
conditions of salinity transfer (Mancera
et al., 1993a
) and for the European sea bass
(Roche et al., 1989
).
The high values of plasma lactate levels in HSW-acclimated gilthead sea
bream agree with data obtained after acclimation of this species
(Sangiao-Alvarellos et al.,
2003; Guzmán et al.,
2004
) and tilapia (Vijayan et
al., 1996
) to HSW. Since lactate can be used in tissues like
gills, kidney and brain to supply their energy requirements
(Mommsen, 1984
;
Mommsen et al., 1985
;
Soengas et al., 1998
), the
increase in plasma lactate levels observed in fish acclimated to LSW or HSW
suggests that this metabolite becomes more important during osmotic
acclimation, presumably reflecting its metabolic use in those organs.
Plasma triglyceride levels increased in the first days of acclimation to
LSW and at the end of acclimation period in fish transferred to HSW,
suggesting a role of this metabolite as a fuel for tissues during osmotic
acclimation. The role of triglycerides in this process has not yet been
addressed, but may be related to a metabolic reallocation of energy resources
once carbohydrate stores have been mobilized. To our knowledge, there are no
comparable studies in the literature, but this result may be in agreement with
the enhanced production of plasma triglycerides already reported in Atlantic
salmon during smoltification (Nordgarden
et al., 2002).
Altogether, changes observed in plasma metabolite levels suggested an
increased availability of fuels, especially during the first days of
transference (adaptive period), which can be used for an enhanced energy
requirement in different tissues of the fish during HSW or LSW acclimation.
However, the possibility that a change in plasma volume may account for some
of the changes in plasma metabolites (like triglycerides) cannot be dismissed.
The availability of fuels is very different in magnitude between HSW and LSW
conditions. In fact, the increases in HSW were much more higher than those in
LSW for all metabolites, especially glucose and lactate during the first
stage, which strongly suggests an increased energy demand in HSW compared with
LSW. Considering the increased plasma levels of cortisol during the adaptative
period (especially in HSW), this hormone may be responsible of at least part
of the metabolic changes observed (Laiz-Carrión et al.,
2002,
2003
).
Liver energy metabolism
Liver glycogen levels declined the first day after transfer to HSW and
throughout the experiment in LSW. The mobilization of liver glycogen would
provide glycosyl units ready to be used to fuel endogenous pathways such as
glycolysis or to be exported to other tissues. However, considering the
changes displayed in HSW by GPase activity, the later rise in glycogen and
fall in lactate levels suggest resynthesis of glycogen from lactate.
An increase in the activity ratio and cofactor activation ratio of liver
PFK was noticed after 3 days of transfer to HSW. In time course studies
reported in the literature, increases were also noticed in liver glycolytic
potential of rainbow trout (Soengas et
al., 1993) and tilapia (Nakano
et al., 1998
) during the first days of acclimation from FW to SW.
In contrast, in gilthead sea bream transferred to LSW we observed a decrease
in this potential from approximately day 7 of experiment onwards. These
changes suggest that a higher glucose use was taking place in liver of
HSW-transferred fish during the first stage of acclimation, whereas in
LSW-transferred fish a reduced use was apparent, especially from day 7 of
acclimation onwards.
Changes displayed by G6Pase activity suggested an increased capacity of liver for exporting glucose on days 1 and 14 in HSW and from day 3 of experiment onwards in LSW. The mobilization of liver glycosyl units from glycogen stores in HSW-transferred fish can be related to the sharp increase of liver and plasma glucose levels at the same time. Thus, liver of gilthead sea bream may have a higher capacity to export glucose to plasma when fish are transferred to HSW, whereas their capacity is lower when transfer is to LSW, especially in the first stages of acclimation.
Modifications observed in liver energy metabolism in gilthead sea bream reinforce the model of a two-stage metabolic response during osmotic acclimation. Interestingly, some of the changes described for both stages behave in a converse way when comparing fish acclimated to HSW and LSW, such as during the first stage for glucose (increase in HSW, decrease in LSW) or G6Pase activity (increase in HSW, decrease in LSW).
Gill energy metabolism
Changes in gill HK activity suggest that an increased use of exogenous
glucose occurs in gills during acclimation to HSW but not to LSW. These
changes in HK activity are reflected in changes in tissue glucose levels that
increased in HSW-acclimated fish and decreased in LSW-acclimated fish. Thus,
at least part of the increased glucose in HSW-transferred fish is apparently
used to store as glycogen in the first days of acclimation. However in
LSW-transferred fish, the increase in glycogen levels from day 3 onwards
suggests synthesis from metabolites other than glucose (maybe lactate), since
tissue glucose levels actually decrease.
