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Water permeability in some euryhaline decapods and Limulus polyphemus
Camp. Biochem.Physiol.,1973,Vol. 44A, pp. 1199to 1213.Pergamon Press. Printed in Great Britain
WATER PERMEABILITY IN SOME EURYHALINE DECAPODS AND LIMULUS POLYPHEMUS JAMES
V. HANNAN
and DAVID
H. EVANS
Division of Functional Biology, Institute of Marine and Atmospheric Sciences and Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables. Florida 33124 (Received 10 JuIy 1972)
Abstract-l. Water turnover rates were measured in Limulq Penaeus and three species of Uca. 2. Limulus was the only species studied that lowered its permeability to water when acclimated to lower salinities. 3. Stress, feeding movements or walking leg autotomy do not affect the water permeability of Uca. 4. The QiO of water infhrx in Litnulus and Uca is approximately 2. 5. All three genera ingest the medium when acclimated to sea water. 6. In Uca pugikator 86 per cent of the water influx is via the gills, 3 per cent is via drinking and 11 per cent is via the exoskeleton.
INTRODUCTION
THE CRUSTACXA arose in sea water and, like the majority of modem marine invertebrates, were probably iso-osmotic to their aquatic environment (Lockwood, 1967). As some members of the group moved onto land they faced the effects of desiccation, i.e. a net loss of water and an increase in haemolymph concentration. Conversely, as other members moved into estuaries and fresh water, they experienced a passive net gain of water and loss of salts. A reduction in integumental permeability to both salts and water could have been one means of diminishing the negative effects of these osmotic problems. Studies on integumental salt permeability in decapods have demonstrated a sequential reduction in the exoskeleton’s permeability to salts in this order: marine, euryhaline, fresh water (Nagel, 1934; Gross, 1957). Through indirect methods involving urine flow studies in fresh-water decapods, Shaw (1959) suggested that water permeability is also lowered in fresh-water crustaceans. Rudy (1967), using tritiated water, examined this aspect of decapod water economy and found a sequential lowering in water permeability paralleling that of salt permeability. Based on these data, it seems reasonabie to postulate that euryhaline crustaceans, when exposed to lower salinities, are able physiologically to decrease their water permeability. Smith (1967, 1970) and Rudy (1967) tested this hypothesis but obtained confiicting results. Using tritiated water, Rudy was unable to find a significant change in the water influx rate for either Carcinus maenas or 1199
1200
J~lll~s V. HANNANANDDAVIDH. EVANS
Palaemonetes virians over a wide salinity range. Smith, in similar experiments using D,O, showed a significant reduction in water permeability in the euryhaline Rithmpmwpeus hti and Car&us maenas. The discrepancy in results for Carcks may have been caused by the difference in experimental temperatures: Rudy adapted and tested his animals at 10°C; Smith adapted them to 15°C and tested at 18°C. Or the discrepancy may be attributable to an isotopic effect. However, King (1969) investigated the water permeability of frog skin to both D,O and TsO and concluded that no isotopic effect was present since the ratio of the permeability coefficients for the influx of both isotopes, D,O/T,O, was 0.9954 f 0.0207. Terrestrial animals lose water continuously to their environment unless the air is saturated. One means of reducing evaporative water loss is to lower the integumental water permeability by physiological adaptations (Potts & Parry, 1963). Among decapods, Herreid (1969) found that the evaporative permeability of the carapace of eight species was correlated to their ecological niche; semiterrestrial forms had the lowest evaporation rate, aquatic forms the highest. The present investigation was conducted to extend the water flux data to other euryhaline arthropods and to study the effects of temperature, stress, feeding, walking leg autotomy, body size, cuticular mineralization, gill chamber irrigation rate and the gill haemolymph flow rate on the hourly water exchange fraction. Also, the water permeability in three species of semiterrestrial, euryhaline decapods is studied in relation to their estuarian distribution. MATERIALS
AND METHODS
The animals studied were the chelicerate Limuh polrphcmw and the decapod crustaceans Uca m&ax, U. pugilator, U. rapax and Pmaeus dfioralu?n. u.?nl+ax was collected in salt marshes of North Carolina; all others were collected in Biscayne Bay, Florida. The salinity of Biscayne Bay fluctuates greatly during the year; therefore, 100% SW was set arbitrarily at 500 mM Na. All solutions were brought to this level by adding Instant Ocean (Aquarium Systems, Inc.) or distilled water, as appropriate. Final concentrations were confirmed with a flame photometer (Instrumentation Laboratory Inc., Model 143). Animals were maintained in indoor S-gal aquaria and, unless otherwise stated, held for at least 5 days prior to experimentation. Minced clam meat and Tetra-Mm, found to be satisfactory foods, were added to those tanks in which the animals had been kept for more than 6 days. Except where otherwise stated, all animals were entire and in intermolt stage C (Drach, 1967). All experiments were conducted at 24°C except for the QtO studies where the animals were held at 14°C for at least 14 days prior to testing. Animals were adapted to the final experimental salinities for at least 72 hr after having been sequentially held in 50, 25, 12, 6 and 3% SW for 2 days each. The adapted animals were then either placed in a radioactive loading bath or the tritium was added directly to their adaptation medium. In all cases the loading bath had a radioactivity of approximately 0.3 /.&i/ml. After 30 min the animals were removed, rinsed in running tap water for 1 min and frozen. Water was extracted from whole animals in a freeze-drying apparatus similar to that described by Rudy (1967). A 100~~1 sample of the extracted radioactive water was then added to scintillation fluid (8 g Omnifluor [New England Nuclear], 120 g naphthaline, made to 1 1. with p-dioxane). The SW loading bath samples were also extracted in the same manner to minimize salt quenching. Activity of the 100~~1 samples was then determined with a Packard tricarb liquid scintillation spectrometer, Model 2003. The samples were
WATER PBRMBA BILITY
IN SOME EURYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1201
counted to at least 10,000 to minimize statistical counting errors. Quenching and background were monitored in all samples and were corrected for. To determine the rate of water influx, the following equation was used:
where Ki is the rate constant in per cent, body water per hr, Co3 is the specific activity of the internal water at isotopic equilibrium and C, is the specific activity of the internal water after a period of time, t, which was 30 min in all cases. Co3 was assumed to be the same as that of the external solution since the bath was so large that dilution by the inactive water from the animal would have a negligible effect. The following method, modified from Gross (1955), was used to determine the water A section of the carapace, hypodermis repermeability of the carapace of Cr. pugilator. moved, was glued to one end of a hollow glass tube with Cenco Soft Seal Tackiwax (Central Scientific Co.). Care was taken to keep the wax out of the tube as this would decrease the surface across which water could move. The tube was filled with a known volume of the appropriate medium and the open end sealed with Vaseline. The tube was then placed in a dish containing the radioactive medium with a specific activity of about 3 &i/ml. A magnetic stirrer, run at low speed, prevented the formation of isotope gradients in the dish. Drinking rates were determined with Gloiil-125 (Iothalamate Sodium I 125) from Abbott Laboratories. This compound has been shown to closely resemble inulin in being non-transportable (Elwood et al., 1967) but has the advantage of being a gamma emitter. The experimental animals were placed into a loading bath with an activity of approximately 50,000 counts/mm per ml. After loading for 2 hr, the animals were placed in a non-radioactive bath for 1 hr to remove the Glofil from the gill chamber. The salinity was the same in all baths for a particular experiment. Finally, the animals were weighed and their radioactivity determined in a Packard Armac gamma tricarb scintillation spectrometer (Model 2001). To transform the water flux data from Ki values to volume/unit of surface area, it was necessary to determine the surface area of the body, legs and gill chamber. Because of their irregular shape, an exact calculation of the surface areas was not attempted. Rather, approximations were made by assuming that the body was a cubic rectangle, the legs were cylinders and the gill chambers were wedges based on Silicon Seal (DuPont) casts. Ecology and life history The horseshoe crab, Limulus polyphemus Linn. (subphylum Chelicerata), inhabits the coastal and estuarian areas of eastern North America. The adults migrate shoreward in spring and summer spawning runs, depositing eggs in the sandy intertidal zone. All developmental stages appear to tolerate a moderate reduction in salinity. For example, McManus (1969) collected them in Delaware Bay, New Jersey, in sea water of 7-30 parts per thousand. Recently, Robertson (1970) has studied osmoregulation in this species. The pink shrimp, Penaeus duorarum Burkenroad (Penaeidae), has a migratory life cycle commencing in deep water on the continental shelf where the benthic adults spawn yearround, releasing buoyant eggs. Postlarvae enter estuaries on the flood tides, settling to the bottom in brackish water nursery grounds. As juveniles and subadults, the shrimp ride ebb tides out of the estuaries toward the deep-water spawning ground, thereby completing the cycle (Hughes, 1969). Farfante (1969) has reviewed the general ecology of this species and Bursey & Lane (1971) have reported on its osmoregulatory capacities. Along the east coast of North America the fiddler crabs (Ocypodidae) are common intertidal animals. Uca minax LeConte, the red-jointed fiddler, occurs on both the Gulf and Atlantic coasts of north Florida. From there it extends northward both into the Gulf states and up the Atlantic seaboard. U. rapax Smith, on the contrary, occurs from south
1202
Jm
V. HANNANAND DAVID H. EVANS
Florida to Rio de Janeiro, being sympatric with U. tinax at least on the Gulf coast of Florida (Salmon, 1967). Distribution of U. pygilatos Bose, the sand-fiddler, completely overlaps the other two species in Florida (Bathbun, 1918). In estuaries the three species occupy distinct habitats. U. minax prefers a muddy substratum and brackish to almost-fresh water, and can survive in fresh water for longer periods of time than either of the other two species (Teal, 1958; Salmon, 1967; personal observations). U. rapux, on the other hand, is observed typically among mangrove roots on mudsand substratum both in the Miami area and in the Caribbean, although Tashian & Vemberg (1958) also found it on sand in northwest Florida. Typically, the adult burrows are located above mean high tide, whereas burrows of U. m&ax are at or below mean high tide. Ucu pug&&r is sympatric with both species but mers ecologicahy in its preference for soil containing more than 40% sand Teal, 1958; Miller, 1961). U. minux is prevented from competing with U. pusihtor in the sandy habitat because its mouth parts lack the spoonshaped setae which enable the latter to remove detritus from coarse material (Miller, 1961; Crane, 1943). RESULTS
AND
DISCUSSION
The integumental water permeability for the arthropods studied is shown in Table 1. In agreement with Rudy’s (1967) findings on other euryhaline decapods, TABLE I-EFFECT OF SALINI~ ON THEWATERINFLUXCONSTANT &
Species
wt. W
% SW
&
u. pl&ator
3.0
100 50 3
0.331 k O-076*(32) 0.359 f 0.062 (22) 0.344 z!z0.073 (23)
u.
