- Email: [email protected]
Osmoregulation of the xanthid crab, panopeus herbstii
Camp. Biochem. fhysiol.
Vol. 90A,
No.
I, pp.
135-139,
1988
Printed in Great
0
OSMOREGULATION OF THE XANTHID PANOPEUS HERBSTII
0300-9629/88 $3.00 + 0.00 1988 Pergamon Press plc
CRAB,
ELIZABETHBLASCOand RICHARDB. FORWARD,JR Duke University Marine Laboratory Beaufort, NC 28516, USA. Telephone: (919) 728-2111; and Zoology Department, Duke University, Durham, NC 28516 USA
(Received 11 September 1987)
Ahstraet-1. After acclimation: the mud crab, Punopeus herbsfii, hyperosmotically regulates between 10 and 28 ppt and osmoconforms above 28 ppt. Chloride ion concentration remained at a relatively constant hyperionic level at lower salinities and became hypoionic at high salinities. 2. The rate of acclimation to hyper and hypoosmotic shocks varied. Upon a sudden change from 27 to 19 ppt, haemolymph osmolality stabilized within 4 hr. Upon a sudden change from 27 to 35 ppt, crabs did not equilibrate their osmolality within 48 hr. Instead, haemolymph osmolality remained hypoosmotic. 3. A gradual 35-23-35 ppt change in salinity over 12 hr resulted in an initial 75 mOsm decrease in osmolality. As salinity increased, the mud crab regulated hyperosmotically until 33 ppt and hypoosmotically thereafter. 4. Upon exposure to a salinity qycle in the field, haemolymph osmolality remained relatively constant. Thus, P. herbstii is capable of hypkr-hypoosmoregulation when exposed to short-term changes in salinity. This conclusion is not suggested by the osmotic relationship upon acclimation.
INTRODUCTION Marine euryhaline decapods which inhabit estuaries are subjected to fluctuating salinities as a result of the tides. Due to the permeable nature of crustaceans’ gill tissues, these salinity changes have the potential to alter the osmotic and ionic concentrations of the crustaceans’ haemolymph. Since osmotic and ionic concentrations affect cell volume, these oscillations in concentrations must be regulated because proper cellular functioning requires a relatively constant cell volume. The degree to which crustaceans can osmoregulate as well as the mechanisms they utilize to cope with salinity fluctuations has been well studied (for reviews, see Schoeffeniels and Gilles, 1970; Gilles and Pequeux, 1983). The effect of sudden hypo and hyperosmotic shocks has also been explored in some detail (Siebers et al., 1972; Engel and Nichols, 1977; Gilles and Pequeux, 1981). In these past experiments, however, the salinity changes were in the order of 20-30ppt which are rare occurrences under normal conditions in estuaries. Thus, while these studies detail the crustacean’s ability to adjust to extreme changes, they do not illustrate how crabs cope with normal salinity changes. Studies using gradual changes in salinity are more realistic, but few in number (Spaargaren, 1974; Findley and Stickle, 1978). Finally, studies on the effect of salinity fluctuations in the field are virtually non-existent. The present investigation was undertaken to compare osmotic responses of a crab to constant and changing external osmolalities. Panopeus herbsrii is an estuarine mud crab and, as such, is potentially subjected to daily fluctuations in salinity (Williams, 1984). In this study, the rates of acclimation to sudden and gradual salinity changes
were determined. In addition, the effect of natural tidal cycle in the field on mud crabs’ haemolymph osmolality was established. It was found that upon acclimation P. herbstii osmoconforms at high salinities and osmoregulates at low salinities. Upon changes in salinity, it can hyper-hypoosmoregulate for short periods of time in salinities where it osmoconforms upon acclimation.
MATERIALSAND METHODS The mud crab, Punopeus herbsrii (H. Milne Edwards), was collected without regards to sex at Beaufort, North Carolina, USA during the months of January-April. The mud crabs were collected l-2 days prior to each experimental period, which lasted no longer than 2 weeks. They were kept unfed in aerated sea-water (18.5”C). Salinities used in the experiments were varied by adding either Instant Ocean or deionized water to sea-water. A refractometer (A.O.) was use& to measure salinity. There were four series of experiments and two sampling techniques. Approximately 0.1 ml of haemolymph was drawn from the chelipeds using a Number 19 syringe needle. Haemolymph samples were frozen immediately and analysed within 3 days. Two different sampling techniques were used. In the first technique, haemolymph samples were drawn from a crab once during the experiment. In the second technique, a single crab’s haemolymph was sampled continuously at every sampling time throughout the course of the experiment. Experiments
(1) The relationship between the crabs’ haemolymph and chloride ion concentration and the external osmolality was determined after acclimating three mud crabs for one week in each of the following salinities: 10, 15, 20, 25, 30, 35, and
135
ELIZABETH BLASCO and RICHARD B. FORWARD JR
136
40ppt. Their haemolymph was sampled using the first sampling technique. (2) The rate of acclimation upon sudden salinity changes was conducted at salinities of 19, 27, and 35 ppt. These salinities were chosen because the mud crabs’ osmoregulatory abilities change from regulating to conforming around 27 ppt (Fig. 1). To determine the haemolymph osmolality upon acclimation to 19 and 35 ppt, three mud crabs were acclimated for 1week at each salinity. Their haemolymph was sampled, and the results averaged. During the same period, thirty mud crabs were acclimated at 27 ppt. After 1 week, I5 crabs were placed in 19 ppt sea-water and 15 crabs in 35 ppt sea-water. Three of the transferred crabs in each salinity were identified by their markings and their haemolymph was sampled using the second sampling technique at 0, 1, 4, 8, I?, 24, and 48 hr after the transfer. The remaining 12 crabs in each salinity were sampled according to the first sampling technique. In this case, two crabs were sampled at each of the following sample hours: I, 4, 8, 12, 24, and 48. The start time zero osmolalities from the marked crabs was used to determine the average haemolymph osmolality upon acclimation to 27 ppt sea-water. (3) To determine the rate of acclimation upon gradual salinity changes, eight large P. herbstii were acclimated for 1 week in 35 ppt sea-water. Five crabs were placed in 35 ppt salt water and salinity was decreased each hour by 2ppt until a minimum salinity of 23 ppt. The salinity was then increased by 2ppt every hour until 35 ppt, the initial salinity. Haemolymph was drawn at time zero and every 3 hr thereafter for a total of 12 hr using the second sample technique. The other three crabs were used as a control and remained in the 35 ppt sea-water for the duration of the experiment. Their haemolymph was sampled at the same time and by the same technique as used for the other five crabs. (4) The osmotic adjustments during a natural tidal cycle were determined for five crabs which were placed in perforated plastic chambers in crab cages suspended from the Duke University Marine Laboratory’s dock. Mud crabs were placed in their containers and suspended from the dock, immediately after collection, 1 day prior to testing. Using the second technique, haemolymph samples were drawn from each crab at time zero and at 3-hr intervals for a total of 12 hr (one tidal cycle). Water samples were collected at the same intervals at the depth of the crabs with a Van Dorn bottle. Salinity was measured with a refractometer and converted to mOsm.
