VOLUME REGULATION IN MOPALIA MUSCOSA (MOLLUSCA: POLYPLACOPHORA)
A Thesis Presented to the Graduate Faculty
of
California State University, Hayward
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Biology
By
Mike Moran
August, 1976 ABSTRACT
Volume regulation ln Mopalia muscosa was
investigated. The chiton does not behave as a perfect
osmometer, and volume regulates in salinities as low
as 60% SW; little volume regulation occurs in hyperosmotic
sea water.
Changes ln tissue free amlno acid concentrations
are accounted for by tissue hydration. Thus, free amino acids are not involved in volume or tissue water + content regulation. Na and Cl are concentrated
considerably in the foot tissue and may be involved ln
volume and tissue water content regulation. ++ . . . Hemolymph Ca lS regulated ln dllute sea water and this may be related to ciliary action (which is
dependent on Ca++) and urine production by the kidney.
Temperatures as high as 19.4°C do not affect
volume regulation, whereas low temperatures of about
7°C affect the response; this is demonstrated by a
decreased water influx and reduced ability to volume regulate.
Fall and Spring volume regulation experiments
indicate the possibility of a seasonal response.
Individuals take up significantly more water in the Fall
ll than in the Spring though further work is needed to verify the possibility of a seasonal response in
M. muscosa.
lll TO: My Mother, Alvin, Winks and Beckie.
v ACKNOWLEDGEMENTS
I would like to sincerely thank Dr. John Martin for use of the atomic absorption spectrophotometer.
Thanks is also glven to Steve Lasley for providing storage space in the chem lab, to Connie Driscoll for typing the rough draft of the thesis and to Lynn
McMasters for drawing Figure l. A thanks is glven to
Dr. Robin Burnette of Hopkins Marine Station for discussions on the biology of M. muscosa and for reading the manuscript. Thanks are given to Dr. Michael Foster for reading the manuscript. A very special thank you goes to my major advisor, Dr. Richard Tullis for guidance in my graduate program. His tremendous teaching abilities are hopefully reflected in this thesis.
Vl TABLE OF CONTENTS
Page
LIST OF TABLES Vlll
LIST OF FIGURES lX
INTRODUCTION . l
MATERIALS AND METHODS 6
RESULTS 16
DISCUSSION 65
SUMMARY 83
LITERATURE CITED 84
Vll LIST OF TABLES
TABLE Page + + ++ ++ l. Na , K , Ca and Mg concentrations of normal sea water and sea water with instant ocean added 18
2. Comparison of observed maximal weight changes to that of a perfect osmometer 38
3. Tissue ion concentrations at different salinities . 44
4. Hemolymph ion concentrations at different salinities . 46
Vlll LIST OF FIGURES
FIGURE Page
1. Description of experimental apparatus 8
2. Fall volume regulation . 20
3. Regression of plate wt. vs. body wt. 24
4. Regression of maximum percent increase in wet wt. of soft parts vs. soft part wet wt. 26
5. Spring volume regulation . 28
6. Volume regulation at different temperatures 31
7. Graphical plot of P (osmotic perme- ability) OS 34
8. Volume regulation of a perfect osmometer 36
9. Foot water content at different salinities 42
10. Tissue NPS at different salinities . 49
11. Hemolymph K+ at different salinities 53
12. Hemolymph Ca++ at different salinities 55
13. Difference between hemolymph and sea water ca++ and K+ at different salinities 57
14. Time Experiment, Cl concentration of the hemolymph 60 + 15. Time Experiment, Na concentration of the hemolymph 62
16. Time Experiment, K+ concentration of the hemolymph 64
lX INTRODUCTION
Intertidal marine invertebrates are subject
to salinity variations. Fresh water runoff, tidal
fluctuations of sea water (SW) in estuaries, and
diluted surface waters after heavy rains which splash
intertidal zones will cause osmotic problems. There-
fore, the ability of an intertidal organism to control
its water content 1n the face of a salinity stress is
extremely important.
Intracellular isosmotic regulation, a term
applied for the ~ontrol of the intracellular osmotic
pressure 1n changing salinities, is an adaptive mech
anlsm many marine invertebrates use to control tissue water content (Pierce, l97la). Intracellular isosmotic
regulation is a process that involves removal of solute
from the cell, resulting'in a decreased osmotic pressure
(Florkin and Schoffeniels, 1969). The solute may be
removed by extrusion or merely rendered osmotically
inactive (Bedford, 1971; Freel, et al., 1973). In
either case the osmotic pressure within the cell 1s
adjusted to approximate that of the body fluids.
In the anisosmotic regulating (i.e., the regula
tion of body fluid osmotic pressure) decapod crustacea,
the necessity for intracellular isosmotic regulation is
l 2
not as great as ln the osmotic conformers (Pierce, l97la). The ability to regulate the body fluid com- position upon the introduction to an altered salinity allows for the maintenance of a small osmotic gradient between the body fluids and the cytoplasm, thus lessening the requirement for osmotic pressure adjustments within the cell.
Anisosmotic regulation is lacking ln marlne molluscs (Webber and Dehnel, 1968; Boyle, 1969, Pierce,
1970; Little, 1972; Simonsen, 1975). Those marine molluscs which are not capable of avoiding a salinity stress, as is true for many lamellibranchs, will have to rely on physiological mechanisms for controlling tissue water content. For long term survival in a reduced salinity, intracellular isosmotic regulation becomes very important to marine osmoconformers. Without this adaptive mechanism a salinity reduction could cause cellular swelling which may lmpalr locomotion, nerve impulse propagation, feeding and reproductive capacity
(Pierce, 1971; Oglesby, 1975).
Duchateau et al. (1952) reported that marlne lamellibranchs had greater concentrations of tissue
e amino acids (ninhydrin positive substance, NPS) than closely related fresh water forms. This suggested a role for amino acids in osmoregulation, and it is now 3
well documented that free amino acids are involved in regulating the cytoplasmic osmotic pressure in inverte- brates. (For review, see Florkin and Schoffeniels, 1969).
The use of ninhydrin positive substance in intracellular isosmotic regulation has been demonstrated in almost all marine invertebrate phyla as well as in fish (Pierce and Greenberg, 1972; Ahokas and Duerr, 1975). The amino acids involved are primarily the nonessential ones
(Claybrook, 1976). For example, in the Chinese crab
Eriocheir sinensis, alanine, arginine, glutamic acid, glycine and the imino acid proline are involved along with trimethyl amine oxide, taurine and betaine (Florkin and Schoffeniels, 1969). In the lamellibranch Modiolus
demissus granosissimus, taurine, glycine, alanine and
proline are the main osmotic effectors (Pierce and
Greenberg, 1972).
Volume regulation, the process by which body
volume or weight is returned towards the original weight
after an osmotic uptake or loss of water, may be achieved
by intracellular isosmotic regulation (Pierce, l97la).
Though free amino acids are usually involved in volume
regulation and the control of tissue water content ln
marine invertebrates, this lS not always the case.
Hoyaux- et a 1 . ' (1976) have shown that a good tissue free
amino acid response in relation to a reduced salinity 4
does not insure greater tissue water content regulation than in an animal lacking such a response. For example, the gastropod Patella vulgatta decreases its tissue free amino acid concentration by 100 mM/kg wet weight and increases its tissue hydration 7.8% when in 50% SW.
The polyplacophoran Acanthochitona discrepans alters its free amino acid pool by 2 mM/kg wet weight when subjected to 50% SW yet its tissue water content changes by only 7.5%. The barnacle Balanus improvisus contains tissue free amino acids plus proline at 211 m-molal in
1000 mOsmol SW (100% SW) (Fyhn and Costlow, 1975); this lS only 20% of the total tlssue osmolarity. The decrease ln NPS plus proline is 115 m-molal when the sea water decreases by 500 mOsmol and tissue water content is regulated away from the line for a perfect osmometer.
