VOL. VII, No. 2 APRIL 1930

CHANGE OF PHOTOTROPIC AND GEOTROPICSIGNS IN DAPHNIA INDUCED BY CHANGES

OF LIGHT INTENSITY

BY G. L. CLARKE.

(Received 28th May 1929.)

(With Eight Text-figures.)

INTRODUCTION.

THE following series of experiments was undertaken in order to obtain furtherinformation in regard to the part played by light in the diurnal migrations ofplanktonic organisms. It is the opinion of many investigators (e.g. Russell, 1926,1927 a) that light is one of the most important factors regulating the verticalposition of the plankton. One method of attacking this problem is observational;another is experimental. In the former, observations upon the vertical distributionof the plankton at different times of day are made by means of tow nets. Then asmuch information as possible is obtained on the light intensities at those depthsand at those times of day, and attempts are made to correlate the movements ofthe plankton with the changes in light intensity. In the experimental method, onthe other hand, plankton animals are brought into the laboratory where the en-vironment can be carefully controlled. Here they are subjected to different con-ditions of illumination, and the resulting movements of the animals may be studied.It is in the belief that investigations of this experimental type can be devised insuch a way as to reveal the fundamental mechanisms of phototropism in particularand of vertical migration in general that I have attempted the work about to bedescribed. Although neither my experiments nor those of previous investigators(cf. Parker, 1902; Ewald, 1910; Dice, 1914; and Rose, 1925) are sufficiently largeeither in number or in scope for generalisations to be made, they indicate the typeof problem susceptible to experimental attack.

1. MATERIAL AND METHOD.

In selecting the material and method for these experiments, two primaryobjects were kept in view. The one was to make a study of the behaviour of theindividual. In this way it was hoped to reach a truer understanding of the basisof light responses and to do so more directly than by drawing deductions fromav^ages of swarms of Daphnia (cf. Esterly, 1919, p. 80). For most of the experi-V one animal was used at a time and the same tests were repeated many times

JEB-VHH 8

no G. L. CLARKE

with the same individual. The other object was to avoid the effect of " shock *much as possible in handling the Daphnia (cf. Esterly, 1919, pp. 66 and 77). ^ ^was accomplished by employing laboratory-raised animals which therefore hadbeen accustomed to aquarium conditions all their lives1. Unless otherwise stated,the experiments which follow were performed on adult specimens of Daphniamagna reared in the laboratory. It was found, however, that wild specimens ofDaphnia pulex reacted to light substantially in the same manner.

The type of apparatus designed for the more careful control of the environmentconsisted essentially of a long glass tube filled with tap water, kept at constanttemperature by a water jacket, and illuminated at one end by a Sheringham Day-light Lamp (150 watts). The glass tube was 6-3 cm. in diameter and 96 cm. long.It was sealed at the end towards the light with a strong piece of plate glass andclosed at the other end by a rubber bung provided with a disc of black groundglass to prevent reflection. Inside this experiment tube two thermometers weresecured by means of copper wire coated with insoluble "Luc" cellulose paint insuch a way that they registered the temperature at the ends of the tube. The waterjacket consisted of a cylindrical museum jar 17 cm. in diameter and 96 cm. longand provided with a tap at each end. Against the bottom of the museum jar insidewas placed a piece of black ground glass, while the top end was provided with athick plate glass cover held firmly in place by six screw clamps. When a rubberwasher coated with vaseline was placed between the glass cover and the lip of themuseum jar, the apparatus was found to be quite water tight and could be usedin either a vertical or a horizontal position. The water which was circulated throughthe water jacket was piped from the laboratory tap. Its temperature could be keptremarkably constant at any desired level by passing the tap water through a coppervessel heated by a gas flame which was regulated by a thermostat device in the usualmanner. The temperature of the water within the experiment tube remained for hourswithin a degree or two of its original value and there was rarely a difference of morethan o-150 C. between one end of the tube and the other. The position of the Daphniawas noted every half minute by taking readings in centimetres from the bottom onthe scale marked on the outside of the water jacket opposite the experiment tube.A red lamp, too dull to affect the movements of the Daphnia and placed upon anadjustable slide behind the apparatus, was employed to make the animal and thecalibration visible. The whole apparatus- was set up in a dark room, but experi-ments could be carried on in the lighted laboratory by enclosing the museum jarand contents in a light-proof box with a sliding front. A peep-hole cut in thismovable front was adjusted precisely opposite the red lamp and the two weremade to move together in following the movements of the Daphnia.

The light intensity of the Sheringham Daylight Lamp could be reduced orincreased by means of a chemical rheostat. This consisted of a tank of tap waterthrough which the current was made to flow by passing between two zinc electrodes.

1 Daphnia may be easily cultured by keeping them in jars each containing about 150 c.c. ofwater at room temperature and feeding them on a dilute solution of rotten egg. Normally feiTproduce broods every ten days. The young require four moults (about ten days) to become ai