7 13008 E 1100 5 4 0 900 I g Z 2 70011 Z 3
/,
/
/
/
/
/
/
/
/
/
/
/
300. ,/’ / / , , , , , , , , , , 300 500 700 900 1100 WATER OSMOLALITY
, 1300
(m0sm)
Fig. 1. Haemolymph osmolality of mud crabs acclimated to various salinities as a function of external osmolality. Each point represents the mean k SE of 3 mud crabs. The dashed line depicts the isosmotic line.
lators below 28ppt and osmoconform at salinities above 28 ppt. Similarly, a relatively constant chloride ion concentration was maintained between 164 (10ppt) and about 492 meq/l (30 ppt) which indicates ionic regulation of the haemolymph (Fig. 2). Haemolymph [Cl-] was hyperionic from 164 (10 ppt) to 316 meq/l (18-19 ppt) whereupon it became hypoionic. After 492 meq/l(30 ppt), [Cl-] parallels the isoionic line but still remains hypoionic by approximately 160 meq/I. Rate of acclimation to sudden salinity changes
When P. herbstii acclimated at 27 ppt were transferred to 19ppt, their haemolymph osmolality decreased by about 60 mOsm in 4 hr, whereupon it stabilized near the value upon acclimation to 19 ppt (Fig. 3). On the other hand, when transferred from 27 to 35 ppt, haemolymph osmolality slowly increased
Analysis
,’
The osmolality and chloride ion concentration of the haemolymph and salt water samples were analysed using a Vapor Pressure Osmometer (Model 5100B, Wescor, Inc.) and a chloride titrator (Radiometer, Model CMTIO) respectively. The haemolymph used for the chloride determinations was diluted IO-fold with deionized water to prevent coagulation in the chloride titrator reaction solution.
/
/
/
1
,’ ,’ ,’
TOO-
s2
,’ ,’
i:_ 5 600
~
,,;,,,,,,,
:_
RESULTS The relationship of haemolymph osmolality and [Cl-] to the external osmolality upon acclimation Between
295 (10 ppt) and 830 mOsm (28 ppt), the
haemolymph osmolality remained relatively constant and hyperosmotic to the external medium (Fig. 1). At about 830mOsm (28 ppt) and higher osmolalities, however, the haemolymph osmolality became isosmotic with the external medium (Fig. 1). These results indicate that mud crabs are good hyperosmotic regu-
,’ 200
’
p I
/”
I 200
J
300
400
500
WATER kIMI
600
700
(mea/L1
Fig. 2. Haemolymph [Cl-] of mud crabs acclimated to various salinities as a function of external [Cl-]. Each point represents the mean k SE of 3 mud crabs. The dashed line depicts the isoionic line.
Crab osmoregulation
g
2 I 8 ‘$
however, the osmolality then decreased by about 50 mOsm (Fig. 4). These trends suggest that: (1) P. her&ii requires longer than 48 hr to completely acclimate to higher salinities, and (2) mud crabs possess an ability to hypoosmotically regulate for at least 48 hr after exposure to an increase in salinity. Since this experiment was also designed to test the haemolymph sampling techniques, it is noteworthy that both techniques (sampling once from each crab and sampling repeatedly from a single crab) provided similar results (Figs 3 and 4). Moreover, the curves depicting the repeated use of a single crab are smoother than the curves of the crabs used once (Figs 3 and 4). These results indicate that repeated sampling from a single crab provides a reliable indication of the crab’s osmotic response upon changes in the external osmolality.
1
880-
3
137
B
I
840-
I
Rate of acclimation to a gradual salinity change
10
-I
1
20
30
TIME
40
50
(hrs)
Fig. 3. Time course of osmotic changes when mud crabs acclimated to 27ppt salt water were suddenly exposed to 19 ppt salt water. Two sampling techniques were used: two different mud crabs were sampled at each time during the experimental period (A) and 3 mud crabs were sampled repeatedly throughout the experiment (B). Points represent the mean & SE. The dashed line indicates the average osmolality of 3 mud crabs which were acclimated to 19 ppt. about 100 mOsm in the course of 24 hr (Fig. 4). At 24 hr, the haemolymph osmolality reached a maximum value of 1OlOmOsm but remained approximately 60 mOsm below the value upon acclimation to 35 ppt. Instead of remaining constant or increasing,
1
0
IO
1
20
30
40
The osmolality of the control crabs remained relatively constant around 1052 mOsm when acclimated to 35 ppt and sampled repeatedly at 3 hr intervals (Fig. 5A) for 12 hr. This again indicates that haemolymph osmolality is not affected by repeated sampling at this interval. When subjected to a gradual change in salinity from 35 (1040 mOsm) to 23 ppt (680 mOsm), haemolymph osmolality steadily but slowly decreased by 75 mOsm (Fig. 5B). However, although the salinity was increased again to 35 ppt, haemolymph osmolality remained relatively constant at 965 mOsm (Fig. 5B). These results indicate the mud crab can regulate hypoosmotically for a period of time at salinities higher than 28 ppt. Osmotic adjustments in the field The haemolymph osmolality remained relatively constant around 910 + 10 mOsm when P. herbstii was exposed to a 5 ppt;dal cycle in the field (Fig. 6). Haemolymph osmolality was hyperosmotic to the external medium until 1700 hr, whereupon it became hypoosmotic by about 35 mOsm. At 1900 hr, hae-
‘50
TIME (hrs)
Fig. 4. Time course of osmotic changes when mud crabs acclimated to 27 ppt salt water were suddenly exposed to 35 ppt salt water. Two sampling techniques were used: two mud crabs were sampled at each time during the experimental period (A) and 3 mud crabs were sampled repeatedly throughout the experiment (B). Points represent the mean + SE. The dashed line indicates the average osmolality of 3 mud crabs which were acclimated to 35 ppt.
