Physiological, biochemical and morphological indicators of osmoregulatory stress in `California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water
1 Department of Zoology, University of British Columbia, 6270 University
Boulevard, Vancouver, BC V6T 1Z4 Canada
2 Department of Biology, San Diego State University, 5500 Campanile Road,
San Diego, CA 92182 USA
3 Department of Biology, University of San Diego, 5998 Alcala Park, San
Diego CA, 92110 USA
* Author for correspondence (e-mail: sardella{at}zoology.ubc.ca)
Accepted 23 January 2004
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Summary |
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Key words: `California' Mozambique tilapia, Oreochromis mossambicus x O. urolepis hornorum, osmoregulatory stress, Salton Sea, chloride cell, salinity challenge
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Introduction |
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Teleosts exposed to hyperosmotic environments experience osmotic water loss
coupled with diffusive ion gains. To offset the loss of water, an increase
drinking rate becomes necessary (Brocksen
and Cole, 1972; Foskett et al.,
1981
; Hwang et al.,
1989
; Cioni et al.,
1991
); in the intestine water is absorbed osmotically, following
active absorption of salts across the epithelium
(Wilson et al., 1996
). In
order to offset the increased ion load, and ultimately survive in saline
waters, fish must actively excrete Na+ and Cl.
Gills are the primary organ involved in osmoregulation in teleost fishes.
Ion-transporting chloride cells, located on the branchial filamental
epithelium, are rich in Na+, K+-ATPase, which drives
excretion mechanisms. Chloride cells are responsible for ion excretion in both
juvenile and adult seawater-acclimated fish
(Foskett et al., 1981
;
Laurent and Dunel, 1980
;
Marshall and Bryson, 1998
;
Perry, 1997
). Chloride cells
in seawater-acclimated fish are generally rich in mitochondria, bear an
extensive basolateral tubular reticulum, and have direct contact with ambient
water through their apical membrane and with blood through the basolateral
cellular membranes (Laurent,
1984
; van der Heijden et al.,
1997
; Wendelaaar Bonga and van
der Meij, 1989
). Four stages of the chloride cell life cycle have
been identified, and include (1) large columnar mature cells, which have an
ultrastructure that is typical of actively functioning cells, (2) slim
crescent-shaped accessory cells, which are less abundant in mitochondria and
have a poorly developed tubular reticulum, (3) tear-shaped immature cells,
structurally intermediate between mature and accessory cells and (4) small and
round degenerating, or apoptotic cells, which have a highly condensed
cytoplasm, multi-lobular and heterochromatic nucleus, and dilated mitochondria
and tubular reticulum (Wendelaar Bonga and van der Meij, 1989). Salinity
tolerance in fishes is dependent upon the appropriate physiological,
biochemical and morphological adjustments to a given salinity.
The salinity tolerance of pure Mozambique tilapia Oreochromis mossambicus has been previously investigated; however, studies have generally focused on seawater acclimation (35 g l1) or hypersaline (>35 g l1) acclimation following large increases in salinity (i.e. direct transfers). This study reports on the ability of a hybrid of this euryhaline species to gradually acclimate to salinities exceeding that of seawater (up to 95 g l1), and incorporates physiological (plasma osmolality, [Na+], [Cl], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical (Na+, K+-ATPase) and morphological (the number of mature, accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress. The ultimate goal of this study was to identify the most appropriate parameters that can be used to model salinity tolerance for fish species that inhabit hypersaline lakes, using a species that is currently known to inhabit such an environment, and to compare the salinity tolerance of this hybrid with that of pure Mozambique tilapia.
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Materials and methods |
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Series I: 28 day acclimation to seawater (35 g l1 salinity)
To determine the time course for seawater-acclimation in this hybrid, two
tanks of fish were kept at 35 g l1 salinity for 28 days, and
seven fish were removed and sampled at 0, 24 h (1 day), 120 h (5 days), 336 h
(14 days), 504 h (21 days) and 660 h (28 days) following transfer for
measurement of plasma osmolality, [Na+], and
[Cl], muscle water content, Hematocrit (Hct) and mean cell
hemoglobin concentration (MCHC). Fish sampled at 14 days were also sampled for
Na+, K+-ATPase activity, and for gill morphological
parameters.
