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First published online June 29, 2007
Journal of Experimental Biology 210, 2518-2525 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005637
Seasonal control of particle clearance by isolated gills from the clam Mercenaria mercenaria
Department of Biological Sciences, The University of Southern Maine, Portland, ME 04104, USA
e-mail: gainey{at}maine.edu
Accepted 2 May 2007
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Summary |
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Key words: clearance, gill, clam, 5-HT, 5-hydroxytryptamine, Mercenaria mercenaria
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Gainey et al., 2003;
Gainey and Greenberg, 2003;
Gainey and Greenberg, 2005).
Recently, I discovered that isolated quarters of a gill not only clear
colloidal graphite from suspension but also transport the cleared particles in
a manner analogous to that in feeding – a complex behavior approaching
that of intact gills. The rate at which isolated gill preparations clear
graphite can be measured and so the pharmacology and seasonality of clearance
can be determined. We can then ask whether the seasonal control of clearance,
an integrated function, can be predicted from information about two critical
components of the system – the lateral cilia and branchial
musculature.
The eulamellibranch gills of Mercenaria are plicate
(Kellogg, 1892). That is,
within each demibranch, adjacent filaments are connected to each other by
interfilament tissue junctions, while the ascending and descending limbs of
some of the filaments are also connected by interlamellar tissue junctions
(the septa), an arrangement that produces the plicae and the water tubes
(Fig. 1).
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;
Morton, 1983); (2) the
latero-frontal cirri, which capture particles
(Beninger et al., 1997;
Silverman et al., 1996;
Silverman et al., 1999)
– although the exact role of these cirri in particle capture is
controversial (Beninger, 2000;
Riisgard and Larsen, 2000;
Silverman et al., 2000;
Ward et al., 2000) – and
(3) the frontal cilia, which move particles to the food grooves
(Purchon, 1968;
Morton, 1983;
Murakami, 1989).
Clearance by the gill, however, is determined not only by the coordinated
activity of the cilia but also by the gill's geometry, which includes the
spacing of the filaments, the shape of the plicae and the cross-sectional area
of the water tubes. These geometric properties are controlled by the branchial
muscles: contraction of the longitudinal muscles narrows the spacing between
adjacent filaments whereas contraction of the water tube muscles not only
narrows the spacing but constricts the water tubes and changes the shape of
the plicae (Gainey et al.,
2003). In an intact clam, of course, the clearance rate is also
related to the degree of valve gape and the cross-sectional area of the
siphons (Jorgensen and Riisgard,
1988; Riisgard et al.,
2003).
In Mercenaria, the lateral cilia are stimulated by
5-hydroxytryptamine (5-HT) and inhibited by dopamine (DA), while the frontal
cilia are inhibited, but not completely stopped, by 5-HT
(Gainey et al., 1999). The
branchial musculature, by contrast, is stimulated by 5-HT, DA and
acetylcholine (ACh) and relaxed by ACh
(Gainey et al., 2003).
Moreover, nitric oxide (NO) potentiates the stimulatory effects of 5-HT, DA
and ACh on the gill muscles from November through June but has no effect from
July through October (the off-season). Indeed, the entire NO signaling cascade
is absent from the branchial musculature during the off-season
(Gainey and Greenberg,
2003).
Here, I report on the measurement of clearance rates by isolated pieces of
gill from Mercenaria mercenaria and the control of clearance by 5-HT
and DA. I presumed that the effects of 5-HT and DA on clearance should reflect
their effects on the lateral cilia and musculature. Thus, I hypothesized that
at low doses 5-HT should increase clearance rates by stimulating the lateral
cilia and should decrease rates at higher doses by contracting the gill
musculature, thus reducing the space between the filaments. In addition, the
effects of 5-HT should be more pronounced during the winter than the summer
due to the seasonal expression of the NO signaling cascade. DA should stop
clearance, not only by inhibiting the lateral cilia but also by contracting
the gill musculature. Finally, because the size and shape of the water tubes
are an indicator of the pumping activity of eulamellibranch gills, I made
morphometric measurements of the cross-sectional area of water tubes and also
the interfilament space in response to 5-HT. Preliminary results of these data
were presented to the Society of Integrative and Comparative Biology
(Gainey and Greenberg,
2007).
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Materials and methods |
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natural seawater from Casco Bay, ME,
USA. Before being used in experiments, the clams were held for at least 3 days
but not more than 2 weeks. During this period, the clams were fed `Coral and
Clam Diet' (Reed Mariculture, Inc., Campbell, CA, USA) every other day.
