The water balance of Modiolus (Mollusca: Bivalvia: Mytilidae): Osmotic concentrations in changing salinities

Comp. Biochem. Physiol., 1970, Vol. 36, pp. 521 to 533. PergamonPress. Printedin Great Britain

THE WATER BALANCE OF M O D I O L U S (MOLLUSCA: BIVALVIA: MYTILIDAE): OSMOTIC CONCENTRATIONS IN CHANGING SALINITIES* SIDNEY K. PIERCE, JR. Department of Biological Sciences, Florida State University, Tallahassee, Florida 32306 (Received 31 March 1970) Abstract--1. Comparative measurements of the osmotic concentrations in three fluid compartments of species of Modiolus from different regions of the marine environment show that the animals are osmotic conformers. However, the body fluids remain hyperosmotic to the environment by a constant amount over the non-lethal salinity range. 2. The hyperosmotic condition appears to be the result of a passive equilibrium rather than active regulation and is probably characteristic of all osmoconformers. 3. The osmotic difference between the blood and the pericardial fluid precludes the possibility of ultrafiltration from the blood into the pericardial cavity using the systolic pressure of the heart as the driving force. INTRODUCTION CLASSICAL studies of osmotic regulation in marine molluscs and worms have indicated that the body fluids of these forms are isosmotic with the environment. T h e data have been reviewed often during the past 30 years (Krogh, 1935 ; Prosser & Brown, 1961 ; Ports & Parry, 1963 ;Potts, 1968). Recently, scattered reports, especially concerning molluscs, suggest that these animals are not isosmotic after all. In some of these studies, the internal freezing point depressions are, as usual, plotted against those of the environment; the resulting curves have the same slope as the isosmotic line but are hyperosmotic to it. For instance, the blood of the gastropods LittoHna littorea, Littorina saxatilis (Remmert, 1968), Limapontia capitata, Limapontia depressa and Assiminea grayana (Seelemann, 1968) all show freezing point depression curves which hyperosmotically parallel the isosmotic line. In another investigation, the freezing point depression curves for the urine of Hydrobia ulvae and Potamopyrgusjenkinsi hyperosmotically parallel the isosmotic line; the osmotic concentration of the urine is less than that of the blood in these snails (Todd, 1964). Freeman & Rigler (1957), although plotting no freezing point depression curves, found the blood of the bivalve Scrobicularia plana to be hyperosmotic to the *Supported, in part, by U.S.P.H.S. Fellowship No. GM-45404 (General Medical Sciences), U.S.P.H.S. Grant No. HE-09283 (National Heart Institute), and by Atomic Energy Commission, Division of Biology and Medicine, Contract No. AT (40-1)-2690. 521

522

SIDNEY K. PmRCE,JR.

environment in low salinities. The pericardial fluid of Xenostrobus securis (formerly Modiola securis) is also slightly hyperosmotic to the environment (Wilson, 1968). On the other hand, Lent (1969) reported that the "pericardial hemolymph" of Modiolus demissus is isosmotic to the environment in salinities from 10 to 45~oo. Recent studies on annelids (Boroffka, 1968; Oglesby, 1968a; Dice, 1969) and sipunculids (Oglesby, 1968b) have also yielded data indicating that over at least some part of the tolerated salinity range of these forms the blood is hyperosmotic to the environment. Remmert (1969) has recently summarized some of these data, noting that the hyperosmotic body fluid may be a characteristic of all osmotically conforming animals, but he could offer no explanation for the phenomenon. The interpretations of these findings have varied. Some authors have ignored the hyperosmotic difference and have persisted in calling the animals isosmotic (e.g. Remmert, 1968), while others have proposed that the phenomenon is an example of hyperosmotic regulation (e.g. Seelemann, 1968). In the first case, if the body fluids are indeed hyperosmotic to the environment, it is obvious that they cannot, at the same time, be isosmotic to the environment. Secondly, regulation implies that the freezing point depression curve for the body fluid deviates in slope from the isosmotic line. If the internal freezing point depression curve parallels the isosmotic line, regulation seems unlikely. However, since most of the above studies were conducted on estuarine or intertidal animals, the possibility still exists that the hyperosmotic body fluid represents a response to environmental stress. The present report compares the osmotic concentrations in the various fluid compartments of bivalves from different regions of the marine environment. The animals of choice for this study were four members of the family Mytilidae: Modiolus demissus demissus (Dillwyn) and Modiolus demissusgranosissimus (Sowerby), are salt marsh dwelling forms; Modiolus modiolus (Linn~) and Modiolus squamosus Beauperthy are subtidal species. Since these four animals are congeneric, the possibility of physiological variation due to specific differences is minimized. This study shows that, regardless of habitat or salinity tolerance, the body fluids of bivalves are indeed hyperosmotic to the environment. Furthermore, this phenomenon is not restricted to any particular fluid compartment, but is found to a varying degree in all of them. MATERIALS AND METHODS Animals and habitats (1) Florida mussels. Florida mussels were obtained from two locations. Modiolus demissus granosissimus were found in the fine grain reduced mud of a salt marsh located on Alligator Point, Franklin County, Florida. Spartina sp. is the main vegetation occurring in the vicinity of the mussels. The animals are buried, usually with only the posterior tip of the valves protruding from the mud. Groups of M. d. granosissimus surround clumps of Spartina which serve as a substrate for byssus thread attachment. Modiolus squamosus were collected from the seaward side of Bay Mouth Bar, an extensive sand bar which runs across the mouth of Alligator Harbor. The animals live in a coarse mixture of gravelly sand and bivalve shells. The byssus threads are attached to other mussels, or to clumps of valves from dead mussels and other bivalves.