Change of Phototropic and Geotropic Signs in Daphnia 111

These electrodes were thin triangular plates of zinc with dimensions of 45 x 45 x^ n n . and held 0-5 cm. apart. Two additional tanks, one above and one below,connected to the central tank by glass tubing fitted with stop-cocks, made itpossible to fill the central tank or to empty it at any desired speed. When thecentral tank was full of tap water1, it added almost no resistance to the electriccircuit; but as the water was drained away the light became dimmer and dimmeruntil the resistance caused by the reduced electrode area resulted in stopping theflow of electricity altogether. Accordingly, the experimenter could obtain a steadychange of light intensity at any speed he wished by merely adjusting the stop-cocks. The resistance of the water could be diminished by adding small quantitiesof washing soda. The light intensity was measured by an A.C. ammeter in theelectric circuit which had been calibrated to show candle power for the daylightlamp used. It was found possible to cut the zinc plates in such a shape that therate of change of light intensity was linear. It is true that the composition of thelight changed as it Was dimmed: at low intensity values there was noticeably morered in the light emitted by the lamp, but this slight change of colour did not appearto interfere with the general results of the experiments in any way2. A skylightprovided with a light-proof door immediately over the apparatus made it possibleto use daylight instead of electric light when required. In this case dimming couldbe produced only by shutting the door of the skylight, but the same general resultswere obtained as with artificial light (cf. Yerkes, 1900). It should be noted that inboth cases all ultra-violet light was excluded by the glass through which the raysof light must pass. Ultra-violet light of wave-length shorter than 3341 A.U. hasbeen shown to be specific for causing negative phototropism in Daphnia pulex(Moore, 1912), but it is doubtful whether enough ultra-violet light reaches plank-tonic animals in nature to affect their vertical movements.

A second type of apparatus was designed particularly to permit lighting fromany direction. It consisted of a glass battery jar (30 cm. long, 20 cm. wide, and30 cm. high) placed upon brackets screwed to the wall with an electric light fixtureon each side, above, and below. Either one or two lights in any position could beused. Sixty-watt bulbs were employed for the most part and dimming was accom-plished by means of the same chemical rheostat.

The usual method of procedure was as follows. A healthy specimen of Daphniamagna was selected and transferred to the experiment tube by means of a largepipette. The water in this tube was tap water which had been allowed to stand forat least several hours. This precaution permitted the escape of the bubbles ofexcess gas which would otherwise form upon the appendages of the Daphnia andprevent it from swimming normally. Moreover, it is of the utmost importancethat the animal be subject to no sudden changes of temperature. Hence by allowingthe tube water to reach room temperature before the transfer of the Daphnia was

1 The concentration of the electrolytes in Cambridge tap water is fairly constant at approxi-mately 0-005 N.^B£ For an account of the different effects of red and blue lights upon the movements of Daphnia,^^pYisch und Kupelwieser, 1913.

i i 2 G. L . C L A R K E

made, "shock" effects could be avoided and any desired lower or higher tem-perature could be reached gradually after the tube had been placed in its w ^ Pjacket. If these precautions were carefully observed, experimentation could beginalmost immediately—otherwise several hours of waiting were necessary before theorganism would behave normally. No change in the behaviour of Daphnia wasobserved for experiments carried out at temperatures of 8° to i8° C , but elaboratetests of the effects of temperature change were not made. No provision for theaeration of the water confined within the experiment tube was necessary since thevolume of water was so comparatively large. Daphnia would live sealed up insidefor a week or more and even produce broods of young, but ordinarily no animalwas used for more than two or three days at a time.

2. PRIMARY SIGN OF TROPISMS.

The great majority of the Daphnia used were primarily negatively phototropicand positively geotropic. That is, these animals swam away from any light re-gardless of its intensity and swam or sank to the bottom of their container in anylight or in darkness. It will be shown that the signs of the tropisms may be tem-porarily changed experimentally, the term primary sign of phototropism or ofgeotropism signifying that sign which the organism always exhibits under constantconditions. There was always a minority of animals which exhibited unusual primaryphototropic and geotropic signs, and I have also observed a few cases of reversal ofprimary sign. For example, one animal seemed to be permanently positive to lightbut a week later it became primarily negative to light. Other specimens appearedto be indifferent to light or to gravity or to exhibit rapid changes of sign for noapparent reason, but such forms were exceptional. When Daphniapulex was used,it was found that the same primary signs existed (i.e. negative phototropism andpositive geotropism), although some investigators have found their experimentalanimals to be permanently positively phototropic (e.g. Yerkes, 1903) or to bepositive to weak light and negative to strong light (Dice, 1914). But whatevervariation in tropisms there may be, we shall deal here only with that type ofDaphnia, by far the most numerous in my material, which is primarily negativelyphototropic and positively geotropic under any constant conditions of illumination.

3. CHANGE OF SIGN.

The hypothesis which seems best to explain the results of the experimentsdescribed below is that changes in the light intensity induce changes in the sign ofthe tropisms. Such changes of tropism signs have been observed by previousinvestigators (cf. Ewald, 1910; Frisch und Kupelwieser, 1913; Dice, 1914; andMast, 1921). The Daphnia under consideration are primarily negatively phototropicand positively geotropic. Following a reduction of the light intensity, the photo-tropism becomes positive and the geotropism becomes negative. These changes^fsign are, however, only temporary, and the temporary signs will be called S i

Change of Phototropic and Geotropic Signs in Daphnia 113

secondary signs. For soon after the light intensity becomes constant, the photo-^ n s m and the geotropism regain their original (primary) signs. An increase inlight intensity strengthens the primary tropisms, or, if the secondary tropisms arestill operative, the return to the primary signs is hastened. Fig. 1 shows the move-ments of a normal animal resulting from such changes in tropism signs when theillumination is from above. While the light intensity remains constant, the Daphniais to be found close to the bottom as a result of its primary negative phototropismand positive geotropism. When the light is dimmed and the change to the secondarytropism signs occurs, the animal is stimulated to swim to the top of the tube(see A, Fig. 1). But soon after the light intensity is again steady, the return to theprimary signs occurs and the Daphnia goes back to the bottom (see B, Fig. 1).Increasing the light intensity to its original value now produces no further effectsince the animal is already at the end of the tube (see C, Fig. 1), but if the light ismade bright again while the animal is still at the top, the return to primary signs,

Bright

DimTop

Bottom

A\r

r/V

V c

\f.V

\1 \ _

LightIntensity

PositionTime

Fig. 1.

hastened by the increase of light intensity, can be demonstrated (see D, Fig. 1).When the tube and light are employed in a horizontal position, the same responsesare observed, but now the movements occur in a horizontal plane. Following adimming of the light, the Daphnia swims to the end of the tube towards the lightsource, and after a short time or when the light is made bright again, it returnsto the far end of the tube. These reactions are phototropic only since geotropism,although present, obviously cannot act when the experiment tube is horizontallyplaced.

