Regulation of urine reprocessing in the maintenance of sodium and water balance in the terrestrial Christmas Island red crab Gecarcoidea natalis investigated under field conditions
1 Morlab, School of Biological Sciences, University of Bristol, Woodland
Road, Bristol, BS8 1UG, UK
2 School of Biological Sciences, University of Sydney, Sydney, NSW 2006,
Australia
* Author for correspondence (e-mail: steve.morris{at}bristol.ac.uk)
Accepted 19 May 2003
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Summary |
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Key words: osmoregulation, water flux, Jnet, Na flux, branchial uptake, serotonin, red crab, Gecarcoidea natalis
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Introduction |
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Land crabs could adjust the salt loss in the P by adjusting both the volume
ultimately voided and the salt composition of the P (Wolcott and Wolcott,
1985,
1991
;
Taylor et al., 1993
). The
crabs could both lower urine filtration rate and/or re-ingest some urine
(Wolcott, 1992
;
Greenaway et al., 1990
;
Taylor et al., 1993
;
Greenaway, 1994
), providing
for an ion-regulatory role for the gut (e.g.
Bliss, 1968
;
Ahearn et al., 1999
). Exactly
how the extent of urine modification is regulated in land crabs is far from
clear. There is growing evidence of hormonal control but considerably more
information is required.
In the terrestrial anomuran B. latro, dopamine, a biogenic amine
released from the pericardial organs, downregulates branchial Na uptake and
Na+/K+-ATPase in the gill epithelial cells
(Morris et al., 2000;
Greenaway, 2003
). By contrast,
evidence from osmoregulating aquatic brachyuran crabs supports a hormonal
upregulation of branchial Na+/K+-ATPase and ion uptake
(reviewed by Morris, 2001
).
Biogenic amines, including octopamine and dopamine, have been linked to an
elevation of cAMP in increasing the Na uptake in diverse marine species (e.g.
Kamemoto and Oyama, 1985
;
Lohrmann and Kamemoto, 1987
;
Sommer and Mantel, 1988
,
1991
). In Chinese mitten crab
Eriocheir sinesis, bioamines promote protein phosphorylation
via a cAMP-dependent protein kinase in the gill tissue
(Trausch et al., 1989
) and
stimulate Na flux (Bianchini and Gilles,
1990
; Detaille et al.,
1992
; Mo et al.,
1998
). This mechanism is also present in freshwater crayfish
(Mo and Greenaway, 2001
) and
is thus ubiquitous in aquatic decapods. Neuropeptides may also prove
important, since, most recently, crustacean hyperglycaemic hormone has been
shown to have marked effects on Na+ transport in crustacean gills
(Spanings-Pierrot et al.,
2000
; Serrano et al.,
2003
).
The possibility that net branchial salt uptake may be acutely adjusted by
alterations in permeability, and thereby the rate of salt loss to a
hypo-osmotic environment, has received relatively little consideration (e.g.
Onken, 1999;
Onken and Reistenpatt, 2002
).
Crabs clearly do adjust overall permeability in response to environmental
circumstances (Péqueux,
1995
). Adjustment of paracellular conductance would alter leak
permeability, which may be altered as much as 10-fold by salinity acclimation
(Onken, 1999
;
Tresguerres et al., 2003
).
Paracellular channels are generally under complex control (e.g.
Anderson and van Itallie,
1995
). In the insect Malpighian tubule, control of paracellular
permeability includes neuropeptide messengers
(Wang et al., 1996
). Hormonal
adjustments to diffusive efflux have apparently been detected in at least one
decapod (Tullis, 1975
).
The present study considered the putative hormonal regulation of salt
reclamation from the urine by the gills in the terrestrial red crab from
Christmas Island, Gecarcoidea natalis (Brachyura: Gecarcinidae),
under field conditions. G. natalis is endemic to the rainforest of
Christmas Island in the Indian Ocean, where the great majority of the crabs
live away from the ocean and do not have ready access to seawater to replenish
body salts (Greenaway, 1994;
Adamczewska and Morris, 2001a
).
In the laboratory, red crabs produce a very dilute P when given freshwater to
drink (Greenaway and Nakamura,
1991
) and in the field manage salt and water balance in response
to the availability of water and dietary salt
(Greenaway, 1994
).
Furthermore, G. natalis held in the laboratory behaved in many ways
as if under semi-xeric conditions. For example, in the field, Na and water
turnover were much higher than in the laboratory whereas filtration rate was
lower, perhaps due to the greater and more diverse availability of water and
salt within the rainforest environment
(Greenaway, 1994
).
Preliminary assessment of data from in situ studies suggested that
the bioamine serotonin, rather than dopamine, has an important stimulatory
role in branchial ion pumping and Na uptake in G. natalis
(Morris, 2001). Subsequent to
the completion of the present study, Taylor and Greenaway
(2002
) provided laboratory
data for regulation of salt balance in G. natalis. That study, of Cl
rather than Na uptake, confirmed modulated branchial Cl uptake from urine in
response to changes in Cl availability and haemolymph concentration, provided
evidence of drinking urine as a potential mechanism of volume and salt
regulation and showed that dopamine can stimulate Cl uptake, but only in crabs
acclimated to drinking 70% seawater. This final finding is consistent with
biogenic amines stimulating branchial ion uptake and with a role in post-renal
urine modification (Taylor and Greenaway,
2002
). However, the ecological and physiological significance of a
mechanism that promotes net salt uptake only into already salt-replete crabs
needs clarification.