It is accepted that gill tissue is able to oxidize lactate
(Mommsen, 1984). In this way
the rise in plasma lactate levels and decrease in gill lactate levels observed
in LSW-acclimated fish could suggest (Le
François et al., 2004
) an enhancement of the use of
exogenous lactate by gills through LDH working in the oxidative direction,
covering the initial stage of LSW acclimation (adaptive period). Considering
that the energy demand of gills produced by
Na+,K+-ATPase activity displayed a U-shape in relation
to environmental salinity, this would also match with an increased use of
other fuels, namely lactate, in extreme salinities (hyper and hypo-osmotic
environments). However, no changes were observed in PK activity in HSW- and
SW-acclimated fish, and an increase was noticed in gill lactate levels during
the adaptive period of acclimation. Therefore the hypothetical increased use
of lactate should be considered only under LSW and not HSW acclimation.
The enhanced capacity of the pentose phosphate pathway after transfer to HSW and LSW, based on changes in G6PDH activity, is interesting, suggesting the enhanced need for reducing power in gills of fish transferred to extreme salinities, which may be related to an increased necessity for them to synthesize lipids.
The changes observed in Na+,K+-ATPase activity in gills are therefore accompanied by two metabolic stages: (i) a first stage, characterized by an important accumulation of glycogen, glucose and lactate in HSW and decreased levels of lactate and glucose in LSW, and (ii) a second stage, where glucose is progressively being used through the pentose phosphate shunt and levels return to normality after 14 days in HSW, whereas an enhanced use of glucose through glycolysis, pentose phosphate and glycogenesis occurs on subsequent days of acclimation in LSW.
Kidney energy metabolism
In kidney, an increase in lactate levels was observed in HSW-transferred
fish, especially in the adaptive period when a return to basal levels of
kidney Na+,K+-ATPase activity was observed, suggesting
an increased importance for this metabolite in kidney of HSW-acclimated fish.
Changes observed in the time course of glycogen and glucose during acclimation
to HSW resemble those measured after acclimation of rainbow trout from FW to
SW (Soengas et al., 1994).
These changes may suggest that an enhanced production of glucose occurs in
kidney (at least part coming directly from glycogen stores) during the first
days of HSW acclimation. However, considering that no changes were apparent
for GPase activity and no G6Pase activity was measured, this must be
interpreted with caution. Changes in kidney HK activity in HSW-acclimated fish
suggest that another important part of the increased glucose levels noticed in
this tissue during the regulatory period could come directly from the blood
stream.
The situation in LSW-transferred fish was almost the converse since: (i)
glucose levels declined at day 1 of transference and increased on day 3, then
returning to levels similar to those of SW-acclimated fish; (ii) HK activity
showed a continuous decline from day 1 onwards, suggesting a decreased
potential in kidney for using exogenous glucose during the first days; and
(iii) a decrease in kidney lactate levels occurred from day 1 onwards, which
may suggest that lactate is increasingly used as a fuel for kidney. In fact,
lactate metabolization may be so high that some of the lactate molecules could
be used through gluconeogenesis to increase glycogen synthesis
(Blasco et al., 2001), which
could match with the increased glycogen levels observed in kidney of
LSW-transferred fish on days 4 and 7. Another converse response was noticed
for glycolytic capacity, which decreased in the first days of acclimation to
HSW and increased at the end of the acclimation period in LSW.
Considering that kidney Na+,K+-ATPase activity sharply increased on the first day of transfer to HSW, the extent of changes in metabolite levels observed at the same time suggest the existence of increased energy demand of this tissue during the first days of acclimation. This energy demand appears to be reduced on the following days. In HSW-acclimated fish, increased excretion of ions by the kidney could be necessary, and thus our metabolic results would suggest an increased use of different metabolites to fuel the increased osmoregulatory work of the kidney during the first days of HSW acclimation. On the other hand, acclimation of gilthead sea bream to LSW should produce increased osmoregulatory work in kidney because of production of a more dilute urine and increased ion reabsorption. Changes observed in metabolic parameters assessed in LSW-transferred fish may also lend support to a lower activation of kidney metabolism during the adaptive period of hypo-osmotic compared with hyperosmotic acclimation.
