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1979
The regulation of body fluids in the estuarine annelid Nereis succinea
James Alan Dykens College of William & Mary - Arts & Sciences
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Recommended Citation Dykens, James Alan, "The regulation of body fluids in the estuarine annelid Nereis succinea" (1979). Dissertations, Theses, and Masters Projects. Paper 1539625054. https://dx.doi.org/doi:10.21220/s2-s9cy-by63
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. THE REGULATION OF BODY FLUIDS u IN THE ESTUARINE ANNELID NEREIS SUCCINEA
A Thesis
Presented To
The Faculty of the Department of Biology
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Master of Arts
by
James A. Dykens ProQuest Number: 10626213
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A /JtMimju Charlotte P. Mangum Q
Eric L. Bradley
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ii TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ...... • iv
LIST OF TABLES ...... v
LIST OF FIGURES ...... vi
ABSTRACT...... vii
INTRODUCTION ...... 2
METHODS AND MATERIALS ...... 4
RESULTS ...... 7
DISCUSSION ...... 12
LITERATURE CITED ...... 16
iii ACKNOWLEDGMENTS
I would like to express my appreciation to Dr. Charlotte P. Mangum for her patience and consistent support throughout the course of this study. I am grateful to Drs. G. Capelli, E. Bradley, and R. Black for their advice and critical readings of the manuscript. The generous supply of amiloride provided by Dr. G. Fanelli of the Merck Institute for Therapeutic Research is appreciated. I thank Mrs. Eddie Albright and Mrs. Karen Dolan for their typing of the manuscript. Finally, my deep appreciation to my wife, Harriet, for her unfailing support and patience.
iv LIST OF TABLES
1. Excretion rates of free amino acids during volume readjustment
v LIST OF FIGURES
1« Time course of volume readjustment
2. Time course of volume readjustment at different temperatures
3. Tissue hydration and time course of volume readjustment in hypoxic sea water
4. Time course of volume readjustment in sea water with 30 mM KT*" or K+ free sea water
5. Time course of volume readjustment in Na free sea water and Cl free sea water * 6. Time course of volume readjustment in divalent cation free sea water
7. Time course of volume readjustment in Amiloride and Diamox
8. Time course of ammonia excretion
vi ABSTRACT
The process of volume readjustment following exposure to dilute sea water was examined in the estuarine polychaete, Nereis succinea. Volume readjustment is an active process that requires O 2 , uninhibited cellular metabolism, and is retarded by cold. The intracellular labile osmolytes lost during volume reduction include free amino acids that are both deaminated and extruded intact into the medium. Losses of intracellular inorganic ions also appear to be involved, but volume readjustment is not affected by ouabain.
The elevated urinary output seen during volume readjustment is not a result of increased intercoelomic hydrostatic pressure. Removal of the supraoesophageal ganglion, the best known neuroendocrine site in annelids, has no effect on volume readjustment.
vii THE REGULATION OF BODY FLUIDS
IN THE ESTUARINE ANNELID NEREIS SUCCINEA INTRODUCTION
Nereis succinea (Frey and Leuckart) is a euryhaline polychaete found
in salinities ranging from 2 °/oo (Filice, 1958) to 70 °/oo (Carpelan and
Linsley, 1961). As in other nereids, changes in the osmolality of its
body fluids conform to those in the ambient medium above 10 °/oo salinity
(Freel, eX_ al., 1973; Oglesby, 1965), but active osmoregulation occurs
below that threshold (Oglesby, 1965). The mechanisms of hyperosmotic
regulation include the reduction of water and ion permeability of the
body wall (Smith, 1964; Oglesby, 1965; Fletcher, 1973) and the active
absorption of NaCl from the medium (Fretter, 1955; Jeuniaux, et al.,
1961; Oglesby, 1972; Smith, 1972; Doneen and Clark, 1974).
