The Potassium Content Gonyaulax Phase Changes Circadian Rhythm of

Plant Physiol. (1974) 53, 337-342

The Potassium Content of Gonyaulax polyedra and Phase Changesin the Circadian Rhythm of Stimulated Bioluminescence byShort Exposures to Ethanol and Valinomycin1

Received for publication August 7, 1973 and in revised form October 29, 1973

BEATRICE M. SWEENEYDepartment of Biological Sciences, University of

ABSTRACT

A circadian rhythm in the intracellular level of K+ inGonyaulax polyedra is reported. When axenic cultures ofGonyaulax in continuous light (60-75 foot candles) are ex-posed for 4 hours to 0.1 or 0.2% ethanol, the subsequent free-running rhythm in stimulated bioluminescence is phase-shifted,the amount and direction of the shift being dependent on thetime in the circadian cycle when cells are treated. The phase-response curve for ethanol closely resembles that for light insimilarly maintained cells. When valinomycin (0.1 or 0.2 ,ugml-') is present in addition to ethanol, the phase of the bio-luminescence rhythm is returned to that of an untreated cellsuspension. Valinomycin thus negates the effect of ethanol onphase. The intracellular K+ level immediately after treatment ofa cell suspension for 4 hours with ethanol (0.1% ) is about halfthat of untreated cells. If valinomycin (0.1 ug ml-') is alsopresent during the 4-hour treatment, the intracellular K+ is onlyslightly lower than in untreated cells. Increasing the externalconcentration of K+ or Na+ for 4 hours has no effect on therhythm of stimulated bioluminescence. These results areinterpreted as support for the hypothesis that the mechanismby which circadian oscillations are generated involves changesin membrane properties.

A number of models for the generation of circadian oscilla-tions have been proposed, but none have proven very satisfac-tory. The most explicit recent hypothesis is the chronon theory(8), which postulates that oscillations arise from the repeatedsequential transcription of a portion of the cellular DNA, pre-sumably composed of genes controlling the various processeswhich show rhythmicity. This model suffers from the fact thatit predicts a severe disruption of rhythmicity by inhibitors oftranscription; although effects of such substances on rhythmscan be observed (12, 21, 22), they are not always present (31,36, 37). For example, actinomycin D inhibits the glow rhythmin Gonyaulax but not the photosynthetic rhythm (21). Such aneffect could be due to uncoupling the glow from the clock orinhibiting the glow. Furthermore, it has not been possible todemonstrate cyclic synthesis and degradation of the enzymesconcerned in the various rhythmic processes such as luciferasein Gonyaulax (25). Recently, it has been suggested that an os-

IThis research was supported in part by Grant GB 8418 fromthe National Science Foundation.

California, Santa Barbara, California 93106

cillation of the physical properties or permeability of mem-branes may mediate the observed rhythms (6, 9, 11). Supportfor such a hypothesis cites the phase changes brought about inPhaseolus by short exposures to substances which can interactwith membranes such as alcohol (3), D20 (4), or valinomycin(6), or by wilting for short times (5). Exposure of Phaseolusplants to ethanol, methanol (23), and D2O (4) increases thelength of the free-running period of the leaf movement rhythm.Similar effects of ethanol and D20 on the period of the tidalrhythm in activity in the isopod Excirolana have also been re-ported (9, 10). The rhythmic firing of the optic nerve in theexcised eye of Aplysia can be phase-shifted by short exposuresto higher than normal concentrations of K+ (11). The rhythmicopening and closing of the leaflets of Albizzia are mediated bymovement of K+ (30). All these effects could be understood astemporary cyclic alterations in membrane properties, althoughno direct measurements of such changes have been reported.

Because the unicellular marine dinoflagellate Gonyaulaxpolyedra is known to show four circadian rhythms, in stimu-lated bioluminescence, in bioluminescent glow, in photosyn-thetic capacity, and in cell division (33), and because theserhythms persist for many cycles in continuous light and con-stant temperature, it is a particularly favorable test object forexamining the role of membrane changes in circadian rhyth-micity. Since valinomycin at low concentrations is known toform a complex with K+, enhancing its transport through bio-logical membranes (14, 27), its effects on rhythms are especiallyinteresting. This paper reports experiments in which cell sus-pensions of Gonyaulax in continuous light were exposed forshort times to ethanol and valinomycin, and the rhythm instimulated bioluminescence was subsequently examined forphase shifts. Determinations of intracellular potassium levelsat different times in the circadian cycle are also reported.