Tilapia were anaesthetized using 0.7 g l1 benzocaine, which was first dissolved in 3 ml of ethanol. Fish were patted dry and weighed, and the caudal peduncle was severed. Blood was collected into heparinized microhematocrit capillary tubes for measurement of Hct and total hemoglobin. Approximately 1.5 g of the left dorsal epaxial muscle was removed to determine muscle water content. Muscle tissue was placed into pre-weighed, plastic scintillation vials and weighed prior to, and following drying to constant mass for 7296 h at 70°C.
The second and third right gill arches and approximately 1.5 cm of duodenal intestine were removed, frozen on dry ice and stored at 80°C for later analysis of Na+, K+-ATPase activity. The second and third left gill arches were removed from the left side of five fish at the 120 h sampling time, and immediately fixed in cold Karnovsky fixative for at least 2 h. The middle third of each gill arch was cut off by a razor blade, and gill arches about 1 mm long, bearing up to 20 filaments in both anterior and posterior rows were used for scanning electron microscopy (SEM), while individual filaments were cut for transmission electron microscopy (TEM) and light microscopy (LM) studies. All specimens were rinsed three times for 10 min each in the phosphate-buffered saline (PBS), and post-fixed in 1% osmium tetroxide for 1 h.
Series II: Direct transfer from seawater to 60 g l1 and 85 g l1 salinity
Thirty seawater-acclimated tilapia were directly transferred to salinities
of 60 and 85 g l1, via a three-quarter tank water
change. Seven fish from each tank were sampled at 0, 3, 24, and 120 h
following transfer. Fish from all sampling times were sampled for measurement
of plasma osmolality, [Na+], [Cl], muscle water
content, Hct and MCHC, while those samples at 120 h were also analysed for
Na+, K+-ATPase activity; sampling was conducted as
described in Series I.
Series III: Gradual salinity increase from seawater to 95 g l1 salinity
Seawater-acclimated tilapia in the remaining tanks were gradually exposed
to increased salinity. Salinity in all tanks was increased 10 g
l1 every 5 days, via a three-quarter water change,
up to 95 g l1; this yielded experimental salinities of 45,
55, 65, 75, 85 and 95 g l1. At each salinity, seven fish
were sampled at 0, 3, 24 and 120 h. Fish from all sampling times were sampled
for measurement of plasma osmolality, [Na+],
[Cl], muscle water content, Hct and MCHC, while those
samples at 120 h were also analysed for Na+, K+-ATPase
activity and morphology; sampling was conducted as described Series I.
Series IV: Oxygen consumption and drinking rate
Oxygen consumption rate
(O2) and
drinking rate were measured in fish acclimated to 35, 55, 75 and 95 g
l1.
O2 was measured
according to the methods of Gonzalez and McDonald
(1994
). Following measurements
at 35 g l1, salinity was increased 10 g l1
every 4 days until the next target salinity was reached. Fish were left at the
target salinity for 1 week before measurements were made, after which salinity
was increased again as previously described. When measuring
O2, six fish
were weighed and transferred to individual 140 ml chambers that were connected
to a 60 l recirculating system filled with water of the appropriate salinity.
Flow rate into individual chambers was approximately 100 ml
min1, and fish were allowed to recover for 24 h before
measurements were taken. To initiate measurements, water samples were
withdrawn from each chamber while water was still flowing. Flow was then
immediately stopped and the chambers were sealed. After 15 min, a second water
sample was taken and flow was restored. The initial and final water samples
were analyzed for PO2 with a Cameron (Guelph,
ON, Canada) O2 meter. Using the initial and final
PO2, and chamber volume, as well as fish mass,
time, and the oxygen absorption coefficient for the target salinity, moles of
O2 consumed per unit time were calculated. Two sets of six fish
were measured on consecutive days.
Drinking rate was measured using a modified method of Wilson et al.
(1996). Fish were placed in
individual 250 ml chambers connected to a recirculating system filled with 50
l of the appropriate salinity at least 24 h prior to the beginning of the
measurement period. 5 µCi (185 kBq) of titrated PEG was added to each
chamber and 5 ml water samples were taken after 4 and 8 h. After 8 h fish were
killed using MS-222, rinsed, weighed and ligated gut removed. The whole guts
were put in 4 ml 8% perchloric acid, homogenized for 1 min, and centrifuged.