Brain energy metabolism
Brain glycogen, which constitutes the major energy store of fish brain
(Soengas and Aldegunde, 2002)
was mobilized during acclimation to HSW and LSW, and this could be related to
a stress effect of salinity on brain metabolism that led this tissue to
activate processes involved indirectly in the osmoregulatory work. These
changes are probably elicited by hormones known to produce metabolic changes
in fish brain (Laiz-Carrión et al.,
2002
,
2003
; Sangiao-Alvarellos et
al., 2004
,
2005a
). Since plasma cortisol
levels increased dramatically during transfer of gilthead sea bream to HSW and
LSW, this hormone is probably the main responsive of changes described. The
important increase observed in HK activity in HSW- and LSW-acclimated fish
also suggests that at least part of the increased glucose within the brain is
coming directly from the blood stream. An increased energy demand in brain
during the first days of acclimation to HSW is also suggested by increased
glycolytic capacity, which can be related to the increase observed in
Na+,K+-ATPase and creatine kinase activities in brain of
tilapia transferred from FW to SW (Weng et
al., 2002
). Another interesting finding was the increase in
lactate levels in HSW-acclimated fish, which indicate that the increased use
of carbohydrates is higher than the energy demand of the brain, resulting in
an accumulation of lactate. The possibility that brain glucose and lactate
levels are at least partly due to blood within the brain cannot be
excluded.
Altogether, changes observed in brain energy metabolism again revealed the
existence of two stages in metabolic changes occurring during
hyper/hypo-osmotic acclimation: a first one of reduced glycogen mobilization
and use of exogenous glucose, followed by a second period of increased
mobilization of glycogen. The enhanced availability of glucose during this
second stage would help to explain the sharp increase in brain free glucose
levels also observed in LSW-acclimated fish. The increased availability of
glucose within brain in the first stage is not apparently used through
glycolysis or the pentose phosphate pathway in LSW-transferred fish since no
important changes were noticed in the activity of selected enzymes from those
pathways, whereas an increased use through glycolysis is apparent in
HSW-transferred fish at that stage. Interestingly, changes in metabolic
parameters in brain are in most cases irrespective of the direction of changes
in salinity, which can be attributed to the action of cortisol
(Laiz-Carrión et al.,
2002,
2003
).
Conclusions
In summary, the time course of acclimation to HSW and LSW in S.
auratus displayed an adaptive and a regulatory period similar to those
previously described for this species when subjected to equivalent transfers
(Laiz-Carrión et al.,
2005a; Mancera et al.,
1993a
). In addition, these periods are characterized by a
tissue-specific reorganization of energy metabolism in two stages. The first
stage involves an increase in the availability and use of fuels in different
organs where an enhanced energy requirement of different osmoregulatory (gills
and kidney) and non-osmoregulatory (liver and brain) tissues involved directly
or indirectly on osmoregulatory work is observed. In the second stage,
osmoregulatory parameters reached homeostasis, and most metabolic parameters
returned to normality. When comparing changes in metabolic parameters between
HSW and LSW-transferred fish, several interesting findings arise such as: (i)
several metabolic parameters (i.e. liver glucose or G6Pase activity) in liver,
gills and kidney, displayed converse responses that are not apparent in brain,
and (ii) the magnitude of changes (as well as the two stages) is less
important in LSW acclimation than in HSW acclimation, suggesting that
acclimation of euryhaline marine species like gilthead sea bream to LSW is
less expensive in terms of energy than acclimation to HSW.
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Blasco, J., Marimon, I., Viaplana, I. and Fernández-Borrás, J. (2001). Fate of plasma glucose in tissues of brown trout in vivo: effects of fasting and glucose loading. Fish Physiol. Biochem. 24,247 -258.[CrossRef]
Boeuf, G. and Payan, P. (2001). How should salinity influence fish growth? Comp. Biochem. Physiol. 130,411 -423.[CrossRef]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Chervinski, J. (1984). Salinity tolerance of young gilthead sea bream Sparus aurata. Bamidgeh. 36,121 -124.
De Boeck, G., Vlaeminck, A., Van der Linden, A. and Blust, R. (2000). The energy metabolism of common carp (Cyprinus carpio) when exposed to salt stress: an increase in energy expenditure or effects of starvation? Physiol. Biochem. Zool. 73,102 -111.[CrossRef][Medline]
Deane, E. E. and Woo, N. Y. S. (2004). Differential gene expression associated with euryhalinity in sea bream (Sparus sarba). Am. J. Physiol. 287,R1054 -R1063.