WATER BALANCEOF .VIODIOLUS

523

All animals were stored at 15-16°C in aquaria containing aerated natural sea water (salinity ~ 31 ~o0) obtained from the Florida State University Marine Laboratory at Turkey Point. T h e water in the storage aquaria was changed every two weeks, but the animals were not fed during either storage or experimentation, in order to prevent osmotic differences due to food break down in the sea water or post absorptive differences in the animals. (2) Massachusetts mussels. Modiolus demissus demissus were collected from a salt marsh, similar to the one described for the southern subspecies, on Penzance Point, Woods Hole, Massachusetts, or were obtained from the Supply Department of the Marine Biological Laboratory at Woods Hole. Modiolus modiolus were collected from the rocky coast of Cape Cod Bay near ~Ianomet, Massachusetts. These mussels are well imbedded below the low water mark in an extremely coarse, rocky sediment to which they attach by byssus threads. Both of the northern species were kept at Woods Hole at 20-22°C, on sea tables, in running sea water from Vineyard Sound (salinity ~ 31 ~oo). Similarly, no provision was made for feeding the Massachusetts animals during storage or experimentation. Since the experiments with the Florida mussels were carried out in Tallahassee, while those with the northern species were done in Woods Hole, exact duplication of experimental procedures was impossible. Minor differences in technique are pointed out in the descriptions of the experiments.

Salinity tolerance (1) Solutions, (a) Florida mussels: Artificial sea water was made in 500-1. batches using the following reagent grade salt concentrations in distilled water: NaC1, 23"48 g/l. ; KC1, 0"66 g/1.; CaCI~, 1-10 g/1.; NaHCOs, 0"192 g/l.; MgCI~, 4"98 g/1.; Na2SO4, 3"92 g/l.; KBr, 0"096 g/1. ; H3BO3, 0"026 g/l. ; SrCI~, 0"024 g/1. ; NaF, 0.003 g/l. (Harvey, 1945). T h e salinity of this solution was 34-36 %0. Artificial sea waters of lower salinity were made by diluting this solution with distilled water. T h e salt concentrations of the recipe were doubled to yield a solution whose salinity, was 70-72%o. This was then appropriately diluted with distilled water to make sea water of salinities ranging from 36-729/oo. T h e salinity of all solutions was determined before use. (b) Massachusetts mussels: Salinities of less than 31~oo were made by appropriate dilution of Vineyard Sound water with distilled water. Salinities greater than 31/oo o, were made by dilution of " 2 X M B L Sea Water" (salinity ~ 64~o) which was obtained from the stock room of the Marine Biological Laboratory (see Cavanaugh, 1964). (2) Criteria for determining survival. Animals surviving in either diluted or concentrated sea water produce byssus threads and appear to siphon actively and regularly. Since both these processes require prolonged opening of the valves, the animals are necessarily exposed to the salinity change for relatively long periods. Therefore, these two activities were used as criteria of normal behavior in, and toleration of, a particular salinity. Gaping animals were considered to have died when the posterior mantle edge and foot failed to contract in response to mechanical stimulation. (3) Procedure of salinity tolerance experiments. Salinity tolerance experiments were carried out in order to determine a suitable non-lethal range over which the osmotic concentrations of each species' body fluids could be tested. (a) Florida mussels: Twelve specimens of each species were taken from 36%0 sea water and placed directly into aquaria, each containing sea water of one of the following salinities : 54, 48, 45, 40, 27, 22, 18, 9, 3 or 1"5~0. In addition, controls were kept at 36~0 sea water. All tolerance experiments were run at 22-23°C. T h e condition of the animals was checked several times daily. Dead animals were counted and removed immediately, and the sea water in which the death had occurred was also changed. In those aquaria in which no deaths occurred, the water was changed weekly. A salinity was considered lethal to a particular species if more than 50 per cent of the experimental animals exposed to it had died within sixty days.

