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Extrarenal modification of urine for ion conservation in ghost crabs, Ucypode quadrata (Fabricius)
J. Exp. Mar. Biol. Ecol., 1985, Vol. 91, pp. 93-107
93
Elsevier
JEM 532
EXTRARENAL MODIFICATION OF URINE FOR ION CONSERVATION IN GHOST CRABS, OCYPODE
THOMAS
G. WOLCOTT
QUADRATA
(Fabricius)’
and DONNA L. WOLCOTT
Department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695 U. S.A
(Received 10 December 1984; revision received 15 April 1985; accepted 16 May 1985) Abstract: When placed under ecologically relevant hypo-osmotic conditions, ghost crabs lose salts at very low rates and osmoregulate well. How can they do this when they, like other brachyuran crabs, produce urine isosmotic with hemolymph? We tested two hypotheses to account for low rates of ion loss: that crabs reduce urine flow (abandon volume regulation); and that they osmoregulate without compromising volume regulation by producing a dilute excretory product through extrarenal modification of urine. Experimental crabs were volume-loaded and hemodiluted by infusion with deionized water; controls were volume-loaded without hemodilution by infusion with isosmotic saline. Crabs were held in containers which allowed the crabs to move fairly freely and to handle wastes normally, and allowed final excretory product to be collected without disturbing the crabs. Crabs infused with deionized water did not reduce urine flow acutely. They produced a dilute excretory fluid containing as little as 10% ofthe total osmolytes, Na, and Cl ofhemolymph and urine. The net effect on osmoregulation was as though they, like other terrestrial animals when water-loaded, produced dilute urine. Key words: terrestrial crab; osmoregulation; salt balance; Ocypode quadrata
INTRODUCTION
Salt-conservation mechanisms were examined in ghost crabs, Ocypode quadrata (Fabricius), because they inhabit hypo-osmotic beach and dune habitats where, given the limitations of crab excretory systems, they should experience rapid salt loss. Salt-depletion has seldom been considered as an environmental threat to crabs invading terrestrial habitats, while water shortage has often been regarded as their principal physiological problem (e.g. Spaargaren, 1975) and has received considerable attention (see e.g. reviews by Bliss, 1968; Mantel, 1979). Much of the available information supports this placement of emphasis. Crabs are relatively permeable animals in environments apparently offering little free water. Evaporation rates correlate well with terrestriality (Bliss, 1968; Herreid, 1969; MacMillen & Greenaway, 1978), as do desiccation tolerances (Bliss, 1968). All of these previous studies stress the importance of water loss, but inputs must also be considered in the water and salt budgets of crabs. Salt shortage could become a ’ Contribution No. 85008 from the MEAS Department, North Carolina State University 0022-0981/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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THOMAS
G. WOLCOTT
AND DONNA
L. WOLCOTT
problem if the crabs had access to abundant but hypo-osmotic water, limited supplies of salts from other sources, and experienced high rates of salt loss due to production of isosmotic urine by the excretory organs. These are the conditions under which ghost crabs appear to exist on the backshore and among the dunes of North Carolina’s Outer Banks. Water available to ghost crabs, with the exception of the sea itself, typically is very dilute. During the summer ghost crabs may spend several days at a time in the interior of the barrier islands (T. Wolcott, unpubl. pers. obs. and radio-tracking data), and, during 5-6 winter months, they do not emerge from their burrows. At these times they have no access to sea water. Instead, they extract interstitial water from sandy burrow walls by a mechanism involving capillary tufts of setae and suction by the gill chambers (Wolcott, 1976, 1984). This system provides water ad libitum wherever the crabs can burrow to within =20 cm of the water table in sandy soil (Wolcott, 1984). However, the ~terstiti~ water resembles sea water only in the foreshore, within a few meters of the surf. Further inland, a layer of fresh water overlies the denser sea water (the “freshwater lens”). It lowers interstitial water concentrations to below 2%, at the foredune on North Carolina beaches and to <0.5%, in ghost crab habitat further from the beach (Hall, 1982; T. Wolcott, unpubl. data). Salt inputs from food are also limited at the times when water sources are hypoosmotic. The primary foods of ghost crabs in North Carolina are osmoconforming invertebrates (Wolcott, 1978) which are as salty as sea water, but these are unavailable inland or underground. Crabs living in the interior of the barrier islands subsist on grasses, seeds and occasional insects (Wolcott, unpubl. stomach analyses), and those overwintering in the burrows do not feed at all. The crabs cannot compensate for the low concentration of ions available in water and food by producing a dilute urine. From a voluminous literature, it is clear that brachyuran antenna1 glands, although capable of modifying urine by selective resorption of ions and part of the water, are unable to produce a final urine differing greatly from the hemolymph in total osmotic concentration (for reviews see Robertson, 1960; Lockwood, 1962; Shaw, 1964; Riegel & Cook, 1975; Mantel, 1979; Mantel & Farmer, 1983). Producing a given volume of isosmotic urine to rid the animal of excess water (or soluble wastes) presumably causes loss of the amount of salts contained in a similar volume of hemolymph. Ocypode quadnm, like other brachyuran crabs, produces isosmotic urine (Gilford, 1962a; Gross, 1964). Nevertheless, ghost crabs are excellent hyperosmotic regulators under ecologically reasonable hypo-osmotic conditions, despite their inability to survive extreme dilutions in traditions immersion experiments (Flemister & Flemister. 1951; Gifford, 1962a; Gross, 1964). When held ~thout food on sand dampened with salinities ranging from full-strength sea water (36x,) to < 1% sea water they survive and regulate hemolymph osmotic and ionic concentrations for 2-6 wk at IO-25 “C (Hall, 1982). How are the crabs able to maintain such low salt iosses in the face of large osmotic gradients? The problems of ion retention are different in terrestrial and aquatic habitats.