The supernatant and water samples were assayed for radioactivity. Drinking
rate was calculated from the average activity per ml H2O over the 8
h and the total number of counts taken up by the gut in that period.
Analyses
Blood parameters
Heparinized microhematocrit capillary tubes were centrifuged at 11 500 revs
min1 for 3 min in a Damon IEC MB (Needham Heights, MA, USA)
microhematocrit centrifuge. Hct was recorded in duplicate or triplicate,
depending on available blood volume, and plasma was expelled into Eppendorf
tubes and frozen at 80°C for analyses of plasma osmolality,
[Na+] and [Cl]. Whole blood total hemoglobin
concentration ([Hb]) was measured using a Sigma total hemoglobin assay kit,
with absorbance measured using a spectrophotometer (Beckman DU640) at 540 nm;
MCHC was calculated as [Hb]/(Hct/100). Plasma osmolality was measured using an
osmometer (Wescor 5500V.P.), and expressed as mOsm kg1
H2O. Plasma [Cl] was measured using the
calorimetric mercuric thiocyanate method
(Zall et al., 1956), and
plasma [Na+] was measured using an atomic absorption
spectrophotometer model 3100A; (Perkin Elmer, Wellesey, MA, USA). Plasma
[Na+] and [Cl] are expressed as mmol
l1 plasma.
Na+, K+-ATPase activity
Gill tissue taken from fish exposed to salinity for 120 h were homogenized
in 1 ml of SEID buffer (250 mmol l1 sucrose, 10 mmol
l1 EDTA·Na2, 50 mmol l1
imidazole, pH 7.3). Na+, K+-ATPase activity at 120 h was
determined using the method of Gibbs and Somero
(1990) and expressed as µmol
l1 ADP h1 mg1 total
protein; protein was determined by the Biuret method
(Gibbs and Somero, 1990
).
Scanning electron microscopy.
Fixed specimens were dehydrated in a graded ethanol series, concluding at
100%, critical-point-dried with liquid CO2, mounted on stubs,
sputter-coated with goldpalladium, and examined using a scanning
electron microscope (Hitachi S 2700; Tokyo, Japan) at the accelerating voltage
of 10 kV. Areas on the trailing (afferent) surface of filament behind the
secondary lamellae and without interlamellar regions were randomly chosen.
Photographs at 2000x magnification of this area (2400 µm2)
from ten different filaments per fish and three fish per group were used for
quantification of apical pits and general examination of the superficial
structure of filament epithelium.
Light microscopy and transmission electron microscopy.
Fixed specimens were dehydrated in a graded ethanol/acetone series with the
final change in absolute acetone before infiltration and embedding in Epon
epoxy resin. Longitudinal semithin (1 µm) and ultrathin (6070 nm)
sections, made parallel to the long axis of filaments, were cut using a
microtome (LKB; Uppsala, Sweden). Semithin sections were mounted on glass
slides, stained with 0.5% Methylene Blue, and examined on a Diastar
microscope. Ultrathin sections were mounted on the copper grids, double
stained with 1% uranyl acetate followed by 0.5% lead citrate, and examined in
an electron microscope (Philips 410; Eindhoven, The Netherlands) at 80 kV. The
ultrastructure of chloride cells was studied in the interlamellar areas of the
filament epithelium. The percentage of mature, accessory, immature, apoptotic
and necrotic chloride cells was determined, following the method described by
Wendelaaar Bonga and van der Meij
(1989), on 20 randomly
selected interlamellar areas, in three filaments per fish, and for four fish
per group.
Statistics
The effects of salinity on Hct, MCHC, plasma osmolality, [Na+],
[Cl], muscle water content and morphological data were
analyzed using one-way analysis of variance (ANOVA) followed by a
post-hoc Dunnet's test.
O2 and drinking
rate were analyzed by one-way ANOVA followed by a post-hoc Tukey
test. ANOVAs were performed using JMP (SAS Institute 2000). A value of
P<0.05 was taken for significance in all statistical tests.
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Results |
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Morphology
Following 2 weeks of exposure to 35 g l1, pavement cells,
a predominant component of the outer layer of filamental epithelium, displayed
both long and short microridges organized into a concentric pattern with a
flat central area. Numerous deep apical pits of multicellular chloride
complexes and few pores of mucous cells were observed on the surface of
filamental epithelium (Fig.