524

SIVNEY K. PIERCE,JR.

(b) Massachusetts mussels: Twelve specimens of both northern species were placed in pans containing water of those salinities which had proved to be non-lethal to the corresponding intertidal or subtidal form in Florida. T h e experiment was performed in the same manner as described for the southern species, except that it was only monitored for 21 days. At the end of this period, any salinity in which 50% or more of the mussels had died was considered to be lethal. All pans were kept on sea tables at the Marine Biological Lab., Woods Hole, in r u n n i n g sea water. This maintained a fairly constant temperature within the pans of about 21°-22°C.

Osmotic concentrations Osmotic concentrations were measured in M. modiolus, M. squamosus and both subspecies of M. demissus. Twelve to 24 animals of each southern species, and 6-8 of each northern species, were placed in those salinities which had proved to be non-lethal in the salinity toleration experiments. They were allowed to acclimate for at least two weeks, or until active siphoning and byssus thread production were occurring. Then, the animals and a sample of water were removed from each aquarium, and the osmotic concentrations of the environment, mantle fluid, pericardial fluid, and blood were determined. (1) Collection of fluids. Samples of mantle fluid were taken by gently prying open the valves and withdrawing the fluid from the mantle cavity with a hypodermic syringe. Two to three milliliters were readily obtained in this manner. Next, the adductor muscles were cut and one valve was removed with as little tissue damage as possible, and especially without opening the pericardial membrane. The external surface of the mantle was blotted dry with filter paper, and the fluid withdrawn from the pericardial cavity by means of a hypodermic syringe inserted through the dorsal pericardial membrane. In the case of M. d. granosissimus and M. modiolus, the pericardial cavity contained sufficient fluid so that individual samples of about 0"25 ml could be obtained. However, no more than 0"10-0"15 ml could be removed from the pericardial cavity of M. d. demissus or M. squamosus; therefore, pericardial fluid from two or three animals of each species was pooled to make the requisite 0'20 ml sample. Following the removal of mantle and pericardial fluids, the soft parts of each animal, still on the half shell, were blotted dry with filter paper to remove any remaining mantle fluid. The mantle, pallial blood sinus and adductor muscles were slashed with a razor blade. The anterior end of the animal was then inserted into the mouth of a flask and blood drained from the cut surfaces into the flask for 5-10 minutes. During this process, both the animal and flask were wrapped in a continuous strip of Parafllm to prevent evaporation and were placed in a refrigerator. Between 1 and 2 ml of blood were obtained from each animal. (2) Measurement of osmotic concentration. Each fluid sample, in a 10 ml Erlemeyer flask sealed with Parafilm, was refrigerated in turn until all the samples for a given experiment had been collected. They were then centrifuged at 2000 x g to sediment cells and debris. Next, a 0.20 ml aliquant of the supernatant fluid was removed from each centrifuge tube and sealed in a sample tube of a freezing point depression osmometer (Osmette; Precision Systems). T h e osmotic concentrations of all samples were measured immediately; three determinations of each sample were made, and the resulting values averaged. Any series of determinations, on a single sample, with a standard error greater than 3"5 mOsm/kg H 2 0 was repeated. Regression analysis and analysis of covariance were calculated on a Control Data Corporation 6400 computer using Bio-medical Computer Programs (Dixon, 1968). RESULTS

Salinity tolerance All species b e h a v e d s i m i l a r l y i n r e s p o n s e to t r a n s f e r f r o m o n e salinity to a n o t h e r . I n i t i a l l y , t h e valves r e m a i n e d s h u t m o s t of t h e time, o p e n i n g i n f r e q u e n t l y a n d o n l y for s h o r t periods. T h e d u r a t i o n of this t y p e of b e h a v i o r varied w i t h the salinity,