I
3
5 TIME
7
9
II
(hrs)
Fig. 5. Time course of osmotic changes upon constant exposure to 35 ppt salt water (A) and upon a gradual change in salinity (B). Mud crabs were repeatedly sampled. The solid line is haemolymph osmolality. ‘The dashed line is the external osmolality. Each point represents the mean + SE of 3 (A) and of 5 mud crabs (B), respectively.
ELIZABETHBLASCOand RICHARD B. FORWARD JR
138
u
I
0900
I
1100
a
1300
TIME
I
I
1500
I
u
1700
18
1900
I
-
I
2100
(hrs)
Changes in osmolality during a natural tidal cycle. The solid line is haemolymph osmolality, while the dashed line is external osmolality. Each point represents the mean k SE of 5 mud crabs.
Fig. 6.
molymph osmolality became hyperosmotic again. Thus, mud crabs can osmotically regulate for periods of time in salinities to which they osmoconform upon week-long acclimation. DISCUSSION
When P. herbstii was acclimated to salinities lower than 28 ppt its haemolymph osmolality was maintained at a constant level (Fig. 1). At salinities higher than 28 ppt, haemolymph osmolality was isosmotic to the external medium (Fig. 1). Thus, P. herbstii is a hyperosmoregulator at low salinities and an osmoconformer at high salinities. This pattern of osmoregulation is common among other marine euryhaline crustaceans, such as Carcinus maenus, Eriocheir sinensis Callinectes sapidus, and (Schoffeniels and Gilles, 1970; Pequeux et al., 1979; Gilles and Pequeux, 1983). In particular, C. sapidus, another estuarine crab, is similar because it also hyperosmotically regulates at salinities lower than 28 ppt (Tagatz, 1971). In dilute media (< 19 ppt), the hyperosmotic concentration of the haemolymph was partially maintained by chloride ions since between 10 and 19 ppt [Cl-] was regulated at a relatively constant hyperionic level (Fig. 2). At salinities between 19 and 30ppt, however, haemolymph [Cl-] was hypoionically regulated (Fig. 2). These results are in agreement with Boone and Claybrook’s (1977) data for P. herbstii’s [Cl-] in a variety of salinities. By comparison, C. sapidus also maintains its [Cl-] hyperionic at low salinities and hypoionic at high salinities (Lynch et al., 1973). When suddenly exposed to 19 ppt after being acclimated to 27 ppt, the mud crabs’ haemolymph osmolality stabilized after 4 hr (Fig. 3). This readjustment of osmolality following a hypoosmotic shock is much quicker than in other euryhaline crustaceans. Most crustaceans required 12-24 hr to stabilize their osmolality (Tagatz, 1971: C. sapidus; Felder, 1972: Callianassa jamaicense; Engel and Nichols, 1977: CaNinectes sapidus). Longer acclimation times, however, may be expected since these other crustaceans were subjected to a 2-4 times greater osmotic shock than that experienced by P. herbstii.
Upon transfer from 27 to 35 ppt, P. herbstii’s haemolymph osmolality slowly increased for 24 hr, reaching a peak 60 mOsm below the acclimation values and then slowly decreased by about 50 mOsm
over the remainder of the experiment (Fig. 4). This indicates that the mud crab can hypoosmotically regulate for short periods of time in salinities greater than 28 ppt. This pattern is not apparent among other crustaceans which have been exposed to a sudden increase in salinity. For example, Engel and Nichols (1977) determined that Callinectes sapidus reached a stable haemolymph conductivity 12- 16 hr after being placed in 35 ppt from 5 ppt sea-water. Furthermore, Siebers (1972) found that Carcinus maenus readjusted its haemolymph osmolality within 24 hr of a 27 ppt salinity increase. Callianassa jamaicerise, though, required longer than 24 hr to reequilibrate: its osmolality reached a stable state 4 days after exposure from 20 to 37ppt even though normal cell volume was restored after 2 days (Felder, 1972). These results are not necessarily incongruous, though, because the mud crab displays an ability to hypoosmotically regulate at salinities higher than 28 ppt only when exposed to small increases in salinity (8 ppt). The crustaceans in the other experiments were exposed to much larger salinity increases (17-30 ppt). A minor goal of this experiment was to determine whether repeated sampling of a single crab’s haemolymph yielded accurate results and how these results compared to the data obtained from sampling a crab once during an experiment, the traditional approach. Repeated sampling of a single crab was found to be a valid technique. The osmolality of repeatedly sampled crabs acclimated at 35 ppt remained relatively constant even though these crabs were sampled five times in 12 hr (Fig. SA). Further, there was no difference between the osmolalities of repeatedly sampled crabs and those of crabs sampled only once (Figs 3 and 4). These findings are supported by Ballard and Abbott (1969) who determined that the daily bleeding of C. sapidus had no significant effect on its haemolymph osmolality. Thus, either technique can be used although, for time course experiments, the repeated sampling technique would be more advantageous because it requires fewer animals. Hyper-hypoosmotic regulating capabilities were again demonstrated by P. herbstii when it was exposed to a gradual 35-23-35 ppt salinity cycle (Fig. 5B). Haemolymph osmolality decreased slightly as external salinity decreased, but then remained constant as salinity increased again (Fig. 5B). When salinity increased past about 33 ppt, haemolymph osmolality, which had been hyperosmotic to the
