Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system
Laboratoire Génome, Populations, Interactions, Adaptation, UMR 5171, Université Montpellier II, Place E. Bataillon, 34095 Montpellier, Cedex 05, France
* Author for correspondence (e-mail: nebelcatherine{at}yahoo.fr)
Accepted 17 August 2005
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Summary |
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Key words: teleost, osmoregulation, gills, kidney, Na+/K+-ATPase, sea-bass, Dicentrarchus labrax
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Introduction |
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Adaptation to various levels of salinity, including FW, involves
coordinated physiological responses based on the function of several
osmoregulatory organs. It is well established that adult euryhaline teleosts
are able to maintain their blood osmolality at about 300350 mOsmol
kg1 in the range of tolerable salinities, due to an
effective hydro-mineral regulation occurring mainly at the gill, urinary
system, intestine and integument levels (see reviews by
Evans et al., 1999;
Greenwell et al., 2003
;
Varsamos et al., 2005
). In FW,
sea-bass are exposed to osmotic water entrance and diffusive ion loss. To
compensate for the passive ion movements, the fish need to limit ion loss and
to (re)absorb ions by specialized cells lining the osmoregulatory epithelia.
These cells, called mitochondria-rich cells (MRC) or chloride cells in the
gills, or more generally ionocytes, are characterized by the abundance of
several ion channels, transporters and enzymes. Among these, the
Na+/K+-ATPase creates ionic and electrical gradients
used for salt uptake from the external medium to the blood. At the cellular
level, the Na+/K+-ATPase is located on a well-developed
tubular system corresponding to an extension of the basolateral cell membrane;
the cells are also called MRCs, due to the numerous mitochondria providing the
energy required by the enzyme. In teleosts, the
Na+/K+-ATPase activity and its abundance have often been
used as indicators of the osmoregulatory ability in adults/juveniles
acclimated to different salinities
(Imsland et al., 2003
) or in
migratory species at specific ontogenetic stages
(Uchida et al., 1996
;
Zydlewski et al., 2003
). In
FW, the main osmoregulatory organs are the gills (for active ion uptake) and
the urinary system (for the production of large amounts of hypotonic urine).
Previous studies have shown that these two organs progressively develop during
the post-embryonic ontogeny of the sea-bass and are functional at the
larva/juvenile transition (Varsamos et
al., 2002a
; Nebel et al.,
2005
), i.e. by the time young fish migrate towards low-salinity
areas and FW.
The objectives of this study were thus: (1) to detect potential differences in the ability of juvenile sea-bass to successfully adapt to FW; (2) to determine different abilities of hyperosmoregulation between the fish assessed as able or unable to live in FW; and (3) to identify the physiological, histological and cellular basis of these differences at the gill and urinary system levels.
This study is a first step towards understanding the meaning of inter-individual variations in response to environmental fluctuations, and their bearings on population differential adaptation.
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Materials and methods |
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Osmolality measurements
Blood and urine osmolalities were measured in the different categories of
fish from FW and SW. Preliminary attempts at urine collection directly
following capture and anaesthesia of fish failed, probably due to
stress-induced urine emission. Therefore, the following procedure was used.
The fish were captured with a hand-net and transferred to a black 10 l plastic
container filled with FW or SW, aerated and covered by a black plastic sheet.
The containers were kept still in a silent room for 2 h. The anaesthetic was
then gently introduced into the water under the plastic cover. This method
allowed about 8090% successful urine collection. The urinary pore and
the branchial chamber of each anaesthetized fish were quickly rinsed with
distilled water and carefully dried with absorbent paper. Urine was collected
following its emission induced by probing the urinary pore and gently
inserting a 2 µl glass micropipette (Drummond microcaps, Bioblock,
Broomall, PA, USA) into the pore. Blood was sampled from the ventral aorta
using a 1 ml syringe. The urine and blood samples were immediately transferred
into mineral oil to avoid evaporation. The osmolality of at least 30 nl of
blood and urine was measured on a Clifton nano-osmometer (Clifton Technical
Physics, Hartford, NY, USA).
