Osmoregulation in the terrestrial Christmas Island red crab Gecarcoidea natalis (Brachyura: Gecarcinidae): modulation of branchial chloride uptake from the urine
1 Department of Zoology, University of Canterbury, Private Bag 4800,
Christchurch, New Zealand
2 School of Biological, Earth and Environmental Sciences, University of New
South Wales, Sydney 2052, Australia
* Author for correspondence (e-mail: h.taylor{at}zool.canterbury.ac.nz)
Accepted 11 July 2002
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Summary |
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Key words: Gecarcoidea natalis, land crab, osmoregulation, chloride reabsorption, excretion, dopamine, cyclic AMP, gill
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Introduction |
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In terrestrial crabs, gas exchange takes place primarily across
branchiostegal lungs (Taylor and Taylor,
1992) but all species retain gills, implicating them in ion
reabsorption. However, contributions to ionoregulation from other epithelia
have not been excluded. Mantel
(1968
) demonstrated uptake of
water and salts by the foregut of G. lateralis and considered this
organ to be important in osmoregulation. In Gecarcoidea natalis, the
posterior gills possess ultrastructural features indicative of active ion
transport (Farrelly and Greenaway,
1992
) but in B. latro, all gills, and also the
branchiostegites, demonstrate high activities of transport ATPases
(Morris et al., 1991
).
Intravascular infusions of water and salines, and experimental manipulation
of the drinking water salinity, indicate that crabs adjust the composition of
P according to the requirements of ionic homeostasis (Wolcott and Wolcott,
1985,
1991
;
Taylor et al., 1993
). However,
our understanding of urine reprocessing as a homeostatic response to altered
salt and water status is incomplete, being based on partial data for only a
few species. More information is needed on the timing, rates and capacity for
adjustment of urine reprocessing following a perturbation. Also, it is unclear
how adjustments in P composition are achieved in vivo. In principle,
crabs could vary the residence time of the urine within the branchial
chambers, its route to the exterior, the perfusion of the osmoregulatory
surfaces with haemolymph, epithelial salt transport rate or a combination of
these. In addition, in B. latro, variations in the volume of P
produced appear to be achieved by adjustments to the filtration rate and by
reingestion of the urine (Greenaway et
al., 1990
; Taylor et al.,
1993
).
There is evidence that urine reprocessing is under endocrine control, but
this is incompletely understood. It is not known whether urine processing is
modulated in response to internal (blood osmolytes, volume) or to extrinsic
(e.g. drinking water composition) stimuli. The pericardial hormone dopamine
inhibits branchial salt reabsorption in the anomuran B. latro
(Morris et al., 2000).
However, this role contrasts with that in hyper-regulating aquatic
brachyurans, in which abundant evidence indicates that dopamine increases salt
uptake by activation of a branchial epithelial
Na+/K+-ATPase
(Zatta, 1987
; Sommer and
Mantel, 1988
,
1991
;
Morris and Edwards, 1995
;
Mo et al., 1998
). Morris et
al. (2000
) suggested that a
state of continuous salt uptake, turned off hormonally, was a better
adaptation to terrestrial life than the converse strategy of switching on salt
uptake as required. However, it is yet to be shown whether a reversed role for
dopamine is a general feature of terrestrial crabs.
This paper examines homeostatic aspects of post-renal reabsorption of
chloride in a terrestrial brachyuran, G. natalis. This crab produces
a dilute final excretory fluid when maintained on a freshwater drinking
regimen (Greenaway and Nakamura,
1991). We measured the steady rates of branchial chloride uptake
attainable by crabs supplied with freshwater and determined the time course of
downregulation of chloride uptake in salt-loaded crabs. Crabs were
salt-loaded: (i) by acclimation to saline drinking water over a period of
weeks, (ii) by self-loading during prolonged perfusion of the branchial
chambers with saline over a period of hours, and (iii) by direct NaCl
injection over a period of minutes. A possible role for dopamine in switching
the uptake system on or off was examined in crabs acclimated to saline and to
fresh drinking water.
In initial experiments, it was noted that there were intermittent periods
of urine output and of the ingestion and regurgitation of the perfusion
saline. It was necessary to account for these accurately in terms of volume
and chloride flux to determine the true branchial flux. A radioactive
filtration marker was used to estimate urine flow, and flow-through branchial
chamber perfusion was employed instead of the recirculating method used
previously (Morris et al.,
1991). The flow-through system permitted observation of temporal
changes in chloride flux and in the movements of urine and oral fluids.