It is important to resolve the roles of monoamines. The present study was conducted entirely in situ on Christmas Island during which different pharmacological approaches to determining the regulatory steps of branchial salt reclamation were used. The study also addressed the quasi-xeric response shown by red crabs in the laboratory by examining the effect of confinement and the availability of drinking water.
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Materials and methods |
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The studies of isotopically labelled crabs were carried out in remote areas within Christmas Island National Park under permit from Parks Australia and the Health Department of Western Australia. All isotopes were shipped directly by Amersham Biosciences (Sydney, Australia).
Seasonal water and salt turnover under natural and confined
conditions
Two treatment groups of crabs were established in each season; free-ranging
animals fitted with radio transmitters and crabs confined in field enclosures.
Crabs were collected from the forest, weighed and fitted with
radio-transmitters (Titley Electronics, Ballina, NSW, Australia) glued to the
carapace, as described previously
(Adamczewska and Morris,
2001a). The crabs were injected through the arthrodial membrane at
the base of the penultimate walking limb with the appropriate isotope, and 2 h
was allowed for equilibration in the body. Dosages of isotopes in all
experiments were: 3H2O, 46.25 Bq g-1 wet
mass; 22NaCl, 52.0 Bq g-1 wet mass;
51Cr-EDTA, 7.4 kBq g-1 wet mass, and the largest fluid
volume injected was 1 µl g-1. After equilibration, a blood
sample (
0.5 ml) was removed and stored in a sealed vial, and the crabs
were released at the point of capture. Dilution of the radiolabels allowed the
calculation of total body water, extracellular fluid volume (ECFV) and Na
space. During the wet season, crabs moved within an extended `home range' and
it was necessary to locate the crabs each day.
The confined animals were treated similarly except that they were returned to enclosures at the point of capture inside the rainforest. The enclosures were made from fuel drums (JET A1 aviation fuel; 200 litre) that had been cut in half midway and from which the ends had been removed to produce open tubes. The steel walls were firmly inserted into the forest floor, so that the animals could contact the normal soil substrate and leaves, and the top was enclosed with wire mesh to prevent marauding robber crabs, B. latro, from preying on the experimental animals. Drinking water vessels were excluded from some enclosures so that the effect of availability of water could be assessed. Haemolymph samples for isotope analysis where taken on days 1, 3 and 6 after release. This required that animals be weighed using a calibrated spring balance in the field (Salter, Bury St Edmunds, UK) and ultimately with an electronic balance. The initial and final samples were in some cases also analysed for major ions and osmotic pressure. The radioactivities of the samples were measured using liquid scintillation counter (Packard Instrument Company, Meridan, USA) or, for 51Cr, using a gamma counter (LKB Wallac Clini Gamma Counter, Turku, Finland).
The daily rate exchange fraction (K) and the biological half-life
(tg) of sodium and water were derived as described
previously for crustaceans (Greenaway,
1980; Morris and van Aardt,
1998
):
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Rates of 51Cr-EDTA clearance, urine production and fluid
resorption within the urinary system were determined where possible. Clearance
(ml h-1) was calculated as K x EDTA space,
determined from dilution of the injected 51Cr-EDTA (Greenaway et
al., 1991; Morris and van Aardt,
1998). Urine samples were obtained by gently deflecting the
nephropore flap and drawing released urine into a fire-drawn pipette. The
ratio of specific 51Cr activity between urine and haemolymph (U:H)
allowed calculation of fluid resorption within the antennal gland.
Drinking rates and the production rate of the final excretory product, P, were determined at the end of the trial periods as well as in some crabs collected directly from the rainforest. This required the recaptured animals to be individually transferred to large plastic chambers (P-chambers) suspended in the lower canopy of the rainforest. Each chamber comprised a bucket that had the base replaced by stainless steel mesh through which fluid but not faeces could pass to be collected in a hydrophobic polyethylene trap. A drinking vessel was included in each chamber. Evaporative loss of water from the drinking containers was determined in the same chambers containing dampened paper towel to simulate crabs. The crabs were weighed before and after their sojourn in the chambers, which was between 18 h and 23 h. The importance of drinking water was further assessed by including a further treatment in which the crabs were provided with empty drinking vessels.
Haemolymph and urine salt composition
The osmotic pressure of the haemolymph and urine, where required, was
determined using a vapour pressure osmometer (Wescor, Logan, USA) or, for
especially dilute solutions, Osmomat (Gonotek; Berlin, Germany)
freezing point depression osmometer. The remaining sample was denatured by
mixing 1:1 with 0.1 mmol l-1 HNO3 and was transported to
the laboratory for further analysis. The Cl concentration was determined using
a chloride titrator (CMT10; Radiometer, Copenhagen, Denmark), and Mg, Ca, K
and Na were measured by atomic absorption spectrophotometry (AAS; GBC 906, GBC
Melbourne, Australia). To suppress interference, samples for measurement of Na
and K were diluted with 5.9 mmol l-1 CsCl2, while for Mg
and Ca measurements samples were diluted with 7.2 mmol l-1
LaCl3.