Morphometric parameters of branchial chloride cells
The first gill arch of the right side of all fish was excised and immersed
for at least 16 h into freshly mixed Champy-Maillet's fixative (0.4% osmium
tetroxyde, 25 mg ml1 iodine and saturated metallic zinc;
Maillet, 1959). After rinsing
with distilled water for at least 3 days, the samples were dehydrated in a
series of ethanol and processed for embedding in Paraplast®. 7 µm-thick
sagittal sections were cut and dewaxed with histochoice LMR. This simple and
rapid method resulted in chloride cells being specifically stained in black
due to their extensive basolateral membrane system
(Hartl et al., 2001
). For all
later analyses, at least 5 animals per group were used.
For chloride cell numbering, 10 filaments per animal were observed to determine the number of positively stained chloride cells. On each filament, the chloride cell number was determined within 10 interlamellar spaces. An interlamellar space is defined as the distance on the filament between two adjacent lamellae. Chloride cell numbers of filaments and lamellae were counted separately.
For the evaluation of cell size and shape, 30 positively stained cells per
animal were identified on photographs and their borders traced manually. Using
a stage micrometer, their area (A), perimeter (P) and shape (S) was calculated
using Optimas software (North Reading, MA, USA). The shape (defined as S=4
AP2) indicates the circularity of the chloride cells. The
shape factor tends to 1 when the cell is spherical
(Zydlewski et al., 2003
).
Histomorphology of the urinary system
Whole animals were immersed into Bouin's liquid for 2448 h. The
fixative penetration was facilitated by longitudinal 2 mm-deep incisions and
sections of the fins. They were washed and dehydrated in an ascending series
of ethanol, and finally embedded in Paraplast®. The whole animals were cut
in longitudinal horizontal 7 µm-thick sections, stained with
Masson-trichrome and observed on a Leica Diaplan microscope (Leitz Wetzlar,
Germany). A precise kidney zone was determined and 23 slides per animal
used for further analysis. From photographs taken at a low magnification
(x10 objective lenses), the borders of the kidney were manually traced
using the Optimas software in order to measure the percentage of the area
occupied by the urinary tubule sections compared to the `total kidney area'.
As the section areas of the dorsal aorta and of the collecting ducts were not
comparable between animals, these areas were subtracted from the total kidney
area in order to standardize the measurements. In this way, the density of
urinary tubules was estimated compared to the `total kidney area' (minus
collecting ducts and dorsal aorta).
Transmission electron microscopy
Freshly dissected gill arches were fixed for 1 h in a solution of 5%
glutaraldehyde buffered at pH 7.4 with 0.2 mol l1 sodium
cacodylate buffer at ambient temperature. Samples were then rinsed in sodium
cacodylate buffer and post-fixed for 1 h in a mixture (v/v) of 2% osmium
tetroxide and 0.45 mol l1 sodium cacodylate buffer at
4°C. After extensive washing in distilled water, the samples were
dehydrated in graded ethanol and embedded in Epon. Ultrathin sections were cut
using a diamond knife on a ReichertJung ultramicrotome (Cambridge, UK),
contrasted with uranyl acetate and bismuth tartrate and examined with a JEOL
1200 EX II transmission electron microscope (Tokyo, Japan), operated at 100
kV.
Immunolocalization of Na+/K+-ATPase in gills and kidney
Sections (4 µm) from Bouin's fixed samples of the gill and the kidney
were immersed into 0.01% Tween 20, 150 mmol l1 NaCl in 10
mmol l1 phosphate-buffered saline (PBS), pH 7.3 for 10 min,
treated for 5 min with 50 mmol l1 NH4Cl (to
screen free aldehyde groups of the fixative), and finally incubated for 10 min
in 1% BSA and 0.1% gelatin in PBS. The slides were incubated for 2 h at room
temperature in a moist chamber with the specific monoclonal mouse antibody
(diluted to 10 µg ml1) raised against the -subunit
of the chicken Na+/K+-ATPase (IgG
5) developed by
Fambrough and purchased from the DSHB (Developmental Studies Hybridoma Bank;
University of Iowa). This antibody has already been successfully used in the
same species (Varsamos et al.,
2002b
; Nebel et al.,
2005
). Control sections were subjected to the same conditions, but
without the monoclonal antibody. After rinsing, all sections were incubated
for 1 h with the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
antibody. The slides were washed, mounted with anti-bleaching mounting medium
and rapidly examined using a Leica Diaplan microscope equipped for
fluorescence with the appropriate filter set (filters of 450 nm to 490 nm) and
coupled to a digital camera and the FW4000 software.
