Max Freshwater Res., 1995,46,947-52
Salinity Tolerance and Osmoregulation in the Silver Perch,
Bidyanus bidyanus Mitchell (Teraponidae), an Endemic
Australian Freshwater Teleost
R. Guo, P. B. at her^ and M. F. Capra
School of Life Science, Queensland University of Technology, Brisbane, Qld 4001, Australia.
*To whom all correspondence should be addressed.
Abstract. Juvenile silver perch, Bidyanus bidyanus, were subjected to direct transfer from fresh water
to various test salinities. No mortality was observed when the fish were transferred from fresh water to
a salinity of 12, but 40% mortality was observed at a salinity of 15 after seven days. Pre-acclimation of
silver perch to a salinity of 12 for seven days resulted in only marginally better survival at higher
salinities. Plasma osmotic concentrations of silver perch rose slightly in salinities below 9 but rapidly at
higher salinities, following the same track as the iso-osmotic line. Minimum body water content was
observed in individuals subjected to a salinity of 15 for 24 h. As found in other freshwater teleosts,
chloride cells were found in the branchial epithelium of silver perch. Accessory cells were observed
beside the chloride cells in both freshwater and salt-water conditions. Fish subjected to a salinity of 12
for seven days showed chloride cells with a more developed tubular system than controls. The length of
the junctions between chloride cells and accessory cells was significantly shorter in fish adapted to a
salinity of 12 than in controls. The ultrastructural feature of 'interdigitations' of accessory cells was not
observed during salt-water adaptation. These data indicate that silver perch is the least tolerant of high
salinities and the most truly freshwater Australian teleost species examined to date.
Introduction
There is little published information on osmoregulation in
Australian freshwater fishes. The silver perch, B, bidyanus,
is a freshwater fish native to the Murray-Darling river
system of southeastern Australia (Merrick and Schmida
1984). Silver perch is a potamodromous species, migrating
wholly within fresh water. The adults undergo extensive
upstream migrations and require an increase in water level to
induce spawning (Cadwallader 1986). Silver perch
generated from hatchery stock have been stocked in many
waterways and ponds in eastern Australia and are also
considered to have high potential for aquaculture (Rowland
and Barlow 1990).
The chloride cells of the gill epithelium are thought to
play a major role in the hydromineral regulation of teleosts
(Keys and Willmer 1932; Philgott and Copeland 1963;
Maetz 1971). It has been well documented that, during
adaptation to salt water, chloride cells increase their cell
number andlor cell volume (Utida et al. 1971; Thomson and
Sargent 1977; Hootman and Philpott 1978; Pisam 1981) and
also show ultrastructurall changes associated with the
development of the tubular system and mitochondria (Doyle
and Epstein 1972; Karnaky et al. 1976; Hossler et al. 1985).
It has also been reported that salt-water adaptation involves
the development of the so-called accessory cells next to, and
in close association with, the apical portion of chloride cells
(Dune1 and Laurent 1973; Sardet et al. 1979; Hootman and
Philpott 1980; Chretien and Pisam 1986). A striking
ultrastructural feature of accessory cells is the presence of
lateral cytoplasmic processes that penetrate the apical
portion of the chloride cell and form numerous plasma
membrane interdigitations during salt-water adaptation
(Dune1 and Laurent 1973; Hootman and Philpott 1980).
Chloride cells and accessory cells are linked by shallow
junctions compared with the tight junctions between
chloride cells and pavement cells (Sardet et al. 1979).
The ability to possess accessory cells and to change the
intercellular organisation and junctional structures has been
suggested as being correlated with the euryhalinity of fish
(Hwang and Hirano 1985).