ION CONSERVATION
IN GHOST
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In air, salt loss through the integument is negligible. Fecal salt losses will be low, even if the fecal fluids are isosmotic with the hemolymph, because the fecal pellets of terrestrial crabs typically contain little water. Furthermore, when feces are being produced, some salts are also presumably being taken in with food. Urine is the principal remaining pathway for salt loss. Therefore we hypothesized that the crabs minimize salt loss by reducing the volume, concentration, or both, of fluid wastes. Reducing production of isosmotic urine would be the simplest mechanism that could account for low ion loss rates from ghost crabs in dilute environments. Consistent with such a mechanism are several pieces of information which suggest that terrestrial crabs minimize the need for urine production by reducing water intake. Integumental permeabilities are low (Herreid, 1969; Greenaway, 1980) in some cases even lower than those of freshwater species (e.g. Rudy, 1967; Thompson, 1970; Subramanian, 1975). This presumably reduces osmotic influx of water. Avoiding contact with hypo-osmotic free water, an option on land, would further minimize excessive influx of water. Terrestrial crabs do decrease urine flow when dehydrated. Gecarcinus lateralis reduces flow by 75 y0 when water is withheld (Harris, 1977) and Ocypode quadrata, Cardisoma guanhumi, C. camifx and Gecarcoidea lalandii virtually cease urine production after several hours in air (Flemister, 1958; Gifford, 1962b; Harris & Kormanik, 1981). Unfortunately, these tests for reduced urine production have only been made under conditions of water shortage, and not in the case of ion shortage with ample water supplies. The hypothesis that ghost crabs curtail urine production when ions are limited has a major weakness. It allows no defense against the osmotic flooding that might occur when heavy rainfall saturates burrows or vegetation through which crabs must move. In other words, it predicts that crabs would not volume regulate when salt-depleted, despite the inevitable hemodynamic problems associated with excess fluid within a rigid exoskeleton. It also predicts that inactive crabs, without ion sources in their winter burrows, would produce no urine for up to 6 months. Too little is known about the metabolism of overwintering crabs to determine whether this constitutes a problem. Reducing the salt concentration of fluid wastes is the second basic ion-conservation strategy. Terrestrial crabs have a novel option for handling urine. Whereas the urine of aquatic crabs is immediately carried away by the medium, that of terrestrial crabs could be directed to other sites (e.g. gills, gut) which, unlike the antenna1 gland, are capable of producing osmotic gradients. These organs could produce a dilute “final excretory product” by either reclaiming salts from the urine, or by secreting additional water into it. This hypothetical modified fluid must be distinguished from primary urine; for convenience we will refer to it in this paper as Product, or P. We tested two hypotheses: that urine volume is reduced, and that final excretory product (P) is hypo-osmotic to hemolymph under conditions of water loading coupled with salt shortage.
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THOMASG. WOLCO-MAND DONNAL. WOLCOTT MATERIALSAND METHODS
The overall strategy for examining salt-conservation mechanisms of the ghost crabs was to load experimental crabs with deionized water (DW), circumventing their normal behavioral control of water intake. To control for effects of volume loading without salt depletion, controls were loaded with isosmotic saline. Ten crabs were assigned to each treatment. Since extrarenal modification of urine depends on the crabs being able to transfer the urine to the appropriate sites (organs), crabs were held under conditions which minimized handling. They were allowed to process urine without disturbance, and the fluid they ultimately discarded (P) was collected. P volume was measured to determine if urine production had been curtailed, and its composition was compared with that of hemolymph samples obtained at the beginning and end of the experiments to determine if extrarenal dilution of urine had occurred. Ghost crabs were collected near the southern tip of Cape Lookout, Carteret Co., North Carolina. They were returned to the laboratory and held for several days individually in l-liter plastic containers containing sand dampened with 10% sea water. This period allowed for gut clearance and thereby minimized fecal contamination of P samples. The salinity of the interstitial water was typical of field conditions on the backshore where crabs commonly burrow. Initial hemolymph samples (Z 100 ~1) were obtained with flame-pointed Pasteur pipettes by puncturing the arthrodial membranes at the base of the 3rd or 4th walking leg. Two 7-~1 subsamples were immediately analyzed for total osmotic concentration (Wescor Model 1500 vapor pressure osmometer) and two 2-~1 samples titrated for determination of chloride (Buchler-Cotlove Chloridometer). The remainder of the sample was expelled into a borosilicate capillary. The ends were sealed with a microoxyhydrogen torch (Water Welder) and the sample frozen pending subsequent cation analyses by atomic absorption spectroscopy (Perkin-Elmer Model 306).
Fig. 1. Infusion site was in the perivisceral hemocoel, located between grooves on the carapace the margins of the anterior foregut (medial to hole) and branchial chamber (lateral).
marking
ION CONSERVATION
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CRABS
The crabs were then fitted with cannulae communicating with the perivisceral hemocoel. A small hole, located so that the cannula would not enter the foregut or the branchial chamber, was drilled nearly through the carapace with a dental burr (Fig. 1). To prevent bleeding during emplacement of the cannula, a thin rubber septum was glued over the site with Dexter Hysol “Superdrop II 2X500” cyanoacrilate. A pointed l-cm length of 18-gauge hypodermic tubing was epoxied into the center of a second septum with z 3 mm of the sharp end protruding through the lower face. This assembly was attached by vinyl tubing to an infusion pump (described below). The lumen was filled with infusate and cyanoacrilate adhesive was applied to the lower face of the septum on the cannula. The cannula was quickly punched through the septum glued to the shell and the predrilled thin spot beneath it, and pressure applied to create a leaktight seal between the two layers of rubber. Fluids were infused at a low rate (0.3 ml * h - ‘) by pumps consisting of 20-ml plastic syringes driven by 24 thread/inch screws attached to 1 RPH synchronous motors (Fig. 2). The syringes were initially sterile and were filled with 0.45~pm filtered solutions. The volume-loaded, ion-depleted group was infused with DW. The control group (volume loaded without ion depletion) received “Gecarcinus Ringers” (after Skinner et al., 1965) diluted to the average osmotic concentration of the initial hemolymph samples. During infusion the crabs were held in funnel-bottom plastic containers (Fig. 2). The fluid dropped by the animals (P) ran down into vials, where it was collected under a
‘NFUSATE
SYRINGE
OIL
LEADSCREW I
’
Y
MOTOR t--l
TRAP
Fig. 2. Experimental apparatus: crabs were infused through indwelling cannulae; tubing from infusion pumps was led through chamber tops, allowing crabs free movement; final excretory product released by crabs was collected under oil in vials at bottom.