Though tissue water content is regulated in Balanus and an active free amino acid response occurs, the magnitude of the response is limited due to the low concentration of free amino acids in the tissue. Other physiological mechanisms are most likely aiding in limiting tissue hydration, or as the authors suggest, some other form of solute is involved along with free amino acids (Fyhn and Costlow, 1975).
Until 1969, osmoregulatory studies in regard to
polyplacophora were for the most part non-existant 5
(Boyle, 1969). Papers of a biochemical nature existed, especially in reference to seasonal biochemical changes
(Vasu and Giesa, 1966; Giesa and Hart, 1967). The reason for the lack of interest in the polyplacophora is certainly puzzling. These hardy molluscs survive well under laboratory conditions and are common inhabitants of the rocky intertidal zone throughout the world (Hyman, 1967).
Mopalia muscosa, the subject of the present
investig~tion, is an abundant inhabitant of the rock
jetty at the mouth of Elkhorn Slough. These chitons will face osmotic stresses ln this habitat. Norris (1969) has reported salinities as low as 20%o (60% SW) in the
surface water in the area where chitons were collected
for this study. This investigation was undertaken to
determine if M. muscosa faced with a salinity stress was:
l) capable of volume regulation;
2) whether or not the chitons behaved as
perfect osmometers;
3) if a volume regulating response was
exhibited, were free amino acids involved;
4) hemolymph and tissue ion regulation;
5) temperature effects on the volume regulating response, if exhibited. MATERIALS AND METHODS
M.. muscosa were collected at lower low water from flat rock surfaces on the south jetty of Elkhorn
Slough. All chitons were taken within the approximate
tidal range of -0.5 to +2.0 feet. Chitons for this
study were restricted to this intertidal range to
limit intra-specific differences due to habitat var-
iation. The animals were pried carefully from the rock with a stainless steel table knife, placed in tupperware containers with SW from the collection site
and transferred ~o the Moss Landing Marine Laboratory.
Once at the laboratory algae were removed from
the chitons and they were placed in a static SW aquarium
(acclimating tank) which held 28 liters of sea water.
The acclimating water came from the collection site and
was unfiltered, though sand particles were allowed to
settle out before the water was placed in the tank. All
chitons were allowed 6-13 days to acclimate to laboratory
cond ions before experiments were performed.
A description of the experimental apparatus is given
ln figure l. Experiments were carried out in two gallon
ss containers with plastic lids or six liter tupperware
containers with form fitting lids. A constant flow of
water past the experimental containers for temperature
6 FIGURE 1. DRAWING OF THE EXPERIMENTAL APPARATUS.
( 1) AIR PUMP, ( 2) ACCLIHATING TANK, ( 3) SIX
LITER EXPERIMENTAL TUPPERWARE CONTAINER,
(4) REFRIGERATED AQUARIUM, (5) LAB SEA WATER
SYSTEM, (6) AIR REGULATORS, (.7) THERMOMETER. 8
-.....:--....__...... ~~~ 9
control was maintained by a gravity feed system ln which water was supplied and cooled when needed by a refrigerated aquarlum. The water for the refrigerated aquarium was supplied by the Marine Laboratory running sea water system. Oxygen was supplied continuously to the acclimating tank and experimental containers.
Salinity, temperature, and pH of the acclimating and experimental sea water were monitored. Salinity was measured with an Induction Salinometer; temperature was recorded twice daily with a mercury thermometer; and pH determinations were performed with a Metrohm/Brinkman
103 digital read out pH meter.
The salinity of the acclimating water ranged from
33.3-34.5%; temperatures were ll-l5°C, the mean temperature being l3°C, and rarely temperatures of l0°C were recorded.
The pH was 7.71-8.11. The experimental temperatures were ll.5-l5.5°C with a mean of l3.3°C; the experimental pH had a range of 7.99-8.31.
Experimental SW was taken from the mouth of
Elkhorn Slough at high tide to insure oceanic conditions.
The water was filtered with a Glass Fiber Type A filter
(Colman Instrument Co.). All experimental SW was
ared by adding approximately ll g of 11 Instant Ocean
ic Sea Salts 11 (Aquarium Systems, Inc.) per
of filtered SW. This produced a salinity of 42.l%o 10
(125% SW). For volume regulation experiments the 125% SW equaled 41.6-41.75%a.
The experimental media were prepared by diluting the 125% sea water with deionized water. Experimental
SW concentrations were 60, 75, 100 and 125% SW. Medium sized chitons, 5-20 g, were used in all experiments; the mean weight was 9.1 g.
In order to monitor weight changes in an altered salinity chitons were removed from the acclimating tanks, placed on plastic petri plates, and allowed 2-3 days to attach to the plate in two liters of acclimating SW. At the start of an experiment, time zero weighings were made which gave the initial weight from which percent changes
in weight could be calculated. The chitons were then weighed at l, 2, 4, 6, 8, 12, and 24 hours after
introduction to two liters of experimental media. In
some cases the one hour weighing was omitted. Weight
changes were measured by removing the petri plate with
the attached chiton, blotting dry with paper towels, and
immediately weighing on a Hettler H20T analytical balance
to the nearest .001 gram. Data from chitons which behaved
abnormally, such as excesslve crawling or curling and
ilure to attach firmly were discarded, though this
rarely happened. During the period when the chitons were ll
being weighed, the temperature of the experimental container was controlled with Blue Ice.
Volume regulatory experiments were also performed at 6.9°C and l9.4°C in 60% SW. These were conducted in the same manner as the l3°C experiments except the high temperature group was continuously aerated during the weighing period.
Hemolymph and tissue ions, along with tissue NPS, were measured after the chitons had spent 48 hours in experimental media. Hemolymph was removed from the pericardium below plate VIII with a 21 gauge needle attached to a l cc tuberculine syringe. The hemolymph was put in small, glass test tubes, covered, sealed immediately with parafilm, and centrifuged at 3000 rpm to remove cells.
The supernatant was then transferred to another test tube, sealed, and stored at 2-3°C until analysis was performed, usually within two days.
For determination of tissue lons and NPS, one cm 2
sections of foot tissue were removed from the center
portion of the foot. These square sections were then
sliced into ten strips one mm thick and treated in the
following manner. NPS was extracted with 30-40 volumes
(v/w) of 100% ethanol (ETOH) for 24 hours ln capped
tic containers after quickly rinsing the foot in
' filtered 100% SW and blotting dry. Preliminary 12
studies have shown that extraction is essentially complete in 2-3 hours. The extract was then poured into a clean plastic container, sealed, and stored at 2-3°C until analysis was performed. For NPS determinations the extract was diluted (1:16) with distilled water. NPS was determined by the method of Rosen (1957), using a
Bausch and Lomb Spectronic 20 spectrophotometer, with optical density readings made at 570 nm. Glycine was used as the standard and free amino acids are reported as mM NP?/kg tissue water. All NPS values were corrected against controls (100% ETOH).
Tissue inorganic ions were extracted with 20 volumes (v/w) of deionized water at 2-3°C for 48 hours.
Na + , K + , Ca++ and Mg ++ determ1nat1ons· · were rna d e w1th· a
Perkin-Elmer 305B Atomic Absorption Spectrophotometer.
Cl determinations were performed with an Oxford Titrator by the method of Schales and Schales (1941), using mercuric nitrate as the titrant. For hemolymph 1ons only single determinations were made, while those of the
tissue and sea water were made in duplicate. An exception
for the hemolymph ion procedure was the Time experiment in
which the 2, 4 and 6 hour samples were run 1n duplicate.