Pharmacological investigations water and sodium balance
Dopamine and serotonin (5-hydroxytryptamine) were administered to crabs by
infusion through the arthrodial membrane of the walking legs at
10-10 mol g-1, and cAMP was administered as the
membrane-permeable dibutyryl-cAMP (db-cAMP) at 10-9 mol
g-1. Infused volumes did not exceed 220 µl and were composed of
an appropriate carrier saline (below) that had been sterilised by boiling. To
avoid oxidation, dopamine was dissolved 10 s prior to infusion in saline
deoxygenated by boiling. A saline control infusion group was included in all
determinations. The saline was modified from that of Greenaway
(1994) and comprised: NaCl,
376 mmol l-1; NaHCO3, 1 mmol l-1;
CaCl2, 12.9 mmol l-1; KSO4, 6.1 mmol
l-1 and MgSO4, 9.24 mmol l-1. The rate of
clearance of 51Cr-EDTA and the efflux of 3H2O
and Na was determined for treated crabs in the forest, and the effects on Na
flux rates were determined also in branchial perfusion of crabs held within
P-chambers (above).
Pharmacological treatment of confined crabs
G. natalis were held for 24 h in enclosures and then infused with
either a monoamine, db-cAMP or saline, as described above. At the same time,
the animals were injected with either 51Cr-EDTA or
3H2O (as described previously). Crabs were re-infused
with the pharmacological trial compound or saline as a control every 8 h over
3 days and sampled for haemolymph every 24 h. At the end of the 3 days, the
51Cr-EDTA-infused animals were sampled for urine, which was also
assessed for 51Cr activity and osmotic pressure. During the dry
season, crabs were assessed for 51Cr-EDTA clearance only, since dry
season water flux rates had been extremely low. During the dry season only, an
extra group was included to test the effects of octopamine. The entire
experiment was duplicated for the dry season except that the animals were
deprived of drinking water. Estimates of drinking and P production could be
obtained only by holding the animals in metabolism chambers, i.e. P-chambers,
within the rainforest. Therefore, additional estimates of water flux were made
over the 24 h period that animals were held in the P-chambers.
Branchial perfusion and Na flux
The branchial perfusion studies determined the net flux of Na from the
urine into the haemolymph (Jnet), the unidirectional flux
into the crab by utilizing 22Na tracer (Jin),
and Jout as the difference between the two
(Jout=JinJnet).
To detect up- and/or downregulation of branchial Na uptake, G.
natalis were either acclimated to drinking 50% seawater (SW) for 2 weeks
or provided with freshwater (FW) (Morris,
2001). The branchial perfusions were carried out using a modified
method from Morris et al.
(1991
,
2000
). The mean mass of the FW
group was 265.8±14.05 g and of the SW group was 288.2±10.90 g.
The carapace (anterior branchiostegite) of the crabs was drilled through into
the branchial chamber with a fine dental drill (battery hobby drill; Dremel)
to allow the insertion of polyethylene cannulae (0.97 mm i.d., 1.27 mmo.d.).
The opening was cauterized, and the 5 cm-long cannula fixed in place with
cyanoacrylate adhesive. The animals were then left to recover for at least 24
h.
The perfusion experiments were carried out at ambient temperature within
the environs of the plateau rainforest. The chambers were those used
previously for collecting P (above) but in which the animal rested above a
funnel to collect overflowing branchial perfusate. Saline solution of ionic
composition close to that of the urine [artificial urine (AU)] was pumped to
both branchial chambers at 0.8 ml min-1 using a peristaltic pump.
The composition of the two different AU used were, for FW and SW respectively,
NaCl, 400 mmol l-1 and 416 mmol l-1; KCl, 16 mmol
l-1 and 12.5 mmol l-1; MgCl2, 7.8 mmol
l-1 and 12 mmol l-1; and CaCl2, 27 mmol
l-1 and 17.5 mmol l-1; both AU contained 50 mmol
l-1 Na2SO4 and 1 mmol l-1
NaHCO3. Both AU were labelled with 22NaCl as a tracer
for unidirectional Na influx. The use of a small fixed extra-corporeal volume
allowed the ready application of Shaw's solutions for unidirectional
22Na flux calculations (Shaw,
1963).
To initiate the perfusion experiments for each animal, a 20 ml volume
of AU was circulated for 20 min to fill the interstices of the branchial
chambers and was then drained to waste. At the same time as the initial
perfusate was supplied, the crab was infused with either sterile saline as a
sham treatment or with saline containing either dopamine at
2x10-4 mol l-1 or db-cAMP at
6.1x10-4 mol l-1. The infusion of the monoamines
very occasionally caused the crabs to regurgitate and, if so, this always
occurred during the preperfusion, allowing the perfusate to be replaced or,
more often, for the animal to be excluded from the experiment. After the 20
min preliminary perfusion, the perfusate was drained and a second measured
volume (
20 ml) was supplied and recirculated. Samples of 0.3 ml were
taken shortly after the initial start time and every 15 min thereafter up to
90 min. The recirculating system can require the judicious inclusion of
monitoring procedures. 51Cr-EDTA injected as a urinary tracer has a
short half-life of 27.7 days, and decay within 22Na-labelled
samples was revealed by counting over several weeks. Very few animals were
discarded, post hoc, from the data set. The crabs ingested a small
volume of perfusate. The amount of Na and 22Na counts thereby
removed during the 90 min perfusion was readily calculated and subtracted from
the branchial uptake rates. Samples were assayed in triplicate for Na and
22Na. The net rate of Na uptake was calculated as described
previously (Morris et al.,
1991
). The rate of 22Na uptake was calculated from the
linear relationship of the decreased loge c.p.m. (counts per
minute) in a fixed volume over time to provide a rate constant
(Morris and van Aardt, 1998
).