THOMAS
98
G. WOLCOTT
AND DONNA
L. WOLCOTT
layer of mineral oil to prevent evaporative concentration. Plastic covers on the containers minimized evaporation from crabs and residual fluid films on funnel walls. The infusion lines were led through holes in the top center of the containers and protected from the crab’s claws by short metal sleeves. The crabs were thus free to maneuver within the confines of their containers, restrained only by the infusion lines entering their backs. The array of 20 infusion pumps and containers was checked periodically for damage to cannulae and for appearance of fluid in the collecting vials. P was collected from beneath the oil and its volume measured with a l-ml pipette. Accuracy, allowing for the oil film in the pipette, was = 5 % . Subsamples were analyzed and stored as described above for hemolymph. To avoid overloading the crabs, the cumulative amount of P collected was checked against the amount of fluid delivered by the infusion pump, taking into consideration any leakage of infusate caused by the crab damaging the infusion line (P samples contaminated with infusate were discarded). If the estimated excess fluid in the crab was > 5 y0 of the initial body weight, the pump was shut off until the crab voided more water. To determine net water gain or loss at the conclusion of the 4-day experiment, the crabs were re-weighed, with a correction applied for the carmula. A final hemolymph sample was taken and treated as above. Significance of changes of hemolymph concentration was determined by paired z-tests. The differences discussed below between hemolymph and P, and between P of DW-infused crabs and saline-infused controls are evident without statistical treatment; values did not overlap in most cases. RESULTS NET WATER
FLUX
Essentially all the water pumped into the animals found its way out again by the end of the experiment; neither group showed significant weight change. Cumulative P output did not account for the total infusate volume, due to discarded contaminated samples and, presumably, some evaporation. The latter was not considered a major source of error because the experimental chambers were tightly closed and because evaporation could only reduce the apparent magnitude of any dilution of P effected by the crabs. Production of P slowed or ceased in some crabs alter several days of infusion (hence the lines ending in the middle of Figs. 3-8). This is partly due to reduced infusion rates; we overestimated the existing volume loads because water was lost by other routes and not accounted for in P collection. Infection was probably a contributing factor; incorporation of antibiotics in infusates has ameliorated this problem in subsequent work.
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OSMOTIC CONCENTRATION
Crabs infused with deionized water reduced P osmotic concentration to 20-50x that of the hemolymph (Fig. 3A). The osmotic concentration of their hemolymph declined (P < 0.001) only an average of 13 % during the experiment. Crabs infused with isosmotic saline produced P isosmotic with hemolymph and all individuals maintained hemolymph concen~ation within 6 y0 of the initial value (mean change for the group not si~~c~t) (Fig. 3B).
IOOO---_
m
c
I cn
---
.
----(
2500.
J 1
A
‘
0
DAYS
INFUSED
4
3
2
I
with
O.W. (co
1000. * FI,
-x’
_
f I
. 0
2
I
DAYS
INFUSED
. 3
with
. 4
SALINE
Fig. 3. Osmotic concentration of hemolymph (dashed line connects initial and final mean values; bars are + SE of mean) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B); each line in this and following figures represents an individual crab.
SODIUM
Crabs loaded with deionized water strongly conserved sodium, reducing its concentration in P to lo-20% that of hemolymph (Fig. 4A). Despite the water loading, concentration of Na in hemolymph showed no signifkant change. Crabs loaded with isosmotic saline initially produced P hyponatremic to hemolymph, but concentrations
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THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
of Na in P gradually approached those of the hemolymph during the course of the experiment (Fig. 4B). During the same period the hemolymph sodium concentrations rose in all sue-infuse crabs, by an average of 15% (P < 0.001).
500
1
0
I
2
DAYS
INFUSED
b
; DAYS
2
INFUSED
4
3
with
. with
D.W.
i
d
SALINE
Fig. 4. Sodium concen~ation ofhemol~ph (dashed line) and P (solid lines) ofcrabs infused with deionized water (A) and isosmotic saline (B).
CHLORIDE
Chloride was also strongly conserved by crabs infused with deionized water (Fig. 5A); as with sodium, the concentration of Cl in P was only lo-50% that in hemolymph. Chloride in hemolymph declined in all crabs infused with DW, but only by an average of 10% (P -=z0.001). Unlike sodium, however, Cl was not conserved by saline-infused crabs (Fig. 5B). Chloride concentration of P was slightly higher than that of the hemol~ph initially, and converged towards hemol~ph values during the course of the experiment; hemolymph chloride rose in all saline-infused crabs by an average of 16% (PC 0.001).
ION CONSERVATION
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IN GHOST CRABS
------
T I E 250
0
2
I
DAYS
INFUSED
4
3
with
D.W.
I
0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 5. Chloride concentration ofhemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
CALCIUM
Like other crabs, Ocypode quadrata tends to conserve calcium. The deionized-water loaded crabs discarded P containing as little as 10% of the concentration of Ca in hemolymph (Fig. 6A). Controls also conserved Ca, but only slightly. Calcium content of P was initially slightly less than that of hemolymph, but converged to hemolymph values during the course of the experiment (Fig. 6B). No significant change in hemolymph Ca was seen in the DW-infused group; this is not surprising given the large Ca depot in the exoskeleton. All crabs in the saline-infused group showed an average increase in hemolymph Ca of 7 %, except one that decreased by 4 1%. Due to this outlier the average increase was not significant. POTASSIUM
Data for this ion were variable; most crabs infused with DW showed concentrations of K in P slightly below those of hemolymph during at least part of the experiment, while
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THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
0
2
I
DAYS
INFUSED
4
3
with
D.W.
BY
0 DAYS
Fig. 6. Calcium concentration
2
I
INFUSED
3
with
4
SALINE
of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
all controls had P values above hemolymph values by the end of the experiment (Fig. 7). Concentration of K in hemolymph declined in all crabs infused with DW (average decrease = 21%, P < 0.001) and in most crabs infused with saline (average decrease = 8 %, not significant due to one outlier that appeared to increase by 40%). MAGNESIUM
Measurements of this ion were highly variable. P values for crabs infused with DW were similar to hemolymph values; those for controls were generally above hemolymph values (Fig. 8). Concentration of Mg in hemolymph decreased slightly in both DW- and saline-infused groups (average decreases = 15% (P< 0.01) and 11% (P< 0.02), respectively).
ION CONSERVATION
0
I
DAYS
103
IN GHOST CRABS
2
3
INFUSED
with
4
D.W.