Crab osmoregulation external medium, became hypoosmotic (Fig. 5B). These results are different from Findley and Stickle’s (1978) findings that the haemolymph osmolality of C. supidus, which was exposed to a 30-l&30 ppt salinity cycle, declined slightly by the 10th hour, then returned to its original concentrations. Findley and Stickle, however, conducted their salinity cycle on a diurnal time scale whereas P. herbstii was exposed to a semidiurnal cycle. On the other hand, when exposed to a low-highlow salinity cycle, mud crabs (Fig. 6) and C. supidus (Findley and Stickle, 1978) had similar osmotic patterns even though the time scale was different. Both maintained constant haemolymph concentrations, which were initially hyperosmotic, but became hypoosmotic as external osmolahty exceeded the haemolymph osmolality (Fig. 6; Findley and Stickle, 1978). Furthermore, as salinity again decreased, both species became hyperosmotic once more. Based on low values for permeability in the range in which Curcinus maenus shows strong regulation of haemolymph electrolyte concentration, Spaargaren (1974) proposed that the effect of salinity fluctuations on haemolymph concentrations would be damped. Furthermore, he predicted, based on permeability values and a mathematical model for sinusoidal fluctuations in salinity, that variations in haemolymph concentrations for C. maenus during a 12-hr tidal cycle would be one third of that of the fluctuations in the external medium. Assuming P. hrrbstii has similar permeability values, the present study supports Spaargaren’s (1974) prediction, since fluctuations in the haemolymph osmolahty of P. herbstii over a gradual salinity change and over a natural tidal cycle are much less than the variations in the external medium (Figs 5B and 6). In fact, P. herbstii haemolymph osmolahty is one fifth of the amount of the external medium upon gradual salinity changes in the laboratory (Fig. 5B) and l/9 that of the external medium in the field (Fig. 6). The lower values found in this study may be a result of differences in permeability or, since mud crabs appear to hypo and hyperosmotically regulate, other mechanisms besides lower permeability. In conclusion, upon long-term acclimation P. herbstii hyperosmotically regulates at salinities less than 28 ppt and osmoconforms at salinities greater than 28 ppt. When subjected to sudden decreases in salinity the crab’s haemolymph osmolality stabilizes within 4 hr. When exposed to sudden increases in salinity, however, haemolymph osmolality does not reequilibrate in 48 hr. Instead, the mud crab appears to hypoosmotically regulate. Hypoosmoregulation is again displayed along with hyperosmoregulation when the mud crabs are subjected to gradual salinity cycles in the laboratory and to tidal cycles in the field.
139
The net result is maintenance of a relatively constant haemolymph osmolality when exposed to a tidal cycle of salinity change. Acknowledgements-This material is based on research supported by the Science Foundation Grant Number OCE-8603945. We thank Dr A. Shanks for critically reading the manuscript.
REFERENCES
Ballard B. S. and Abbott W. (1969) Osmotic accommodation in Callinectes sapidus (Rathbun). Comp. Biochem. Physiol. 29, 67 l-687. Boone W. R. and Claybrook D. L. (1977) The effect of low salinity on amino acid metabolism in the tissues of the common mud crab, Panopeus herbstii (Milne-Edwards). Comp. Biochem. Physiol. SlA, 99-106. Engel D. W. and Nichols C. D. (1977) A method for continuous in viuo measurement of haemolymph conductivity in crabs. J. exp. Mar. Biol. Ecol. 26, 203-209.
Felder D. L. (1978) Osmotic and ionic regulation in several western Atlantic Callianassidae (Crustacea, decapoda, Thalassinidae). Biol. Bull. (Woods Hole, Mass.) 154, 409429. Findley A. M. and Stickle W. B. (1978) Effects of salinity fluctuations on the haemolymph composition of the blue crab, Callinectes sapidus. Mar. Biol. 46, 9-15. Gilles R. and Pequeux A. (1983)Interaction of chemical and osmotic regulation with the environment. In The Biology offhe Crusfacea. (Edited by Vernberg F. J. and Vernberg W. B.), pp. 1099178. Academic Press, New York. Gilles R. and Pequeux A. (1981) Cell volume regulation in crustaceans: Relationship between mechanisms for controlling the osmolality of extracellular and intracellular fluids. J. exp. Zool. 215, 351-362. Lynch M. P., Webb K. L. and Van Engel W. A. (1973) Variations in serum constituents of the blue crab Callinectes sapidus: chloride and osmotic concentrations. Comp. Biochem. Physiol. 44A, 719-734. Pequeux A., Vallota A. C. and Gilles R. (1979) Blood proteins as related to osmoregulation in Crustacea. Comp. Biochem. Physiol. 64, 433175. Schoffeniels E. and Gilles R. (1970) Osmoregulation in aquatic arthropods. In Chemical Zoology (Edited by Florkin M. and Scheer B. T.), Vol. 5, _ pp. . 255-286. Academic Press, New York. Siebers D.. Lucu C.. Soerline K.R.. and Eberlein K. (1972) Kinetics’ of osmoregulatioi in the crab Carcinus mbenus: Mar. Biol. 17, 291-303. Spaargaren D. H. (1974) A study on the adaptation of marine organisms to changing salinities with special references to the shore crab Carcinus maenus. Comp. Biochem. Physiol. 47, 499-5 12. Tagatz M. E. (1971) Osmoregulatory ability of the blue crabs in different temperature-salinity combinations. Chesapeake Sci. 12, 14-17. Williams A. B. (1984) Shrimps, Lobsters and Crabs of the Atlantic Coasr of the Eastern United Slates, Maine IO Florida. pp. I-550. Smithsonian Institution Press, Washington D.C.