The requirement to exclude some data eventually produced a balanced design
with N=8 for each of the treatments.
Determination of branchial Na+/K+-ATPase
activity and the effects of serotonin
Na+/K+-ATPase activity was determined in the gill
tissue of G. natalis previously infused with serotonin or saline
(control). 30 min after the infusion, the crabs were cooled at 4°C until
completely torpid, the gills were removed from one side of the animal, and the
animals were then rapidly frozen. The gills were weighed and homogenized in 25
mmol l-1 Tris/acetate buffer containing phenylmethylsulphonyl
fluoride (0.2 mmol l-1), dithiothreitol (0.1 mmol l-1)
and aprotinin (100 units ml-1). Protein concentration was
determined for the gill homogenate using a test kit (protein assay 500-0001
Kit 1; Bio-Rad, Hemel Hempsted, UK) calibrated using bovine gamma globulin in
the concentration range of 01.57 mg ml-1. Each homogenate
assay was replicated, and absorbances were determined at 595 nm. For assay,
the homogenates were 89 mg ml-1. ATPase activity was
determined in (1) a buffer of the following composition
MgCl2, 6 mmol l-1; NaCl, 100 mmol l-1; KCl,
10 mmol l-1; Tris, 25 mmol l-1, adjusted to pH 7.4 with
acetic acid and (2) in the same buffer without KCl but containing 3.5
mmol l-1 ouabain, which specifically inhibits
Na+/K+-ATPase. The difference between the ATPase
activity in the two buffers could then be attributed to
Na+/K+-ATPase activity. The reaction was started by the
addition of vanadium-free ATP (9.1 mmol l-1) and stopped after 20
min at 25°C by the addition of trichloracetic acid (0.6 mol
l-1). After centrifugation at 10 000 g for 10 min,
the inorganic phosphate (Pi) concentration was determined in the
supernatant using the method of Fiske and Subbarow (kits 661-11 and 661-8;
Sigma). Absorbance was determined at 660 nm using a transportable
spectrophotometer (Novaspec II; PharmaciaBiotech, Uppsala, Sweden).
Statistical analyses
Homogeneity of variances was verified using Bartletts 2
test prior to one- and two-way analysis of variance (ANOVA). Post hoc
testing was by Tukey's test for one-way analyses and by A Matrix Contrast
analyses when significant differences were indicated by two- and three-way
ANOVA.
Where it was required to determine if means differed from zero, a one-sample t-test was employed. Analysis used Systat packages.
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Results |
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Providing drinking water in the enclosures made no difference to urine composition, and the only effect was a dilution of Na concentration of the haemolymph of crabs with access to water (Table 2). However, in contrast to the wet season, confinement during the dry season had further effects. Most noticeable was a general reduction in Mg concentrations in confined crabs but also a tendency to a specific increase in urinary Mg not seen in the wet season (Table 2). The lower urinary Ca concentration (9.5±1.3 mmol l-1) seen in free-ranging crabs during the wet season was also observed during the dry season (6.6±2.1 mmol l-1). Apparently, Ca was always reabsorbed from the urine of free-ranging crabs but not from that of crabs confined in enclosures.
Water turnover
The crabs lost some mass and, although the largest relative loss of 8.14%
was in the crabs confined during the dry season, the loss could not be
correlated with season or generally with any effect of confinement
(Table 3). Curiously, the
lowest loss rates were in free-ranging crabs in the dry season
(Table 3). There were also some
significant decreases in the water space, but again these were not correlated
with season or treatment (P=0.24) and could not simply account for
any mass loss (Table 3). There
was no difference in the half-life for water turnover of confined animals
compared with free-ranging G. natalis, which in the dry season was
5.29±0.55 days and 4.81±0.47 days, respectively. In comparison,
there was a very marked decrease in the half-life of water in G.
natalis in the wet season, to 1.75±0.21 days for confined crabs
and to 2.24±0.39 days for free-ranging animals. The rate of water
efflux in wet season animals was approximately double that of dry season
animals, with overall mean rates of 272.13±34.97 ml kg-1
day-1 and 140.34±10.31 ml kg-1 day-1
(Fig. 1). The water efflux
rates were completely unaffected by confinement in field enclosures.
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51Cr-EDTA clearance and urine flow
The 51Cr-EDTA space was significantly smaller in the dry season
crabs (22.7% body mass) than in those in the wet season (27.9%) but did not
vary between crabs confined in enclosures and those ranging in the surrounding
forest. The most remarkable result was the more than doubling of the clearance
rate during the wet compared with the dry season
(Fig. 2). In free-ranging
crabs, the rate increased from 53.45±8.74 ml kg-1
day-1 in the dry season to 111.26±23.76 ml kg-1
day-1 in the wet season. Despite an apparent reduction in the dry
season, confinement had no significant effect on clearance rate or, as far as
could be determined, on urine flow rate
(Fig. 2). Urine flow rate was
determined from the urine-to-haemolymph ratio (U:H) for 51Cr-EDTA,
which in wet season crabs was 1.23±0.07 in free-ranging animals and
1.44±0.46 in confined animals. These values were not different from
each other (P=0.14) nor were they different from values of one
(t-test). Thus, no significant concentration of the urine occurred
within the antennal gland. It was impossible to obtain urine from any of the
crabs in the dry season and thus to determine urine 51Cr-EDTA. A
range of urine flow values was estimated for dry season crabs by using the
values of U:H=1.04 from Greenaway
(1994) and from the wet season
animals above (Fig. 2). In any
case, urine flow rates were not different from the corresponding clearance
rate (Fig. 2).
