Rheotaxis in Planaria Alpina by R

Home , Rheotaxis

RHEOTAXIS IN PLANARIA ALPINA BY R. S. A. BEAUCHAMP. (Assistant Naturalist at the Laboratory of the Freshwater Biological Association of the British Empire.)

(Rectivd 4th October, 193a.)

(With Seven Text-figures.)

INTRODUCTION. THE rheotactic responses of fresh-water planarians have been studied more than those of any other invertebrate. Yet these records are for the most part incomplete, and at variance with each other. The only conclusions that can be drawn from them are that the stream-living forms are more inclined to give the positive rheotactic response than the lake-living forms. Also the stream-living forms are not always positively rheotactic, at least under experimental conditions. The earliest observation is by Johnson (182a), who records having seen a number of " planarians " migrating upstream. It seems that Johnson's observations refer either to PUmaria alpma or PofyceHt contuta. In 1900 Volz made a similar observation on PI. alpma in a spring near Aarberg. In 1903 Pearl worked in America with PI. dorotocephala, a species which is found only in streams. He says: "The planarian is positively rheotactic to very weak currents (as delivered by a fine capillary tube), the form of the reaction being precisely the same as that given to other weak stimuli." Working with the same species, Allen (1915) came to the conclusion that it was positively rheotactic only in strong currents. Before considering the more recent experimental work, reference should be made to the environmental factors which control the spread of these animal*. The three European species, PI. alpma, PoL contuta and PL gonocephala, are all limited to the upper reaches of streams, since the summer temperature of the lower reaches is fatal to them. Temperature is the chief controlling factor, but the rate of flow is also important (Beauchamp and Ullyott, 1932). Any fluctuation in the temperature of the stream should shorten or lengthen the area over which these nnimaia occur, dependent on whether the temperature of the stream is raised or lowered. The difference between the summer and winter distribution shows this to be the case (Wilhelmi, 1904 and Carpenter, 1928). Voigt (1907) showed that PI. gonocephala migrated up tributary streams, which had previously been colonised only by PI. alpma and Pol. contuta, on the warming of the water, following the cutting down of woods. The simple maintenance of position in a stream of water, quite apart from these observed migrations, demands some form of rheotactic response on the part of these ii4 R. S. A. BEAUCHAMP animals. This response was first demonstrated by Voigt (1904). He placed a number of planarians (species not stated) in a large tube with two small tubes coming in at the top; one brought pure water, the other water to which bait juices had been added. When the latter tube was turned on the animals were " alarmed " and crawled up the tube. He could obtain no response when the anifnuia were tested with pure water. In 1913 Steinmann, using PL alpina, demonstrated positive rheotaxy in ordinary water. The apparatus used was a sloping glass tube. Steinmann *s conclusions were that 80 per cent of all the triclads tested were positively rheotactic. Kafka (1914) and Olmstead (1917) have shown that PL gonocephala and PL maculata are geotactic. Olmstead showed in PL metadata that the positively geotactic animal became negatively geotactic after feeding. The writer has con- firmed this observation for PL alpina. PI. alpina is strongly negatively geotactic for 6 or 7 days after feeding. In the light of this knowledge all Steinmann's experiments are open to the criticism that they measure geotaxis as well as rheotaxis, since he used a sloping tube. Doflein (1925) made a careful study of the chemotaxis of PI. alpina, PI. gonocephala, PL htgubris and Dendrocochtm lacteum. She found, that in still water, food substances stimulated PL alpina to move about and to make "search movements." But, unlike the other three species, PL alpina was unable to orientate itself to the food. Similarly in running water PL alpina could not appreciate the direction from which the food stimulus was received. Doflein's rheotactic experiments were done on a gently sloping glass plate, so of necessity there must be some confusion between geotaxis and rheotaxis. She found that 80 per cent of the PI. alpina were positively rheotactic, and considered that those which showed the negative response were either weakly or damaged. Doflein states that the rheotactile organs are situated in the head. Using a pipette, she found that the nnimaia only responded to a current of water when it was directed on to the head region. She further assumed that the receptors were situated in the pair of head tentacles. Koehler (1926) disproved this assumption by removing the head tentacles and finding that the animals responded normally. His current consisted of a stream of water on an inclined plane and also of a jet of water from a fine pipette. In 1932 he again concluded that PL alpina was strongly positively rheotactic and that the rheoceptora were distributed over the whole body. In these later experiments he used only the pipette method for making his current. Hubault (1927) experimented with PL alpina using a circular dish. He made his current by means of a jet at the side; the water was removed from the centre of the dish by a siphon. He stated that nearly 80 per cent were positively rheotactic. The current strengths used were very great judging from the fact that about 14 per cent of the animals were washed away. None of his experiments lasted longer than 5 min. Voute (1928) found that all the PL alpina collected from a stream and lakes near Oo8terbeck were decidedly negatively rheotactic. But putting food into the water made them react positively. No description is given of the apparatus used. Rheotaxis in Planaria alpina 115 Carpenter (1927) found that PI. alpina was always strongly negatively rheotactic. She also stated that if the animals were kept in still water for several days they no longer responded to a current of water. It is possible that these observation* are the result of unsatisfactory experimental conditions. It seems unlikely that any stream- living animal could be persistently negatively rheotactic, without sooner or later deserting its normal habitat. Summarising we may say that the majority of workers have found that a large percentage of the PI. alpina tested show a positive response to a current of water. The rate or extent of this reaction has in no case been determined. No work has been done on the responses given by the same individual over a long period of time. Nor has any attempt been made to correlate the condition of individuals with their behaviour.