525

WATER BALANCE OF M O D I O L U S

lasting from only a few hours in salinities close to 36~oo, to as much as 7-10 days in the most dilute and most concentrated sea water. This intermittent opening was followed, either by the resumption of extended periods of siphoning and byssus production, or by death. Modiolus demissus granosissimus survived in all salinities between 3~ooand 48%0. In all cases, normal siphoning behavior and byssus production were evident. Animals in 1"5%o began to die within 5 days from the start of the experiment. Although some animals in this salinity were still alive at the end of two weeks, the valves appeared to have remained tightly shut during this period. No animal survived in 1"5~oofor more than 16 days. Similar lethal results were obtained using sea water of 54~oo. Modiolus squamosus survived in salinities ranging from 22%0 to 41%o. On either side of this range (i.e., 18~o and 45%o), at least 50% of the animals died and these salinities were considered to be lethal limits. M. d. demissus died after 7 days in 3~o, but survived for 21 days in salinities ranging from 8%0 to 48~o. M. modiolus survived for 21 days in salinities ranging from 27~o to 41~oo. These salinity limits were used as standard ranges in the osmotic concentration experiments and their magnitude for each species is evident from Fig. 1-4.

Osmotic concentration of body fluids Osmotic measurements were made on three fluid compartments in each of the four mussels. The mean osmotic concentrations of the mantle fluid, pericardial fluid and blood are shown for M. d. granosissimus in Table 1 ; for M. squamosus in TABLE 1--MEAN OF

OSMOTIC CONCENTRATION OF MANTLE FLUID~ PERICARDIAL FLUID AND BLOOD

M. d. granosissimus M A I N T A I N E D

I N SEA WATER OF VARIOUS OSMOTIC PRESSURES

Sea water (mOsm/kg H~O)

Mantle fluid Pericardial fluid Blood

113

290

503

779

1076

1145

1350

115" (_+1-12) 120 (_+1"22) 132 (_+1"64)

292 (_+0"76) 299 (_+0"71) 311 (_+1"06)

507 (_+0"43) 512 (_+1.25) 520 (+2"36)

779 (_+2"28) 781 (_+3"14) 800 (_+2"99)

1077 (+_2"84) 1079 (_+1"73) 1092 (_+2"86)

1145 (_+0"70) 1147 (_+1"46) 1154 (-+3"07)

1351 (_+1"01) 1361 (_+1-98) 1377 (-+1"13)

*Osmotic pressure in mOsm/kg H20 (+ standard error). Table 2; for M. d. demissus in Table 3; and for M. modiolus in Table 4. The regression lines calculated from these data are shown, respectively, in Figures 1-4. The F-ratios obtained from the analysis of covariance indicate that the fluids are significantly more concentrated than the sea water (0.01 level) in all salinities tested.

1500

I 500

I 500

I 700

l 900

I . riO0

I 15 O 0

./

External osmotic concentration, mOsm/kg H~O

I0010~/0

300t/,

5OO

Mantle fluid Pericardiol fluid , ~

F l a . 1. R e g r e s s i o n lines f o r t h e o s m o t i c c o n c e n t r a t i o n s o f t h e f l u i d c o m p a r t m e n t s o f M. d. granosissimus o v e r t h e n o n - l e t h a l s a l i n i t y r a n g e . R e g r e s s i o n l i n e f o r m u l a e a r e : m a n t l e fluid, Y = 0 " 9 9 8 x + 2"735; p e r i c a r d i a l fluid, Y = 0 - 9 9 8 x + 7"546; b l o o d , Y = 0 " 9 9 6 x + 18"856.

Hc

o "6 c

.°.9. 70O

o

L 9O0 g u

g

E

E I lO0

"I-

o

70C

0 I0100

500

I

500

I

700

I

I

I100

/

///

mOsm/kg H20

900 External osmotic concentration,

I

300

Mantle fluid-.PericordiaJBio_od f l u /

I

1300

FIo. 2. Regression lines for the osmotic concentrations of the fluid compartments of M. squamosus over the non-lethal salinity range. Regression line formulae are: mantle fluid, Y = 0-999x + 3"069; pericardial fluid, Y = 1-011x + 0"455 ; blood, Y = 1-015x + 4"420.

c ~

el}

-6 c

E 500

c o

c

.9

E E 90O

0 II00 =7

1300

t~

m

to o~

L

Ioo,b',

300

500

70C~

90(3

5007001

External osmotic concentration,

3(~0

...

mOsm/kg

9~)0 ....