Gill preparation
The two gills were dissected away from the body wall distal to the visceral
ganglion, and the branchial nerves removed. Each gill was split in two along
its dorsal cut edge to produce four demibranchs per clam. Each demibranch was
then cut into anterior and posterior halves, and a loop of thread was tied to
the free (uncut) end of the ventral margin. Finally, the isolated pieces of
gill were placed in a dish of artificial seawater [ASW; 1000 mosmol
kg–1, 10°C; made according to the MBL (Woods Hole) recipe
(http://www.mbl.edu/BiologicalBulletin/COMPENDIUM/Comp-ASW.html)].
Measurement of clearance rates
Aquadag (Acheson Colloids, Port Huron, MI, USA), a paste of graphite
particles with a median particle diameter of 0.8 µm, was made up as a 4%
colloidal suspension in distilled water; 4.7 ml of this stock was then diluted
with ASW to a final volume of 1 liter and stored at 10°C. This suspension
was aerated for at least 5 min before each clearance experiment both to mix
and aerate it.
To measure the clearance of colloidal graphite, pieces of gill were hung from aluminum hangers by a loop of thread in glass vials (4 cm high x 2 cm diameter) containing 10 ml of the ASW/Aquadag suspension. Samples (0.5 ml) were removed with a pipettor at the beginning of the experiment, and every 3 min thereafter for 15 min; the pipettor tip was placed 2 cm into the vial adjacent to the piece of gill. The relative concentration of graphite in the solution was determined with a spectrophotometer set to a wavelength of 500 nm. Four or six pieces of gill were used in each experiment. Two experiments, each with four vials lacking gills, were performed to measure the rate of spontaneous settling of the graphite. Over the 15 min duration of the experiments, no settling in these vials could be measured.
Clearance rates were calculated by regressing the ln(fractional initial absorbance) vs time (min). The rate constants – the slopes of the regression lines – were estimated with either SAS JMP v.5.1 (SAS Institute, Cary, NC, USA) or Systat v.11 (Systat Software, Inc., San Jose, CA, USA). Rate constants with a 1-tailed P value of >0.05 were considered statistically insignificant and entered as 0. Mass-specific clearance rates (ml min–1 g–1 dry mass) were calculated by multiplying the rate constants by 8.5 ml (the average volume of fluid in the vials) and dividing by the dry mass of the gills, which was obtained after the tissues had been dried overnight at 60°C.
Design of clearance experiments
Maximal autonomous clearance rate
For these rate determinations, the gills were dissected and the loop of
thread attached. The gills were then allowed to recover overnight. Before
these measurements, the gills were examined with a microscope to ensure that
the lateral cilia were active, the ostia open and the water tubes
inflated.
Basal rate
Before exposure to 5-HT, DEANO (diethylamine/nitric oxide complex) or 5-HT
+ L-NAME (nitro-L-arginine methyl ester), clearance
rates were first determined on untreated pieces of gill to measure the basal
clearance rate; these gills had been dissected 1–2 h before use. Before
this initial trial, five random patches on each piece of gill were inspected
with a microscope to determine if the lateral cilia were active. Pieces of
gill with more than two patches of active lateral cilia were rejected; the
rationale being that only gills clearing graphite at the basal rate would show
maximal stimulation in response to an agonist.
Treatment rate
After determining the basal rates, the gills were transferred, on their
hangers, to a second vial of ASW containing the appropriate agent for the
following times: 5-HT for 5 min, DEANO for 15 min and L-NAME for 20
min. After the allotted time, the gills were transferred to a third vial
containing ASW/Aquadag and the appropriate agents, and water samples were
removed as in the initial run. One or two pieces of gill were left untreated
for the second run and served as untreated controls.
A different repeated-measures design was used to measure the inhibitory
effects of DA. Rather than a basal rate determination, pieces of gill were
first exposed to 10–5 mol l–1 5-HT to ensure
that they were clearing graphite at their maximal rate. After this maximal
rate determination, the pieces of gill were suspended for 5 min in a second
vial containing only ASW and DA and were then transferred to a third vial
containing ASW/Aquadag and DA for the treatment rate measurement. To retard
the oxidation of DA, an ascorbic acid buffer (see
Malanga, 1975) was added to
the ASW. In these experiments, data are expressed as the ratio of treatment
rate/maximal rate. Regression lines were fitted to these data with a logistic
function of the form:
response=1/[1+exp(ß0+ß1xlog DA)], where
ß0 and ß1 are intercept and slope parameters,
respectively. The concentrations of agonist giving half-maximal responses
(EC50) were estimated according to the following formula:
EC50=10^(–ß0/ß1). These
non-linear regressions were done in Systat v.11.