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Materials and methods |
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Effects of the salinity of the drinking water on the [Cl-]
of the final excretory fluid
Crabs were transferred from the freshwater culture to individual chambers
constructed from 101 plastic buckets fitted with lids and stainless-steel mesh
bottoms. A conical plastic bag, containing 10 ml of paraffin oil, attached
below the bucket, trapped excreted fluids (P), which were removed with a
syringe and cannula via a small hole. Crabs were provided with 20 ml
of drinking water (dyed to reveal spillage), which was changed daily, and a
leached cottonwood leaf. The drinking water was initially tapwater and was
switched to 70% seawater ([Cl-]=360 mmol l-1) on day 4;
P was collected for a further 5 days, and the volume and [Cl-] of
the samples were recorded. At the final collection time, a haemolymph sample
(approximately 200 µl) was removed for [Cl-] analysis, using a
syringe and 21-gauge needle via a predrilled hole in the carapace
above the pericardial sinus.
Measurement of chloride uptake from saline-perfused branchial
chambers
At least 1 day before perfusion, the carapace was predrilled for
pericardial blood-sampling and tracer injection, and holes approximately 1 mm
in diameter were drilled bilaterally through the dorsal carapace over the roof
of each branchial chamber and cauterized to prevent blood loss. A polyethylene
cannula (5 cm; 1 mm o.d.) was inserted approximately 1 mm into each chamber,
glued (with cyanoacrylate) into place and temporarily plugged. At least 1 h
before the first haemolymph sample, approximately 10 MBq 100 g-1
body mass of a filtration marker (51Cr-EDTA; Amersham, UK) in
saline (50 µl 100 g-1 body mass) was slowly injected into the
pericardial sinus and allowed to circulate. A haemolymph sample was taken 30
min before perfusion commenced, and the total [Cl-] of the
perfusion saline was adjusted to that of the haemolymph by addition of NaCl
(other cations as chlorides, in mmol l-1: K+, 6;
Ca2+, 14; Mg2+, 7). At the start of the experiment, the
cannulae were connected to the perfusion lines, and the crab was placed into a
covered plastic box and allowed to settle for approximately 1 h. Saline was
then infused into both branchial chambers using two channels of a peristaltic
pump at a total flow rate of approximately 0.6 ml min-1 (calibrated
at the start and end of each run). After a delay of several minutes, fluid
emerged at the leg bases and collection commenced. This fluid drained into a
funnel, from where it was continuously removed and distributed into timed
fractions (manually or using a Gilson fraction collector). A second haemolymph
sample was taken at the end of the infusion. The volume, [Cl-] and
radioactivity of each outflow fraction and haemolymph sample were
measured.
Calculation of branchial, urinary and oral chloride fluxes
The rate of branchial net uptake of chloride (JCl,Gill)
was estimated for each collected fraction as:
JCl,Gill=chloride input in saline + chloride input in
urine chloride in outflow or:
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The volume of saline retained R by the crab in each sampling
interval was:
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In preliminary experiments, the filtration marker was not used and the branchial chambers were perfused with a hypoosmotic saline ([Cl-=200 mmol l-1, other ion concentrations as given above), simulating partially processed urine. The rate of branchial chloride uptake (uncorrected for urine flow) was estimated from the volume of fluid collected in each interval and the difference in the [Cl-] of the input and output fluids. This method generally gave results similar to those obtained using the marker. However, an apparent inhibition of branchial chloride uptake following certain treatments was shown to be caused by the release of urine hyperosmotic to the perfusate.
Effects of salt loading on chloride fluxes
Chloride fluxes in crabs acclimated to fresh drinking water during
prolonged saline perfusion of the branchial chambers
The branchial chambers of crabs taken from the freshwater cultures were
perfused with saline for 5-8 h. This established the time course of the onset
of chloride uptake, and the effects of prolonged self-loading with salt on
chloride transport, urine flow and the ingestion of branchial fluid. The
relationship between the branchial chloride fluxes and the [Cl-1]
of the haemolymph was also examined.
Chloride fluxes in crabs acclimated to saline drinking water
These experiments were designed to determine the effect of chronic
salt-loading on branchial chloride transport. Crabs were maintained in
cultures supplied with 70% seawater ([Cl-]=360 mmol l-1)
for drinking for 7-9 days before commencing perfusion.