2B). Chloride complexes combined mature, immature and accessory
cells that were linked by short apical tight junctions and followed desmosomes
(Fig. 3A,B). No
interdigitations between apexes of chloride cells within the multicellular
complexes were found. Aging chloride cells degrading by apoptosis displayed a
reduced size, highly condensed cytoplasm, a markedly indented nucleus with
condensed chromatin, and numerous enlarged mitochondria surrounded by
distended tubules of the network (Fig.
3D). Numbers of mature, accessory, immature, and apoptotic cells
are presented in Table 1.
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Series II: Direct transfer from seawater to 60 g l1 and 85 g l1 salinity
Direct transfer from 35 g l1 to 85 g l1
salinity resulted in 100% mortality. In contrast, all fish survived direct
transfer from 35 g l1 to 60 g l1, but
significant effects on sub-lethal indicators of osmoregulatory stress were
observed. Plasma osmolality, [Na+] and [Cl]
increased only qualitatively 3 h post-transfer, while significant increases
over pre-transfer values were observed 24 h after, but only in osmolality
(Fig. 4). Plasma osmolality,
[Na+] and [Cl] all returned to near pre-transfer
values by 120 h. Na+, K+-ATPase activity significantly
increased from 2.46±0.48 to 8.87±1.62 µmol
h1 µg1 protein between 0 and 120 h,
respectively, which represents a near 260% increase in activity following
transfer from 35 to 60 g l1 salinity.
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Series III: Gradual salinity increase from seawater to 95 g l1 salinity
Plasma osmolality, [Na+] and [Cl]
With the exception of minor mortality observed at 85 g l1
salinity, tilapia hybrids survived a 10 g l1 salinity
increase every 5 days up to a salinity of 95 g l1. Plasma
osmolality values were not significantly different, relative to those at 45 g
l1, in fish exposed to 5565 g l1
salinity, and were consistent with values obtained from 35 g
l1 fish sampled at 14 and 28 days. However, in fish exposed
to 75 g l1 salinity, osmolality was significantly increased
at 24 and 120 h following transfer. At the 3 h sampling time, plasma
osmolality was significantly increased relative to 45 g l1
in fish exposed to salinities of 85 and 95 g l1
(Fig. 5). Plasma
[Na+] and [Cl] followed similar trends, and were
significantly elevated at 85 and 95 g l1, with the exception
of [Cl] at 3 h, which was only significantly increased at 95
g l1 salinity (Fig.
6).
|
|
Na+, K+-ATPase activity
Branchial Na+, K+-ATPase activity remained relatively
constant in gills exposed to 3565 g l1 salinity, but
was significantly increased at 75 and 85 g l1
(Fig. 7). Gut Na+,
K+-ATPase activity averaged 5.30±0.41 mmol ADP
h1 mg1 protein over the range of
salinities, and did not vary significantly (data not presented).
|
Morphology
The superficial pattern of pavement cells was largely unchanged from that
of a typical seawater-acclimated epithelium in fish exposed to 4565 g
l1 salinity (Figs
2,
8A), although the first
appearance of interdigitated junctions between mature and accessory chloride
cells was in fish exposed to 55 g l1 salinity
(Fig. 9). However, at
7595 g l1 salinity the surface of pavement cells bore
fewer marginal microridges, and cells had an expanded flat central area
(Fig. 8BD). The number
of apical pits per 2400 µm2 remained relatively consistent in
the epithelia of tilapia exposed to 3555 g l1, but
then was significantly increased from 6595 g l1
salinity (Table 1). No mucous
cells were observed in the filamental epithelium of fish exposed to 75 g
l1 salinity or greater. The first appearance of
interdigitated mature and accessory chloride cells was noted in fish exposed
to 55 g l1 salinity (Fig.
9A).
