notes on the choanoflagellate, codosiga botrytis, ehrbg. · notes on the choanoflagellate, codosiga...

Notes on the Choanoflagellate, Codosigabotrytis , Ehrbg.

By

Geoffrey Lapagc, M.Sc, M.B., Ch.B.,

Lecturer in Zoology, the Victoria University of Manchester.

With 10 Text-figures.

C O N T E N T S .

PAGE

1. MATERIAL AND METHODS 471

2. OUTLINE OF THE MORPHOLOGY o r C O D O S I G A B O T R Y T I S . 472

3. PREVIOUS V I E W S o r THE STRUCTURE OF THE COLLAR AND OF THE

MODE OF F E E D I N G . . . . . . . . 479

4. T H E MUCOUS ENVELOPE . . . . . . . 486

5. T H E M O D E OF F E E D I N G 491

6. T H E FLAGELLUM AND THE CURRENTS IT PRODUCES . . . 494

7. T H E CONTRACTILE VACUOLES . . . . . . 503

8. SUMMARY . . . . . . . . . . 506

9. LITERATURE 507

1. M A T E R I A L A N D M E T H O D S .

DURING the summer of 1924 some material containingnumerous Mastigophora was brought into the laboratory byMr. J. T. Wadsworth from ponds near High Lane in Derbyshire,and among these the Choanoflagellate genera Codosiga andSalpingoeca were identified. My attention being directed byProfessor S. J. Hickson to the conflicting statements thathave been made about the structure and function of thecollar in the Choanonagellata, I adopted his suggestion thatI should attempt to clear up this question. The speciesCodos iga b o t r y t i s was selected for the purpose, a furtherabundant supply of it having been obtained from a pond nearStyal, Cheshire, where it was living upon the stalks of Lemna.

472 GEOFFREY LAPAGE

Little can be seen of the structure of this organism unlessthe illumination is very carefully adjusted. I have usedthroughout the work a flask filled with a solution of copperacetate and interposed between the electric light and thecondenser of the microscope. This gives a light-green back-ground which does not tire the eye so quickly as the nakedlight, and it gives also much better definition, especially ifall the blinds are drawn and a dark cloth is put over the headto shade the eye,s.

For the observations upon the flagellum and its currentsI have used carmine particles suspended in the pond-waterin which the animals were living. Carmine is "also useful fordetermining the extent and shape of the collar, since theparticles usually adhere quickly to the collar and collectespecially at its upper margin, helping to define this.

For the observations upon the ingestion of food, however,1 did not use carmine. It is quite easy to observe the animalfeeding upon its normal diet, which consists largely of cocciin the pond-water, and these are large enough to be followedwithout difficulty. I thought that the results obtained in thisway would be more reliable than any that would be obtainedwith such an unusual diet as carmine. I found, in fact, thatCodosiga does not ingest carmine particles at all readily.

I have used throughout the work a Zeiss apochromatic2 mm. oil immersion lens, with various eye-pieces, numbers2 and 4 being most useful for general observation. The com-pensating 6, 12, and 18 are useful for confirming and extendingthe observations made with the lower powers. I have alsofound the Zeiss stereoscopic binocular eye-piece of very greatservice, especially in observations upon the mucous envelopein which the three-dimensional view of the whole organismwhich it gives is invaluable.

2. OUTLINE OF THE MORPHOLOGY OF C O D O S I G A

B O T R Y T I S .

Codosiga b o t r y t i s is a minute organism, possessinga protoplasmic body that is normally more or less oval in

NOTES ON CODOSIGA

TEXT-FIG. 1.

473

Fl

Diagram of Codosiga b o t r y t i s , Elirbg. A portion only ofthe flagellum (Fl) is shown. C, collar; Nu, nucleus; ME,mucous envelope ; 0 V, the three small vacuoles which will laterfuse to form the left contractile vacuole ; CV, the right con-tractile vacuole shown in diastole; A, the large non-contractilevacuole of unknown significance at the base of the body; S,stalk.

NO. 275 ii

474 GEOFFREY LAPAGB

outline, with a truncated anterior end (Text-fig. 1). The bodymay, however, assume a more spherical outline and globularshape, or it may, especially in young individuals, be drawn outinto a more elongate oval (Text-fig. 3). The body is filled,usually, with numerous vacuoles, among which are two con-tractile vacuoles, situated at different levels in the posteriorhalf of the body and upon opposite sides of it. These contractilevacuoles pulsate alternately, the interval between successivesystoles of either vacuole being normally about thirty secondsor rather longer.

The body is enclosed in a delicate, transparent case, describedby earlier authors as a mucous envelope (Text-fig. 1, ME),which is continued below into a rigid stalk (S), by which theanimal is attached to the substratum upon which it lives.This substratum may be the stalk of a water-plant or thestalk of a Vorticella or the surface of a Eotifer or other aquaticanimal. Probably the organism attaches itself to any surfacethat is firm enough to afford it the light foothold that it needs,provided that sufficient food is to be found in the vicinity.

The body bears upon its free end the structure that has beencalled the collar (Text-fig. 1, C). This structure is, in thisspecies, a clear, transparent, protoplasmic membrane whichhas the shape, when it is fully expanded, of a hollow, truncatedcone with sides that are slightly concave outwards. Thenarrower, truncated end of the cone is based upon the freeend of the animal's body, and the wider end is expanded freelyin the water.

While the collar normally takes this form, its shape may,under certain conditions, be altered considerably and quickly.Its upper margin may be contracted until the free end of thecone is narrower than its base (Text-fig. 6), and its height mayalso be reduced. The collar may, in fact, be retracted altogetherinto the body and be again extruded, the process having ledsome authors to compare it to a hollow pseudopodium (SavilleKent, 10). It seems to be correct, therefore, to regard thecollar as a protoplasmic extension of the body, which can beretracted or extended by the organism according to its needs.

NOTES ON CODOSIGA

TEXT-FIGS. 2 AND 3.

475

Fl

ME-

Fig. 2.—Diagram showing the mucous envelope (ME), whose outlineis emphasized. The collar (C) is shown in optical section. Fl, flagel-lum ; CV and CV', the two contractile vacuoles ; A, the non-contractile vaeuole at the base of the body; S, stalk; Nu, nucleus.

A -CV'

Fig. 3.—One of the more slender, younger individuals. The outlineof the mucous envelope (ME) is heavily lined in. Fl, flagellum ;C, collar seen in optical section ; A, non-contractile vaeuole at thebase of the body; C V, the right contractile vaeuole; Nu, nucleus.

476 GEOFFREY LAPAGB

In optical section the collar presents the appearance of twofine, slightly diverging lines which arise from the sides of thefree end of the body, and look, at first sight, like accessoryflagella or setae (Text-figs. 2 and 3, C). It is exceedingly diffi-cult to see the collar itself, because it is so delicate and trans-parent ; and it cannot be seen at all without very carefulmanipulation of the illumination. It is very easy, therefore,to make mistakes about its extent and shape.

The flagellum (Text-fig. 1, Fl), which is very long and moreeasily seen than the collar, arises from the free end of thebody in the middle of the area enclosed by the base of the collar.It extends out into the water for a distance equal to at leasttwo or three times the height of the collar. The length of theflagellum is one of the most remarkable features of the organism.