7.0
100 50 3
0.331 k 0.054 (17) 0.338 f 0.059 (11) 0.300 + 0.067 (6)
U. rapax
2.6
100 50 3
0.213 f 0.047 (23) 0.215 f 0@40 (18) 0.210 f 0.045 (20)
P. duorarum
5.8
100
0.765 f 0.093 (17)
L. polyphemus
5.9
100 50 20
2.025 & 0.403 (28) 1.395 f 0.306 (26) 1.596 f 0.336 (23)
?tli?mx
* Mean f S.D. (N)
the three species of Uca do not demonstrate a significant (t = > 0.1) change in their water permeability in hypoosmotic media. The chelicerate Limulus, on the other hand, does reduce its sea water-adapted integumental water permeability by 20 per cent to 30 per cent when adapted to brackish water for 1 week or more (P= < 0.01).
WATER PERMEABILITY
DECAPODS AND LIMULUS
IN SOME EUBYHALINS
POLYPHEMUS
1203
Water permeabiIity within a decapod genus Variations in the integumental water permeability within some sympatric members of the genus Uca are seen in Table 1. The permeability of U. rapax was the lowest of the three species, which is probably indicative of its greater adaptation to the terrestrial environment. This conclusion is supported by the work of Herreid (1969), who found that the rate of evaporative loss in eight species of decapod is directly related to the degree of terrestrialness. That is, evaporative loss is the highest in aquatic species and lowest in the most terrestrial species. However, the low water permeability in this species could be an adaptation to fresh water since its Ki is equal ti; that of Astucus (Rudy, 1967), a fresh-water crayfish, If this is the case, then U. rapax would be expected to survive better in fresh water than U. pugizator, and at least as well as U. m&ax, which occurs in the headwaters of estuaries. Survival in fresh water was tested by comparing the time it took for half of a group of crabs to die when kept in distilled water (DW). The time interval for U. minax, U. rapax and U. pugilator was 3 weeks (Teal, 1958), 3 days and 3 days, respectively. Therefore, the low Kt in U. rapax is probably due to its terrestrial existence and is not an adaptation to fresh water. The ability of U. minax to survive about seven times longer in fresh water than U. rapax despite its greater integumental water permeability indicates that survival in fresh water involves something in addition to decreased integumental permeability to water. This other factor is probably a mechanism to replace ions lost by passive diffusion, as has been demonstrated in the Gammaridae by Sutcliffe (1968). Evans (1969) has shown that several laboratory and ecological parameters will alter the water permeability in teleosts. Therefore, it was necessary to study the potential. effect of these parameters on the water flux in crustaceans. The effect of stress Transferring animals (termed “stress” in Table 2) from the holding tanks to the loading bath has been shown to increase the rate of water flux in large trout, Salmo trutta, but to have the reverse effect on the plaice, Platichthys platessa TABLE~--EFFECT
OF sTRBSS ON THE
K( IN SBA
WATER-AD-
ANIMALS
wt.
Species
k)
Stress +
Pf??W.?US P.??UWUS Limulus Limulus u. pugilator u. pugdator
3.7 4.5
+
0.765 f 0*093t(17) 0.854 + 0.082 (7)
5.4 5-o 3.2 2.9
+ +
1.883 2.102 0.337 0.331
* - , No stress; + , stress. f Mean 5 S.D. None of the means is the 0.05 level of the Student t-test.
+ 0.305 + 0.379 + 0.036 rt 0.076
(10) (18) (7) (25)
significantly different within a species at
1204
Jmm V. HANNAN ANDDAVIDH. EVANS
(Evans, 1969). Analogous experiments
were conducted on U. pugilator, Penaeu.~ and Linulus to determine the potential effects of stress on the water permeability of these arthropods. Since none of the values for any one species in Table 2 is significantly different at the 5.0 per cent level of the Student t-test, it is evident that stress did not affect the integumental water permeability in these species. The eflect of feeding movements Since the fiddler crabs were fed only twice a week, many would commence to make feeding movement with the chelipeds when placed in the loading bath. This involved sweeping the chelipeds several times through the water and then bringing them to the mouth. Despite the fact that the loading bath contained no sand or material other than the appropriate medium, it was impossible to say whether or not the fiddlers were able to filter something out of the water and subsequently swallow it. If some of the loading medium were swallowed it would obviously increase the calculated water influx. To determine if the feeding movement affected the K,, an attempt was made to induce a group of crabs to make feeding movements while in the loading bath. This was done by introducing (into the radioactive medium) a small amount of water that had been washed over their customary food, minced clams (called “food-water” in Table 3). Care was taken to admit no visible food particles. This method, however, was unsatisfactory because not all of the animals would begin to feed and those that did would stop within minutes. Therefore, another method was employed. Eyestalkless crabs were observed to be hardy, active, and could be induced to make feeding movements for long periods of time when exposed to “food-water”. The eyestalks were cut off with a pair of scissors without further measure as described by Abramowitz et al. (1940). Post-operative mortality was about 10 per cent the first 2 days with few deaths thereafter. To determine if eyestalk ablation would significantly affect the water influx, the KI of an intact group of U. pug&or was compared with the & of an eyestalkless group. Since no significant difference was found between the two groups (unpublished data), eyestalkless crabs were used to test the effects of feeding movement on the water influx constant. Crabs with eyestalks removed on the same day were kept in an aquarium until the day prior to testing. They were then randomly divided into two groups for testing, One group was loaded with tritium by the standard method; the other group was induced to make feeding movements by introducing small amounts of food-water into the loading bath. The results (Table 3) show that the Ki values for all groups were not significantly different from the water influx constant in normal U. pugilutor (P< O-1). The fleet of walking leg autotomy When a walking leg is autotomized, a thin, preformed membrane is left to cover the stub. To test the effect of a missing leg on the water influx constant,
WATER PERMEABILITYIN SOME EURYHALJNEDBCAPODSAND LIMULUS TABLE ~-EFFECT
POLYPHEMUS
1205
OF FEEDINGMOVEhBNTS ON THR & OF BYE-ABLATEDANIMALS (Further explanation in text)
wt. Species
(g)
% SW
Food *
&
U. pugilator U. pugilator
2.4 2.3
loo 100
+
O-336 + 0.069+(3) 0.385 + 0.102 (3)
* - , No food stimulus; t Mean + S.D. (N)
+ , food stimulus present.
specimens of U. pugilator were induced to autotomize one walking leg. After a l-week recovery period, no significant effect on the Kt (n = 5 ;Ki = 0.311) was apparent in SW-adapted animals (I’< O-1). The effect of shell mineralization
Calcification of the new cuticle commences soon after ecdysis and continues through most of the intermolt cycle. To test the relationship between the degree of cuticular mineralization and water permeability, horseshoe crabs were collected and kept 4 days in a common aquaria in the laboratory. They were then divided into two groups: papershell and hardshell. The carapace of the former buckled under slight pressure; the carapace of the latter group was very resistant to pressure, indicating a greater degree of calcification. The papershell individuals maintained this condition for weeks when kept in the laboratory, and all readily ate minced clam meat. Therefore, they probably do not represent either Stage A or B in Drach’s (1967) intermolt cycle, for crustaceans rapidly pass through these stages and do not feed. As seen in Table 4, the water permeability in both groups was essentially the same, indicating that the degree of mineralization of the carapace is not a factor in TABLE ~-EFFECTS OF SHELLHARDNESSAND SIZE ON Ki IN Limulus
wt.
63) Hard shell Paper shell Small Large * Meanf
S.D.
5.4 5.4 3.6 12.7
% SW 100 100 50 50
Ki 1.969 1.797 1.354 1.339
f f f f
0*185*(S) 0.395 (5) 0*255 (4) 0.149 (3)
(N)
controlling the integumental water permeability. This supports the conclusion of Yonge (1936) that it is the thin epicuticle, which is usually not calcified and contains no chitin, that is mainly responsible for controlling the cuticular permeability.
Jm
1206
V. HANNANAND DAVID H. EVANS
The effect of boa’y size The relationship between body size and the total water influx can be quantified by plotting these two parameters in a manner similar to that used by Evans (1969) for the water flux in fish. A line drawn through the resulting data points could be expressed by the equation M = uWx, where M is the water influx in ml water/ animal per hr, a is the Y intercept, W is the body weight in grams and x is the slope of the line. If the flux is directly related to the body surface: volume ratio x will equal 0.67; if the flux is directly related to the body weight, x will equal 1.00. From the data (Table 4) the value for x can be calculated to be 0.89 for Limulus. Recalculating Smith’s data (1970) on decapods gives 0.81 for Rithro+vpeus and 0.73 for Carck.s. Consequently, in these species the water flux is not directly related to either the body weight or to the body surface area : volume ratio. The same conclusion was reached by EVANS(1969) for teleosts. Integumental water permeability as a jimdun
of temperature
The effect of temperature on water movement in crustaceans has never been examined. This is necessary because the studies on direct measurements of water flux in crustaceans (Rudy, 1967; Smith, 1967, 1970) have been conducted at different experimental temperatures. Q10 values for the experimental temperature range 14-24°C are given for U. pugibztw and Limdus in Table 5. The former exhibits a variable QlO, depending on the external salinity, ranging from 2.1 in sea TABLE 5--p10 VALUESFOBWATT INFLUXIN Two mcxm OF AKR-IROPOD ADAPTED FOR 14 day8 TO Two TEMPQmTmEREGIMBS
Species
u. pugihtor
L. polyphemus
l
water to
wt. (8) 3
5-9
y&SW
“C
100 100 50 50 3 3
24 14 24 14 24 14
0.331& O-158 f 0.359 f o-209 f O-344 + O-200 f
0*076*(32) 0.051 (17) o-062 (22) oa48 (12) O-073 (23) 0.022 (6)
2.10
100 100
24 14
2.025 f 0403 (28) O-781 + 0.227 (12)
2.59
JG
910
1.71 1.73
Mean it S.D. (IV)
1.7 in 3-5Oo/o SW. Limulus has a Qi,, of 2.6 over the same temperature range in sea water. In contrast, the Qi,, values for water permeability of SkIis (Insecta) larval cuticle is between 3 and 3.8 (Shaw, 1955); for the frog and Petromyzon it is about 2-O (Potts & Parry, p. 172) ; for three species of teleost the average was 190 with a range of 1.77-2.12 (Evans, 1969). To compare water flux values, data from many sources are typically converted This is done with Q10 values, such as to a common experimental temperature.