4. PHOTOTROPISM AND GEOTROPISM DIFFERENTIATED.

To show that both phototropic and geotropic forces are present, experimentswere performed using the battery jar apparatus in which the Daphnia were freeto move in any direction. In this case illumination was provided by the SheringhamDaylight Lamp directed horizontally as shown in Fig. 2 a. The large size of the

ctor of the lamp made it certain that the light entering the water was uniformitensity and evenly distributed over the whole surface of the exposed side of

i i 4 G. L . C L A R K E

the tank. As long as the light intensity was maintained constant, the Daphniaalways found at corner (i)—the result of their primary negative phototropism Ipositive geotropism. When the light was dimmed, however, the tropism signs werereversed: the phototropism became positive and the geotropism became negative.As a result the animals swam to corner (3). Soon after the light intensity againbecame constant, the phototropic and geotropic responses regained their originalprimary signs, and consequently the Daphnia returned to corner (i)1. The pathtaken from (1) to (3) depends of course upon the relative strengths and speeds ofthe two forces acting upon each animal. The positive phototropism tending tocause the organism to move towards the light usually acts sooner than the negativegeotropism producing the upward movement. Course B is followed by thoseanimals in which the positive phototropism is much stronger and faster in itseffect. Course A is followed by those in which these forces are about equal, or

IYXI \A V! \

1 •1 1

t:1 11 1

i '

t

1

1

11

1

tI

t111

iB1

10

Fig. 2 a. Fig. 2 b.The arrow indicates the direction of the light.

more frequently, in which the geotropic reaction is the stronger. The curves ofCourse A upward and downward, although slight, are in the same direction asin Course B. This is because in Course A the phototropic effect is felt first andmasks to some extent the fact that the geotropic response is really stronger asshown by the next experiment.

That these differences in relative strengths do in fact exist is substantiated byexperiments in which the light source is placed below the tank. The phototropicand geotropic forces are now directly antagonised. Those animals (about three-eighths of the total number) in which the phototropism is stronger than the geo-tropism (these followed Course B in Fig. 2 a) will be found at the top of the tank,because their phototropism is primarily negative, while those specimens in whichthe geotropism is stronger than the phototropism (these followed Course A inFig. 2 a) will stay near the bottom because their geotropism is primarily positive.If the light intensity is now reduced, the signs of both the phototropism and the

1 For another example of the resolution of phototropic and geotropic forces, see CrozierWolf, 1928.

Change of Phototropic and Geotropic Signs in Daphnia 115

Jropism are changed. The result is that those Daphnia which were at the topto the bottom, and those which were at the bottom swim to the top. And,

as we should expect, increasing the light sends both types back to their originalpositions (see Fig. 2 b).

The situation is represented in Table I. In each case the stronger tropismis shown in heavy print:

Table I.

Type A

Type B

PrimarySecondaryPrimarySecondary

Phototropism

1 + 1 +

Fast reaction

Geotropism

1 +

1 +

Slow reaction

r

• I . - * -

'"'ase ax.

Fig. 3 a.

XCase aa.

When two opposing lights are thus employed, all phototropic effects areneutralised and phototropism may be considered as absent. Since geotropism aloneremains as an effective force, all the Daphnia move directly upward when the lightis dimmed (secondary negative geotropism) and directly downward when it is madebright again (return to primary positive geotropism) (see Fig. 3 a). In Case al

there is no diagonal course as there was when one horizontal light was used; inCase a% the two types of Daphnia are not separated out as they were when thelower light alone was used.

is possible to separate the phototropic responses and the geotropic responsest other ways. When the rate of dimming the light is made very slow, the

was dimmed at such a slow rate that the animal was never stimulated tobottom. In Case b2 the light was switched out. Accordingly, the Daphnia under-went an instantaneous change from bright illumination to complete darkness.Since there was no light present to orientate the animal when it began to swim,a phototropic response could not occur. The result was that the animal swamdirectly upwards, and then back to the bottom when the light was switched onagain. The geotropic response alone was effective.

! t

•:--«::

Case &,.Fig. 3 b.

Case b2.

XBright

XDim

XDim

2 min. after dimmingFig. 3 c.

