QUANTITATIVE STUDIES ON THE CILIATE

GLAUCOMA

II. THE EFFECTS OF STARVATION

BY J. P. HARDING, P H . D .

Zoological Laboratory, Cambridge

(Received 2 February 1937)

(With Five Text-figures)ALTHOUGH it is a matter of common observation that ciliates and other Protozoawhen starved become smaller in size, and often more slender in shape, very littlequantitative work has been done on these changes. It is the purpose of this paperto deal in a quantitative way with the effects of the starvation of Glaucoma; inparticular with the changes that result in size and shape. It is hoped that thisstudy may throw some light on the mechanism by which the shape of ciliates iscontrolled.

Cultures of Glaucoma were starved by centrifuging them free from the Pseudo-monas on which they had been fed, and placing them in a solution of non-nutrientsalts. It was impossible to remove all the bacteria in this way, but they werereduced to insignificant numbers and the few that remained were eaten by theciliates in a very short time.

The length and breadth of an individual were measured with a micrometereyepiece, and as the transverse section was circular the volume or the surface-area

Table I. Changes in size and shape induced by starving Glaucoma for a month

Time

0 hours1

3 ,5 ,7 ,9 ,

11 ,15 .2 da1 ,5 ,7 ,

12 ,18 ,25 ,32 .

ys

No. percu.mm.

2'74 15-56-77-67-87 67-77-37-89 0

9 38 1—

H I

I I - 2

Length

35937-439740-135-834-734'93 3 22 9 928-426-525'42 3 120-51 9 2168

Breadth

2 5 62 1 71 6 614-41 3 4i 3 ' i1 3 31 3 91 2 81 2 813-11 3 212-5n - 61 1 7

" 4

Shape-index

00

7 i584 235373838424345495252576168

VolumeM*

I2,49O9,8255,6904,33°3,4153,2503,33O3,37O2,59O2,4752,4902,3351,8301,4801,4201,160

Surface

26302240I74O1405125011801200122010109629359067766506 1 0

534

Totalvolume

per mm.'

33,60040,2503i,3OO29,00026,00025,40025,30025,90018,90019,30022,40021,60014,800

—

15,80013,000

Totalsurface

per mm.'

7100918095609800950092008520940074607500840084206290

—67606100

1EB-XIVIV

432 J. P. HARDING

could be calculated with a fair degree of accuracy from the formulae for a prolatespheroid.1 Twenty individuals were measured from each sample. Other detailsof technique have been described in a previous paper (Harding, 1937).

Number

18Time in hours

Fig. i. Changes in the dimensions of Glaucoma following the removal of thebacterial food supply from a well-fed culture.

Table II. Changes in size and shape induced by the starvationof Glaucoma for 36 hours

36

Time

02

468

1 012

243°36

No. ofGlaucoma

per cu. mm.

10-316-432-3*4-8—

3 5 03 2 0—

35O35"7

Length

52'85 2 952'549346-745-845"538'04 0 9373

Breadth

3 1 62 5 819-017-51 6 61 5 0J4'31 4 515-71 5 *

Shape-index

0

6 1 2

48-937-335'53563 2 93 1 438-a3 8 242-4

Volume1 1 *

28,80017,50011,1008,5007,ioo5,7405,1504,4705,58o4,93O

1 Formulae for the prolate spheroid: „

Volume = T̂T a.b*; surface-area = 2n j6* + a" v / ~ i ^ , , • s

where a and b are the long and short semi-axes respectively.