Vol. 90A,
No.
I, pp.
135-139,
1988
Printed in Great
0
OSMOREGULATION OF THE XANTHID PANOPEUS HERBSTII
0300-9629/88 $3.00 + 0.00 1988 Pergamon Press plc
CRAB,
ELIZABETHBLASCOand RICHARDB. FORWARD,JR Duke University Marine Laboratory Beaufort, NC 28516, USA. Telephone: (919) 728-2111; and Zoology Department, Duke University, Durham, NC 28516 USA
(Received 11 September 1987)
Ahstraet-1. After acclimation: the mud crab, Punopeus herbsfii, hyperosmotically regulates between 10 and 28 ppt and osmoconforms above 28 ppt. Chloride ion concentration remained at a relatively constant hyperionic level at lower salinities and became hypoionic at high salinities. 2. The rate of acclimation to hyper and hypoosmotic shocks varied. Upon a sudden change from 27 to 19 ppt, haemolymph osmolality stabilized within 4 hr. Upon a sudden change from 27 to 35 ppt, crabs did not equilibrate their osmolality within 48 hr. Instead, haemolymph osmolality remained hypoosmotic. 3. A gradual 35-23-35 ppt change in salinity over 12 hr resulted in an initial 75 mOsm decrease in osmolality. As salinity increased, the mud crab regulated hyperosmotically until 33 ppt and hypoosmotically thereafter. 4. Upon exposure to a salinity qycle in the field, haemolymph osmolality remained relatively constant. Thus, P. herbstii is capable of hypkr-hypoosmoregulation when exposed to short-term changes in salinity. This conclusion is not suggested by the osmotic relationship upon acclimation.
INTRODUCTION Marine euryhaline decapods which inhabit estuaries are subjected to fluctuating salinities as a result of the tides. Due to the permeable nature of crustaceans’ gill tissues, these salinity changes have the potential to alter the osmotic and ionic concentrations of the crustaceans’ haemolymph. Since osmotic and ionic concentrations affect cell volume, these oscillations in concentrations must be regulated because proper cellular functioning requires a relatively constant cell volume. The degree to which crustaceans can osmoregulate as well as the mechanisms they utilize to cope with salinity fluctuations has been well studied (for reviews, see Schoeffeniels and Gilles, 1970; Gilles and Pequeux, 1983). The effect of sudden hypo and hyperosmotic shocks has also been explored in some detail (Siebers et al., 1972; Engel and Nichols, 1977; Gilles and Pequeux, 1981). In these past experiments, however, the salinity changes were in the order of 20-30ppt which are rare occurrences under normal conditions in estuaries. Thus, while these studies detail the crustacean’s ability to adjust to extreme changes, they do not illustrate how crabs cope with normal salinity changes. Studies using gradual changes in salinity are more realistic, but few in number (Spaargaren, 1974; Findley and Stickle, 1978). Finally, studies on the effect of salinity fluctuations in the field are virtually non-existent. The present investigation was undertaken to compare osmotic responses of a crab to constant and changing external osmolalities. Panopeus herbsrii is an estuarine mud crab and, as such, is potentially subjected to daily fluctuations in salinity (Williams, 1984). In this study, the rates of acclimation to sudden and gradual salinity changes
were determined. In addition, the effect of natural tidal cycle in the field on mud crabs’ haemolymph osmolality was established. It was found that upon acclimation P. herbstii osmoconforms at high salinities and osmoregulates at low salinities. Upon changes in salinity, it can hyper-hypoosmoregulate for short periods of time in salinities where it osmoconforms upon acclimation.
MATERIALSAND METHODS The mud crab, Punopeus herbsrii (H. Milne Edwards), was collected without regards to sex at Beaufort, North Carolina, USA during the months of January-April. The mud crabs were collected l-2 days prior to each experimental period, which lasted no longer than 2 weeks. They were kept unfed in aerated sea-water (18.5”C). Salinities used in the experiments were varied by adding either Instant Ocean or deionized water to sea-water. A refractometer (A.O.) was use& to measure salinity. There were four series of experiments and two sampling techniques. Approximately 0.1 ml of haemolymph was drawn from the chelipeds using a Number 19 syringe needle. Haemolymph samples were frozen immediately and analysed within 3 days. Two different sampling techniques were used. In the first technique, haemolymph samples were drawn from a crab once during the experiment. In the second technique, a single crab’s haemolymph was sampled continuously at every sampling time throughout the course of the experiment. Experiments
(1) The relationship between the crabs’ haemolymph and chloride ion concentration and the external osmolality was determined after acclimating three mud crabs for one week in each of the following salinities: 10, 15, 20, 25, 30, 35, and
135
ELIZABETH BLASCO and RICHARD B. FORWARD JR
136
40ppt. Their haemolymph was sampled using the first sampling technique. (2) The rate of acclimation upon sudden salinity changes was conducted at salinities of 19, 27, and 35 ppt. These salinities were chosen because the mud crabs’ osmoregulatory abilities change from regulating to conforming around 27 ppt (Fig. 1). To determine the haemolymph osmolality upon acclimation to 19 and 35 ppt, three mud crabs were acclimated for 1week at each salinity. Their haemolymph was sampled, and the results averaged. During the same period, thirty mud crabs were acclimated at 27 ppt. After 1 week, I5 crabs were placed in 19 ppt sea-water and 15 crabs in 35 ppt sea-water. Three of the transferred crabs in each salinity were identified by their markings and their haemolymph was sampled using the second sampling technique at 0, 1, 4, 8, I?, 24, and 48 hr after the transfer. The remaining 12 crabs in each salinity were sampled according to the first sampling technique. In this case, two crabs were sampled at each of the following sample hours: I, 4, 8, 12, 24, and 48. The start time zero osmolalities from the marked crabs was used to determine the average haemolymph osmolality upon acclimation to 27 ppt sea-water. (3) To determine the rate of acclimation upon gradual salinity changes, eight large P. herbstii were acclimated for 1 week in 35 ppt sea-water. Five crabs were placed in 35 ppt salt water and salinity was decreased each hour by 2ppt until a minimum salinity of 23 ppt. The salinity was then increased by 2ppt every hour until 35 ppt, the initial salinity. Haemolymph was drawn at time zero and every 3 hr thereafter for a total of 12 hr using the second sample technique. The other three crabs were used as a control and remained in the 35 ppt sea-water for the duration of the experiment. Their haemolymph was sampled at the same time and by the same technique as used for the other five crabs. (4) The osmotic adjustments during a natural tidal cycle were determined for five crabs which were placed in perforated plastic chambers in crab cages suspended from the Duke University Marine Laboratory’s dock. Mud crabs were placed in their containers and suspended from the dock, immediately after collection, 1 day prior to testing. Using the second technique, haemolymph samples were drawn from each crab at time zero and at 3-hr intervals for a total of 12 hr (one tidal cycle). Water samples were collected at the same intervals at the depth of the crabs with a Van Dorn bottle. Salinity was measured with a refractometer and converted to mOsm.
