Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation
1 Department of Zoology, University of British Columbia, Vancouver BC,
Canada V6T 1Z4
2 Department of Biology, McMaster University, Hamilton ON, Canada L8S
4K1
* Author for correspondence (e-mail: scott{at}zoology.ubc.ca)
Accepted 7 June 2004
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Summary |
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Key words: killifish, gene expression, gill morphology, Na+/K+-ATPase, ion flux, kidney, evolution
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Introduction |
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Species within the genus Fundulus are suggested to have arisen
from brackish water ancestors, and there is substantial variation in both the
salinity of their native habitats (ranging from freshwater to seawater) and
their salinity tolerance (Griffith,
1974). Intraspecific differences in salinity tolerance and
distribution also appear to exist within some Fundulus species. For
example, northern populations of F. heteroclitus have higher
fertilization success and larval survival in hyposmotic salinities than
southern populations (Able and Palmer,
1988
). Furthermore, the proportion of northern genotypes increases
in freshwater habitats, even at latitudes and temperatures that are typical
for the southern subspecies (Powers et
al., 1993
). It is therefore likely that molecular or physiological
differences exist within F. heteroclitus that form the basis for
variation in freshwater tolerance.
Past habitat availability may have selected for differences in freshwater
tolerance between northern and southern individuals of F.
heteroclitus. After previous glaciation events, new freshwater and
estuarine habitats would have opened due to glacial retreat from previously
ice-covered areas (Powers et al.,
1986). F. heteroclitus populations able to colonize
northern habitats therefore faced opportunities for freshwater invasion
without competition from indigenous fish species, in contrast to the situation
further south. The role of natural selection in freshwater invasion events has
been explicitly demonstrated (Lee and
Petersen, 2002
), and numerous accounts attest to the selective
advantage of euryhalinity (Lee and Bell,
1999
). However, the physiological adaptations necessary for
brackish water and marine fish to invade freshwater are unclear. Intraspecific
comparison of the mechanisms maintaining ion balance in F.
heteroclitus populations in freshwater may identify factors of selective
importance for freshwater adaptation.
The hyposmotic nature of freshwater environments (typically <10 mOsmol
l-1) favours ion efflux from fish, because they maintain
substantially higher body fluid osmolarity (300-350 mOsmol l-1).
The largest component of ion efflux in freshwater fish probably occurs across
the gills due to their high surface area. Fish in freshwater therefore
decrease the paracellular permeability across the gill epithelium, primarily
by increasing the thickness of tight junctions between mitochondria-rich (MR)
cells and neighbouring cells (Sardet et
al., 1979; Ernst et al.,
1980
). Fish also decrease transcellular permeability by
inactivating ion secretion pathways (Marshall et al.,
1993
,
1998
,
2000
;
Scott et al., 2004
).
To counteract ion efflux and maintain ionic homeostasis, fish in freshwater
absorb ions across the gills (see reviews by
Wood and Marshall, 1994;
Perry, 1997
;
Evans et al., 1999
;
Marshall, 2002
). Sodium
absorption by killifish gills is likely to involve a basolateral
Na+/K+-ATPase and an apical
Na+/H+-exchanger
(Patrick and Wood, 1999
;
Claiborne et al., 2002
;
Scott et al., 2004
) but may
also involve basolateral H+-ATPase
(Katoh et al., 2003
). Unlike
the majority of fish in freshwater, F. heteroclitus does not actively
absorb chloride and thus maintains chloride balance through unique and yet
undefined mechanisms (Patrick and Wood,
1999
).
The objective of the present study was to compare the ionoregulatory ability of individuals from northern and southern populations of F. heteroclitus after direct salinity transfer. The ionoregulatory ability of each population was assessed by measuring survival, plasma ions, mRNA expression, protein activity, ion flux, paracellular permeability, gill morphology and aspects of renal function after transfer from near-isosmotic brackish water (10 g l-1) to freshwater.
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Materials and methods |
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Salinity transfer experiment
Some of the data collected in this salinity transfer experiment have been
previously reported for the northern subspecies alone
(Scott et al., 2004). Eight
northern and southern control fish were sampled after acclimation to 10 g
l-1, after which fish from each population were quickly transferred
by net to aquaria containing freshwater (0 g l-1; composition as
above) or brackish water (10 g l-1). Individual fish were
subsequently sampled by netting at 3 h, 8 h, 24 h, 96 h, 14 days and 30 days
after transfer from brackish water. The fish were stunned by cephalic blow,
blood samples were collected in heparinized capillary tubes from the severed
caudal peduncle, and the fish were then killed by rapid decapitation. Blood
was centrifuged at 13 000 g for 10 min, and plasma was frozen
in liquid nitrogen. Second and third gill arches were immediately frozen in
liquid nitrogen. All tissues were stored at -80°C until analysed.