The organism is small. The smallest individuals that I haveseen measured 7-07/x long by 5-05 ix broad at their widest part.Individuals measuring 16-16/^ long by 9-09/x broad were amongthe largest seen. The majority measured 101 p long by 8-08/xbroad. The collar is usually about the same height as thelength of the body ; but, as I have stated above, its heightmay vary. Its width at its upper end also varies, but isnormally wider than the width of the body. At its base, onthe other hand, the collar is not so wide as the greatest widthof the body, since it arises from the tapering anterior end.In one individual whose greatest breadth was 8-08/x the collarmeasured at its base 606ft. In an individual 12-12p. longby 8-08 n broad, with a collar 1212/x high, the flagellum wasbetween 40-4ju, and 50-5/x long. In another individual 101 fj.long by 7-07/x broad the flagellum appeared to reach 30-3or 40ju.. Another had a body 12-12/x long hy 8-08/A broad anda flagellum about 40-4/x long. But it is very difficult to measurethe flagellum accurately because it is hard to locate the extremetip, especially in the living organism, in which the tip, at anyrate, is generally in motion. In carmine preparations thedisturbance of the particles in the water helps one to gauge itslength.

The stalk varies a good deal in length according to the age

NOTES ON CODOSIGA 477

of the individual. It may be quite short in solitary formswhich have recently settled down, while in older colonies itmay reach as much as four or five times the length of the body.In solitary individuals the stalk is usually of the same diameterthroughout, expanding only at its base, where it is attachedover an area about twice as broad as its diameter. I have notbeen able to find anything like a basal disc of attachment.The stalk simply broadens out over the substratum, its basebeing more like that of a tree trunk than like the foot of a wine-glass (Text-figs. 1 and 2).

Distally the stalk appears to be directly continuous withthe mucous envelope of the body. Fisch (7) states that he hasseen the body plasma pass into the stalk, which is, he thinks,hollow, as Biitschli (1) also suggests. I have never seen any-thing like this. The stalk certainly looks like a tube, but Ihave not been able to prove that it is one. In some individualsit appears to have been twisted upon itself round its long axis(Text-fig. 1,8). In solitary forms the stalk passes graduallywithout any marked change of its diameter into the mucousenvelope. But in older individuals that have divided and inwhich, therefore, if the statements of earlier workers are correct,the distal portion of the stalk has divided with them, thereis an abrupt difference in diameter, just as Biitschli (1) hasfigured (Text-fig. 4), between the stout main stalk of the colonyand the much more slender stalks of the separate individuals.

According to earlier workers the organism multiplies bybinary fission, the nucleus, body, and collar all being splitinto two after retraction of the flagellum. The daughterindividuals then put out new flagella, and the distal portion ofthe stalk thereupon divides also. Repetition of binary fissionmay give rise to colonies of more than two individuals. Clark (4)says that he has seen colonies of as many as eight individuals,and Ehrenberg, quoted by Biitschli (1), reports as many as ten.Usually, however, the number of individuals in any particularcolony does not exceed six. New colonies are begun by thedetachment of single individuals, which swim away baseforemost, the flagellum acting as a pulsellum and the collar

478 GEOFFREY LAPAGB

being directed backwards. The free individuals settle downelsewhere, secreting a new stalk and living an independentlife.

This brief outline of the structure of Codosiga is based partlyupon my own observation and partly upon the accounts givenby earlier workers. But I have intentionally avoided anydetailed description either of the mucous envelope in whichthe body is enclosed or of the collar, because it is chiefly intheir statements about these that the earlier workers disagree.Previous workers may, in fact, be divided into two groupsaccording to their views about the structure of the collar.

On the one hand Clark, Stein, Saville Kent, Biitschli, Fisch,and the more recent worker Burck, take the view of the collarwhich is usually expressed in the text-books. They regard itas a closed membrane, conical in shape, or pitcher-like ortrumpet-shaped when it is fully expanded, its upper rim,when it is viewed from above, showing as a closed ring.

The other group adopts the view that was apparently firstput forward by Entz (6) and supported by Franze (9), Ehiiich(5), and Schouteden (11). These workers regard the collaras being the upper vertically expanded portion of a spirallycoiled membrane which begins on the surface of the body atone of the two vacuoles which were described by earlier workersas contractile vacuoles. Franze, at any rate—and in this heapparently agrees with Entz—thinks that this particularvacuole from which he believes that the alleged spiral membranestarts, is not a contractile vacuole at all. He calls it a ' Schling-vacuole ', or gullet-vacuole, and he thinks that all food particlesare ingested by it. He believes, further, that what have beendescribed as pulsations of this vacuole are in reality swallowingmovements of it (' Schluckbewegungen '). According to him,the spiral membrane of which he believes the collar to becomposed, starts at this alleged ' Schlingvacuole ' upon itsspiral course around the free end of the body and, when itreaches the upper surface of the body, expands above that toform the collar. Franze, and those who agree with him, donot therefore regard the collar as a close membrane. According

NOTES ON OODOSIGA 479

to them the rim of it, seen from above, would not appear asa closed ring, but would show a gap at one point where the edgeof the spirally coiled membrane was free ; and in a lateral viewthere would be a corresponding slit all down one side of thecollar. It would be exceedingly difficult to see such a gap orslit in a structure which is, even as a whole, hard enough todiscern. But I may as well say at once that, although I was atone stage of my observations much inclined to agree with thisview of the structure of the collar, I was subsequently compelledto abandon it. It is impossible, however, to explain the reasonsfor that conclusion until the behaviour of the food particles,when they are being ingested, has been described. And beforeI give my own observations it will be advantageous to sum-marize briefly those of previous workers.

8. PREVIOUS VIEWS OF THE STRUCTURE OF THE COLLAR

AND OF THE MODE OF FEEDING.

Codos iga b o t r y t i s was first adequately described byJames Clark (4), and he correctly interpreted the collar as a' highly flexible and retractile ' closed membrane ; but hisotherwise excellent account of it is marred by a misconceptionof the mode of feeding. He says that the flagellum ' is usuallyrigid, except at the tip, which is constantly occupied in throwingparticles of various kinds toward the mouth by vigorousspasmodic movements or jerks '. It is true that the flagellumdoes execute such movements (cf. below) ; but it is extremelydoubtful whether they are of any use in providing the animalwith food. Certainly Clark failed altogether to understandthe normal mode of feeding. He confesses, in fact, that hedid not observe the actual ingestion of food, in the followingpassage : ' That the mouth . . . is situated near the base ofthe flagellum . . . is rendered certain by the fact that particlesof food are thrown by that organ directly against the area uponwhich it is based and are taken within the body somewherein that region ; but, on account of the minute size of themorsels, and the rapidity with which they are swallowed, ithas not been possible to determine precisely at what point.'

480 GEOFFREY LAPAGE

About the precise function of the collar itself Clark does notseem to have formed any opinion.

Saville Kent (10), on the other hand, elaborated an ingenioushypothesis about the function of the collar. Studying theanimal by means of carmine particles suspended in water,he came to the conclusion that the collar is a closed ' infundi-buliform film of sarcode that may be protruded from or with-drawn at will into the general substance of the monad's body',like a pseudopodium. With so much we may readily agree. Buthe says further that a circulation of the substance of the collar' is constantly being maintained, flowing upwards on the outside,over the distal edge of the rim, and downwards on the innersurface, at the base of which it again comes in contact andmerges with the protoplasmic substance of the body '. SavilleKent further states that the flagellum creates a strong centri-fugal current bringing particles which strike against the collarand, adhering to it, are carried along ' with the circulating currentof the collar's substance, up the outside and down the insideuntil, on reaching the base of its inner surface, they areengulfed within the sarcode substance. . . . The indigestibleresidua are eventually liberated from the area, limited by thebase of the collar, within which they primarily gained access.'

