Water balance of a euryhaline sea anemone, Diadumene leucolena

Comp. Biochm.

WATER

Physiol.,1974, Vol. 49A, pp. 159 to 167. PergnmonPress. Printed in Great Britain

BALANCE OF A EURYHALINE SEA ANEMONE, DIADUMENE LEUCOLENA*t

SIDNEY

K. PIERCE,

JR.

and LEO L. MINASIAN,

JR.

Department of Zoology, University of Maryland, College Park, Maryland 20742, U.S.A. (Received 1 August 1973)

Abstract-l. Diadumene leucolena is an osmoconformer in salinities ranging from 6 to 33 x0. 2. Over the entire non-lethal salinity range, tissue hydration varied by only 3.5 per cent. 3. Intracellular concentrations of taurine, glutamic acid, glycine and alanine increased by an amount far in excess of that predicted by tissue hydration changes. 4. Volume regulation in D. leucolena is accomplished by the regulation of an intracellular, osmotically active solute : the free amino acid pool.

INTRODUCTION ALTHOUGH

considerable work has been forthcoming on volume regulating mechanisms in fresh water coelenterates, this process has scarcely been examined in marine species. In company with most other fresh water forms, fresh water coelenterates utilize ions to prevent cellular swelling resulting from continual water influx from the hypoosmotic environment (Koblick & Yu-Tu, 1967; Hazelwood et al., 1970; Benos & Prusch, 1972, 1973). In marine animals, a similar process of cellular volume regulation by solute extrusion obtains but, in all species tested, the osmotic solute source is intracellular free amino acids rather than ions (see Emerson, 1969; Pierce, 1971, for reviews). Amongst the coelenterates, the free amino acid pool size has been reported for several species (siphonophores, Ceccaldi & Dumas, 1967, Dumas & Ceccaldi, 1973 ; scyphozoans, Severin et al., 1972; Webb et al., 1972; actinians, Kittredge et al., 1962, Severin et al., 1972). Most of these studies have ignored the effects of salinity on the free amino acid pool size. However, a direct relationship has been found between intracellular free amino acid concentration and salinity in the polyps of Amelia aurita and Chrysaora quinquecirrha (Webb et al., 1972). Unfortunately, Webb et al. did not measure changes in tissue water content. Therefore, although intracellular free amino acid pools exist in coelenterates and, in the scyphozoan polyp at least, vary in size with salinity, no * Supported by N.S.F. Grant No. GB-36347. t Contribution No. 25 from the Tallahassee, Biological Association. 159

Sopchoppy

and Gulf Coast Marine

160

SIDNEY K. PIERCE, JR. ANDLEO L. MINASIAN, JR.

study to date has compared tissue water changes, free amino acid pool size and salinity variation in a marine coelenterate. The study reported here investigated the effects of salinity change on the water balance and intracellular free amino acid pool size of Diadumene leucolena (Verrill), a small, euryhaline sea anemone common throughout the Chesapeake Bay system (Cory, 1967). Th e results of this study show that D. Zeucolena, although an osmoconformer, regulates cell volume in increasing salinities utilizing the intracellular free amino acids taurine, glutamic acid, glycine and alanine. MATERIALS

AND

METHODS

Animals and solutions Diadumene leucolena were collected from the surfaces of oyster shells which were suspended in wire mesh trays at a mean depth of 1 m in the mouth of the Patuxent River near Solomons, Maryland. The anemones were maintained in aerated artificial sea water (11x,) at 15°C under constant lighting at the College Park campus. All animals were kept at this salinity for at least 7 days prior to use in any experiment and they were starved throughout. All experimental solutions were prepared by diluting full-strength (36x0 = 1029 mOsmole/kg H,O) artificial sea water (Instant Ocean) with distilled water. The osmotic concentrations of all solutions were measured before use with a freezing point depression osmometer (Osmette, Precision Systems). Salinity