I 0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 7. Potassium concentration of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
DISCUSSION
An acute shutdown in urine production at the expense of volume regulation does not appear to be a major mechanism for minimizing salt loss in hemodiluted ghost crabs. Both hemodiluted and control animals regulated volume, producing P in response to infusion. The gradual reduction in P output was, as noted above, probably artifactual. When chronically exposed to hypo-osmotic conditions in nature, the crabs probably do reduce urine production without compromising volume regulation, by behaviorally limiting water influx. When overwintering, they have access only to dilute interstitial water for several months, with no salt input from other sources. They survive these conditions for weeks in the laboratory (Hall, 1982). It seems likely that inactivity and low metabolic rates at winter temperatures generate few wastes and permit the animals to function largely as closed systems. To determine their responses when volume loaded, we denied them the option of regulating water inthrx in our experiments. Production of markedly hypo-osmotic P is the major mechanism of ion conservation
THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
104
0
2
I
DAYS
4
3
with
INFUSED
D.W.
40. i I
E 4
. 20.
I
, B
*
0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 8. Magnesium concentration of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
in hemodiluted ghost crabs. Despite inability of crabs to produce hypo-osmotic urine, substantial @O-90%) reductions in ion losses are achieved when dilute environmental conditions cause volume loading, hemodilution and (presumably) diuresis. The ratios of the concentrations of each ion in P and hemolymph can be compared, analogous to the urine: blood (U : B) or urine : hemolymph (U : H) ratios commonly given in the literature for the usual method of excretion. It is evident that under dilution stress Cl and Na, which are the principal osmolytes, are strongly conserved (P: hemolymph ratios of z 1: 10; Figs. 4 and 5), and the P accordingly is hypo-osmotic to hemolymph by a similar ratio (Fig. 3). Two explanations may be advanced for the surprising conservation of Na by control crabs, under conditions which were not intended to impose ion depletion (Fig. 4). The first is simply that the crabs were slightly ion-depleted by the holding conditions prior to the experiment and were rebuilding their Na pool by keeping a portion of the infused sodium. Some aspects of the data support this notion. The hemolymph sodium concentration did climb slightly during the experiment, while the sodium concentration
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of P gradually rose until it was approximately isoionic. On the other hand, the holding conditions were well within the normal range of field values, suggesting that ion depletion should have been minor. Alternatively, there is some indication that the sodium pump of ghost crabs is continually “turned on”. The crabs could be strongly hyper-regulating most of the time in nature, despite their hemolymph being hypo-osmotic to sea water (Gifford, 1962a). Their exposure to sea water during feeding is brief; much of their time is spent in contact with water substantially hypo-osmotic to the hemolymph. Typically, terrestrial and semi-terrestrial crabs show high levels of gill Na’ -K + -ATPase which are unresponsive to changes in osmotic environment (Towle, 1984). The enzyme of 0. quadrata has been characterized by C. W. Holliday, working in our laboratory and at Lafayette College, and its activity shows no significant change after ghost crabs are held 1 month on sand dampened with either deionized water or 50% sea water (Holliday, pers. comm.). The slight rise in hemolymph Na in the saline-infused group during the experiment (Fig. 4) could therefore be interpreted as either a regulatory response to pre-existing hyponatremia, or an inescapable consequence of a sodium uptake pump which cannot be shut down completely. The data for total osmotic concentration suggest that the saline-infused crabs were not initially hemodiluted, whether or not they were sodium-depleted. Their P was isosmotic with hemolymph (Fig. 3B). It contained slightly more chloride than hemolymph (P : H ratios of 1.2: 1; Fig. 5B), but this is a consequence of the Ringer’s solution using Cl as the “generic anion” in place of all the undefined negatively charged solutes in hemolymph. The “extra” Cl in the saline infusate (530 mM/l vs. 371 mM/l in hemolymph) was the major difference in ionic composition between the two, and represented a Cl load that had to be excreted. The remaining ions analyzed in this experiment are minor osmolytes, although of great physiological importance. As expected from the existing literature for aquatic crabs, the control ghost crabs tended to conserve Ca (Fig. 6B) and excrete Mg (Fig. 8B). The P concentrations of these ions in DW-infused crabs were offset lower, such that Ca was conserved more obviously and Mg, present in concentrations similar to those of hemolymph, was excreted less rapidly than in controls. Potassium, a major ion in the herbivorous diet, was excreted by saline-infused crabs (P : H ratios of z 2 : 1 by the end of the experiment) and weakly conserved by most DW-infused crabs. Nevertheless, hemolymph K declined in both groups. Possibly K efllux partially offsets the strong ion difference caused by resorption of more Na than Cl. However the production of dilute P is accomplished, it has important implications. In effect, the ghost crabs can produce dilute urine like other terrestrial animals and thereby regulate hemolymph Na, Cl and osmotic concentration. This implies that salt limitation is not as severe a limiting factor as previously assumed, and accounts for the crabs’ ability to exist in habitats so markedly hypo-osmotic to their hemolymph. The ability of ghost crabs to survive in extremely hypo-osmotic environments has not become evident in previous studies (e.g. Flemister, 1958) because the experimental
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THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
conditions (immersion in various salinities) were unrealistic. Ghost crabs almost never have access to standing water other than the sea. Away from the surf their sole source of water is the soil. These conditions permit behavioral regulation of water influx (via both passive osmosis and active “volitional” pathways) and hence of volume of urine produced; and allow manipulation of urine extrarenally before final loss. These adaptations in turn permit very effective ion conservation, allowing the “semiterrestrial” ghost crabs to exist in habitats as hypo-osmotic as those of fresh water crustaceans.
ACKNOWLEDGEMENTS
This study was supported in part by NSF grant no. PCM-8310465. Our thanks are due to Drs. Jeng-sheng Huang and C. Boss for use of their atomic absorption spectrophotometers.
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RUDY, P. P., JR., 1967. Water permeability in selected decapod Crustacea. Comp. Biochem. Physiot., Vol. 22, pp. 581489. SHAW,J., 1964. The control of salt balance in the Crustacea. Symp. Sot. Exp. Biol., Vol. 18, pp. 237-256. SKINNER, D. M., D. J. MARSH & J. S. COOK, 1965. Physioio~cal salt solution for the land crab Gecarc~~as fateralis. &of. Bull. (Woods Hole, Muss.), Vol. 129, pp. 355-365.