Immunoblotting
Immunoblotting was carried out in gill and kidney homogenates according to
the method of Pomport-Castillon et al.
(1997), with modifications.
The gills (without the gill arch) of the left side of the animals and the
mid/posterior part of the kidney (urinary and collecting tubules/ducts) were
dissected and stored in SEI buffer (150 mmol l1 sucrose, 10
mmol l1 Na2EDTA, 50 mmol l1
imidazole) at 80°C until use. The tissue was homogenized in 300
µl (kidney) and 500 µl (gills) ice-cold MIIM buffer (250 mmol
l1 sucrose, 5 mmol l1 MgCl2, 5
mmol l1 Tris/Hepes, pH 7.3), centrifuged at 4000
g for 5 min (4°C), and the supernatant assayed for protein
content by the Bradford method (Bradford,
1976
). These homogenates were used for immunoassays and
Na+/K+-ATPase activity measurements. Samples were
diluted (1:8, 1:10) with PBS (phosphate buffered saline, pH 7.3). A 2 µl
sample from each dilution was deposited in triplicate on a 0.45 µm-thick
nitrocellulose membrane. Five known quantities of canine
Na+/K+-ATPase (Sigma-Aldrich, St Louis, MO, USA), 0.3125
µg, 0.625 µg, 1.25 µg, 2.5 µg and 5 µg, were deposited in
triplicate on the same membrane to standardize the measurements. After
saturation in 5% skimmed milk (SM) in PBS at 37°C for 30 min, the SM
powder was removed by washing the membrane twice with PBS. The strips were
incubated for 2 h with the monoclonal mouse antibody (diluted at 3 µg
ml1 in 0.5% SM-PBS) raised against the
-subunit of
the chicken Na+/K+-ATPase (IgG
5). After washing,
the avidine peroxidase conjugate (Pierce Interchim; Rockford, IL, USA) at 2
µg ml1 was added to the membrane for 1.5 h. The membrane
was washed and the fractions were developed with
2-chloro-naphtol
acetate (Sigma). Color development was stopped by rinsing the membrane with
distilled water. The membrane was dried at room temperature and rapidly
scanned. The immunoblots were analyzed using the Software Scion Image and the
colour intensity measured for each blot.
Na+/K+-ATPase activity measurements
Gill and kidney Na+/K+-ATPase activity was determined
according to the method developed by Flik et al.
(1983).
Na+/K+-ATPase activity measurements, expressed in
µmol Pi mg1 protein h1, were based on
the differences in ATP hydrolysis in the presence and absence of ouabain (1.4
mmol l1). The same homogenates as those for the immunoassays
were used.
Statistical comparisons
Results are expressed as mean ±
S.E.M. Student's t-tests were used
for statistical comparisons of mean values.
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Results |
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Osmoregulation
Compared to SWS, blood osmolality was slightly but significantly lower (by
12%) in FWS. Most strikingly, blood osmolality was much lower in FWU (by 32%;
214±14 mOsmol kg1) compared to FWS (316±20
mOsmol kg1). By contrast, in SWS, urine (360±12
mOsmol kg1) was isotonic to blood (360±13 mOsmol
kg1). In FWS, urine (227±43 mOsmol
kg1) was markedly hypotonic to the blood (316±20
mOsmol kg1).
In contrast, urine in FWU fish was isotonic to the blood (213±15 mOsmol kg1 vs 214±14 mOsmol kg1). The results are illustrated in Fig. 1.
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Gills
Branchial chloride cell distribution, morphometrical parameters and ultrastructural observations
Representative Champy-Maillet-prepared gill sections of SWS, FWS and FWU
are shown in Fig. 2AC.
During the salinity challenge of the sea-bass from SW to FW, significant
changes in branchial chloride cell distribution
(Fig. 2AC), abundance
(Figs 2AC,
3;
Table 1) and morphology
(Table 1) were noted. The
chloride cells of SWS were exclusively located on the gill filaments
(Fig. 2A). In the FWS and FWU
groups, chloride cells were also located on the filaments, but other, more
elongated chloride cells (shape factor decreased to 0.6;
Table 1) were observed on the
lamellae (Fig. 2B,C).
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The mean total number of branchial chloride cells (including filamentary and lamellar chloride cells) within a distance of 10 interlamellar spaces increased significantly after the FW challenge, by 56% in FWS and by 167% in FWU, compared to SWS (Fig. 3). The mean number of filamentary chloride cells in FWU was significantly higher by 32% compared to FWS (F=2.965) and by 35% compared to SWS (F=1.517).