METHOD AND APPARATUS. All the experiments described in this paper were done with PI. alpina. Since it is convenient to have a large number of animal* available in the laboratory a stock supply was kept Moreover, it is an advantage to have the animaia under known conditions previous to their being tested. The water in which these stock animals were kept was well aerated or else supplied with a current of water from the tap. The temperature was kept below io° C. At first some difficulty was experienced in keeping the animals in a healthy condition; later work showed that tap water was unsuitable owing to the presence of dissolved iron. In the end the most satisfactory conditions were found to be given by keeping not more than fifty animals in a shallow open basin of spring water. Under these conditions it was found unnecessary to aerate or circulate the water. A few stones were provided for the «nima1« to crawl under. The temperature was kept at a low and constant value by leaving the basin in a cellar. Owing to the slope of the ground the cellar received light from a window on one side, this ensured normal diurnal light variations. It was found that there was marked periodicity in the activities of the animals. During the day they remained under the stones, and in the evening they started to move about But, under the quiet conditions in the basin, this periodicity was lost after a few days although the daily changes of light were unaltered. The rheotactic responses of these animnjn were tested in a long glass trough. This trough was 120 cm. long and 10 cm. wide. It was shaded from all sources of direct light and received only diffuse light from the interior of the room. At first all the experiments were done with animals which were collected from the top of the streams. These animal* might well be expected to be positively rheotactic. Yet in almost every case the animals moved downstream; that is to say they were negatively rheotactic. At the time it was felt that these results were the outcome of unsatisfactory conditions. The discovery that the tap water was injurious to these animals led to a radical n6 R. S. A. BEAUCHAMP change in the method employed. Clearly it was necessary to use only spring water from where the animals were collected. This made it necessary to devise a circulating apparatus which would give a fairly considerable and steady flow. It was essential that there should be no metal included in the apparatus which might contaminate the water. This precluded the use of any sort of motor or even a mercury valve. The general form of this apparatus is best understood by reference to Fig. i. From the vessel A the water flows through the trough used for the rheotactdc experiments. From this trough the water pours into the periodic syphon B. When the level of the water in B rises to the top of the siphon, the vessel automatically empties itself into the basin C.

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Fig. I. Diagrammatic view of the apparatus. The arrows indicate die direction in which the water Bow*.