SPAARGAREN,D.H., 1975. Notes on the osmotic and ionic regulation of some brachyuran crabs from Curacao. Neth. J. Sea Res., Vol. 9, pp. 273-286. SUBRAMANIAN,A., 1975. Sodium and water permeabilities in selected Crustacea. PhysfoI. Zool., Vol. 48, pp. 398-403. THOMPSON,L.C., 1970. Osmote@ation of the freshwater crabs Metopaulius depressus (Grapsidae) and Pseudothelphusa jouyi (Pseudothelphusidae). Ph.D. thesis, University of California, Berkeley, 101 pp. TOWLE,D. W., 1984. Membrane-bound ATPases in arthropod ion-transporting tissues. Am. Zool., Vol. 24, pp. 177-185. Wo~con, T.G., 1976. Uptake of soil capillary water by ghost crabs. Na~re (random), Vol. 264, pp. 756-757. WOLCOTT,T. G., 1978. Ecological role of ghost crabs, Ocypode quadrata (Fabricius) on an ocean beach: scavengers or predators? J. Exp. Mar. Biol. Ecol., Vol. 3 1, pp. 67-82. WOLCOTT,T. G., 1984. Uptake of soil interstitial water: mechanisms and ecological significance in the ghost crab Ocypode quadrata and two gecarcinid land crabs. Physioi. Zool., Vol. 57, pp. 161-184.
93
Elsevier
JEM 532
EXTRARENAL MODIFICATION OF URINE FOR ION CONSERVATION IN GHOST CRABS, OCYPODE
THOMAS
G. WOLCOTT
QUADRATA
(Fabricius)’
and DONNA L. WOLCOTT
Department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695 U. S.A
(Received 10 December 1984; revision received 15 April 1985; accepted 16 May 1985) Abstract: When placed under ecologically relevant hypo-osmotic conditions, ghost crabs lose salts at very low rates and osmoregulate well. How can they do this when they, like other brachyuran crabs, produce urine isosmotic with hemolymph? We tested two hypotheses to account for low rates of ion loss: that crabs reduce urine flow (abandon volume regulation); and that they osmoregulate without compromising volume regulation by producing a dilute excretory product through extrarenal modification of urine. Experimental crabs were volume-loaded and hemodiluted by infusion with deionized water; controls were volume-loaded without hemodilution by infusion with isosmotic saline. Crabs were held in containers which allowed the crabs to move fairly freely and to handle wastes normally, and allowed final excretory product to be collected without disturbing the crabs. Crabs infused with deionized water did not reduce urine flow acutely. They produced a dilute excretory fluid containing as little as 10% ofthe total osmolytes, Na, and Cl ofhemolymph and urine. The net effect on osmoregulation was as though they, like other terrestrial animals when water-loaded, produced dilute urine. Key words: terrestrial crab; osmoregulation; salt balance; Ocypode quadrata
INTRODUCTION
Salt-conservation mechanisms were examined in ghost crabs, Ocypode quadrata (Fabricius), because they inhabit hypo-osmotic beach and dune habitats where, given the limitations of crab excretory systems, they should experience rapid salt loss. Salt-depletion has seldom been considered as an environmental threat to crabs invading terrestrial habitats, while water shortage has often been regarded as their principal physiological problem (e.g. Spaargaren, 1975) and has received considerable attention (see e.g. reviews by Bliss, 1968; Mantel, 1979). Much of the available information supports this placement of emphasis. Crabs are relatively permeable animals in environments apparently offering little free water. Evaporation rates correlate well with terrestriality (Bliss, 1968; Herreid, 1969; MacMillen & Greenaway, 1978), as do desiccation tolerances (Bliss, 1968). All of these previous studies stress the importance of water loss, but inputs must also be considered in the water and salt budgets of crabs. Salt shortage could become a ’ Contribution No. 85008 from the MEAS Department, North Carolina State University 0022-0981/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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THOMAS
G. WOLCOTT
AND DONNA
L. WOLCOTT
problem if the crabs had access to abundant but hypo-osmotic water, limited supplies of salts from other sources, and experienced high rates of salt loss due to production of isosmotic urine by the excretory organs. These are the conditions under which ghost crabs appear to exist on the backshore and among the dunes of North Carolina’s Outer Banks. Water available to ghost crabs, with the exception of the sea itself, typically is very dilute. During the summer ghost crabs may spend several days at a time in the interior of the barrier islands (T. Wolcott, unpubl. pers. obs. and radio-tracking data), and, during 5-6 winter months, they do not emerge from their burrows. At these times they have no access to sea water. Instead, they extract interstitial water from sandy burrow walls by a mechanism involving capillary tufts of setae and suction by the gill chambers (Wolcott, 1976, 1984). This system provides water ad libitum wherever the crabs can burrow to within =20 cm of the water table in sandy soil (Wolcott, 1984). However, the ~terstiti~ water resembles sea water only in the foreshore, within a few meters of the surf. Further inland, a layer of fresh water overlies the denser sea water (the “freshwater lens”). It lowers interstitial water concentrations to below 2%, at the foredune on North Carolina beaches and to <0.5%, in ghost crab habitat further from the beach (Hall, 1982; T. Wolcott, unpubl. data). Salt inputs from food are also limited at the times when water sources are hypoosmotic. The primary foods of ghost crabs in North Carolina are osmoconforming invertebrates (Wolcott, 1978) which are as salty as sea water, but these are unavailable inland or underground. Crabs living in the interior of the barrier islands subsist on grasses, seeds and occasional insects (Wolcott, unpubl. stomach analyses), and those overwintering in the burrows do not feed at all. The crabs cannot compensate for the low concentration of ions available in water and food by producing a dilute urine. From a voluminous literature, it is clear that brachyuran antenna1 glands, although capable of modifying urine by selective resorption of ions and part of the water, are unable to produce a final urine differing greatly from the hemolymph in total osmotic concentration (for reviews see Robertson, 1960; Lockwood, 1962; Shaw, 1964; Riegel & Cook, 1975; Mantel, 1979; Mantel & Farmer, 1983). Producing a given volume of isosmotic urine to rid the animal of excess water (or soluble wastes) presumably causes loss of the amount of salts contained in a similar volume of hemolymph. Ocypode quadnm, like other brachyuran crabs, produces isosmotic urine (Gilford, 1962a; Gross, 1964). Nevertheless, ghost crabs are excellent hyperosmotic regulators under ecologically reasonable hypo-osmotic conditions, despite their inability to survive extreme dilutions in traditions immersion experiments (Flemister & Flemister. 1951; Gifford, 1962a; Gross, 1964). When held ~thout food on sand dampened with salinities ranging from full-strength sea water (36x,) to < 1% sea water they survive and regulate hemolymph osmotic and ionic concentrations for 2-6 wk at IO-25 “C (Hall, 1982). How are the crabs able to maintain such low salt iosses in the face of large osmotic gradients? The problems of ion retention are different in terrestrial and aquatic habitats.