Saville Kent gives, as a frontispiece to his ' Manual of theInfusoria ', an excellent figure of this remarkable conceptionof the mode of feeding. But, since he, like Clark, supposesthat the food particles are ingested inside the collar, his repre-sentation of the course taken by the food particles is incorrectand his figure must be abandoned. Saville Kent is, however,correct in stating that defoecation occurs from the area enclosedby the collar. Clark before him; and Butschli and others afterhim, have noted the same fact.

Butschli (1) came much nearer to the truth about the modeof feeding when he recognized that the food particles are notingested by the area enclosed by the collar. He saw that,although they adhere to the outer surface of the collar asSaville Kent described, yet they pass down, and not up, itsouter surface to be ingested outside the collar, near to its base,


by what Biitschli called a vacuole which he described as wander-ing round the free end of the body in this region (Text-fig. 4, X).This so-called ' vacuole ' is easily seen. It appears, just asBiitschli says, alternately at opposite sides of the body justbeneath the base of the collar. It is also easy to confirmBlitschli's observation that the food particles pass into it.The only error in Biitschli's account is, in fact, his interpreta-

TEXT-FIG. 4.

Tracing from Biitsehli (1, PI. xi, a portion of fig. 1, a) toshow the outline of his so-called travelling vacuole (X) in opticalsection. MS, main stalk of the colony ; JS, S, the stalks of theseparate individuals.

tion of the structure as a ' vacuole '. Entz, Franze, andEhrlich evidently doubted this interpretation, and were ledby their own observations to formulate a different viewof it.

Briefly stated, the view taken by Entz, Franze, and Ehrlichis that the structure which Biitschli described as a ' vacuole 'is not a vacuole at all, but the profile aspect of a portion of thespiral membrane of which they believe the collar to be com-

482 GEOFFREY LAPAGE

posed. They think, as Ehrlich (5) says, that this membrane,either as a result of' an active wave movement' passing alongit, or as the result of the passage along it of foreign bodieswhich set it into passive movement, successively applies itselfto the body and then stands away from that; and that themembrane is seen in profile aspect at the side of the body at themoments when it stands away from the body. Since its courseis supposed to be a spiral one, it is seen thus alternately atopposite sides of the body at slightly different levels. Ehrlich

TEXT-FIG. 5.

Tracing from Ehrlich (5, Text-figs. 4 and 4 a) to illustrate theEntz-Franze view of the collar as a spiral membrane. 4 a, theorganism in optical section.

especially gives clear figures in support of this view (Text-fig. 5).But, ingenious and attractive as the hypothesis is, and muchinclined as I was at one stage of my observations to supportit, I have been compelled to abandon it, because no fact couldbe found in favour of it.

If the membrane were in reality there, it should be possibleto see it as a fine line as it passes over the body from theso-called ' gullet vacuole ' towards the base of the collar. Itis certainly possible to see a fine line taking a course like this(Text-figs. 1, 2, 3, 7, &c), and I was for some time misled by

NOTES ON C0D0SI6A 483

this. But, as we shall see later, this line is not the border ofa spiral membrane, but the margin of the mucous envelopein which the body is enclosed, a structure whose essentialimportance in the feeding process neither Entz, Pranze, norEhrlich has realized.

Secondly, if Biitschli's so-called ' vacuole ' were, as this

TEXT-FIG 6.

Tracing from Burok (3, PL xii, figs. 3 a, 3 6, 3 c) showing his view ofthe mucous envelope (ME) and of the ingestion of food par-ticles {F).

hypothesis suggests, merely a portion of a spiral membraneseen in optical section as it passes round the base of the collar,then it would be visible alternately on b o t h sides only incertain aspects of the animal. There would be aspects in whichit would be visible on one side only. But careful observation

484 GEOFFREY LAPAGE

shows that it can be seen always on both sides in every indivi-dual, from whatsoever aspect the organism is viewed.

Thirdly, there is no doubt, as my own observations upon themode of feeding given below and those also of Burck (3) show,that food is not taken in always at the same point as theEntz-Franze hypothesis requires, namely at the alleged' Schlingvacuole ' ; and that the structure that Franze callsthe ' Schlingvacuole ' does not actually exist. Whether he hasmistaken one of the contractile vacuoles for it or has regardedone of the numerous vacuoles with which the body is usuallyfilled as a ' Schlingvacuole ', I cannot say.

Fourthly, Franze evidently thinks, if I rightly understandhis view from Burck's quotation of it (3) and from his ownshorter paper (8), that the course of the food particles down thecollar is a spiral one. He seems to think that the food particlespass down in the gutter between the spiral membrane, of whichhe thinks the collar is composed, and the body, and thus reachthe ' Schlingvacuole '. Observation shows, however, that theydo not take a spiral course at all. They always pass verticallydown the collar, as Burck, Butschli, and others correctlystate (Text-figs. 6, 8) ; and they are certainly not alwaysingested at the same point. They may be taken in at oppositesides of the body of the same individual or at different levelson the same or opposite sides.

Fifthly, the collar, when it is viewed from above, alwaysappears as a closed ring. I have carefully studied manyindividuals from various angles and with every kind of illumina-tion and, although I am aware that it is difficult to be certainthat, in a structure so delicate and hard to see, there may notbe a minute gap in the rim of the collar such as the Entz-Franze hypothesis requires, yet I am convinced that thereis no such gap.

Lastly, there is the consideration that it is difficult to under-stand how or why an organism relatively so primitive in otherrespects should have developed a structure so complex as thespiral membrane which the hypothesis postulates. The otherview that the collar is a protrusion of the protoplasm of the


body in the form of a hollow cone is much more readilyconceivable ; and it is a simpler hypothesis.

I agree, therefore, with Burck (3) that the view of the struc-ture of the collar put forward by Entz, Franze, and Ehrlichmust be definitely abandoned. Burck has adduced argumentssimilar to some of those given above ; but I did not unfor-tunately see his paper until I had arrived independently atthe same conclusion as his in this respect.

If Ave return now to the summary of the views of previousauthors about the structure of the collar and the mode offeeding, we find that Fisch (7) came still nearer to the truththan did Biitschli, because he gave more attention to themucous envelope with which the body is clothed. Afterdiscussing Saville Kent's view of the mode of feeding he agreeswith Blitschli that the food is taken in at the base of the collarand outside it, by ' vacuole-like projections ' with which thefood particles come into contact. He does not agree, however,that these vacuoles wander round the free end of the body,as Biitschli thought; and he insists that the food particlescoming down the collar do not come into contact with these' vacuole-like projections ' at the base of the collar, but thatthe ' vacuoles ' appear, not directly at the base of the collar3

but ' immer ziemlich weit unterhalb desselben ' (cf. Text-fig. 5,copied from Ehrlich, who makes a similar statement and adducesit in support of his hypothesis, and p. 493 below, where thisappearance is explained). Fisch further states that he couldnot make out any movement of the particles adhering to thecollar.