The number of lamellar ionocytes was much higher (by 245%) in the FWU compared to the FWS (Table 1). The ratio of the mean number of lamellar ionocytes to the mean number of filamentary ionocytes was 0.53 in the FWS and 0.97 in the FWU, which illustrates the much higher number of lamellar chloride cells in the FWU.
The mean area and perimeter of the filamentary chloride cells were
significantly higher in the SWS than in the FWS. There was no significant
difference in the filamentary cells morphometry (area, perimeter and shape
factor) between the FW groups (FWS and FWU;
Table 1), but the lamellar
cells of the FWU had a larger area and perimeter than the FWS cells.
Ultrastructural observations showed that the chloride cells of the FWU
displayed the same features as observed in FWS (results not shown) and
described in previous studies (Varsamos et
al., 2002b). No sign of cell degeneration was detected in
filamentary (Fig. 4A) and
lamellar FWU chloride cells. The chloride cells of FWU contained numerous
mitochondria characterized by an electron-dense matrix
(Fig. 4A,B). The apical part of
the cells, which was devoid of mitochondria, displayed a few microridges and
presented a dense vesiculotubular system
(Fig. 4A,C). An extensive
tubular system, distributed throughout the whole cytoplasm, was continuous
with the basolateral cell membrane (Fig.
4A). Tight junctions were observed between chloride cells and
pavement cells (Fig. 4C).
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Branchial Na+/K+-ATPase immunofluorescence, immunoassays and specific activity
Control sections without primary antibody showed no immunolabeling (results
not shown). In SWS, the filamentary chloride cells were densely immunostained
(Fig. 2D). The filaments and
the lamellae of all FW-exposed fish observed presented positively
immunostained chloride cells (Fig.
2E,F).
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Following the salinity challenge of D. labrax from SW to FW, the branchial specific Na+/K+-ATPase activity was increased by 56% in FWS and by 168% in FWU (Fig. 5B). The Na+/K+-ATPase activity was 72% higher in the FWU than in the FWS (Fig. 5B).
Urinary system
Morphometric analyzis of the urinary system
Examinations of sea-bass kidney sections revealed differences between the
three groups of fish. In the SWS kidney, the lumen of the collecting ducts
seems larger than in FWS and FWU (Fig.
6B,D,F). More strikingly, the renal tissue appears less dense in
FWU (Fig. 6E,F) compared to FWS
(Fig. 6C,D) and SWS
(Fig. 6A,B). In order to
quantify the latter observations, the area of the kidney tubules was measured
and compared to the total kidney area (minus the ducts and the dorsal aorta)
in the three groups of fish. The resulting data show similar values in SWS and
FWS, but they reveal a lower tubular density (1823% lower), in the FWU
kidney (Fig. 7).
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Discussion |
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Blood osmolality measurements were carried out in order to estimate the
osmoregulatory ability of the different sea-bass groups. In the
well-acclimated sea-bass juveniles (SWS and FWS), the blood osmolality was
maintained within a range of 320360 mOsmol kg1 in FW
and SW, respectively, demonstrating an efficient hyper- or
hypo-osmoregulation. These values are in agreement with other studies carried
out in the sea-bass (Lasserre,
1971; Varsamos et al.,
2001
) and in other euryhaline teleosts such as striped bass
Morone saxatilis (Jackson et al.,
2005
), Chanos chanos
(Lin et al., 2003
) and
Crenimugil labrosus (Lasserre,
1971
). In the FWU, the blood osmolality decreased by 3040%
to 213 mOsmol kg1, an osmotic imbalance which may be
considered a symptom of non-adaptation
(Franklin et al., 1992
)
leading to death. In similar experiments, the transfer of sea-bass to FW by
Jensen et al. (1998
) resulted
in a loss of Na+ and Cl and in a decrease of the
plasma osmolality to 240 mOsmol kg1; the fish died within 10
days. The sea-bass used by these authors are physiologically close to the FWU
fish in this study. A similar blood osmolality value was measured in
Tetraodon nigroviridis in FW (Lin
et al., 2004
), where no mortality was detected, and in
hypophysectomized Morone saxatilis maintained in FW
(Jackson et al., 2005
). In the
latter, an injection of prolactin was followed by a recovery of the blood
osmolality to normal values. A possible hormonal deficiency might cause the
osmoregulatory failure in the FWU and should be considered in further studies.