MATERIALS AND METHODS

Cultures of a strain of Gonyaulax polyedra Stein isolated atScripps Institution of Oceanography in 1960 were used in theseexperiments. In all cases where cells were exposed to ethanol,axenic cultures of this strain purified by Dr. R. R. L. Guillardand designated "Gonyaulax 60P" were employed, because, ifbacteria were present, they used ethanol as a carbon source,multiplied excessively, and killed the Gonyaulax. The mediumfor cultures with bacteria was that employed previously (35),in which sea water was diluted to 75% salinity and enrichedwith nitrate, phosphate, iron, EDTA, and soil extract. Foraxenic cultures, "f' medium (15) with half-strength nutrientsand the addition of 2% v/v soil extract was used. Cultureswere grown to stationary phase (cell density of 5,000-10,000cells ml') in alternating light (300 ft-c) and darkness, each of

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12 hr duration at 22 C. All light sources were cool white fluo-rescent lamps.

For experiments, cells were transferred to continuous light(60-75 ft-c, 22 C) at the end of a light period. In one experi-ment, a large sample of "red tide," composed of more than99.5% Gonyaulax polyedra, was collected at night from SantaBarbara Harbor, divided into liter aliquots, and transferred tocontinuous light at 0700 the next morning. Liter samples wereharvested at intervals over 2 days for the determination of in-tracellular potassium.

In experiments where the effects of valinomycin were to beobserved, 100-ml samples were withdrawn from a culture ofGonyaulax in continuous light, and valinomycin dissolved in95% ethanol was added. The concentration of ethanol in thecell suspension was 0.1 or 0.2% v/v. Four hours later, cellswere sedimented by centrifugation at 30g for 30 sec in an In-ternational clinical centrifuge, the medium containing valino-mycin was decanted, and the cells were resuspended in mediumwithout additive. Two-milliliter aliquots were then pipettedinto sterile shell vials and replaced in continuous light for themeasurement of stimulated luminescence. Ethanol at the sameconcentration as that present in the valinomycin was added toanother sample of the culture and removed in the same manneras valinomycin. A third sample of the cell suspension withoutthe addition of either ethanol or valinomycin was centrifugedand resuspended as a further control. Experiments were re-

peated at least twice.Experiments were carried out at different circadian times

after the cell suspensions were transferred to continuous lightat 1200 circadian time. The time of the 4-hr exposure is givenaccording to the convention of a circadian time scale of 24 hr,in which 0 hr is taken as biological dawn (25). In one experi-ment, the effects of continuous exposure to ethanol and valino-mycin were also examined.The bioluminescence of aliquots was measured by stimulat-

ing a fresh sample of the cell suspension in a photomultiplierphotometer previously described (35). Mechanical stimulationwas provided by a small motor-driven stirrer like that employedby Hamman and Seliger (16) and was continued for 1 min. Thetotal light emitted from each of two or three samples was re-

corded at 3-hr intervals over three or four cycles of the rhythmin stimulated bioluminescence. Valinomycin (mol wt 1111.3)was obtained from Cal Biochemical Co.

Experiments in which the sodium or potassium concentrationin the cell suspension was increased by 100 mm by the additionof NaCl or KCl, and the medium replaced after 4 hr, were car-

ried out as described for ethanol and valinomycin.For the determination of the potassium content of Gonyau-

lax, cells were harvested by filtration on a No. 1 filter, 2.5cm in diameter, which had been prewashed three times indouble-distilled water. Cells were washed from the ifiter withdouble-distilled water and were extracted by freezing and thaw-ing and grinding in a glass homogenizer. Cell debris was re-

moved by centrifugation, and the K+ and Na+ in the super-natant were determined with an Eppendorf flame photometer.The extent of contamination by the ions from the medium was

estimated by adding "C-inulin (0.5 ptc) to the cell suspensionjust before harvesting, and determining the radioactivity ofboth the original cell suspension and the supernatant after ex-

traction, in a Packard scintillation counter in 5 ml of POPOPin toluene. The protein content of the extract was determinedby the Lowry procedure (24), after hydrolyzing the extract andthe bovine serum albumin used as a standard in 0.5 N NaOHovernight at room temperature. In some experiments, thepacked cell volume was estimated by centrifugation in cali-brated tubes. The standard error for the determinations of,umoles K+ per mg protein by this procedure was + 3.5%.