It was not, however, until Burck (3) published his paper thatthe part played by the mucous envelope was given its trueimportance. I have copied, in Text-fig. 6, such details of Burck'sfigures as are relevant to this question. They show that hecorrectly interpreted the whole process. Burck has realized,as Fisch nearly did, that the structure which Biitschli thoughtwas a vacuole travelling rhythmically round the upper end ofthe body is in reality not a vacuole at all, nor is it the opticalsection of a spiral membrane. It is simply the upper portion

486 GEOFFREY LAPAGE

of the mucous envelope that clothes the body. The uppermargin of this mucous envelope, as Burck says, ' periodischvon dem Kragenbasis abhebt . . .', and is seen only in profileview at the sides of the body and only when it stands awayfrom the surface of the body there. When a food particlereaches the base of the collar it passes into the spacebetween the body of the animal and the outstanding mucousenvelope, and when the envelope again lies close to the bodysurface the food particle appears to be enclosed in a vacuole.This vacuole then moves basalwards and is eventually taken intothe substance of the body.

With this view I agree ; but I must differ from Burck's viewof the mechanism by means of Avhich the process is effectedand also with regard to other points of minor importance.

4. THE MUCOUS ENVELOPE.

It is not easy to convince oneself that Burck is right in inter-preting Biitschli's ' vacuole' as the upper margin of themucous envelope seen in profile at the sides of the body atthose moments when it stands away from the surface of thebody there. It is perhaps easiest to make sure of this bystudying younger individuals which are more elongate-ovalin outline, since the body of these individuals does not com-pletely fill the mucous envelope and the envelope is thereforemore easily seen (Text-fig. 3). It will be seen from this figure,and from most of my other figures, that the upper margin ofthe mucous envelope is in reality much lower than Burckshows it (cf. Text-fig. 6, copied from Burck's figures). Inevery individual that I have studied I have always seen itlower than the apex of the body and never high up on thecollar as Burck shows it, although I have patiently looked forit there. It is true that most of the individuals that I chosefor study were viewed obliquely from above rather than inlateral view, and that one would expect the upper border ofthe mucous envelope to appear, in such a view, lower downon the body than it would appear in a lateral view such asBurck figures. But I have not seen it so high up as he shows


it, even in lateral view. As a matter of fact Burck confessesthat he has not actually seen the upper border of the envelope.He says (3) that ' Am lebenden Individuen lasst sich der freieBand der Schleimhiille nicht erkennen, ebensowenig wie derfreie Kragenrand. Deshalb scheint es hier, als wenn sich einefreie Lamelle an der Kragenbasis erhebe, die urspriinglich kurzist und sich spiiter nach hinten gegen die Basis des Tiers ver-langert '. He thinks, in other words, that, since the upperborder of the mucous envelope is so delicate that it cannotbe seen, the side wall of it appears like a free lamella standingout from the side of the body at the base of the collar, exactly,in fact, as Ehrlich shows it (Text-fig. 5). I agree that this isthe appearance that one usually sees, and that it is very difficultindeed to see the upper border of the envelope. But I have beenable to distinguish it with the help of the Zeiss stereoscopicbinocular eye-piece and after several days of prolonged observa-tion with careful manipulation of the illumination.

It was, however, especially difficult to follow it over the sur-face of the body. In individuals full of vacuoles and food Icould not do this. At one time I thought that what I saw wasnot the upper margin of the envelope, but either a secondcollar or the margin of a case more like that which encloses thebody of Salpingoeca for example. But both these conjecturesproved to be wrong. I am convinced that the upper margin ofthe mucous envelope is as I have shown it in my text-figures.

It is to be looked for, especially in individuals seen obliquelyfrom above rather than in profile view, not over the base of thecollar, but on the body, below the base of the collar. In someindividuals, indeed, which are seen more obliquely still, itpasses over the body quite low down and then shows itselfas a fine line in this situation. I have no doubt that it is thisline that Entz and Franze have seen, and that they have inter-preted it as the border of a spiral membrane starting from one ofthe vacuoles in the body. When the animal is seen in sucha position that the margin of the envelope crosses a vacuolein the interior of the body, it would be easy to make sucha mistake.

GEOFFREY LAPAGE

When the mucous envelope is seen in profile view as it standsaway from the body at the sides, it appears exactly as Butschlifigured it (Text-fig. 4), like a vacuole that appears at oppositesides of the body alternately. The average time betweenthe moment when the ' vacuole ' is largest at one side and themoment when it is largest at the other is 1-5 minutes. Thisinterval is fairly constant, but variations occur. The intervalmay be shortened to one minute or it may be lengthened to

TEXT-FIG. 7.

a. 2-50p.m

^2-54 g. 2-55 h.2-55k j.2-56 k. 2-57^p.Timed camera lucida sketches, drawn with ocular 4, T. L. 146, Zeiss

2 mm. apochromatic oil immersion, to show the passage of therhythmical wave of contraction round the free end of the body andthe consequent illusion that the mucous envelope stands awayfrom the body.

as long as five minutes on rare occasions. Usually one findsthat these delays are associated with the ingestion of a foodparticle in the manner to be described below.

I gave particular attention to an attempt to find out thereason why the upper portion of the mucous envelope shouldstand away from the surface of the body as it does. Threepossible explanations were considered :

1. A wave of contraction might be passing round the upperpart of the mucous envelope itself, so that a bulge was beingproduced in the even contour of its upper margin, which, when


it was seen in profile at the side of the body, appeared as the' vacuole '. This is, I understand, the view adopted by Burck.I cannot, however, agree with it.

2. The whole body of the animal might be slowly rotatingaround its base at the bottom of the mucous cup in which itlies, the rotation being caused, perhaps, by the rotatorymovements of the flagellum, very much as an egg might becaused to rotate inside an egg-cup that was a little too bigfor it. If this were happening the rim of the envelope wouldbe invisible wherever the body of the organism was at anyparticular moment in contact with the envelope ; but elsewherethere would be, at the same moment, a sjiace between thesurface of the body and the envelope which would, in profileview, appear like a ' vacuole '. Also the margin of the envelopewould appear to move up the body when the body was movingtowards the observer, and down it when the body was movingaway from the observer. The margin of the envelope would,however, remain in a fixed position, while the outline of thebody would move alternately away from and towards it.

I endeavoured by means of a scale in the eye-piece adjustedsuccessively over the flagellum, the margin of the collar, theoutline of the body-surface, and the margin of the envelope,to find out whether any of these movements were occurring.The collar, flagellum, and mucous envelope remained in thesame position. Movement was occurring at the body-surfaceonly. I have to conclude, therefore, that the body was notrotating as a whole inside the mucous envelope.

3. The movement noted at the body-surface led me to thethird and, I believe, the correct explanation. It suggestedthat, although it was not a ' vacuole ' that was passing rhythmi-cally round the free end of the body as Biitschli thought, nora wave of contraction in the envelope, it might well be arhythmical contraction of the body that was doing so. Ireflected, further, that such a rhythmical wave of contractionwould be more likely to occur in the protoplasmic body thanin the mucous envelope, and that its occurrence was well inaccord Avith the various records of the occurrence of amoeboid

NO. 275 K k

490 GEOFFREY LAPAGE

movements in the body that have been made by Saville Kent(10) and others. Such a rhythmical wave of contraction maybe compared not inaptly with the ' euglenoid ' movementsthat occur in other Hastigophora. Observations upon the modeof feeding supported this view also. Finally, I noticed the fact,illustrated in all my figures, that, at the moment when the' vacuole ' is widest at the side of the body, there is also onthe same side a distinct groove or indentation in the outline ofthe body. On the opposite side, on the contrary, the outlineof the body shows at that moment no such groove ; but itappears there when the ' vacuole ' in due course arrives atthat side. This groove is suggested in one of Burck's figures(cf. Text-fig. 6 and Burck's PI. xii, fig. 3 a), and also in one ofPisch's figures (PI. xiii, fig. 73) ; but neither of them commentsupon it.