WATER PERMEA BILITY
IN SOMZ EIJRYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1207
those in Table 2. However, this may yield misleading numbers because the decapods that have been studied are of tropical, subtropical, temperate or a combined distribution. This is an important aspect, because it is obviously invalid to convert data for the tropical-subtropical zone U. rapax, tested at 24”C, to the experimental temperature of 10°C used for the temperate zone Cu~cinu.s. One generality, however, may be inferred from the water flux data on decapods : the integumental water permeability is lowest in fresh-water species, intermediate in euryhaline species and highest in marine species. Drinking rates The drinking rates for U. pugilator, Penaeu.~and Limuh are given in Table 6. Uca and Limulus show essentially the same rate as that obtained for three species of marine decapods by Dal1 (1967) using colloidal silver (AguO). From these data, the TAECE ~-DRINKING RATESOF SEVERAL ARTHROPODS Species
% SW
u. pugilator
100
u. pugdator
PcMl?US LiWl.ldUS
5 100 100
Drinking rate 6.0 $/g 2-S p/g 17.3 /d/g 7.3 p/g
per per per per
hr hr hr hr
f f f f
l-4*(6) 0.6 (5) 5.1 (6) l-9 (5)
* Mean + S.D. (N)
percentage of the hourly water exchange fraction that is due to drinking can be calculated by comparing the total quantity of water exchanged to the amount of water entering by drinking per unit time. The water content of U. pug&or was determined by comparing the wet weight with the ash weight. Seventy-one per cent of six females was water compared to 67 per cent of nine males. The mean of all animals was 69 per cent, which is essentially the same as the 68 per cent (n = 13) determined by Guyselman (1953). Therefore, U. pugdator has approximately 700 PI/water per g. Assuming that all the water is equally exchangeable, 231~1 (33 per cent) will exchange per hour with the environment. The drinking rate of 6-O $/g per hr will thus constitute about 3 per cent of the total water influx. Drinking in Limuh is 7-3 PI/g per hr (Table 6). Assuming that 70 per cent of the body is water, then 1400 J/g (200 per cent) will exchange with the environment in 1 hr. Therefore, in this species, drinking contributes less than 0.5% to the total water influx. Exoskeleton water permeability in U. pugilator By attaching a piece of the carapace to a glass tube, the water permeability of the carapace with the hypodermis removed was found to be the same in sea water and distilled water (Table 7). This is to be expected because the Kt values for U. pugiZator are the same in these two media. Herreid (1969) determined the
1208
JAMIUV. HANNANANDDAVIDH. EVANS
exoskeleton’s evaporation rate to be about 3 pl/cma per hr for U. pugilator, which is essentially the same as that determined in aqueous media (2.7 &ma per hr) (Table 9). He also found that the rate was the same for living and dead animals, indicating that the living hypodermis is not a rate-limiting factor in controlling exoskeleton water flux. In addition, there was no change in the rate of water loss when the arth&ial membranes were covered, suggesting they contribute an insignificant amount to the total water flux. TABLE ~-CARAPACE WATERPRIMEABILITY Medium
IN u.
~lA&UtOY
mg HOH/cms per hr $/pa per set 2.6 f 1*05*(6) 2.7 f 1.08 (4)
Distilled water Sea water
7.2 7.5
* Mean + S.D. (N)
Since the cuticle varies in thickness and hardness over the body and legs, the above value of 2.7 &ma per hr may not be representative of the water flux over the entire exoskeleton (exclusive of the gills). To determine this, the body was divided into three regions-the body proper, the chela and the walking legs. By comparing the percentage of the total surface area that a region contained to the percentage of the total water flux that passes through that region (as adapted from Herreid, 1%9), it was found that the cuticle (exclusive of the gills) is approximately uniformly water permeable (Table 8). This is to be expected because (1) the data BODY TABLE S-COMPARISON OF THE REUTIVE WATERPERhQIABILITYOF TWRBlB REGIONSIN Curdisomcr (ADAPTED FROM HERRIBD,1960)
Region
% of the total evaporation rate
% total body evaporation lossi
% of total surface area1
Body proper
21
36
36
Walking legs
22
38
40
Chela
1s
26
24
Total
58*
100
100
* The remainder presumably lost via the gill chamber. t This was calculated using 58 per cent as 100. 1 Includes only the three body regions considered.
on Limulus (Table 4) shows that there is no difference in water flux between papershell and hardshell animals, and (2) the permeability of the cuticle may be chiefly a function of the thin epicuticle (Yonge, 1936).
WATER PERMEAB ILITY
IN SOME BIJRYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1209
Quantzfkation of the water injhx paths in U. pugilator By combining the data on carapace water permeability with the drinking rate and the total hourly water exchange fraction, it is possible to quantify all components of the water influx into U. pugihtol in aqueous media (Table 9). TABLE 9-@JANTIFICATION
OF THE PATHS OF WATER INFLUX INTO A FBMALE FIDDLER CRAB, u.
pugilntor
Path
Area (cm*/g)
Water influx oll/mD per hr)
Total &l/g/hr)
% in&x
Body and legs
8.3
3.0
25.0
11.0
Gill5
6.2
31.0
200.0
86.0
Drinking
-
-
6.0
3.0
14.5
-
231.0
100.0
Total
As stated above, 6 ~1 of the 231 pi/g that are exchanged per hour will enter by drinking. Therefore, about 225 pi/g of water will enter the crab across the hypodermis. The body surface area of a female has approximately 8.3 cm2/g. At a rate of 3 $/cm2 per hr (Table 7), about 25 pll/g per hr will enter the crab via the body and legs. This means that the remainder, 200 pi/g per hr will enter via the gills. The gill surface area is 6.24 cm2/g for this species (Gray, 1957). Therefore, 32 #me per hr of water will enter across the gills. In semiterrestrial crabs such as Uca, the gill chamber is large and serves as an accessory respiratory surface (Wolvekamp & Waterman, 1960). Due to its respiratory function it may be more permeable than the outer body cuticle, and therefore its possible contribution to the water influx must be estimated. The area for both chambers in a 3-g female is about 2.5 cm2. However, microscopic examination of the gill chamber reveals that less than 50 per cent of this area is vascularized and therefore would be expected to exhibit a permeability similar to that of the gills. Adding these data to those in Table 9 changes the influx percentage by about 1 per cent, which is not considered significant within the lirnitations of this study. Converting the above data to pa/p2 per set gives 7.5 pa/t*.”per set for the carapace and 83.0 pa/p2 per set for the gills of U. pugilator. The latter value compares favorably to 69 pa/p2 per set for the gills of Artemih (Smith, 1969). In the alder fly larva, SiaZis Zutaria, the permeability is between 0.04 and 0.05 pa/p2 per set (Shaw, 1955) while in Vahmia ventricosa the permeability is 2.4 p2/p2 per set (Gutknecht, 1967). The ovarian eggs of the salmon, SaZmo salar, have an initial permeability of 0*2-0.06 p3/p2 per set but within 3 hr the eggs became “water hardened” and the permeability declined to
1210
JAMESV. HANNANAND DAVIDH. EVANS
The high water permeability of the gills of the two crustacean species studied to date may be a consequence of pore canals. Smyth (1942) noted that these canals penetrate the cuticle of the gills of C. maenas and extend deep into the underlying epidermal cell. He postulated that they may promote gas exchange. However, they could also enhance the movement of water across the gills. Potential effects of changes in gill haemolymph flow and the gill irr&ation rate Smith (1970) in using the term “apparent water permeability” pointed out that the reduction in the hourly water exchange fraction he noted in brackish water could result from (1) a change in the circulation of the haemolymph through gills, or (2) a change in the irrigation rate of the gills, as well as from (3) a reduction in cuticular or epithelii water permeability. Since the first two factors could increase or decrease the Kt in any media, their potential effect must be determined. (a) Gill haemoljvnphfz ow rate. If the tritium in the gill haemolymph approaches equilibrium with the loading bath, then the calculated Ki would be reduced due to back-diffusion, i.e. the diffusion from the animal back to the loading bath. This would happen if the circulation through the gills were slow enough to permit the small volume of haemolymph in the gills-approximately 50 4 in a 3-g female U. pug&or (Pearse, 1929 )-to approach equilibrium before it re-enters the body. To determine if this is happening, the time it would take to equilibrate the volume of blood in the gills must be compared with the rate of circulation through the gills.