RESULTS

If the circadian rhythms that are observed in Gonyaulaxhave a common origin in cyclic differences in membrane prop-erties, then a rhythm in intracellular ion levels might be ex-pected. A preliminary examination of the potassium concentra-tion in Gonyaulax cells showed changes over the course of theenvironmental light-dark cycle, cells containing about twice asmuch K+ at the end of the light period as at its beginning (Ta-ble I). Since such a change could be the result of ion accumu-lation in photosynthesis during the light period, measurementsof the ion content in cells in continuous light were made. Anintensity was chosen where the circadian rhythm of photosyn-thesis is not expressed, so that the rate of photosynthesis wasconstant over time (32). These measurements also showed amaximum in intracellular potassium at the end of the biologi-cal day phase, at 12 hr circadian time (Table II). Two cycles inpotassium content were observed in the "red tide" sample incontinuous light (Table III). Thus, differences in K+ persist incontinuous light and represent another circadian rhythm inGonyaulax. While a rhythm in intracellular K+ is consistentwith the hypothesis that the overt rhythms in Gonyaulax havea common origin in cyclic membrane permeability, it does notconstitute proof of such an hypothesis.

Table I. Intracellular Cation Concentration of Gonyaulax polyedraat Different Times in a Light-Dark Cycle

The light intensity was 300 ft-c and the temp was 22 C. Thelight-dark cycle was 12:12 hr. Ion concentrations are mm.

Circadian Time Cells SampledIon Medium

O hr 6 hr 12 hr 18 hr 24 hr

K+ 9 15 24 31 18 16Na+ 315 54Mg2+ 51 27 30

Table II. Intracellular Potassium Ion Concentration of Gonyaulaxpolyedra at Different Times in Continuous Light

A culture was divided and half (No. 2) was rephased by 1800,so that all sample preparation could be carried out during a 12-hrtime space. The concentration of K+ in the medium was 12 mm.The light intensity was 75 ft-c, and the temperature was 22 C.

Circadian Time Cells SampledMedium

O hr 6 hr 12 hr 18 hr 24 hr

No. Amoles/mg protein1 5.7 7.8 8.52 8.9 6.9 7.1

Table III. Intracellular Potassium Ion Concentration of a

Gonyaulax "Red Tide" at Different Times afterTransfer to Continuous Light at Circadian Time 0

The concentration of K+ in the sea water was 10 mm. The tem-

perature was 22 C, and the light intensity was 75 ft-c.

Time Cells Sampled afterTransfer to Continuous 8 11 14 17 26 31 36 39Light (hr)

Concn K+ (mM) 23 23 21 21 17 19 20 14,smoles/mg protein 4.4 5.0 4.3 3.9 3.2 4.9 5.2 3.7

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GONYAULAX RHYTHM: K+, ETHANOL, VALINOMYCIN

Gonyaulax does not contain a large central vacuole, and cellsexamined by electron microscopy (34) are seen to consistlargely of membrane-bound organelles including the large nu-cleus, chloroplasts, mitochondria, trichocysts, and vesicles ofunknown nature. The total intracellular potassium is onlyslightly higher than that in the medium, but the concentrationwithin small vaculoes could be much higher, and could changeby a greater fraction over a cycle than the total potassium.Thus nothing can be said at present concerning the accumula-tion of K+ in Gonyaulax. Although measurements of Na+ andMg`+ in extracts were made, the contamination of the extractswith the medium, which contain high concentrations of theseions, prevented accurate determinations of intracellular levels.

Increasing the potassium or the sodium content of the me-dium by 100 mm for 4 hr had no effect on the phase of therhythm in stimulated bioluminescence in Gonyaulax, irrespec-tive of the time in the cycle when the high ion pulse occurred.However, Gonyaulax can tolerate rather large changes in thesalinity of the medium, and did not lose motility on the addi-tion of either ion, so it is likely that this cell is able to regulatethe internal ion concentration irrespective of the external me-dium.