30 min. after dimming

5. DURATION OF TROPISM REACTIONS.

As we have seen, the Daphnia respond to a dimming of the light by swimmingupwards, and towards the light source. Soon after dimming has ceased andthe intensity is held at a constant low value, the primary signs of the tropismsare regained and consequently the Daphnia swim to the bottom of their containerand move as far away from the light source as possible. If the intensity of thelight is very low, these responses are weak, but although not as marked as withstronger illumination, a negative phototropism and a positive geotropism are alvregained. If the light is extinguished, phototropism necessarily disappears,

Change of Phototropic and Geptropic Signs in Daphnia 117

ct that the majority of animals returned to the bottom even in complete darknessthat geotropism is present and positive as before. Fig. 3 c expresses these

facts in diagrammatic form.The effect of light upon geotropism in various planktonic organisms has been

studied by many investigators. Attention is called particularly to the work ofDice (1914) on Daphnia pulex. Following Experiment 12, "Persistence of NegativeGeotaxis in Darkness," in which data are given to show a persistence of 4^ hours'duration, Dice says: "We have shown that in Daphnia pulex increase of lightintensity causes a tendency toward positive geotaxis, while decrease of intensitycauses a tendency toward negative geotaxis. This tendency seems to be strongerthe greater the change in intensity. It seems also that these tendencies are per-sistent for a considerable length of time." And in the summary he says: "Thediurnal movements of Daphnia pulex are caused chiefly by variations in geotaxisinduced by changes in light intensity." My results agree with Dice's as far asthe effects of increased and decreased illumination are concerned, but I neverobserved the secondary negative geotropism to persist for more than a few minutes.It is true that in my material the fraction of the total number of animals which didnot return to the bottom was larger in D. pulex than in D. magna, yet with bothforms I found that the majority of animals always returned to the bottom in lightor in darkness. Hence it is doubtful how far this photo-geotropic effect will befound to explain the diurnal migrations of plankton in general.

6. "TIME-CHANGE" AND "PLACE-CHANGE."

Enough evidence has now been given to establish the fact that Daphnia isstimulated by a change in light intensity. It is to be noted next that this changein intensity may occur in two different ways. The organism may be subjected toa change in time or to a change in space. If the Daphnia is stationary in one placeand the source of light is dimmed, then the animal experiences a "time-change"—the light intensity at its particular position in the water changes with time. This isthe type of light intensity change we have been considering thus far, and, as wehave seen, the animal is caused to move by such a stimulus. But if the intensityof the illumination is held constant, and the animal swims about, then as theanimal moves to or from the light source or swims into shadows or bright regions,it experiences a "place-change" in the light intensity. Whether or not Daphniacan perceive and is stimulated by low gradient "place-changes" is a matter fordiscussion. It is hard to believe that "time-change" and "place-change" do notcome to the same thing. If an animal swims from a region of high intensity to aregion of low intensity, why does it not receive the same stimulus as if the lightis dimmed in time over the same range? But in the experiments described, theDaphnia do not appear to be stimulated by the change in intensity which they mustexperience in swimming from one end of the tube to the other. Following a re-( • i o n of the light, the Daphnia swims towards the light source. After a short timeat a constant low intensity the animal swims back again to the far end of the tube,

n8 G. L. CLARKE

as we have seen. Why is it that upon reaching this region of still lower ^ ^the animal is not stimulated again to seek the light? It may be because the anirrralis never able to swim fast enough to produce an effective change of intensity (see"Rate of Change" below). The fact is that the animal ordinarily remains quietlyat the far end of the tube. This matter needs further investigation and carefulmeasurements of the rates of change involved.

Sheet of tlim paper

P\ Region of1 Reduced/•Light

Q I Intensity

Fig. 4 a. Experiment 35. February 27th, 1929. Temperature n-2° C.

t!'\ 1 '\ • /

VCase 6,. Case b2.

Fig. 46 . Experiment 33. February 26th, 1929, 100-watt blue bulb used. Temperature 13-7° C.

But if an abrupt "place-change" of light intensity is encountered, the Daphniawill receive a stimulus. Experiment 35 will serve as one example of this (seeFig. 4 a). Following a dimming of the light, the Daphnia swims diagonally up-wards. The abrupt decrease in light encountered at Q speeds up the reaction whilethe abrupt increase in light at P holds the response in check for some time. Thesame phenomena are observed during the return trip following a brightening ofthe light source.

Another example of the perception of "place-change" is taken from the geo-tropic responses. As we have seen above, the geotropism of Daphnia is such thatany increase of light (regardless of direction) tends to send the animals down ^ma decrease to send them up. A square hole is cut in a black sleeve placed around

Change of Phototropic and Geotropic Signs in Daphnia 119

liddle of a museum jar standing in the diffuse daylight of the room. Wheneverthe Daphnia swam in front of the opening from any point, they were

immediately stimulated to swim directly downward although in many cases theycould have reached darkness much sooner by returning in the direction whencethey had come.

7. EFFECT OF INTENSITY AND DIRECTION OF LIGHT.Now that reactions due to changes in the light intensity have been considered,

we may deal with the effect of the direction of the light. Holt and Lee (1901)have shown that the distinction sometimes made between photopathy (sensitivenessto intensity of light) and phototaxis (sensitiveness to direction of light) as differentforms of irritability is unwarranted. It is made clear that the intensity of the lightdetermines the sign of the response (positive or negative), while the part of thebody stimulated—determined by the direction of the light—decides the ultimateorientation of the animal. Enough experiments have already been given to establishthe fact that a change of light intensity stimulates the Daphnia to move. The directionin which the animal responds' is determined by the force of gravity and by thedirection of the rays of light falling upon it. The response to gravity we havealready considered. Up to this point the phototropic responses discussed havebeen simple movements directly to or from the light source. Other experi-ments demonstrate clearly that the direction of the light rays does not stimulatethe organism, but merely orientates it after it has been urged to move by achange in the light intensity. If the animal is negatively phototropic, it turns inthe direction from which it is receiving the least amount of light until it comesto be moving directly away from the light source. Conversely, a positively photo-tropic animal is orientated by the light falling upon it to move directly towards thelight source.