ION CONSERVATION
IN GHOST
CRABS
95
In air, salt loss through the integument is negligible. Fecal salt losses will be low, even if the fecal fluids are isosmotic with the hemolymph, because the fecal pellets of terrestrial crabs typically contain little water. Furthermore, when feces are being produced, some salts are also presumably being taken in with food. Urine is the principal remaining pathway for salt loss. Therefore we hypothesized that the crabs minimize salt loss by reducing the volume, concentration, or both, of fluid wastes. Reducing production of isosmotic urine would be the simplest mechanism that could account for low ion loss rates from ghost crabs in dilute environments. Consistent with such a mechanism are several pieces of information which suggest that terrestrial crabs minimize the need for urine production by reducing water intake. Integumental permeabilities are low (Herreid, 1969; Greenaway, 1980) in some cases even lower than those of freshwater species (e.g. Rudy, 1967; Thompson, 1970; Subramanian, 1975). This presumably reduces osmotic influx of water. Avoiding contact with hypo-osmotic free water, an option on land, would further minimize excessive influx of water. Terrestrial crabs do decrease urine flow when dehydrated. Gecarcinus lateralis reduces flow by 75 y0 when water is withheld (Harris, 1977) and Ocypode quadrata, Cardisoma guanhumi, C. camifx and Gecarcoidea lalandii virtually cease urine production after several hours in air (Flemister, 1958; Gifford, 1962b; Harris & Kormanik, 1981). Unfortunately, these tests for reduced urine production have only been made under conditions of water shortage, and not in the case of ion shortage with ample water supplies. The hypothesis that ghost crabs curtail urine production when ions are limited has a major weakness. It allows no defense against the osmotic flooding that might occur when heavy rainfall saturates burrows or vegetation through which crabs must move. In other words, it predicts that crabs would not volume regulate when salt-depleted, despite the inevitable hemodynamic problems associated with excess fluid within a rigid exoskeleton. It also predicts that inactive crabs, without ion sources in their winter burrows, would produce no urine for up to 6 months. Too little is known about the metabolism of overwintering crabs to determine whether this constitutes a problem. Reducing the salt concentration of fluid wastes is the second basic ion-conservation strategy. Terrestrial crabs have a novel option for handling urine. Whereas the urine of aquatic crabs is immediately carried away by the medium, that of terrestrial crabs could be directed to other sites (e.g. gills, gut) which, unlike the antenna1 gland, are capable of producing osmotic gradients. These organs could produce a dilute “final excretory product” by either reclaiming salts from the urine, or by secreting additional water into it. This hypothetical modified fluid must be distinguished from primary urine; for convenience we will refer to it in this paper as Product, or P. We tested two hypotheses: that urine volume is reduced, and that final excretory product (P) is hypo-osmotic to hemolymph under conditions of water loading coupled with salt shortage.
96
THOMASG. WOLCO-MAND DONNAL. WOLCOTT MATERIALSAND METHODS
The overall strategy for examining salt-conservation mechanisms of the ghost crabs was to load experimental crabs with deionized water (DW), circumventing their normal behavioral control of water intake. To control for effects of volume loading without salt depletion, controls were loaded with isosmotic saline. Ten crabs were assigned to each treatment. Since extrarenal modification of urine depends on the crabs being able to transfer the urine to the appropriate sites (organs), crabs were held under conditions which minimized handling. They were allowed to process urine without disturbance, and the fluid they ultimately discarded (P) was collected. P volume was measured to determine if urine production had been curtailed, and its composition was compared with that of hemolymph samples obtained at the beginning and end of the experiments to determine if extrarenal dilution of urine had occurred. Ghost crabs were collected near the southern tip of Cape Lookout, Carteret Co., North Carolina. They were returned to the laboratory and held for several days individually in l-liter plastic containers containing sand dampened with 10% sea water. This period allowed for gut clearance and thereby minimized fecal contamination of P samples. The salinity of the interstitial water was typical of field conditions on the backshore where crabs commonly burrow. Initial hemolymph samples (Z 100 ~1) were obtained with flame-pointed Pasteur pipettes by puncturing the arthrodial membranes at the base of the 3rd or 4th walking leg. Two 7-~1 subsamples were immediately analyzed for total osmotic concentration (Wescor Model 1500 vapor pressure osmometer) and two 2-~1 samples titrated for determination of chloride (Buchler-Cotlove Chloridometer). The remainder of the sample was expelled into a borosilicate capillary. The ends were sealed with a microoxyhydrogen torch (Water Welder) and the sample frozen pending subsequent cation analyses by atomic absorption spectroscopy (Perkin-Elmer Model 306).
Fig. 1. Infusion site was in the perivisceral hemocoel, located between grooves on the carapace the margins of the anterior foregut (medial to hole) and branchial chamber (lateral).
marking
ION CONSERVATION
IN GHOST
91
CRABS
The crabs were then fitted with cannulae communicating with the perivisceral hemocoel. A small hole, located so that the cannula would not enter the foregut or the branchial chamber, was drilled nearly through the carapace with a dental burr (Fig. 1). To prevent bleeding during emplacement of the cannula, a thin rubber septum was glued over the site with Dexter Hysol “Superdrop II 2X500” cyanoacrilate. A pointed l-cm length of 18-gauge hypodermic tubing was epoxied into the center of a second septum with z 3 mm of the sharp end protruding through the lower face. This assembly was attached by vinyl tubing to an infusion pump (described below). The lumen was filled with infusate and cyanoacrilate adhesive was applied to the lower face of the septum on the cannula. The cannula was quickly punched through the septum glued to the shell and the predrilled thin spot beneath it, and pressure applied to create a leaktight seal between the two layers of rubber. Fluids were infused at a low rate (0.3 ml * h - ‘) by pumps consisting of 20-ml plastic syringes driven by 24 thread/inch screws attached to 1 RPH synchronous motors (Fig. 2). The syringes were initially sterile and were filled with 0.45~pm filtered solutions. The volume-loaded, ion-depleted group was infused with DW. The control group (volume loaded without ion depletion) received “Gecarcinus Ringers” (after Skinner et al., 1965) diluted to the average osmotic concentration of the initial hemolymph samples. During infusion the crabs were held in funnel-bottom plastic containers (Fig. 2). The fluid dropped by the animals (P) ran down into vials, where it was collected under a
‘NFUSATE
SYRINGE
OIL
LEADSCREW I
’
Y
MOTOR t--l
TRAP
Fig. 2. Experimental apparatus: crabs were infused through indwelling cannulae; tubing from infusion pumps was led through chamber tops, allowing crabs free movement; final excretory product released by crabs was collected under oil in vials at bottom.