To insure that all ions would read on the
spectrophotometer within the range of standards used, + following dilutions were made: SW and hemolymph Na 13
were diluted 1:401 with a solution of 1000 ppm (parts per
million) K+ to reduce ionization effects and l% HN0 ; 3 + . . + 0 K was dlluted 1:41 wlth 1000 ppm Na and l~ HN0 ; 3 ++ ++ . + Ca and Mg were dlluted 1:81 with 1000 ppm K , l% HN0 , 3 ++ and l% La to reduce phosphate interference. La++ had ++ ++ also been added to the Ca and Mg standards so that
standards and samples would contain the same concentration.
Blanks were run in all cases.
Tissue ions were treated ln the following manner: + . + ++ ++ Na was dlluted 1:6; K , l:ll; Ca , 1:4.3; and Mg , 1:6
with the above mentioned solutions. The method of
Schales and Schales was modified to measure tissue Cl
The Cl standard was diluted 1:6 with deionized water to
glve a concentration of l6.67mEq, and titrant was
diluted 1:4. To justify this modification SW samples
were diluted 1:20 and then titrated with the modified
titrant. This essentially gave the same results as with
undiluted reagents and standard, therefore it was felt
that this modification did not affect the sensitivity of the procedure.
Tissue water content determinations were made with
60-llO mg portions of foot tissue. The foot section was
~ quickly blotted dry, placed on preweighed filter paper,
and weighed to the nearest .01 mg. The section was then
oven dried at 96°C for 20 hours. The percent water 14
content was calculated ln the following manner:
wet wt. - dry wt. x = % wet wt. 100 H20 content
A small correction was made for water lost from the filter paper.
Whole body water content was determined by blotting dry four chitons, and drying to constant weight
To observe the effect of a reduced salinity on
Na + , K+ and Cl over time, a specific experiment was designed, hereafter referred to as the Time experiment.
Chitons at time zero were removed from the acclimating tank and placed in three liters of 60% SW. Three chitons
from the acclimating tank had hemolymph withdrawn and
were designated the time zero samples. Thereafter,
three chitons had hemolymph removed at 2, 4, 6, 8, 12 and
24 'hours. At the time chitons were removed, a sample of
sea water (0.2 ml) was also taken for ion analysis.
Means were computed from the data and compared
with Student's t-test after determining that the variances
were similar with an F-test (Sakal and Rolf, 1969). In
cases where the variances were significantly different,
non-parametric Mann Whitney U test was used to
lish significance between two sets of measurements. 15
Regression analysis was performed by the method of least squares. The significance of the regression coefficient, when tested, was performed by a t-test (Box 11.4,
Sokal and Rolf, 1969). At-test was used to determine the significance level unless otherwise stated. In all graphs and tables, means are given and variability in the data is represented by one standard error of the mean. RESULTS
Before experiments were performed, dilutions of normal SW and that with Instant Ocean (IO) added were + + ++ ++ prepared and the Na , K , Ca and Mg concentrations determined (Table 1). This was done to determine if IO changed the ion composition of experimental media ln comparison to that of normal sea water. IO did not change the lon concentrations considerably, thus justifying the use of IO in preparing experimental sea water.
Two sets of volume regulation experiments were performed. The first took place in September 1975 (Fall volume regulation experiments) and the second in March and April 1976 (Spring volume regulation experiments).
The salinity of the acclimating sea water at the time
chitons were transferred to experimental media was
33.7-34.3%a.
The results of the Fall experiments are shown in
Figure 2. ~· muscosa volume regulates in salinities as
low as 6 0 96 S\IJ. In both 60 and 75% SW there is a
decrease in weight of approximately 6% after a maximal
weight gain. After twenty-four hours in dilute sea
water the chitons were returned to 100% SW and their
Weights monitored over the next twenty-four hours. The
16 17
TABLE 1. COMPARISON OF ION CONCENTRATIONS IN NORMAL SEA WATER TO THAT OF SEA WATER WITH INSTANT OCEAN ADDED. SALINITY OF THE ]\lORMAL 100% SW WAS 33.60%o, WHILE THE SALINITY OF SEA WATER WITH (IO) ADDED WAS 33.68%o· 18
ION % sw. NORMAL sw. EXPTL. sw. (mEq) (mEq)
- - -
125 ------575.7
100 444.5 459.7 + Na 75 328.5 338.6
60 262.9 257.9
125 ------11.1
100 8. 8 8. 6 K+ 75 5.8 5.6
60 4.8 4.5
125 ------133.9
100 103.1 109.5 Hg++ 75 82.5 84. 5
60 66.6 69.9
I 125 ------29.3
100 21. 6 23.4 Ca ++ 75 16.1 17.2
60 12.1 12.8 - I--' ill
FIGURE 2. VOLUME REGULATION IN M. MUSCOSA AT
DIFFERENT SALINITIES. THE CHITONS WERE RETURNED TO 100% SWAT THE ARROWS. FOR 60% SW, N = 6;
75% SW, N = 6; 100% SW, N = 4; 125% SW, N = 3. 20
0 '\t
-..._(/) .... -+ '\t C\J I ~ ~ uj J. .AOOS 1'11101 NI3!>N'VH~% N'I13W 21 overshoot ln weight after returning the chitons to 100% SW is due to a loss of solute from the animals. If only water was movlng the chitons would have regained their original weight on return to normal SW (Pierce, l97lb). The gradual gain in weight after the ove~shoot towards the original weight implies a net water uptake or flux inward. The exact mechanism by which this is accomplished in M. muscosa is not known. It may be the passlve diffusion of salts into the chitons followed by osmotically obligated water. In 125% SW little volume regulation takes place, there is only a passive water loss to the media. Lange and Mostad (1967) have criticized volume regulatory experiments in which whole animals were used. The authors state that weight variations may be caused by excretion, loss of fecal pellets, etc. This may be true, but excretion may also be an inherent mechanism in volume regulation (Fletcher, l974b). Volume regulation cannot even in part be accounted for by defecation ln ~· muscosa, and defecation will in fact not affect the variability in the data. Few fecal pellets were seen in the experimental containers during the course of an experiment, plus the wet weights of fecal pellets from acclimating tank were determined. The pellets had an average weight of 1 mg. A single chiton would have had 22 to pass several fecal pellets for a barely noticeable change in weight and this never occurred. Figure 3 shows a regresslon of plate weight on body weight. Using this regresslon line the soft tissue wet weight of a given chiton could be determined, which is the osmotically active tissue. All statistical procedures in the volume regulation experiments are based on soft parts, though all graphs except Figure 7, show changes in total body wet weight. yigure 4 ls a regressi6n of maximal percent increase in soft tissue wet weight from Fall 60% SW chitons on soft tissue initial wet weight. The regression coefficient is not significantly different from zero. This suggests that the percent increase ln weight in dilute media is independent of size. Spring volume regulation experiments were initially performed to determine if ~· muscosa was capable of fur~her reducing its weight beyond 24 hours in 60% SW. An interesting point came out in the data (Figure 5): Spring 60 and 75% SW chitons gained less weight than the Fall group; this is highly significant (P=.Ol) in both cases. The Ca++ concentration of the experimental media was found ~ to be the same ln both the Fall and Spring ++ experiments. Thus, Ca which lS known to affect membrane Permeability (Alexander, Teorill and Aborg, 1939; Kimizuka FIGURE 3. PLATE WEIGHT OF M. MUSCOSA AS A FUNCTION OF TOTAL BODY WET WEIGHT. 