Total RNA extraction and reverse transcription
Total RNA was extracted from tissues (20 mg) using Tripure isolation
reagent (Roche Diagnostics, Montreal, QC, Canada) following the manufacturer's
instructions. RNA concentrations were determined spectrophotometrically, and
RNA integrity was verified by agarose gel electrophoresis [
1%
agarose:Tris-acetate EDTA (w/v)]. Extracted RNA samples were stored at
-80°C following isolation. First-strand cDNA was synthesized by reverse
transcribing 3 µg total RNA using 10 pmoles of oligo(dT18)
primer and 20 U RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas
Inc., Burlington, ON, Canada) following the manufacturer's instructions.
Real-time PCR analysis of gene expression
Primers for killifish Na+/K+-ATPase
1a (accession no. AY057072), cystic fibrosis transmembrane
conductance regulator (CFTR) Cl- channel (accession no. AF000271),
Na+/K+/2Cl- cotransporter 1 (NKCC1; accession
no. AY533706) and elongation factor 1
(EF1
, expression control;
accession no. AY430091) were designed using Primer Express software (version
2.0.0; Applied Biosystems Inc., Foster City, CA, USA) and are reported in
Scott et al. (2004
). Gene
expression was quantified using quantitative real-time PCR (qRT-PCR) on an ABI
Prism 7000 sequence analysis system (Applied Biosystems Inc.). PCR reactions
contained 1 µl of cDNA, 4 pmoles of each primer and Universal SYBR green
master mix (Applied Biosystems Inc.) in a total volume of 21 µl. All
qRT-PCR reactions were performed as follows: 1 cycle of 50°C for 2 min and
95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C
for 1 min (set annealing temperature of all primers). PCR products were
subjected to melt curve analysis to confirm the presence of a single amplicon,
and representative samples were electrophoresed to verify that only a single
band was present. Control reactions were conducted with no cDNA template or
with non-reverse-transcribed RNA to determine the level of background or
genomic DNA contamination, respectively. Genomic contamination was below 1:49
starting cDNA copies for all templates.
A randomly selected control sample was used to develop a standard curve for
each primer set, and all results were expressed relative to these standard
curves. Results were then standardized to EF1, a gene for which mRNA
expression in the gills does not change following salinity transfer (data not
shown), and were expressed relative to the matched brackish water controls
within each time point. All samples were run in duplicate (coefficients of
variation were
10%). Because significant changes in gene expression due to
freshwater transfer were observed in the northern population only at 24 h, 96
h and 14 days post-transfer by Scott et al.
(2004
), gene expression was
only quantified at these time points (and pre-transfer) in the southern
population.
Na+/K+-ATPase activity
Na+/K+-ATPase activity was determined by coupling
ouabain-sensitive ATP hydrolysis to pyruvate kinase- and lactate
dehydrogenase-mediated NADH oxidation as outlined by McCormick
(1993). For this assay, second
and third gill arches were homogenized in 500 µl of SEI buffer (150 mmol
l-1 sucrose, 10 mmol l-1 EDTA, 50 mmol l-1
imidazole, pH 7.3) containing 0.1% Na-deoxycholate and centrifuged at
5000 g for 30 s at 4°C. Supernatants were immediately
frozen in liquid nitrogen and stored at -80°C until analysed. ATPase
activity was determined in the presence or absence of 0.5 mmol l-1
ouabain using 10 µl supernatant thawed on ice and was normalized to total
protein content (measured using the bicinchoninic acid method; Sigma-Aldrich,
Oakville, ON, Canada). All samples were run in triplicate (coefficients of
variation were always
10%). Na+/K+-ATPase activity,
measured as ouabain-sensitive ATPase activity, is expressed as µmol ADP
mg-1 protein h-1.