Quantitative Studies on the Ciliate Glaucoma 433

The exact course of the changes induced by starvation depends on the initialtate of the ciliates, their size and their content of food vacuoles, which in turn

depends on the intensity of feeding previous to starvation. In all the experimentsthe food vacuoles were digested and disappeared in 4-5 hours. Multiplication of

Well fed Starved1 hour 3 hours 5 hours

lOOu

30 hours 5 days 10 days 17 days 25 days

Fig. a. Camera lucida drawing* of starving Glaucoma fixed in neutral formalinand stained with Sudan III. g, fatty globule: v, food vacuole.

the Glaucoma usually ceased after about 6 hours; but in one case it lasted for 12hours. The volume of the individuals decreased very rapidly at first because of themultiplication of the ciliates and the disappearance of the food vacuoles. After theseinfluences had ceased the volume decreased only very slowly. When the Glaucomahad been starved for a month the volume was about one-tenth that of the originalindividuals. Although they were still able to swim actively most of the individuals

28-3

434 J- P- HARDING

settled at the bottom after a few days; but none of them died for at least a month.The apparent increase in the numbers between the 12th and the 25th days inTable I was probably due to concentration of the culture by evaporation.

The changes in the shape of the Glaucoma were very pronounced, the animalsat first became relatively slender, and later tended to return to their normal morerounded shape. The length at first actually increased, the rapid decrease in volumeat this time was due to the decrease in breadth which was considerable. After anhour or so of starvation the length began to decrease and continued to do so regu-larly. The breadth on the other hand ceased to decrease after a time; so that the

KMI/Z

Well fed Starved'.\ hour*

Starved.") lionr-i

Fig. 3. Camera lucida drawings showing the effects of starvationon the size and shape of adult Glaucoma.

Glaucoma became more rounded. The comparatively slender shape was found to becharacteristic only of slightly starved Glaucoma. The different behaviour of thelength and the breadth as starvation proceeded is clearly shown by the changes inthe ratio of breadth to length. The higher this ratio, which will be referred to as the"shape-index", the more round and "plump" are the ciliates regardless of theirabsolute size. During the early stages of starvation there was a rapid decrease inthe shape-index until a minimum was reached at about the time that multiplicationceased; then the index increased again and finally the ciliates regained their originalshape.

The changes in shape were not artefacts due to fixation of the ciliates in differentphysiological states because the changes were sufficiently pronounced to be followed

Quantitative Studies on the Ciliate Glaucoma 435

by eye in the living cultures. Nor was the initial decrease in the average shape-indexof a starving culture due to the fact that as the ciliates stopped multiplying feweryoung individuals were present. This is evident because adult Glaucoma which wereundergoing fission were found to have a much higher shape-index when well fedthan when slightly starved (Fig. 3).

For measurements of the mega-nucleus it was necessary to fix the Glaucomain Schaudinn's fluid and stain with Feulgen's nuclear reagent. Schaudinn'sfixative caused some contraction of the cell and of the nucleus; but this was the onlycombination found that would stain the nucleus so that it could be distinguishedfrom the food vacuoles. The nuclei were considerably more heavily stained thanthe food vacuoles, although the latter contained Pseudomonas which took up thestain. The approximate volume of the nucleus was calculated from the semi-length,a, and the semi-breadth, b, by the formula V=$v (a.by. When the size of theGlaucoma decreased on starvation the nuclear volume decreased in a similar way,in so far as the diminution was rapid at first and slower when multiplication ceased.The decrease in the volume of the nucleus, however, was relatively less than thatof the ciliates. The nucleus at first occupied about 2-5 per cent of the volume of theciliate; but after 14 hours of starvation it occupied nearly 5 per cent (Table III).

Table III . The decrease in volume of the mega-nucleus induced by starvation

Relativevolume ofnucleus^ours

0

369

14

No. ofGlaucoma

per cu. mm.

3°63

119 -5—1 1 8

VolumeGlaucoma

87205190345°26752200

Volumenucleus

/*'