Blood flows directly from the gills to the pericardial sac in %marus (Burger & Smythe, 1953) and C arcinus (Blat&ford, 1971). Since the gills are the only source of blood for the pericardial sac, which in turn is the source of blood for the heart, the cardiac output must represent the volume of haemolymph flowing through the gills. The minute volume for Car&us (Blatchford, 1971) is 5-34 per cent of the body weight, or 50-340 pi/g per min. The water flux across the gills of U. pugilator is 200 &g per hr or 3.3 I*.l/g per min. Therefore, 3.3 ~1 (l-7 per cent) of the 50-340 pll/g per min that circulates through the gills will exchange with the loading bath. From these data it is evident that the rate of flow of haemolymph through the gills is not a limiting factor in determining the hourly water exchange fraction in decapod crustaceans. (b) Gill chamber irrigation rate. It is assumed, in calculating the K,, that the specific activity of the gill chamber water is equal to that of the loading bath. However, since the net flow of tritium is into the gills, the specific activity of the gill chamber water is constantly diminished. The amount of the reduction will be inversely related to the gill irrigation rate; the slower the flow the greater the reduction in the specific activity of the gill chamber water. As stated above, water will exchange between the gill haemolymph and the gill chamber water (loading bath) at a rate of 200 pi/g per hr. The ventilation volume for small individuals of Car&us is about 90 ml/g per hr (Arudpragasam & Naylor, 1964). Assuming a similar rate in Uca, the percentage of the gill chamber water that will exchange with the gill haemolymph water per unit time will be 0.2 ml/90
WATER
PEFtMBAB1LI-N
ml or O-2 per cent. determining the &.
IN
SOME
BUBYHALINB
DBCAPODS
AND
LIMULUS
POLYPHEMUS
1211
Thus, gill chamber irrigation rate is not a limiting factor in
SUMMARY
1. Measurements were made of the flow of tritiated water across Limulus, Penaeus and three species of Uca. The effects of several laboratory and environmental parameters on the hourly water exchange fraction (K,) were determined. 2. The Ki values for Uca pugilator, U. rapax, U. minax and Peruzeus duorarum are O-33, O-21, 0.33 and O-77, respectively. These values are related to the ability to osmoregulate and estuarian distribution. 3. While the Ki in Limulus is reduced from 2.0 in sea water to 1.5 in 20-50s SW, none of the three species of Uca altered its integumental water permeability in response to lowering salinity. 4. Stress, feeding movements and walking leg autotomy do not significantly affect the K, in Uca. Further, neither the haemolymph flow rate in the gills nor the gill chamber irrigation rate is a limiting factor in determining the Kt in this species. 5. The degree of calcification of the cuticle in Limbs has no significant effect on the Ki. 6. The water influx is related to the O-89 power of the body weight in Limulus. 7. The Qi,, of the KS in Limulw is 2.6 in sea water; that of Uca is 2.1 in sea water and 1.7 in 3-5Oo/o SW. 8. The drinking rate in Limuh is 7.3 pi/g per hr; that of Penaeus is 17.3 PI/g per hr. Uca shows a decline in the drinking rate from 6 $/g per hr in sea water to 2.5 Ill/g per hr in 3% SW. 9. In U. pugihtor the water permeability of the exoskeleton is 7.5 $/$ per set; that of the gills is 83.0 pa/~s per sec. Also, 11 per cent of the water influx enters by the exoskeleton, 3 per cent by drinking and 86 per cent via the gills. Acknowledgements-Ttis-This research was supported by N.I.H. training grant No. HDOO187 to the Laboratory for Quantitative Biology (J. V. H.) and N.S.F. grant No. GB16839 (D. H. E.). REFERENCES R. & ABRAMOWITZA. (1940) Moulting, growth and survival after eye&& removal in Uca pugilator. Biol. Bull. mar. biol. Lab., Woo& Hole 78, 179-188. AR~DPRAGASAM K. D. & NAYLOR E. (1964) Gill ventilation volumes, oxygen consumption and respiratory rhythms in Car&w maenas (L.). J. exp. Biol. 41, 309-322. BLATCHFORDJ. G. (1971) Haemodynamics of Car&us maenar (L.). Camp. Biochem. Physiol. 39, 193-202. BURGERJ. W. & SMYTHE C. (1953) The general form of circulation in the lobster, Homarus. J. cell. cot@. Physiol. 42, 369-383. BURSEY C. R. & LANE C. E. (1971) OsmoreguIation in the pink shrimp Penaeus duolawn Burkenroad. Camp. Biochem. Physiol. 39, 483-493. CRANE J. (1943) Display, breeding and relationships of fiddler crabs (genus Uca) in the field. Zoologica 29, 161-168. DALL W. (1967) Hypo-osmoregulation in Crustacea. Con@. Biochem. Physiol. 21,653-678. AJSRAMOWITZ
1212
JAMESV. WAN
ANDDAVIDH. EVANS
DICK D. A. (1959) Osmotic properties of living ceils. Int. Rev. Cytol. 8, 388448. DRACH P. & T~HERNI~~~TZEFF C. (1967) Sur la methode de determination des stades d’intermue et son application generaie aux crustaces. Vie Milieu 18, 595-606. ELW~~D C. M., SIGMANE. M. 8z Tnwxrt C. (1967) The measurement of glomeruiar filtration rate with lUI-eodium iothalamate (Conray). Br. J. Radiol. 48, 581-591. EVANS D. H. (1969) Studies on the permeability to water of selected marine, freshwater and euryhaiine teleosta. J. exp. Biol. 58, 689-703. FARFNQJZ I. P. (1969) Western Atlantic shrimps of the genus Penaeus. Fishery Bull. Fish Wildl. Serv. U.S. 67,461-591. GRAY I. E. (1957) A comparative study of the gill area of crabs. Biol. Bull. mar. biol. Lab., Woo& Hole 112, 3442. GROSSW. J. (1955) Aspecta of osmoregulation in crabs showing the terrestrial habit. Am. Nat. 89, 205-222. GROSSW. J. (1957) An analysis of response to osmotic stress in selected decapod crustacea. Biol. Bull. mar. biol. Lab., Woo& Hole 112, 43-62. GUTKNXC~ J. (1967) Membrane of Valouia uentricosa: apparent absence of water-filled pores. Science N. Y. 158,787-788. Gs J. B. (1953) An anaiyaia of the molting process in the fiddler crab, Ucapugilatot. Biol. Bull. 104, 115-136. HERREID C. F., II (1969) Integumental permeability of crabs and adaptation to land. Camp. B&hens. Physiol. 29,423-429. HUGHESD. A. (1%9) Responses to salinity change as a tidal transport mechanism of pink Biol. Bull. mar. biol. Lab., Wooa% Hole 136, 43-53. shrimp, Penaeus dswamm. KING V. (1969) A study of the mechanism of water transfer across frog skin by a comparison of the permeability of the skin to deuterated and tritiated water. J. Physiol., Lond. 288, 529-538. LOCKWOOD A. M. P. (1967) Aspects of the Physiology of Crustacea. Freeman, San Francisco. MCMANUSJ. J. (1969) Osmotic relations in the horseshoe crab, Limulus polyphemus. Am. Midl. Nat. 81, 569-573. MILLERD. C. (1961) The feeding mechanism of fiddler crabs, with ecological considerations of feeding adaptations. Zooiogica 44,89-102. NAGEL H. (1934) Die Aufgaben der Exkretionsorgane und der kiemen bei der Osmoregulation von Car&us maenas. 2. uergl. Physiol. 21, 468-491. PFZARSE A. S. (1929) The ecology of certain estuarian crabs at Beaufort, N.C. J. EZisha Mitchell Sci. Sot. 44, 230-237. POTTS W. T. W. & PARRYG. (1963) Onotic and Ionic Regulation in Animals. Pergamon Press, Oxford. POTTSW. T. W. & RUDY P. P. (1970) Water balance in the eggs of the Atlantic salmon, Salmo salar. J. exp. Biol. 50, 223-237. R.ATI-IJXTN M. J. (1918) The Grapsoid Crabs of America. Spec. Bull. U.S. Nat. Mus. No. 97, l-461. ROBERTSON J. D. (1970) Osmotic and ionic regulation in the horseshoe crab, Limulus polyphemus (Linnaeus). Biol. Bull. 138, 157-183. RUDY P. P. (1967) Water permeability in selected decapod crustacea. Con@. Biochem. Physiol. 22, 581-589. SALMON M. (1967) Coastal distribution, display and sound production by Florida fiddler crabs (genus Uca). Anim. Behau. 15,449-W. SHAW J. (1955) The permeability and structure of the cuticle of the aquatic larva of Sialis lutaria. J. exp. Biol. 32, 330-252. SHAW J. (1959) Salt and water balance in the East African freshwater crab, Potamon niloticus (M. Edw.). J. ex$. Biol. 36,157-176. SMITH P. G. (1969) The ionic relations of Artemiu sulina (L.)-II. Fluxes of sodium, chloride and water. J. exp. Biol. 51, 739-757.
WATERPERMEABILITYIN SOMEEURYHALINEDECAPODS AND LIMVLVS
POLYPHEMVS
1213
SMITH R. I. (1967) Osmotic regulation and adaptive reduction of water permeability in a brackish-water crab, Rithropanopeus harrisi (Brachyura, Xanthidae). Biol. Bull. mar. biol. Lab., Woods Hole 133, 643-658. SMITH R. I. (1970) The apparent water permeability of Car&us maenas (Brachyura, Portunidae) as a function of salinity. Biol. Bul1.m ar. biol. Lab., Woods Hole 139, 351-362. SMYTH J. D. (1942) VII. A note on the morphology and cytology of the branchiae of Carcinus maenas. Proc. Roy. Sot. Irish Acad. B48, 105-118. SUTCLIF~~ D. W. (1968) Sodium regulation and adaptation to fresh water in gammarid crustaceans. J. exp. Biol. 48, 459-480. TEAL J. M. (1958) Distribution of fiddler crabs in Georgia salt marshes. Ecology 39,185-193. WOLVEKAMPH. P. & WATERMANT. H. (1960) Respiration. In the Physiology of Crustacea (Edited by WATERMANT. H.). Academic Press, New York. YONCE C. M. (1936) On the nature and permeability of chitin-II. Permeability of the uncalcified lining of the foregut of Homarus. Proc. R. Sot. B 120, 15-41. Key Word IndewWater Limulus; Penaeus; Uca.
turnover; influx; drinking rates; Qi,,; permeability to water;
WATER PERMEABILITY IN SOME EURYHALINE DECAPODS AND LIMULUS POLYPHEMUS JAMES
V. HANNAN
and DAVID
H. EVANS
Division of Functional Biology, Institute of Marine and Atmospheric Sciences and Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables. Florida 33124 (Received 10 JuIy 1972)
Abstract-l. Water turnover rates were measured in Limulq Penaeus and three species of Uca. 2. Limulus was the only species studied that lowered its permeability to water when acclimated to lower salinities. 3. Stress, feeding movements or walking leg autotomy do not affect the water permeability of Uca. 4. The QiO of water infhrx in Litnulus and Uca is approximately 2. 5. All three genera ingest the medium when acclimated to sea water. 6. In Uca pugikator 86 per cent of the water influx is via the gills, 3 per cent is via drinking and 11 per cent is via the exoskeleton.