Mont/e ftuid

H20

t. 1100

. ~ /

-i 1500

FIG. 3. Regression lines for the osmotic concentrations of the fluid compartments of M. d. demissus over the non-lethal salinity range. Regression line formulae are: mantle fluid, Y = 1"004x-2"191; pericardial fluid, Y = 1.008x+3"072; blood, Y = 1"012x+6.111.

~: H

c

E

"5

u

".7_

E

~ 0

E

£

0

l tO0

1300

0

O~oo

500

500

700

900

~00

l

500

700

__1

External osmotic corlcentration,

I

300

/

PericordioIBiood fluid

Mantle fluid /

mOsm/kg

I

900

/

I

H20

I I00

I_

1300

Fro. 4. Regression lines for the osmotic concentrations of the fluid compartments of M. modiolus over the non-lethal salinity range. Regression formulae are: mantle fluid, Y = 0.996x+5'0775; pericardial fluid, Y = 0"991x+12"989; blood, Y = 1"008x+4"051.

c ~

E o

o

u

o o

E

0

£

1300

~q to

SIDNEY K. PIERCE,JR.

528

TABLE 2--MEAN OSMOTIC CONCENTRATIONOF MANTLEFLUID, PERICARDIALFLUID AND BLOOD OF d~. $ q ~ m o $ ~ MAINTAINEDIN SEAWATEROF THE VARIOUSOSMOTICCONCENTRATIONSSHOWN Sea water (mOsm/kg H~O) 720

807

995

1102

Mantle fluid

725 * ( + 0.41)

807 ( + 0.68)

996 ( + 0.80)

1106 ( + 0-57)

Pericardial

731 (+0-37)

815 (+1"31)

1006 (+0.31)

1117 (+1.61)

Blood

738 ( _+1"50)

823 ( + 1-35)

1009 ( + 1.55)

1128 ( _+2.60)

* Osmotic concentration in m O s m / k g FIfO ( + standard error). TABLE 3--MEAN OSMOTICCONCENTRATIONSOF MANTLEFLUID, PERICARDIALFLUID AND BLOOD OF M. d. demissus MAINTAINED IN SEA WATER OF THE VARIOUS OSMOTIC CONCENTRATIONS SHOWN

Sea water (mOsm/kg H 2 0 ) 246

488

712

934

1087

1279

Mantle fluid

248* ( _+0-73)

485 ( +_0-57)

712 ( _+0"50)

934 ( _+0"64)

1090 ( _+0"79)

1284 ( _+0"65)

Pericardial fluid

254 ( _+0-57)

494 ( + 1 "14)

719 ( _+0"59)

939 ( + 1"00)

1110 ( _ 1"22)

1291 ( +_0"51)

Blood

259 (+0.41)

499 (+0.60)

724 (+0.82)

945 (+1.90)

1114 (+1.70)

1302 (+1.41)

* Osmotic concentration in m O s m / k g H~O ( + standard error). TABLE 4 MEANOSMOTIC CONCENTRATIONS OF MANTLEFLUID, PERICARDIAL FLUIDAND BLOOD OF M . m0d/o/u$ MAINTAINEDIN SEA WATEROF THE VARIOUSOSMOTIC CONCENTRATIONS SHOWN

Sea water (mOsm/kg H 2 0 ) 717

939

1079

M a n t l e fluid

720* ( _+0.71)

939 ( _ 0.57)

1081 ( _+1.48)

Pericardial fluid

724 ( _ 0"40)

943 ( _+1.19)

1083 ( _+0"51)

Blood

727 ( + 0-61)

951 ( + 1.35)

1092 ( + 1.80)

* Osmotic concentration in m O s m / k g H~O ( + standard error).