Parts of the Murray-Darling system are naturally saline,
but since the development of extensive irrigation the
salinities of some rivers have increased (Collett 1978). To
date, there has been no evidence of significant effects of
increasing salinities on the fish fauna. Guo et al. (1993)
reported that there were no significant effects of salinities
below 9 on development of silver perch eggs. Larvae
hatched at a salinity of 6 had a better survival rate than those
hatched in fresh water during the first 20 days after hatching
(Guo et al. 1993). It is likely that during the course of their
evolution many species associated with the Murray-Darling
system have had to contend with large natural fluctuations in
salinity (Cadwallader 1986). The present study examines the
R. Guo et al.
salinity tolerance and osmoregulation of silver perch, a
freshwater teleost species that has had to contend with both
natural and artificial fluctuations in salinity.
Materials and Methods
The salinity units used in this paper follow the Practical Salinity Scale
of 1978 (PSS 78). Juvenile silver perch between 6 and 8 cm total length
were purchased from a local freshwater fish hatchery. The fish were kept in
tanks supplied with recirculated fresh water for at least four weeks before
use. Fish were fed twice a day with a pelleted commercial fish food. Water
temperature ranged from 22°C to 27°C during this period.
Salinity Tolerance of Silver Perch
Two experiments were carried out. Experiment 1 was to determine the
effects of direct transfer of juvenile silver perch from fresh water to a
number of test salinities. The experiment was conducted in four
recirculated-water systems; each system contained three experimental
tanks (12 L) with a header tank (25 L) and a large bottom tank (50 L). Water
was pumped from the bottom tank to the header tank and then fed by
gravity through the experimental tanks. Overflow water then flowed into
the bottom tank. Seven test salinities were used (0,6, 12,15,18,21 and 24).
generated by mixing sea water (30) with aged tap water. Experiments were
divided into two sets. First the test salinities 0, 6, 12 and 24 were used,
followed by the test salinities 0, 15, 18 and 21. Each test salinity occupied
one recirculated system. Therefore, the overall design of this experiment
consisted of seven salinities x three replicates, or 21 individual
experimental tank trials. Photoperiod was maintained in a 1ight:dark
regime of 12 : 12 with light from 0700 to 1900 hours, and water temperature
was kept at 24°C & 1°C by controlling room temperature. Ten fish were
transferred directly from a freshwater rearing tank to each individual
experimental tank. Survival was recorded each hour for the first 6 h and
then at 12, 18,24,48,72,96, 120, 144 and 168 h after the start of each trial.
Half volumes of water in each system were changed during the
experimental period, and the fish were fed as normal (feeding activity was
generally low). The experiment was terminated after individuals had been
subjected to the individual test salinities for seven days.
The aim of Experiment 2 was to determine the effects of transferring
juvenile silver perch (which were acclimated to a salinity of 12 for
seven days) to higher salinities. The experiment was canied out in
recirculated-water systems similar to those described above. Three test
salinities were used (12, 18 and 24). Sets of 10 juvenile silver perch that
had been acclimated to a salinity of 12 were transferred into individual test
tanks. Survival rate was monitored over the next seven days.
Efjects of Salinity on Plasma Osnzoric Concentration and Body Water
Content
Six tanks (12 L) were set up with salinities of 0,6,9, 12, 15 and 18. Ten
juvenile silver perch chosen randomly were transferred directly from a
freshwater rearing tank into each experimental tank. At 24 h after transfer,
fish were sampled to measure plasma osmotic concentration and body
water content. Individuals exposed to a salinity of 18 were sampled earlier
at 15 h after transfer to avoid death before the experiment concluded.
Sampled fish were first anaesthetized in MS-222 (Sigma Chemical Co.)
(1 : 10000) for 5 min, and then the blood from five individuals per
treatment was sampled by severing the caudal peduncle and collecting the
free-flowing blood into 1.5-mL Epindorf tubes. Individual blood samples
were then centrifuged to isolate plasma. The osmotic concentration of
individual plasma samples was measured with a Fiske freezing-point
osmometer. The remaining five individuals per treatment were sacrificed
and dried with filter paper. Individuals were weighed, then dried in a 105°C
oven until they reached constant dry weight.