Freshwater flux experiments
Killifish were held at McMaster University for at least 30 days before
experimentation in 10 g l-1 brackish water (made up in
dechlorinated Hamilton city tap water) at room temperature (21°C) and
were maintained at a 14 h:10 h L:D photoperiod. Freshwater was prepared to
approximate Vancouver city tap water by mixing appropriate amounts of
dechlorinated Hamilton water and reverse-osmosis water (measured final
composition: [Na+], 0.17 mmol l-1; [Cl-],
0.20 mmol l-1). Static polyethylene flux chambers were fitted with
a lid and aeration line and were wrapped in black plastic to minimize
disturbance of fish. Each chamber contained 270 ml of freshwater and 37 kBq of
either 22Na (NEN Life Science Products Inc., Boston, MA, USA) or
36Cl (ICN Biomaterials, Irvine, CA, USA) isotope at the start of
each flux period.
Unidirectional and net Na+ flux rates and net Cl- flux rates were measured over the first 8 h after transfer to freshwater and at 1 and 4 days post-transfer in one experiment, and also at 8 and 14 days post-transfer in a second experiment. Unidirectional Na influx was measured by monitoring the disappearance of 22Na isotope from the water. In the first experiment, 10 fish from each population were transferred to individual freshwater flux chambers containing isotope, and duplicate water samples (2x5 ml for 22Na radioactivity measurements) were taken every 30 min for the first 8 h after transfer. Additional flux measurements were made 1 and 4 days after transfer on the same fish. For these flux periods, duplicate water samples were taken every hour for 4 h after the addition of isotope. In the second experiment, 10 killifish from each population were first transferred to static aerated freshwater tanks for 7 days and then moved to flux chambers. Flux periods at 8 and 14 days after transfer were then conducted as before, with water samples being taken every hour for 4 h after the addition of isotope on days 8 and 14. In both experiments, water in the flux chambers was replaced between flux periods with clean freshwater (containing no radioactivity, at least once daily) to remove waste and excess isotope.
Unidirectional Cl- influxes from F. heteroclitus in
freshwater are normally extremely low and cannot be determined by measuring
the disappearance of isotope from the water. They must instead be measured by
quantifying isotope appearance in the fish, which is a more sensitive
technique (Patrick et al.,
1997; Wood and Laurent,
2003
). Unidirectional Cl- influx was therefore
determined 8 days after freshwater transfer by measuring whole-animal uptake
of 36Cl. Eight killifish from each population were first held in
static aerated freshwater tanks for 8 days, then moved to flux chambers
containing isotope. Water samples were taken immediately after the addition of
36Cl (just before fish were added), and fish and water were sampled
4 h later. At the end of both 22Na and 36Cl experiments,
fish were rinsed by allowing live animals to ventilate their gills (for at
least 5 s) in freshwater containing no radioactivity and were then sacrificed.
Radioactivity (counts per minute, c.p.m.) was determined in each fish and in
each water sample, as were total water Na+ and Cl-
concentrations.
Unidirectional Na+ influx rates (Jin,Na) in
µmol kg-1 h-1 were calculated as:
![]() | (1) |
where is the slope of the regression line of radioactivity
versus time (in c.p.m. h-1), SA is the mean
external specific activity (in c.p.m. µmol-1) and M is
the body mass (in kg). Unidirectional Cl- influx rates
(Jin,Cl) were calculated as:
![]() | (2) |
where fish is the total radioactivity in the fish (in
c.p.m.) and t is the flux period duration (in h).
Mean net Na+ and Cl- flux rates
(Jnet,ion) in µmol kg-1 h-1 were
calculated as:
![]() | (3) |
where [ion]i and [ion]f are the concentrations of
Na+ or Cl- in the water at the start and end of the flux
period (in µmol l-1), respectively, and V is the mean
flux chamber volume (in litres). By conservation of mass, unidirectional
efflux rates (Jout,ion) in µmol kg-1
h-1 were calculated as:
![]() | (4) |
Because the above calculations assume there is no `backflux' of
radioisotope from the fish into the surrounding media, which can be a
significant source of error, internal specific activity within the fish must
remain low compared with external specific activity of the medium. At the end
of each flux experiment, the ratio of internal to external specific activity
was therefore verified to be <10%
(Kirschner, 1970).
A method adapted from Curtis and Wood
(1991) was employed to study
the diffusive permeability of the fish to a paracellular permeability marker 8
days after freshwater transfer. The technique monitors the appearance of
radiolabelled polyethylene glycol ([3H]PEG-4000) in the external
water relative to the radioactivity of a terminal plasma sample, from which
PEG-4000 clearance rates can be calculated. Radioactivity appearing in
discrete pulses represents bouts of urination from the urinary bladder (renal
PEG-4000 clearance), whereas radioactivity not appearing in pulses represents
diffusion across the gills and body surface (extra-renal PEG-4000 clearance).