The blood osmolality recorded in all these fish including the FWU D.
labrax (200240 mOsmol kg1) may be considered as
the lower limit of tolerable blood osmolality in the sea-bass.
These observations raise the question of the physiological cause(s) of the
osmoregulatory failure in the FWU fish. The decrease in plasma osmolality
might originate from a failed regulation of the passive ion and water flow
between their blood and the external medium. In other fish species, changes in
body water content have been reported, which caused severe osmotic water loss
at high salinities (Sclafani et al.,
1997; Moustakas et al.,
2004
). Thus, in the FWU, an osmotic water entrance might result in
a lower blood osmolality, but their ability to limit water entrance was not
examined. In our study, the possible causes of osmoregulatory failure were
addressed at the gill and urinary system levels.
Owing to their numerous chloride cells and their enzymatic equipment, the
gills are considered as the major osmoregulatory site in euryhaline teleosts
(Evans et al., 1999;
Wilson and Laurent, 2002
; Lin
et al., 2003
,
2004
). In the successfully
FW-adapted sea-bass (FWS) the FW challenge induced changes in the chloride
cells, which were smaller and more numerous than in SW, particularly through
their occurrence on the lamellae. At the ultrastructural level, the surfaces
of their apical openings increased and the leaky junctions between adjacent
cells were replaced by tight junctions
(Varsamos et al., 2002b
). In
all analyzed fish, the branchial chloride cells displayed high amounts of
Na+/K+-ATPase, apparently located within the deep
basolateral membrane infoldings, as shown through immunofluorescence. A
significantly higher Na+/K+-ATPase abundance was
measured through immunoblotting in FWS compared to SWS fish, and the
Na+/K+-ATPase activity followed the same trend. In the
same species, a similar increase in enzyme activity has been reported in FW
(Jensen et al., 1998
),
probably related to the higher number of chloride cells. According to Jensen
et al. (1998
), the branchial
enzyme activity of D. labrax is minimum in an iso-osmotic environment
and increases after transfer to hypo- and hyperosmotic media. This pattern of
regulation of gill Na+/K+-ATPase activity, also reported
in Tetraodon nigroviridis (Lin et
al., 2004
), seems to be mostly found in euryhaline species living
in an environment where salinity fluctuates
(Jensen et al., 1998
). This
reaction differs from the majority of teleosts in which the
Na+/K+-ATPase activity increases with salinity
(McCormick et al., 1989
;
Uchida et al., 1996
). These
examples illustrate the generally accepted view that changes in
Na+/K+-ATPase activity correlate with transepithelial
transport, although this enzyme is found in all animal cells for volume
regulation.
As the osmoregulatory failure observed in FWU might hypothetically have
originated from branchial chloride cell dysfunction or degeneration, the gill
structure was compared between FWS and FWU fish. But, contrary to this
hypothesis, the FW challenge induced a significantly higher increase in
filamentary, and mainly in lamellar, chloride cell numbers in the FWU compared
to the FWS. The resulting increase in total chloride cell number might
generate higher ion transporting activities in the branchial epithelium of
FWU. It is worth noting that the ultrastructure of chloride cells in FWU did
not show any sign of abnormality or degeneration compared to similar cells in
FWS. FWU thus clearly possess more apparently normal functioning gill chloride
cells than the FWS. Whether this increase in number results from a
signal-induced proliferation (possibly hormonal, involving cortisol;
Dang et al., 2000;
Sloman et al., 2001
) remains
to be investigated. Although tight junctions were observed here between FWU
chloride and pavement cells, a temporary leakiness of the branchial epithelium
during the chloride cell proliferation cannot be excluded and would contribute
to the decrease in blood osmolality.