tolerance experiments

Twenty-five anemones were placed into finger bowls containing 1500 ml of sea water of various salinities (5, 6, 7, 11, 30, 31, 32, 33 or 34x,,). The experiment was run in duplicate so that a total of fifty animals were exposed to each salinity. All bowls were sealed with plastic wrap to minimize evaporation. The osmotic concentrations of the experimental solutions changed by no more than 6 mOsmole/kg H,O (0.2x,) during the course of the study. The anemones were examined daily for 8 days for mortality. Mortality was determined by the failure of the column and the tentacles to contract in response to mechanical stimulation with a metal probe (Sassaman & Mangum, 1970). Those salinities in which deaths exceeded 50 per cent were considered lethal. Osmotic acclimation of the coelenteron fluid A group of anemones were removed from 165 mOsmole/kg H,O, blotted and placed into an osmotic concentration of 100, 296, 490 or 695 mOsmole/kg H,O. Three 0.2-ml samples of coelenteron fluid were obtained at several time intervals (5,15,30,60,180,360, 720,144O min). The samples were centrifuged at 1230 g for 5 min and the osmotic concentration of the supernatant was determined with the osmometer. Fluid from the coelenteron was obtained by one of two procedures: the anemone was blotted and either allowed to contract, expelling the fluid contents of the coelenteron which were collected, or the body wall was slit open with a sharp blade and the fluid contents drained. Usually, fluid from several anemones was required to obtain the O-2-ml sample. The osmotic concentrations of the three samples from each salinity at each time interval were averaged. Volume and amino acid regulation Anemones were acclimated to four osmotic concentrations (200, 465, 69.5 and 890 mOsmole/kg HzO) for at least 2 weeks. Following acclimation, the animals were removed from the test solution and the oral disc, including the tentacles, was cut from each animal and discarded. Next, the coelenteron was slit open longitudinally and blotted to remove all external fluid. The tissue was weighed, frozen on dry ice, lyophilized overnight and then

SEA

WATERBALANCEOFAEURYHALINE

161

ANEMONE

reweighed. The percentage of water in the tissues was then calculated by dividing the difference between wet and dry weights by the wet weight (Pierce, 1971). The freeze-dried tissue was then homogenized in 3 ml distilled water using a motordriven Teflon homogenizer. The homogenate was centrifuged at 20,000 g for 10 min at 0°C to remove tissue debris. Protein was precipitated and removed from the supernatant by the addition of an equal volume of 20% aqueous sulfosalacylic acid and centrifugation at 20,000 g for 30 min. This supernatant was then frozen and lyophilized. The residue was dissolved in an appropriate volume of sodium citrate buffer (pH 2.20) and the amino acid composition of this final solution was determined with an amino acid analyser (JEOL JLC6AH). Finally, in order to test for the effects of starvation on the amino acid pool size, the above amino acid analysis procedure was done using freshly collected anemones. The field salinity at the time of collection was 9.5 x0.

RESULTS

Salinity tolerance At least 50 per cent between

6 and 33x,.

exceeded

these

practice, lethal

limits.

the criteria

initial period

of the D. Zeucolena survived in each of the test salinities was less than 20 per cent in the salinities which No mortality occurred in the control salinity (11x,). In

Survival

used to determine

of acclimation.

range,

the

death

proved

to be unreliable

In many cases, especially

anemones

stimulation

during

the initial

contractile

response

returned

would

show

4 days.

no contractile

After

or the animals

during

the

near the limits of the nonresponse

to mechanical

the end of that period, had obviously

begun

however,

the

to deteriorate.

Osmotic concentration of the coelenteronjluid and tissue water variation D. Zeucolena is clearly an osmoconformer. tions

of the coelenteron

external

osmotic

fluid were

concentrations

time course

of the acclimation

with the environment

~-TIME

After acclimation, equal

of the coelenteron

osmotic

to, or slightly

over the non-lethal

equilibrium TABLE

either

salinity

concentra-

hyperosmotic

range

(Fig.

fluid is rapid, reaching

in less than 3 hr in all salinities

to The

an osmotic

(Table

COURSE OF THE OSMOTIC ACCLIMATION OF THE COELENTERON kUCOk?ul IN VARIOUS SALINITIES

1). 1).

FLUID OF D.

External osmotic concentration (mOsmole/kg J&O)

0

5

15

30

60

180

360

720

1440

100 296 490 695

166* 168 166 183

155 219 271 306

142 245 324 386

137 271 439 464

130 257 454 596

120 299 492 697

115 303 492 713

107 298 493 715

106 296 501 699

Time after transfer to test solution (min)

* Osmotic concentration of coelenteron fluid (mOsmole/kg H,O). 6

162

SIDNEY 0

K. PIERCE, JR. AND LEO L. MINASIAN,JR.

800'

I"

.o

300.

s E 6

200.

ii :

IOO-

/:'

E

0-T

0 External

200

300

osmotic

400

500

concentration,

600

700 mOsm/kg

800 HP0

FIG. 1. Osmotic concentrations of the coelenteron fluid of D. leucolena following acclimation to various salinities. Dotted line represents isosmotic values. Solid line connects actual data points.