When the ' vacuole ' disappears it is obliterated, not bya contraction of the mucous envelope towards the surface ofthe body but by a distinct bulging out of the body-surfacetowards the envelope. A distinct and quite sudden surge ofthe protoplasm towards the envelope undoubtedly occurs.

The so-called ' vacuole ' results therefore, not because themucous envelope stands away from the body as Burck suggests,but because the body contracts away from the envelope. Thewave of contraction that passes slowly and with a fairlyregular rhythm round the free end of the body, just beneaththe base of the collar, draws the body away from the mucousenvelope as it passes round, producing thus a space betweenthe envelope and the body. It is this space which, when it isseen at what is, in any particular profile aspect of the organism,one side, looks like a vacuole at that point. And, as we shallsee presently, it is into this space that the food particles dropwhen they have reached the base of the collar.

When the contraction passes on the space disappears with it,because it is filled up by the bulge of the body-surface whichfollows the contraction, just as another bulge precedes it.The clear space cannot be seen again until the wave of contrac-tion has reached the opposite side of that particular profile


view. It will be further obvious that this hypothesis explainsin a simple way why it is that the ' vacuole ' is always seenalternately at opposite sides of the body, and only at the sides,whatever the position may be from which the animal is viewed.

5. THE MODE OF FEEDING.

The explanation of the alleged ' vacuole ' given above issupported by the behaviour of the body when food particlesare being ingested.

When a food particle has passed down the outside of thecollar, and has arrived at its base, it lies just above the upperrim of the mucous envelope. Here, if the ' vacuole ' is notbelow it at that moment, it can be seen to remain (Text-fig. 8, A). It is as if the particle awaited the appearance ofthe ' vacuole ' below it.

Then when the ' vacuole ' does appear below it the particlepasses down into it. That is to say, it passes down into thespace that is created between the body and the mucousenvelope by the rhythmical contraction of the upper end of thebody. At this stage the particle appears, as Burck says, tobe enclosed by the ' vacuole ' (Text-fig. 8, C, D, J, K). Thereis, as a matter of fact, a stage preceding this at which theparticle can be seen to be not inside the mucous envelopebut just level with its rim (Text-fig. 8, B and H). A little laterstill, as the contraction passes on and the ' vacuole ' begins tobe obliterated again by the bulge that follows it, the body-wallinterposes itself between the particle and the upper margin ofthe envelope. It moves across to meet the envelope so thatthe particle is completely shut in (Text-fig. 8, E and L). Oneof Burck's figures (3, PI. xii, fig. 3&, of which my fig. 6 showsa tracing) illustrates this stage admirably, showing the inter-position of the body between the particle and the upper rimof the envelope. The particle is therefore now enclosed inwhat is not even yet a true vacuole, but only a space betweenthe body and the envelope.

As the bulge that follows the contraction advances and stillfurther fills up the ' vacuole ', the particle is thrust further

492 GEOFFREY LAPAGB

down into the mucous cup. Its presence there together witha certain amount of water taken in with it, between therelatively more rigid mucous envelope and the softer body-wall, causes an indentation in the latter (Text-fig. 8, E and L).This indentation becomes very evident as the particle descendslower and lower. The clear space in which the particle lies

TEXT-FIG. 8.

6.5-7|p.m H.5-3 J. 5-95 K. 5-loi £.5-1»z /M. 5/2p.m.

Timed freehand sketches to illustrate the ingestion of food particlesby two different individuals.

may, indeed, extend in some cases almost to the base of theanimal (Text-fig. 8, L) ; but more usually it does not extendso far. Possibly its extent is determined by the amount ofwater taken in with the particle.

In any case, whatever its extent, its presence there may giverise to the appearance of a free lamella there, standing outfrom the side of the body, since usually only the margin of the


space, formed by the mucous envelope, is seen. It is probablysuch appearances as these that were interpreted by Ehrlichas the border of his spiral membrane (cf. Text-fig. 5), and whichled Pisch also to state that the ' vacuole ' appears always somedistance below the base of the collar.

Finally, by what appears to be still an amoeboid movementof the body rather than a contraction of the mucous envelope,the particle is pressed into the body with sudden force. It isas if the body were, throughout the whole process, constantlyendeavouring to press itself close to the envelope again. Itcan only do this by forcing the particle, together with thewater taken in with it, into its own interior. The explosiveforcing of the food particle into the body is quite a remarkableoccurrence. The particle creates, as it enters, a noticeabledisturbance in the protoplasm, as Burck also noted.

The final stage is the complete obliteration of all traces of thespace between the mucous envelope and the body-wall. Theparticle now lies in a true food vacuole in the interior of thebody (Text-fig. S, F and M), and it should be noticed that itis not until it has reached this stage that the particle is enclosedin a true vacuole at all. By this time the wave of contractionround the free end of the body has usually reached the otherside of the animal. I should add that I have once or twice seen,when a particle was being ingested in this way, a distinctbulging out of the normally even contour of the mucous envelopearound the ' vacuole ' with its contained particle, very muchas Burck shows it (Text-fig. 6). At first I thought that thiswas an indication that the mucous envelope was indeed ableto expand and contract, and that Burck's explanation mightbe correct after all. But the occurrence was not at all common,and I came to the conclusion that what I had seen was merelya local and passive bulging of the envelope, probably due tothe ingestion, with the particle, of a greater amount of waterthan usual.

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6. THE FLAGELLUM AND THE CURRENTS IT PRODUCES.

The flagellum is a very striking structure, readily seen withproper illumination. It is very long. It projects beyondthe collar for at least a distance equal to two or three times theheight of that structure, that is to say, it is at least three orfour times as long as the height of the collar (cf. measurementsgiven above, p. 476). We shall see below that a study of thecurrents produced by the flagellum suggests that there maybe a definite correlation between the extraordinary length ofthe flagellum and the height of the collar.

M o v e m e n t s of t h e Flagellum.—When the organismis in a normal healthy state and is actively feeding the flagelluniis not so readily seen, because it is constantly rotating aroundits own long axis. When this action is most vigorous it isextremely difficult to see the flagellum at all, and its actionthen sets the whole organism in rapid vibration upon its stalkAt other times, however, only the distal portion of the flagellumrotates, the proximal portion being held stiff and straight.This portion is therefore readily seen within the collar and fora short distance above it, and the organism itself does notvibrate.

Sometimes, on the other hand, the flagellum is quite motion-less, when it usually lies in the sigmoid curve well illustratedby Clark (4). Or it may execute lazy, sinuous movementswhich pass along it from base to tip. These sinuous movementsmay increase in vigour until they become lashing movementsof greater or less power. On other occasions still the flagellummay execute spasmodic jerking movements, bending about itsmiddle and recoiling again like a steel spring. It is probablymovements such as this which Clark describes in the passagequoted above, and which led him to think that the flagellumwas, by means of them, throwing food into the interior of thecollar. I think it probable that these latter jerking move-ments are abnormal, and may have been the expression of thecondition produced in the organism by evaporation from thepreparation in which it was being examined. The slow, sinuous


movements increasing to lashing are more characteristic ofthe resting phases, when the organism is not actively feeding.The rotatory movement is characteristic of the active feedingphase, and also, one may add, of the free-swimming, detachedorganism.