Branclzial Chloride Cells of Silver Perch
Gills from silver perch reared in fresh water and a salinity of 12 for
seven days were dissected out and pre-fixed with 3% glutaraldehyde (in
cacodylate buffer, pH 7.4) for 1 h at room temperature. They were then
stained by a Mn-Pb staining technique (based on Pisam et al. 1987, except
with lead nitrate instead of lead citrate; Pisam, personal communication)
for 2 h at room temperature. Tissue samples were post-fixed by a technique
based on ferrocyanide-reduced osmium (Kamovsky 1971) (1% osmium
tetroxide, 1.5% K4Fe,(CN),.3H,O, buffered with cacodylate, pH 7.4) for
1 h at room temperature. After being washed with distilled water, the
post-fixed gills were then dehydrated through a graded ethanol and acetone
series and embedded in Spun's resin (Spurr 1969). Ultrathin sections, of a
golden colour, were obtained with the aid of a Reichert 0 M U 2
ultramicrotome with glass knives, and with a lead citrate stain (2-5 min).
Observations of prepared specimens were made with a JEM-1200EX
transmission electron microscope.
Results
Salinity Tolerance
After direct transfer to test salinities, juvenile silver perch
showed 60% survival at a salinity of 15 but could not
tolerate higher salinities (Table 1). Fish in salinities higher
than 15 died within 18 h (Table 1). Survival of individuals
pre-acclimated to a salinity of 12 for seven days was only
Table 1. Mortality of silver perch (%) after direct transfer from fresh
water to various test salinities
Time after test
(h)
0
6
12
Test salinity
15
18
21
24
Table 2. Mortality of pre-acclimated silver perch (%) after direct
transfer froma salinity of 12 to higher salinities
Silver perch had been acclimated to a salinity of 12 for seven days
Time after test
(h)
12
Test salinity
18
24
Osmoregulation in the Silver Perch
marginally better in higher salinities (Table 2). All
pre-acclimated individuals transferred to salinities higher
than 15 died within 24 h (Table 2).
All individuals showed stress responses following
transfer to salinities above 15. For the first 2 h individuals
were generally calm, swimming slowly near the bottom of
the tanks, following which they moved up, close to the
water surface, where many attempted to gulp air. Later,
individuals turned black in colour and some developed skin
haemorrhages. Finally, individuals lost the ability to swim
and lay on the bottom until breathing activity ceased
completely.
Salinity Effects on Plasma Osmotic Concentration and
Body Water Content
Plasma osmotic concentrations of individuals exposed to
different test salinities showed that with an increase in
environmental salinity, plasma osmotic concentration also
rose at salinities between 0 and 9. Further increases in
salinity resulted in a rapid increase in plasma osmotic
concentration that followed the same track as the
iso-osmotic line (Fig. 1). Individuals exposed to a salinity
of 18 for 15 h had virtually the same osmotic concentration
(570 mOsm kg-' H20) as the external medium. Analysis of
the body water content of silver perch exposed to the
different test salinities showed that individuals displayed
some dehydration even after exposure to a salinity of 6 (Fig.
1). Minimum body water content was recorded in fish
0
6
12
exposed to a salinity of 15, where it reached a value of
67.5%, which was 2.5% less than that of freshwater control
fish (Fig. 1).
Characterization of the Branchial Chloride Cells of Silver
Perch
Chloride cells were observed in the branchial epithelium
of silver perch on the trailing edge of the filaments. All
chloride cells showed the same basic morphology.
Mitochondria were numerous and evenly distributed
throughout the cytoplasm. An abundant smooth-walled
tubular system opened to the basolateral plasma membrane,
which was densely ramified throughout the cytoplasm. Most
chloride cells also had their apical membranes directly
exposed to the outer medium. Part of the basal membrane
made close contact with the basal lamina. Chloride cells
were located either at the base of the secondary lamellae or
in the interlamellar region. In most instances, two or more
chloride cells occurred together and formed a multicellular
complex.