Data distribution and transformation
The clearance rates are highly skewed, having a distribution approximating
an exponential. Accordingly, the data were ln-transformed to give a normal
distribution (Kolmogorov–Smirnov one-sample test 2-tailed
P=0.17, N=367). Because clearance rates of 0 are undefined
by this transformation, 1 was added to all basal clearance rates for
statistical testing. Not surprisingly, the ratios of the treatment rate to the
basal rates were also skewed and were ln-transformed to achieve normality
(Kolmogorov–Smirnov one-sample test 2-tailed P=0.67,
N=159). All statistical tests were performed on ln-transformed data,
but the results are presented untransformed in figures and tables for
clarity.
Morphometry
Pieces of gill were dissected and pinned to the bottom of Petri dishes (50
mm diameter), the bottoms of which were coated with Sylgard® (Dow
Chemical, Midland, MI, USA); the dishes contained 5 ml of ASW. To measure the
cross-sectional area of the water tubes, small pieces of gill (about 1 mm high
and 5–10 mm long) were cut perpendicular to the plane of the gill
filament so that the cut open ends of the water tubes were visible from above.
To measure the interfilament spaces, an entire demibranch was pinned flat upon
the bottom of the dish, with the outer face of the demibranch facing up. These
gill preparations were allowed to relax for 60 min and were then exposed to
increasing concentrations of 5-HT. After the addition of 5-HT, the solution
was mixed with a Pasteur pipette, and then 5 min later the gills were
photographed with a Spot RT color CCD camera (Diagnostic Images, Sterling
Heights, MI, USA) at a magnification of 50x for the measurements of
water tube areas or 100x for the measurements of gill filament spacing.
After another 5 min, 5-HT was again added to the gills to make the next higher
concentration, and the process was repeated. The cross-sectional areas of the
water tubes and the interfilament spaces were measured with Spot Advanced
image analysis software (v.3.5; Diagnostic Images).
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Basal clearance rates
The mean mass-specific clearance rate of untreated pieces of gill was
14.51±0.79 ml min–1 g–1 dry mass
(s.e.m.; N=589 pieces of gill from 144 clams). Because the lateral
cilia on these pieces were largely inactive, this basal clearance rate is due
mostly to the activity of the latero-frontal and frontal cilia. A nested
analysis of variance (ANOVA), with individual clam nested within month as an
error term, revealed no significant differences in monthly basal clearance
rates (P=0.22).
The effects of 5-HT
Clearance
Irrespective of the season, 5-HT had a biphasic effect upon clearance
rates: low doses stimulated clearance rates, whereas higher doses decreased
the maximal rate. During the winter, the threshold for 5-HT stimulation was
10–6 mol l–1; the response peaked at
10–5 mol l–1 and then declined, so that
doses of 5x10–5 and 10–4 mol
l–1 had no net effect upon clearance
(Fig. 3). At
10–5 mol l–1 5-HT, the mean clearance rate
increased by 8.8 to 31.82 ml min–1 g–1 dry
mass (s.e.m.=7.09; N=10). During the summer, the gills were less
responsive to 5-HT and the threshold increased from 10–6 mol
l–1 to 5x10–6 mol l–1
(Fig. 3). Although the maximal
stimulation was still at 10–5 mol l–1 5-HT,
the mean clearance rate increased by only 2.3 to 18.49 ml
min–1 g–1 dry mass (s.e.m.=3.81;
N=14). This increase is significantly lower than that during the
winter (1-tailed P=0.02; N=24).
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The effects of NO
During the winter, NO (generated by 10–6 mol
l–1 DEANO) tripled the basal clearance rate, but during the
summer, NO only doubled this rate. The clearance rates of untreated controls
increased by a factor of 1.2. ANOVA revealed that the means of rate ratios
(i.e. treatment rate/basal rate) are significantly different
(P=0.049; N=74); post-hoc analysis of these data
with Dunnett's test revealed that the rate ratios of the gills during the
winter are significantly greater than the untreated controls
(Fig. 6). Thus, NO stimulated
clearance during the winter but had no significant effect during the
summer.