The C u r r e n t s p roduced by the Flagellum.—Ifthe organism is studied in its active feeding phase in preparationsin which fine particles of carmine have been suspended thecurrents produced by the flagellum are well seen. Suchpreparations show that the action of the flagellum, when itrotates as a whole, is to create, above the collar, a column ofupward suction like a spout in the centre of which the flagellumitself moves. As Saville Kent says (10), it causes the waterto sweep in from the sides of the animal and below it on allsides, and to sweep away from the organism along the axis ofthe flagellum itself. Text-fig. 9 is designed to illustrate this.It represents an optical section of the more important currents.

Particles may be brought to the region of either the collar orbody of the animal by any of the currents A, B, C, D, E, F, G.Some of these currents are, however, more rapid than others ;and observation of the carmine particles shows that thecurrents may be divided into two groups.

1. P r i m a r y C u r r e n t s such as those labelled A, B, C aremuch more rapid. These are directed towards the upper thirdor so of the collar (e. g. C), or towards the area immediatelyabove its upper margin (e.g. A and B). Since these primarycurrents are so rapid particles caught in them can rarely ornever be taken in as food. The great majority of such particles,in fact, after approaching the collar relatively slowly, are sweptsuddenly and with great rapidity into the central centrifugalstream above the collar. Passing up this they emerge abovein any of the upper currents labelled A'-G'. Those whichemerge along A' may, if the flagellum be active enough andthe current therefore sufficiently strong, be swept round andre-enter the current A below. Or they may enter the secondaryeddies, such as H and J, and so repeat their journey up thecentral spout. Those which emerge along the upper currents

496 GEOFFREY LAPAGE

such as D', E', F', and G', for example, are generally thrownoff altogether, the force of the current being generally insuffi-cient to bring them round again to a position below the rim ofthe collar. Particles taking a course internal to G and G'pass, of course, practically parallel to the long axis of theflagellum and tend to be thrown off vertically upwards, oftenbeing whisked away by coming into contact with the upperend of the flagellum.

It often happens, of course, that particles caught in theprimary rapid currents impinge upon the upper third or so ofthe collar. They would do so, for example, if they werecaught in the current 0. But the pace of these primary currentsis so rapid that, although particles caught in them may actuallyadhere to the collar for a short time, they are quickly swept upto its rim and are there cast off into the main central spout.This explains what I could not understand until I had studiedthe currents, namely, why some particles pass rapidly up thecollar and are immediately thrown off, while others pass downit more slowly. Those that pass up the collar rapidly are alwaysthose that strike the collar at its upper third or so, and therapidity of their passage is entirely due to the rapidity of thecurrent that brings them, the adhesiveness of the collar notbeing sufficient to retain them against the force of this current.

The main function, therefore, of the rapid primary currents,such as those labelled A, B, and C, is not to feed the animaldirectly. Their function is threefold.

First, they induce the secondary, slower currents D, E, F,G, &c, which are the true feeding currents.

Secondly, they keep the rim of the collar clean. They prevent,for example, the permanent clogging of the collar by suchaccumulations of matter as may temporarily cover it whenthe water is filled with carmine particles. I have also seen therim of the collar clogged for a while with accumulations ofbacteria and other debris (as Biitschli also describes, 1) ; butthese accumulations were always finally swept away by theaction of the primary rapid currents concentrated upon thisvery area.


Thirdly, the primary currents maintain a rapid centrifugalcurrent above the collar, which sweeps away all particles thrownoff by that structure, including, of course, the defoecated

TEXT-FIG. 9.

Diagram of the optical section of the system of currents created bythe rotatory movement of the flagellum.

matter which is thrown out of the body inside the collar atthe base of the nagelluni. It is worth while to mention herein passing that I have often seen defoecation occur, and canfully confirm the statements of Burck, Btitschli and others,

498 GEOFFREY LAPAGE

who insist that defoecation always occurs within the collarfrom the area enclosed by its base. Fisch is certainly wrongwhen he asserts that it may occur from any point on thesurface of the body. Defoecated matter, indeed, voided intothe interior of the collar, affords a very striking demonstrationof the power of the central centrifugal current, being suckedout of the collar and whisked away almost instantaneously.

2. The S e c o n d a r y F e e d i n g C u r r e n t s * such asthose labelled D, E, F, G in Text-fig. 9, are slower. Theybecome, in fact, progressively slower the farther they areremoved from the primary currents A, B, and C.

Particles caught in them amble along in a leisurely waytowards the lower two-thirds or so of the collar, to which theyadhere. Subsequently, and presumably only if they arepalatable, they pass slowly down the collar and are ingestedin the manner described above.

Some of these particles may impinge first upon the body andbe drawn up over it, rolling over its surface, before they reachthe base of the collar. It is quite easy to see bacteria doing thisand, since the outer surface of the mucous envelope is more orless sticky, some of these particles may adhere to it. Probablythe condition described by Butschli, in which the whole bodyseemed to be covered with bacteria, is to be explained in thisway, such adherent matter having been brought to the surfaceof the mucous envelope by the slow currents in the regionof G (Text-fig. 9).

The force of the secondary currents is not strong enough toovercome the resistance offered by the adhesive surface of thecollar ; nor is it sufficient to overcome whatever force it is thatcarries the food down the collar to the body.

What the nature of this latter force is I cannot say. SavilleKent asserts that the particles of food are carried over thesurface of the collar by means of protoplasmic streams in itssubstance, which he says he has seen. But, since no otherobserver has succeeded in seeing these streams, and sinceSaville Kent thought that their course was up, not down, theoutside of the collar, his statement must be abandoned.


The force of gravity could not be responsible, as Burck alsopoints out, because the particles pass straight down whatevermay be the position of the animal. They do so even when itis orientated with the long axis of the collar perpendicularto the direction of the action of the force of gravity.

Nor are they carried down, as I at one time thought that theymight be, by the backwash created by the upwardly directedfeeding currents. In carmine preparations I could not detectany trace of such a backwash; and, in any case, even if itwere there, it would not carry the particles always in a straightline down the collar, whatever the position at which theyadhere. Nor would it explain why some of them pass firstdown the collar for a certain distance and then up again.

That such oscillations of food particles up and down thecollar do occur I have often noticed. In one instance a particleeven reached the base of the collar, and hovered for a whileover the rim of the mucous envelope ; but it finally passed upthe collar again and was thrown off. Such oscillations may beexplained, I think, by the supposition that there is constantlygoing on an antagonism between whatever force it is thattends to carry the particles down the collar and the upwardforce of the feeding currents. In addition to these two activeforces, the adhesiveness of the collar must also play a part.Certainly this hypothesis may explain why particles maysometimes remain at the same place upon the collar for longperiods, showing only a rapid vibration. In such cases itis conceivable that the power of the upward feeding currentsis exactly balanced by whatever force it is that carries theparticles down the collar and by the adhesiveness of the collar'ssurface. A position of rest results, therefore, and is maintainedso long as the flagellum does not either slacken or increase itsvigour. If the flagellum increases its vigour one supposes thatthe particle is carried up the collar and thrown off at its rim ;if it slackens the downward force prevails, and the particle is,if it is palatable, ingested. The vibration shown by theparticles may be either ordinary Brownian movement or anoscillation due to the antagonism of the forces acting upon it.