If a change in the permeability to potassium is a componentof the process by which oscillations are generated, then artifi-cially altering the intracellular potassium would be expectedto alter the phase of the observable rhythms. Valinomycin isknown to change the permeability of many biological mem-branes to potassium (27). Therefore, cells were exposed forshort times to valinomycin and the rhythm of stimulated bio-luminescence was measured over several cycles in continuouslight. Since valinomycin is relatively insoluble in water, stocksolutions were made up in ethanol and ethanol controls wereincluded in all experiments. Phase shifts were observed in cellstreated with valinomycin, as compared with ethanol controls,and the amount and direction of phase shift depended on thecircadian time of exposure, confirming the findings of Binningand Moser (6) in Phaseolus. However, considerable phase shiftsoccurred in the ethanol controls, as compared with the lumi-nescence rhythm in cells which had merely been centrifugedand resuspended in fresh medium. Changing the medium hadno effect on phase, confirming previous experience (18). Phaseshifts with ethanol also depended on the time when the cellswere treated. For example, exposing a Gonyaulax cell suspen-sion to 0.1% ethanol for 4 hr between 12 and 16 hr circadiantime resulted in a 3-hr phase delay in the bioluminescentrhythm (Fig. 1). The presence of valinomycin (0.1 jug ml-') inaddition to ethanol (0.1%) returned the phase to that of thecontrol, in which the medium was replaced but no additionswere made. Thus, as compared with the ethanol control, va-linomycin-treated cells were phase-advanced by 3 hr. Givenlater in the circadian cycle, ethanol alone advanced rather thandelayed phase (Fig. 2), and again valinomycin reversed this ef-fect, bringing the phase back to that of the untreated control.Neither valinomycin nor ethanol had any effect on the phaseof the rhythm in bioluminescence between 5 and 9 hr circadiantime, the biological day phase. The time-dependent phasechanges caused by 4-hr exposures to ethanol are summarizedin Figure 3A, a phase-response curve for ethanol. A similarphase-response curve for the effect of pulses of valinomycinas compared to those of ethanol alone is given in Figure 3B. Acomparison of these curves shows that they are mirror imagesof each other and documents the opinion that valinomycin re-verses the phase changes expected from the ethanol in whichit is dissolved.The effects of 0.2% ethanol were indistinguishable from

those of 0.1% ethanol and were reversed by 0.2 ,ug ml-' valino-mycin. A range of valinomycin concentrations from 0.02 to 10

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FIG. 1. Effect of exposure of Gonyaulax polyedra strain 60P toethanol and valinomycin for 4 hr at 12 to 16 hr circadian timeon the phase of the rhythm in stimulated bioluminescence in con-tinuous light (60 ft-c.) at 22 C. Data points are average of totallight emitted by two or three 2-ml samples of cell suspension.Luminescence of cells exposed to 0.1 Ag ml-' valinomycin and0.1% ethanol (-X-); luminescence of cells exposed to 0.1%ethanol (-A-); luminescence of cells resuspended in new mediumwithout other treatment (-O-). The time of maximum lumi-nescence is shown by arrow.

jAg mP was assayed for phase changes. Valinomycin at 0.25,ug ml-' or more damaged cells so extensively that a rhythm inbioluminescence could not be measured. The effects on phasewere concentration-dependent in the concentration range 0.05to 0.2 Pg ml' (Table IV).The intracellular K+ of cells extracted at the end of a 4-hr

exposure to ethanol from 12 to 16 hr circadian time was abouthalf that of the untreated control cells, while the K+ content ofcells exposed to valinomycin in ethanol was only slightly lowerthan that of the untreated control (Table V). Thus valinomycinpartially reverses the effect of ethanol on intracellular K+. It isinteresting to note that the greatest phase delay after ethanoltreatment occurs at about 12 hr circadian time, at the peak ofthe rhythm in intracellular K+ in untreated cells.

Gonyaulax cell suspensions continuously exposed to valino-mycin (0.2 jug ml') in 0.2% ethanol showed no rhythm in bio-luminescence over 3 days in continuous light. The level ofluminescence was high and the cells were motile during thistime. This observation requires repetition, however.