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The proportion of cells within the four stages of the chloride cell life cycle also changed with salinity. The number of cells in mature, accessory, immature or apoptotic stages quantified within 20 interlamellar regions, remained relatively consistent in fish exposed to 3555 g l1 salinity (Table 1). At 6595 g l1 salinity, the number of accessory and apoptotic chloride cells significantly increased, and at 75 g l1 the number of immature cells became significantly increased, while the number of mature cells was reduced. Changes in cellular parameters are summarized in Table 1, and TEMs representing different stages of chloride cells exposed to various salinities are presented in Figs 10, 11, 12. Finally, several cells from epithelia of fish exposed to 95 g l1 salinity exhibited features that are considered signs of cellular necrosis, including electron-transparent vacuolated cytoplasm, swollen mitochondria possessing fragmented cristae, an irregularly organized tubular reticulum, and a nucleus with a low electron density (Fig. 12FG).
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Series IV: Oxygen consumption rate and drinking rate
Oxygen consumption rate
(O2) decreased
with salinity. In fish acclimated to 95 g l1 salinity,
O2 decreased
40.5% relative to fish acclimated to seawater. Relative decreases in
O2 at 55 and 75
g l1 salinity were 17.6 and 29.5%, respectively
(Fig. 13). Drinking rate
remained constant at 35 and 55 g l1 salinity, but was
significantly increased at 75 g l1 salinity
(Fig. 14). Fish acclimated to
95 g l1 for 2 weeks did not show an increased drinking rate,
but these fish were in poor condition and means are based on N=3 due
to mortality.
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Discussion |
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When tilapia hybrids are gradually transferred to increased salinity, with 120 h between transfers, they survive exposures to salinity as great as 65 g l1 before showing any signs of osmoregulatory stress. Tilapia no longer maintained a steady state with respect to osmoregulation above 65 g l1 salinity. Changes in gill morphology were most sensitive, and first appeared after 120 h of exposure to 55 g l1 salinity, indicating that they may be good indicators of osmoregulatory stress in this species. More specifically, it was concluded that the number of apoptotic chloride cells would best serve as an indicator of stress, due to their sizable increase immediately prior to other signs of osmoregulatory failure such as elevated plasma osmolality, [Na+], [Cl], and branchial Na+, K+-ATPase.
Series I: Seawater acclimation of `California' Mozambique tilapia
Physiological, biochemical and morphological data indicate that 2 weeks is
sufficient for tilapia hybrids to become acclimated to seawater. Transfer from
20 to 35 g l1 salinity resulted in increases plasma
osmolality, [Na+] and [Cl] 24 h following
transfer (Fig. 1), but plasma
osmolality decreased to a relatively constant level, near 340 mOsm
kg1, when measured at 2 and 4 weeks. Although plasma
[Na+] and [Cl] remained significantly elevated
over the 4 week period, concentrations consistently remained near 180 and 160
mmol l1, respectively, from 2 to 4 weeks. Hwang et al.
(1989) found that
seawater-acclimated Mozambique tilapia had a plasma osmolality and
[Cl] of 343.8±17 mOsm kg1 and
156±8.8 mmol l1, respectively, and Uchida et al.
(2000
) found
seawater-acclimated tilapia had an osmolality of approximately 330 mOsm
kg1. Furthermore, the general morphology and ultrastructure
of the branchial epithelium of tilapia hybrids acclimated to 35 g
l1 salinity were similar to those previously observed in
other seawater-acclimated species of tilapia
(Cioni et al., 1991
;
Hwang, 1987
;
van der Heijden et al., 1997
;
Wendelaaar Bonga and van der Meij,
1989
).
Series II: Mortality and sub-lethal effects of direct transfer to increased salinity
Survival was 100% following direct transfer from 35 g l1
to 60 g l1 salinity; however, significant effects on plasma
osmolality, [Cl] (Fig.
4) and Na+, K+-ATPase activity were
observed. Plasma [Na+] and [Cl] followed a
similar pattern to plasma osmolality, which was significantly elevated at 24 h
following transfer but returned to near pre-transfer values by 120 h
(Fig. 4). The return of plasma
osmolality to pre-transfer levels, in spite of a greatly increased osmotic
gradient, is probably due to the nearly 260% increase in Na+,
K+-ATPase activity that was observed 120 h following transfer.
Kultz et al. (1992) also
observed large increases in enzyme activity when transferring Mozambique
tilapia from 10 g l1 to 45 g l1, and 60 g
l1 salinity.