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The dry season crabs drank considerably more than the crabs in the wet season, and the animals that had been confined drank all of the available water, more than 48 ml kg-1 day-1 (Fig. 2). For example, free-ranging crabs in the dry season drank 38.4±7.07 ml kg-1 day-1 whereas those in the wet season drank only 7.17±1.94 ml kg-1 day-1. The mean drinking rate of free-ranging crabs in the wet season was not significantly different from a rate of zero, i.e. not drinking at all (one-sample t-test, P=0.058). However, the seasonal difference in drinking of approximately 31 ml kg-1 day-1 was a small fraction of the difference in urine flow (Fig. 2) or, especially, the 127 ml kg-1 day-1 difference in overall water efflux (Fig. 1). The rate of P flow was reciprocal to the trend in drinking rate so that crabs that drank the largest amount during the dry season produced little, or more often, no P (Fig. 2). At the other extreme, during the wet season free-ranging crabs produced P at a rate (37.81±7.4 ml kg-1 day-1) approaching 50% of the urine flow rate (88.0±21.12 ml kg-1 day-1) and, importantly, more than five times greater than the drinking rate (Fig. 2). G. natalis that had been confined had both higher drinking rates and lower rates of P release than the free-ranging animals.
Sodium turnover
In the wet season of February 1997, there was rainfall on every day of the
trials, with 52 mm falling on the second day. There were few significant
effects of confinement alone on the Na status but some changes were associated
with the season (Table 4). The
Na space was clearly largest in animals confined in enclosures during the dry
season (37.8% body mass) and smallest in G. natalis ranging in the
forest during the wet season (26.5% body mass), which was associated with a
reciprocal trend in both the biological half-life of Na in the animal and the
rate constant for Na turnover (Table
4). For example, in crabs confined in enclosures during the dry
season, tg was 11.9 days but was only half this (5.5 days)
in free-ranging crabs during the wet season. The same trend was less obvious
in the total body Na of G. natalis
(Table 4), despite scaling to a
standard mass of 250 g. Nonetheless, the lowest value of 16.53 mmol was
recorded in the wet season, and the highest value of 36.44 mmol during the dry
season. Consequently, there were no differences in Na efflux between seasons
or as a result of confinement (Table
4), and the overall mean efflux was 10.49±0.96 mmol
kg-1 day-1.
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Pharmacological trials Cr-EDTA clearance and water flux
The large decrease in clearance rate when the crabs were deprived of
drinking water was confirmed but there was no effect of dopamine, octopamine
or db-cAMP on mass, ECFV or Cr-EDTA clearance
(Table 5). By contrast, during
the dry season serotonin induced an increase in clearance rate of
1617%. However, in wet season trials, neither dopamine
(10-10 mol g-1), serotonin (10-10 mol
g-1) nor db-cAMP (10-9 mol g-1) produced any
significant change in the clearance of 51Cr-EDTA, the flow of urine
or the water efflux rates of G. natalis
(Table 6).
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Neither serotonin, dopamine nor db-cAMP altered water efflux in either the freshwater (FW) or seawater (SW) groups. Acclimating the crabs to drinking 50% SW increased the water flux by 113% and at the same time also reduced P production, in some cases to zero (Table 7). There was no effect of either acclimation (P=0.26) or drug infusion (P=0.24). Both dopamine and serotonin caused marked reductions in P production by FW-acclimated crabs to 4.5 ml kg-1 day-1 and 4.4 ml kg-1 day-1, respectively, as compared with 17.6 ml kg-1 day-1 in the saline-infused controls. This effect was apparently not mediated by cAMP (Table 7). In these crabs, water flux was determined during the 24 h period that crabs were held in the P-chambers, and the rates were much lower than water flux and clearance rates otherwise determined in situ.
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Branchial modification of urine
Different results of pharmacological infusions were obtained from G.
natalis that had been drinking FW compared with those acclimated to
drinking 50% SW (Figs 3,
4). In the FW crabs, serotonin
increased Na Jnet significantly by 57% from
197.2±31.1 mmol kg-1 day-1 to 309.9±17.6
mmol kg-1 day-1, which was primarily the result of an
increase in active uptake so that unidirectional Na influx
(Jin) increased from 242.6±29.7 mmol
kg-1 day-1 to 321.1±22.2 mmol kg-1
day-1 (Fig. 3). By
contrast, dopamine infusion resulted in a Jin of
210.4±20.5 mmol kg-1 day-1, which was not
significantly different from the control, and neither was the rate in
cAMP-infused crabs (Fig. 3).
There were no significant fluctuations in the Jin of
SW-drinking G. natalis.