500 GEOFFREY LAPAGB

The study of the behaviour of the food particles suggestsa further interesting point. For, if the account of the modeof feeding given above is correct, it is obvious that the very lifeof the organism depends entirely upon the position at whichthe primary currents are brought to a focus. Normally, aswe have seen, they are brought to a focus at, or just above,the rim of the collar. But, if that focus were, for example,much lower than this, then the rapid primary currents wouldplay over a greater area of the collar, less of its surface wouldbe exposed to the slower feeding currents, and most of the foodwould be swept away by the very vigour of the mechanismthat was securing it. It is conceivable, indeed, that, if thefocus of the rapid primary currents were much below themiddle of the collar, the animal would be unable to retain anyfood at all upon the collar. All the food would be swept awayfrom here, and the slow-feeding currents would bring it onlyto the surface of the mucous envelope covering the body,through which it could not be ingested. The organism would,in these circumstances, starve.

We have to ask ourselves, therefore, how it is that suchfailures of the feeding mechanism as these are prevented.The answer is probably to be found in the study, first, of therate at which the flagellum rotates, and, secondly, of the re-lation of the length of the flagellum to the height of thecollar. It has been established by earlier observers that theheight of the collar can be varied by the organism. The lengthof the flagellum is, as we have shown above, remarkable for thesize of the organism. And we have further seen that its rotatorymovements may vary in vigour, being sometimes relativelyfeeble when only the tip rotates, at other times much morevigorous when the whole flagellum takes part in the movement.

We may suggest, therefore, that there is probably a delicatecorrelation, always in action, between these three factors,the length of the flagellum alone remaining relatively constantin any one individual. To this relatively constant factor,both the rate and character of the rotatory movement of theflagellum and also the height of the collar are adjusted, in such


a manner that the focus of the rapid primary currents is keptat, or just above, the rim of the collar; or, in other words,in such a manner that the greatest possible area of the collaris exposed to the vitally important secondary slow-feedingcurrents.

The course of the currents also explains why it is that theorganism, when it is detached, usually swims with the collarbackwards. As Saville Kent realized, the flagellum thenacts as a pulsellum, creating centrifugal currents away from thecollar and body, so that the organism is propelled base foremostthrough the water. Schouteden (11) says that he has on rareoccasions seen it also swim collar foremost, but that such amode of progression seems to be effected with difficulty and tooccur by jerks. He also says that when the creature, movingin this way, encountered any obstacle, it reverted at once tothe normal mode of progression and escaped base foremost.Possibly during such a jerking progression collar foremost theflagellum was not rotating, but executing undulatory or jerkingmovements such as those described above, the effect of whichwas to cause the flagellum to act as a tractellum and to drawthe organism after it.

I have endeavoured to depict the system of currents as athree-dimensional figure in Text-fig. 10. In it one of theprimary rapid currents, A, is shown in its course towards andaway from the collar on all sides. Certain of the others, suchas C", G' and the currents H' internal to G', are shown on theupper side only. Eeference to the optical section of the systemshown in Text-fig. 9, in which the lettering is the same, anda realization that the currents shown there are repeated on allsides of the animal, enables us to understand that the currentsboth enter and emerge along an infinite series of trumpet-shaped cones, the sides of which are concave in successivelydecreasing degrees so that they all fit into one another. I haverepresented, in addition to one of the primary currents, A,two of these cones only. The currents labelled H', internalto G', and nearest to the flagellum, have, as described above,a course practically parallel to the long axis of the flagellum.

502 GEOFFREY LAPAGE

In other words, they continue in the direction of the long axisof the main centrifugal spout above the collar, and I haverepresented them as a cylinder with slightly concave sides.

TEXT-FIG. 10.

Diagram to show a three-dimensional view of some of the currentsshown in Text-iig. 9.

The figure is, of course, only a convention. It selects onlya few of the currents, and it is quite beyond my powers to giveany adequate representation of the fluidity of the currents.But even so imperfect a convention as this may perhapshelp the reader to realize something of the complexity ofthe commotion which this creature sets up in the water.


7. THE CONTRACTILE VACUOLES.

Contradictory statements have been made by previousauthors about the number of contractile vacuoles possessed bythis organism. Clark originally stated that, although in indivi-duals preparing for division he had seen three contractilevacuoles, yet there are normally only two, both of which aresituated in the posterior third of the body, but at oppositesides. He said that systole occurs in each every thirty seconds,that they pulsate alternately, and that each vacuole at com-plete diastole raises ' the surface of the body in a quite per-ceptible bulge '.

Saville Kent said that two contractile vacuoles are usuallypresent, and that their pulsation occupies sixty seconds, a muchlonger interval.

Biitschli confirmed Clark's account, and stated further thatthe two contractile vacuoles are not in the same transversesection of the body, but that one is situated farther forward,approximately at the middle of the body. He never saw athird, and thought that Clark had mistaken for a third con-tractile vacuole one of the many vacuoles with which the bodyis often filled. I am inclined to agree with this view. Biitschlifurther very accurately noted that the two contractile vacuolescontract alternately, that their systole is relatively slow, andthat, when each vacuole is formed anew, there first appearsan elongated area of fluid, probably built up by the fusion ofseveral smaller vacuoles, and that this becomes spherical justbefore systole. Biitschli (2) further criticizes as improbablethe view of Entz that one of the contractile vacuoles is a' Schlingvacuole ' and the elongated area of fluid a gullet,a criticism with which we have already agreed above.

Franze, who appears to share the view of Entz, is howevercorrect, according to my own observations, when he statesthat the contractile vacuole begins as first one, then two andthree small vacuoles which run together. The resulting vacuolebecomes oval, corresponding at this stage to Biitschli's elongatedarea of fluid, and then swells out the plasma wall, as Clark

504 GEOFFREY LAPAGE

described. This I can certainly confirm, and I have representedit in Text-fig. 1. But I have never been able to see either thesmall canals which Franze says lead to the growing vacuoleor the outlet canal by means of which he says the contractilevacuole empties itself to the exterior. Franze gives thirtyseconds as the interval behveen successive contractions of thevacuole.

Fisch agrees with Clark and Biitschli that there are usuallytwo contractile vacuoles, and he gives a figure of the row of smallvacuoles whose fusion to form an elongated area of fluid givesrise to the mature vacuole. He says that there may be asmany as six of these preliminary vacuoles, but I have neverseen more than three (Text-fig. 1). Fisch says that the con-tractile vacuoles contract ' twice or thrice a minute'.

Burck finds two contractile vacuoles in Codosiga and con-firms Biitschli's view of their mode of origin ; but he cannotconfirm, any more than I can, the existence of the canalsleading to the growing vacuole.