Over the non-lethal salinity range, tissue water content changed by approximately 3.5 per cent while external osmotic concentrations varied by 77 per cent (Table 2). Thus, D. Zeucolena maintains an almost constant cellular water content despite the lack of an extracellular means of osmoregulation. TABLE

2--TOTALWATERASPERCENTWETWEIGHTINTHETISSUEOF TO VARIOUS OSMOTIC CONCENTRATIONS

D. leucolena ACCLIMATED

Osmotic concentration (mOsmole/kg H,O) Total H,O as y0 wet weight* (_+ S.D.) 200 465 688 885

83.8 82.6 80.5 79.1

(f (+ (+ (?

1.25) 3.60) 1 .lO) 2.12)

* Total water as per cent wet weight = [(wet weight - dry weight)/wet weight] x 100. Amino acid regulation

A total of twenty-one intracellular free amino acids were identified in the tissues of freshly collected D. Zeucolena along with three small unknown peaks (Table 3).

WATER TABLE

BALANCE

OFAEURYHALINB

SEA ANEMONE

163

~--LIST OF INTRACELLULARFREEAMINO ACIDS IDENTIFIED IN THETISSUESOFFRESHLY COLLECTED D.leucolma (OSMOTIC CONCENTRATION = 277mOsmole/kgH,O)

Amino acid Lysine Histidine Ornithine Arginine Taurine Aspartic acid Threonine Serine Glutamic acid* Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine B-Al anine Aminoisobuytric acid Three unknown peaks

Concentration @M/g dry wt.) 17.30 1.49 Trace 6.56 31.96 Trace 7.85 Trace 61.56 9.86 17.75 15.00 2.85 3.21 Trace 3.01 3.33 1.86 2.16 Trace Trace

* Sum of glutamic acid and glutamine.

Since comparable amino acid concentrations were found in the tissues of experimental anemones acclimated to similar salinities starvation is not a factor affecting observed amino acid changes, at least over the acclimation period used. Those amino acids present in concentrations less than 10 PM/g dry weight did not vary Similarwith salinity in preliminary experiments and were not considered further. ly, the preliminary experiments indicated very little variation in concentrations of basic amino acids. As a result, amino acid analysis was done only for acidic and neutral amino acids in subsequent experiments. The variation of intracellular free taurine, glutamic acid, glycine and alanine concentrations with external osmotic concentration is shown in Fig. 2. While the rest of the intracellular free amino acid pool remains virtually constant, regardless of salinity, the concentrations of these four amino acids increase markedly with increasing salinity. The glutamic acid concentrations reported are actually a In our chromatograms, glutamine mixture of glutamic acid and glutamine. appeared in high, but extremely variable concentrations in all salinities. This variation is due to the conversion of glutamine to glutamic acid which occurs at significant rates even if the samples are stored frozen (Jeppsson & Karlsson, 1972).

164

SIDNEY K. PIERCE, JR. AND LEO L. imNASIAN, JR.

2%

I

22c 3 0

1

u i

I

80 60 40

Osmotic

concentration,

mOsm/kg

H,O

FIG. 2. Variation in the osmotically active intracellular free amino acids of l3. leucolena acclimated to various osmotic concentrations. Glu* is the sum of glutamic acid + glutamine. Error bars are standard errors of the mean.

Thus, the glutamic acid and glutamine concentrations are reported here as a summed value (Fig. 2, Glu*). The histograms of Fig. 2 also show that the individual, osmotically active amino acids do not all increase in concentration at the same rate in response to increasing external osmotic pressure. For instance, in sea water of 200 mOsmole/Kg H,O, glutamic acid is the most abundant amino acid. At 465 mOsmole/kg H,O, glycine predominates. Glycine concentrations level off in 695 mOsmole/kg I-i20 and do not change significantly up to 890 mOsmole~kg HsO. Taurine concentrations show a pattern similar to glycine. On the other hand, the largest increases in glutamic acid and alanine concentrations occur between 695 and 890 mOsmole/kg H,O. Figure 3 demonstrates that the increase of intracellular concentrations of taurine, glutamic acid, glycine and alanine are much greater than the concentration changes which would be expected to result from the decrease in tissue water. These expected amino acid concentration increases due to tissue water changes can be calculated from the following equation:

where C, is the expected amino acid concentration In a given osmotic concentration, Pt is the amount of water in the tissues as a percentage of the wet weight in the same osmotic concentration, P, is the amount of tissue water in the lowest osmotic

WATER BALANCE OF A EURYHALINE

165

SEA ANEMONE

i .c"300.s x k 0 zoo? a.