From C the water is sucked into the chamber D from which the air is exhausted by means of a filter pump. During this process the exit tube c is closed by the glass valve. When the amount of water delivered by the periodic siphon has been drawn up from C into the vessel D, the long sloping tube d fills with air. This releases the negative pressure in D and the water escapes from this chamber by way of tube c through the glass valve into the vessel E. From E it siphons over into A. The vessel X serves to minimise the changes of level in A. In addition the siphon between E and A damps the rise in level following the emptying of chamber D. Rheotaxis in Planaria alpina 117 The efficient working of this apparatus is dependent on a number of small details. The design of the periodic siphon is important Its internal opening must be wide. This ensures that the column of water is broken at the end of the emptying process and replaced by air. On the other hand the rest of the siphon must not be too wide, otherwise the water will start to flow out as soon as it reaches the bottom of the curve at the top, that is to say at the level x. It will be seen that for greater efficiency the bore of the siphon should be varied according to the current circulating. The greater the current the greater should be the bore of the siphon tube. In this particular case where the flow was usually about 1200 c,c. per min., the bore of the siphon tube was fin. The internal opening was 1 in. The vessel itself was a Terry sweet-jar with the bottom removed. Removing the bottom is easily done by standing the jar in } in. of cold water and then pouring a little boiling water into it Rubber bungs size 1^ in. fit the mouths of these jars. It will be seen in Fig. 1 that the long sloping tube dis held at the bottom end by a cord. It is of the greatest importance that a small length of elastic be included in this attachment; without it the apparatus works very inefficiently1. In the chamber D it was found necessary to bend the tube b slightly to one side away from the tube a, as water, falling off the roof of this chamber, was liable to be sucked down a. The glass valve was constructed by sealing a glass tube on to the side of a small reagent bottle, of the type possessing a ground glass stopper combined with a pipette. Both ends of this combined stopper and pipette were closed, but first the lower half of the pipette was filled with mercury to weigh it down. If this were not done the stopper was apt to be blown right out of the bottle on the release of the water from chamber D. It sometimes happened that the stopper returned to its seating so abruptly that the weight of water in D was not sufficient to reopen it. This difficulty was overcome by pulling out the stem of the pipette until it almost touched the bottom of the bottle and then fitting the end with a small rubber pad. Without the rubber pad either the pipette or the bottom of the bottle was liable to break. Experience has shown that this apparatus will function satisfactorily for an indefinite length of time. If the pressure of the tap water is low and if the apparatus is to be worked to its fullest capacity it may be advisable to use two filter pumps in parallel. This apparatus can circulate about 3 litres of water per minute. Using this apparatus and the glass trough, better results were obtained and a large percentage of the animpin tested showed the positive response. But the straight sides of the trough made it possible for the animals to find shelter in the angle between the sides and the bottom. In addition, one was uncertain whether the behaviour of an animal crawling on the vertical sides of the trough was comparable with its behaviour on the bottom.

1 It ha* been found poaaible to dispense with the periodic siphon B, since a long elastic attachment causes excursions of d sufficient to maintain the periodicity necessary for the working of the apparatus. u8 R. S. A. BEAUCHAMP Finally the glass trough was exchanged for a long wooden one. This trough was 150 cm. long by 12 cm. wide, the depth was approximately 1 cm. The bottom was flat and the sides were gradually curved up so that there were no corners or vertical sides. Fig. 2 shows one end of the trough, seen from above and in longitudinal section. The water wells up from the bottom of the shallow pool at one end and overflows into the main part of the trough by way of a gentle slope. The outflow is exactly similar to the inflow. This arrangement eliminated eddies, at least over the range of currents used in these experiments. The direction of current could be reversed by a system of glass Y-pieces and clamps. The speed of the current had no appreciable effect on the behaviour of the ; probably this is partly accounted for by the fact that changes in the current,