[3H]PEG-4000 is the marker of choice for glomerular filtration rate
in fish (Beyenbach and Kirschner,
1976
), and its renal clearance rate is considered equivalent to
the glomerular filtration rate (GFR). On day 7, approximately 16 h prior to
measurements on day 8, killifish were injected intraperitoneally with 1 µl
g-1 of [3H]PEG-4000 (NEN Life Science Products) in 140
mmol l-1 NaCl (111 kBq µl-1) and left in their
individual containers for the label to equilibrate overnight throughout the
extracellular compartment. On day 8, the water was changed, the volume set to
250 ml, the aeration set to produce good mixing, and the fish left to settle
for a further 60 min. Thereafter, a 1 ml water sample was drawn from each fish
container at exactly 5-min intervals for the next 6-7 h, after which a blood
sample was taken and plasma separated as described earlier.
Plots of total water [3H] radioactivity against time revealed
clear step-wise increases attributable to bouts of urination, and the sum of
all radioactivity (c.p.m.) appearing in these pulses (by renal excretion) was
subtracted from the total appearance (c.p.m.) over the 6-7 h period to yield
extrarenal excretion (c.p.m.). Dividing each of these values by plasma
radioactivity (c.p.m. µl-1), time (h) and body mass (kg) yielded
the renal clearance rate of PEG-4000 (equivalent to GFR; in ml kg-1
h-1) and the extrarenal clearance rate of PEG-4000 (in ml
kg-1 h-1), the latter providing an index of the
diffusive permeability of the fish to a paracellular permeability marker. As
terminal urine samples for radioactivity counting could not be obtained from
these small fish, urine flow rate could not be determined (cf.
Curtis and Wood, 1991), but
urination frequency (bursts h-1) could be calculated.
Ion and radioactivity measurements
Sodium concentrations of plasma and water samples were determined using
flame atomic absorption spectrophotometry (SpectrAA-220FS; Varian, Mulgrave,
VC, Australia) with Fisher Scientific (Nepean, ON, Canada) certified
standards. Chloride concentrations of plasma and water samples were measured
colorimetrically (Zall et al.,
1956) with Radiometer (Copenhagen, Denmark) certified standards.
22Na radioactivities in fish and water samples were determined
using a Minaxi Autogamma 5000 counter (Packard Instruments, Downers Grove, IL,
USA). For 36Cl radioactivities in whole fish, animals were digested
in three volumes of 1 mol l-1 HNO3 at 60°C for 48 h.
These samples were centrifuged, supernatants (1 ml) were added to 5 ml of an
acid-compatible scintillation cocktail (Ultima Gold; Packard Bioscience,
Meriden, CT, USA), and radioactivity was measured by scintillation counting
(Rackbeta 1217; LKB Wallac, Turku, Finland). 36Cl radioactivities
in water samples (5 ml) were also measured by scintillation counting in 10 ml
of scintillation fluid (ACS; Amersham, Piscataway, NJ, USA), and data were
corrected for the slight difference in counting efficiencies between the two
scintillation fluors. 3H radioactivities in water (1 ml) and plasma
(10-20 µl) samples were measured by scintillation counting in a standard
volume ratio of 1 ml of water (or diluted plasma) to 5 ml ACS (quench was
shown to be uniform across samples).
Scanning electron microscopy
Second and third gill arches of killifish from each population were sampled
before (N=3) and 8 days after transfer to Vancouver freshwater
(N=5), as described above. Gills were fixed for 24 h in 0.1 mol
l-1 phosphate-buffered saline (pH 7.4) containing 2%
paraformaldehyde and 2% glutaraldehyde. After fixation, samples were
post-fixed in 0.1 mol l-1 cacodylate buffer (pH 7.4) containing 1%
OsO4. Tissues were dehydrated progressively in ethanol (70, 85, 95
and 100%) for 10 min in each solution. Gill arches were dried in
hexamethyldisilazane and sputter-coated with gold.
Images collected by scanning electron microscopy were analyzed using a
method similar to Daborn et al.
(2001). Random locations on the
afferent-vascular edge of gill filaments were observed at 3000x
magnification. Apical crypts, freshwater-type MR cells ('chloride cells') and
`intermediate' cells were counted for at least 10 different locations
throughout the gills. Averages were calculated for each fish and expressed as
density mm-2.