The effects of NaCl injection on chloride fluxes in
freshwater-acclimated crabs
This series examined responses to acute elevation of haemolymph [NaCl]. The
branchial chambers of crabs from the freshwater culture were perfused for
approximately 1 h to establish steady uptake. Branchial chamber perfusion was
then stopped for approximately 5 min while 5 mol l-1 NaCl (800
µl 100 g-1 body mass) was infused via a pre-drilled
hole into the pericardial sinus. Branchial chamber perfusion was then
restored, and the outflow was collected for a further 2 h. The
[Cl-] of the perfusate (mean 424 mmol l-1) was adjusted
to be approximately midway between the measured initial
[Cl-]h and the estimated [Cl-]h
after the injection. The distribution of injected chloride and elevation of
[Cl-]h were similar to those in perfused crabs absorbing
chloride over several hours (see Results), indicating that circulation was
maintained. Mild impairment of locomotor function was evident in two
individuals, and only four crabs were subjected to this treatment. The
response to injection of NaCl was compared statistically with that of control
crabs injected with iso-ionic saline (next section).
Effects of dopamine and cyclic AMP on chloride fluxes
Freshwater-acclimated crabs
Crabs were taken from the freshwater culture and their branchial chambers
were perfused for 0.5-1 h until a steady uptake had been established. Without
interruption of perfusion and fluid collection, perfusion saline containing
1.0 mmol l-1 dopamine (250 µl 100 g-1 body mass), was
injected into the pericardial sinus. The concentration of dopamine,
distributed throughout the extracellular space (24.3±1.0 ml 100
g-1 body mass) (Greenaway,
1994) was therefore approximately 10 µmol l-1. The
injection took approximately 1 min, and a further 1 min elapsed before
removing the needle and sealing the hole. To reduce oxidation, dopamine was
bubbled with nitrogen until use. In a similar series, crabs were injected with
saline (250 µl 100 g-1 body mass) containing 6 mmol
l-1 dibutyryl cyclic AMP (dbc-AMP, final haemolymph concentration
approximately 60 µmol l-1). Control crabs were injected with a
similar volume of perfusion saline.
Saline-acclimated crabs
Branchial chambers of crabs from cultures supplied with saline drinking
water (70% seawater) for 30-50 days were perfused for 60 min before injecting
dopamine as above. Other conditions were as for the freshwater-acclimated
crabs injected with dopamine.
Chemical analyses
Samples of haemolymph, P and the afferent and efferent branchial chamber
perfusion salines were analyzed for [Cl-] within 24 h using an
electrometric chloride titrator (Radiometer CMT 10). Haemolymph samples were
diluted immediately with distilled water (1:3) to reduce coagulation in the
titration vessel. The radioactivity of fluid samples was measured using a
gamma counter (Packard or LKB-Wallac Minigamma). Samples were adjusted to a
standard volume with distilled water and corrected for background
radioactivity. Urine and haemolymph samples were diluted and analyzed for
metal atoms by flame atomic absorption spectroscopy (GBC, Avanta; Na and K,
airacetylene; Ca and Mg, N2Oacetylene; CsCl used to
suppress ionization). Osmolalities were determined by vapour pressure
osmometry (Wescor 5520).
Data analyses
Values in the text, table and graphs are means ± S.E.M.; see figure
legends for N values. Urinary and oral chloride fluxes are converted
to approximate flow rates on the right-hand axes of graphs, which divide the
chloride flux by the mean [Cl-] of the perfusion saline. Flows
given in the text were individually calculated for each crab and sample
time.
Time courses of flux changes are shown at a resolution of 0.25-0.5 h, for several hours, but for statistical analysis of the effects of injected substances, the mean flux rate for each crab during 1 h following the injection was compared with a baseline rate established during 1 h prior to the injection (repeated-measures ANOVA; Statistica software, Statsoft, Tulsa, OK, USA). As there were significant time-dependent changes in the control FW crabs, individual `responses' were calculated (mean rate after treatment minus mean rate before) and compared among treatments by one-way analysis of variance (ANOVA). Tukey's HSD test was used for multiple post-hoc contrasts. Repeated-measures ANOVA was used for analysis of changes in P concentration and production rate and for comparison of haemolymph and urine ion concentrations in FW and SW crabs. A probability P of <0.05 was taken as statistically significant unless stated otherwise.