III: Sub-lethal effects of graded transfer to increased salinity
The increase in salinity from 35 g l1 to 65 g
l1 represents a more than twofold increase in the osmotic
gradient between the water and the blood, yet no significant increases in
plasma osmolality, [Na+] or [Cl] (Figs
5,
6) were observed, nor was there
an increase in Na+, K+-ATPase activity
(Fig. 7). Kultz and Onken
(1993) observed a dramatic
reduction in chloride cellaccessory cell leaky junction conductance
over the range of salinities from 35 g l1 to 60 g
l1, and suggested that Mozambique tilapia reduce their whole
body permeability in hypersaline water up to 60 g l1, which
could offset the cost of osmoregulation. This hypothesis is consistent with
the data obtained in the current study. The formation of interdigitation
junctions in fish exposed to 55 g l1 salinity may indicate
that the fish are approaching the limit for effective osmoregulation
via epithelial permeability reduction, and that a more traditional
mechanism of salt excretion, such as an increase in Na+,
K+-ATPase activity or drinking rate, is needed
(Laurent, 1984
). A similar
phenomenon was described in Mozambique tilapia after 5 days of acclimation to
full-strength seawater (Wendelaar Bonga and van der Meij, 1989).
Another possible explanation for the lack of change in plasma osmolality, [Na+] and [Cl] could be that the measured level of Na+, K+-ATPase activity is sufficient to deal with environmental salinities up to 65 g l1. Furthermore, in spite of the lack of change in gut Na+, K+-ATPase, the role of the gut in water absorption should not be ruled out; drinking rate was unchanged between 35 and 55, but increases in water absorption rate still may have occurred.
At exposure to salinities greater than 65 g l1, there are significant changes in physiological, biochemical and morphological indicators of osmoregulatory stress, which are probably indicative of osmoregulatory failure at the highest salinities. Plasma osmolality became significantly elevated, relative to seawater, 3 h following transfer to 85 g l1 and 95 g l1 salinity and at 24 and 120 h following transfer to 7595 g l1 salinity; with plasma [Na+] and [Cl] following similar trends (Figs 5, 6). These increases in osmolality and ion levels coincide with a dramatic increase in Na+, K+-ATPase activity at 75 g l1 and 85 g l1 (Fig. 7), which is a good indication of an osmoregulatory challenge.
Exposure to salinities greater than 65 g l1 increased the
turnover rate of branchial chloride cells. Changes in physiological and
biochemical parameters in fish exposed to 6595 g l1
were associated with wide-scale morphological alterations along the branchial
epithelium. In 65 g l1-exposed fish, the number of accessory
cells and apoptotic chloride cells increased nearly twofold and threefold,
respectively, but the number of mature cells was similar to that in fish
exposed to 3555 g l1 salinity. Exposure to
7595 g l1 salinity resulted in dramatic changes in
the ratio between different subtypes of chloride cells. Mature chloride cells
constituted only 11% of the total number of cells in 75 g
l1-exposed fish, and only 5% in 95 g
l1-exposed fish; in contrast, mature cells constituted
3236% of all chloride cells in fish exposed to 3555 g
l1 salinity (Table
1). Similar results observed in freshwater-acclimated tilapia,
exposed to a number of stressful factors (e.g. high salinity, low pH or
cadmium), were attributed to an increased turnover of chloride cells due to
rapid aging (van der Heijden et al.,
1997; Wendelaaar Bonga and van
der Meij, 1989
).
TEM photos of the tilapia epithelium exposed to 75 g l1 in the current study reveal partial and completely covered apical pits (Fig. 11B,C), which probably occurred at other high salinities as well. It is unclear to what end tilapia in hypersaline water are using a low-salinity acclimation strategy, but it may represent an attempt to seal off chloride cell leaky junctions to prevent further gain of salts or loss of water to the external environment. A more detailed investigation into this phenomenon will certainly prove interesting.
Fish exposed to salinities above 65 g l1 face a further
osmoregulatory challenge, as the branchial epithelium may have a drastically
reduced capacity for ion transport. It has been previously shown that the
ion-transporting capacity of the epithelium is dependent on the number of
mature cells more so than on total number
(Kultz et al., 1992).
Functional activity of accessory and immature cells should be lower relative
to mature cells, due to their poorly developed tubular reticulum and a limited
number of mitochondria. Furthermore, a very low ion-transporting capacity, if
any, may be expected from degenerating cells, and chloride cells occluded by
pavement cells may be considered functionally silent. Significant increases in
plasma osmolality, [Na+] and [Cl] are observed in
concert with a significant decrease in the number of mature chloride cells.