IOO-

ho

2bo

360

Osmotic

460

560

6b0

concentration,

760

Ed0

mOsm/

9&O

Id00

kg HP

FIG. 3. Comparison of expected changes in free amino acid concentration calculated from tissue hydration values (dotted line) and observed concentrations (solid line) in D. leucolena acclimated to various salinities.

concentration and C,, is the measured intracellular amino acid concentration in the lowest osmotic concentration. Application of the data in Table 2 and the amino acid concentrations in 200 mOsmole/kg H,O (Fig. 2) result in a predicted increase in intracellular free amino acids due to tissue water changes (dotted line, Fig. 3) which is far less than the observed increase (Fig. 3, solid line). This difference between expected and observed amino acid concentration changes indicates that the increases in intracellular concentrations of taurine, glutamic acid, glycine and alanine are probably due to a regulatory process, not to a passive equilibrium. DISCUSSION

D. leucolena is a euryhaline osmoconformer. However, in spite of being unable to maintain extracellular osmotic concentrations apart from the environment, this anemone is a good volume regulator. The mechanism of cell water control is much like that found in other marine invertebrates: intracellular free amino acid regulation. Thus, as external salinities increase intracellular solute concentrations are increased in order to prevent water loss to the environment and subsequent cell shrinkage. The amino acids utilized for this regulatory process are those commonly glutamic acid, glycine and alanine (again, see used by other species: taurine, Emerson, 1969; Pierce, 1971 for reviews). The mechanism of water balance in marine coelenterates is different from that found in fresh water coelenterates. Hydra littoralis (Benos & Prusch, 1972) and the medusa, Craspedacusta sowerbyi (Fleming & Hazelwood, 1967; Hazelwood et al.,

SIDNEY K. PIERCE, JR. AND LEO L. MINASIAN, JR.

166

1970), control cell volume by active isosmotic extrusion of intracellular Na+ and K+ into the coelenteron as osmotic solute. K+ is actively reabsorbed into the cells lining the coelenteron, thus creating a coelenteron fluid which is hypoosmotic to the cells. The net effect of this process is that water leaves the cells and cell volume is maintained (Benos & Prusch, 1972). The coelenteron is periodically emptied of excess fluid; Hydra expells the fluid contents of the coelenteron more frequently as external osmotic pressures are decreased (Benos & Prusch, 1973). The coelenteron in fresh water coelenterates, then, acts in the manner of a distal-type (SchmidtNielsen, 1965) of excretory tubule. On the other hand, if the coelenteron of D. Zeucolena has any osmoregulatory function, it must act as a proximal-type (SchmidtNielsen, 1965) of tubule since coelenteron osmotic pressures are very close to those of the environment following acclimation. D. leucolena provides further support for the hypothesis that the degree of euryhalinity exhibited by a marine animal is directly related to the size of the intracellular free amino acid pool (Pierce, 1971). It is obvious from both its ecological distribution (Cory, 1967) and the salinity tolerance experiments reported here, that D. Zeucolena is one of the most euryhaline species of Coelenterata. By comparison the free amino acid pool size is considerably larger in D. leucolena than in other more stenohaline anthozoan species in similar salinities (Kittredge et al., 1962; von Holt, 1968; Severin et al., 1972). On the other hand, the pool size of D. leucolena compares favorably with those of other euryhaline species (e.g. Emerson, 1969; Pierce, 1971; Severin et al., 1972). Finally, the source of the intracellular free amino acids which appear in increasing salinities should be considered. There are two obvious possibilities : first, amino acids may be taken up from the surrounding water or from the diet. For instance, A. au&a polyps take up glycine from the external medium at a rate dependent on external salinity (Shick, 1973). Similarly, Fungia scutaria will rapidly exhaust 1962). external concentrations of lysine, aspartic acid and glycine (Stephens, In our study, however, exogenous amino acids are an unlikely source for the increased intracellular concentrations since the anemones were starved throughout the experiments. The second possibility is that the amino acids are produced as a result of increased intracellular protein catabolism in increasing salinities. There is some evidence for this process in gastropod molluscs (Bedford, 1971). Currently, we are examining other species for this regulatory mechanism.