The mechanisms of body volume regulation in annelids have not been as well studied, although the chronology of the process is more complete
ly described than in other animal groups (Oglesby, 1965, 1978). Essen
tially, three phases can be distinguished: (1) a brief period of
swelling lasting from 1-2 hours after a salinity decrease; (2) a plateau that lasts from 2-8 hours; (3) a gradual weight loss over 1-3 days. The presence of this latter phase of slow weight reduction is considered by
Brown, _et al. (1972) to be indicative of volume regulation. Although body volume regulation is not perfect, the magnitude of weight loss during the readjustment phase is impressive. Both intracellular and extracellular fluid regulation is involved in this process, although their relative contribution to the observed trends in body weight is not clear (Oglesby, 1978). While the basic mechanism of intracellular 2 3
readjustment is believed to be the extrusion of osmolytes and with them
osmotically obligated water, the processes responsible for extrusion
have not been investigated. Freel, et al. (1973), who attempted to
identify the labile osmolytes in muscle tissue of N. succinea, concluded
that both intracellular NaCl and ninhydrin positive substances (free
amino acids) are involved. Some possible mechanisms for the readjust ment of intracellular fluid volume include: (1) active extrusion of
inorganic ions; (2) passive efflux of inorganic ions; (3) efflux of free
amino acids; (4) catabolism of free amino acids.
The readjustment of extracellular fluid volume clearly involves an
increase in urine output, observed during volume readjustment (Smith,
1970; Oglesby, 1972; Fletcher, 1974). The effector of enhanced urine
flow, however, is unknown. Brown (1972) proposed that the increased nephridial clearance rates could result from a passive rebound effect of the body wall connective tissue. If there is a passive elastic response, however, it is not clear why the time course of readjustment always includes an initial period of swelling. Therefore, a more plausible hypothesis would include the active contraction of body wall musculature after 1-3 hours in a dilute medium, the point at which weight begins to return toward the initial value. Fletcher (1974), however, calculated that flow rates generated by increased activity of the nephridial cilia could account for the observed fluid loss, necessitating no change in coelomic fluid pressure. A third possibility, not previously proposed, is the secretion of a diuretic hormone by the best known neuroendocrine organ in annelids, the supraoesophageal ganglion complex.
These mechanisms of intracellular and extracellular volume regula tion have been examined in N. succinea. METHODS & MATERIALS
Specimens of Nereis succinea were collected at Indian Field Creek on the York River Estuary in Virginia in salinities varying from 12 °/oo to
20 °/oo. Animals were provided with glass tubes and kept at 15°C in water of 14 °/oo for at least five days prior to experimentation.
Neither heteronereids nor gravid individuals were used.
Worms were placed in small volumes (ca. 15 ml) of experimental media, removed, blotted, and then weighed on a Bethlehem Torsion Balance (± 1 mg).
Although the initial weight did not vary by more than 3 mg, it was taken twice, two hours apart, and the mean used as the 100% value for wet weight computations.
Low salinity water was prepared by adding distilled water to natural sea water from the Eastern Shore of Virginia (34 °/oo). The ion substitu tion media were prepared using the ion ratios of M.B.L. Formula #4
(Cavanaugh, 1956). Divalent cation free media were prepared according to Cavanaugh (1956). Sodium free media were prepared by direct substitu tion of NaCl with osmotically equivalent amount of either CsCl, RbCl, or
LiCl. Osmolality was verified by freezing point depression (Osmette,
Precision Systems). PO2 was measured with Yellow Springs Instrument Co.
0^ electrode model 54. Ammonia excretion was measured by confining individual animals in 30 ml vessels for 36 hours and measuring the change in ammonia with the phenol hypochlorite method (Soloranzo, 1969).
Coelomic fluid was obtained via body wall puncture with drawn out cap illary tubes and pH measured on a Radiometer Blood Gas Analyzer BMS 1. 4 5
Ninhydrin positive substances (TNPS) were measured using the follow ing adaptation of the method of W,D, DuPaul and K.L. Webb (personal comm unication) : A A N NaAcetate buffer was made by dissolving 164.05 g anhydrous NaAcetate in 500 ml ammonia free water. To obtain a pH of
5.5, 50 ml acetic acid was added and the buffer stored in dark bottles at 5°C. Solution A was made by adding 0.2602 g hydrazine sulfate plus
1 drop I^SO^ to 800 ml ammonia free water and then bringing the volume to 1 1. To make solution B, 16.5 ml acetate buffer plus 1 g. ninhydrin were added to 50 ml methyl cellosolve (ethylene glycol monomethyl ether).