Interestingly, Na+, K+-ATPase activity was highest at 75
g l1 and 85 g l1 salinity
(Fig. 7); the source of the
increase in enzyme activity in a largely degenerating epithelium is another
point of interest for further study. The significance of the increased
turnover rate of chloride cells with respect to survival of the animal is also
unclear, but it is unlikely that fish could survive long exposure periods at
salinities above 5565 g l1, in particular at 95 g
l1 when signs of cellular necrosis in the epithelium become
apparent.
Series IV: Oxygen consumption rate and drinking rate
Oxygen consumption rate
(O2) decreased
with salinity, by as much as 40.5% from 35 g l1 to 95 g
l1 (Fig.
13). Although the majority of previous work on how salinity
affects
O2 has
focused on changes during acclimation from freshwater to seawater
(Farmer and Beamish, 1969
;
Morgan et al., 1997
), Swanson
(1998
) observed a reduction in
O2 at 55 g
l1 salinity relative to 35 g l1 in
milkfish Chanos chanos, and attributed it to a reduction in activity
level, allowing for greater use of metabolic energy for osmoregulation. An
alternative hypothesis was proposed by Haney and Nordlie
(1997
), when sheepshead minnow
Cyprinodon variegatus
O2 decreased
nearly 33% over a range of salinities from 40 g l1 to 100 g
l1; this decrease is relatively comparable to that observed
in the current study. Haney and Nordlie
(1997
) suggested that the drop
in
O2 was
related to a change in the permeability of the branchial epithelium, as
described by Kultz and Onken
(1993
), as the reduction in
branchial permeability would result in less oxygen diffusion into the animal.
These hypotheses are not mutually exclusive, and it is probable that both play
a role in tilapia hybrid osmoregulation.
Drinking rate was not significantly changed at salinities below 75 g l1 (Fig. 14). The lack of an increase over this salinity range indicates that the tilapia are not losing excess water to the environment in spite of the increase from 35 g l1 to 55 g l1, and the large increase in osmotic gradient between the blood and the water. This is consistent with the hypotheses formed in Series III that suggest a preventative strategy of osmoregulation in hypersaline water up to a salinity of 65 g l1 via a reduction in branchial permeability.
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Conclusions |
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Morphology, in particular the number of apoptotic chloride cells in the epithelium, appears to be the most sensitive and dramatic indicator of osmoregulatory stress. Gill morphology remained relatively unchanged until fish were exposed to a salinity of 55 g l1, but plasma parameters tended not to change until fish were exposed to salinities greater than 65 g l1. To better visualize these changes, morphological parameters were plotted on a scale of relative change, with values obtained at 55 g l1 set to 1.0, and the same with plasma parameters obtained at 65 g l1 that were also set to 1.0 (Fig. 15). This interpretation provides a good template for potential salinity tolerance modeling for this species when exposed to graded salinity increases. Whether or not the use of gill morphology as an osmoregulatory stress indicator is effective over longer durations of exposure to hypersalinity remains to be seen.
|
The final goal of this study was to gain insight into the salinity
tolerance of `California' Mozambique tilapia, which has ecological relevance
considering the continual increase in salinity of the Salton Sea; it may be
concluded that with all other variables under strict control, these hybrids
can tolerate salinities up to 65 g l1 and show little to no
change in physiological parameters, with the possible exception of the
decreased activity level that was indicated by a reduced
O2 values.
Furthermore, these tilapia hybrids seem to employ a strategy involving a
reduction in permeability in hypersaline conditions, which has been previously
described by Kultz and Onken
(1993
). Above 65 g
l1 salinity, changes in physiological, biochemical and
morphological indicators of osmoregulatory stress become apparent, indicating
a limit to salinity tolerance via permeability reduction. To further
add to this model, the effects of other abiotic factors such as temperature or
hypoxia, which have been observed to fluctuate in saline lakes such as the
Salton Sea, need to be included; temperature in particular has been shown to
greatly reduce salinity tolerance in this tilapia hybrid (Sardella et al.,
2003), and reduce osmoregulatory ability
(Al Amoudi et al., 1996
).
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Acknowledgments |
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References |
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