The famous experiments of Loeb (1918) showing the mechanical nature of thisphototropic orientation in various animals are well known. Holt and Lee (1901)devised experiments with Infusoria using a trough of water through which a bandof light passed. A prismatic screen was placed perpendicularly to the beam oflight so that a grading of the light intensity from bright at one side to dim at theother was produced at right angles to the direction of the light. Yerkes (1903)working on Daphnia pulex produced a similar optical condition by another type ofapparatus. In both investigations it was found that negative animals moved intothe dark end of the trough and positive animals into the bright end although inboth cases the animals were required to move at right angles to the direction of thelight. The mechanism by which this result is produced and its agreement withthe theory that intensity stimulates and direction orientates are clearly set forthby Holt and Lee. That this mechanism of orientation functions in planktonicanimals regardless of the fact that it may lead them into even more unsuitableconditions is shown by experiments using converging and diverging beams of light.

Kassing the light through a cylinder of water, Moore (1909) succeeded ining the light in such a way that a "caustic " was produced in a second cylinder

120 G. L . C L A R K E

in which nauplii of Balanus were swimming. He found that the animalseither directly toward the light or directly away from it (according to thetheir phototropism) although for part of each journey the light intensity was in-creasing and for part it was decreasing. He says on p. 18:

At first sight it looks proven from this that intensity of light is of no effect,and the direction of incidence the whole matter, because the organisms appear toswim in one direction indifferently, whether the illumination is increasing or de-creasing. In reality, however, such a conclusion would be fallacious, for in orderthat, say, a. positive organism should turn when it began to swim in light of graduallydecreasing intensity, it would be necessary for it to turn its sentient surface awayfrom the light, and that would plunge it into darkness.

And on p. 33:

Movement in converging and diverging light is shown to be explicable on thebasis of intensity of light alone, and that direction produces its effects in a secondarymanner on account of the light and shade effects of the animal's own body.

In my investigations with Daphnia magna a similar experiment was performedas shown in Fig. 4 b. A lens 16 cm. in diameter was used to produce a cone oflight in the experiment chamber. When the light was dimmed the Daphnia movedtowards the light source although it was thereby moving into a region of weakerlight, and when the light intensity was increased, the animal moved away althoughthis meant swimming into a region of greater and greater intensity. Following areduction of the light intensity, the phototropism becomes positive as we haveseen. Presumably this means that the organism is stimulated to seek again thesame light intensity. This would ordinarily mean moving towards the light and theresponse to that stimulus has come to be a turning towards the direction of thelight and hence a movement toward the light source. In this experiment the samereaction occurs. The Daphnia is stimulated to turn towards the side of its bodywhich is most strongly illuminated, that is, the side towards the light since the otherside is in shadow. The animal may perceive that it is getting into weaker andweaker light, but the mechanism of the response is such that it is forced to movein that direction. Exactly the same argument holds for the return trip. When thelight is made brighter, the animal is compelled to move away from it. As it reachesregions of greater and greater intensity, it may be stimulated to swim faster andfaster, but it cannot turn about and move back to a more favourable position.

8. ABSENCE OF ABSOLUTE OPTIMUM LIGHT INTENSITY.

There is, then, no "absolute optimum" light intensity for these Daphnia. Theydo not seek any particular intensity of illumination. The animals become adaptedto the light intensity which exists at that time and place—this is for them a "relativeoptimum." If the intensity rises or falls below the value to which they are thenaccustomed, the organisms are stimulated to move accordingly. Soon after thelight intensity becomes constant at its new value, the Daphnia have become ad^Adto the new conditions and the original primary tropisms come into play once more.

Change oj fnototropic and (Jeotropic Signs in Daphnia 1 2 1

situation is in no way altered by using different kinds of lights or differentdirVRons nor by making the water less transparent (through the agency of mud,"Aquadag," Bismark brown, etc.) and thus increasing the rate of change of lightintensity in space (t.e. "place-change").

Many previous investigators have discussed the possibility of there being anabsolute optimum light intensity for planktonic organisms. Of those who giveevidence for believing such an absolute optimum to exist, special attention is calledto Russell (1927 a, pp. 247 and 253). Evidence against such a belief is given byYerkes (1903, p. 362), Moore (1909, p. 32), and Ewald (1910, p. 15 and 1912,p. 594). Special attention is called to the experiments of Yerkes already discussedin the preceding section of this paper. It will be remembered that the Daphnia

•oeUS

O

140

120

100

80

60

40

20

§ 9 0

§ eo•° 70

<H 50

8 40

a 30

• | 2 0

LightIntensity

Positionof

Daphnia

11:35 11:40 11:45 11:50 n:55 12:00 12:05 12:10 12:15

Time

Fig. s. Experiment 20. February 6th, 1929, Apparatus Type 1, vertical. Temperature 11-9° C.

was placed in a band of light of graded intensity, the grading being at right angles tothe direction of the light. Since the animal under consideration was positivelyphototropic, it moved towards the highly illuminated end of the trough. If therehad been an absolute optimum of light intensity, stimulation would have ceased whenthat absolute value had been reached and the animal would have stopped swimmingin that direction. The fact that the Daphnia continued to move into greater andgreater light intensities and even into regions of lethal thermal conditions provesthat there is no absolute optimum in this case.

Proofs that the relative intensity and not the absolute intensity is important indetermining the position of Daphnia are to be found in most of the graphs of re-corded experiments. Attention is called particularly to Experiment 20; a graphof 4^t of this is shown above in Fig. 5. Here it will be seen that when the lightintensity is reduced from 126 c.p. to 33, the Daphnia responds by swimming to

122 G. L. C L A R K E

the top of the tube. A further reduction from 33 to 9 keeps the animal atBut when the light is increased from 9 to 33, the Daphnia swims to the bdSince the illumination at the top of the experiment tube is necessarily muchbrighter than that at the bottom (due to absorption in the water), the organismcannot be said to be in an optimum light intensity in both places. Yet with alight source of 33 c.p. the animal is first at the bottom and then at the top. Theeffect such an intensity has upon the movements of the animal depends not uponits absolute value but upon whether it is reached by a dimming or a brighteningof the light. The sign of the stimulus received depends upon the history of theenvironmental changes and not upon the situation at the moment.