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Results |
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Effects of drinking saline on [Cl-] and rate of release of
final excretory fluid (P)
Individual daily P production by crabs supplied with fresh water varied
between 0 and 17.5 ml. The mean rate of P production during the first day was
5.11±1.77 ml day-1 (approximately 3.4% of body mass per day)
and fell to 1.61±0.47 ml day-1 (approximately 1.1% of body
mass per day) over 4 days (Fig.
1). On switching the drinking water to 70% seawater, P production
increased again to approximately 4.5 ml day-1 (repeated-measures
ANOVA; days 5 and 6 significantly greater than days 3 and 4).
|
Crabs supplied with freshwater produced dilute P, mean [Cl-] decreasing from 12.46±2.51 mmol 1-1 on day 1 to 8.83±0.95 mmol l-1 on day 4. On switching to seawater, the [Cl-] of the P increased to 376±18 mmol 1-1 on day 9 (Fig. 1; mean values on days 6-9 were all significantly greater than those on days 1-5; repeated-measures ANOVA). On day 9, the [Cl-] of the P was not significantly different from the [Cl-] of the drinking water (360 mmol 1-1) but was still hypo-ionic to the haemolymph (436±36 mmol 1-1 for the six crabs that produced P on day 9; paired t-test, P<0.01). Crabs differed greatly in the rate of adjustment of the [Cl-] of the P, which increased to half the blood concentration in less than 1 day in some crabs to more than 5 days in others.
Time course of chloride uptake from the branchial chamber perfusate
in crabs acclimated to fresh drinking water
On commencing perfusion with saline of the branchial chambers of crabs from
the freshwater culture, branchial chloride uptake was initially low but
increased to a maximum rate over approximately 1 h
(Fig. 2) and then slowly
declined. In some crabs, chloride uptake ceased completely within 5 h, but
others maintained chloride uptake for more than 8 h. The mean rate of
branchial chloride uptake (JCl,Gill) over the period
0.5-1.5 h was 8.68±0.60 mmol kg-1 h-1; between
4.5 and 5.5 h, it was 4.42±1.19 mmol kg-1 h-1
(significantly different; paired t-test, P=0.005). The
difference in [Cl-] between the perfusion inflow and outflow was
20-50 mmol 1-1 at a perfusion rate of approximately 0.6 ml
min-1.
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The individual maximum rate of chloride uptake (10.12±0.45 mmol kg-1 h-1, averaged over 0.5 h) occurred at times between 0.5 and 2.5 h in different individuals. At the start of perfusion, the haemolymph chloride concentration, [Cl-]h, of these crabs ranged from 307 to 408 mmol 1-1 (mean 365±37 mmol 1-1). The peak uptake rate was not correlated with the initial [Cl-]h and was quite constant (Fig. 3). However, after 4.5 h of perfusion, a significant negative correlation (r2=0.68, P<0.01) existed between JCl,Gill and the initial [Cl-]h; i.e. branchial chloride uptake was more rapidly attenuated in crabs with high haemolymph chloride concentrations. Haemolymph chloride concentration rose by a mean of 81 mmol 1-1 to 446±31 mmol 1-1 (range 412-494 mmol 1-1) during the 5-8 h of perfusion.
|
Urine flow increased steadily from a mean rate of 1.54±0.46 ml kg-1 h-1 in the period 0.5-1.5 h to 7.69±1.56 ml kg-1 h-1 between 4.5 and 5.5 h (Fig. 2; the initial high value is probably an artefact caused by wash-out of residual tracer from the branchial chambers) (P<0.01, paired t-test).
Crabs typically retained a large volume of the saline during the first hour or so, partly because of initial filling of the branchial chambers and partly because of ingestion, and continued to ingest fluid at a decreasing rate thereafter (oral flow rates, Fig. 2).
Effects of acclimation to saline drinking water on chloride
fluxes
Crabs that had been drinking 70% seawater for 7-9 days showed approximately
zero branchial chloride uptake during 2.5 h of branchial perfusion
(Fig. 4; most individuals
showed a small overall net loss). The initial [Cl-]h of
these crabs was higher than that of crabs on the freshwater regimen
(497.5±8.7 mmol 1-1) but did not rise significantly during
perfusion (final [Cl-]h=502.4±8.9 mmol
1-1, paired t-test). The rate of urine output in the
saline-acclimated crabs was initially higher than in the freshwater group but
it did not increase during 2.5 h of perfusion. Ingestion (and occasional
regurgitation) of the perfusate occurred throughout the period of perfusion
(Fig. 4; periodic negative oral
flows, representing regurgitations, are obscured in the mean values). Fluid
was retained at approximately double the rate observed in the
freshwater-acclimated crabs and exceeded urine output. This was also evident
from swelling of the arthrodial membranes and pericardial sacs in some
crabs.