THOMAS
98
G. WOLCOTT
AND DONNA
L. WOLCOTT
layer of mineral oil to prevent evaporative concentration. Plastic covers on the containers minimized evaporation from crabs and residual fluid films on funnel walls. The infusion lines were led through holes in the top center of the containers and protected from the crab’s claws by short metal sleeves. The crabs were thus free to maneuver within the confines of their containers, restrained only by the infusion lines entering their backs. The array of 20 infusion pumps and containers was checked periodically for damage to cannulae and for appearance of fluid in the collecting vials. P was collected from beneath the oil and its volume measured with a l-ml pipette. Accuracy, allowing for the oil film in the pipette, was = 5 % . Subsamples were analyzed and stored as described above for hemolymph. To avoid overloading the crabs, the cumulative amount of P collected was checked against the amount of fluid delivered by the infusion pump, taking into consideration any leakage of infusate caused by the crab damaging the infusion line (P samples contaminated with infusate were discarded). If the estimated excess fluid in the crab was > 5 y0 of the initial body weight, the pump was shut off until the crab voided more water. To determine net water gain or loss at the conclusion of the 4-day experiment, the crabs were re-weighed, with a correction applied for the carmula. A final hemolymph sample was taken and treated as above. Significance of changes of hemolymph concentration was determined by paired z-tests. The differences discussed below between hemolymph and P, and between P of DW-infused crabs and saline-infused controls are evident without statistical treatment; values did not overlap in most cases. RESULTS NET WATER
FLUX
Essentially all the water pumped into the animals found its way out again by the end of the experiment; neither group showed significant weight change. Cumulative P output did not account for the total infusate volume, due to discarded contaminated samples and, presumably, some evaporation. The latter was not considered a major source of error because the experimental chambers were tightly closed and because evaporation could only reduce the apparent magnitude of any dilution of P effected by the crabs. Production of P slowed or ceased in some crabs alter several days of infusion (hence the lines ending in the middle of Figs. 3-8). This is partly due to reduced infusion rates; we overestimated the existing volume loads because water was lost by other routes and not accounted for in P collection. Infection was probably a contributing factor; incorporation of antibiotics in infusates has ameliorated this problem in subsequent work.
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OSMOTIC CONCENTRATION
Crabs infused with deionized water reduced P osmotic concentration to 20-50x that of the hemolymph (Fig. 3A). The osmotic concentration of their hemolymph declined (P < 0.001) only an average of 13 % during the experiment. Crabs infused with isosmotic saline produced P isosmotic with hemolymph and all individuals maintained hemolymph concen~ation within 6 y0 of the initial value (mean change for the group not si~~c~t) (Fig. 3B).
IOOO---_
m
c
I cn
---
.
----(
2500.
J 1
A
‘
0
DAYS
INFUSED
4
3
2
I
with
O.W. (co
1000. * FI,
-x’
_
f I
. 0
2
I
DAYS
INFUSED
. 3
with
. 4
SALINE
Fig. 3. Osmotic concentration of hemolymph (dashed line connects initial and final mean values; bars are + SE of mean) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B); each line in this and following figures represents an individual crab.
SODIUM
Crabs loaded with deionized water strongly conserved sodium, reducing its concentration in P to lo-20% that of hemolymph (Fig. 4A). Despite the water loading, concentration of Na in hemolymph showed no signifkant change. Crabs loaded with isosmotic saline initially produced P hyponatremic to hemolymph, but concentrations
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THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
of Na in P gradually approached those of the hemolymph during the course of the experiment (Fig. 4B). During the same period the hemolymph sodium concentrations rose in all sue-infuse crabs, by an average of 15% (P < 0.001).
500
1
0
I
2
DAYS
INFUSED
b
; DAYS
2
INFUSED
4
3
with
. with
D.W.
i
d
SALINE
Fig. 4. Sodium concen~ation ofhemol~ph (dashed line) and P (solid lines) ofcrabs infused with deionized water (A) and isosmotic saline (B).
CHLORIDE
Chloride was also strongly conserved by crabs infused with deionized water (Fig. 5A); as with sodium, the concentration of Cl in P was only lo-50% that in hemolymph. Chloride in hemolymph declined in all crabs infused with DW, but only by an average of 10% (P -=z0.001). Unlike sodium, however, Cl was not conserved by saline-infused crabs (Fig. 5B). Chloride concentration of P was slightly higher than that of the hemol~ph initially, and converged towards hemol~ph values during the course of the experiment; hemolymph chloride rose in all saline-infused crabs by an average of 16% (PC 0.001).
ION CONSERVATION
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101
IN GHOST CRABS
------
T I E 250
0
2
I
DAYS
INFUSED
4
3
with
D.W.
I
0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 5. Chloride concentration ofhemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
CALCIUM
Like other crabs, Ocypode quadrata tends to conserve calcium. The deionized-water loaded crabs discarded P containing as little as 10% of the concentration of Ca in hemolymph (Fig. 6A). Controls also conserved Ca, but only slightly. Calcium content of P was initially slightly less than that of hemolymph, but converged to hemolymph values during the course of the experiment (Fig. 6B). No significant change in hemolymph Ca was seen in the DW-infused group; this is not surprising given the large Ca depot in the exoskeleton. All crabs in the saline-infused group showed an average increase in hemolymph Ca of 7 %, except one that decreased by 4 1%. Due to this outlier the average increase was not significant. POTASSIUM
Data for this ion were variable; most crabs infused with DW showed concentrations of K in P slightly below those of hemolymph during at least part of the experiment, while
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THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
0
2
I
DAYS
INFUSED
4
3
with
D.W.
BY
0 DAYS
Fig. 6. Calcium concentration
2
I
INFUSED
3
with
4
SALINE
of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
all controls had P values above hemolymph values by the end of the experiment (Fig. 7). Concentration of K in hemolymph declined in all crabs infused with DW (average decrease = 21%, P < 0.001) and in most crabs infused with saline (average decrease = 8 %, not significant due to one outlier that appeared to increase by 40%). MAGNESIUM
Measurements of this ion were highly variable. P values for crabs infused with DW were similar to hemolymph values; those for controls were generally above hemolymph values (Fig. 8). Concentration of Mg in hemolymph decreased slightly in both DW- and saline-infused groups (average decreases = 15% (P< 0.01) and 11% (P< 0.02), respectively).