Chloride cells, whose function has been extensively studied in fish (see
reviews by Evans et al., 1999;
Marshall, 2002
;
Varsamos et al., 2005
), may be
differently involved in osmoregulation according to their lamellar or
filamentary location in some species
(Shikano and Fujio, 1998
). In
D. labrax, only filamentary cells have been observed in SW (this
study), suggesting their function as ion excretory sites. We hypothesize that
lamellar chloride cells are involved in hyper-osmoregulation in FW, in FWS and
in FWU (this study). They would also be involved in hypo-osmoregulation at
doubly concentrated seawater (Varsamos et
al., 2002b
). Since the lamellae are also the site responsible for
O2 and CO2 exchanges, a high proliferation of chloride
cells, as observed in FWU, may result in a reduced lamellar area for
respiration, as shown in Oncorhynchus mykiss
(Bindon et al., 1994
). In fact,
gas and ion transfer is closely linked in FW teleosts, as reported by Randall
and Brauner (1998
). According
to Perry (1998
), lamellar
chloride cell proliferation is a common response of freshwater fish to enhance
the ion transporting capacity, but also has negative effects on respiratory
gas transfer because of a thickening of the blood-to-water diffusion barrier.
Moreover, the lamellar chloride cells of FWU are larger than those of FWS,
which supports this hypothesis. In the sea-bass, these circumstances may
contribute to the FWU mortality. A much higher
Na+/K+-ATPase abundance and activity were recorded in
FWU compared to FWS, thus generating an increased energy expenditure
associated to hyperosmoregulation in FWU. This highly energy-demanding
process, competing with other physiological requirements, may also compromise
survival.
In summary, the gill chloride cells of FWU seem to possess all required features to efficiently hyperosmoregulate. Their higher number in FWU compared to FWS suggests a compensatory process that will be discussed later. But this abundance of chloride cells may in turn reduce the surface available for gas exchanges, and thus negatively interfere with respiration. Concurrently, the increased branchial Na+/K+-ATPase abundance and activity may worsen the respiratory problem through increased O2 requirement. The high amounts of energy expenditure for ion absorption from FW may contribute to the high mortality recorded in the FWU. But since the results discussed above tend to rule out any direct degeneration process of gill chloride cells as a contributive factor to the osmoregulatory failure in FWU, further investigations were performed on the urinary system.
The kidney is known to play an important role in osmoregulation in
euryhaline teleosts, by changing the rate of urine flow and controlling the
balance between ion secretion and reabsorption according to the environmental
salinity (see reviews by Hickman and
Trump, 1969; Dantzler,
1992
). As reported in this study, urine is isotonic to blood in
SWS; the FWS are able to produce hypotonic urine, starting at least in
2-month-old juveniles (Nebel et al.,
2005
), probably through active ion reabsorption by the cells
lining the urinary epithelia. Other euryhaline teleosts like Platichthys
flesus (Lahlou, 1967
) and
Paralichthys lethostigma (Hickman
and Trump, 1969
) also possess this capacity to vary the urine
osmolality according to salinity. In contrast, the FWU not only show a low
blood osmolality, but have also lost the ability to save ions at the kidney
level since their urine is isotonic to their blood. We suggest that the FWU
urinary system is unable to actively reabsorb ions, particularly
Na+ and Cl, in order to produce hypotonic
urine.
Experiments were carried out to test whether this decreased capacity of ion
reabsorption from the filtrate originated from a lower amount and/or activity
of the Na+/K+-ATPase within the urinary epithelia. This
enzyme is located within the deep basolateral membrane infoldings of the cells
lining the urinary tubules and ducts at both salinities, and of the dorsal
part of the bladder in FW (Nebel et al.,
2005). The localization of Na+/K+-ATPase
revealed by immunofluorescence varies according to the kidney section; in this
study, a mostly basal cell location was observed in all urinary tubules,
whereas the collecting ducts presented homogeneously distributed cell
fluorescence. These characteristics have also been observed during the
ontogeny of the sea-bass (see discussion in
Nebel et al., 2005
). The high
amount of Na+/K+-ATPase in the collecting ducts and
dorsal bladder in FW suggests their increased involvement in active ion
transport. The tubular fluid has been shown to become progressively diluted
along the distal tubule, the collecting tubule/collecting duct system (CT/CD)
and the succeeding urinary bladder through Na+ and
Cl reabsorption (review in
Hentschel and Elger, 1989
). In
SWS sea-bass, the ducts seemed to be less stained and the urinary bladder
showed no staining, which helps to explain the lower
Na+/K+-ATPase content and activity reported in kidney
homogenates. At high salinities, the physiological need for ion retention
decreases and instead the fish secrete/excrete ions (mainly divalent ions) in
order to avoid ion invasion.