24 .. (\J - 1'1') ro ()) ()) II co ... (\J (f) :a (!) ._ "\t" !-= \.9. (\J ~ ' >- 0 ~ 0 f:\. m ,, 0 (\J --1 ,.. <:( 1-- 0 1-- -\0 - (\J- J__ l__ L_ -~I ____[_ - 0 (\J N (J1 FIGURE 4-. MAXIMUl'1 PERCENT INCREASE IN SOFT PART WET WEIGHT IN FALL 60% SW M. MUSCOSA, AS A FUNCTION OF SOFT PART WET WEIGHT. 26 0 1.() -. II \,. co r.n E <.9 X N • t-= - ' 3: 1"- 1- <;t w r-...: N 3 II (j) )- 1- n:: <.o <( Q_ 1- lL 0 (j) 1.() 0 0 0 0 ~ t0 (\J '.L 1.-1 .1. 3 111 1 CJ 'r! d 1 .::! 0 S N I 3 S 'r! 3 Cf aN I % X 'r! W FIGURE 5. VOLUME REGULATION IN SPRING M. MUSCOSA AT DIFFERENT SALINITIES. FALL EXPERIMENTS SHOWN FOR COMPARISON OF POSSIBLE SEASONAL RESPONSE. (!) SPRING CONTROLS; (~) FALL CONTROLS. SPRING CHITONS, N = 5 FOR 60% SW; N = 7 FOR 75% SW; N = 5 FOR SPRING CONTROLS, 100% SW. N VALUES FOR FALL CHITONS ARE GIVEN IN FIGURE 2. 28 ·~ ' ~ (J) ~ cr) 0~ ~ 0 () <.0 <.o <.9 ~ 0 (J) -J z C\J -J ~ ..q: 0 u_ !.() I'- <.9 (!) u; z '- I 0:: o_ w (J) (J) ::::::>: ~ 1- C\J !.() r--- -J _J <:t lt_ co 'l!,\ l 3 /,\ A 0 0 8 l \11 0 l '3 9 N V H J %, N V 3 V'J 29 and Koketsu, 1962; Palek, et al., 1971 a+b; Rorive and Kleinzeller, 1972) is not a factor. Also as mentioned above, the chitons were collected from a restricted vertical zone in the rocky intertidal, therefore intraspecific differences are probably not a major factor. The basis for the difference in the amount of water uptake may be a seasonal one. Q· muscosa's volume regulating response might vary on a seasonal basis, though further work is clearly needed to verify this point. The effect of temperature on volume regulation has been for the most part neglected in marine inver tebrates, except for Boyles' study (1969). Norris (1969) has shown that temperatures range seasonally from 4-l5°C at the mouth of Elkhorn Slough. Therefore the effect of this parameter on volume regulation was investigated (Figure 6). The l3°C curve is that of the Spring 60% SW chitons already shown 1n Figure 5. High temperatures l9.4°C do not affect the response, the obvious overlap in the standard error bars between 13° and l9.4°C experiments show that no significant differences occurred at any given time. Significant differences between 6.9° and l9.4°C chitons occurred at 2, 6, 8, 12, and 24 hours, and between 6.9° and l3°C at 2, 8, and 12 hours. There was no statistical difference in maximal weight gain between any of the three temperature groups. w 0 FIGURE 6. VOLUME REGULATION IN 60% SW, OF SPRING M. HUSCOSA AT THREE TEMPERATURES. X DENOTES SIGNIFICANT DIFFERENCE BETWEEN 6.9°C AND l3.0°C CURVE. Y REPRESENTS SIGNIFICANT DIFFERENCES BETWEEN 6.9°C AND 19.4°C CURVE. 31 ~M A008 1~101 Nl 3~N~H~ % N~3~ 32 The osmotic permeability p was calculated OS according to Fletcher (l974a) and the formula follows: p (l) OS p lS ln kg H 0/kg animal per hour per unit osmolal OS 2 concentration difference, lS the osmolality of the c0 acclimatisation medium (100% SW), c 0 ~ the osmolality of the chiton hemolymph in the acclimating medium, is the z0 water content of the animal (based on soft parts) kg H 0/kg animal when acclimated to , is the osmolality 2 c0 c1 of the experimental medium (60% SW) and f is the fractional increase in weight t hours after transfer to the experimental media. To obtain f, all observed experimental values for percent increase in weight (soft parts) were divided by 100. Wherever the term c 0 ~-c 0 occurred in the equation it was cancelled, since M. muscosa is an osmotic conformer and this was confirmed by direct osmotic pressure measurements of hemolymph and sea water. Using experimentally determined values of f, P at a given time t could be determined (Figure 7). OS M. muscosa does not behave as a perfect osmometer (Figure 8). The theoretical increase in the weight of soft parts at a given time of chitons behaving as if they were perfect osmometers was also calculated according to FIGURE 7. OSMOTIC PERMEABILITY (P ) IN FALL OS 60% SW M. MUSCOSA AS A FUNCTION OF TIME, BASED ON SOFT PART WET WEIGHT. 34 0 (\J co - v _J____ J. ______.L ___ co FIGURE 8. COMPARISON OF THE RATE OF WEIGHT CHANGE OF A PERFECT OSMOMETER (DASHED LINE) WITH THAT OF FALL 60% SW M. MUSCOSA (SOLID LINE). BOTH CURVES ARE BASED ON CHANGES IN SOFT PARTS. THE SINGLE DATA POINT (Z), MEAN~ 1 S.D., IS THE CALCULATION OF THE MAXIl'1UJ:vl WEIGHT CHANGE OF A PERFECT OSJ:vlOMETER USING ONLY BODY WATER CONTENT AND SEA WATER SALINITY. 36 ___ I ~- C\J N 0 C\J 0 :.?: (/) 0 C\J I I \ \ \ I \ \ \ \ \ \ \ ' I () 0 () 0 0 lc; fi-) (\J ~- - \j _, ":'i ! "'\ ~' ? f} -(:/ ~-J \ •• j t;' :t /1 1~ 71 /,\ J. \f d _l OS fjJ -' ..:.,. j' J I~ N 1/ DVJ 37 TABLE 2. OBSERVED WEIGHT CHANGES (IN %) IN FALL M. MUSCOSA, COMPARED TO EXPECTED WEIGHT CHANGES OF A PERFECT OSMOMETER. THE EXPECTED WEIGHT CHAI'l"GES WERE CALCULATED BY USING SEA WATER SALINITY AND BODY WATER CONTENT (SOFT PARTS). 38 OBSERVED EXPECTED % sw. N X S.E. N X S.E. p 39 Fletcher (l974a) and derivation lS glven ln the same paper. The formula is: t = ( 2 ) Values of P before active regulation begins must be OS used to calculate the theoretical curve, this resulted in the pooling and averaging of the l and 2 hour p OS values Slnce they were not significantly different. This average P value was then used to calculate the . OS theoretical curve.· Table 2 shows observed maximal weight changes (soft parts) for Fall chitons and the expected weight changes if they were perfect osmometers as calculated from body water content (soft parts) and sea water salinity. The maximal weight gain calculated by this much simpler method, 46.4%, agrees well with the asymptotic value 48.7% calculated according to equation 2. The 60% SW chitons as already shown in Figure 8 deviate significantly from a perfect osmometer (P=.OOl) as do the 75% SW chitons (P=.OOl). The 125% SW chitons appear to behave as perfect osmometers though the observed value is slightly lower than expected. Since the number of chitons used was only three, further work is needed before conclusive statements 40 about ideal osmotic behavior (whole animal) ln hyperosmotic solutions can be made. Additional evidence for deviation from a perfect osmometer was obtained by the data shown in Figure 9 for foot tissue water content. In hypo or hyperosmotic media the water content of the foot deviates significantly from ' that of a perfect osmometer (P=.02 for 125% and 75% SW; P=.OOl for 60% SW). Thus, mechanisms at the cellular level are implicated in the control of body water content. The water content of soft parts ranged from 65.8 to 74%, mean 69.3% for four chitons. In general all ions vary ln a somewhat linear manner in foot tissue with changing SvJ salinity (Table 3). K+ concentration is an order of magnitude greater than that of the hemolymph, reflecting high intracellular concentrations, common throughout the animal kingdom ++ ++ (Prosser, 1973). Ca and Mg concentrations are approximately half the hemolymph values (compare Tables + 3 and 4). Na and Cl concentrations are lower than hemolymph values, though these two ions appear to be concentrated considerably in~- muscosa foot tissue. These + high values for Na and Cl may be due to high intra- cellular values or a large extracellular space. Tissue NPS (Figure 10) makes up approximately 4.3% of the tissue osmotic pressure in 100% SW, 46.9 mM/kg FIGURE 9. WATER CONTENT OF THE FOOT OF M. MUSCOSA (SOLID CIRCLES) AS A FUNCTION OF SALINITY. THE NUMBER IN PARENTHESIS IS THE N VALUE. THE WATER CONTENT OF A PERFECT OSMOMETER IS SHOWN BY THE OPEN CIRCLES. 