J. exp. Biol. (1977), 6SU 57-67tyithT figure*Printed in Great Britain

URINE PRODUCTION RATE ANDWATER BALANCE IN THE TERRESTRIAL CRABS

GECARCINUS LATERALIS AND CARDISOMAGUANHUMI

BY R. R. HARRIS

Department of Zoology, School of Biological Sciences,University of Leicester, Leicester LEi jRH

(Received 21 October 1976)

SUMMARY

1. The rate of urine production in the terrestrial crabs Gecarcinus lateraUs(Freminville) and Cardisoma guanhitmi Latreille maintained on s.w.-moistsand was measured by inulin clearance.

2. The inulin U/B ratios in both species rose to about unity after 48 h, andremained at this level. It was concluded that neither species withdrew waterfrom the primary urine.

3. The relatively high rate of inulin clearance in G. lateraUs (10*69 % bodywt day"1) was considerably reduced when animals were transferred to drysand (2-15 % body wt day-1).

4. The rate of water loss on dry sand was measured in G. lateraUs andchanges in the haemolymph and urine osmolality recorded.

5. The prevention of possible reabsorption of water from voided urine didnot appear to affect significantly the rate of water loss in conditions of waterdeprivation.

6. The maintenance of urine production rates measured on s.w.-moistsand would double the water loss of G. lateraUs in dry conditions.

INTRODUCTION

Land crabs of the family Gecarcinidae occur widely in the tropical and subtropicalAmericas and also in the Indo-Pacific region. The genus Gecarcinus is considered tobe the most terrestrial of this family and is found in dry burrows, sometimes far inland(Bliss, 1963, 1968). Cardisoma generally lives in low-lying swampy areas and burrowsdown to ground water (Gifford, 1962; Feliciano, 1962; Herreid & Gifford, 1965).

Bliss (1968) has discussed some aspects of salt and water balance in her review ofthe adaptations of terrestrial decapods. In adult land crabs, water uptake from moistsubstrates is thought to occur via the abdominal setae to pericardial sacs, branchialchamber and thence to the haemolymph. Water uptake has been shown to be underhormonal control (Bliss, Wang & Martinez, 1966). Mantel (1968) has shown that the gutof Gecarcinus is permeable to water and salts, which may be taken up from food.Behavioural adaptations are important to ensure that water loss is kept to a minimum.

Because the haemolymph and urine of land crabs are isosmotic, it has been suggested

58 R. R. HARRIS

that the antennal gland plays no significant role in the conservation of water (Gross,1963; Bliss, 1968). This suggestion is based on little independently derived evidencesuch as that which might be provided by studies of the excretion of filtration-markersubstances. For example, it has been shown in Potamon edulis, a semi-terrestrialfreshwater crab, that U/B ratios of the marker substance inulin are greater than 1,even though haemolymph and urine are isosmotic. It is suggested that reabsorption ofwater with some sodium and chloride uptake could account for this (Harris & Micallef,1971; Harris, 1975). Flemister (1958) found that injected inulin was cleared from thehaemolymph of Gecarcinus lateraUs kept on sand, indicating that urine was formedduring exposure to dry conditions. Flemister was unable to collect urine from thenephropore of bis crabs and he assumed that the inulin was retained within theantennal gland. This suggested that complete reabsorption of the primary urineoccurred in these conditions. However, in the semi-terrestrial crab Uca mordax,urine inulin concentrations did not exceed those of the haemolymph even underconditions where water reabsorption from the urine would be expected (Schmidt-Nielsen, Gertz & Davis, 1968).

In the present study, the rate of clearance of inulin and inulin U/B ratios of Cardi-somaguanhumi and Gecarcinus lateraUs were measured. This was in order to determinethe rate of urine production under different experimental conditions and to assessthe importance of urinary reabsorption in the water economy of the land crabs.

MATERIALS AND METHODS

Adult G. lateraUs and C. guanhttmi were collected by hand from burrows nearBellairs Research Institute, Barbados (Chace & Hobbs, 1969). They were kept in thelaboratory at 22 °C on sand moistened with sea water into which they burrowed.They were fed on coconut flesh.