Statistical analyses
Data are expressed as means ±
S.E.M. Kruskal-Wallis H
non-parametric analysis of variance (ANOVA) was used to determine overall
differences as a function of time (for each salinity) for each variable except
survival. Mann-Whitney U non-parametric comparisons were then used to
compare between salinities (within populations at each time) or between
populations (at each salinity and time). Survival between sampling times was
compared using the one-tailed Fisher's exact test. Statistical analyses were
conducted using SPSS version 10.0, and a significance level of
P<0.05 was used throughout.
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Results |
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Plasma ions
The effect of freshwater transfer on plasma ions differed between
populations in a manner consistent with the differences in mortality
(Fig. 2). Freshwater transfer
decreased plasma Na compared with brackish water controls in both populations,
but this only occurred 1 day after transfer of northern killifish while
southern killifish had decreased plasma levels 3 h, 8 h and 1 day after
transfer to freshwater (Fig.
2A). Plasma Na balance was re-established 4 days after transfer in
both populations.
|
Differences in plasma Cl between populations after freshwater transfer were more pronounced than differences in plasma Na (Fig. 2B). Northern killifish maintained plasma Cl balance at all times after freshwater transfer. By contrast, southern killifish in freshwater had lower plasma Cl at 1, 4 and 14 days after transfer compared with brackish water controls.
Gene expression and protein activity
Na+/K+-ATPase regulation in the gills after
freshwater transfer differed between northern and southern populations of
F. heteroclitus. There was a prolonged increase in the relative
Na+/K+-ATPase 1a mRNA expression in
individuals from the northern population, which peaked at 4 days in freshwater
at nearly 6-fold above time-matched brackish water controls
(Fig. 3A). Individuals from the
southern population increased Na+/K+-ATPase
1a expression to a lesser extent, to only 4-fold that of
controls at 4 days after transfer. Unlike northern killifish, southern
killifish did not increase expression at 1 or 14 days into freshwater.
|
Changes in Na+/K+-ATPase activity in the gills as a result of freshwater transfer also differed between populations. Activity increased 2-fold at 1 and 4 days following freshwater transfer in northern killifish (Fig. 3B), while no significant increases occurred after transfer in southern killifish (Fig. 3C). Activity also reached higher absolute levels in northern killifish (3.5±0.5 and 2.9±0.3 µmol mg-1 protein h-1 at 8 h in northerns and southerns, respectively).
Relative expression of the seawater ion transporters NKCC1 and CFTR decreased at 1 and 4 days after freshwater transfer in both populations, dropping to approximately 2.5- and 10-fold below brackish water controls 1 day after transfer, respectively (Table 1). Decreased expression of these genes persisted longer in southern individuals, however, remaining below that of controls 14 days after freshwater transfer.
|
Ion fluxes and PEG-4000 clearance rates
There were only small differences in Na+ fluxes between northern
and southern populations following freshwater transfer. Both northern and
southern fish decreased Na+ efflux rapidly after freshwater
transfer (Fig. 4A).
Unidirectional efflux and negative net flux were significantly decreased 4-8 h
after transfer compared with the 0-4 h flux period, and this decrease
persisted until at least 14 days after transfer. Both populations also
increased unidirectional Na+ influx progressively after freshwater
transfer. By 14 days after freshwater transfer, however, northern killifish
had significantly higher unidirectional influx and net flux than southern
killifish. In fact, northern fish increased Na+ influx by 5-fold 14
days post-transfer compared with the 0-4 h flux period, while southerns
increased influx by only 3.3-fold. Net flux was also slightly higher in
northern fish after 96 h in freshwater.
|
We observed large differences in net Cl- flux between northern and southern killifish (Fig. 4B). Although fish from both populations decreased Cl- loss initially, northern killifish eliminated loss by 1 day after transfer and this was maintained after at least 14 days in freshwater. By contrast, southern killifish did not appear to decrease Cl- loss below 100 µmol kg-1 h-1 and significantly differed from northern killifish at 1 and 14 days after transfer. Unidirectional Cl- influx was small (less than 3% of unidirectional Na+ influx; Fig. 4A) and was identical between populations at 8 days post-transfer (Table 2).
|
Patterns of PEG-4000 clearance differed substantially between killifish populations at 8 days post-transfer (Table 2). Extrarenal clearance rates, which represent the general paracellular permeability of the gills and body surface, were 3-fold higher in southern killifish. Renal clearance was higher in northern killifish, which had 1.6-fold greater glomerular filtration rates (i.e. renal PEG-4000 clearance rates) and 1.5-fold more frequent bursts of urination, although actual urine flow could not be calculated.