WATER BALANCE OF

529

MODIOLUS

Application of the Studentized range (Q distribution) to the data shows that the mantle fluid is not significantly different from sea water, that the pericardial fluid is hyperosmotic to the mantle fluid (0-01 level), and that the blood is significantly hyperosmotic to the pericardial fluid (0-01 level). The analysis of covariance further shows that the slopes of the regression lines for the fluids in M. d. demissus, as well as in M. modiolus, are significantly different (0.01 level). However, no significance was found for the difference in slopes of the regression lines calculated from the data for M. d. granosissimus and M. squamosus. In summary, both subspecies of M. demissus are poikilosmotic and euryhaline, while M. modiolus and M. squamosus are poikilosmotic and stenohaline, but the body fluids of none of the mussels is isosmotic to the environmental solution. Although the central finding of this report is the difference in osmotic concentration between the blood and the environment, the mussels used were too small to permit direct sampling of the blood from the sinuses or from the heart. Therefore, experiments were carried out to assure that the method by which the blood was obtained did not result in its contamination. The blood collection method was tested with two other species: Mercenaria mercenaria (Linn6) and Ensis directus (Conrad). Both are large bivalves, readily available at the Marine Biological Lab oratory, Woods Hole. Blood was removed from the ventricle of M. mercenaria, and from the blood sinus in the foot of E. directus, by means of a hypodermic syringe. In addition, blood was collected from these two species by slashing the tissue and draining, as described for the mussels. The osmotic concentrations of the blood collected directly from the circulatory system, and from the slashed tissue, were determined with the osmometer, and are presented in Table 5. The concentrations of the blood COMPARISON OF THE OSMOTIC CONCENTRATIONS OF THE BLOOD OF M . mercenaria E. directus COLLECTED DIRECTLY FROM THE CIRCULATORY SYSTEM AND BY SLASHING THE

TABLE 5--A AND

TISSUE*

Species M. mercenaria

E. directus

Blood from ventricle

Blood from slashed tissue

956t ( + 0"67)

956 ( + 0"26)

Blood from foot sinus 956 ( +_1"11)

Blood from slashed tissue 960 ( + 0"63)

* Both species were kept in sea water of 943 mOsm/kg H20. 1" Osmotic concentration in mOsm/kg H~O ( + standard error). collected by both procedures are similar; thus, the blood concentration observed in the mussels is not a function of the collection method. Furthermore, in the salinity tested, the bloods of M. mercenaria and E. directus were hyperosmotic to the sea water, illustrating that the phenomenon is not unique to mytilids.

530

SIDNEYK. PIERCE,JR.

DISCUSSION The data presented here show that the three species of Modiolus are osmoconformers; that is, the osmotic concentration of the body fluids varies linearly with the concentration of the environment. However, in Modiolus, as well as in other bivalves, the body fluids are hyperosmotic to the environment over the non-lethal salinity range. Since the range of variation of salinities in the intertidal and subtidal environments is different, the hyperosmoticity of the body fluids is not a response to salinity stress in a given habitat. It is the lethal salinity limits which are a function of normal habitat; the salinity tolerances of intertidal organisms are characteristically greater than those shown by subtidal forms (Mayes, 1962; Vernberg, Schlieper & Schneider, 1963; Born, 1968). Therefore, it is not unexpected that the marsh dwelling subspecies of M. demissus show larger salinity tolerances. The relatively hyperosmotic concentrations of the blood and pericardial fluid might be explained in two ways. Firstly, active concentration processes might be occurring. However, the slope of the regression line for the osmotic concentration of the blood of these mussels is constant and parallels the line for seawater (Fig. 1-4); but active osmoregulation necessarily implies a freezing point depression curve differing in slope from the isosmotic line over at least some of its length. Therefore, the concentration differences found here cannot be the result of active processes. The second possibility is that the osmotic difference is caused by a passive Gibbs-Donnan type of equilibrium. In this case, the only requirements are a membrane, impermeant charged protein molecules on one side of the membrane, and a freely diffusible ion species. The net result of such a system would be a difference in osmotic pressure between the inside and outside of the membrane depending on the protein concentration and the number of charges on it. This second possibility seems to fit the data best. Notwithstanding the existing data on animals considered to be isosmotic with respect to their environment (Potts & Parry, 1963; Robertson, 1964; Potts, 1968), any animal having protein in solution in its extracellular water and permeable external membranes must be hyperosmotic to its environment. The osmotic difference between the blood and the pericardial fluid is of considerable importance. The pericardial fluid is hypothesized to be an ultrafiltrate of the blood, produced across the ventricle wall by the systolic pressure of the heart (Picken, 1937; Smith & Davis, 1965). For this hypothesis about the site and mechanism of ultrafiltration to be correct, the hydrostatic pressure of the heart must exceed the osmotic pressure difference between the blood and the pericardial fluid. A range of ventricular systolic pressures, 0.2-1.9 mm Hg (0.00020.0025 atm), for several species of bivalves is known (Smith & Davis, 1965). The concentration difference which would generate an equivalent osmotic pressure can be calculated from the van t'Hoff equation: rr = ACRT, where ~r is osmotic pressure in atmospheres, AC is the difference in the osmolal concentration across the membrane, R is the gas constant and T is the absolute temperature. Using the lower of Smith & Davis' (1965) values, a concentration difference of 8"18 x 10 -7