Rates of inulin clearance were determined using (hydroxy-f^CJmethyl) inulin(Radiochemical Centre, Amersham). Gecarcinus Ringer solution, 01 ml (Skinner,Marsh & Cook, 1965), containing o-8 /*Ci of radioisotope was injected into eachweighed animal by Agla microsyringe. The injection site was a predrilled hole, coveredby a rubber membrane to prevent leakage, in the dorsal carapace above the heart.One and a half hours after injection, 0-5 ml of haemolymph was removed from eachanimal and mixed with 0-5 ml of distilled water on a planchette. The sampleswere air-dried and counted, using a Milliard MX 168 G-M counter and Panaxsealer. Samples of urine were prepared similarly for counting, o-i ml of urinewas collected directly from the nephropore by temporarily displacing the 3rdmaxilliped, which almost completely covered the opening. Standards of knowndilutions, with distilled water, of the original injection medium and known volumesof inactive haemolymph and urine were used to prepare quench curves. Samplecounts were corrected to 100% efficiency using these curves. The degree ofinitial dilution after injection, and therefore the inulin space, was calculated, andknowing individual space values, the rate of clearance, C (in ml day"1), was determinedfrom the slope of a semi-logarithmic plot of haemolymph \}lC\ activity against time.The urine production rate, V (ml day-1), was calculated from the relationship C =U/B. V, where U/B is the final ratio of inulin activity of urine haemolymph. V was,also expressed as % body weight day1. All animals were weighed on a Mettlet

Urine production and water balance in crabs 59

balance accurate to o*i g. A standard, timed weighing procedure was used whichincluded the removal of sand grains adhering to mouthparts, chelae and paraeopods.Regression lines were fitted to inulin clearance data by the least squares methodusing a Wang 720 C programmable calculator. The statistical significance of thedifference between means was calculated using Student's t test.

To investigate the possibility that water was reabsorbed from the urine of G. later altsto an extent significant in the water economy of the animal, released urine was divertedlaterally by means of rubber strips bonded to the carapace by Eastman 610 adhesive(Fig. 46). The effectiveness of the seal between carapace and rubber strip was tested.Animals were injected with Gecarcinus Ringer containing indigo-carmine which iscleared by the antennal gland and colours the urine. Observations showed that theanimals released urine as discrete droplets which were retained on the rubber stripsand were dispersed laterally from the region surrounding the branchial exhalant open-ing. On s.w.-moist sand, animals operated on in this way maintained a constant bodyweight. Groups of control and experimental animals with strips in position wereinjected with [1*C]inuUn and transferred to dry sand (R.H. 55%; T= 22 °C). Haemo-lymph and urine ["CJinulin activities and osmolalities were measured. The weightof each animal was recorded initially and subsequent weighings made and correctedfor weight loss due to haemolymph removal. Osmolality was determined by cryoscopyusing an Advanced Osmometer 3W. 0-25 ml samples of haemolymph and urine,diluted 1 :1 with distilled water, were prepared. Standards were treated similarly.

RESULTSThe rate of urine production

Semi-logarithmic plots of ["Cjinulin activity against time in G. lateralis and C.guanhumi are shown in Fig. 1. Rates of inulin clearance expressed as ml day-1 and %body weight day"1 are shown in Table 1. From these values and mean final U/B ratiosthe rates of urine production have been calculated (Table 1).

The mean rate of urine production in G. lateralis on s.w.-moist sand was 10-47± 1-53 (S.E.M.) % body weight day"1. The rate of C. guanhumi was i'89±o>34%day"1 in animals kept in similar conditions.

In dry conditions G. lateralis showed a marked decrease in the rate of inulin clear-ance (Fig. 1). The animals were maintained on dry sand in glass containers (R.H.55%; T = 22 °C) and allowed no access to water. During the experiments theanimals lost weight. The mean clearance was 2-15 ±o-68% body weight day-1. Therate of urine production was not calculated since a final inulin U/B ratio could notbe determined before the animal died.

There appeared to be a reduction in the rate of urine filtration in G. lateralis main-tained in dry conditions. Rates of inulin clearance in C. guanhumi on dry sand werenot measured.