This mixture was brought to 100 ml with ammonia free water. A 0.1 ml sample of the unknown was added to 0.3 ml solution A followed by 0.8 ml solution B. The tubes were covered, inverted several times, heated at
95°C for 15 minutes and then cooled. Four ml of 60% ethanol were added and the absorbance at 570 nm determined with a Bausch and Lomb Spectronic
20 spectrophotometer. A standard curve of four graded solutions of glycine ranging in concentration from OmM to 25mM resulted in AlmM amino nitrogen = A0.42 absorbance. Because this TNPS assay is also sensitive to NH^, ammonia was monitored simultaneously and the values subtracted from TNPS.
Tissue hydration was estimated by placing excised body wall muscu lature onto porous nylon packed into centrifuge tubes with holes through the bottom. The tubes were centrifuged at 500 g. for 10 minutes in a
Sorvall model GLC-1 centrifuge. The tissue was weighed on a Sartorious balance (±0.1 mg), dried at 60°C for 24 hours and then re-weighed.
Intercoelomic hydrostatic pressure was measured with a Statham
Laboratories Inc. Physiological Pressure Transducer model P23Dc connected to a Grass Model 7B Polygraph. A hypodermic needle was placed into the coelom through the dorsal surface at a point approximately one third of 6 the body length from the anterior. Pressure was continuously monitored for several hours prior to a salinity decrease and for at least 30 hours afterwards.
The supraoesophageal ganglion was isolated and removed via a longi tudinal incision through the prostomium and the following eight to ten anterior segments. Mock surgery consisted of locating the ganglion but leaving it undisturbed. Less precisely, the anterior eight to ten segments were totally removed. Animals were also forced to autotomize their tail segments by applying prolonged, gentle pressure at the mid section. All animals and isolated tail sections were allowed to recover at least 24 hours prior to exposure to a salinity change. RESULTS
Time Course
Upon exposure to dilute sea water, IN. succinea swells and gains
weight. This initial phase of swelling lasts from 45 minutes to two
hours at 15°C and is followed by a plateau of approximately one hour
after which the weight declines toward the original value (Fig, 1),
Both the magnitude of the weight gain and the timing of volume readjust
ment are a function of the magnitude of the salinity shock, and the
salinity tolerance of this species; in other, more stenohaline annelids,,
swelling is greater and weight readjustment slower (Oglesby, 19781, At
15°C, N_. succinea requires 12 to 16 hours to return to 10% of initial
weight following a salinity shock of 14 °/oo to 8 °/oo and 48 hours
following a decrease of 34 °/oo to 8 °/oo (Fig, 2A), Even after 80
hours at 8 °/oo body weight remains at least 7% greater than the initial value.
The process of volume readjustment is sensitive to temperature; it
takes twice as long for animals to return to 10% initial weight at 5°C
than at 15°C (Fig. 2B).
Hydration of Tissues and Intact Animals
The water content of body wall muscle tissue changes in dilute media.
The time of greatest hydration (60.3% ± 1,2) coincides with that of maxi mum wet weight of intact animals. Tissue hydration begins to return to initial values just as whole animal weight begins to decline (Fig, 3A),
7 Water content of intact animals, including blood and coelomic fluid is greater at 8 °/oo (90.5% ± 2.5 N=10) than at 14 °/oo (82.3% ± 2.9
11=10), in agreement with other studies of hydration in nereids (Oglesby,
65a, 1978; Freel, 1973).
Under hypoxic conditions IN. succinea loses not only its ability to adjust volume after a salinity decrease, but also its ability to maintain volume with no salinity change (Fig. 3B). In 14 °/oo with no salinity change, wet weight of whole animals increases after 48 hours to 24.8% of original value and remains elevated even after 90 hours. During this same time period, however, the change in the hydration of excised body wall tissue is not significant (p = .005). At 14 °/oo and in aerated water 49.5% (± 2.0 N=10) of the musculature is water. In the same salinity, at P 0^ < 1 Torr, the percent hydration after 48 hours is
52.8% (± 3.6 N=10). However, the percent hydration increases to 65.4%
(± 2.9 N=10) 48 hours after transfer to hypoxic water of lower salinity
(8 °/oo).