ION CONSERVATION
0
I
DAYS
103
IN GHOST CRABS
2
3
INFUSED
with
4
D.W.
I 0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 7. Potassium concentration of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
DISCUSSION
An acute shutdown in urine production at the expense of volume regulation does not appear to be a major mechanism for minimizing salt loss in hemodiluted ghost crabs. Both hemodiluted and control animals regulated volume, producing P in response to infusion. The gradual reduction in P output was, as noted above, probably artifactual. When chronically exposed to hypo-osmotic conditions in nature, the crabs probably do reduce urine production without compromising volume regulation, by behaviorally limiting water influx. When overwintering, they have access only to dilute interstitial water for several months, with no salt input from other sources. They survive these conditions for weeks in the laboratory (Hall, 1982). It seems likely that inactivity and low metabolic rates at winter temperatures generate few wastes and permit the animals to function largely as closed systems. To determine their responses when volume loaded, we denied them the option of regulating water inthrx in our experiments. Production of markedly hypo-osmotic P is the major mechanism of ion conservation
THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
104
0
2
I
DAYS
4
3
with
INFUSED
D.W.
40. i I
E 4
. 20.
I
, B
*
0
2
I
DAYS
INFUSED
3
with
4
SALINE
Fig. 8. Magnesium concentration of hemolymph (dashed line) and P (solid lines) of crabs infused with deionized water (A) and isosmotic saline (B).
in hemodiluted ghost crabs. Despite inability of crabs to produce hypo-osmotic urine, substantial @O-90%) reductions in ion losses are achieved when dilute environmental conditions cause volume loading, hemodilution and (presumably) diuresis. The ratios of the concentrations of each ion in P and hemolymph can be compared, analogous to the urine: blood (U : B) or urine : hemolymph (U : H) ratios commonly given in the literature for the usual method of excretion. It is evident that under dilution stress Cl and Na, which are the principal osmolytes, are strongly conserved (P: hemolymph ratios of z 1: 10; Figs. 4 and 5), and the P accordingly is hypo-osmotic to hemolymph by a similar ratio (Fig. 3). Two explanations may be advanced for the surprising conservation of Na by control crabs, under conditions which were not intended to impose ion depletion (Fig. 4). The first is simply that the crabs were slightly ion-depleted by the holding conditions prior to the experiment and were rebuilding their Na pool by keeping a portion of the infused sodium. Some aspects of the data support this notion. The hemolymph sodium concentration did climb slightly during the experiment, while the sodium concentration
ION CONSERVATION
IN GHOST CRABS
105
of P gradually rose until it was approximately isoionic. On the other hand, the holding conditions were well within the normal range of field values, suggesting that ion depletion should have been minor. Alternatively, there is some indication that the sodium pump of ghost crabs is continually “turned on”. The crabs could be strongly hyper-regulating most of the time in nature, despite their hemolymph being hypo-osmotic to sea water (Gifford, 1962a). Their exposure to sea water during feeding is brief; much of their time is spent in contact with water substantially hypo-osmotic to the hemolymph. Typically, terrestrial and semi-terrestrial crabs show high levels of gill Na’ -K + -ATPase which are unresponsive to changes in osmotic environment (Towle, 1984). The enzyme of 0. quadrata has been characterized by C. W. Holliday, working in our laboratory and at Lafayette College, and its activity shows no significant change after ghost crabs are held 1 month on sand dampened with either deionized water or 50% sea water (Holliday, pers. comm.). The slight rise in hemolymph Na in the saline-infused group during the experiment (Fig. 4) could therefore be interpreted as either a regulatory response to pre-existing hyponatremia, or an inescapable consequence of a sodium uptake pump which cannot be shut down completely. The data for total osmotic concentration suggest that the saline-infused crabs were not initially hemodiluted, whether or not they were sodium-depleted. Their P was isosmotic with hemolymph (Fig. 3B). It contained slightly more chloride than hemolymph (P : H ratios of 1.2: 1; Fig. 5B), but this is a consequence of the Ringer’s solution using Cl as the “generic anion” in place of all the undefined negatively charged solutes in hemolymph. The “extra” Cl in the saline infusate (530 mM/l vs. 371 mM/l in hemolymph) was the major difference in ionic composition between the two, and represented a Cl load that had to be excreted. The remaining ions analyzed in this experiment are minor osmolytes, although of great physiological importance. As expected from the existing literature for aquatic crabs, the control ghost crabs tended to conserve Ca (Fig. 6B) and excrete Mg (Fig. 8B). The P concentrations of these ions in DW-infused crabs were offset lower, such that Ca was conserved more obviously and Mg, present in concentrations similar to those of hemolymph, was excreted less rapidly than in controls. Potassium, a major ion in the herbivorous diet, was excreted by saline-infused crabs (P : H ratios of z 2 : 1 by the end of the experiment) and weakly conserved by most DW-infused crabs. Nevertheless, hemolymph K declined in both groups. Possibly K efllux partially offsets the strong ion difference caused by resorption of more Na than Cl. However the production of dilute P is accomplished, it has important implications. In effect, the ghost crabs can produce dilute urine like other terrestrial animals and thereby regulate hemolymph Na, Cl and osmotic concentration. This implies that salt limitation is not as severe a limiting factor as previously assumed, and accounts for the crabs’ ability to exist in habitats so markedly hypo-osmotic to their hemolymph. The ability of ghost crabs to survive in extremely hypo-osmotic environments has not become evident in previous studies (e.g. Flemister, 1958) because the experimental
106
THOMAS G. WOLCOTT AND DONNA L. WOLCOTT
conditions (immersion in various salinities) were unrealistic. Ghost crabs almost never have access to standing water other than the sea. Away from the surf their sole source of water is the soil. These conditions permit behavioral regulation of water influx (via both passive osmosis and active “volitional” pathways) and hence of volume of urine produced; and allow manipulation of urine extrarenally before final loss. These adaptations in turn permit very effective ion conservation, allowing the “semiterrestrial” ghost crabs to exist in habitats as hypo-osmotic as those of fresh water crustaceans.
ACKNOWLEDGEMENTS
This study was supported in part by NSF grant no. PCM-8310465. Our thanks are due to Drs. Jeng-sheng Huang and C. Boss for use of their atomic absorption spectrophotometers.
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