When comparing the FWS and FWU urinary systems, no apparent difference in
Na+/K+-ATPase immunostaining was noted in the urinary
tubule, duct and bladder between the two fish categories. But the enzyme
abundance and activity were noticeably lower in FWU compared to FWS. The
relative tubular density in the different sea-bass groups was thus
investigated and found to be lower in the FWU than FWS. This observation means
either a lower number or a lower length of nephrons in FWU than in FWS. Cell
apoptosis might occur in the FWU kidney, as reported in the proximal tubules
of Cyprinus carpio (Fischer and
Dietrich, 2000). In the present study, no distinction was made
between the different tubule sections, so which of these is likely to be less
represented in FWU is therefore not known. The urinary tubules enclose at
least proximal tubules I and II, as well as collecting tubules
(Nebel et al., 2005
), which
have different functions (Hickman and
Trump, 1969
). The low blood osmolality in the FWU suggests a
possible degeneration of the urinary tubules mainly involved in ion
reabsorption. These may be the first proximal tubules in the sea-bass, since
their cells possess a dense system of apical tubules, endocytotic vesicles and
vacuoles, all suggesting a high activity of reabsorption
(Nebel et al., 2005
). These
tubules are known to be the site of ion reabsorption in Pseudopleuronectes
americanus (Elger et al.,
1998
). Ultrastructural observations of the FWU tubules are thus
necessary.
We also note that the Na+/K+-ATPase content and activity were similar in SWS and FWU fish. The apparent similarity between these data may lead to the hypothesis that the proper (possibly hormonal) signal for FW adaptation was not given, or not received, at the kidney level, in the FWU sea-bass. However, Na+/K+-ATPase immunolocalization is quite different between SWS and FWU, particularly in the collecting ducts and the dorsal bladder. In addition, the renal tubule density is much lower in FWU than in SWS fish. Thus, according to these available data, the signal hypothesis may be ruled out.
The kidney, and particularly the distal segments, play an essential role in
the regulation of water balance (Nishimura
et al., 1983). Active ion reabsorption is often coupled to an
osmotic water transport to the blood, depending on the water permeability of
the urinary epithelium. The collecting ducts are lined by a membrane
impermeable to water, and the rate of water transport depends on hormonal
stimulation, on the presence of aquaporins and on the rate of water delivery
by the nephrons (reviewed by Nishimura and
Fan, 2003
). In FW teleosts, the water permeability of the
collecting ducts is very low (Nishimura et
al., 1983
). In FWU, an increased permeability of the collecting
ducts to water cannot be ruled out, and might be another factor contributing
to the decrease in blood osmolality.
In summary, the ion transporting pump is less abundant and less active in the FWU urinary system, because of a lower density of urinary tubules, possibly due to their partial degeneration. If the reabsorbing tubules are less numerous or damaged, this may cause, over several weeks, a decrease in the net active ion reabsorption from the filtrate, resulting in a net loss of ions by the organism. This altered function may in turn result in a decreased blood osmolality, which must be readjusted by ion absorption from the external medium by other osmoregulatory organs like the gills. This physiological attempt at osmoregulatory compensation would explain the high number and activity of the branchial chloride cells in active ion transports in FWU. During chronic exposure to FW, the branchial ion uptake is not sufficient to compensate for the urinary ion loss, resulting in an osmoregulatory imbalance leading to a decrease of the blood osmolality to critical levels and, finally, to the death of the FWU.
In the wild, a fraction of the juvenile sea-bass population is believed to
undergo seasonal low salinity and/or to migrate to FW and there is indirect
evidence of differential mortality
(Lemaire et al., 2000). These
differential mortalities observed in FW probably result from the existence, in
the sea-bass stock from which our experimental animals were drawn, of a
polymorphism for osmoregulatory capacity, as suggested by Allegrucci et al.
(1994
). The existence of this
polymorphism and the ensuing selective pressure when juveniles enter brackish
waters would explain the genetic divergence observed between marine and lagoon
sea-bass samples in the western Mediterranean
(Lemaire et al., 2000
).
Correlates of these differential physiological abilities at the genetic level
will be investigated in future studies. It still remains to be determined
whether individuals select their habitat as a function of their osmoregulatory
ability (in this case, fish found upstream rivers would be assimilated to the
FWS in our study), or passively undergo the selective mortalities following
their migration to highly unpredictable environments such as lagoons, in which
case local adaptation would be achieved at a high genetic cost.
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