Inorganic Ions
Removal of K+ , the major intracellular cation, from the medium significantly increases swelling and prolongs both the plateau and the phase of volume readjustment (Fig. 4A) . Increasing the concentration -f- of ambient K increases swelling, prolongs the plateau and prevents readjustment (Fig. 4B). Replacement of external Na by Rb , Cs or Li is lethal, the period required for death of half of the animals varying
*4* 4“ *4 from 18 hours for Rb to 24 hours for Cs to 30 hours for Li . Prior to death, there is no evidence of volume readjustment or of an end to swell ing in two more lethal media (Fig. 5A). Replacement of external Cl ions -2 by SO^ also results in a total loss of the ability to readjust body 9
volume (Fig. 5B). +2 As noted earlier by Ellis C1937), the presence of Ca is necessary
for volume readjustment to proceed (Fig, 6A & B), Removal of external +2 Mg does not prevent volume readjustment entirely, but the process is
significantly (p = .05) retarded. Even after 48 hours, worms remain
swollen by 20% initial weight (Fig, 6C)«
Metabolic Inhibitors
The addition of 10 ouabain to a medium containing intact animals decreases ammonia excretion (Mangum, et al,, 1978) and the addition of -3 10 M ouabain to a medium containing microsomes from several epithelia inhibits ATPase activity ( M a n g u m , et al,, in prep.). Howevery ouabain has no significant effect on volume regulation (Mangum, personal communi cation). Amiloride (N-amidino^-3, 5, - diamino-6^chloropyrazine-carboxamide),
■b b a less specific inhibitor of Na + K ATPase activity than ouabain
(Kirschner, et al., 1973), tends to retard the readjustment phase even at 10 ^M (Fig. 7A). The carbonic anhydrase inhibitor Diamox slightly _2 retards the restoration phase; worms in 10 M Diamox take 50 hours to reach 10% initial weight (Fig. 7B), Blocking energy production with 2,
4-dinitrophenol irreversibly inihibits volume readjustment (Mangum,, personal communication).
Nitrogen Excretion
Within 30 minutes of a salinity change of 34 °/oo to 8 °/oo, ammonia excretion increases 150% and then slowly returns towards the initial rate.
The time course closely resembles that for whole animal wet weight (Fig.
8A). Concomitant with increased excretion, the coelomic fluid pH rises from 7.06 (± 0.08 N=5) in 34 °/oo to 7.28 (± 0.07 N=6) after 36 hours 10
In 8 °/oo. During the same period, the ammonia concentration of the coelomic fluid increases from 0,06 mM/L body water C± 0,02 N-5) to
0.18 mM/L body water Cl 0,03 N=6) or from 0,28 pM/g dry wt, C± 0,09) to 1.43 pM/g dry wt. (± 0.23)
The total free amino acid pool (TNPS - ammonia) in'Ny succinea decreases with lowered salinities from 621,10 pM/g dry wt, in 34 °/oo to 49.45 pM/g dry wt, in 4 °/oo (Mangum, personal communication).,
Ninhydrin positive substances appear in the medium during volume read justment. Control worms held at 14 °/oo lose 124,47 pM/g dry wt ’ 24 hours (Table 1), However, during the same time period following a salin ity change from 14 °/oo to 8 °/oo, N, succinea loses 206,03 pM/g dry wt
(Table 1).
There seems to be a "pulse" of NPS extrusion that lasts for several hours but occurs at different times in each, worm. This variable time of rapid NPS extrusion is reflected by the large initial standard errors in
Table 1. During a given time interval some worms extrude much NPS while others lose very little. There does not appear to be a clear relationship between the period of greatest NPS excretion and most rapid rate of volume readjustment. However, after 24 hours, all worms return to initial weight,
NPS efflux decreases substantially, and the standard errors become more indicative of the accuracy of the assay. In 10 ~*M ouabain NPS efflux at the same rate (223.65 pM/g dry wt * 24 hours, ± 20,03 N-10) as in the absence of ouabain.
Effects of Removal of Anterior Segments
After forced autotomy of tails, both tailless segments and tails were kept at 14 °/oo for 24 hours. Within the same time required for intact animals to return to 10% initial weight, all (N=20) tailless 11 segments and tall sections transferred to 8 °/oo return to 10% initial weight. After recovery from removal of the supraoesophageal ganglion by either precise surgery or general removal of the first eight to ten seg ments, worms were transferred to 8 °/oo. The weight of worms subjected to surgery but with intact supraoesophageal ganglia as well as worms without ganglia returns to 10% initial value in the same 24 hours required by intact animals. Autotomized and decapitated animals not subjected to a salinity transfer neither gain nor lose weight.