2 1 81 5 21 2 61 1 8108

2-529336644475

THE FEEDING OF STARVED GLAUCOMAGlaucoma which had been starved for 48 hours were fed again by adding a few

bacteria to the culture (Table IV, Fig. 4). In about 6 hours most of the bacteriahad been eaten, and the ciliates had trebled in volume. The Glaucoma did not beginto multiply until after the bacteria had disappeared, then they did so for 12-15 hours,with the result that there was an immediate decrease in their average volume. Bythe time the Glaucoma stopped multiplying their volume had been reduced to thatof the original starved ciliates. The maximum breadth and the maximum shape-index coincided in time with the maximum size of the ciliates; but the length didnot reach its maximum until about 6 hours later. The Glaucoma at the beginning ofthe experiment had been starved for 48 hours and those at the end for about 20 hoursand it is instructive to compare the two. Although the average volume was aboutthe same the ciliates starved for 20 hours were longer and more slender than thosestarved for 48 hours, the shape-indices being 39 and 57 per cent respectively.Fig. 6 shows that the dimensions of the Glaucoma at the end of the experiment were

436 J. P. HARDING

approaching the dimens;ons of those that had been starved for longer with which the

experiment was started.

Number

\ Bacteria

5 2010 15Time in hours

Fig. 4. The effects of feeding Glaucoma with one small meal of bacteria.

25

Table IV. The effects of feeding starved Glaucoma with one small meal

Timehours

oi

2

3456

I9

IOI I

12

131 41516182 02 428

Bacteriaper cu. mm.

I33,ooo———

100,000—

75,ooo36,00014,0008,5003,5oo1,9001,6001,900——————

Glaucomaper cu. mm.

6 56-75-86-87 0

8-56 99 0

9 515-41 8 02 3 0

26-033-5—

3 1 0

27629-037-03 5 O39O

Length

"

266527-42 6 32 9 93 2 133-834734-43 5 13 7 137-437738938938-43 7 93 8 13 8 037'937-434'9

Breadth

1 5 31 6 917-81 8 92 2 72 4 62 4 824-42 3 022'519-21 7 7IS'S14-914-41 4 1

1 3 91 3 51 3 0

13-013-5

Shape-

0//o

576268637i757271666152474 0

38383736535'5343539 . J

Volume/•'

3,4OO4,4004,7005,8oo9,000

11,00011,10010,60010,0009,9007,35O6,3005,0004,6004,2004,1003,9OO3,6503,4003,4OO3,4OO

Quantitative Studies on the Ciliate Glaucoma 437

When Glaucoma which had been starved for 27 days were placed in a suspensionof 4,000,000 Pseudomonas per cu. mm. they fed on the bacteria and increased insize (Fig. 5). After 9 hours they began to reproduce so that dividing individualscould be found in the samples. There was a considerable lag in the formation offood vacuoles. At the end of the first hour most of the individuals contained no foodvacuoles and after 3 hours of feeding there were only three or four food vacuolesper ciliate. Well-fed Glaucoma form about ten food vacuoles under these conditions.

Starvedfor 27 days

Fed for1 hour

3 hours6 hour*

100//

9 hours 12 hours

Fig. 5. Camera lucida drawings of various stages of recovery from prolonged starvation.

It appeared that the oral ciliary apparatus had degenerated considerably so that fewbacteria were captured until it had grown again.

The shape-index of the Glaucoma was higher when they were feeding again afterstarvation than it was at any other time (i.e. they were more nearly spherical).Compare the ciliates of Fig. 5 with the one that had been well fed for several genera-tions in Fig. 2 (1st drawing). Even the short period of feeding described earlierwas sufficient to produce a shape-index of 75 per cent which is higher than that fornormal well-fed Glaucoma.

438 J. P. HARDING

DISCUSSION

The starvation of mass cultures of Colpidium colpoda was studied by Vieweger(1925). The behaviour of Glaucoma was similar to that of Colpidium in that theciliates decreased rapidly in volume at first, when the ciliates were still multiplying,and that later the decrease in size was very slow. There were also differences:Colpidium continued to multiply for 8 days while Glaucoma ceased to do so in aboutas many hours. It is probably for this reason that Colpidium decreased to one-fifteenth of its original volume in less than a week, while Glaucoma took a monthto decrease to one-tenth of its volume. Vieweger's measurements of the nucleus ofColpidium showed that the decrease in its volume did not begin until some daysafter the food was removed and the volume of the ciliate had greatly decreased.On the other hand the nucleus of Glaucoma decreased rapidly in volume as soonas starvation began and decreased only slowly after the Glaucoma had stoppedmultiplying.