7 13008 E 1100 5 4 0 900 I g Z 2 70011 Z 3
/,
/
/
/
/
/
/
/
/
/
/
/
300. ,/’ / / , , , , , , , , , , 300 500 700 900 1100 WATER OSMOLALITY
, 1300
(m0sm)
Fig. 1. Haemolymph osmolality of mud crabs acclimated to various salinities as a function of external osmolality. Each point represents the mean k SE of 3 mud crabs. The dashed line depicts the isosmotic line.
lators below 28ppt and osmoconform at salinities above 28 ppt. Similarly, a relatively constant chloride ion concentration was maintained between 164 (10ppt) and about 492 meq/l (30 ppt) which indicates ionic regulation of the haemolymph (Fig. 2). Haemolymph [Cl-] was hyperionic from 164 (10 ppt) to 316 meq/l (18-19 ppt) whereupon it became hypoionic. After 492 meq/l(30 ppt), [Cl-] parallels the isoionic line but still remains hypoionic by approximately 160 meq/I. Rate of acclimation to sudden salinity changes
When P. herbstii acclimated at 27 ppt were transferred to 19ppt, their haemolymph osmolality decreased by about 60 mOsm in 4 hr, whereupon it stabilized near the value upon acclimation to 19 ppt (Fig. 3). On the other hand, when transferred from 27 to 35 ppt, haemolymph osmolality slowly increased
Analysis
,’
The osmolality and chloride ion concentration of the haemolymph and salt water samples were analysed using a Vapor Pressure Osmometer (Model 5100B, Wescor, Inc.) and a chloride titrator (Radiometer, Model CMTIO) respectively. The haemolymph used for the chloride determinations was diluted IO-fold with deionized water to prevent coagulation in the chloride titrator reaction solution.
/
/
/
1
,’ ,’ ,’
TOO-
s2
,’ ,’
i:_ 5 600
~
,,;,,,,,,,
:_
RESULTS The relationship of haemolymph osmolality and [Cl-] to the external osmolality upon acclimation Between
295 (10 ppt) and 830 mOsm (28 ppt), the
haemolymph osmolality remained relatively constant and hyperosmotic to the external medium (Fig. 1). At about 830mOsm (28 ppt) and higher osmolalities, however, the haemolymph osmolality became isosmotic with the external medium (Fig. 1). These results indicate that mud crabs are good hyperosmotic regu-
,’ 200
’
p I
/”
I 200
J
300
400
500
WATER kIMI
600
700
(mea/L1
Fig. 2. Haemolymph [Cl-] of mud crabs acclimated to various salinities as a function of external [Cl-]. Each point represents the mean k SE of 3 mud crabs. The dashed line depicts the isoionic line.
Crab osmoregulation
g
2 I 8 ‘$
however, the osmolality then decreased by about 50 mOsm (Fig. 4). These trends suggest that: (1) P. her&ii requires longer than 48 hr to completely acclimate to higher salinities, and (2) mud crabs possess an ability to hypoosmotically regulate for at least 48 hr after exposure to an increase in salinity. Since this experiment was also designed to test the haemolymph sampling techniques, it is noteworthy that both techniques (sampling once from each crab and sampling repeatedly from a single crab) provided similar results (Figs 3 and 4). Moreover, the curves depicting the repeated use of a single crab are smoother than the curves of the crabs used once (Figs 3 and 4). These results indicate that repeated sampling from a single crab provides a reliable indication of the crab’s osmotic response upon changes in the external osmolality.
1
880-
3
137
B
I
840-
I
Rate of acclimation to a gradual salinity change
10
-I
1
20
30
TIME
40
50
(hrs)
Fig. 3. Time course of osmotic changes when mud crabs acclimated to 27ppt salt water were suddenly exposed to 19 ppt salt water. Two sampling techniques were used: two different mud crabs were sampled at each time during the experimental period (A) and 3 mud crabs were sampled repeatedly throughout the experiment (B). Points represent the mean & SE. The dashed line indicates the average osmolality of 3 mud crabs which were acclimated to 19 ppt. about 100 mOsm in the course of 24 hr (Fig. 4). At 24 hr, the haemolymph osmolality reached a maximum value of 1OlOmOsm but remained approximately 60 mOsm below the value upon acclimation to 35 ppt. Instead of remaining constant or increasing,
1
0
IO
1
20
30
40
The osmolality of the control crabs remained relatively constant around 1052 mOsm when acclimated to 35 ppt and sampled repeatedly at 3 hr intervals (Fig. 5A) for 12 hr. This again indicates that haemolymph osmolality is not affected by repeated sampling at this interval. When subjected to a gradual change in salinity from 35 (1040 mOsm) to 23 ppt (680 mOsm), haemolymph osmolality steadily but slowly decreased by 75 mOsm (Fig. 5B). However, although the salinity was increased again to 35 ppt, haemolymph osmolality remained relatively constant at 965 mOsm (Fig. 5B). These results indicate the mud crab can regulate hypoosmotically for a period of time at salinities higher than 28 ppt. Osmotic adjustments in the field The haemolymph osmolality remained relatively constant around 910 + 10 mOsm when P. herbstii was exposed to a 5 ppt;dal cycle in the field (Fig. 6). Haemolymph osmolality was hyperosmotic to the external medium until 1700 hr, whereupon it became hypoosmotic by about 35 mOsm. At 1900 hr, hae-
‘50
TIME (hrs)
Fig. 4. Time course of osmotic changes when mud crabs acclimated to 27 ppt salt water were suddenly exposed to 35 ppt salt water. Two sampling techniques were used: two mud crabs were sampled at each time during the experimental period (A) and 3 mud crabs were sampled repeatedly throughout the experiment (B). Points represent the mean + SE. The dashed line indicates the average osmolality of 3 mud crabs which were acclimated to 35 ppt.