Intercoelomic Pressure
Hydrostatic pressure of the coelomic fluid in worms acclimated to
14 °/oo is 5.5 cm H^O (± 0.4 N=5). There is no increase in coelomic hydrostatic pressure following a downwards salinity transfer. In fact, within 1.5 hours following a transfer from 14 °/oo to 8 °/oo the pressure drops slightly to 5.0 cm H^O (± 0.3 N=5) and remains at this new level for up to 36 hours. The magnitude of the minute to minute pressure changes due to movement by the worm (± 0.2 cm H^O) remains constant regardless of external salinity. DISCUSSION
Maintainence of body volume in N, succinea and its readjustment following a salinity decrease are active processes. Volume regulation shows a thermal sensitivity characteristic of many metabolic processes and is dependent upon the presence of 0^ and uninhibited cellular metab olism, If ATP phosphorylation is blocked by 2, 4-^dinitrophenol, neither the plateau nor the restoration phases occur. Without 0^, the animal cannot perform the reactions necessary to maintain body volume, much less to adjust to a salinity change. Forty-eight hours after transfer to hypoxic water with no salinity change, the wet weight of N, succinea increases substantially (24,8%), yet the level of tissue hydration is unaltered. The increased weight must, therefore, be due to an increase in the extracellular fluid volume. However, 48 hours after transfer to hypoxic water of lower salinity, the higher body weight is accompanied by greater tissue hydration. Thus body volume maintenance may change independently of tissue hydration, but control of tissue hydration appears to be an integral component of body volume readjustment.
Following a transfer to dilute media, inorganic salts are rapidly lost both through the general body epithelium and via urine CSmith, 1970;
Oglesby, 1972; Machin, 1975), After acclimation to dilute salinities, the ■4* concentration gradient across the cell membranes always drives Na into + the cells (Freel, et al,, 1973), That intracellular Na is lowered with decreasing salinities, i.e,, is expelled against a concentration gradient, strongly implicates that an active transport mechanism is responsible for 12 13
at least some of the Na loss. Doneen and Clark (1978) found in
N. succinea an active influx of Na and a decreased permeability to Na
across the body wall at 7 °/oo, a salinity at which body fluid osmolality -J" is regulated. Ouabain, although influencing ammonia excretion and Na +
K ATPase activity, has no effect on volume readjustment, suggesting
that Na uptake or extrusion may not play a crucial role in volume read-
justment. Amiloride, a non-specific inhibitor of Na + K ATPase, _2 retards the volume readjustment phase at 10 M. However, amiloride may
induce other deleterious metabolic effects not directly related to mem
brane ion movements.
As in red blood cells (Kregenow, 1971), the elimination of intra
cellular osmolytes in response to decreased salinity could be dependent
on passive efflux of inorganic ions along existing concentration gradients.
Increasing the concentration gradient across the membranes should facil
itate volume readjustment. Conversely, reversing the concentration
gradient for passive loss should retard volume readjustment. When 4* ambient K is removed, thus increasing the tendency for K to efflux passively, the readjustment phase is not shortened. When external K+
concentration is increased, readjustment is blocked. Replacement of 4* 4* "I* external Na by other group 1 cations (Cs , Rb , and Li ) both increases
the potential for intracellular Na+ to efflux and provides competition + + + for the Na binding site of the Na + K ATPase. In all three cases,
the ability to readjust volume is lost, not potentiated. Removal of the | | |_ j_ divalent cations Ca and Mg also blocks volume readjustment. However, ++ if Mg alone is removed, there is some readjustment present. Animals in
Cl free media show a weight loss following no salinity change and a lack of volume readjustment after exposure to dilute media. This weight loss could be due to a passive loss of Cl without the simultaneous influx of 14 the sulfates from the medium. Regardless of the dynamics, volume readjustment does not appear to be heavily dependent on passive inorganic ion efflux.