9. THE LATENT PERIOD AND THE EFFECT OF RATE OF CHANGE.

These changes of tropism signs do not take place immediately following a re-duction of the light—there is always a certain latent period or "lag" which isinterposed between the stimulus and the response. There may well be two suchlatent periods concerned with every response. First, there is the interval betweenthe initiation of light reduction and the perception by the Daphnia of this dimming,and second, there is the interval between the perception and the response (swim-ming movement) which follows:

Lag LagDIMMING s- PERCEPTION s- MOVEMENT

In view of the fact that we have no information upon the integral parts of thewhole reaction, but can observe only the times of beginning and ending, the twopossible reactions are taken together, and the latent period is denned as the timewhich elapses between the start of the dimming of the light and the start of move-ment of the animal:

Latent PeriodDIMMING BEGUN > MOVEMENT BEGUN

This latent period always exists whatever the absolute light intensity and the rateof change may be. But the duration of the latent period depends upon the speedat which the light intensity is changed. Thus, the slower the dimming of the light,the longer the latent period lasts—in other words, the slower the rate of change oflight intensity, the more time elapses between the beginning of the dimming andthe beginning of the swimming movements in response. It might be supposedthat a certain amount of intensity change was required to stimulate the Daphnia torespond—that, starting from a certain high value of illumination, a certain lowvalue had to be reached before the animal was caused to move. Obviously, sucha situation would produce qualitatively the same effect because at a slower rateof dimming a longer time would be required for the requisite amount of intensitychange to occur—for the certain low value to be reached. But when the data fromactual experiments are consulted, it is seen that such an explanation ^fc

satisfy all the facts. For, the Daphnia does not respond after a certain amouniof

Change of Phototropic and Geotropic Signs in Daphnia 123

intejgity change has occurred—on the contrary, the swimming is initiated at adif^^i t intensity value for every different rate of dimming. In general, the fasterthe rate of intensity change, the smaller the amount of change required and hencethe greater the absolute intensity existing when the organism responds. Thesefacts are represented diagrammatically in Fig. 6. The two responses shown,X and Y, occur after a dimming of the light over the same range but atdifferent speeds. A glance at this diagram will make clear that response X, resultingfrom a slow rate of dimming, occurs after a long latent period and at a low intensityvalue, whereas response Y, resulting from a rapid rate of dimming, occurs after ashort latent period and at a relatively high intensity value.

Fig. 7 is the graphical expression of actual observations—the data of Experi-ment 14. In this experiment the responses of one individual Daphnia were watchedduring a long series of tests in which the light intensity was changed many times overthe same range but at different speeds. Although such a long series of observationsupon the responses of one animal under these circumstances was carried out only

Intensityfor Y

Bright

LightIntensity

Bottom

Time Latent periodfor Y

Fig. 6.

Latentperiodfor Y

once, nevertheless the results seem trustworthy since the animal was still behavingnormally at the end of this experiment and since other shorter experiments confirmthe general conclusions. The conditions were most carefully controlled. The experi-ment tube was placed within a light-proof box in addition to the usual light leakageprecautions, rest periods of at least 15 minutes each were allowed between tests, andthe temperature was delicately regulated as shown by the values given at the topof the graph.

From a study of the graph of Experiment 14 Table II has been drawn up showingthe duration of the latent period (in minutes) and the absolute light intensity(measured by the candle power of the light source) at the time of response as func-tions of the rate (c.p./min.) at which the light is dimmed. For example, consultthe graph at 2.30 p.m. (Response G). Here the light intensity is reduced at therate of 19 c.p./min. Three minutes after dimming has begun, the animal respondedby starting to swim directly to the top of the tube. At the moment when this

occurred, the light strength was 50 c.p. Contrast this with the succeeding3.30 p.m. (Response H). In this case the rate of dimming was 37 c.p./min.,

6 CHI 20

55-|llo|— Light Intensity

3CH

25

9:00 1020 10.30 1040 10 50 TTTOO l t iO 11.50 11:30 TT40 11'50 12:00 12:10a m - Time A B C D

1220 12:30 1.2:40

Fig. 7-

Change of Phototropic and Geotropic Signs in Daphnia 125

period 22-5 min., and the light strength was 23 c.p. From the datain Table II the curves of Fig. 8 were constructed. This graph shows that

as the rate of dimming is increased, the duration of the latent period is decreased,while the magnitude of the absolute light intensity at the time of response isincreased (i.e. the amount of intensity change required is decreased).

Table II.

Intensity and latent period as functions of rate.

Response

/HFAEDGJCB

Ratec.p ./min.

i-63-76-67-S

16-01 8 0

19-029-041-054-o

Light intensityc.p.

7-S2 3 02 3 019542-070-050-033°65-084-0

Latent periodmin.

63-522-51 2 51 1 54-02 - 0

3-o2-5I-O

°SExperiment 14. January 23rd, 1929. The "Light Intensity" is measured by the strength in

candle power of the light source.

Intensity- 90 C.p.

Timemin

70

60

50

40

30

20

to

•j

/kbsQ

/J m

V"1 \l_atent Perio

Absolute IntensityCurve

10 20 30 50 60 c.p./min.