REFERENCES BEDFORD J. J. (1971) Osmoregulation intracellular isosmotic regulation. BENOSD. & PRUSCH R. (1972)

in Melunopsis trifasciutu-IV. The possible Comp. Biochem. Physiol. 40A, 1015-1028.

Osmoregulation

in fresh-water Hydra.

control

of

Comp. Biochem. Physiol.

43A, 165-171. BENOS D. & PRUSCH R. (1973) Osmoregulation in Hydra: column contraction of external osmolarity. Comp. Biochem. Physiol. 44A, 1397-1400.

as a function

CECCALDI H. J. & DUMAS R. (1967) Etude comparative siphonophores. Comp. Biochem. Physiol. 22, 487-493.

de quelques

des acides

amines

WATERBALANCEOF A EURYHALINESEAANEMONE

167

CORY R. L. (1967) Epifauna of the Patuxent River Estuary, Maryland, for 1963 and 1964. Chesapeake Sci. 8, 71-89. DUMAS R. & CECCALDIH. J. (1973) Acides amines libres et proteiques chez le siphonophore Forskalia edwardsi. Comp. Biocha. Physiol. 45B, 43-50. EMERSOND. N. (1969) Influence of salinity on ammonia excretion rates and tissue constituents of euryhaline invertebrates. Camp. Biochem. Physiol. 29, 1115-1133. FLEMING W. R. & HAZELWOODD. H. (1967) Ionic and osmoregulation in the fresh water medusa, Craspedacusta sowerbyi. Comp. Biochem. Physiol. 23, 911-915. HAZELWOODD. H., POTTS W. T. W. & FLEMING W. R. (1970) Further studies on the sodium and water metabolism of the freshwater medusa, Craspedacusta sowerbyi. 2. vergl. Physiol. 67, 186-l 91. VON HOLT C. (1968) Uptake of glycine and release of nucleoside-polyphosphates by zooxanthellae. Comp. Biochem. Physiol. 26, 1071-1079. JEPPSSON J. 0. & KARLSSON I. M. (1972) Ion-exchange chromatography of physiological sulphur amino acids on a highly crosslinked resin. J. Chromatogr. 72, 93-103. KITTREDCE J. S., SIMONSEN D. G., ROBERTS E. & JELINEK B. (1962) Free amino acids of marine invertebrates. In Amino Acid Pools (Edited by HOLDEN J. T.), pp. 176-186. Elsevier, New York. KOBLICK D. C. & Yu-Tu L. (1967) The osmotic behavior of digestive cells of Chlorohydra viridissima. J. exp. Zool. 166, 325-330. PIERCE S. K., JR. (1971) A source of solute for volume regulation in marine mussels. Comp. Biochem. Physiol. 38A, 619-635. SASSAMANC. & MANCUMC. P. (1970) Patterns of temperature adaptation in North American Atlantic coastal actinians. Mar. Biol. 7, 123-130. SCHMIDT-NIELSEN B. (1965) Comparative morphology and physiology of excretion. In Ideas in Modern Biology (Edited by MOORE J. A.), pp. 393-425. Natural History Press, New York. SEWRIN S. E., BOLDYREVA. A. & LEBEDEVA. V. (1972) Nitrogenous extractive compounds of muscle tissue of invertebrates. Comp. Biochem. Physiol. 43B, 369-381. SHICK J. M. (1973) Effects of salinity and starvation on the uptake and utilization of dissolved glycine by Amelia aurita polyps. Biol. Bull. mar. biol. Lab., Woods Hole 144, 172-179. STEPHENS G. C. (1962) Uptake of organic material by aquatic invertebrates-I. Uptake of glucose by the solitary coral, Fungia scutaria. Biol. Bull. mar. biol. Lab., Woods Hole 123, 648-659. WEBB K. L., SCHIMPF L. & OLMON J. (1972) Free amino acid composition of scyphozoan polyps of Aurelia aurita, Chrysaora quinquecirrha and Cyanea capillata at various salinities. Comp. Biochem. Physiol. 43B, 653-663. Key Word Index-Free amino acid regulation; volume regulation; salinity tolerance; Diadumene leucolena; water balance.

osmoregulation;