Fig. a. One end of the trough, teen from above and in longitudinal section. The two ends of the trough are exactly similar. as measured by floating pieces of cork, were not followed to the same degree by changes in the current immediately above the bottom of the trough. As measured by floating pieces of cork, the current used in all experiments was approximately 2 J m. per minute. The temperature of the water during all experiments was kept below io° C. This could only be brought about by cooling the whole laboratory. This was easily done as the experiments were carried out during the winter months. The initial experiments with this trough proved unsatisfactory. This was the result of toxic substances dissolving out of the wood. The trough was made of teak and the difficulty was overcome by giving it a number of coats of cellulose paint. Black paint was used; this provided an additional safeguard against light errors. The whole apparatus, consisting of the circulating apparatus and the painted trough, now proved entirely satisfactory. Rheotaxis m Planaria alpina 119 Earlier workers studied the reactions of a large number of animals and expressed their results as percentage number of positively or negatively rheotactic forms. It was thought better to study the reactions of individual pninrmifr over a long period of time and discover whether changes occurred in their behaviour. The actual movements of the animals were copied on long strips of paper, the same size as the trough. Usually, animals were tested for several days in succession and then transferred to a labelled dish and later tested again. Occasionally an individual was kept as long as 6 weeks in the trough. During this time changes in its behaviour might occur, as explained later, but on no occasion did changes occur owing to unsuitable experimental conditions. The experimental conditions were undoubtedly entirely satisfactory for the health of the animals; in no case did an animal become moribund or out of condition while in the trough.

EXPERIMENTAL DATA.

(a) THE SEXUAL CYCLE. Once the above apparatus had been constructed satisfactory experimental data were obtained. Errors due to possible changes in the chemical content of the water were eliminated and the animals were kept in water of optimum composition. The trough was evenly illuminated and kept in a low light intensity, although the rheotactic response was always stronger than the phototactic. The temperature of the water was controlled. With these precautions, if the nnimpin re-orientated themselves to the current when the direction of the flow was reversed, it was considered that the response could only be attributed to rheotaxis. As a rule the animals were tested in the evening and sometimes late into the night, that is to say during their normal period of activity. The individual which was being tested was allowed during the day to rest under a stone in the trough. In the evening when it had come from under the stone of its own accord, the stone was removed and the current turned on. Before turning on the current the movements of the animal in the still water were recorded. Fig. 3 shows the actual path of an individual. The dotted line at A shows its movements before the current was turned on. These movements were quite at random and demonstrated clearly that the animal was under no external influence which might have tended to direct its movements. The continuous line shows the path taken with the current on. The animal now moved upstream in a straight line. At C and at D the direction of the current was reversed and the animal responded by turning round and moving upstream in the new direction. At E the current was shut off, with the result that the animal again moved about in an entirely unonentated manner, as at the beginning of the experiment The arrows indicate the direction of the animal's movements and are marked in at minute intervals. During the months of December and January it was found that almost all of the animals collected from the top of the streams were positively rheotactic. An individual was considered to be positively rheotactic only if it re-orientated itself each 120 R. S. A. BEAUCHAMP time the current was changed and moved a minimum distance of three metres against the stream. The rate of movement is dependent on the temperature. At o° C. the nnimaia move at the rate of a cm. per minute. The rate of movement goes up as the temperature is raised, at io° C. it is 8 cm. per minute. One of the individuals collected in January from the top of the stream was tested to see how far it would move upstream before becoming fatigued. This test was carried out at a temperature of 6° C, the rate of movement being 5 cm. per minute. Each time the animal reached the top of the trough the current was reversed, the animal then re-orientated itself and proceeded in the opposite direction. The experiment was concluded after the animal had travelled 20 m. upstream in a continuous effort lasting 7^ hours. The discrepancy between the rate of movement given, namely 5 cm. per minute, and the rate as calculated from the total distance gone, divided by the total time taken, is accounted for by the time taken re- orientating each time the current was reversed. Moreover, the 20 m. refers only to A J