DISCUSSION

The phase of all circadian rhythms that have been examinedin this respect is altered by short exposures to light. The amountand direction of these phase changes is dependent on when inthe circadian cycle the cells are exposed to light and may be

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FIG. 2. Effect of exposure of Gonyaulax polyedra strain 60P toethanol and valinomycin for 4 hr at 20 to 24 hr circadian timeon the phase of the rhythm in stimulated bioluminescence in con-tinuous light (60 ft-c.) at 22 C. Data points are averages of totallight emitted by two or three 2-ml samples of cell suspension.Luminescence of cells exposed to 0.1 ,ug mP valinomycin and 0.1%ethanol (--- x---); luminescence of cells exposed to 0.1% ethanol(-A-); luminescence of cells resuspended in new medium with-out other treatment (---0---). The time of maximum luminescenceis shown by arrow.

represented by a phase response curve (28). Many attempts havebeen made to duplicate this phase-response curve by treatingorganisms with various biologically active substances for shorttimes, with the hope of identifying components of the bio-chemical processes by which oscillations are generated. Untilrecently, these have been remarkably unsuccessful, Most meta-bolic inhibitiors are ineffective in this respect (2, 18), althoughsome appear to render biological timekeeping more erratic thanusual. Specific inhibitors of RNA and protein synthesis like-wise fail to duplicate the effects of light on phase (21, 22). Ofa long list of substances assayed for phase-shifting in the Gony-aulax glow rhythm (18), only arsenite appeared to delay phasesomewhat. More recently, however, some success in experi-ments of this type has been achieved. A high concentration ofethanol (25%) given to shoots of Phaseolus via the transpira-tion stream was reported to delay phase by 3 hr if given for 4to 5 hr some time after the middle of the biological nightphase (3). Temporary deuteration of Phaseolus is followed bytransient cycles in leaf movement, but stable phase delays are

also obtained (4). In the Euglena phototactic rhythm also, ex-posure for 24 hr or more to D20 is followed by phase delays(1). Unfortunately, a complete phase-response curve for etha-nol or deuterium pulses cannot be constructed for eitherPhaseolus or Euglena from the published data. Phaseolus plantsallowed to wilt for 6 hr responded with phase changes in theleaf movement rhythm, the nature of the response dependingon when wilting occurred (5).

In Phaseolus, valinomycin appears to be one of the mostsuccessful substances in imitating the phase changes caused bylight (6). The effectiveness of this substance, both in Phaseolusand in Gonyaulax, suggests the implication of K+ in the gen-

eration of rhythmicity. This is supported by experiments witha circadian rhythm in a very different system, the isolatedAplysia eye (11). WVhen the level of K+ in the medium sur-

rounding this tissue is temporarily increased, the rhythm in the

firing of the optic nerve is phase-shifted, and the amount anddirection of the change in phase is dependent on the time ofthe high K+ pulse. It is also interesting in this connection thata circadian rhythm in intracellular K+ can be observed in Go-nyaulax. Since the results of experiments with valinomycin inPhaseolus and increased K+ in Aplysia have been plotted oncoordinates of time and phase shift different from each otherand from those used in this paper, the data from these experi-ments have been replotted (Fig. 4), to allow a comparison be-tween them, and with the data from Gonyaulax. Phase changeswith increased potassium levels in Aplysia follow a patternquite similar to the phase response curve for valinomycin

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FIG. 3. Phase-response curves for the effects on the phase ofthe circadian rhythm in stimulated bioluminescence in Gonyaulaxpolyedra strain 60P brought about by 4-hr exposures to 0.1%ethanol (A) and to 0.1 ,ug ml-" valinomycin plus 0.1% ethanol calcu-lated with reference to ethanol only (B). Note that phase advancesare considered positive in sign and are plotted upward from zeroon the ordinate. The abscissa is circadian time, using the con-

vention that dawn equals 0 hr. The time of the dark period of theprevious entraining light-dark cycle is shown as a shaded bar on

the abscissa. Cells in all experiments were in 60 to 75 ft-c. con-

tinuous light at 22 C.

VALINOMYCINO.1 Lg/ml 20-24c.t.