INTRODUCTION
THE CRUSTACXA arose in sea water and, like the majority of modem marine invertebrates, were probably iso-osmotic to their aquatic environment (Lockwood, 1967). As some members of the group moved onto land they faced the effects of desiccation, i.e. a net loss of water and an increase in haemolymph concentration. Conversely, as other members moved into estuaries and fresh water, they experienced a passive net gain of water and loss of salts. A reduction in integumental permeability to both salts and water could have been one means of diminishing the negative effects of these osmotic problems. Studies on integumental salt permeability in decapods have demonstrated a sequential reduction in the exoskeleton’s permeability to salts in this order: marine, euryhaline, fresh water (Nagel, 1934; Gross, 1957). Through indirect methods involving urine flow studies in fresh-water decapods, Shaw (1959) suggested that water permeability is also lowered in fresh-water crustaceans. Rudy (1967), using tritiated water, examined this aspect of decapod water economy and found a sequential lowering in water permeability paralleling that of salt permeability. Based on these data, it seems reasonabie to postulate that euryhaline crustaceans, when exposed to lower salinities, are able physiologically to decrease their water permeability. Smith (1967, 1970) and Rudy (1967) tested this hypothesis but obtained confiicting results. Using tritiated water, Rudy was unable to find a significant change in the water influx rate for either Carcinus maenas or 1199
1200
J~lll~s V. HANNANANDDAVIDH. EVANS
Palaemonetes virians over a wide salinity range. Smith, in similar experiments using D,O, showed a significant reduction in water permeability in the euryhaline Rithmpmwpeus hti and Car&us maenas. The discrepancy in results for Carcks may have been caused by the difference in experimental temperatures: Rudy adapted and tested his animals at 10°C; Smith adapted them to 15°C and tested at 18°C. Or the discrepancy may be attributable to an isotopic effect. However, King (1969) investigated the water permeability of frog skin to both D,O and TsO and concluded that no isotopic effect was present since the ratio of the permeability coefficients for the influx of both isotopes, D,O/T,O, was 0.9954 f 0.0207. Terrestrial animals lose water continuously to their environment unless the air is saturated. One means of reducing evaporative water loss is to lower the integumental water permeability by physiological adaptations (Potts & Parry, 1963). Among decapods, Herreid (1969) found that the evaporative permeability of the carapace of eight species was correlated to their ecological niche; semiterrestrial forms had the lowest evaporation rate, aquatic forms the highest. The present investigation was conducted to extend the water flux data to other euryhaline arthropods and to study the effects of temperature, stress, feeding, walking leg autotomy, body size, cuticular mineralization, gill chamber irrigation rate and the gill haemolymph flow rate on the hourly water exchange fraction. Also, the water permeability in three species of semiterrestrial, euryhaline decapods is studied in relation to their estuarian distribution. MATERIALS
AND METHODS
The animals studied were the chelicerate Limuh polrphcmw and the decapod crustaceans Uca m&ax, U. pugilator, U. rapax and Pmaeus dfioralu?n. u.?nl+ax was collected in salt marshes of North Carolina; all others were collected in Biscayne Bay, Florida. The salinity of Biscayne Bay fluctuates greatly during the year; therefore, 100% SW was set arbitrarily at 500 mM Na. All solutions were brought to this level by adding Instant Ocean (Aquarium Systems, Inc.) or distilled water, as appropriate. Final concentrations were confirmed with a flame photometer (Instrumentation Laboratory Inc., Model 143). Animals were maintained in indoor S-gal aquaria and, unless otherwise stated, held for at least 5 days prior to experimentation. Minced clam meat and Tetra-Mm, found to be satisfactory foods, were added to those tanks in which the animals had been kept for more than 6 days. Except where otherwise stated, all animals were entire and in intermolt stage C (Drach, 1967). All experiments were conducted at 24°C except for the QtO studies where the animals were held at 14°C for at least 14 days prior to testing. Animals were adapted to the final experimental salinities for at least 72 hr after having been sequentially held in 50, 25, 12, 6 and 3% SW for 2 days each. The adapted animals were then either placed in a radioactive loading bath or the tritium was added directly to their adaptation medium. In all cases the loading bath had a radioactivity of approximately 0.3 /.&i/ml. After 30 min the animals were removed, rinsed in running tap water for 1 min and frozen. Water was extracted from whole animals in a freeze-drying apparatus similar to that described by Rudy (1967). A 100~~1 sample of the extracted radioactive water was then added to scintillation fluid (8 g Omnifluor [New England Nuclear], 120 g naphthaline, made to 1 1. with p-dioxane). The SW loading bath samples were also extracted in the same manner to minimize salt quenching. Activity of the 100~~1 samples was then determined with a Packard tricarb liquid scintillation spectrometer, Model 2003. The samples were
WATER PBRMBA BILITY
IN SOME EURYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1201
counted to at least 10,000 to minimize statistical counting errors. Quenching and background were monitored in all samples and were corrected for. To determine the rate of water influx, the following equation was used:
where Ki is the rate constant in per cent, body water per hr, Co3 is the specific activity of the internal water at isotopic equilibrium and C, is the specific activity of the internal water after a period of time, t, which was 30 min in all cases. Co3 was assumed to be the same as that of the external solution since the bath was so large that dilution by the inactive water from the animal would have a negligible effect. The following method, modified from Gross (1955), was used to determine the water A section of the carapace, hypodermis repermeability of the carapace of Cr. pugilator. moved, was glued to one end of a hollow glass tube with Cenco Soft Seal Tackiwax (Central Scientific Co.). Care was taken to keep the wax out of the tube as this would decrease the surface across which water could move. The tube was filled with a known volume of the appropriate medium and the open end sealed with Vaseline. The tube was then placed in a dish containing the radioactive medium with a specific activity of about 3 &i/ml. A magnetic stirrer, run at low speed, prevented the formation of isotope gradients in the dish. Drinking rates were determined with Gloiil-125 (Iothalamate Sodium I 125) from Abbott Laboratories. This compound has been shown to closely resemble inulin in being non-transportable (Elwood et al., 1967) but has the advantage of being a gamma emitter. The experimental animals were placed into a loading bath with an activity of approximately 50,000 counts/mm per ml. After loading for 2 hr, the animals were placed in a non-radioactive bath for 1 hr to remove the Glofil from the gill chamber. The salinity was the same in all baths for a particular experiment. Finally, the animals were weighed and their radioactivity determined in a Packard Armac gamma tricarb scintillation spectrometer (Model 2001). To transform the water flux data from Ki values to volume/unit of surface area, it was necessary to determine the surface area of the body, legs and gill chamber. Because of their irregular shape, an exact calculation of the surface areas was not attempted. Rather, approximations were made by assuming that the body was a cubic rectangle, the legs were cylinders and the gill chambers were wedges based on Silicon Seal (DuPont) casts. Ecology and life history The horseshoe crab, Limulus polyphemus Linn. (subphylum Chelicerata), inhabits the coastal and estuarian areas of eastern North America. The adults migrate shoreward in spring and summer spawning runs, depositing eggs in the sandy intertidal zone. All developmental stages appear to tolerate a moderate reduction in salinity. For example, McManus (1969) collected them in Delaware Bay, New Jersey, in sea water of 7-30 parts per thousand. Recently, Robertson (1970) has studied osmoregulation in this species. The pink shrimp, Penaeus duorarum Burkenroad (Penaeidae), has a migratory life cycle commencing in deep water on the continental shelf where the benthic adults spawn yearround, releasing buoyant eggs. Postlarvae enter estuaries on the flood tides, settling to the bottom in brackish water nursery grounds. As juveniles and subadults, the shrimp ride ebb tides out of the estuaries toward the deep-water spawning ground, thereby completing the cycle (Hughes, 1969). Farfante (1969) has reviewed the general ecology of this species and Bursey & Lane (1971) have reported on its osmoregulatory capacities. Along the east coast of North America the fiddler crabs (Ocypodidae) are common intertidal animals. Uca minax LeConte, the red-jointed fiddler, occurs on both the Gulf and Atlantic coasts of north Florida. From there it extends northward both into the Gulf states and up the Atlantic seaboard. U. rapax Smith, on the contrary, occurs from south
1202
Jm
V. HANNANAND DAVID H. EVANS
Florida to Rio de Janeiro, being sympatric with U. tinax at least on the Gulf coast of Florida (Salmon, 1967). Distribution of U. pygilatos Bose, the sand-fiddler, completely overlaps the other two species in Florida (Bathbun, 1918). In estuaries the three species occupy distinct habitats. U. minax prefers a muddy substratum and brackish to almost-fresh water, and can survive in fresh water for longer periods of time than either of the other two species (Teal, 1958; Salmon, 1967; personal observations). U. rapux, on the other hand, is observed typically among mangrove roots on mudsand substratum both in the Miami area and in the Caribbean, although Tashian & Vemberg (1958) also found it on sand in northwest Florida. Typically, the adult burrows are located above mean high tide, whereas burrows of U. m&ax are at or below mean high tide. Ucu pug&&r is sympatric with both species but mers ecologicahy in its preference for soil containing more than 40% sand Teal, 1958; Miller, 1961). U. minux is prevented from competing with U. pusihtor in the sandy habitat because its mouth parts lack the spoonshaped setae which enable the latter to remove detritus from coarse material (Miller, 1961; Crane, 1943). RESULTS
AND
DISCUSSION
The integumental water permeability for the arthropods studied is shown in Table 1. In agreement with Rudy’s (1967) findings on other euryhaline decapods, TABLE I-EFFECT OF SALINI~ ON THEWATERINFLUXCONSTANT &
Species
wt. W
% SW
&
u. pl&ator
3.0
100 50 3
0.331 k O-076*(32) 0.359 f 0.062 (22) 0.344 z!z0.073 (23)
u.