Fig. 3. The actual path of tn individual. The dotted line at A thowa it» movements in still water. At of the current was reversed. At E the current was stopped. The arrows indicate the direction of the indicate the direction of the current. the "progress" made in an upstream direction. Actually there were very few deviations, the animal moved up in almost a straight line, as in Fig. 3. When individuals were tested for several evenings in succession, they were not removed from the trough. The water was allowed to flow out from the pools at the two ends, so that the animal* were left isolated in the centre part of the trough. A stone was put into the trough, under which they were always to be found during the daytime. But in the evening they always came out from under the stone of their own accord. That is to say the normal periodicity of the animals was continued when they were stimulated to move about during the evenings. As already pointed out this periodicity is lost when the nnimnU are kept for a number of days in quiet water. Animals tested on consecutive nights were quite consistent in their responses, that is to say those animals which were positively rheotactic one evening were positively rheotactic the next The majority of those collected during December and January were positively rheotactic but a few were found to be negatively rheotactic. These animals were, as a rule, just as consistent in their behaviour as were the positively rheotactic individuals; that is to say they always went downstream. Rheotaxis in Planaria alpina 121 As yet no one has studied the negative response; in some cases workers have considered rheotaxis as synonymous with positive rheotaxis and have ignored entirely the negative response, considering it to be the result of damage or weakness. Many individuals which were consistently negatively rheotactic were in no sense weakly. They were often bigger than those which showed the positive response; they were undamaged, while damaged animals were often positively rheotactic. Their rate of movement at particular temperatures was usually slightly higher than the rate of movement of positively rheotactic individuals at the same temperatures. This negative response could not be explained on the grounds of unsuitable water or other experimental conditions, since positively rheotactic individuals were often put into the trough at the same time as the negatively rheotactic ones. The former would migrate upstream while the latter would migrate, down. When the direction of the current was reversed, both lots of animals would re-orientate themselves and make their way respectively up and down the current

B the current mi turned on; the continuous line ihowa the path taken. At C and at D the direction animal's movements and are marked in at minute intervals. The arrows immediately below the letters

Serial sections were cut of a number of these persistently negatively rheotactic individuals, and in all cases they were found to be mature. The testes were large, both the testes and the vasa deferentia contained spermatozoa, but as a rule the testes were not full and appeared to be past their period of maTimal production. The condition of the ovaries was always very much more characteristic. In a normal fully sexual individual each of the two spherical ovaries measure about 120/* in diameter; in all the negatively rheotactic individuals they were reduced in size to about 40^, and were clearly exhausted, showing that die animals had already laid their cocoons. Since all these animals had been collected from the very top of the stream it is evident that at one time they must have been positively rheotactic Examination of the positively rheotactic individuals showed them to be either fully developed or else beginning to develop sexually. From this it seems clear that animal* which are developing sexually become positively rheotactic and remain positively rheotactic until the sexual cycle is complete. When the cocoon is laid the behaviour of the animal changes and it becomes negatively rheotactic. This change over was witnessed in a particular case when the animal laid a cocoon while in the experimental trough. Previous to laying the cocoon it had been positively rheotactic, after laying it became negatively rheotactic. JM-Xii 9 122 R. S. A. BEAUCHAMP