I
3
5 TIME
7
9
II
(hrs)
Fig. 5. Time course of osmotic changes upon constant exposure to 35 ppt salt water (A) and upon a gradual change in salinity (B). Mud crabs were repeatedly sampled. The solid line is haemolymph osmolality. ‘The dashed line is the external osmolality. Each point represents the mean + SE of 3 (A) and of 5 mud crabs (B), respectively.
ELIZABETHBLASCOand RICHARD B. FORWARD JR
138
u
I
0900
I
1100
a
1300
TIME
I
I
1500
I
u
1700
18
1900
I
-
I
2100
(hrs)
Changes in osmolality during a natural tidal cycle. The solid line is haemolymph osmolality, while the dashed line is external osmolality. Each point represents the mean k SE of 5 mud crabs.
Fig. 6.
molymph osmolality became hyperosmotic again. Thus, mud crabs can osmotically regulate for periods of time in salinities to which they osmoconform upon week-long acclimation. DISCUSSION
When P. herbstii was acclimated to salinities lower than 28 ppt its haemolymph osmolality was maintained at a constant level (Fig. 1). At salinities higher than 28 ppt, haemolymph osmolality was isosmotic to the external medium (Fig. 1). Thus, P. herbstii is a hyperosmoregulator at low salinities and an osmoconformer at high salinities. This pattern of osmoregulation is common among other marine euryhaline crustaceans, such as Carcinus maenus, Eriocheir sinensis Callinectes sapidus, and (Schoffeniels and Gilles, 1970; Pequeux et al., 1979; Gilles and Pequeux, 1983). In particular, C. sapidus, another estuarine crab, is similar because it also hyperosmotically regulates at salinities lower than 28 ppt (Tagatz, 1971). In dilute media (< 19 ppt), the hyperosmotic concentration of the haemolymph was partially maintained by chloride ions since between 10 and 19 ppt [Cl-] was regulated at a relatively constant hyperionic level (Fig. 2). At salinities between 19 and 30ppt, however, haemolymph [Cl-] was hypoionically regulated (Fig. 2). These results are in agreement with Boone and Claybrook’s (1977) data for P. herbstii’s [Cl-] in a variety of salinities. By comparison, C. sapidus also maintains its [Cl-] hyperionic at low salinities and hypoionic at high salinities (Lynch et al., 1973). When suddenly exposed to 19 ppt after being acclimated to 27 ppt, the mud crabs’ haemolymph osmolality stabilized after 4 hr (Fig. 3). This readjustment of osmolality following a hypoosmotic shock is much quicker than in other euryhaline crustaceans. Most crustaceans required 12-24 hr to stabilize their osmolality (Tagatz, 1971: C. sapidus; Felder, 1972: Callianassa jamaicense; Engel and Nichols, 1977: CaNinectes sapidus). Longer acclimation times, however, may be expected since these other crustaceans were subjected to a 2-4 times greater osmotic shock than that experienced by P. herbstii.
Upon transfer from 27 to 35 ppt, P. herbstii’s haemolymph osmolality slowly increased for 24 hr, reaching a peak 60 mOsm below the acclimation values and then slowly decreased by about 50 mOsm
over the remainder of the experiment (Fig. 4). This indicates that the mud crab can hypoosmotically regulate for short periods of time in salinities greater than 28 ppt. This pattern is not apparent among other crustaceans which have been exposed to a sudden increase in salinity. For example, Engel and Nichols (1977) determined that Callinectes sapidus reached a stable haemolymph conductivity 12- 16 hr after being placed in 35 ppt from 5 ppt sea-water. Furthermore, Siebers (1972) found that Carcinus maenus readjusted its haemolymph osmolality within 24 hr of a 27 ppt salinity increase. Callianassa jamaicerise, though, required longer than 24 hr to reequilibrate: its osmolality reached a stable state 4 days after exposure from 20 to 37ppt even though normal cell volume was restored after 2 days (Felder, 1972). These results are not necessarily incongruous, though, because the mud crab displays an ability to hypoosmotically regulate at salinities higher than 28 ppt only when exposed to small increases in salinity (8 ppt). The crustaceans in the other experiments were exposed to much larger salinity increases (17-30 ppt). A minor goal of this experiment was to determine whether repeated sampling of a single crab’s haemolymph yielded accurate results and how these results compared to the data obtained from sampling a crab once during an experiment, the traditional approach. Repeated sampling of a single crab was found to be a valid technique. The osmolality of repeatedly sampled crabs acclimated at 35 ppt remained relatively constant even though these crabs were sampled five times in 12 hr (Fig. SA). Further, there was no difference between the osmolalities of repeatedly sampled crabs and those of crabs sampled only once (Figs 3 and 4). These findings are supported by Ballard and Abbott (1969) who determined that the daily bleeding of C. sapidus had no significant effect on its haemolymph osmolality. Thus, either technique can be used although, for time course experiments, the repeated sampling technique would be more advantageous because it requires fewer animals. Hyper-hypoosmotic regulating capabilities were again demonstrated by P. herbstii when it was exposed to a gradual 35-23-35 ppt salinity cycle (Fig. 5B). Haemolymph osmolality decreased slightly as external salinity decreased, but then remained constant as salinity increased again (Fig. 5B). When salinity increased past about 33 ppt, haemolymph osmolality, which had been hyperosmotic to the