42 () -(\J --() () -() 0::: LIJ () .... 0) <:{ S: 0:: lJ.j <:{ w h. 0 (/) lJ.j co ~ C) * ~ Cr) () c !'-.. ~L- l J 1 J C\1 () co {() '\t C\1 () co 00 Q) !'- 1'- ,...... !"-- 1'.. (J) (.LOO.:l) .LN3J.NOO CJ3l_\IM % N'ri3W 43 TABLE 3. FOOT TISSUE ION CONCENTRATIONS (mEq/Kg tissue water) IN M. MUSCOSA AS A FUNCTION OF SALINITY. C1 Na+ % SW. N X S.E. X S.E. X S.E. Ca++ % SW. N X S.E. N X S.E. ==-=-=-=;;::--=-~=---===--==-=~-=-=-::.=:=-..=---=-=.-=--~~=· 125 3 13.9 ± 0.3 3 52.7 ± 5.9 100 2 10.0 ± 0.8 2 51. 3 ± 2.6 75 3 8. 6 ± 1.1 2 45.9 ± 0 . 3 60 3 6. 8 ± 1.1 3 31.7 ± 0 . 9 TABLE 4. HEMOLYMPH AND SEA WATER ION CONCENTRA TIONS (mEq) AS A FUNCTION OF SALINITY. TABLE 4 IS CONTINUED ON THE FOLLOWING PAGE. 46 HEMOLYHPH SEA WATER - ION % sw. N x S.E. N X S.E. p - 0 I 125 3 6 7 3. 2 ± 10.0 2 6 8 6. 2 ± 3. 7 N. S. I 100 3 5 31. 2 ± 9.1 2 s 4 3. ·s ± 5.5 N.S. I - C1 75 3 407.5 ± 1.7 2 402.4 ± 2.1 N.S. 60 3 313.1 ± 3.8 2 340.2 ± 1.1 .02 125 3 590.8 ± 3.5 2 604.5 ± 1.0 N.S. 100 3 483.2 ± 13.0 2 479.2 ± 6.0 N.S. Na + 75 3 374.9 ± 5.1 2 377.0 ± 8.1 N.S. 60 3 303.4 ± 4.9 2 318.8 ± 6.0 N.S. 125 3 11.6 ± . 55 2 11.0 ± . 2 5. N.S. 100 3 10.2 ± .50 2 8. 6 ± .20 0.1 + K 75 3 7. 3 ± .12 2 6 . 9 ± .05 0.1 60 3 5.8 ± .07 2 5.5 ± .OS 0.05 4-7 ION % sw. N X S.E. N X S.E. p F' '====~- 125 3 27.3 ± . 54- 2 26.7 ± .05 N.S . 100 3 21. 8 ± .29 2 20.8 ± .01 0.1 ++ Ca 75 3 17.5 ± .13 2 14-.8 ± 0 .001 60 3 15.4- ± .18 2 12.7 ± .01 .001 125 3 119.5 ± 2.75 2 122.9 ± .20 N.S. 100 3 . 9 6. 9 ± 1.18 2 97.2 ± 1.6 N.S. ++ Mg 75 3 75.2 ± . 13 2 75.1 ± .20 N.S . 60 3 61. 2 ± . 2 3 2 59.9 ± 0 .05 FIGURE 10. FOOT TISSUE NPS (SOLID LINE) AS A FUNCTION OF SALINITY. THE DASHED LINE REPRESENTS THE EXPECTED CONCENTRATION CALCULATED ACCORDING TO CLARK (1968). EACH OBSERVED DATA POINT IS THE MEAN OF THREE CHITONS. 49 0 fl) 0 C\l 0 0 co 0 r-- 0 w _..._l ___l __ --~---'----·1 __-:; LO 0 0 LO LO v ~31.tltl\ 30SSI.l O>t/SdN S9/0W-W so tissue water. This low value suggests that free amlno acids are not involved in osmoregulation in M. muscosa. Changes in NPS concentration with salinity are completely accounted for by tissue hydration. The expected NPS concentrations were calculated according to Clark (1968) by the following formula: original NPS X amount of tissue H ln 100 g. of 2 o tissue (100% SW) % dry wt. (100% SW) X % water content (dilute SW) % dry wt. (dilute SW) = Expected NPS concentration. In general ~· muscosa shows the typical molluscan + trend in ionic regulation (Table 4). Na conforms to the SW concentration at all salinities tested. Cl lS significantly lower than the sea water value in 60% SW but the SW value may be an artifact of experimental technique, and Cl appears to conform to SW concentrations + at 'all salinities. Na hemolymph and sea water values for 60% SW and Cl 60% SW values are somewhat high. Cl 60% SW has a value of 340.. 2 meq, when this lS divided by .6 a value of 567 meq lS obtained and when compared to the Cl 100% SW value of 543.5 meq a 4.3% error arises. The Na+ hemolymph value from 60% SW is 303.4 meq, divided 51 by .6 this glves a value of 505 meq compared to the hemolymph 100% SW value of 483.2 meq, a 4.5% error. The Na+ 60% SW value of 318.8 divided by 0.6 gives 531 meq + an 11% error when compared to the Na 100% SW concentra- + tion. The Na 60% SW value shows the largest disagreement with expected values, therefore statements based on the Na+ 60% SW concentration will have to be made with considerable caution. ++ + Both Ca and K hemolymph values are elevated above th~t of the SW between 100 and 60% SW (Figures ll and 12). The manner in which the two ions are maintained above the sea water concentration appears to differ ++ (Figure 13). The difference between hemolymph and SW Ca increases as the salinity decreases. Conversely the + difference between hemolymph and sea water K decreases. Except for 100% SW the K+ difference is fairly uniform at ++ any salinity, while this clearly is not so for Ca . Linear regression analysis for hemolymph K+ on SW K+ gives the following equation y = .151 + l.072X. The slope 1.072 is not significantly different from 1.0 and suggests ++ a passive distribution. The shape of the Ca curve did not lend itself to linear regression analysis. If intracellular isosmotic regulation were taking Place as part of the volume regulatory process, solute extrusion may occur from the cells to the hemolymph. To 52 z 0 H f--l u z ;::J ~ [!) ~ f--l r-l H '-?' r-l <"·~ H r.Ll ~ p:; 0 C\l 0 0 0 a:: IJ.J I- 0 <:( m S: <:( IJ.J (/) 0 co ~ 0 <.0 + ){ b3W ++ FIGURE 12. HEMOLYMPH Ca CONCENTRATION AS A FUNCTION OF SALINITY IN M. MUSCOSA. 0 C\1 -0 0 0 a:: w 1- _.,.. 0 0 <.D --~----1 J.__ Q) 0 C\1 C\.1 FIGURE 13. DIFFERENCE BET\~EEN HE:tv10LYt1PH AND SEA WATER K+ AND Ca ++ AS A FUNCTION OF SALINITY IN M. MUSCOSA. 57 0 t() 0 C\1 0 0 0 Q: lu..... <:( ~ 0 - (j) <:( lu (/) ~ 0 co 0 I'- 0 <.0 ~_j ___l.!..-,._=J~~L~~=l"~-~-~~""'".L~-·~.....,d 0 0 1.(). 0 l"t) C\1 + >I 0 N \t ~ .j. o :J 'IX S 0 N \1 H d ~"J A-~ 0 ~-L3 H N33A\J.38 b3w Nl 3:JN3CJ3.d.:JIQ 58 test this hypothesis, hemolymph concentrations over time - + + of Cl , Na and K in 60% SW were measured after transferring chitons from 100% SW to 60% SW (Figures 14-16). No solute extrusion appears to take place, though this clearly does not rule out sequestering of ions (Oglesby, 1975). Equilibration times were established by pooling the SW values at 4, 6, 8, 12, and 24 hours and testing for significance between hemolymph values at each time sampled and the pooled SW values. Cl reaches equilibrium in 8 hours, though the 4 hour value is not significantly different from that of the SW (Figure 14). Na+ is in equilibrium within 5-7 hours (Figure 15). K+ appears not to reach equilibrium in 24 hours (Figure 16). K concentra- tions of chitons which had been in 60% SW for L~8 hours show values closer to the experimental SW concentration than those sampled after 24 hours in the Time experiment. (Jl w FIGURE 14. C1 CONCENTRATION (LOWER SOLID LINE) OF THE HEMOLYMPH IN M. MUSCOSA AFTER TRANSFER AT TIME 0 FROM 100% SW TO 60% SW. FOR THE CONTROLS (UPPER SOLID LINE) EACH DATA POINT IS REPRESENTED BY 1 CHITON. THE DASHED LINE IS THE SEA WATER Cl CONCENTRATION. 60 0 C\l Cf) _J ill 0 0::: r- z 0 (_) I !/) -1.., I J:: C\l i- -w I ~ I 1- I \ \ b ' ' ' ' '.;:, I I -::::.?-:.-_. -~··-~-'~L _____ J.~·~--ML~=J._6__ .t.--....JJ 0 0 0 0 0 0 0 ~ ~ 0 ~ W ~ N ~ ~ ~ ~ ~ ~ ~ _I:J b 3W + FIGURE l5. Na CONCENTRATION (SOLID LINE) OF THE HEMOLYMPH IN M. MUSCOSA AFTER TRANSFER AT TIME 0 FROM 100% TO 60% SW. THE DASHED LINE IS THE SEA + WATER Na CONCENTRATION. 62 0 N 1/) -I-. N -::t: I I 6 - ro I I I J I I I ' \ - '¢ \ \ ~ I \ I _1-:~~~~'-'··--L __( __ ~l _.....;;t.tJ \\ 0 0 co co '¢ C\1 FIGURE 16. K+ CONCENTRATION (SOLID LINE) OF THE HEMOLYMPH IN M. MUSCOSA AFTER TRANSFER AT TIME 0 FROM 100% SW TO 60% SW. THE DASHED LINE IS THE SEA WATER K+ CONCENTRATION. 64 0 I 0 C\.1 lO I 1 I I N (/) ? -< -I... -::t: lU I ~ I 1- ~ w I I 0 I J { --;t \ \ 'o I I -.:f:::,_____ L_~-~~-~J. ___ ,. __ [_~~~"____l6_. 