Gill morphology
The gills of both northern and southern killifish had a similar morphology
in brackish water (Fig. 5A,B).
Apical crypts were abundant in both populations (2000 mm-2),
while freshwater-type MR cell density remained low
(Fig. 6). `Intermediate' cells,
characterized by features that are midway between seawater (apical crypt) and
freshwater (flat surface equipped with microvilli) morphologies were equally
abundant between populations and salinities.
|
|
Northern and southern killifish had different gill morphologies after
transfer to freshwater (Fig.
5C,D). Apical crypt density in northern killifish gills was
15-fold lower in freshwater than in brackish water, while southern killifish
in freshwater had an apical crypt density only 3-fold lower than those in
brackish water (Fig. 6). The
abundance of freshwater-type MR cells increased significantly in both
populations after freshwater transfer (3000-4000 mm-2).
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Discussion |
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Regulation of Na+ balance
There were small differences in the ability of northern and southern
killifish populations to regulate Na+ balance after freshwater
transfer. Plasma Na+ levels in individuals from the southern
population decreased for a longer period after transfer when compared with
those from the northern population, and this decrease appeared to be of
greater magnitude. The cause of this difference is unclear, as Na+
fluxes did not differ between populations until 96 h after transfer.
Differences in water flux across the gills
(Robertson and Hazel, 1999),
drinking rates (Potts and Evans,
1967
) or renal function (see below) might have accounted for
differences in plasma Na+ between populations.
Rapid re-establishment of plasma Na+ balance in northern
killifish after freshwater transfer has been observed numerous times,
demonstrating the excellent euryhalinity of these animals. Transfer from
seawater to freshwater initially decreases plasma Na+ as early as 4
h after transfer, but pre-transfer seawater levels are quickly restored 1-2
days into freshwater (Jacob and Taylor,
1983; Katoh and Kaneko,
2003
). Northern killifish transferred from freshwater to seawater
reestablish plasma Na+ with equal rapidity
(Jacob and Taylor, 1983
;
Marshall et al., 1999
). We
have observed similar results after transfer from near-isosmotic brackish
water to either freshwater or seawater
(Scott et al., 2004
),
suggesting that individuals of the northern population can freely move between
freshwater and seawater environments.
Northern killifish increased both the relative mRNA expression
(1a-isoform) and protein activity of gill
Na+/K+-ATPase by a greater magnitude and for a longer
duration after freshwater transfer than did southern killifish.
Mitochondria-rich cells in killifish gills are known to proliferate after
freshwater transfer (Katoh and Kaneko,
2003
). An increase in the abundance of cells expressing high
levels of Na+/K+-ATPase may have therefore contributed
to the Na+/K+-ATPase upregulation in freshwater.
Furthermore, differences in relative Na+/K+-ATPase
expression between populations may have arisen from lower cell proliferation
in southern fish after freshwater transfer. In support of this hypothesis,
northern killifish gills had a high density of freshwater-type MR cells (as
previously reported by Hossler et al.,
1985
), while density appeared to be lower in the gills of southern
killifish in freshwater.
The regulation of ion flux across fish gills by changes in ion transporter
expression is generally assumed but infrequently tested (e.g. Sullivan et al.,
1995,
1996
). In this regard, we
speculate that the differences in Na+/K+-ATPase gene
regulation between northern and southern killifish were at least partly
responsible for the observed differences in Na+ flux. Northern
killifish had greater changes in Na+/K+-ATPase
expression, which occurred concurrent with more positive net flux at 4 and 14
days and greater unidirectional Na+ influx at 14 days after
freshwater transfer. Therefore, these data provide evidence for how active ion
flux can be modulated by ion transporter gene regulation.
Even though differences in Na+/K+-ATPase expression
and activity appear to exist between F. heteroclitus populations
after freshwater transfer, the resultant differences in Na+ flux
were small. Both populations initially suffered high Na+ efflux, at
rates comparable with other Fundulus species
(Pang et al., 1974);
interestingly, our initial rates in F. heteroclitus are intermediate
between F. diaphanus, a freshwater species, and F. majalis,
a seawater species. Efflux decreased rapidly after freshwater transfer,
however, such that net loss was nearly eliminated by 24 h after transfer in
both F. heteroclitus populations. Similarly rapid reductions in
Na+ efflux have been previously observed for F.
heteroclitus (Motais et al.,
1966
; Potts and Evans,
1967
; Pic, 1978
;
Wood and Laurent, 2003
) as
well as F. kansae (Potts and
Fleming, 1971
) after transfer from saline water to freshwater.