(b) THE EFFECT OF STARVATION AND FEEDING. It is well known that planariana can resist the effects of starvation for a very long time. They absorb their gonads and become reduced in size. A number of positively rheotactdc animals were kept for two months without food. It was then noticed that they no longer gave their consistent positive responses. At times they went upstream but rather more often they went down. Often when the current was reversed they did not re-orientate, but continued in the direction they had previously been going, now of course the opposite direction with regard to the current When travelling downstream these animals tended to keep a very much straighter course than when going up. Often when going up they would zig-zag from side to side of the trough. This resulted in much greater progress being made in the downstream direction than in the up. This change in behaviour should be contrasted with that noticed on completion of the full sexual cycle, when the animals become decidedly negatively rheotactic. Examination of the starved individuals showed that the gonads had been absorbed, but as yet the size of the am'maU was only slightly reduced. If these animals were fed with a piece of Gammons or Caddis larva, they were as a rule very disinclined to move for 24 hours. But after that time they would react positively to the current This reaction would last for about 6 days, and then they would occasionally go downstream again, reverting to their previous undecided behaviour. After a second feed the animal continued positively rheotactic for a very considerable time, that is to say well over a month, after which time the effects of starvation would be repeated. The first reaction after feeding, namely, the positive rheotaxy lasting 6 or 7 days may be called " temporary " positive rheotaxy. The reaction after a second feed may be called "permanent" positive rheotaxy. Often a single feed would be sufficient to render the animal "permanently" positively rheotactic. Feeding an individual, which had laid a cocoon and become persistently negatively rheotactic, resulted in it too becoming "temporarily" positively rheotactic. This reaction lasted 6 or 7 days, as in the case of starved individuals; or, sometimes it lasted for rather a shorter time, the animal then reverting to its previous behaviour. It was possible to produce "permanent" positive rheotaxy in these individuals. But it took longer than in individuals that had been starved and had never completed their sexual cycle. Clearly there are two effects of feeding negatively rheotactic animals. The first is almost immediate and results in the ^nimg1« becoming positively rheotactic for about 6 d^ys. The second is the development of "permanent" positive rheotaxy, brought about, presumably, by the re-development of the sexual characters responsible for positive rheotaxy. Rheotaxis in Planaria alpina 123 The first, namely "temporary" rheotaxy, may be compared with the negative geotaxy induced by feeding. This latter reaction was observed by Olmstead (1917), working with PL maculata. Olmstead noted that the duration of this response was about 5 days. The writer has observed an exactly similar reaction in PL alpina; in this case the reaction usually lasted 6 or 7 days. We may suppose that the absorption of the digested food acts as a general stimulus; L. Hyman (1919) has shown that the rate of metabolism of the whole animal goes up after feeding. This general stimula- tion appears, in some way, to affect the geotactic and rheotactic responses. The "permanent" positive rheotaxy induced by feeding starved animals is less difficult to understand. Starvation caused these animals to de-differentiate and lose their positive rheotaxy. Feeding enabled them to re-develop those characters which were responsible for their original positive rheotaxy. This positive response is closely associated with sexual development and the negative response following the completion of the sexual cycle is very striking, yet it was found that feeding induced the "permanent" positive response before the animals had re-developed their gonads. It seems, therefore, that the factor responsible for the positive response develops before the gonads are differentiated. It was noted above that it took longer to induce "permanent" positive rheotaxy in individuals which had completed their sexual cycle and become negatively rheotactic than in individuals which had been starved. It is evident from this that after laying a cocoon some factor is developed which inhibits the positive response and causes the persistent negative response to reappear once the immediate and temporary effect of feeding has worn off. It is equally clear that this inhibiting factor is gradually lost, since the "permanent" positive response can be induced after a certain length of time. The negative response following the laying of a cocoon, though definitely observed in a large number of cases is not absolutely invariable. One individual which was well fed previous to laying did not become negatively rheotactic after laying its cocoon. This animal was seen copulating on February 6th; on February 29th it laid a cocoon. All this time the animal had been positively rheotactic. It had been fed at intervals of about 7 days. This amount of food is greatly in excess of what the animal« are likely to get in their normal habitat, and may explain why the usual change to negative rheotaxy did not occur.

OBSERVATIONS AND EXPERIMENTS IN THE FIELD. During the autumn, winter and spring three streams in the immediate neighbourhood of the laboratory were kept under observation. They were typical mountain streams. Two of them flowed into Windermere and the other into Blelham Tarn. These three streams which contained PL alpina appeared to have a more constant water supply than other similar streams which did not contain these animals. It was found impossible to visit all three streams at frequent intervals. It was decided, therefore, to visit one of the streams more frequently than the other two. The more occasional visits (every 3 or 4 weeks) to the other two sufficed to show that I24 R. S. A. BEAUCHAMP exactly similar migrations occurred in them as occurred in the stream which was more closely observed. The latter was visited about every 10 days. It was thought that more frequent visits would disturb the stream too much. Since there are no permeable rocks in which a supply of water can be stored, the source is not very constant, either with regard to the quantity of water or to temperature. But the water-holding capacity of the soil is sufficient to insure that the stream does not dry up in dry weather. Fig. 4 shows the variations in temperature. The curve with the points marked by triangles shows the temperature of the water at the source. The curve with the points marked by squares shows the temperature of the water 300 m. from the