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During the winter, pretreating pieces of gill with 10–5 mol l–1 L-NAME inhibited the effects of 10–5 mol l–1 5-HT (1-tailed P=0.03; N=17; Fig. 7). In addition, ANOVA revealed that the clearance rates of pieces of gills exposed to L-NAME and 5-HT during the winter were equal to the rates of pieces of gill exposed only to 5-HT during the summer (P=0.98; N=113). During the summer, L-NAME had no effect upon the clearance rates of pieces of gill treated with 10–5 mol l–1 5-HT (1-tailed P=0.29; N=18).
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). The
responses to both agents are markedly seasonal, thus the gills are more
sensitive to 5-HT and DA during the winter than during the summer.
Additionally, the seasonal effects of 5-HT are modulated by an NO signaling
cascade, which is present during the winter but not the summer. Finally, the
results show that the rate of graphite clearance is, in part, the result of
the interaction between the activity of the lateral cilia and the gill
musculature. Thus, the behavior of the gill is predictable from the behavior
of its components.
DA and the inhibition of clearance
The effect of DA upon clearance is completely inhibitory, irrespective of
the season. This was expected because DA not only inhibits the lateral cilia
(Gainey et al., 1999) but also
stimulates the gill musculature (Gainey et
al., 2003). As was the case for the stimulation of clearance rates
with 5-HT, the gills are more sensitive to DA during the winter. The
dose–response curves for the effects of DA also reveal another effect
– that there is little inhibitory modulation of clearance rates. During
summer, clearance rates declined abruptly from maximal to minimal between
5x10–6 and 10–5 mol
l–1 DA. The effect is not as pronounced during the winter as
the mean clearance rate declined to around 50%, then became minimal. The
dose–response curve for the effects of DA on the lateral cilia has a
similar shape; the cilia beat maximally and then abruptly stop between
10–6 and 10–5 mol l–1 DA
(Gainey et al., 1999).
5-HT and the regulation of clearance
Irrespective of the season, 5-HT has a biphasic effect upon clearance:
concentrations lower than 10–5 mol l–1 5-HT
stimulate clearance, while higher concentrations inhibit it. The biphasic
nature of this response readily follows from the effects of 5-HT on the
lateral cilia and the gill musculature. 5-HT stimulates quiescent lateral
cilia of Mercenaria with a threshold from 10–7
(Aiello, 1970) to
10–6 mol l–1
(Gainey et al., 1999) and it
also stimulates the gill musculature to contract, again with a threshold of
10–6 mol l–1
(Gainey et al., 2003;
Gainey and Greenberg, 2003).
But between 10–6 and 10–5 mol
l–1 5-HT, the beating of the lateral cilia is stronger than
the contraction of the gill muscles, and microscopic observation of the water
tubes shows that their cross-sectional area increases, i.e. the water tubes
become inflated when pumping begins. Similar observations on the relationship
between the activity of the lateral cilia and inflation of the water tubes
have been made in vitro on excised gills of Juxtamusium
maldivense (Pectinidae) (Jorgensen,
1976) and in vivo, via endoscopy, on the gills of
Pyganodon cataracta (Unionidae)
(Tankersley, 1996). This
behavior is analogous to that of sea anemones that have expelled the water
from their coelenteron by contraction of their musculature: reinflation of the
coelenteron and extension of the musculature is accomplished by cilia in the
siphonoglyphs. At concentrations of 5-HT above 10–5 mol
l–1, contraction of the gill musculature is strong enough to
counteract the beating of the lateral cilia, so the diameter of the water
tubes is reduced, as is the interfilament space. Thus, the rate of clearance
is reduced due to changes in gill geometry.
The current paper reports that clearance rates in actively pumping gills
vary from 5 to 60 ml min–1 g–1 mass –
a 12-fold range – but the beat of the lateral cilia varies from 7 to 27
Hz – only a 
4-fold range
(Gainey et al., 1999). A
limited range in the rate of lateral ciliary beating has also been reported in
Mytilus edulis (Catapane et al.,
1978). Jorgensen et al.
(Jorgensen et al., 1986;
Jorgensen et al., 1988),
Riisgard and Larsen (Riisgard and Larsen,
1995) and Grunbaum et al.
(Grunbaum et al., 1998) have
shown, in Mytilus edulis, on both experimental and theoretical
grounds, that the width of the interfilament space is the primary factor
regulating flow through a fillibranch gill. In addition, Silverman and
colleagues (Gardiner et al.,
1991; Medler and Silverman,
1997; Medler and Silverman,
2001) proposed that the gill musculature regulates flow in
eulamellibranch gills. The striking difference between the rates of lateral
ciliary beat and clearance in the Mercenaria gill, when taken
together with the changes in interfilament space and water tube
cross-sectional area (also reported here), support this hypothesis.