Along with reductions in passive Na+ efflux, Na+
influx increased progressively in both populations after freshwater transfer
and, by 14 days, reached levels 3- to 5-fold above the initial influx. These
influx rates are somewhat lower than previous reports in northern killifish,
both shortly after transfer (Wood and
Laurent, 2003) and after freshwater acclimation (Potts and Evans,
1966
,
1967
;
Patrick and Wood, 1999
). This
difference is undoubtedly explained by the lower Na+ levels in our
freshwater (0.17 mmol l-1) compared with these other studies
(2-5-fold higher), because Na+ influx is critically dependent on
environmental Na+ in this concentration range
(Patrick et al., 1997
). Taken
together, the results discussed above suggest that differences in
Na+ regulation between populations of F. heteroclitus are
small and are unlikely to account for the pronounced differences in mortality
in freshwater.
Regulation of Cl- balance
Northern and southern killifish populations differ significantly in their
ability to regulate Cl- in freshwater, which probably contributes
to the large differences in mortality they experienced after transfer.
Northern fish actually appear to regulate Cl- levels more strictly
than Na+ levels, as plasma Cl- was maintained for at
least 30 days after freshwater transfer. This is in agreement with previous
reports for northern killifish. For example, in a study by Jacob and Taylor
(1983), transfer from seawater
to freshwater decreased serum osmolality transiently, which was almost
entirely accounted for by changes in serum Na+. By contrast, we
observed that southern killifish rapidly lost Cl- balance after
freshwater transfer, as plasma levels fell quickly after transfer and were not
re-established. It is possible that these decreases in Cl- levels
were sufficient to cause mortality: similar decreases in plasma Cl-
levels have been observed in long-horned sculpin (Myoxocephalus
octodecimspinosus) after transfer to hyposmotic environments, and these
decreases were associated with greater mortality
(Claiborne et al., 1994
). In
addition, Cl- imbalance would have created a `strong ion'
difference in the plasma of southern killifish (i.e.
[Na+]>[Cl-]). To maintain charge neutrality,
compensatory increases in plasma [HCO3-] and pH may have
occurred in southern fish, so the resulting blood alkalosis might have also
contributed to the differences in mortality between populations.
The observed differences in plasma Cl- between northern and
southern killifish were probably due to differences in Cl- flux.
Because unidirectional Cl- influx is extremely small in F.
heteroclitus in freshwater (present study;
Patrick et al., 1997;
Patrick and Wood, 1999
;
Wood and Laurent, 2003
),
changes in total Cl- flux after freshwater transfer are primarily
dictated by changes in unidirectional Cl- efflux. Shortly after
transfer, individuals from both populations reduced passive Cl-
loss, which is consistent with previous results
(Pic, 1978
). Northern
populations continued to decrease Cl- efflux, such that
Cl- loss was eliminated by 24 h following freshwater transfer and
remained negligible thereafter. This rapid elimination of Cl- loss
is likely to account for the ability of northern killifish to maintain plasma
Cl- balance in spite of negligible branchial uptake. Southern
killifish did not eliminate Cl- efflux, which remained consistently
higher than 100 µmol kg-1 h-1, so they were unable to
preserve Cl- balance.
In order to eliminate Cl- efflux, killifish entering freshwater
must eliminate both active and passive routes of Cl- excretion.
Active secretion of ions across fish gills as occurs in seawater involves a
basolateral Na+/K+-ATPase, a basolateral
Na+/K+/2Cl- cotransporter (NKCC1) and an
apical cystic fibrosis transmembrane conductance regulator (CFTR)
Cl- channel (see reviews by
Wood and Marshall, 1994;
Perry, 1997
;
Evans et al., 1999
;
Marshall, 2002
). One and four
days after freshwater transfer, individuals from both populations decreased
gill mRNA expression of the seawater transporters NKCC1 and CFTR. In fact,
this suppression actually persisted for a longer duration in southern
killifish, so differences in active Cl- secretion are unlikely to
account for the observed differences in Cl- efflux.