Schouteden (11), who has studied the contractile vaeuoleswith the special object of confirming or denying Franze'sstatement that one of the contractile vacuoles is in realitya ' Schlingvacuole ', concludes, like the other workers quotedand myself, that Franze is wrong, and that there are twocontractile vacuoles. He also confirms Biitschli's and Fisch'sstatements about their mode of formation, and agrees withFisch that they void to the exterior. He has timed the vaeuolescarefully, and he shows that they pulsate more or less alternately,at intervals varying between forty and fifty seconds. Hefinds, however, that it is rare to find equal intervals betweenthe alternative pulsations of the two vacuoles, but that, inspite of this, the pulsations continue in the same rhythm or doso with very small variations. My own timing of the contractilevacuoles shows, however, that there is very little variationin the intervals between the systoles of the right and leftvacuoles respectively. In other respects my results confirmhis in the main. They show that the intervals between thesuccessive systoles of either vacuole are fairly regular. In one

NOTES ON CODOSIOA 505

series of observations the average interval between successivesystoles works out at 57 seconds, the quickest pulsation requir-ing 55 seconds, the slowest 70 seconds. This is a relativelyslow beat and may have been caused by local conditions onthe slide. But it corresponds more or less with the 60 secondsgiven by Saville Kent, and is not much slower than Schoute-den's times of 40 seconds for one vacuole and 50 seconds forthe other. In other series of observations the average timebetween successive systoles was 32-5 seconds, one vacuolerecording a complete beat in 24 seconds, its longest beat beingone that lasted 42 seconds. This is, I think, nearer the normalrate of the beat in active, healthy individuals, and it agreesbetter with the statements of Clark and Franze that the vacuolopulsates every 30 seconds and with Fisch's ' twice or thricea minute '.

To sum up, I can fully confirm the observations of all thoseauthors who believe that there are normally two contractilevacuoles in Codosiga b o t r y t i s , one of which is situatedmore anteriorly near the middle of the body on one side, theother more posteriorly near the base of the body on the oppositeside. Both vacuoles are often difficult to see, because they areobscured by the numerous food and other vacuoles withwhich the body is often filled. The posterior vacuole, as othershave also noted, is often very close to a large, clear vacuole ofunknown significance that is usually present at the base of theanimal over the stalk, and which Clark described as the' reproductive organ ' (Text-figs. 1, 2, 3, A). What its signifi-cance really is I have not been able to determine, any morethan have other workers. It never contains food. Perhaps itperforms some kind of hydrostatic function. It certainly isnot contractile and is quite independent of the contractilevacuoles.

The two contractile vacuoles pulsate alternately at intervalsnormally of about thirty seconds or longer, and the rhythm oftheir pulsation is fairly regular. I can also confirm the state-ments of Pranze, Fisch, and Biitschli that the vacuoles beginby the formation of several smaller vacuoles which fuse to

NO. 275 L 1

506 GEOFFREY LAPAGE

form an elongated area of fluid. I have never seen more thanthree of these preliminary vacuoles. The elongated area offluid becomes more oval as it fills, and remains oval until shortlybefore systole, when it quickly becomes spherical. At thisstage, as both Clark and Franze saw, it raises the surfaceof the body into a distinct rounded projection. Systole isrelatively slow. The contractile vacuoles must empty them-selves through the mucous envelope.

In conclusion, I should like to thank Professor S. J. Hickson,at whose suggestion the work was undertaken, for kindlyinterest and constant advice. Mr. J. T. Wadsworth has alsogiven me much useful criticism and help. His long experienceof microscopical observation renders his assistance of especialvalue in work of this kind, which depends so much uponaccuracy of observation and correct interpretation of thephenomena observed.

8. SUMMARY.

1. This paper records observations upon living specimensonly of the Choanoflagellate Codosiga b o t r y t i s , Ehrbg.

2. The study of the organism does not support the viewput forward by Entz, Franze, and Ehrlich, that the collar isa spiral membrane.

3. It shows that the collar of Codosiga b o t r y t i s is, asearlier workers correctly stated, a protoplasmic, flexible,retractile, conical membrane, closed on all sides except on itsupper, free surface. Its function as an apparatus for catchingfood is discussed.

4. The flagelluni and the currents it produces are described,and the part played by them in bringing food to the collar, andtheir other functions, are explained.

5. It is shown that the food, when it has been caught bythe collar, passes down the outside of that structure to beingested at or below the middle of the body and not at itsupper end and never by the area enclosed by the collar. Defoe-cation, however, invariably occurs from the area enclosed bythe collar.


6. The actual ingestion of food is effected by means of arhythmical contraction which passes round the upper end ofthe body just below the collar, acting in conjunction with themucous envelope in which the body is enclosed. As this waveof contraction passes round it creates a space between the bodyand the envelope at successive points along its course. It isthis space which, being seen only in profile at the sides of thebody, has been interpreted either as a travelling vacuole(Biitschli) or as a portion of a spiral membrane (Bntz, Franze',Ehrlich). It is into this space that the food particle dropswhen it has reached the base of the collar. It is then sealed upin it, so to speak, as the wave of contraction passes on, by thereturn of the body-surface to close apposition to the envelopeabove it. Being now interposed between the body and themucous envelope, it appears to lie in a vacuole in this situation ;and it is eventually pressed into the interior of the body when,by a continuance of the amoeboid movement, the body againmoves into close contact with the mucous envelope over thearea occupied by this apparent vacuole.

7. There are normally two contractile vacuoles situated atdifferent levels and on opposite sides of the posterior half of thebody. Entz and his school are wrong in interpreting one ofthese as a so-called ' gullet vacuole ' at which food is said tobe ingested.

8. No individuals have as yet been seen that show eitherdivision, encystment, or conjugation.

LlTEBATURE.

1. Biitschli, O. (1878).—" Beitriige zur Kenntniss der Flagellaten undeiniger venvandten Organismen.", ' Zeitschrift fur wissenschaft-liche Zoologie ', Bd. 30.

2. (1883-7).—' Protozoa in Bronn's Klassen und Ordnungen desThierreichs'. Abtheilung 2, " Mastigophora ". Leipzig undHeidelberg.

3. Burck, Carl (1909).—" Studien iiber einige Choanoflagellaten",' Archiv fiir Protistenkunde', Bd. 16. (This work contains a goodliterature list.)

L l 2

508 GEOFFREY LAPAGE

4. Clark, H. James (1868).—" On the Spongiae Ciliatae as InfusoriaFlagellata", ' Annals and Magazine of Natural History', series i,vol. 1, pp. 191-9, &o.

5. Ehrlich, Richard (1908).—" Ein Beitrag zur Frage von der Membrander Choanoflagellaten", ' Biologisches Centralblatt', Bd. 28.

6. Entz, Geza (1883).—" A tordai 6s szamosfalvi sostavakostoross(Flagellata) ", „ Termiszetiajzi Fiizetek" VII K. and Tanulm.aveglenjek korebol. I. Budapest. 1886.

7. Fisch, C. (1885).—" Untersuohungen iiber einige Flagellaten undvervvandte Organismen ", ' Zeitschrift fur wissenschaftliche Zoo-logie', Bd. 42.

8. Franze, R. H. (1893).—" t)ber die Organisation der Choanoflagellaten(Vorlaufige Mittheilung) ", ' Zoologischer Anzeiger ', Bd. 16.

9. (1897).—' Der Organismus der Craspedomonaden '. Budapest.10. Kent, W. Saville (1880).—' A Manual of the Infusoria ', vol. 1.

London, David Bogue.11. Schouteden, H. (1904-5).—" Notes sur quelques Amibes et Choano-

flagellates " , ' Archiv fur Protistenkunde ', Bd. 5.12. Stein, F. Ritter von (1878).—' Der Organismus der Infusionsthiere ',

Abtheilung 3, Halfte 1. Leipzig.

notes on the choanoflagellate, codosiga botrytis, ehrbg. · notes on the choanoflagellate, codosiga...

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