Rate of change of intensity of lightFig. 8. Experiment 14. January 23rd, 1929.

The graph of Experiment 14, as well as many other observations I have made,shows that in general when once the stimulus has taken effect, when once thereaction has started, the response proceeds at a maximum rate and continues toa Maximum magnitude. Take, for example, the two cases in Experiment 14(fl^onses G and H) referred to in the preceding paragraph. Here although the

jEB-vnii 9

126 G. L. CLARKE

rate of dimming is very different, when once started the animal swims in bpthcases to the very top and at its maximum speed. To be sure this is not alwaj^^hecase, as can be seen in the earlier part of this same graph where submaximal andirregular responses are to be found. Usually, however, the response is maximal.Moreover, the Daphnia generally swims straight to the top of the tube even ifthe dimming of the light is stopped soon after the upward movement begins. Insome cases, however, a continuation of the dimming is necessary to send theDaphnia to the very top. If the rate of change is very fast, however, the dimmingmay have ceased before the response has even begun. Whether or not the animalwould respond while the light intensity is held constant following a dimming ofthe light down to an intensity just above the value at which a response is usuallyevoked at the same rate of reduction has not been determined. Such an experi-ment would give us information upon the nature of the latent period and thepossibility of there being two " lag " periods for each response. But the experimentsappear to show definitely that a certain minimum amount of intensity change musttake place in order to produce a response; a change of intensity over a rangesmaller than this minimum will not stimulate the organism to move, no matterhow fast the rate of change may be. Many more observations are needed in thisfield and experiments especially designed to test each particular problem must beperformed before conclusions can be drawn.

10. FATIGUE.

The question of fatigue is an important one and has been discussed byother workers {e.g. Yerkes, 1900 and 1903). In all the experiments describedthus far ample time between the various tests has been allowed for rest. But inExperiment 21 (graph not reproduced here) in which the apparatus described onp. n o was used in a horizontal position the light was dimmed four times over thesame range (116-3 C-P-) a n ^ a t t n e same speed without any interval between thetests. The experimental animal responded maximally and in practically an identicalmanner after the first three stimuli. But the fourth response was of approximatelyonly \ magnitude. Further stimulation evoked even smaller responses and a restof about 20 minutes was not sufficient to restore the animal to its normal condition.But a 3-hour period of complete darkness (the animal remaining at the bottomduring this time) is followed by a normal response when the light is subsequentlyincreased and dimmed.

11. SPECIMENS WITH REVERSED PRIMARY SIGNS.

Thus far I have been dealing exclusively with animals which are primarilynegatively phototropic and positively geotropic. A small number of Daphnia magnaare found to be primarily positively phototropic and negatively geotropic.^t isimportant to note that these individuals react to changes in light intensl^rin

Change of Phototropic and Geotropic Signs in Daphnia 127

the same way as the more usual forms of Daphnia except that the tem-secondary tropism signs are evoked by an increase of the illumination

instead of by a decrease. When the light intensity is reduced, the primary signsare strengthened, but increasing the light intensity results in the temporary estab-lishment of the secondary signs. When experimenting with these animals, then,a dimming of the light produces no effect as they are already as close to the sourceof light as possible, due to their primary positive phototropism, but as soon as thelight intensity is increased they become temporarily negatively phototropic andstart swimming away from the light. As before, moreover, this change of signdoes not last long and the Daphnia soon come back to their starting-point close tothe light source. In complete darkness the animals are found at the top of thetube as a result of the primary negative geotropism existing in this type of Daphnia.

12. ADAPTATION.

In all this work, adaptation suggests itself as the explanation of the phenomenaobserved (see Crozier and Wolf, 1928, and Adrian, 1928). The optimum lightintensity for an animal at any given moment is that intensity to which the organismis then adapted. If the environmental conditions are changed, that is if the illumi-nation is subsequently increased or diminished, the Daphnia is stimulated to swimeither away from the light source or towards it. But as soon as the animal hasbecome adapted to the new intensity value, it responds to further stimulation inexactly the same way as it did at the old intensity value. That adaptation is not aslow process is indicated by the uniform results obtained after each of three similarreductions of the light with no interval allowed for rest between. This is shownclearly in Experiment 21 referred to above. If adaptation were slow, one wouldexpect to find a progressive change in the three responses (such as summation,etc.) instead of the identical curves obtained in the graph. But on the other hand,adaptation is not instantaneous for the animal is stimulated to move towards thelight the whole time until the process of adaptation is complete. Evidently,adaptation has not been accomplished during the time which elapses while theDaphnia is swimming towards the light and while it remains at the top of the tube—that is, several minutes.

By means of experiments in which the light intensity was switched instan-taneously from one value to another it has been shown that the rate of change ofintensity cannot be too fast for the Daphnia to be stimulated by it. Further supportof this theory of adaptation is found in the fact that it is usually possible to dimthe light at such a slow rate that no response is obtained, although the animaldoes react to a change of light intensity over the same range when this changetakes place at a high speed. This suggests that at the slower rate adaptation inDaphnia is able to keep pace with the change of light intensity: the organism isalways adapted and therefore never stimulated to move. I have never succeededin^mionstrating conclusively that the light can be dimmed over its whole rangefr^m full bright to complete darkness without producing a response, although this

9-2

128 G. L. CLARKE

can probably be done when the difficulty of preventing the animal fromto other stimuli over such a long period of time is overcome. But that arpart of the light range can be traversed without causing a movement of the animalby such a slowing of the rate of change has been shown many times—for example,see Response / , Experiment 14, Fig. 7. One important phase of the observedresponses of Daphnia for which this theory of adaptation does not seem to pro-vide an adequate explanation is the return of the primary signs of the tropisms.Following a reduction of the light intensity the animal swims towards the lightapparently stimulated to move again into a region of intensity to which it has beenadapted. No matter how small a light change has taken place, the Daphnia usuallyswims to the very end of the tube, as we have seen, although in so doing, in thecase of very small reduction, it must pass by the intensity value which had existedat its first position. When the animal reaches the end of the tube nearest the lightsource, it remains there supposedly until it has become adapted to the new intensity.,When this adaptation is complete, the Daphnia starts swimming away again.Apparently, this return to primary negative phototropism is not a phase of adapta-tion, but is due to a different stimulus produced by a steady light.