The U/B ratio of inulin

The U/B ratio of injected inulin increased in G. lateralis to a mean value of 0-98± 0-02 (S.E.M.) after 48 h and was maintained at this level during the course of theexperiment (166 h) (Fig. 2). The relationship between P*C]inulin activity in the

6o R. R. HARRIS

Cardisoma guanhumi

Gecarcimu lateralis

(dry sand)

40 60Time (h)

80 100

Fig. i. The decrease in ["C]inulin activity of the haemolymph of Gecardmts lateralis andCarcUsoma guanhumi. Co is the initial activity of the haemolymph and C, is the activity of thehaemolymph after t hours.

Table i . Rates of imdin clearance, inulin U/B ratios and rates of urine productionof G. lateralis and C. guanhumi

(n = no. of animals. The mean and standard error of the mean are given.)

Inulin clearance rateFinal

Urineproduction rate

G. lateralis (s.w.-moist sand)

ml day"1 % body wt day"1 U/B ratio (% body wt'day1)

6-85 10-69 0-98 10-4711-53

G. lateralis (dry sand) 1-45G. lateralis (dry-sand-experimental 1-78

animals)*C. guanhumi (s.w.-moist sand) 8-36

a-i5±o-68a-5° ±<>77

093

( )(n = 4)

1-89 ±034

• See text.

haemolymph and the urine (Fig. 3) shows that there was no significant differencebetween them after 48 h.

Inulin U/B ratios close to unity were also recorded in C. guanhumi on s.w.-moistsand (0-93 ± 0*07) and the time course of the changes in U/B ratio were similar.In G. lateralis on dry sand it became difficult to obtain urine samples large enough todetermine P*C] activity after 24 h (Fig. 2).

In marine decapods, inulin U/B ratios generally exceed 1 (Riegel, 1972), suggestingan ability to withdraw water from the primary urine. In these land crabs there was noappreciable withdrawal of water and even a slight dilution of the primary urine seemsto have taken place, particularly in C. guanhumi. However, it is more likely thatiexperimental error would account for the ratios deviating slightly from 1.

Urine production and water balance in crabs 61

Time(h)

Fig. a. Timlin U/B ratios after injection in G. lateralis and C. guanhwni. • , Ratios on s.w.-moistsand and, d ratios on dry sand in G. lateralis; • , ratios on s.w.-moist sand in C. guanhum.

10

a

i o-5

1Is

20 40 60 80

Time(h)100 120 140 160

Fig. 3. The relationship between haemolymph and urine ["C]inulin activities in G. lateralis.• , Activity of the haemolymph; O, activity of the urine.

The reabsorption of voided urine

In G. lateraUs the nephropore is aknost completely covered by the 3rd maxilliped.Beneath the nephropore lies a pad of setae bordering the groove running ventro-laterally to the margin of the 3rd maxilliped (Fig. 4a). Carson (1974) has suggestedthat in Gecarcinus ruricola the copious growth of micro-organisms found in theinterstices of the setal pad may be important in removing nitrogenous compounds from

3 EZB 68

62 R. R. HARRIS

1 ant.

e.b.o.

2 m.

2 ant.

Cut base 3 m.

\m.

1 ant.

3 m.

md.2m.

2 ant.

Cut base 3 m.

Fig. 4(0) Diagram illustrating the position of the nephropore of G. later alts. The right 3rd max-illiped (3m) has been removed, int., First maxilliped; 2m., second maxilliped; md., mandible;e.b.o., exhalant branchial opening; n., nephropore; 3 ant., antenna; 1 ant., antennule. s.p., setalpad. (6) Diagram illustrating the position of rubber strips to divert urine released from thenephropore of G. lateraHs Labelling as (a), rt., rubber strip.

Urine production and water balance in crabs 63

100

80

70

s.w. -moist (control)

Dry sand(control)

Dry sand (experimental)

20 40 60 80 100Time (h)

120 140

Fig. s. Changes in the body weight of G. lateralis after transfer to dry sand. Animals with rubberstrips in position are referred to as experimental. The relativery constant body weight of animalson s.w.-moist sand is also shown.