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Effects of dopamine, cyclic AMP and NaCl injection on chloride fluxes
in freshwater-acclimated crabs
Dopamine and dbc-AMP did not change the rate of branchial chloride uptake
by FW crabs. Controls injected with iso-ionic saline decreased
JCl,Gill from 7.23±0.67 mmol kg-1
h-1 in the hour before injection to 6.24±0.61 mmol
kg-1 h-1 in the hour following
(Fig. 5A). Similar small
decreases in JCl,Gill followed pericardial infusion of
dopamine (Fig. 5B; from
6.86±0.89 to 6.43±0.88 mmol kg-1 h-1) and
of dbc-AMP (Fig. 5C; from
7.37±0.79 to 6.64±0.91 mmol kg-1 h-1).
Although statistically significant overall (P<0.0001;
repeated-measures ANOVA, contrast analysis) and of similar magnitude to the
decline in JCl,Gill observed in non-injected crabs
(Fig. 2), these responses were
not significantly different from each other (ANOVA, Tukey).
|
By contrast, infusion of hyperionic NaCl over 5 min caused an abrupt 80% depression of branchial chloride uptake (Fig. 6; from 10.01±0.49 to 1.95±0.80 mmol kg-1 h-1). This was significantly greater than the control response (P<0.0001, ANOVA, Tukey) and persisted for more than 2h. During NaCl infusion, [Cl-]h increased by 78 mmol l-1 (from 380.0±34.6 to 458.5±20.3 mmol l-1), a change similar to that observed in perfused crabs freely absorbing chloride over 5-8h (see above), corresponding to a chloride distribution volume of 0.510 l kg-1.
|
The rate of urine production increased steadily in all groups of injected FW crabs (Figs 5, 6). In saline-injected controls, the increase, from 1.05±0.20 ml kg-1 h 1 before injection to 2.26±1.05 ml kg-1 h-1 after the injection, was similar to the change over the corresponding period in non-injected crabs (from 1.85±0.67 to 2.85±0.90 ml kg-1 h-1). Larger increases in urine production followed injection of dopamine (from 1.58±0.47 to 10.58±1.96 ml kg-1 h-1), dbc-AMP (from 3.39±0.91 to 9.53±2.53 ml kg-1 h-1) and hyperionic NaCl (from 1.32±0.27 to 10.11±2.5 ml kg-1 h-1). Within all three experimental groups, the increases were statistically significant (P<0.001, P<0.05, P<0.05, respectively; repeated-measures ANOVA, Tukey), but only the dopamine response differed significantly from the control response (P<0.05, one-way ANOVA, Tukey).
An increase in urine output also followed injection of dopamine in the preliminary series, which did not employ the filtration marker. This was inferred from an apparent reduction in the branchial chloride uptake as the more concentrated urine entered the perfusion flow [see Materials and methods: dopamine reduced the uncorrected uptake from 6.83±0.67 to 3.56±1.16 mmol kg-1 h-1 (mean ± S.E.M., N=9)], which was significantly different (t-test) from the control response (from 6.26±0.93 to 6.27±1.18 mmol kg-1 h-1, mean ± S.E.M., N=6).
The crabs ingested perfusion fluid throughout the experiment. Both the dopamine group and the saline controls regurgitated fluid immediately following the injection (Fig. 5).
Effects of dopamine on branchial chloride fluxes in saline-acclimated
crabs
In crabs acclimated to saline drinking water for 30-50 days, mean branchial
chloride flux JCl,Gill was negative, indicating a small
net loss (Fig. 7). Dopamine
caused a highly significant switch from net loss in the hour before to net
uptake in the hour following the injection (from -0.11±0.32 to
1.12±0.63 mmol kg-1 h-1), but there was no change
in the saline-injected control crabs (from -0.56±0.27 to
-0.65±0.35 mmol kg-1 h-1) (P<0.01,
P>0.99 respectively; repeated-measures ANOVA, Tukey).