CONCLUSION.

A discussion of the possible bearing of the results of this paper upon theproblem of the vertical migration of plankton animals in general would be pre-mature. In the first place, many more experiments must be performed uponDaphnia itself. Secondly, observations upon the behaviour of a large number ofdifferent genera of plankton animals must be made. And thirdly, some methodmust be devised for proving that results obtained in the laboratory may legitimatelybe applied to the behaviour of the organisms in nature. Still the responses ofDaphnia to changes of light intensity are suggestive. Diurnal migration may bedue simply to a positive phototropism and a negative geotropism produced by therising of the sun in the morning and to a reversal of those tropisms when the sunsets at night. But we have seen that Daphnia exhibits complete power of adapta-tion to light—it is primarily negative to any intensity of illumination. What wouldprevent such an animal from swimming down and down indefinitely? How canthese responses be reconciled with the fact that the plankton has been observed tobe distributed at definite levels in the water? If it could be shown that planktonorganisms in nature, unlike laboratory Daphnia, have no power of adaptation tolight, or have a very limited power, it would be conceivable that they be stimulatedto seek a region of fixed light intensity which was for them an absolute optimum.Were this found to be the case, a theory that the plankton follows an optimumlight region as that region moves down into the depths and up to the surface againduring the course of each day would be tenable. As it is, experimental results donot seem to agree satisfactorily with inferences from field observations. Evidentlythe mechanism of the responses of these animals must be much more thoroi^Myinvestigated before the causes of diurnal migration can be conclusively ascertanrcd.

Change of Phototropic and Geotropic Signs in Daphnia 129

SUMMARY.

1. A method is described for studying the responses of Daphnia to changes oflight intensity with special attention to the behaviour of the individual and to theavoidance of " shock " effects. The types of apparatus used provide for rigid controlof the temperature, for illumination from any direction, and for an adjustable rateof change of the light intensity by means of a chemical rheostat.

2. The great majority of Daphnia magna and Daphnia pulex were found to beprimarily negatively phototropic and positively geotropic. That is, they alwaysexhibited those tropistic signs under constant conditions of illumination.

3. A reduction of the light intensity causes a temporary reversal of the tropismsigns. The secondary signs thus produced are positive phototropism and negativegeotropism.

4. The presence of both phototropic and geotropic forces is proved by experi-ments in which illumination is (1) from one side, (2) from beneath, and (3) fromtwo opposing sides or from above and below simultaneously. In these tests andin others in which very slow and very fast rates of dimming are used the photo-tropic and geotropic forces are resolved, antagonised, and neutralised in succession.The responses of the Daphnia indicate that there are two types of animals whichexhibit exactly the same tropisms, but in one type phototropism is the strongerwhile in, the other geotropism is the stronger.

5. In this material it was found that the temporary secondary tropistic signspersisted only a few minutes while the primary signs persisted for hours, althoughthis effect was somewhat less marked in weak light or in darkness.

6. The difference between "time-change" and "place-change" of light in-tensity is pointed out. Daphnia is stimulated by both types of change if the rateof change is sufficiently great.

7. That photosensitive animals are stimulated to respond to changes in theintensity of light only and are merely orientated by the direction of the light isshown in the work of previous, investigators as well as in this paper. The rigidityof this mechanism is indicated by experiments in which the light is graded inintensity at right angles to its direction and in which the light is rendered con-verging and diverging by a lens.

8. Evidence is given for believing that there is no "absolute optimum" lightintensity for Daphnia but that a "relative optimum" exists which is the intensityto which the animals are adapted at the moment.

9. The interval between the inception of the reduction of the light intensityand the beginning of swimming movements in response is called the latent period.The faster the rate of dimming, the shorter is the duration of the latent period.A minimum, amount of intensity change is required to produce any response, atany speed, but beyond that the slower the rate of dimming, the greater is theamount of change required and hence the lower is the absolute intensity at which

tesponse takes place. Ordinarily, the response is maximal in respect to bothand magnitude.

130 G. L. CLARKE

10. Fatigue will interfere with experimentation unless guarded against.11. Specimens of Daphnia with reversed primary signs gain temporary

dary signs following an increase of light intensity; otherwise they behave like themore usual forms.

12. The possibility that the processes of adaptation in Daphnia may accountfor the photic responses observed is discussed. Support for this theory is derivedfrom the fact that it is possible to dim the light over a given range at such a slowrate that no response is produced.

The importance, as well as the difficulty, of applying the results of laboratoryexperiments of this type upon the responses of Daphnia to the general problem ofthe behaviour of plankton animals in vertical diurnal migration is stressed.

I am indebted to Professor J. S. Gardiner and to Mr J. T. Saunders for theirsustained assistance and advice during the progress of this investigation at theZoological Laboratory of Cambridge University. I am also indebted to Dr C. G.Lamb of the Engineering Laboratory for many helpful suggestions and the loanof apparatus.

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