The magnitude of the reduction of the intracellular pool of free amino acids (FAA) with decreasing salinity certainly indicates its role as a major source of labile osmolytes, Mangum (personal communication} has measured a difference in total free amino acids of 572 yM/g dry wt, between worms adapted to 34 °/oo and 4 °/oo. This corresponds to a diff^- erence in FAA of 114 yM/g dry wt, between the salinities of 14 °/oo and
8 °/oo. The rapid increase in coelomic fluid pH and ammonia excretion following salinity reduction support an hypothesis of an increased rate of FAA catabolism. Given that the change in FAA concentration is complete within 24 hours after a salinity decrease and assuming that the excretion of 1 yM of ammonia represents the loss of 1 yM of FAA, then the increase in ammonia excretion rates (30 yM/g dry wt, *2 4 hours} accounts for about 26% of the expected FAA reduction of 114 yM/g dry wt. Intact amino acids appear in the medium during volume readjustment, a process not seen in the more stenohaline species N, virens (Haberfield, 1977} but apparent in animals of other phyla (Pierce, 1971). The measured rates of FAA excretion and the total efflux during volume readjustment C82 yM/g dry wt. 24 hours) are sufficient to account for the remaining 70% of the expected reduction of the FAA pool.
Urine output increases with decreasing salinity and during volume readjustment (Smith, 1970; Oglesby, 1972; Fletcher, 1974), This increase is apparently not brought about by an increase in coelomic fluid hydro static pressure as proposed by Brown (1972), The possibility of increased ciliary activity in the nephridia remains to be examined.
Water balance in annelids as well as other animals has been shown to 15 be partly under hormonal control (Kamemoto, et al., 1966; Golding, 1974).
Removal of the most important neuroendocrine organ (supraoesophageal ganglion) in N. succinea has no effect on volume readjustment. Moreover, isolated tail sections have no difficulty in reestablishing weight after transfer to dilute media. Therefore, if a neurohormone is involved it must originate in the smaller clusters of neurosecretory cells often noted in the ventral nerve cord (Tashiro, 1978; Dorsett, 1978).
In conclusion, N_. succinea relies heavily upon its intracellular pool of free amino acids as a source of labile osmolytes. These free amino acids are both deaminated and extruded intact into the medium during volume readjustment. Reduction of extracellular fluid volume via increased urine output is not due to an increase in coelomic fluid hydrostatic pressure and volume readjustment proceeds in the absence of the supraoesophageal ganglion. TABLE 1
Rates of amino acid efflux (yM/g dry wt * hr) into the medium following a salinity decrease of 14 °/oo to 8 °/oo. Mean ± S.E.;
N = 20; Temp - 15°C.
Hours after decrease Salinity decrease No salinity decrease
3 11.07(± 2.46) 6.23(± 2.09)
6 9.99(± 2.32) 6.83(± 1.44)
10 8.28(± 2.42) 6.46(± 2.14)
18 7.85(± 1.12) 4.74(± 1.21)
24 8.83(± 0.79) 3.58(± 0.74)
36 2.94(± 0.67) 2.50(± 0.72)
Total FAA efflux 206.03(±19.53) 124.47(±18.60) (yM/g dry wt * 24 hr)
Change in FAA 81.59 (yM/g dry wt) Fig. 1 Time course of volume readjustment in sea water,
Mean ± S.E., N=35, 15°C.
O = salinity decrease of 34 °/oo to 8 °/oo
□ = no salinity change HOURS Fig* 2 Time course of volume readjustment in sea water.
Mean ± S.E.
X - salinity decrease of 34 °/oo to 8 °/oo
O - salinity decrease of 14 °/oo to 8 °/oo
□ = no salinity change
A - 15°C, N=45
B — 5°C, N=30 % % WEIGHT CHANGE 6 40 80 2 60 80 2 40
0 0 0 . 50 HOURS 100 4 8“2 4 A T15 0 _ ■■ B 8
o « - 0 Fig. 3 A - Time course of tissue hydration and of volume readjustment
following a salinity decrease of 14 °/oo to 8 °/oo.
Mean ± S.E., N=30s 15°C.
□ = tissue hydration
O = volume readjustment
B - Time course of volume readjustment in hypoxic water
(P02 £ 3 torr).
Mean ± S.E., N=30, 15°C.
O salinity decrease of 14 °/oo to 8 °/oo
□ = no salinity change % % WEIGHT CHANGE % WEIGHT CHANGE 40r 30 20 4 4 0 0 8 6 20
0 12 HOURS *o- 3 6 3 4 82 4
6 6
0 7 2 60 50 3 A 8
% TISSUE HYDRATION 0 Fig. 4 Time course of volume readjustment.