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Changes in the overall loss component (Jout) were important in the SW-acclimated crabs (Fig. 4). Acclimation of G. natalis to drinking 50% SW reduced the Jnet rate compared with FW-infused control crabs by 29% to 139.7±16.5 mmol kg-1 day-1 (Fig. 4). The decline in Jnet was due not to any change in the active uptake component (Jin) but instead to a significantly greater passive loss, as shown by Jout=-111.9±24.6 mmol kg-1 day-1 as compared with only -45.4±12.8 mmol kg-1 day-1 in the FW control crabs. In the SW G. natalis, both dopamine and serotonin increased Jnet (247.8±21.5 mmol kg-1 day-1 and 223.5±38.4 mmol kg-1 day-1, respectively) and re-established a net Na uptake rate similar to that in FW saline control crabs (Figs 3, 4). Jin remained unaffected and similar to that of the control crabs (Fig. 4). Rather, the primary effect of dopamine and especially serotonin in SW G. natalis was to reduce the relatively elevated loss such that Jout was lower (dopamine, -62.9±26.6 mmol kg-1 day-1; serotonin, -21.0±19.8 mmol kg-1 day-1) than that of SW control crabs (Fig. 4). Infusion of db-cAMP also had effects in SW crabs but these were not apparent in Jnet, which remained unchanged due to a decrease in Jin that balanced a lower Jout (Fig. 4).
The rate of drinking and thus the uptake of Na by ingestion was highly variable between individual crabs and it was not possible to discern any difference with respect to either the salinity of drinking water acclimation (FW, 230.6 mmol kg-1 day-1; SW, 239.9 mmol kg-1 day-1; P=0.61) or pharmacological infusion (P=0.36).
Na+/K+-ATPase and serotonin
Serotonin infusion of G. natalis drinking FW promoted a 92%
increase in Na+/K+-ATPase activity to 1.71±0.32
µmol Pi mg-1 protein h-1 compared
with control crabs (0.89±0.16 µmol Pi
mg-1 protein h-1). In view of the similarity between the
Jin values for SW and FW crabs perfused with saline only
(Figs 3,
4), it would seem important
that the Na+/K+-ATPase activity of SW crabs
(0.79±0.03 µmol Pi mg-1 protein
h-1) was unchanged compared with that of FW G.
natalis.
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Discussion |
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Seasonal water balance
Water turnover of red crabs doubled from 140 ml kg-1
day-1 in the dry season to
280 ml kg-1
day-1 in the wet season, demonstrating a considerable flexibility
in water management in the field. The dry season water turnover rate in
situ was almost identical to the 138 ml kg-1 day-1
previously measured on Christmas Island at the end of the dry season but was
considerably greater than the 71 ml kg-1 day-1 in red
crabs driven by the dry season into burrow retreats
(Greenaway, 1994
).
Significantly, the flux rates of crabs measured in P-chambers rapidly declined
to 30 ml kg-1 day-1 (21% of the dry season rate in
situ). In the laboratory, G. natalis took up water at a rate
between 13% and 22% of that in crabs in the field
(Greenaway, 1994
). The water
turnover rates of Birgus latro in the laboratory were as little as
one-third of those measured in the field
(Greenaway et al., 1990
;
Taylor et al., 1993
;
Greenaway, 2001
). The
difference might be variously explained by re-ingestion of urine
(Greenaway, 2001
), recycling
the tritium label, and by depriving the crabs of the water in their food. For
laboratory-held red crabs, urine flow was 85 ml kg-1
day-1 while the final P flow was less than 10 ml kg-1
day-1 (Greenaway,
1994
). It is apparent that confining the crabs in P-chambers
induced a semi-xeric response similar to that seen in laboratory-held
crabs.
Red crabs drank from the branchial perfusate during the current
investigation and also in other studies during which oral flow increased from
37 ml kg-1 day-1 to 184 ml kg-1
day-1 (Taylor and Greenaway,
2002). In B. latro, EDTA clearance in the field was
similar to clearance rates determined in the laboratory, leading Greenaway
(2001
) to speculate that
primary urine formation might be held constant and water balance managed by
regulated drinking of the urine. In red crabs on Christmas Island, the reduced
dry season water turnover was managed by a significantly reduced clearance:
50 ml kg-1 day-1 compared with >100 ml
kg-1 day-1 in the wet season as well as an almost
complete cessation of P production. Drinking rate was similar to urinary flow
rate and thus water from the food made little contribution during the dry
season.
During the wet season, red crabs drank less and had high urine flow and
water turnover. Greenaway
(1994) comments on the large
water content of food during the wet season and this, together with direct
absorption of water during heavy downfalls (red crabs shelter from heavy rain;
S. Morris and M. D. Ahern, personal observation), probably leads to the
excessive water uptake requiring the production of large volumes of a dilute
P. Water turnover rates were higher than clearance rates in both confined and
free-ranging crabs, and the possibility of some isotopic exchange during the
wettest weather contributing to elevated rates must be recognised
(Greenaway, 1994
). Lowered
activity of semi-fossorial red crabs during the dry season may allow both
generally lowered metabolic rate (Adamczewska and Morris,
2000
,
2001a
,b
)
and water conservation (Greenaway,
1994
). Under dry season circumstances in which almost no P is
produced there is thus no opportunity for post-renal modification of
urine.