7.0
100 50 3
0.331 k 0.054 (17) 0.338 f 0.059 (11) 0.300 + 0.067 (6)
U. rapax
2.6
100 50 3
0.213 f 0.047 (23) 0.215 f 0@40 (18) 0.210 f 0.045 (20)
P. duorarum
5.8
100
0.765 f 0.093 (17)
L. polyphemus
5.9
100 50 20
2.025 & 0.403 (28) 1.395 f 0.306 (26) 1.596 f 0.336 (23)
?tli?mx
* Mean f S.D. (N)
the three species of Uca do not demonstrate a significant (t = > 0.1) change in their water permeability in hypoosmotic media. The chelicerate Limulus, on the other hand, does reduce its sea water-adapted integumental water permeability by 20 per cent to 30 per cent when adapted to brackish water for 1 week or more (P= < 0.01).
WATER PERMEABILITY
DECAPODS AND LIMULUS
IN SOME EUBYHALINS
POLYPHEMUS
1203
Water permeabiIity within a decapod genus Variations in the integumental water permeability within some sympatric members of the genus Uca are seen in Table 1. The permeability of U. rapax was the lowest of the three species, which is probably indicative of its greater adaptation to the terrestrial environment. This conclusion is supported by the work of Herreid (1969), who found that the rate of evaporative loss in eight species of decapod is directly related to the degree of terrestrialness. That is, evaporative loss is the highest in aquatic species and lowest in the most terrestrial species. However, the low water permeability in this species could be an adaptation to fresh water since its Ki is equal ti; that of Astucus (Rudy, 1967), a fresh-water crayfish, If this is the case, then U. rapax would be expected to survive better in fresh water than U. pugizator, and at least as well as U. m&ax, which occurs in the headwaters of estuaries. Survival in fresh water was tested by comparing the time it took for half of a group of crabs to die when kept in distilled water (DW). The time interval for U. minax, U. rapax and U. pugilator was 3 weeks (Teal, 1958), 3 days and 3 days, respectively. Therefore, the low Kt in U. rapax is probably due to its terrestrial existence and is not an adaptation to fresh water. The ability of U. minax to survive about seven times longer in fresh water than U. rapax despite its greater integumental water permeability indicates that survival in fresh water involves something in addition to decreased integumental permeability to water. This other factor is probably a mechanism to replace ions lost by passive diffusion, as has been demonstrated in the Gammaridae by Sutcliffe (1968). Evans (1969) has shown that several laboratory and ecological parameters will alter the water permeability in teleosts. Therefore, it was necessary to study the potential. effect of these parameters on the water flux in crustaceans. The effect of stress Transferring animals (termed “stress” in Table 2) from the holding tanks to the loading bath has been shown to increase the rate of water flux in large trout, Salmo trutta, but to have the reverse effect on the plaice, Platichthys platessa TABLE~--EFFECT
OF sTRBSS ON THE
K( IN SBA
WATER-AD-
ANIMALS
wt.
Species
k)
Stress +
Pf??W.?US P.??UWUS Limulus Limulus u. pugilator u. pugdator
3.7 4.5
+
0.765 f 0*093t(17) 0.854 + 0.082 (7)
5.4 5-o 3.2 2.9
+ +
1.883 2.102 0.337 0.331
* - , No stress; + , stress. f Mean 5 S.D. None of the means is the 0.05 level of the Student t-test.
+ 0.305 + 0.379 + 0.036 rt 0.076
(10) (18) (7) (25)
significantly different within a species at
1204
Jmm V. HANNAN ANDDAVIDH. EVANS
(Evans, 1969). Analogous experiments
were conducted on U. pugilator, Penaeu.~ and Linulus to determine the potential effects of stress on the water permeability of these arthropods. Since none of the values for any one species in Table 2 is significantly different at the 5.0 per cent level of the Student t-test, it is evident that stress did not affect the integumental water permeability in these species. The eflect of feeding movements Since the fiddler crabs were fed only twice a week, many would commence to make feeding movement with the chelipeds when placed in the loading bath. This involved sweeping the chelipeds several times through the water and then bringing them to the mouth. Despite the fact that the loading bath contained no sand or material other than the appropriate medium, it was impossible to say whether or not the fiddlers were able to filter something out of the water and subsequently swallow it. If some of the loading medium were swallowed it would obviously increase the calculated water influx. To determine if the feeding movement affected the K,, an attempt was made to induce a group of crabs to make feeding movements while in the loading bath. This was done by introducing (into the radioactive medium) a small amount of water that had been washed over their customary food, minced clams (called “food-water” in Table 3). Care was taken to admit no visible food particles. This method, however, was unsatisfactory because not all of the animals would begin to feed and those that did would stop within minutes. Therefore, another method was employed. Eyestalkless crabs were observed to be hardy, active, and could be induced to make feeding movements for long periods of time when exposed to “food-water”. The eyestalks were cut off with a pair of scissors without further measure as described by Abramowitz et al. (1940). Post-operative mortality was about 10 per cent the first 2 days with few deaths thereafter. To determine if eyestalk ablation would significantly affect the water influx, the KI of an intact group of U. pug&or was compared with the & of an eyestalkless group. Since no significant difference was found between the two groups (unpublished data), eyestalkless crabs were used to test the effects of feeding movement on the water influx constant. Crabs with eyestalks removed on the same day were kept in an aquarium until the day prior to testing. They were then randomly divided into two groups for testing, One group was loaded with tritium by the standard method; the other group was induced to make feeding movements by introducing small amounts of food-water into the loading bath. The results (Table 3) show that the Ki values for all groups were not significantly different from the water influx constant in normal U. pugilutor (P< O-1). The fleet of walking leg autotomy When a walking leg is autotomized, a thin, preformed membrane is left to cover the stub. To test the effect of a missing leg on the water influx constant,
WATER PERMEABILITYIN SOME EURYHALJNEDBCAPODSAND LIMULUS TABLE ~-EFFECT
POLYPHEMUS
1205
OF FEEDINGMOVEhBNTS ON THR & OF BYE-ABLATEDANIMALS (Further explanation in text)
wt. Species
(g)
% SW
Food *
&
U. pugilator U. pugilator
2.4 2.3
loo 100
+
O-336 + 0.069+(3) 0.385 + 0.102 (3)
* - , No food stimulus; t Mean + S.D. (N)
+ , food stimulus present.
specimens of U. pugilator were induced to autotomize one walking leg. After a l-week recovery period, no significant effect on the Kt (n = 5 ;Ki = 0.311) was apparent in SW-adapted animals (I’< O-1). The effect of shell mineralization
Calcification of the new cuticle commences soon after ecdysis and continues through most of the intermolt cycle. To test the relationship between the degree of cuticular mineralization and water permeability, horseshoe crabs were collected and kept 4 days in a common aquaria in the laboratory. They were then divided into two groups: papershell and hardshell. The carapace of the former buckled under slight pressure; the carapace of the latter group was very resistant to pressure, indicating a greater degree of calcification. The papershell individuals maintained this condition for weeks when kept in the laboratory, and all readily ate minced clam meat. Therefore, they probably do not represent either Stage A or B in Drach’s (1967) intermolt cycle, for crustaceans rapidly pass through these stages and do not feed. As seen in Table 4, the water permeability in both groups was essentially the same, indicating that the degree of mineralization of the carapace is not a factor in TABLE ~-EFFECTS OF SHELLHARDNESSAND SIZE ON Ki IN Limulus
wt.
63) Hard shell Paper shell Small Large * Meanf
S.D.
5.4 5.4 3.6 12.7
% SW 100 100 50 50
Ki 1.969 1.797 1.354 1.339
f f f f
0*185*(S) 0.395 (5) 0*255 (4) 0.149 (3)
(N)
controlling the integumental water permeability. This supports the conclusion of Yonge (1936) that it is the thin epicuticle, which is usually not calcified and contains no chitin, that is mainly responsible for controlling the cuticular permeability.
Jm
1206
V. HANNANAND DAVID H. EVANS
The effect of boa’y size The relationship between body size and the total water influx can be quantified by plotting these two parameters in a manner similar to that used by Evans (1969) for the water flux in fish. A line drawn through the resulting data points could be expressed by the equation M = uWx, where M is the water influx in ml water/ animal per hr, a is the Y intercept, W is the body weight in grams and x is the slope of the line. If the flux is directly related to the body surface: volume ratio x will equal 0.67; if the flux is directly related to the body weight, x will equal 1.00. From the data (Table 4) the value for x can be calculated to be 0.89 for Limulus. Recalculating Smith’s data (1970) on decapods gives 0.81 for Rithro+vpeus and 0.73 for Carck.s. Consequently, in these species the water flux is not directly related to either the body weight or to the body surface area : volume ratio. The same conclusion was reached by EVANS(1969) for teleosts. Integumental water permeability as a jimdun
of temperature
The effect of temperature on water movement in crustaceans has never been examined. This is necessary because the studies on direct measurements of water flux in crustaceans (Rudy, 1967; Smith, 1967, 1970) have been conducted at different experimental temperatures. Q10 values for the experimental temperature range 14-24°C are given for U. pugibztw and Limdus in Table 5. The former exhibits a variable QlO, depending on the external salinity, ranging from 2.1 in sea TABLE 5--p10 VALUESFOBWATT INFLUXIN Two mcxm OF AKR-IROPOD ADAPTED FOR 14 day8 TO Two TEMPQmTmEREGIMBS
Species
u. pugihtor
L. polyphemus
l
water to
wt. (8) 3
5-9
y&SW
“C
100 100 50 50 3 3
24 14 24 14 24 14
0.331& O-158 f 0.359 f o-209 f O-344 + O-200 f
0*076*(32) 0.051 (17) o-062 (22) oa48 (12) O-073 (23) 0.022 (6)
2.10
100 100
24 14
2.025 f 0403 (28) O-781 + 0.227 (12)
2.59
JG
910
1.71 1.73
Mean it S.D. (IV)
1.7 in 3-5Oo/o SW. Limulus has a Qi,, of 2.6 over the same temperature range in sea water. In contrast, the Qi,, values for water permeability of SkIis (Insecta) larval cuticle is between 3 and 3.8 (Shaw, 1955); for the frog and Petromyzon it is about 2-O (Potts & Parry, p. 172) ; for three species of teleost the average was 190 with a range of 1.77-2.12 (Evans, 1969). To compare water flux values, data from many sources are typically converted This is done with Q10 values, such as to a common experimental temperature.