W A T E R BALANCE OF M O D I O L US

531

Osm/1. is calculated. If the actual measured difference in osmotic concentration between the blood and pericardial fluid exceeds this calculated value, the hydrostatic pressure within the bivalve heart will be insufficient to cause filtration. Even a blood pressure as high as 5mm Hg--well above either Picken's (1937) value for Anodonta cygnea or the pressures reported by Smith & Davis (1965)--would allow for a concentration difference no larger than 3 x 10 -~ Osm/l. However, 3 x 10 -~ Osm/l. is much smaller than the difference in osmotic concentrations between the blood and pericardial fluid found here, and also less than the difference recorded by Picken (1937) in A. cygnea. Any consideration of ultrafiltration must include membrane resistance to fluid flow. A pressure of 20 mm Hg across the glomerular membrane of the dog is necessary to overcome the viscous resistance of the membrane (Winton, 1956). Although the filtration membrane in molluscan systems, if any exists, has not been found (Ports, 1969), it must be a structure with extremely low resistance to flow in view of the low molluscan systolic pressures. In conclusion, ultrafiltration across the heart wall, from the blood to the pericardial cavity, driven by intracardiac hydrostatic pressure, cannot occur in Modiolus. Probably it cannot occur in other bivalves either. Why has the hyperosmoticity of the body fluid generally been unrecognized ? Most students of osmotic control have used melting or freezing points as measures of osmotic concentration. The average difference between the blood of M. d. granosissimus and sea water is approximately 20 mOsm/kg H~O, a freezing point depression difference of 0-036°C. The pericardial fluid is about 6 mOsm/kg H~O more concentrated than sea water, a freezing point depression difference of only 0.0108°C. These differences are within the accuracy of the osmometer employed in the experiments reported here. However, earlier authors have usually used methods involving Beckmann or Heidenhain type thermometers. In many cases, the accuracy of the measurement was not reported. Ramsay (1948) describes a method for measuring freezing points to the nearest 0.005°C; Ports, who employed this method (1954), published temperatures only to the second decimal place. On the basis of freezing point depression measurements, Picken (1937) considered the blood and pericardial fluid of A. cygnea to be isotonic. He then compared nonmineral refractive index values of A. cygnea blood with data from human serum in order to arrive at a colloidal osmotic pressure difference between the blood and pericardial fluid. It seems, therefore, that the previous methods employed may not have been able to resolve the small differences reported here consistently enough to prevent statistical manipulations from cancelling them out. Finally, the use of osmotic pressure measurements in the description of the relationship between an animal and its environment depends, at the outset, upon the choice of an appropriate body fluid for measurement. Wilson (1968) and Lent (1969) used pericardial fluid rather than blood as a measure of the internal osmotic concentration of bivalves. While the pericardial fluid might be considered to be a blood filtrate (Potts, 1967), it is not, in fact, blood, and bathes only the pericardial membranes and the external surface of the heart. Since pericardial fluid does not

532

SIDNEY K. PIERCE,JR.

c o m e in c o n t a c t w i t h t h e v a s t m a j o r i t y o f t h e cells, it is p r o b a b l y n o t u s e f u l as a m e a s u r e of i n t e r n a l o s m o t i c c o n c e n t r a t i o n .