Seasonal salt balance
Haemolymph osmotic pressure was approximately 50 mOsm higher in
free-ranging G. natalis crabs during the dry season (805 mOsm)
compared with those in the wet season. This was not a large difference when
compared with 1100 mOsm in red crabs during an unseasonably dry breeding
migration (Greenaway, 1994
).
Osmotic pressure in red crabs on Christmas Island typically ranges between 670
mOsm and 810 mOsm (Adamczewska and Morris,
2000
,
2001b
) and is somewhat below
this range in laboratory-held crabs
(Taylor and Greenaway,
2002
).
The antennal glands are of low importance in ion regulation except for some Ca reabsorption. The dry season increase in osmotic pressure was generally reflected in increased haemolymph ion concentration. The lower osmotic pressure, [Na] and [Mg] in both haemolymph and urine of confined versus free-ranging red crabs may be explained by a relatively higher drinking rate without a corresponding increase in clearance in the former.
The Na space of dry season crabs tended to be greater than that in the wet
season crabs, particularly in the confined crabs, but this was not reflected
in the total body Na. While the Na space values were similar, the trend is
opposite to that previously found when comparing wet and dry conditions
(Greenaway, 1994). It is
significant that red crabs in the dry season of 1997 had a mean ECFV of 21.9%,
similar to the dry season value of 24.3% reported by Greenaway
(1994
), while in the wet
season the ECFV value was much higher at 31.7% and much closer to that of the
freshwater amphibious crabs Holthuisana transversa (Taylor and
Greenaway, 1994) and Potamonautes warreni
(Morris and van Aardt, 1998
).
Thus, the Na space is enlarged in the wet season by increases in the
relatively Na-rich ECFV. Confined crabs in the dry season had the greatest
biological half-life for Na of tg=12 days, which is
nonetheless considerably shorter than reported previously
(Greenaway, 1994
). The
consequence of the greater Na body content but greater half-life of Na in dry
season animals is an efflux that was constant at all times. Greenaway
(1994
) reported a doubling of
Na efflux from 2.31 mmol kg-1 day-1 to 4.39 mmol
kg-1 day-1 when comparing sampling periods with and
without rainfall. The lowest rate in the present study was 8.37 mmol
kg-1 day-1. The rate of 2.31 mmol kg-1
day-1 was associated with a water turnover rate of 71 mmol
kg-1 day-1, which was also lower than the in
situ rates reported here, as too are the clearance rates. If the urine
represents a major route for Na loss, then reduced clearance and increased
urine ingestion would promote Na retention (reduced efflux). Crabs during the
dry season appear to be primarily in water conservation mode and to balance
evaporative water loss by drinking whatever water is available in addition to
almost all of their own urine. The evidence is that crabs held under
laboratory conditions are most like dry season crabs but even more like those
held in P-chambers. Red crabs in the wet season appear to experience an excess
of water that must be excreted without excessive loss of ions. A situation of
unavoidable high water throughput, and thus high clearance and thereby high
potential salt loss, would be that most requiring branchial urine
reprocessing; i.e. during the wet season.
Pharmacological trials
Pharmacological trials on confined G. natalis had few effects on
whole-body indices of water and salt management. The elevation of clearance
rate promoted by serotonin was apparent only during the dry season but
occurred even when the crabs were deprived of drinking water. It seems likely
that very high clearance rates during the wet season (>150 ml
kg-1 day-1) represent maximum rates to deal with
super-availability of water. If so, the capacity for serotonin to promote an
increase in clearance will exist only in dry season crabs when clearance rate
is inherently lower.
Water flux over 24 h in crabs in P-chambers was low [29.5 ml
kg-1 day-1 (FW) and 41.4 ml kg-1
day-1 (SW)] compared with control crabs (132.7 ml kg-1
day-1). In the SW group, the total body water declined to 53.9%
compared with 67.1% in free-ranging crabs and to 63.0% in FW crabs. These
lowered flux rates were very similar to those of Greenaway
(1994) and indicate a rapid
response by red crabs on being moved into the experimental chambers. Resolving
this response will require further in situ field investigations of
water movements in unconfined crabs. Nonetheless, acclimating the crabs to
drinking 50% SW resulted in almost zero P production and thus removed any
scope for further downregulation of branchial ion reabsorption. In the
laboratory, increased salinity of the drinking water of red crabs produced a
dynamic change in the volume of P over 5 days
(fig. 1 in
Taylor and Greenaway, 2002
).
In the FW crabs, both serotonin and dopamine also reduced the volume of P
released, presumably partly due to re-ingestion of the urine. The present
in situ study was unable to establish any additional effect of
dopamine on urine production (Taylor and
Greenaway, 2002
; but see discussion of passive permeability
below).
Branchial mechanisms
The branchial perfusion experiments confirmed FW-acclimated red crabs to be
in a very different physiological state to those acclimated to drinking 50%
SW. The marked effect of serotonin in increasing net branchial Na uptake by FW
G. natalis (Morris,
2001) was confirmed, as was the absence of any effect of either
dopamine or db-cAMP (Taylor and Greenaway,
2002
). The upregulation by serotonin in red crabs is similar in
principle to that in osmoregulating marine brachyuran crabs
(Morris, 2001
for review) but
differs in detail. A dopaminergic, cAMP-mediated upregulation of branchial ion
pumping appears ubiquitous in the aquatic brachyuran species
(Lohrmann and Kamemoto, 1987
;
Sommer and Mantel, 1988
,
1991
;
Bianchini and Gilles, 1990
;
Detaille et al., 1992
;
Morris and Edwards, 1995
).