Silver perch chloride cells were large and lightly stained,
and their apical surfaces were endowed with short microvilli
(Figs 2 and 3). Numerous membrane-bound bodies with
electron-dense contents were present in the apical regions
(Fig. 3). Inside the multicellular complex, smaller accessory
18
Test salinity
body water content in silver perch
Pig. 1. (0)Plasma osmolality and (0)
24 h after direct transfer from fresh water to various test salinities. The
dashed line is the iso-osmotic line. Data points are means + s.e., sample size
is 5 for each data point. Standard errors less than 5 (mOsm kg-' H20) are
not shown.
Fig. 2. Low-magnification photographs of the gill epithelium of silver
perch in freshwater condition. Three chloride cells (cc) are observed at the
base of secondary lamellae, in close contact with the pillar capillaries (P).
Chloride cells are associated with an accessory cell (ac), which is darker in
colour. Scale bar, 2 y m.
950
cells were observed beside chloride cells (Figs 2, 3 and 4).
Accessory cells could be easily distinguished from chloride
cells by their smaller size, less developed tubular system,
and darker colouration. Many isolated glycogen particles
were distributed throughout the accessory cell cytoplasm
(Figs 3 and 4), but no membrane-bound bodies were
observed in the apical regions except for some small
vesicular and tubular elements. No apical interdigitations
were observed between individual chloride cells and their
accessory cells.
Fig. 3. Chloride cells (CC) of silver perch from fresh water. The tubular
system is heterogeneous and consists of loosely anastomosed membranous
tubules (t). Many membrane-bound bodies (mb) are encountered in the
apical portion of the chloride cell. In an accessory cell, a few glycogen
particles (g) are observed. m, mitochondrion; er, endoplasmic reticulum;
PC, pavement cell; AC, accessory cell; j, apical junction between CC and
AC. Scale bar, 0.5 pm. Fig. 4. Chloride cell (CC) of silver perch exposed
to a salinity of 12 for seven days. The tubular system is more developed
than under freshwater conditions (Fig. 3) and consists mostly of a network
of tightly anastomosed membranous tubules (t). m, mitochondrion;
er, endoplasmic reticulum; g, glycogen particles; j, apical junction between
CC and AC. Scale bar, 0.5 pm.
R. Guo et al.
The chloride cells in the gills of silver perch exposed to a
salinity of 12 for seven days did not show any cytoplasmic
interdigitations as have been reported occurring in sea-water
or sea-water-adapted euryhaline fish. Their tubular system
developed extensively to form a tight network of
anastomosed membranous tubules (Fig. 4). There were
fewer microvilli on apical surfaces, and all the
membrane-bound bodies disappeared from apical regions
(Fig. 4). The length of the apical junction binding both
chloride cells and accessory cells was shorter: 2 0 4 0 nm in
length (Fig. 4) compared with 80-140 nm in control fish
(Fig. 3).
Discussion
The ability to maintain a suitable stable internal
environment is a necessary requirement for animals to
survive in osmotically unfavourable external environments
(Eckert et al. 1988). In fish, this ability has evolved over a
long period of time and is achieved by several
osmoregulatory organs involving the gill, gut and renal
systems. Endocrine regulation is also involved in fish
osmoregulation.
Silver perch occur naturally throughout the
Murray-Darling river system except in the cool, high, upper
reaches of streams (Pollard et al. 1980; Merrick and
Schmida 1984). So far, only 22 native species of fish have
been found to complete their life cycles in fresh water
within the Murray-Darling system (Cadwallader 1986).