WATER PERMEA BILITY
IN SOMZ EIJRYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1207
those in Table 2. However, this may yield misleading numbers because the decapods that have been studied are of tropical, subtropical, temperate or a combined distribution. This is an important aspect, because it is obviously invalid to convert data for the tropical-subtropical zone U. rapax, tested at 24”C, to the experimental temperature of 10°C used for the temperate zone Cu~cinu.s. One generality, however, may be inferred from the water flux data on decapods : the integumental water permeability is lowest in fresh-water species, intermediate in euryhaline species and highest in marine species. Drinking rates The drinking rates for U. pugilator, Penaeu.~and Limuh are given in Table 6. Uca and Limulus show essentially the same rate as that obtained for three species of marine decapods by Dal1 (1967) using colloidal silver (AguO). From these data, the TAECE ~-DRINKING RATESOF SEVERAL ARTHROPODS Species
% SW
u. pugilator
100
u. pugdator
PcMl?US LiWl.ldUS
5 100 100
Drinking rate 6.0 $/g 2-S p/g 17.3 /d/g 7.3 p/g
per per per per
hr hr hr hr
f f f f
l-4*(6) 0.6 (5) 5.1 (6) l-9 (5)
* Mean + S.D. (N)
percentage of the hourly water exchange fraction that is due to drinking can be calculated by comparing the total quantity of water exchanged to the amount of water entering by drinking per unit time. The water content of U. pug&or was determined by comparing the wet weight with the ash weight. Seventy-one per cent of six females was water compared to 67 per cent of nine males. The mean of all animals was 69 per cent, which is essentially the same as the 68 per cent (n = 13) determined by Guyselman (1953). Therefore, U. pugdator has approximately 700 PI/water per g. Assuming that all the water is equally exchangeable, 231~1 (33 per cent) will exchange per hour with the environment. The drinking rate of 6-O $/g per hr will thus constitute about 3 per cent of the total water influx. Drinking in Limuh is 7-3 PI/g per hr (Table 6). Assuming that 70 per cent of the body is water, then 1400 J/g (200 per cent) will exchange with the environment in 1 hr. Therefore, in this species, drinking contributes less than 0.5% to the total water influx. Exoskeleton water permeability in U. pugilator By attaching a piece of the carapace to a glass tube, the water permeability of the carapace with the hypodermis removed was found to be the same in sea water and distilled water (Table 7). This is to be expected because the Kt values for U. pugiZator are the same in these two media. Herreid (1969) determined the
1208
JAMIUV. HANNANANDDAVIDH. EVANS
exoskeleton’s evaporation rate to be about 3 pl/cma per hr for U. pugilator, which is essentially the same as that determined in aqueous media (2.7 &ma per hr) (Table 9). He also found that the rate was the same for living and dead animals, indicating that the living hypodermis is not a rate-limiting factor in controlling exoskeleton water flux. In addition, there was no change in the rate of water loss when the arth&ial membranes were covered, suggesting they contribute an insignificant amount to the total water flux. TABLE ~-CARAPACE WATERPRIMEABILITY Medium
IN u.
~lA&UtOY
mg HOH/cms per hr $/pa per set 2.6 f 1*05*(6) 2.7 f 1.08 (4)
Distilled water Sea water
7.2 7.5
* Mean + S.D. (N)
Since the cuticle varies in thickness and hardness over the body and legs, the above value of 2.7 &ma per hr may not be representative of the water flux over the entire exoskeleton (exclusive of the gills). To determine this, the body was divided into three regions-the body proper, the chela and the walking legs. By comparing the percentage of the total surface area that a region contained to the percentage of the total water flux that passes through that region (as adapted from Herreid, 1%9), it was found that the cuticle (exclusive of the gills) is approximately uniformly water permeable (Table 8). This is to be expected because (1) the data BODY TABLE S-COMPARISON OF THE REUTIVE WATERPERhQIABILITYOF TWRBlB REGIONSIN Curdisomcr (ADAPTED FROM HERRIBD,1960)
Region
% of the total evaporation rate
% total body evaporation lossi
% of total surface area1
Body proper
21
36
36
Walking legs
22
38
40
Chela
1s
26
24
Total
58*
100
100
* The remainder presumably lost via the gill chamber. t This was calculated using 58 per cent as 100. 1 Includes only the three body regions considered.
on Limulus (Table 4) shows that there is no difference in water flux between papershell and hardshell animals, and (2) the permeability of the cuticle may be chiefly a function of the thin epicuticle (Yonge, 1936).
WATER PERMEAB ILITY
IN SOME BIJRYHALINE
DECAPODS AND LIMULUS
POLYPHEMUS
1209
Quantzfkation of the water injhx paths in U. pugilator By combining the data on carapace water permeability with the drinking rate and the total hourly water exchange fraction, it is possible to quantify all components of the water influx into U. pugihtol in aqueous media (Table 9). TABLE 9-@JANTIFICATION
OF THE PATHS OF WATER INFLUX INTO A FBMALE FIDDLER CRAB, u.
pugilntor
Path
Area (cm*/g)
Water influx oll/mD per hr)
Total &l/g/hr)
% in&x
Body and legs
8.3
3.0
25.0
11.0
Gill5
6.2
31.0
200.0
86.0
Drinking
-
-
6.0
3.0
14.5
-
231.0
100.0
Total
As stated above, 6 ~1 of the 231 pi/g that are exchanged per hour will enter by drinking. Therefore, about 225 pi/g of water will enter the crab across the hypodermis. The body surface area of a female has approximately 8.3 cm2/g. At a rate of 3 $/cm2 per hr (Table 7), about 25 pll/g per hr will enter the crab via the body and legs. This means that the remainder, 200 pi/g per hr will enter via the gills. The gill surface area is 6.24 cm2/g for this species (Gray, 1957). Therefore, 32 #me per hr of water will enter across the gills. In semiterrestrial crabs such as Uca, the gill chamber is large and serves as an accessory respiratory surface (Wolvekamp & Waterman, 1960). Due to its respiratory function it may be more permeable than the outer body cuticle, and therefore its possible contribution to the water influx must be estimated. The area for both chambers in a 3-g female is about 2.5 cm2. However, microscopic examination of the gill chamber reveals that less than 50 per cent of this area is vascularized and therefore would be expected to exhibit a permeability similar to that of the gills. Adding these data to those in Table 9 changes the influx percentage by about 1 per cent, which is not considered significant within the lirnitations of this study. Converting the above data to pa/p2 per set gives 7.5 pa/t*.”per set for the carapace and 83.0 pa/p2 per set for the gills of U. pugilator. The latter value compares favorably to 69 pa/p2 per set for the gills of Artemih (Smith, 1969). In the alder fly larva, SiaZis Zutaria, the permeability is between 0.04 and 0.05 pa/p2 per set (Shaw, 1955) while in Vahmia ventricosa the permeability is 2.4 p2/p2 per set (Gutknecht, 1967). The ovarian eggs of the salmon, SaZmo salar, have an initial permeability of 0*2-0.06 p3/p2 per set but within 3 hr the eggs became “water hardened” and the permeability declined to
1210
JAMESV. HANNANAND DAVIDH. EVANS
The high water permeability of the gills of the two crustacean species studied to date may be a consequence of pore canals. Smyth (1942) noted that these canals penetrate the cuticle of the gills of C. maenas and extend deep into the underlying epidermal cell. He postulated that they may promote gas exchange. However, they could also enhance the movement of water across the gills. Potential effects of changes in gill haemolymph flow and the gill irr&ation rate Smith (1970) in using the term “apparent water permeability” pointed out that the reduction in the hourly water exchange fraction he noted in brackish water could result from (1) a change in the circulation of the haemolymph through gills, or (2) a change in the irrigation rate of the gills, as well as from (3) a reduction in cuticular or epithelii water permeability. Since the first two factors could increase or decrease the Kt in any media, their potential effect must be determined. (a) Gill haemoljvnphfz ow rate. If the tritium in the gill haemolymph approaches equilibrium with the loading bath, then the calculated Ki would be reduced due to back-diffusion, i.e. the diffusion from the animal back to the loading bath. This would happen if the circulation through the gills were slow enough to permit the small volume of haemolymph in the gills-approximately 50 4 in a 3-g female U. pug&or (Pearse, 1929 )-to approach equilibrium before it re-enters the body. To determine if this is happening, the time it would take to equilibrate the volume of blood in the gills must be compared with the rate of circulation through the gills.