Guzmán, J. M., Sangiao-Alvarellos, S., Laiz-Carrión, R., Míguez, J. M., Martín del Rio, M. P., Soengas, J. L. and Mancera, J. M. (2004). Osmoregulatory action of 17ß-estradiol in the gilthead sea bream Sparus auratus.J. Exp. Zool. 301,828 -836.[CrossRef]
Holmes, W. N. and Donaldson, E. M. (1969). The body compartments and the distribution of electrolytes. In Fish Physiology, Vol. 1 (ed. W. S. Hoar, and D. J. Randall), pp. 1-89. San Diego: Academic Press.
Jensen, M. K., Madsen, S. S. and Kristiansen, K. (1998). Osmoregulation and salinity effects on the expression and activity of Na+,K+-ATPase in the gills of european sea bass, Dicentrarchus labrax (L.). J. Exp. Zool. 282,290 -300.[CrossRef][Medline]
Kelly, S. P. and Woo, N. Y. S. (1999a). Cellular and biochemical characterization of hypo-osmotic adaptation in a marine teleost, Sparus sarba. Zool. Sci. 16,505 -514.[CrossRef]
Kelly, S. P. and Woo, N. Y. S. (1999b). The response of seabream following abrupt hypo-osmotic exposure. J. Exp. Biol. 55,732 -750.
Kelly, S. P., Chow, I. N. K. and Woo, N. Y. S. (1999). Haloplasticity of black seabream (Mylio macrocephalus): Hypersaline to freshwater acclimation. J. Exp. Zool. 283,226 -241.[CrossRef]
Keppler, D. and Decker, K. (1974). Glycogen. Determination with amyloglucosidase. In Methods of Enzymatic Analysis (ed. H. U. Bergmeyer), pp.1127 -1131. New York: Academic Press.
Laiz-Carrión, R., Sangiao-Alvarellos, S., Guzmán, J. M., Martín del Rio, M. P., Míguez, J. M., Soengas, J. L. and Mancera, J. M. (2002). Energy metabolism in fish tissues related to osmoregulation and cortisol action. Fish Physiol. Biochem. 27,179 -188.[CrossRef]
Laiz-Carrión, R., Martín del Río, M. P., Míguez, J. M., Mancera, J. M. and Soengas, J. L. (2003). Influence of cortisol on osmoregulation and energy metabolism in gilthead sea bream Sparus aurata. J. Exp. Zool. 298,105 -118.
Laiz-Carrión, R., Guerreiro, P. M., Fuentes, J., Canario, A. V. M., Martín del Rio, M. P. and Mancera, J. M. (2005a). Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus. J. Exp. Zool. 303,563 -576.[CrossRef]
Laiz-Carrión, R., Sangiao-Alvarllos, S., Guzmán, J. M., Martín del Río, M. P., Soengas, J. L. and Mancera, J. M. (2005b). Growth performance of gilthead sea bream Sparus aurata in different osmotic conditions: implications for osmoregulation and energy metabolism. Aquaculture. In press.
Lasserre, P. (1971). Increase of (Na++K+)dependent ATPase activity of gills and kidney of two euryhaline marine teleosts, Crenimugil labrosus (Risso, 1826) and Dicentrarchus labrax (Linnaeus, 1758), during adaptation to fresh water. Life Sci. 10,113 -119.[CrossRef]
Le François, N. R., Lamarre, S. G. and Blier, P. U. (2004). Tolerance, growth and haloplasticity of the Atlantic wolfish (Anarhichas lupus) exposed to various salinities. Aquaculture 236,659 -675.[CrossRef]
Lin, C. H., Tsai, R. S. and Lee, T. H. (2004). Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge. Comp. Biochem. Physiol. 138,287 -295.
Maetz, J. (1974). Aspects of adaptation to hypo-osmotic and hyper-osmotic environments. In Biochemical and Biophysical Perspectives in Marine Biology (ed. D. C. Malins and J. R. Sargent), pp. 1-167. New York: Academic Press.