Crab osmoregulation external medium, became hypoosmotic (Fig. 5B). These results are different from Findley and Stickle’s (1978) findings that the haemolymph osmolality of C. supidus, which was exposed to a 30-l&30 ppt salinity cycle, declined slightly by the 10th hour, then returned to its original concentrations. Findley and Stickle, however, conducted their salinity cycle on a diurnal time scale whereas P. herbstii was exposed to a semidiurnal cycle. On the other hand, when exposed to a low-highlow salinity cycle, mud crabs (Fig. 6) and C. supidus (Findley and Stickle, 1978) had similar osmotic patterns even though the time scale was different. Both maintained constant haemolymph concentrations, which were initially hyperosmotic, but became hypoosmotic as external osmolahty exceeded the haemolymph osmolality (Fig. 6; Findley and Stickle, 1978). Furthermore, as salinity again decreased, both species became hyperosmotic once more. Based on low values for permeability in the range in which Curcinus maenus shows strong regulation of haemolymph electrolyte concentration, Spaargaren (1974) proposed that the effect of salinity fluctuations on haemolymph concentrations would be damped. Furthermore, he predicted, based on permeability values and a mathematical model for sinusoidal fluctuations in salinity, that variations in haemolymph concentrations for C. maenus during a 12-hr tidal cycle would be one third of that of the fluctuations in the external medium. Assuming P. hrrbstii has similar permeability values, the present study supports Spaargaren’s (1974) prediction, since fluctuations in the haemolymph osmolahty of P. herbstii over a gradual salinity change and over a natural tidal cycle are much less than the variations in the external medium (Figs 5B and 6). In fact, P. herbstii haemolymph osmolahty is one fifth of the amount of the external medium upon gradual salinity changes in the laboratory (Fig. 5B) and l/9 that of the external medium in the field (Fig. 6). The lower values found in this study may be a result of differences in permeability or, since mud crabs appear to hypo and hyperosmotically regulate, other mechanisms besides lower permeability. In conclusion, upon long-term acclimation P. herbstii hyperosmotically regulates at salinities less than 28 ppt and osmoconforms at salinities greater than 28 ppt. When subjected to sudden decreases in salinity the crab’s haemolymph osmolality stabilizes within 4 hr. When exposed to sudden increases in salinity, however, haemolymph osmolality does not reequilibrate in 48 hr. Instead, the mud crab appears to hypoosmotically regulate. Hypoosmoregulation is again displayed along with hyperosmoregulation when the mud crabs are subjected to gradual salinity cycles in the laboratory and to tidal cycles in the field.
139
The net result is maintenance of a relatively constant haemolymph osmolality when exposed to a tidal cycle of salinity change. Acknowledgements-This material is based on research supported by the Science Foundation Grant Number OCE-8603945. We thank Dr A. Shanks for critically reading the manuscript.
REFERENCES
Ballard B. S. and Abbott W. (1969) Osmotic accommodation in Callinectes sapidus (Rathbun). Comp. Biochem. Physiol. 29, 67 l-687. Boone W. R. and Claybrook D. L. (1977) The effect of low salinity on amino acid metabolism in the tissues of the common mud crab, Panopeus herbstii (Milne-Edwards). Comp. Biochem. Physiol. SlA, 99-106. Engel D. W. and Nichols C. D. (1977) A method for continuous in viuo measurement of haemolymph conductivity in crabs. J. exp. Mar. Biol. Ecol. 26, 203-209.
Felder D. L. (1978) Osmotic and ionic regulation in several western Atlantic Callianassidae (Crustacea, decapoda, Thalassinidae). Biol. Bull. (Woods Hole, Mass.) 154, 409429. Findley A. M. and Stickle W. B. (1978) Effects of salinity fluctuations on the haemolymph composition of the blue crab, Callinectes sapidus. Mar. Biol. 46, 9-15. Gilles R. and Pequeux A. (1983)Interaction of chemical and osmotic regulation with the environment. In The Biology offhe Crusfacea. (Edited by Vernberg F. J. and Vernberg W. B.), pp. 1099178. Academic Press, New York. Gilles R. and Pequeux A. (1981) Cell volume regulation in crustaceans: Relationship between mechanisms for controlling the osmolality of extracellular and intracellular fluids. J. exp. Zool. 215, 351-362. Lynch M. P., Webb K. L. and Van Engel W. A. (1973) Variations in serum constituents of the blue crab Callinectes sapidus: chloride and osmotic concentrations. Comp. Biochem. Physiol. 44A, 719-734. Pequeux A., Vallota A. C. and Gilles R. (1979) Blood proteins as related to osmoregulation in Crustacea. Comp. Biochem. Physiol. 64, 433175. Schoffeniels E. and Gilles R. (1970) Osmoregulation in aquatic arthropods. In Chemical Zoology (Edited by Florkin M. and Scheer B. T.), Vol. 5, _ pp. . 255-286. Academic Press, New York. Siebers D.. Lucu C.. Soerline K.R.. and Eberlein K. (1972) Kinetics’ of osmoregulatioi in the crab Carcinus mbenus: Mar. Biol. 17, 291-303. Spaargaren D. H. (1974) A study on the adaptation of marine organisms to changing salinities with special references to the shore crab Carcinus maenus. Comp. Biochem. Physiol. 47, 499-5 12. Tagatz M. E. (1971) Osmoregulatory ability of the blue crabs in different temperature-salinity combinations. Chesapeake Sci. 12, 14-17. Williams A. B. (1984) Shrimps, Lobsters and Crabs of the Atlantic Coasr of the Eastern United Slates, Maine IO Florida. pp. I-550. Smithsonian Institution Press, Washington D.C.