Mean ± S.E., N=30, 15°C.
0 = salinity decrease of 14 °/oo to 8 °/oo
□ = no salinity decrease
A - sea water containing 30 mM K *4* B - K free sea water % WEIGHT CHANGE 80- 60 - 60 0 O 0 0 0 0 ,42 HOURS 7|2 3 6 3 6 48 60 4 A 8
4 Figo 5 Time course of volume readjustment.
Mean ± S.E., N=30, 15°C.
Closed symbol = salinity decrease of 14 °/oo to 8 °/oo
Open symbol - no salinity change
A — sea water with Na+ replaced by other group 1 cations
☆ = Li+ O = Cs+ □ = Rb+
B - sea water with Cl replaced by sulfates
O = salinity decrease of 14 °/oo to 8 °/oo
□ = no salinity change % WEIGHT CHANGE -20 -2 80 60 40 40 6 20 20
0 0 - HOURS 2
4 5 36 Fig. 6 Time course of volume readjustment in divalent cation free
sea water.
Mean ± S.E., N=30, 15°C.
O * salinity decrease of 14 °/oo to 8 °/oo
□ = no salinity change
++ ++ _ A - Ca and Mg free
B - Ca"H” free j I C - Mg free % WEIGHT CHANGE 60 6 8 80 40 2 20 20 6 0 80
0 0 0 0 - .6— .{>- - - ■
HOURS 36 4 4 3
6 '4 48 48
8 '' 100 Fig. 7 Time course of volume readjustment in presence of metabolic
inhibitors.
Mean ± S.E., N=*30, 15°C.
Closed symbol = salinity decrease of 14 °/oo to 8 °/oo
Open symbol * no salinity change
A — Amiloride □ = 10 3 M
O = 10"4 M
☆ = 10”5 M
B - Diamox □ ~ 10 ^ M
O = 10~3 M % WEIGHT CHANGE -10 -10 20 30 30 20 —a, □— 1 □ HOURS 2
4 3*6 3
6 0 6 60 Fig. 8 A - Time course of ammonia excretion into sea water following
a salinity decrease of 34 °/oo to 8 °/oo.
Mean ± S.E., N=45, 15°C.
B - Time course of volume readjustment during ammonia
excretion experiment. % % WEIGHT CHANGE NH4 EXCRETION (jiM-gm dry wt.-hr.) 100 20 0 6 0 8 40 20 30 1
o]> 1 2 12 HOURS 2 4 2 4 8 6 3 36 LITERATURE CITED
Beadle, L.C., 1937. Adaptation to changes of salinity in the poly-
chaetes. I. Control of body volume and of body fluid concentration
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Brown, S.C., J.B. Bdzil, and H.L. Frisch, 1972. Responses of Chaetopterus
variopedatus to osmotic stress with a discussion of the mechanisms of
isosomatic volume regulation. Biol. Bull., 143: 278-295.
Carpelan, L.H., and R.H. Linsley, 1961. The Pile Worm, Neanthes succinea
(Frey and Leukart). Pages 35-47 in B.W. Walker, Ed., The Ecology of
the Salton Sea, California, in Relation to the Sport Fishery. Bull
etin No. 113, State of California, Department of Fish and Game.
Cavanaugh, G.M., 1956. Formulae and Methods V. of_ the Marine Biological
Laboratory Chemical Room. M.B.L., Woods Hole, Mass.
Clark, M.E., 1968a. Free amino-acid levels in the coelomic fluid and
body wall in polychaetes. Biol. Bull., 134: 35-47.
Clark, M.E., 1968b. A survey of the effect of osmotic dilution on free
amino acids of various polychaetes. Biol. Bull., 134: 252-260.
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James Alan Dykens
Born on May 5, 1951 in Worcester, Massachusetts. Graduated from
Lawrence Academy in Groton, Massachusetts, 1969. Entered University of
Vermont 1969 and transferred to University of New Hampshire, 1971.
B.A. degree in English Literature from University of New Hampshire,
1973. Attended Boston University, 1974 to 1976. M.A. candidate,
College of William and Mary 1976 to 1979 and graduate teaching assistant in Department of Biology 1977-1978. Presently a Ph.D. candidate at
University of Maine at Orono.