JCl,Gill peaked at 1.72±0.81 mmol kg-1
h-1 approximately 1 h after dopamine injection and was sustained
for more than 2 h. An increase in urine output was observed in both
dopamine-injected and control-injected crabs, but the responses were not
statistically different from each other.
|
Relationship between the rate of chloride uptake and the increase in
haemolymph chloride concentration
The haemolymph chloride concentration increased in perfused crabs that
showed net uptake of chloride and decreased in those showing a net loss. The
ratio of the branchial chloride flux (corrected for urinary contribution) to
the rate of change of haemolymph [Cl-] gives an estimate of the
mean volume of distribution of the absorbed chloride (`chloride space'). There
were no significant differences in the chloride space among
freshwater-acclimated crabs either uninjected or injected with saline,
dopamine or cAMP, for which the pooled mean was 0.501±0.026 l
kg-1 (N=25).
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Discussion |
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Branchial processing of urine was clearly adjusted to the requirements of
ionic homeostasis. Switching the drinking water of non-perfused crabs from
freshwater to saline, caused the [Cl-] of the P to increase
40-fold. Haemolymph osmolality and ion concentrations were elevated in SW
crabs and may provide a signal to switch off branchial salt uptake. This is
supported by zero net uptake of chloride observed in SW crabs, by abrupt
inhibition of chloride uptake on infusion of NaCl into FW crabs and by
downregulation of branchial chloride uptake during prolonged saline perfusion
of FW crabs. Similar negative feedback control of sodium and chloride fluxes
has been proposed for freshwater crayfish
(Shaw, 1964;
Mo and Greenaway, 2001
).
However, the observation that the rate of down-regulation of
JCl,Gill, rather than initial
JCl,Gill, was dependent on [Cl-]h
merits further investigation. The relative importance of endocrine mediation
(as discussed below) versus `autoregulatory' modulation of ion fluxes
in response to internal osmotic change
(Onken, 1996
) must also be
considered.
Downregulation of chloride uptake prevented swamping of the haemolymph with
salt during experimental saline irrigation of the gills, but its importance
for crabs in the rainforest is less clear. Normally, the quantity of chloride
reclaimed would be limited by the volume of urine entering the branchial
chambers and by the increasing gradient from haemolymph to P. However, such a
mechanism would be advantageous during breeding migrations when red crabs
immerse themselves in seawater for many hours
(Hicks et al., 1990). This
`dipping' behaviour facilitates rehydration and replenishment of salts lost
during the migration (Greenaway,
1994
).
Experimentally perfused crabs were unable bypass the absorptive surfaces so that changes in the [Cl-] of the perfusate must have been due to changes in branchial chloride transport. However, direct shedding of urine remains a possible means of increasing salt loss for crabs in the field.
On saline perfusion of FW crabs, the rate of chloride uptake rose to a
maximum over approximately 1 h. This might reflect the time taken for fluid to
percolate between the gill lamellae, although they are provided with elaborate
spacing structures to facilitate this process
(Farrelly and Greenaway, 1992).
Or perhaps chloride transport is activated only when fluid enters the
branchial chambers. Both G. natalis and B. latro detect the
entry of fluid into the branchial chambers, adopting a characteristic stance
at the onset of perfusion, apparently to assist retention of fluid during
processing.
Stimulation of branchial chloride uptake by dopamine in saline-acclimated
G. natalis is consistent with the osmoregulatory role of this
pericardial neurohormone in aquatic brachyurans
(Sommer and Mantel, 1988;
Morris and Edwards, 1995
;
Mo et al., 1998
;
Morris, 2001
). Thus, in the
evolution of a terrestrial lifestyle, G. natalis has retained an
endocrine mechanism present in its marine brachyuran ancestors. By contrast,
dopamine has an inhibitory effect on the uptake of chloride and other ions in
the terrestrial anomuran B. latro
(Morris et al., 2000
).
Dopamine did not restore chloride uptake rates of SW crabs to the level in
FW crabs. However, dopamine appears to regulate the existing
Na+/K+-ATPase
(Sommer and Mantel, 1988;
Morris and Edwards, 1995
;
Mo et al., 1998
;
Morris, 2001
;
Towle et al., 2001
). Red crabs
maintained on the saline regimen for several weeks completely shut down net
chloride uptake. The small response may reflect dedifferentiation of
ionocytes, associated with reduced membrane infoldings and levels of transport
enzymes, as observed in seawater-acclimated euryhaline crabs
(Towle et al., 1976
;
Péqueux et al., 1984
;
Compere et al., 1989
). Such
changes might be reversed slowly. Although dopamine had no effect on the rate
of chloride uptake in FW crabs in the present study, another bioamine,
serotonin stimulated sodium transport in freshwater-acclimated G.
natalis (Morris, 2001
).