Other than the work of Trausch et al.
(1989
) on Eriocheir
sinesis, there has been little suggestion of a role for serotonin but
this too was dependent on a cAMP-activated protein kinase. Red crabs without
access to seawater utilize serotonin as a primary messenger, but independently
of cAMP, and thus appear quite different to their marine ancestors.
The serotonin-induced elevation in branchial Jnet for
Na was due to an increase in Jin, and none of the
treatments altered Jout in FW crabs. The importance of the
serotonin message to the branchial Na pumps in G. natalis is clear in
the large response of the branchial Na+/K+-ATPase. There
is little doubt as to the pivotal regulatory role of the branchial pumping
system (e.g. Asselbourg et al.,
1991; Mo et al.,
1998
; Towle et al.,
2001
; Henry et al.,
2002
).
G. natalis almost completely cease branchial Cl uptake when given
70% seawater to drink (Taylor and
Greenaway, 2002). A similar response was evident in the Na uptake
of B. latro drinking seawater
(Taylor et al., 1993
). On
Christmas Island, G. natalis showed a less extreme downregulation,
possibly since the water was less saline and also because there was some
rainfall during the acclimation period. The reduced uptake was clearly not as
any result of decreased Jin since, somewhat unexpectedly,
the pumping component of the branchial epithelia remained unchanged. Instead,
the lowered Jnet was consequent on increased Na leak
permeability, i.e. in Jout. The branchial
Na+/K+-ATPase activity was likewise undiminished by SW
acclimation, which strongly supports this conclusion.
A relatively small upregulation of Cl uptake by G. natalis was
reported by Taylor and Greenaway
(2002). The upregulation of
branchial Na Jnet by dopamine in G. natalis in
situ was not due to any change in unidirectional influx of Na since
Jin remained at the rate found in FW- and SW-acclimated
control crabs. The elevation in Jnet was entirely due to
amelioration of the elevated Na leakage (Jout) in SW crabs
and most likely due to effects on paracellular conductances. An almost
identical response was determined for serotonin in SW red crabs. The net
result in both cases was to return Jnet to close to that
in FW control crabs. This rate was well below the elevated rate caused by
serotonin in FW crabs. In the SW crabs there was some evidence that the
effects on Jout may be mediated by cAMP but that cAMP may
also be involved in downregulation of Na influx, but in such way that
Jnet is unchanged.
The mechanisms of salt reclamation from the urine of Christmas Island red
crabs are considerably more complex than described by preliminary models
(Morris, 2001;
Greenaway and Taylor, 2002
).
Serotonin has a potentially adaptive role in stimulating branchial
Na+/K+-ATPase and branchial Na uptake from the urine in
crabs with low Na intake. G. natalis drinking freshwater and with a
low Na diet can produce a very dilute P
(Greenaway, 1994
), suggesting
that the branchial uptake of Na should be close to maximum with little
capacity for serotonin to stimulate uptake. The experimental crabs in the
rainforest had access to the normal leaf-litter diet and reduced the osmotic
pressure of the P from 392 mOsm in SW crabs to 73 mOsm in FW crabs (
90%
absorption relative to the haemolymph) compared with more than 97% Cl
reabsorption exhibited in the laboratory
(Taylor and Greenaway, 2002
).
Thus, in the present study, the wet season animals, with their high turnover
rates, apparently still had significant unutilized uptake capacity that could
be activated by serotonin.
In SW-acclimated animals, branchial uptake mechanisms would be expected to be proportionately less active. In other words, uptake would be `turned down' and thus increase the capacity for biogenic stimulation and upregulation. The mechanisms revealed are inconsistent with this simple model. Decreased net uptake in SW-acclimated crabs is not achieved by any acclimatory downregulation of Na+/K+-ATPase or of Na Jin but by an increased loss. The stimulatory effect of serotonin is blocked in SW red crabs and replaced by modulation of leak permeability. In this regard, improving net salt uptake by reducing loss with no increase in pumping work seems advantageous.
There appear to be two suites of responses. Small alterations in leak permeability and thus Jnet might represent very straightforward mechanisms for assisting the voiding of excess salt gained perhaps from the diet. The serotonergic responses of FW-acclimated animals seem useful if they are elicited in addition in crabs with very low dietary Na uptake and in crabs that must promote urinary salt reclamation beyond that routinely required.
The terrestrial gecarcinid was established to be quite different to the
anomuran B. latro and this is consistent with a separate evolution to
life on land (Morris, 2001,
2002
;
Taylor and Greenaway, 2002
;
Greenaway, 2003
). However, the
branchial reprocessing system in G. natalis is not otherwise very
similar to that in marine crabs or, consequently, to that of a putative marine
ancestor. The multilevel response and the complex interaction of changes in
branchial Na pumping and modulated Na leakage may well be derived from a
marine species but provide features especially suited to life on land where
salt is usually limiting and water must be conserved without excessive salt
loss. Work on other terrestrial species and their nearest aquatic relatives is
urgently required to resolve both the function and phylogenetic origins of
these control mechanisms.
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Acknowledgments |
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