The interpretation of the changes in shape of starving Glaucoma is by no meanssimple. It is mathematically true that the surface of the Glaucoma in the earlystages of starvation was greater than it would have been if the ciliates had retainedtheir normal shape as they decreased in volume, and it is tempting to speculatefurther. The interpretation is complicated by the fact that the Glaucoma were stillmultiplying at this stage. It may be shown mathematically that if the total volume ofthe ciliates were to remain constant, and the shape to remain the same, the totalsurface of the ciliates would increase ^ 2 (=1-26) times every generation time.Since the ciliates were observed to become more slender in shape, either the totalvolume was decreasing or else the total surface increased more than 1-26 timesevery generation time, or both.

From the data of Table I, where the first generation time was 3 hours, it isfound that the total volume decreased in this time from 33,600/x3 to 31,300 fx3

per cu. mm. and the total surface increased from 7100 /x2 to 9560 fj.2, which ismore than 1-26 times (9560 + 7100= 1-35); so that both these factors seem to playa part in decreasing the shape-index of the Glaucoma.

The daughter cells of a dividing Glaucoma are almost spherical in shape. Thisis what would be expected, since a great increase in the surface is necessary tocover the ciliate as it becomes constricted into two, and any deficiency in the surfacearea would result in an appraoch to the spherical shape. This however is not truefor all ciliates. Jennings (1908) found that in Paramecium the length increasedrapidly as the constriction of fission deepened and the breadth decreased to someextent until the time of separation. When Paramecium divides the pellicle mustincrease rapidly in area and instead of there being a deficiency of surface there isactually an excess. The processes which cause fission to take place must also inducea rapid increase of the pellicle in Paramecium; this is probably to a lesser extent alsotrue for Glaucoma.

The decrease in the shape-index is most pronounced in the early stages ofstarvation when the ciliates are assimilating the food in the food vacuoles that are

Quantitative Studies on the Ciliate Glaucoma 439

still present. The area of the surface pellicle is probably in some way related to theamount of living protoplasm. When the volume of the Glaucoma decreased owing tothe disappearance of the food vacuoles, there was not a corresponding decrease inthe volume of the living protoplasm (this may have been increasing). The surfacewas therefore in a sense "too large" for the volume and, as a result, the shapedeparted farther from the spherical. It is reasonable to suppose that later, when theGlaucoma have used up all the food of the food vacuolea and are dependent onreserves, the substance of the pellicle is used as a source of nourishment. Thiswould result in a decrease in the ratio of surface to volume so that the ciliates wouldapproach the normal, more spherical shape again.

The very rounded shape of the Glaucoma that are being fed after starvation,compared with individuals that have been fed for several generations, suggeststhat the increase in volume takes place so rapidly that the surface is unable to keepup sufficiently for the shape to be normal. This may be because a higher proportionof the volume of the ciliates consists of undigested food than is normally the case.

SUMMARY

1. Glaucoma may be starved for at least a month without any deaths occurring.2. Starved Glaucoma stop multiplying in from 6 to 12 hours according to the

extent of feeding before starvation.3. The food vacuoles are all eliminated by about 5 hours of starvation.4. The starving individuals become smaller and smaller, the decrease in size

being most rapid at first while the ciliates are still multiplying.5. The nucleus becomes smaller in the same way as the volume of the ciliate,

but to a relatively less extent.6. During the early stages of starvation the Glaucoma become long and slender,

and later they tend to return to their normal more spherical shape.7. The changes in shape induced by starvation are interpreted in terms of the

relative changes in the surface and volume of the ciliates.

REFERENCES

HARDING, J. P. (1937). J. exp. Biol. 14, 422.JENNINGS, H. S. (1908). Proc. Amer. pHl. Soc. 47, 393-546.VIEWEGER, J. (1925). Arch. Biol. 34, 479-506.