Interrelationships between these two bioamines and osmoregulation require
clarification.
Besides regulating branchial ion transporters in decapods, dopamine and
other biogenic amines have widespread vasomotor effects, influencing cardiac
function (Wilkens and McMahon,
1992; Wilkens and Mercier,
1993
; Wilkens,
1999
), flow through the cardio-arterial valves (Kuramoto and
Ebara, 1994; Kuramoto et al.,
1992
,
1995
), arterial resistance
(Wilkens, 1997
) and branchial
resistance (Taylor et al.,
1995
). Branchial resistance changes occur in `efferent valves',
which are located in the gill lamellae of aquatic crabs
(Taylor, 1990
); Taylor and
Taylor, 1986
,
1991
,
1992
) and are also observed in
the gills of G. natalis (Farrelly
and Greenaway, 1992
). Thus, endocrine modulation of branchial ion
transport in vivo might involve both direct effects on epithelial
transport and changes in gill perfusion.
Dopamine acutely increased the rate of urine formation in FW crabs, and
serotonin has been shown to have a similar effect when chronically injected
into red crabs in the field (Morris,
2001). Whether these are components of an integrated response that
regulates salt and water balance, or a more generalized stress reaction,
remains to be established. The widespread effects of bioamines suggest they
may not be primarily agents of osmoregulation in crabs. The eyestalks have
been implicated in the control of osmoregulation in decapods
(Berlind and Kamemoto, 1977
;
Mantel, 1985
;
McNamara et al., 1990
;
Charmantier-Daures et al.,
1994
; Pierrot et al.,
1994
; Eckhardt et al.,
1995
; Onken et al.,
2000
; Spanings-Pierrot et al.,
2000
), including Gecarcinus lateralis
(Mantel, 1968
), and thus
deserve more attention in other terrestrial crabs.
Red crabs consistently imbibed the perfusion saline and regurgitated
intermittently, indicating that they could adjust P volume by reingestion of
urine, as proposed for B. latro
(Greenaway et al., 1990;
Taylor et al., 1993
). Mantel
(1968
) demonstrated
iso-osmotic absorption of saline in the foregut of G. lateralis, and
proposed an osmoregulatory role for this organ. However, the foregut did not
contribute to dilution of the perfusate in the present study. Calculation of
branchial chloride fluxes assumed that regurgitated fluids had the same
[Cl-] as the perfusate. Chloride uptake in the foregut would have
been evident as pulses, synchronized with regurgitations. Examination of the
time course of chloride fluxes in individual crabs indicated that this was not
the case (data not shown). Presumably, in G. natalis, the site of
reabsorption of ions is located in the posterior gills, in which
ion-transporting epithelia predominate
(Farrelly and Greenaway,
1992
).
The compositions of the haemolymph and urine of FW and SW crabs confirm
that the antennal organs are unimportant in osmotic and ionic regulation, as
in B. latro (Taylor et al.,
1993), emphasizing the importance of post-renal processes.
Haemolymph osmolality, [Na+], [Ca2+] and
[Cl-] were all elevated by approximately 45% after several weeks of
the 70% seawater regimen and were similarly increased in the urine. Haemolymph
[K+] was better regulated (28% rise), but this was not associated
with an appropriate change in the urine. Renal handling of Mg2+
appears to be regulatory in G. natalis. Although haemolymph
[Mg2+] doubled in SW crabs, urinary [Mg2+] increased
fourfold. G. natalis resembles marine Brachyura in this
respect (Robertson, 1949
,
1953
;
Lockwood and Riegel, 1969
) but
differs from the anomuran B. latro, in which urinary and haemolymph
[Mg2+] are similar and which is unable to excrete magnesium during
prolonged seawater exposure (Taylor et
al., 1993
).
Although G. natalis lacks a diluting segment its renal system, branchial and oral processing of the urine provide a versatile system for salt and water regulation. Despite their evolutionary divergence 160 million years ago, and independent emergence onto land, anomuran and brachyuran terrestrial crabs have acquired essentially similar osmoregulatory systems, although differing with respect to the role of dopamine. It remains to be investigated whether this difference is characteristic of the Anomura or of coenobitids or whether it is unique to coconut crabs.
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