Mancera, J. M., Laiz-Carrión, R. and Martín del Río, M. P. (2002). Osmoregulatory action of PRL, GH and cortisol in the gilthead sea bream (Sparus aurata L.). Gen. Comp. Endocrinol. 129,95 -103.[CrossRef][Medline]
Mancera, J. M., Pérez-Fígares, J. M. and Fernández-Llebrez, P. (1993a). Osmoregulatory responses to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata). Comp. Biochem. Physiol. 106,245 -250.[CrossRef]
Mancera, J. M., Fernández-Llebrez, P., Grondona, J. M. and Pérez-Fígares, J. M. (1993b). Influence of environmental salinity on prolactin and corticotropic cells in the euryhaline gilthead sea bream (Sparus aurata L.). Gen. Comp. Endocrinol. 90,220 -231.[CrossRef][Medline]
Mancera, J. M., Pérez-Fígares, J. M. and Fernández-Llebrez, P. (1994). Effect of cortisol on brackish water adaptation in the euryhaline gilthead sea bream (Sparus aurata L.). Comp. Biochem. Physiol. 107,397 -402.[CrossRef]
Mancera, J. M., Pérez-Fígares, J. M. and Fernández-Llebrez, P. (1995). Effect of decreased environmental salinity on growth hormone cells in the euryhaline gilthead sea bream (Sparus aurata L.). J. Fish Biol. 46,494 -500.
Marshall, W. S. (2002). Na+, Cl-, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospectives synthesis. J. Exp. Zool. 293,264 -283.[CrossRef][Medline]
Marshall, W. S., Emberley, T. R., Singer, T. D., Bryson, S. E.
and McCormick, S. D. (1999). Time course of salinity
adaptation in a strongly euryhaline estuarine teleost, Fundulus
heteroclitus: a multivariable approach. J. Exp.
Biol. 202,1535
-1544.
McCormick, S. D. (1993). Methods for nonlethal gill biopsy and measurement of Na+,K+-ATPase activity. Can. J. Fish. Aquat. Sci. 50,656 -658.
McCormick, S. D. (1995). Hormonal control of gill Na+,K+-ATPase and chloride cell function. In Fish Physiology, Vol. 14 (ed. C. M. Wood and T. J. Shuttleworth), pp. 285-315. San Diego: Academic Press.
McCormick, S. D. (2001). Endocrine control of osmoregulation in teleost fish. Am. Zool. 41,781 -794.
Mommsen, T. P. (1984). Metabolism of the fish gill. In Fish Physiology, VolXB (ed. W. S. Hoar and D. J. Randall), pp.203 -238. New York: Academic Press.
Mommsen, T. P., Walsh, P. J. and Moon, T. W. (1985). Gluconeogenesis in hepatocytes and kidney of Atlantic salmon. Mol. Physiol. 8,89 -100.
Mommsen, T. P., Vijayan, M. M. and Moon, T. W. (1999). Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation Rev. Fish Biol. Fish. 9, 211-268.
Morgan, J. D. and Iwama, G. K. (1991). Effects of salinity on growth, metabolism, and ion regulation in juvenile rainbow trout (Oncorhynchus mykiss) and fall Chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. Aquat. Sci. 48,2083 -2094.
Morgan, J. D., Sakamoto, T., Grau, E. G. and Iwama, G. K. (1997). Physiological and respiratory responses of the Mozambique tilapia (Oreochromis mossambicus) to salinity acclimation. Comp. Biochem. Physiol. 117,391 -398.[CrossRef]
Nakano, K., Tagawa, M., Takemura, A. and Hirano, T. (1997). Effects of ambient salinities on carbohydrate metabolism in two species of tilapia: Oreochromis mossambicus and O. niloticus. Fish. Sci. 63,338 -343.
Nakano, K., Tagawa, M., Takemura, A. and Hirano, T. (1998). Temporal changes in liver carbohydrate metabolism associated with seawater transfer in Oreochromis mossambicus. Comp. Biochem. Physiol. 119,721 -728.[CrossRef]
Nordgarden, U., Hemre, G. I. and Hansen, T. (2002). Growth and body composition of Atlantic salmon (Salmo salar L.) parr and smolt fed diets varying in protein and lipid contents. Aquaculture 207,65 -78.[CrossRef]
Richards, J. G., Semple, J. W., Bystriansky, J. S. and Schulte,
P. M. (2003). Na+/K+-ATPase
-isoform switching in gills of rainbow trout (Oncorhynchus
mykiss) during salinity transfer. J. Exp. Biol.
206,4475
-4486.
Roche, H., Chaar, K. and Pérès, G. (1989). The effect of a gradual decrease in salinity on the significant constituents of tissue in the sea bass (Dicentrarchus labrax, Pisces). Comp. Biochem. Physiol. 93,785 -789.[CrossRef]
Rush, J. W. E. and Spriet, L. L. (2001).
Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine
nucleotides and caffeine. J. Appl. Physiol.
91,2071
-2078.