Acknowledgements--The author wishes to thank Dr. Michael J. Greenberg of the Florida State University, in whose laboratory this work was carried out, for his help and encouragement. Further thanks are due to Dr. Gerald van Belle for advice in the interpretation of statistical treatments, and to Dr. Richard Mariscal for reviewing the manuscript. REFERENCES BORN J. W. (1968) Osmoregulatory capacities of two caridean shrimps Syncaris pacifica and Palaemon macrodactylus. Biol. Bull. 134, 235-244. BOROFFKA I. (1968) Osmo- und Volumenregulation bei Hirudo medicinalis. Z. vergleich. Physiol. 57, 348-375. CAVANAUCHG. M. (Editor) (1964) Formulae and Methods V. Marine Biological Laboratory, Woods Hole, Mass. DICE J. F., JR. (1969) Osmoregulation and salinity tolerance in the polychaete annelid Cirriformia spirabrancha (Moore, 1904). Comp. Biochem. Physiol. 28, 1331-1343. DIxoN W. J. (Editor) (1968) B M D Biomedical Computer Programs. Univ. of Calif. Publications in Automatic Computation, No. 2. University of California Press, Berkeley. Fm~EMAN R. F. H., & RIGLEI~F. H. (1957) The responses of Scrobicularia plana (DaCosta) to osmotic pressure change. 3t. Mar. Biol. Ass. U.K. 36, 553-567. HARVEY H. W. (1945) Recent Advances in the Chemistry and Biology of Sea Water, p. 164. Cambridge University Press, London. KROGHA. (1965) Osmotic Regulation in Aquatic Animals. Dover, New York. LENT C. M. (1969) Adaptations of the ribbed mussel Modiolus demissus (Dillwyn), to the intertidal habitat. Am. Zool. 9, 283-292. MAYES P. A. (1962) Comparative investigations of the euryhaline character of Littorina and the possible relationship tointertidalzonation. Nature, Lond. 195, 1269-1270. OCLESBY L. C. (1968a) Responses of an estuarine population of the polychaete Nereis limnicola to osmotic stress. Biol. Bull. 134, 118-138. OGLESBY L. C. (1968b) Some osmotic responses of the sipunculid worm Themiste dyscritum. Comp. Biochem. Physiol. 26, 155-177. PICKEN L. E. R. (1937) T h e mechanism of urine formation in invertebrates. II. T h e excretory mechanism in certain mollusca..7, exp. Biol. 14, 20-34. POTTS W. T. W. (1954) T h e inorganic composition of the blood of Mytilus edulis and Anodonta cygnea, jY. exp. Biol. 31,376-385. POTTS W. T. W. (1967) Excretion in the molluscs. Biol. Rev. 42, 1-41. POTTS W. T. W. (1968) Osmotic and ionic regulation. Ann. Rev. Physiol. 30, 73-104. POTTS W. T. W. (1969) Aspects of excretion in the Molluscs. Studies in the Structure, Physiology and Ecology of Molluscs (Edited by FRETTER V.), pp.187-192. Academic Press, New York. POTTS W. T. W. & PARRY G. (1963) Osmotic and Ionic Regulation in Animals. Pergamon Press, New York. PROSSEa C. L. & BROWN F. A., JR. (1961) Comparative Animal Physiology. Saunders, Philadelphia. RAMSAY J. A. (1949) A new method of freezing point determination for small quantities. .7. exp. Biol. 26, 57-64. REMMEaT H. (1968) Die Littorina-Arten: Kein Modell ftir die Entstehung der Landschnecken. Oecologia2, 1-6. REMMEnT H. (1969) l~ber Poikilosmotie und Isoosmotie. Z. vergl. Physiol. 65, 424 427. ROBERTSON J. D. (1964) Osmotic and ionic regulation. In Physiology of MoUusca (Edited by WILBUR K. M. & YONGE C. M.) Vol. 1, pp. 283-312. Academic Press, New York.

W A T E R BALANCE OF M O D I O L U S

533

SEELEMANN U. (1968) Zur ~rberwindung der biologischen Grenze M e e r - L a n d durch M o l l u s k e n - - I I . Untersuchungen an Limapontia capitata, Limapontia depressa und Assiminea grayana. Oecologia 1,356-368. SMITH L. S. & DAVIS J. C. (1965) Haemodynamics in Tresus nuttallii and certain other bivalves, y. exp. Biol. 43, 171-180. TODD M. E. (1968) Osmotic balance in Hydrobia uh, ae and Potamopyrgus jenkinsi (Gastropoda : Hydrobiidae). ~t. exp. Biol. 41, 665-677. VERNBERG F. J., SCHLIEPER C. & SCHNEIDER D. E. (1963) The influence of temperature and salinity on ciliary activity of excised gill tissue of molluscs from North Carolina. Comp. Biochem. Physiol. 8, 271-285. WILSON B. R. (1968) Survival and reproduction of the mussel Xenostrobus securis (Lain.) (Mollusca: Bivalvia: Mytilidae) in a Western Australian estuary. Part 1. Salinity tolerance. ~. Nat. Hist. 2, 307-328. WINTON F. R. (1956) T h e pressures and flows of blood and urine within the kidney. In Modern Views on the Secretion of Urine (Edited by WINTON F. R.), pp. 61-95. Little, Brown & Co., Boston.

Key Word Index--Bivalves; molluscs; osmosis ; osmotic conformers; salinity tolerance ; Modiolus; bivalve excretion; ultrafiltration; mussels; Mytilidae.