0 co lO DISCUSSION Volume regulation ls an adaptive physiological mechanism. The reduction ln body volume due to loss of water will aid in the maintenance of normal physiological functions. The water uptake when placed in dilute SW, will interfere with locomotion and consequently feeding due to a swollen foot. By reducing body volume a more firm attachment to the substrate may be maintained. In regulating foot tissue water content, the chitons will not have· to deal with excessively swollen cells, therefore membrane structure will not be severely altered and the chiton membranes will be able to carry out the numerous physiological functions. For M. muscosa this is most likely of short term significance; salinity variations when they occur; probably do not last for more than twenty-four hours, if that long, at the mouth of Elkhorn Slough. Deviation from a perfect osmometer (total body weight) has been shown in two other chitons, Cyanoplax hartwegii (McGill, 1975) and Nuttalina californica (Simonsen, 1975). The volume regulating response of ~· hartwegii is very similar to that of Fall ~· Muscosa; both chitons gain between 10-12% in body weight within 65 66 two hours after introduction to 75% SW, and then bring their weight down to approximately 4% of their original weight after twenty-four hours. N. californica exhibits little volume regulation, but certain mechanisms are involved in limiting water uptake or loss in an altered salinity, since this chiton does not behave as a perfect osmometer (Simonsen, 1975). These mechanisms are probably the loss of salts, passively, through the body wall and bound water within the cytoplasm. Classically, marine molluscs are considered osmotic conformers. Though this ls still true in most cases, the body fluids appear to be slightly hyperosmotic to a given SW osmotic pressure (Pierce, 1970; Magnum and Johansen, 1975). M. muscosa is also an osmoconformer + and that is seen in that the major ions Na and Cl of the hemolymph, which primarily determine the osmotic pressure, conform close~y to the sea water concentration (Table 4). Osmoconformity in the polyplacophora has been demonstrated by Boyle (1969) and Simonsen (1975). Regulation of Ca++ is evident in M. muscosa and not uncommon in marine molluscs. Webber and Dehnel (1968) also report Ca++ regulation in the limpet Acmaea scutum Cnow Notacmaea scutum) but only in those from estuaries. Piper (1975) reports Ca++ regulation in the chiton Nuttalina californica from the rocky intertidal of 67 Monterey Bay, Callfornla.. . The reasons f or Ca ++ regulatlon. may be numerous since this lon is involved ln many complex physiological and biochemical mechanisms, and ln light of Fletcher's work (l974b), a possible role for Ca++ in volume regulation will be put forward below. ++ . + . . Ca regulatlon and K elevatlon ln the hemolymph of M. muscosa have negligible effects on the hemolymph osmotic pressure. The total increase above isosmotic conditions are less than 3 mOsmols in 60% SW, assuming both ions are 100% ionized. A small amount of protein binding·may occur for both ions, thus the increase in osmotic pressure is less than expected. K+ appears to be distributed passively between + . . hemolymph and sea water. The K elevatlon lS roughly constant at all salinities and may be due to a negatively charged blood protein (Pierce, 1971). As previously stated M. muscosa ls an osmoconformer, therefore the decreased hemolymph osmotic pressure in a reduced salinity will cause the development of a large osmotic gradient between the cytoplasm and hemolymph. A tissue acting as if it were a perfect osmometer under these conditions would take up water until considerable swelling and stretching of cellular membranes had taken place. In this case the calculated theoretical water content of a perfect osmometer would equal the observed 68 water content. ~· muscosa regulates its tissue water content by possibly two factors, bound water in the cytoplasm and intracellular isosmotic regulation. Water within the cell is not in simple solution (Ha~elwood, et al., 1969; Hinke, 1970); as much as 25% of the water in the myoplasm of the giant barnacle Balanus nubilus does not act as solvent (Hinke, 1970). The existence of bound water will result in a reduced solvent volume within the cell and this in effect will limit the water uptake or loss ln an altered salinity. Therefore, since a solute source has not been specifically implicated in intracellular isosmotic regulation in M. muscosa, the extent of the role played by bound water or intracellular isosmotic regulation in the regulation of tissue water content cannot be stated at this time. In a number of marine invertebrates, tissue water content and volume regulation are accounted for by free amlno acids (Allen, 1961; Lynch and Wood, 1966; Virkar, 1966; Emerson, 1969; Virkar and Webb, 1970; Marique and Gilles, 1970; Pierce, 197la; Pierce and Greenberg, 1972 and 1973; Gilles, 1972; Dall, 1975; Bartberger and Pierce, 1976). In cases where NPS concentrations are low, for instance 50 mM NPS/kg tissue water in 100% SW (hypothetical example), this in itself suggests little role for NPS in intracellular isosmotic 69 regulation. Over a wide salinity range, especially ln dilute sea water, NPS is simply limited in the effect it can have on cytoplasmic osmotic pressure. If a salinity reduction is 500 mOsmol and assuming approx imately equal osmotic pressures in the cytoplasm and hemolymph, it's obvious that the above hypothesized NPS concentration can do little to relieve a large oSmotic gradient between the hemolymph and cytoplasm. The imino acids proline and hydorxy proline give a yellow color with ninhydrin instead of the usual purple and cannot be measured at 570 nm (Light, 1974). In the mollusca, proline may be involved in intracellular osmoregulation but it is not the major amino acid contributing to the cytoplasmic osmotic pressure. Gilles (1972) reports a value of 0.4 mOsmol proline per kg tissue water in the chiton Acanthochitona discrepans in 100% SW. Proline plays little role 1n intracellular osmoregulation in the bivalve Modiolus demissus granosissimus where proline concentrations are low, less than 20 uM/g dry weight and changes with salinity are negligible (Pierce and Greenberg, 1972). The clam Mya arenaria has less than 3 mM proline per kg tissue H in 30% SW (Virkar and Webb, 1970). Proline 2 o could be involved 1n intracellular osmoregulation in M. muscosa but comparative data indicate this is not 70 so and proline concentrations ln M. muscosa are most likely low. High levels of tissue free amlno acids are not only found in those euryhaline organisms that use them in intracellular osmoregulation, they are also found in stenohaline marine invertebrates (Clark, 1968). Since free amino acid pools appear to be ubiquitous in marine invertebrate tissues, what is the primary role of these small organic molecules? Shick (1975) provid~s evidence of a nutritional role for dissolved free amino acids ln the coelenterate Aurellia aurita. Starved and fed groups of~- aurita were supplied with 14 C-glycine and the production of 14 was monitored over time. After co 2 three hours the starved group showed significantly increased 14 C0 production over that of the fed group. 2 This indicated that catabolism of glycine was taking place, though the Kreb's Cycle lS not involved (Shick, 1975). Glycine in ~- aurita is catabolized via the glycine cleavage system, with one mole of glycine producing a mole each of NADH and NADPH (Shick, 1975). 2 2 Additional experiments with ~- aurita show that the percent strobilation by polyps is affected by amino acids. Starved~- aurita resulted in only 22.5% of the polyps strobilating, while fed, starved plus alanine, and starved plus glycine showed 100% strobilation. 