Sangiao-Alvarellos, S., Laiz-Carrión, R., Guzmán, J. M., Martín del Río, M. P., Míguez, J. M., Mancera, J. M. and Soengas, J. L. (2003). Acclimation of Sparus aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs. Am. J. Physiol. 285,R897 -R907.
Sangiao-Alvarellos, S., Lapido, M., Míguez, J. M. and Soengas, J. L. (2004). Effects of central administration of arginine vasotocin on monoaminergic neurotransmitters and energy metabolism of rainbow trout brain. J. Fish Biol. 64,1313 -1329.[CrossRef]
Sangiao-Alvarellos, S., Míguez, J. M. and Soengas, J. L. (2005a). Actions of growth hormone on carbohydrate metabolism and osmoregulation of rainbow trout. Gen. Comp. Endocrinol. 141,214 -225.[CrossRef][Medline]
Sangiao-Alvarellos, S., Guzmán, J. M., Laiz-Carrión, R., Martín del Río, M. P., Míguez, J. M., Mancera, J. M. and Soengas, J. L. (2005b). Actions of 17ß-estradiol on carbohydrate metabolism in liver, gills and brain of gilthead sea bream Sparus auratus during acclimation to different salinities. Marine Biol. 146,607 -617.[CrossRef]
Scott, G. R., Richards, J. G., Forbush, B., Isenring, P. and Schulte, P. M. (2004). Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am. J. Physiol. 287,C300 -C309.[CrossRef]
Soengas, J. L. and Aldegunde, M. (2002). Energy metabolism of fish brain. Comp. Biochem. Physiol. 131,271 -296.[CrossRef]
Soengas, J. L., Barciela, P., Fuentes, J., Otero, J., Andrés, M. D. and Aldegunde, M. (1993). The effect of seawater transfer in liver carbohydrate metabolim of domesticated rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 105,337 -343.[CrossRef]
Soengas, J. L., Fuentes, J., Andrés, M. D. and Aldegunde, M. (1994). Direct transfer of rainbow trout to seawater induces several changes in kidney carbohydrate metabolism. J. Physiol. Biochem. 50,219 -228.
Soengas, J. L., Aldegunde, M. and Andrés, M. D. (1995a). Gradual transfer to seawater of rainbow trout: effects on liver carbohydrate metabolism. J. Fish Biol. 47,466 -478.[CrossRef]
Soengas, J. L., Barciela, P., Aldegunde, M. and Andrés, M. D. (1995b). Gill carbohydrate metabolism of rainbow trout is modified during gradual adaptation to sea water. J. Fish Biol. 46,845 -856.[CrossRef]
Soengas, J. L., Strong, E. F. and Andrés, M. D. (1998). Glucose, lactate, and ß-hydroxybutyrate utilization by rainbow trout brain: changes during food deprivation. Physiol. Zool. 71,285 -293.[Medline]
Tintos, A., Míguez, J. M., Mancera, J. M. and Soengas, J. L. (2005). Development of a microtitre plate indirect ELISA for measuring cortisol in teleost fish, and evaluation of stress responses in rainbow trout and gilthead sea bream. J. Fish Biol. In press.
Venturini, G., Cataldi, E., Marino, G., Pucci, P., Garibaldi, L. and Bronz, P. (1992). Serum ions concentration and ATPase activity in gills, kidney and oesophagus of European sea bass (Dicentrarchus labrax, Pisces, Perciformes) during acclimation trial to fresh water. Comp. Biochem. Physiol. 103,451 -454.[CrossRef]
Vijayan, M. M., Morgan, J. D., Sakamoto, T., Grau, E. G. and
Iwama, G. K. (1996). Food-deprivation affects seawater
acclimation in tilapia: hormonal and metabolic changes. J. Exp.
Biol. 199,2467
-2475.
Weng, C. F., Chiang, C. C., Gong, H. Y., Chen, M. H. C., Huang,
W. T., Cheng, C. Y. and Wu, J. L. (2002). Bioenergetics of
adaptation to a salinity transition in euryhaline teleost (Oreochromis
mossambicus) brain. Exp. Biol. Med.
227, 45-50.
Woo, N. Y. S. and Fung, J. C. (1981). Studies on the biology of the red sea bream Chrysophrys major II. Salinity adaptation. Comp. Biochem. Physiol. 69,237 -242.[CrossRef]
Woo, N. Y. S. and Murat, J. C. (1981). Studies on the biology of the red sea bream Chrysophrys major III. Metabolic response to starvation in different salinities. Marine Biol. 61,255 -260.[CrossRef]