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pulses in the leaf movement rhythm in Phaseolus. Althoughthe change from delay to advance is more sudden in the Ap-lysia data, the maximum phase advance occurs in both systemsat 24 hr circadian time. The phase-response curve for ethanolin Gonyaulax (Fig. 3A) is also similar in general form, andshows a maximum phase advance at 24 hr circadian time. Thissimilarity suggests that all three effects share a common mecha-nism, possibly a transitory change in intracellular K+.

In Gonyaulax, valinomycin clearly negates the effect of eth-anol alone, both in phase shifting the rhythm of stimulatedbioluminescence and in changing the total intracellular K+.Both ethanol and valinomycin would be expected to exert ef-fects on the structure and permeability of biological mem-branes. Ethanol is a lipid solvent and has been shown to sta-bilize red blood cell membranes against hemolysis (20, 26).Valinomycin is known to act as an ionophore for K+ and hasrecently been found to substitute for the K+ requirement of amembrane-bound K+ and Na+-dependent ATPase active in iontransport (19). The biochemical basis for the opposite effect ofethanol and valinomycin in Gonyaulax awaits further experi-mentation on isolated membrane systems. Spin-label and othersuch studies are in progress in this laboratory.

In Gonyaulax, the phase-response curve for the effect ofshort exposures to dilute ethanol mimics that to light reason-ably closely. Phase changes are of about the same magnitudeas those produced by bright light pulses in cells in continuouslight (7). The effects of both light and ethanol pulses changefrom a phase delay to a phase advance in the early part of thebiological night phase (7). Exposure to light or to ethanol in themiddle of the biological day phase has no effect on the phaseof the subsequent rhythm in stimulated bioluminescence. Lighthas been found to alter the electrical properties of Acetabularia(13, 17, 29). Thus it is possible that the phase-shifting effectsof light may be membrane-mediated.

Table IV. Phase Shifts after Treatment of Gonyaulax polyedraStrain 60P by Valinomycin

The organism was treated for 4 hr with different concentrationsof valinomycin from 8 to 12, or from 12 to 16 hr circadian time.Phase shifts are calculated relative to the ethanol control.

Phase ShiftValinomycin Concn

at 8-12 c.t.1 at 12-16 c.t.

pg mU' hr

0.02 00.05 1.90.1 3 30.2 3.80.25 toxic

1 Circadian time.

Table V. Intracellular Potassium Concentration of Gonyaulaxpolyedra Strain 60P after a 4-hr Exposure to Etha/nol or to

Valinomycin and Ethanol from 12 to 16 hrCircadian Time

K+ in

ExperimentUntreated control Ethanol (0.1%) ethanol (0om mcin+

pmoles/mg protein

I 3.0 1.6 2.7II 3.3 1.8 2.1

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FIG. 4. Phase-response curves for the effect of 0.1 ,ug ml[' va-linomycin in 0.1% ethanol on the phase of the leaf movementrhythm in Phaseolus coccineus calculated with reference to the eth-anol control, from Buinning and Moser (6), (-O-); and for theeffects of high potassium ion (100 mM) pulses on the phase of therhythm in firing rate of the optic nerve of isolated eyes of AplysiaCalifornia, from Eskin (11) (-X--). The data have been replottedso that circadian time 0 is dawn in both cases and phase advancesare plotted upward. The time of the dark period of the previousentraining light-dark cycle is shown as a shaded bar on the abscissa.

The phase and the period of a circadian rhythm are proper-ties of the underlying oscillator, and are the only measurablefeatures which can be unequivocally assigned to this oscillatorat present, as distinct from the physiological processes whichthis oscillator controls. Substances which affect phase or periodare thus indicators of the nature of this oscillator. The findingsthat the circadian rhythm in stimulated bioluminescence inGonyaulax is phase-shifted by ethanol, that this phase shift isreversed by the presence of valinomycin in low concentrations,and that the intracellular K+ in untreated cells shows a cir-cadian rhythm, strengthen the hypothesis that the mechanismby which circadian rhythms are generated involves an oscilla-tion in the physical properties of biological membranes, or in-tracellular ion concentration.

Acknowledgments-The author wishes to express her appreciation to J. Nel-son and A. Swerdloff for their excellent technical assistance, to R. R. L. Guillardfor the axenic culture of Gonyaulax, strain 60P, and to P. Laris for the use of hisflame photometer.

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