71 The manner in which marine invertebrates obtain free amino acids has also been investigated. Most soft bodied marine invertebrates are capable of reducing the amino acid concentration of an experimental solution (Stephens, 1968). In vivo synthesis of amino acids also takes place through amination reactions. The enzyme glutamate dehydrogenase (GDH) which catalyzes the fol lowing reaction, a KGA + NH E---~ glutamate 4 NADH ~NAD appears to be pivotal (Hochachka and Somera, 1973). Therefore, the free amino acid pool is maintained by absorption (digestive and through the body wall) and synthesis. The uptake of amino acids has not only been shown for whole animals but also for bivalve gill (Wright et al., 1975; Anderson, 1975). Wright et al. (1975) have shown that the uptake of cycloleucine into in vitro preparation of Mytilus californianus gill follows Michaelis-Henton kinetics. The uptake lS also against tremendous concentration gradients and strongly suggests active transport. The gill of the bivalve Rangia cuneata 72 appears to actively absorb glycine, and this uptake is dependenf on the Na+ concentration in the experimental + media. Na -free experimental media inhibit uptake, which indicates the involvement of a cotransport system (Anderson, 1975). The above evidence, along with the ubiquitous nature of free amino acid pools in marine invertebrates, suggest that the main role of these small organlc molecules has yet to be determined and that it need not be osmoregulation (Clark, 1968; Freel et al., 1973). From a biochemical point of view the use of free amino acids in intracellular osmoregulation does make good sense. Inorganic ions, specifically Cl and K+ are needed for enzyme activity (Schoffeniels, 1974; Hochachk~ and Somera, 1973). One may speculate that already existing free amino acid pools became involved in intra- cellular osmoregulation to lend a "sparing effect" on intracellular ion concentrations. In inorganic ion concentrations could be maintained intracellularly, then it's likely enzyme activity would not be affected. The use of free amino acids in intracellular isosmotic regulation can be ruled out as a mechanism for volume regulation in~· muscosa, though salts, primarily + - Na and Cl may be involved. Freel et al. (1973) have demonstrated an active intracellular decrease in Na+ and 73 Cl with decreasing salinity in the polycheate Neanthes SUCClnea:. The Chinese crab Eriocheir sinensis decreases its intracellular ion concentration to a greater extent than it does free amino acids, when animals adapted to fresh water and sea water are compared (Florkin and Schoffeniels, 1969). Other possible mechanisms for volume regulation are: l) active transport of salts across the body wall followed by osmotically obligated water; 2) increased output of urine by the kidney; 3) the "volumestat" mechanism of Brown et al. (1972). The "volumestat" hypothesis states that the kinetic energy of water influx is transferred to the integument of the organism as .a result of stretching elastic elements in the integumental membrane. When isosmotic conditions have been achieved, the energy stored in the stretched membrane due to the increased hydrostatic pressure, forces water and salts through the membrane into the environment. The "volumestat" hypothesis can also be ruled out as a mec.hanism for volume regulation ln H. muscosa slnce this theory is dependent on the lack of viscous integumental .elements. + The high concentrations of Na and Cl ln the foot tissue of H. muscosa may reflect high concentrations intracellularly. Extracellular space (ECS) determinations would need to be made with inulin. Once ECS had been 74 determined at each experimental salinity, calculations would then reveal if intracellular concentrations of Na+ and Cl had actually decreased. This in itself is subject to criticism, since an immobile compartment of inorganic ions exists in muscle (Dunham and Gainer, 1968). Even though immobile ion compartments exist, extracellular space determinations would be of some value and could give some indication if Na+ and Cl involvement in intracellular osmoregulation is actually taking place. ~oss of salts from an animal's body fluids followed osmotically by water, after introduction to a hyposmotic medium will contribute to volume control. Volume control may be thought of as the limit to the percent increase in weight after introduction to a hyposmotic medium. Volume control may be considered separate from active volume regulation; the loss in weight after a maximal uptake of water in dilute SW. In actuality active volume regulation may begin before it manifests itself as a loss in weight, and may occur during the period of volume control. The physiological mechanisms responsible for volume regulation may in part, be responsible for volume control. The loss of salts implies the body wall is permeable to salts and this has been proven by Boyle (1969) who placed the chiton Sypharochiton ~lliserpentis in sea water-sucrose 1 75 1 1 mixtures isosmotic to sea water. A loss 1n weight occurred which indicated a loss of salts from the 1 chiton's body fluids followed by water. Thus, simply the 1 loss of salts from body fluids before active volume 1 regulation begins cannot account for the volume 1 regulation response but merely volume control. If salts are lost passively during the period of volume 1 regulation, as indicated by hemolymph ion concentrations 1 which have not reached equilibrium with the experimental 1 media, then volume regulation can in part be accounted 1 for by passive diffusion of salts. Once ion equilibrium is established and assuming potential differences across 1 the body wall in osmoconformers are negligible, active transport of ions will have to take place for the continuation of the volume regulating response. In Fall 60% SW ~· muscosa, the passive loss of salts will only account for a reduction in weight from approximately l~-18% (Figure 2). Indeed, another 5% reduction occurs from 18-13%. The situation is different for Spring 60% SW chitons. The passive loss of salts could possibly explain the loss in weight from 13-9% and the steepest part of the volume regulation curve, though another 6% reduction in weight occurs over the next sixty-four hours (Figure 5). The further reduction in weight after 1on equilibrium is reached will have to be due to active transport, assuming 76 no other mechanisms for volume regulation exist. If active transport of salts occurred across the body wall, and an efflux of salts was taking place simultaneously from the cell interior, the hemolymph concentration might not reflect the efflux from the cells...... ++ Marlne lnvertebrates placed ln lsosmotlc Ca free media have been shown to swell excessively. This, along ++ with experimental evidence that Ca affects membrane configuration and permeability, has led to the theory that inCa++ free media permeability lS increased and the swelling due to an increased water influx. Recent ++ evidence indicates that Ca caused configurational changes in membranes and the resultant decreased passive permeability are due to contractible proteins which possess Ca++_ATPase activity (Palek et al., 1971; Rorive and Kleinzeller, 1972). Fletcher (l974b) working with the polycheate Nereis diversicolor, demonstrated swelling by this annelid ln. Ca ++ f ree medla.. The swelling was not due to an lncrease· d water ln· f lux! Af· ter twenty-f our h ours ln Ca+ + free media, the diffusional water influx measured with tritiated water was not different from controls. Ciliary action is in part under the control of . ++ lntracellular Ca concentration (Eckert, 1972; Murakami and Eckert, 1972). Fletcher (l974b) has reached the 77 conclusion that the swelling observed in ~· diversicolor ++ . in Ca free SW is not due to increased water influx but due to the cessation of urine