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8EPT. OCT. NOV. DEC. JAN. FEB. MAR. APR. MAY JUNE JULY 1931 1932 Fig. 4. Temperature records. The point* marked by triangle* are temperature* recorded at the source. The square* are temperature* recorded 300 m. from the source.

source. The temperature variations at the source, though they would be considered Urge for a true spring, are considerably less than those at 300 m. The number of PL aJpma found at particular stations down the stream were recorded and especial care was taken to determine the lowest limit at which this species was found. During October and November a few individuals could always be found about 400 m. from the source. But by December none could be found so far down the stream, and by the end of January the lowest limit was about 225 m. from the source. That is to say, the lowest point at which this species could be found was by the end of January about 175 m. higher up the stream than it was during October. There are two possible explanations to this; either that the individuals below 225 m. from the source had been killed or else they had migrated upstream. The huge increase in the numbers found at the source show the latter Rheotaxis in Planaria alpina 125 suggestion to be correct Fig. 5 is a graph showing both the lowest limh of P/.a^>oia and alto the number of animals found on a particular area (roughly \ sq. m.) at the top of the stream. These two graphs alone make it quite clear that during the winter months there was a general migration upstream. The average size of the animals at the top of the stream during December and January was larger than the average size of the animals 200 m. from the top. That is to say, the larger animals were the ones which had migrated upstream. By the end of January a few newly hatched individuals were to be found; these were all in the upper part of the stream.

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OOT. NOV. DEO. JAN. FEB. MAR. APfl. 1031 1032 Fig. 5. The top graph show* the lowest point in the stream where Pi. atpata was found at different time* of the year; the bottom graph show* the number of anirniU found on a particular area at the top of the stream.

The great number of animal* at the top of the stream during January and February was very remarkable. On one occasion as many as a6o were found on the under surface of a single stone; this surface measured about 250 sq. cm. As already stated experiments showed that almost, all of the anirnaia collected from the top of the stream during January were positively rheotactic. But by the middle of February only about 50 per cent of them were positively rheotactic, and by the first week in March only 5 per cent were positively rheotactic. The great majority of the remainder proved to be individuals which had been starved and had de-differentiated and had then lost their positive rheotaxy, as explained in the preceding section. The rest were individuals which had completed their sexual cycle and showed the more definite negative response. 126 R. S. A. BBAUCHAMP One would naturally suppose that as soon as these animals became negatively rheotactic they would start to migrate downstream. They were prevented from doing so in the early part of this year by the shortage of water in the stream. From the last week in January till the first week in March there was scarcely any rain and in consequence by March the streams were very dry. But on March 5th and 6th there was heavy rain, and immediately afterwards it was found ttujt almost all the planarians had disappeared from the top of the stream. This is shown at the point X on the bottom graph, Fig. 5. Most of these animals migrate downstream of their own accord. But some get washed away and carried down to where the stream runs more quietly or to where it flows into the lake. Here they are deposited. An account is given by the writer (1932) of the conditions under which these individuals may establish themselves on die lake shore. In short, during December and January there was a general migration upstream on the part of the PL alpma population. During February and the beginning of

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OOT NOV. DEO. JAN. FEB. APR. Fig. 6. The length of stream occupied by PL alpata. The width of the black area* indicate* the density of the population. By April the population was smaller, since some individuals were washed downstream.