Clearance is regulated in isolated gills of Mercenaria by
endogenous transmitters, implying that clearance is a regulated and not an
autonomous process. That clearance is regulated in response to environmental
variables, such as seston concentration and oxygen tension, has been
championed by Bayne and colleagues (Bayne
et al., 1993; Bayne,
1993; Bayne, 1998;
Bayne, 1999;
Bayne, 2000;
Hawkins et al., 1999;
Cranford and Hill, 1999;
Cranford, 2001;
Widdows, 2001). The opposing
view, i.e. that clearance is an autonomous process and is partially dependent
upon the physical properties of seawater, has been championed by Jorgensen and
Riisgard (Jorgensen et al.,
1986; Jorgensen et al.,
1988; Jorgensen et al., 1990;
Jorgensen, 1990;
Jorgensen, 1996;
Riisgard, 2001a;
Riisgard, 2001b;
Riisgard et al., 2003). If
allowed to recover overnight from dissection, unstimulated gills – in
October – had clearance rates that are statistically indistinguishable
from gills in the summer that had been stimulated by 5-HT. This implies that
clearance is an autonomous process. But the regulation of clearance over a
12-fold range by 5-HT implies that clearance is a regulated process. Taken
together, these two sets of seemingly contradictory observations suggest that
the isolated, unstimulated gill clears at the maximal summer rate and that the
visceral ganglion – removed in the preparations used for this study
– regulates clearance by inhibition of the maximal rate.
NO and seasonal rate compensation
Although NO stimulates particle clearance during the winter, it has no such
effect during the summer. Furthermore, the nitric oxide synthase (NOS)
inhibitor L-NAME reduces the effects of 5-HT during the winter, and
this effect is statistically indistinguishable from the effects of 5-HT during
the summer, when NO signaling has no effect on clearance. These results are
consistent with an earlier study of the immunohistochemical localization of
NOS and soluble guanylate cyclase (sGC) in the gills
(Gainey and Greenberg, 2003).
We reported that, during the autumn, NOS is found in the gill musculature,
which penetrates each gill filament, but during the summer, NOS is confined to
the base of the gill filaments in the interfilament tissue junctions.
Similarly, sGC is localized in the muscles and the gill filament epithelium
during the winter but is restricted to the tips of the filaments in the area
of the frontal cilia during the summer. On the other hand, when whole gills
were loaded with the NO-fluorescent probe DAF-diacetate
(4,5-diaminofluorescein-diacetate), microscopic observation showed NO
pervading the gills in both seasons (L.F.G., unpublished). However, the
absence of sGC from the area of the lateral cilia and muscle is consistent
with the lack of an effect of NO on particle clearance during the summer.
Although the gills synthesize NO during the summer, this substance has no
effect upon particle clearance, because sGC, a downstream effector in the
signaling pathway, is absent. NO has no direct effect upon the gill
musculature (Gainey and Greenberg,
2003) but during the autumn and winter it stimulates quiescent
lateral cilia to beat (L.F.G., unpublished). Thus, the effect of NO on the
gills during the winter is twofold: it directly stimulates the lateral cilia
and potentiates the effects of 5-HT upon the branchial muscles.
The seasonal difference between the clearance rates of gills is striking.
One of the characteristics of animals showing this type of seasonality, i.e.
seasonal rate compensation, is that animals acclimatized to winter conditions
will have a higher rate than those acclimatized to summer conditions, provided
that both are tested at the same temperature
(Hochachka and Somero, 1973;
Prosser, 1973). The
5-HT-induced clearance rates of Mercenaria gills show such a
response. Thus, at 10–5 mol l–1 5-HT –
the concentration producing maximal clearance – winter gills had a mean
clearance rate of 31.82 ml min–1 g–1 dry
mass whereas summer gills had a mean clearance rate of only 18.49 ml
min–1 g–1 dry mass. Moreover, winter gills
treated with the NOS inhibitor L-NAME – again at
10–5 mol l–1 5-HT – had a mean
clearance rate of 14.01 ml min–1 g–1 dry
mass, which is statistically equal to the summer rate. Therefore, if NO
production during the winter is inhibited, seasonal rate compensation is
abolished. I therefore propose that the NO signaling cascade and its seasonal
expression are the mechanisms of seasonal clearance rate compensation in
Mercenaria gills, again implying that clearance is a regulated, as
opposed to an autonomous, process.
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Acknowledgments |
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