flow, which is dependent on ciliary action. The kidneys of the polyplacophora are lined with cilia, and the cilia serve the purpose of moving urine ++ (Hyman, 1967). The fact that M. muscosa reguJates Ca and that urine flow is dependent on ciliary action, can only lead to the conclusion that the kidney may be involved in volume regulation. This is only speculation and further research will be necessary to either prove or disprove this hypothesis, but until this question has been resolved, the above mentioned role of the kidney cannot be ruled out. Stickle and Howey (1975) state that a reduction ln excretory function may take place in the kidney of Thais haemostoma during salinity fluctuations due to reduced ++ Ca . T. haemostoma failed to volume regulate in reduced salinities. It's unfortunate that the effect of temperature on volume regulation has been only slightly studied. Salinity fluctuations surely occur concurrently with temperature fluctuations in the environment. Temperature effects on respiration are well documented and expressed by the well known Q relationship. Boyle (1969) has 10 studied temperature and its effect on volume regulation 78 1n the chiton ~· pelliserpentis. The experiments were conducted at 10°, 20° and 30°C in 50% SW. The chitons at 30°C gained considerably more weight than those at 10° and 20°C which gave almost identical curves for weight ga1n over time. High temperatures, l9.4°C, did not affect the volume regulating response of M. muscosa. Low temperatures, 6.9°C, did have a serious affect on the response. Influx of water is reduced, as shown by the reduced gain 1n weight at two hours (Figure 6), when compared to the controls (l3°C curve). If the volume regulating response is due to water movement across the general body surface, then outflux of water 1s also decreased since the ability to volume regulate lS reduced. If the volume regulating response is due to urine production by the kidney, then this mechanism has been affected. In any case, water influx and outflux has been reduced by low temperatures. The experiment may suggest that the volume regulating response is due to water movements across the general body surface. The reduced influx and outflux of water in the 6.9°C experiment may simply be due to the reduced diffusional permeability of water in the cold (House, 1974). The diffusional permeability to water for frog skin is reduced by low temperatures (House, 1974) and Evans (1969) has shown that in three species of teleosts 79 that water flux is reduced 1n the cold (l0°C) when compared to 20°C. The Q for Carassius auratus is 10 2.12, for Phoxinus phoxinus 1.77 and Platichthys platessa 1.81. It could be that water movements across the body wall and through the kidney are taking place as mentioned above and that both mechanisms are affected by the cold. The ability to volume regulate does not neces sarily imply that the organism is euryhaline (Lange, 1972). It is the salinity range in which the animals are capable of volume regulation and osmoregulation that determines euryhalinity and stenohalinity (Lange, 1972). The salinity range at which euryhaline and stenohaline are differentiated has not been stated and the two terms certainly contain a large amount of ambiguity. In an attempt to establish whether or not l1. muscosa is eury haline or stenohaline a survival experiment 1n dilute SW was performed. Six M. muscosa were used 1n this experiment in,GO% SW in which the water was changed every four to six days. One chiton became detached after two weeks and another after four weeks. The other four chitons remained attached and active for an additional two weeks, at the end of this two week period they were returned to normal SW. The four chitons survived the return to normal SW for four weeks at which time the experiment was terminated. The survival experiment clearly indicated that M. muscosa ls 80 capable of survival in a wide range of salinity and that these chitons are euryhaline. The problem with this has already been mentioned; the concept of euryhaline and stenohaline has not been clearly defined in terms of absolute salinity. Recognizing this problem still leads me to conclude ~· muscosa is euryhaline. Euryhalinity is in part dependent on intra cellular isosmotic regulation and as mentioned above, involves free amino acids in most marine invertebrates (Lange, 1972). Clearly, in ~· muscosa free amino acids are not involved and intracellular isosmotic regulation may involve salts. In the molluscan genus Modiolus, volume regulation occurs in a reduced salinity. These bivalves when returned to normal SW overshoot their original weight and this is due to solute extrusion from cells, notably the free amino acids (Pierce, l97lb). Modiolus fails to gain weight after the overshoot on return to normal SW, in other words, the volume response is unidirectional (Pierce, l97lb). Pierce (l97lb) has concluded that the volume regulating response which 1s due to intracellular isosmotic regulation has allowed bivalves to invade fresh waters and indeed they have. The unidirectional response has not allowed for a reinvasion of the marine environment. Once the solute 81 is lost from the animal's tissue it lS committed to its tissues being diluted. In M. muscosa, reintroduction to normal SW after a salinity stress also results in an overshoot in weight. M. muscosa differs from Modiolus though, in that the original weight is nearly attained within twenty-four hours after return to normal SW (Figure 2). This weight gain after the overshoot does not commit M. muscosa to a more diluted state as in Modiolus, and after being subjected to a reduced salinity, M. muscosa can attain its original weight when returned to normal SW. This return to the original weight when reintroduced to normal SW would seem to favor those animals which are penetrating estuaries, where salinity fluctuations are extreme and common. Modiolus did not survive beyond two weeks after return to normal SW from a reduced salinity (Pierce, 197lb). M.muscosa survived four weeks and showed no ill affects, the chitons were active, and a few fecal pellets were defecated. The evidence presented for M. muscosa clearly indicates this osmoconforming Mollusc ls capable of penetrating into estuaries and surviving severe salinity fluctuations. Though invasion of fresh water has not been accomplished by the polyplacophora (Hyman, 1967), it may be that the use of free amino acids in intracellular isosmotic regulation is a necessary 82 prerequisite. The utilization of free amino acids would allow for a larger solute loss and a greater adaptability to fresh water conditions. Animals relying on salts can only allow a certain amount to be lost before enzyme activity is severely impaired. SUMHARY 1. ~· muscosa is an osmotic conformer, though ++ . . + . . the Ca lon lS regulated, and K elevatlon ln the hemolymph appears to be due to a passive distribution. 2. The ability to volume regulate is demonstrated in hyposmotic sea water, and M. muscosa does not behave as a perfect osmometer. 3. Tissue water content lS regulated and deviates from an ideal osmometer ln both hypo- and hyperosmotic sea water. 4. Volume regulation and tissue water content regulation are not due to free amino acids as in many marine invertebrates. 5. Volume regulation may be accounted for by active transport of salts across the body wall and the kidney. 6. Ca++ regulation ln relation to ciliary action, urine flow, and volume regulation is discussed. 7. 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