Table 2. The rates of water loss (T) from G. lateralis after transfer to dry sandexpressed as a percentage of body weight per day

(T = A^IOO/R7)*, where x is the rate of water loss in g per animal day"1 and W is the weight of theanimal. The mean ± S.K.M. are given, n = no. of animals.)

ControlExperimental

Mean body wet weight(g)

66-485-S

(% body wt* day1)

s-78±o-36(n = 6)4'97±o-37(n = 6)

the urine before fluid is reabsorbed into the haemocoel at the base of the carapacegroove that supports the pad. In G. lateralis the arrangement of the nephropore andsetal pad is similar. The released urine was diverted to investigate reabsorption ofurine (Methods and Fig. 46). The rates of ["CJinulin clearance of control and experi-mental animals were not significantly different (P>o-io) (Table 1).

It had been found in preliminary experiments that diversion of urine in animalskept on s.w.-moist sand did not affect the animals' ability to maintain water balance.The possibility that reabsorption or urinary water might be important in drierconditions was investigated.

On dry sand G. lateralis showed a decrease in body weight (Fig. 5). This wasassumed to be solely due to water loss and to be linear in the first 50 h. The rate ofthe loss was expressed as a percentage of the initial body weight per day corrected fordifferences in surface area for different animal weights by the 'two-thirds rule'

S-a

64

140

130

i t 1 2 0

1* 1,0

100

90

R. R. HARRIS

Dry sand (control)

Dry sand (experimental)

S.W.-moist (control)

100Time(h)

200

Fig. 6. The changes in osmolality of the haemorymph of G. lateralit after transfer on to drysand. Animals with rubber strips positioned beneath the nephropore are referred to as experi-mental. The mean and standard error of the mean are indicated.

1-5

10

0-5

100 200

Time(h)

Fig. 7. The relationship between haemorymph and urine osmolality (expressed as a ratio U/B)of G. lateralis after transfer onto dry sand compared with animals mnintaini-H on s.w.-moistsand. • , U/B osmolality ratios of G. lateralis maintained on S.w.-moist sand O, A, Controland experimental dry sand G. lateralis respectively. • , Ratios of C. guankumi on s.w.-moistsand.

(Table 2). There were no significant diflFerences in the rates of water loss betweencontrol and experimental animals (P>o-io).

On s.w.-moist sand the haemolymph osmolality of G. lateraUs was maintainedrelatively constant. In animals on dry sand it increased sharply and continued to riseuntil death (Fig. 6). After 70 h the difference between haemolymph osmolalities in

Urine production and water balance in crabs 65

control and experimental groups of G. lateralis on dry sand was not significant(P>o-io). An unexpected longer survival time (between 100-120 h) was recorded inthe experimental group. This may be due to the higher mean body weight of theseanimals, although Bliss (1968) noted that size did not influence survival in this species.She found a mean survival time of 89 h at, however, a higher temperature (30 °C) andrelative humidity (78 %).

Determination of both haemolymph and urine osmolalities during desiccation inG. later alts showed that the U/B osmolality ratio remained at I-OI + 0-05 (S.E.M.) up to24 h after transfer to dry conditions (Fig. 7). Urine samples could not be collectedafter this time. This was not significantly different from the mean ratio of animalskept on s.w.-moist sand (1*04±0*02) (P>o-io) (Fig. 7).

Although the possibility remains that] the experimental procedure was not com-pletely effective in preventing water reabsorption from the released urine, it appearsthat this process is not an important water conservation mechanism in G. lateralisin dry conditions.

DISCUSSION

Gecarcinus lateralis living in s.w.-moist sand is apparently in water balance andproduces urine at a relatively high rate (10-47% body weight day"1). Rates recordedin many freshwater and estuarine decapods are similar, e.g. Astacus fluviatilis 8-22 %,Bryan (i960); Eriochevr sinensis 3-6-18-7%, Scholles (1933), De Leersnyder (1967);Carcinus maenas 4-4 % Binns (1969). These rates can be contrasted with those recordedin potamonids, freshwater semi-terrestrial crabs with an Old World distribution, e.g.Potamon niloticus 0-05-0-6% (Shaw, 1959); Potamon edulis 0-58% (Harris 1975).The rate found in C. guanhunri was slightly faster than the latter values at 1 -89 % bodyweight day 1 on s.w.-moist sand. This was considerably slower than that of G. lateralisin similar conditions. This is surprising since the former species is usually consideredto be less well adapted to terrestrial conditions (Bliss, 1968; Gifford, 1968). Thesemi-terrestrial ghost crab Ocypode aVricans has a rate not dissimilar to that of C.guanhumi of 2-3 % body weight day"1 (Flemister, 1958). No values have been pre-viously reported for the rate of urine production in land crabs, although Flemister(1958) measured inulin clearance rates in G. lateralis.