Populations of F. heteroclitus appear to differ in their ability
to eliminate passive Cl- loss. The higher extrarenal clearance rate
of PEG-4000 in southern killifish indicates that the paracellular permeability
of their gills is higher; increased Cl- loss may have therefore
occurred through the paracellular pathway. The observed differences in gill
morphology between populations are consistent with this hypothesis. A typical
morphological feature of the gills of fish in seawater is the presence of
apical crypts, which are formed by multicellular complexes of MR cells that
share shallow junctions with high solute permeability. Both populations have
equal abundance of apical crypts in brackish water, at densities approximately
7-8-fold lower than in seawater-acclimated animals
(Hossler et al., 1985).
However, southern killifish have significantly more apical crypts in
freshwater and thus maintain morphological features of the seawater gill.
After transfer of northern killifish to hyposmotic environments, apical crypts
are either covered over by pavement cells
(Daborn et al., 2001
) or are
widened to uncover freshwater-type MR cells equipped with microvilli
(Katoh and Kaneko, 2003
).
Freshwater MR cells typically form tight junctions with neighbouring cells
that have low paracellular permeability to solutes
(Sardet et al., 1979
;
Ernst et al., 1980
). These
morphological transformations occur in conjunction with rapid reductions in
Cl- secretion (Daborn et al.,
2001
). More apical crypts on the gills of southern killifish
therefore suggests that fewer crypts were covered and/or converted into
freshwater-type MR cells; incidentally, there was a trend towards greater
freshwater MR cell density in northern killifish.
As well as the structural reorganization of cell-cell junctions that occurs
during transformation between seawater and freshwater gill morphologies,
associated changes occur in the actin cytoskeleton within MR cells. These
morphological alterations to cytoskeletal elements are important for the
changes in transepithelial conductance that occur after salinity change
(Daborn et al., 2001).
Interestingly, interactions between actin and
Na+/K+-ATPase appear essential for tight junction
formation in epithelia (Rajasekaran and
Rajasekaran, 2003
), so differences in
Na+/K+-ATPase gene regulation between killifish
populations may be related to differences in Cl- efflux through the
paracellular pathway.
In addition to greater Cl- efflux and paracellular permeability,
southern killifish exhibited lower glomerular filtration rates and lower
urination burst frequencies than did northerns in freshwater. High glomerular
filtration rates and urination frequencies are characteristic of freshwater
fish, while the opposite are characteristic of seawater fish (e.g.
Hickman and Trump, 1969;
Curtis and Wood, 1991
;
Sloman et al., 2004
). These
observations suggest that slower or incomplete acclimation of renal function
may have been another factor contributing to the poorer ionoregulatory
performance and survival of southerns in freshwater.
Possible mechanisms of freshwater adaptation
Our data suggest that southern killifish are less tolerant of freshwater
transfer than are northerns because the coordinated response of their gills
and kidney is less effective at maintaining ion balance. This may include
small differences in Na+ regulation, suggested by small differences
between populations in plasma Na+,
Na+/K+-ATPase expression and activity in the gills, and
ion flux and possibly gill MR cell abundance. A more convincing cause for
differences in mortality, however, is the differences in Cl-
regulation. Southern killifish experience large decreases in plasma
Cl-, have significant Cl- efflux and maintain a moderate
density of apical crypts after freshwater transfer. By contrast, northern
killifish maintain plasma Cl-, eliminate Cl- efflux,
have lower paracellular permeability, have very few apical crypts in
freshwater and have more typical freshwater renal function. Taken together,
southern killifish seem to preserve some elements of seawater ionoregulatory
physiology, while northerns are better able to make the necessary adjustments
for freshwater acclimation.
A great deal of evidence suggests that northern populations of F.
heteroclitus have evolved greater freshwater ionoregulatory ability than
have southern populations. This is indicated by differences in distribution
patterns (Powers et al.,
1993), reproductive success
(Able and Palmer, 1988
) and
adult survival (present study) in hyposmotic environments. These differences
may have arisen from selection acting on preexisting variability within F.
heteroclitus or, possibly, from introgression of alleles from sympatric
freshwater species (e.g. F. diaphanus;
Dawley, 1992
). Although the
evolutionary pressures accounting for these differences in salinity tolerance
are still uncertain, our data suggest that minimizing Cl- imbalance
was an essential evolutionary step allowing northern killifish to survive in
freshwater. Freshwater acclimation was possible in northern killifish despite
no apparent mechanism for branchial Cl- uptake. These animals may
instead be able to survive in freshwater habitats and maintain Cl-
balance by minimizing Cl- efflux and meeting Cl- demands
through the diet (Wood and Laurent,
2003
).
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Acknowledgments |
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References |
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