Blood flows directly from the gills to the pericardial sac in %marus (Burger & Smythe, 1953) and C arcinus (Blat&ford, 1971). Since the gills are the only source of blood for the pericardial sac, which in turn is the source of blood for the heart, the cardiac output must represent the volume of haemolymph flowing through the gills. The minute volume for Car&us (Blatchford, 1971) is 5-34 per cent of the body weight, or 50-340 pi/g per min. The water flux across the gills of U. pugilator is 200 &g per hr or 3.3 I*.l/g per min. Therefore, 3.3 ~1 (l-7 per cent) of the 50-340 pll/g per min that circulates through the gills will exchange with the loading bath. From these data it is evident that the rate of flow of haemolymph through the gills is not a limiting factor in determining the hourly water exchange fraction in decapod crustaceans. (b) Gill chamber irrigation rate. It is assumed, in calculating the K,, that the specific activity of the gill chamber water is equal to that of the loading bath. However, since the net flow of tritium is into the gills, the specific activity of the gill chamber water is constantly diminished. The amount of the reduction will be inversely related to the gill irrigation rate; the slower the flow the greater the reduction in the specific activity of the gill chamber water. As stated above, water will exchange between the gill haemolymph and the gill chamber water (loading bath) at a rate of 200 pi/g per hr. The ventilation volume for small individuals of Car&us is about 90 ml/g per hr (Arudpragasam & Naylor, 1964). Assuming a similar rate in Uca, the percentage of the gill chamber water that will exchange with the gill haemolymph water per unit time will be 0.2 ml/90
WATER
PEFtMBAB1LI-N
ml or O-2 per cent. determining the &.
IN
SOME
BUBYHALINB
DBCAPODS
AND
LIMULUS
POLYPHEMUS
1211
Thus, gill chamber irrigation rate is not a limiting factor in
SUMMARY
1. Measurements were made of the flow of tritiated water across Limulus, Penaeus and three species of Uca. The effects of several laboratory and environmental parameters on the hourly water exchange fraction (K,) were determined. 2. The Ki values for Uca pugilator, U. rapax, U. minax and Peruzeus duorarum are O-33, O-21, 0.33 and O-77, respectively. These values are related to the ability to osmoregulate and estuarian distribution. 3. While the Ki in Limulus is reduced from 2.0 in sea water to 1.5 in 20-50s SW, none of the three species of Uca altered its integumental water permeability in response to lowering salinity. 4. Stress, feeding movements and walking leg autotomy do not significantly affect the K, in Uca. Further, neither the haemolymph flow rate in the gills nor the gill chamber irrigation rate is a limiting factor in determining the Kt in this species. 5. The degree of calcification of the cuticle in Limbs has no significant effect on the Ki. 6. The water influx is related to the O-89 power of the body weight in Limulus. 7. The Qi,, of the KS in Limulw is 2.6 in sea water; that of Uca is 2.1 in sea water and 1.7 in 3-5Oo/o SW. 8. The drinking rate in Limuh is 7.3 pi/g per hr; that of Penaeus is 17.3 PI/g per hr. Uca shows a decline in the drinking rate from 6 $/g per hr in sea water to 2.5 Ill/g per hr in 3% SW. 9. In U. pugihtor the water permeability of the exoskeleton is 7.5 $/$ per set; that of the gills is 83.0 pa/~s per sec. Also, 11 per cent of the water influx enters by the exoskeleton, 3 per cent by drinking and 86 per cent via the gills. Acknowledgements-Ttis-This research was supported by N.I.H. training grant No. HDOO187 to the Laboratory for Quantitative Biology (J. V. H.) and N.S.F. grant No. GB16839 (D. H. E.). REFERENCES R. & ABRAMOWITZA. (1940) Moulting, growth and survival after eye&& removal in Uca pugilator. Biol. Bull. mar. biol. Lab., Woo& Hole 78, 179-188. AR~DPRAGASAM K. D. & NAYLOR E. (1964) Gill ventilation volumes, oxygen consumption and respiratory rhythms in Car&w maenas (L.). J. exp. Biol. 41, 309-322. BLATCHFORDJ. G. (1971) Haemodynamics of Car&us maenar (L.). Camp. Biochem. Physiol. 39, 193-202. BURGERJ. W. & SMYTHE C. (1953) The general form of circulation in the lobster, Homarus. J. cell. cot@. Physiol. 42, 369-383. BURSEY C. R. & LANE C. E. (1971) OsmoreguIation in the pink shrimp Penaeus duolawn Burkenroad. Camp. Biochem. Physiol. 39, 483-493. CRANE J. (1943) Display, breeding and relationships of fiddler crabs (genus Uca) in the field. Zoologica 29, 161-168. DALL W. (1967) Hypo-osmoregulation in Crustacea. Con@. Biochem. Physiol. 21,653-678. AJSRAMOWITZ
1212
JAMESV. WAN
ANDDAVIDH. EVANS
DICK D. A. (1959) Osmotic properties of living ceils. Int. Rev. Cytol. 8, 388448. DRACH P. & T~HERNI~~~TZEFF C. (1967) Sur la methode de determination des stades d’intermue et son application generaie aux crustaces. Vie Milieu 18, 595-606. ELW~~D C. M., SIGMANE. M. 8z Tnwxrt C. (1967) The measurement of glomeruiar filtration rate with lUI-eodium iothalamate (Conray). Br. J. Radiol. 48, 581-591. EVANS D. H. (1969) Studies on the permeability to water of selected marine, freshwater and euryhaiine teleosta. J. exp. Biol. 58, 689-703. FARFNQJZ I. P. (1969) Western Atlantic shrimps of the genus Penaeus. Fishery Bull. Fish Wildl. Serv. U.S. 67,461-591. GRAY I. E. (1957) A comparative study of the gill area of crabs. Biol. Bull. mar. biol. Lab., Woo& Hole 112, 3442. GROSSW. J. (1955) Aspecta of osmoregulation in crabs showing the terrestrial habit. Am. Nat. 89, 205-222. GROSSW. J. (1957) An analysis of response to osmotic stress in selected decapod crustacea. Biol. Bull. mar. biol. Lab., Woo& Hole 112, 43-62. GUTKNXC~ J. (1967) Membrane of Valouia uentricosa: apparent absence of water-filled pores. Science N. Y. 158,787-788. Gs J. B. (1953) An anaiyaia of the molting process in the fiddler crab, Ucapugilatot. Biol. Bull. 104, 115-136. HERREID C. F., II (1969) Integumental permeability of crabs and adaptation to land. Camp. B&hens. Physiol. 29,423-429. HUGHESD. A. (1%9) Responses to salinity change as a tidal transport mechanism of pink Biol. Bull. mar. biol. Lab., Wooa% Hole 136, 43-53. shrimp, Penaeus dswamm. KING V. (1969) A study of the mechanism of water transfer across frog skin by a comparison of the permeability of the skin to deuterated and tritiated water. J. Physiol., Lond. 288, 529-538. LOCKWOOD A. M. P. (1967) Aspects of the Physiology of Crustacea. Freeman, San Francisco. MCMANUSJ. J. (1969) Osmotic relations in the horseshoe crab, Limulus polyphemus. Am. Midl. Nat. 81, 569-573. MILLERD. C. (1961) The feeding mechanism of fiddler crabs, with ecological considerations of feeding adaptations. Zooiogica 44,89-102. NAGEL H. (1934) Die Aufgaben der Exkretionsorgane und der kiemen bei der Osmoregulation von Car&us maenas. 2. uergl. Physiol. 21, 468-491. PFZARSE A. S. (1929) The ecology of certain estuarian crabs at Beaufort, N.C. J. EZisha Mitchell Sci. Sot. 44, 230-237. POTTS W. T. W. & PARRYG. (1963) Onotic and Ionic Regulation in Animals. Pergamon Press, Oxford. POTTSW. T. W. & RUDY P. P. (1970) Water balance in the eggs of the Atlantic salmon, Salmo salar. J. exp. Biol. 50, 223-237. R.ATI-IJXTN M. J. (1918) The Grapsoid Crabs of America. Spec. Bull. U.S. Nat. Mus. No. 97, l-461. ROBERTSON J. D. (1970) Osmotic and ionic regulation in the horseshoe crab, Limulus polyphemus (Linnaeus). Biol. Bull. 138, 157-183. RUDY P. P. (1967) Water permeability in selected decapod crustacea. Con@. Biochem. Physiol. 22, 581-589. SALMON M. (1967) Coastal distribution, display and sound production by Florida fiddler crabs (genus Uca). Anim. Behau. 15,449-W. SHAW J. (1955) The permeability and structure of the cuticle of the aquatic larva of Sialis lutaria. J. exp. Biol. 32, 330-252. SHAW J. (1959) Salt and water balance in the East African freshwater crab, Potamon niloticus (M. Edw.). J. ex$. Biol. 36,157-176. SMITH P. G. (1969) The ionic relations of Artemiu sulina (L.)-II. Fluxes of sodium, chloride and water. J. exp. Biol. 51, 739-757.
WATERPERMEABILITYIN SOMEEURYHALINEDECAPODS AND LIMVLVS
POLYPHEMVS
1213
SMITH R. I. (1967) Osmotic regulation and adaptive reduction of water permeability in a brackish-water crab, Rithropanopeus harrisi (Brachyura, Xanthidae). Biol. Bull. mar. biol. Lab., Woods Hole 133, 643-658. SMITH R. I. (1970) The apparent water permeability of Car&us maenas (Brachyura, Portunidae) as a function of salinity. Biol. Bul1.m ar. biol. Lab., Woods Hole 139, 351-362. SMYTH J. D. (1942) VII. A note on the morphology and cytology of the branchiae of Carcinus maenas. Proc. Roy. Sot. Irish Acad. B48, 105-118. SUTCLIF~~ D. W. (1968) Sodium regulation and adaptation to fresh water in gammarid crustaceans. J. exp. Biol. 48, 459-480. TEAL J. M. (1958) Distribution of fiddler crabs in Georgia salt marshes. Ecology 39,185-193. WOLVEKAMPH. P. & WATERMANT. H. (1960) Respiration. In the Physiology of Crustacea (Edited by WATERMANT. H.). Academic Press, New York. YONCE C. M. (1936) On the nature and permeability of chitin-II. Permeability of the uncalcified lining of the foregut of Homarus. Proc. R. Sot. B 120, 15-41. Key Word IndewWater Limulus; Penaeus; Uca.
turnover; influx; drinking rates; Qi,,; permeability to water;