On s.w.-moist sand the maintenance of a constant body weight implies that therelatively high rates of urine production in G. lateralis, together with water loss byother routes, are balanced by water and salt uptake. Bliss (1963) found that G. lateraliswas able to take up water from damp substrates and suggested that water was trans-ported from abdominal setae in contact with the substrate to haemolymph circulatingthrough the gills via the pericardial sacs. Spaargaren (1975) suggested that water wastaken up from food in G. ruricola.

The tendency to dismiss the antennal gland as an organ important in osmoregulationin land crabs ignores the possibility of isosmotic withdrawal of water and salts fromthe primary urine. Production of copious urine isosmotic with the haemolymphrepresents a potential drain on the body salt content. However, judging from thepresent results, there appears to be no net water or salt reabsorption from the primaryurine in either G. lateralis or C. guanhumi.

The evidence presented here suggests that the urinary water recycling mechanism

66 R. R. HARRIS

suggested by Carson (1974) is not an important component of the total water lossin dry conditions. The setal pad situated beneath the nephropore also guards theentrance to the exhalant branchial canal and may prevent the entry of sand grainsduring burrowing. The presence of micro-organisms in the interstices of the setaemay be regarded as a neutral association with the crab. Gifford (1968) has suggestedthat in Cardisoma urine drains mainly into the branchial exhalant canal also. However,comparison of the position of the nephropore in C. guanhumi with that of G. lateraUsshows that the arrangement is quite different in the former. Here the nephroporeopens directly onto the carapace and is not covered by the 3rd maxilliped. Urine drainsaway from the oral region onto laterally situated setal pads.

In dry conditions G. lateraUs is capable of decreasing the rate of clearance of inulinfrom the haemolymph to just over a quarter of the rate on s.w.-moist sand. Flemister(1968) also found that inulin was cleared more slowly in dry conditions. Rates ofclearance calculated from his data are slower than those reported here on s.w.-moistsand but faster than the dry sand values. It is assumed that the decrease in clearancerate represents a decrease in the rate of filtration into the coelomic end-sac. Two possi-bilities can be considered here. Firstly, that there is a decrease in the permeabilityof the basal lamina to water and permeant solutes such as inulin (Schmidt-Nielsen et al.1968). Secondly, that permeability remains unchanged but the haemolymph pressurein the antennal artery or adjacent haemocoel is reduced. If the hydrostatic pressurein the coelomic end-sac remains constant and the colloid osmotic pressure of haemo-lymph is a small component, the net filtration pressure will be reduced (Belman,1976). It would be interesting to investigate these possibilities in G. lateraUs.

The effect on the water balance of the animal of a reduction of the urinary waterloss in dry conditions can be assessed by comparing urine production rates and waterloss rates. Assuming that inulin clearance represents urine production rate, thenurinary water loss is nearly 40 % of the total on dry sand. Maintenance of the urineproduction rates found on s.w.-moist sand would almost double the water loss ofanimals in dry conditions. This decrease in rate may be an adaptation to reduce waterloss when the animal exhibits nocturnal running in low humidities (Bliss, 1968).

This work was supported in part by the Browne Research Fund of the RoyalSociety, and by the Research Fund of the University of Leicester. Radioisotopecounting facilities were kindly provided by the Department of Physics, Universityof the West Indies, Cave Hill, Barbados. I would like to thank the Director, Dr FinnSander, of the Bellairs Institute of McGill University, St James, Barbados, for hishospitality, and Dr A. P. M. Lockwood, of the University of Southampton, for criti-cism of the manuscript.

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