Salinity tolerance studies of native freshwater fish have
shown that fish from the Murray-Darling river system can
tolerate relatively high salinities, e.g. golden perch
(Macquaria ambigua) has been found to tolerate salinities
up to 36 (Dulhunty and Merrick 1976; Langdon 1987) and
four smaller species (Retropinna semoni, Craterocephalus
stercusrnuscarum,
Hypseleotris
klunzingeri
and
Melanotaenia splendida) have their LD50 at salinities of
58.7,43.7,38 and 29.8, respectively (Williams and Williams
1991). Spangled perch (Leiopotherapon unicolor), a close
relative of silver perch also found in the Murray-Darling
river system, can tolerate salinities up to 35.5 (Beumer
1979). The present experiments demonstrate that silver
perch have a relatively poor ability to tolerate raised
salinities (above 15) in comparison with other common
native species in the Murray-Darling river system.
Freshwater species such as Retropinna semoni, Hypseleotris
klunzingeri and Craterocephalus stercusmuscarum are also
found naturally in saline lakes in Victoria with salinities
(Chessman and
1974).
perch
occur naturally in the Bohle River drainage (northern
Queensland), where salinities range from 4 to 15.2 (Beumer
1979). ~
~perchlis alsod found~in ~~k~
~ E ~ ~ often
~ ,
contains hypersaline water (Dulhunty and Merrick 1976;
Glover and Sim 1978).
Bsmoregulation in the Silver Perch
Silver perch have never been reported from natural
systems with salinities that exceed 3. They do occur,
however, in a few tidally influenced coastal streams in New
South Wales, where they may have been introduced (Lake
1971). Generally, fishes adapted solely to fresh water (water
salinities <3) cannot regulate plasma ion concentrations
when external osmolality rises above plasma osmolality
(Williams and Williams 1991). Most stenohaline freshwater
fish, e.g. the catfish (Clarias lazera), carp (Cyprinus carpio),
and goldfish (Carassius auratus) die at external salinities
between 10 and 20 (Chervinski 1984; Geddes 1979;
Threader and Houston 1983). Death is due to abnormal ion
ratios in the body fluids, resulting in neuromuscular
malfunction and dehydration (Holliday 1971).
Laurent and Dunel (1980) claimed that accessory cells
never occur in freshwater fish. During a study on the
freshwater rainbow trout (Salmo gairdneri), Pisam et al.
(1989) reported that numerous accessory cells could also be
found in freshwater-adapted fish. Hwang and Hirano (1985)
reported that smaller, more electron-opaque cells often
occurred beside chloride cells in freshwater carp (Cyprinus
carpio) and freshwater ayu (Plecoglossus altivelis); these
smaller cells resemble accessory cells but do not
interdigitate with neighbouring chloride cells. The chloride
cells of silver perch observed in the present study were
similar to those reported by Hwang and Hirano. Accessory
cells are thought to have a role in the osmoregulation of
sea-water-adapted fishes (Sardet et al. 1978, 1979; Hwang
1987, 1988; Pisam et al. 1988, 1990; Hwang et al. 1989;
Pisam and Rambourg 1991). The development of
interdigitations and leaky junctions at the apexes of chloride
cells has been suggested as being associated with the
requirement for larger ion exchange in sea-water-adapted
gills (Sardet et al. 1979; Laurent and Dunel 1980; Hwang
and Hirano 1985; Hwang 1987). In silver perch, although
there were some modifications of the tubular systems and
the Qunctional structures, the important cytological feature
of 'interdigitations' was not observed during salt-water
adaptation. The biological function(s) of accessory cells in
freshwater fish remain unknown.
Salinities in the Murray River of south-eastern Australia
now rarely exceed 1.2 (Mackay et al. 1988). It is believed
that, before impoundment, the Murray River was degraded
during very dry seasons to a series of pools where salinities
reached 6 (Mackay et al. 1988). The fact that silver perch
possess accessory cells on their gills may indicate that this
species or some immediate ancestor has been subjected to
relatively high salinities in inland waters in the recent past
andlor that the presence of this characteristic is simply a
legacy of the marine or estuarine ancestry of the species.
Whatever the origins, silver perch appear in many respects
to have evolved further towards a purely freshwater life
cycle than have other modern native species found in the
Murray-Darling river system.
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Manuscript received 23 January 1995; revised and accepted 28 March 1995