March these animals either completed their sexual cycle or else were starved, with the result that they became negatively rheotactic. But by this time the streams were so dry that they were unable to start migrating downstream until the rain came on March 5th and 6th. Fig. 6 shows the length of stream occupied by PI. alpma between October and April. The width of the black areas indicates the density of the population. Laboratory experiments (see above) showed that if individuals, which had been starved and which had de-differentiated and lost their positive rheotaxy, were fed they regained their positive rheotaxy. This return to positive rheotaxy was so decided that an experiment was attempted to show the effect of feeding in the field. The result was very striking. The experiment was done on the stream which had previously been the most closely observed. Feeding was commenced almost immediately after the sudden migration downstream on March 5th and 6th. Every 2 or 3 days for a fortnight about 150 insect larvae and Gammons were collected from other streams. These were killed and put into the stream. The first lot of food was put in at the lowest Rheotaxis in Planaria alpina 127 point where PL alpina mi to be found, at that time about 300 m. from die source. Successive lot* of food were put in, higher and higher upstream, with the idea that, in this way, the greatest use would be made of the food. The counts of the number of animals found on the particular area at the top of the stream were continued (see Fig. 7, a, the continuous line, with the points marked by triangles). At the start of the experiment this area and 5 m. of the stream below it were cleared of all PL alpina. This was done so as to eliminate errors due to the wandering of individuals in the immediate neighbourhood. On each occasion when the counts were made, all the animals found were either taken back to the laboratory for examination or else were killed. In this way it was hoped to obtain a really accurate idea of the numbers migrating upstream.

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20°"

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FEB. MAR. APR. Fig. 7. Number of Pi. alpina recorded from particular area* at the top of two stream*. a m fed during the period of time marked by a thick black line. The first sign of a migration upstream was evident towards the end of a fortnight. But, as shown by the graph (Fig. 7, a), the full effect was not shown till later. The dotted curve, b, shows numbers of PL alpina counted at the top of one of the other streams which was used as a control. A comparison of the two curves a and b shows the effect of feeding. The thick black line indicates the time when stream a was supplied with food. During the whole of this experiment no food was put into the top 50 m. of the stream. This invalidates any suggestion that food substances in solution " attracted " the animals to the top of the stream. With the exception of a few very recently hatched individuals, all the planarians found at the top were well fed. One can only suppose that they had taken the food lower down the stream and had redeveloped and, becoming positively rheotactic, were forced to migrate upstream. 128 R. S. A. BEAUCHAMP

CONCLUSIONS. Sexual development in PL alpine is associated with low temperatures, see Steinmann (1913) and Carpenter (1928). It is certain that sexual individuals are rarely found at temperatures above 10° C. In cold springs where the temperature is less than io° C. sexual individuals can usually be found at any time of the year. That is to say, there is no fundamental seasonal rhythm in the reproduction of this species. But in the winter, when the temperature of the whole stream is lowered, sexual development is made possible for the whole population. It has been shown that positive rheotaxy accompanies sexual development Consequently, if the planarian population of a stream develops sexually, a general migration upstream is bound to follow. This migration leads to dense overcrowding at the top of the stream, with the result that there is a shortage of food. Consequently, most of the animals which reach the top of the stream are eventually starved. A few are able to complete their sexual cycle and these lay cocoons and then become negatively rheotactic. Both those that have been starved and those that have completed their sexual cycle migrate downstream. The starved animals if they find food, soon re-develop and become positively rheotactic again; they then migrate upstream again and may lay cocoons or they may again be starved. Temperature and the amount of available food are clearly the two most important factors which control the sexual development of PL alpina and its migrations up and down the stream.

SUMMARY. 1. PI. alpina is normally active in the evening and quiescent during the day. 2. PI. alpina is shown to become positively rheotactic when developing sexually, but becomes negatively rheotactic on completing the sexual cycle. 3. Starvation leads to de-differentiation and the loss of positive rheotary. 4. Feeding produces a temporary positive rheotaxy and a temporary negative geotaxy. 5. Continued feeding leads, if temperature conditions are suitable, to the re- development of those characters which produce positive rheotaxy in sexual individuals. 6. The development and consequently the behaviour of PL alpina is controlled by temperature and the food supply. 7. Considerable migrations up and down the stream are brought about by changes in the animal's behaviour. Rheotaxis in Planaria alpina 129

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