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Full text of "Opalina : its anatomy and reproduction, with a decription of infection experiments and a chronological review of the literature"

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FOR THE PEOPLE 
FOR EDVCATION 
FORSCIENCE ' 






LIBRARY 

OF 

THE AMERICAN MUSEUM 

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NATURAL HISTORY 





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J/'V/Zr the compliments of Maynard M. Metcalf. 



Opalina. 



Its Anatomy and Reproduction, with a Description of Infection Experiments and 
a Chronological Review of the Literature. 



By 
Maynard M. Metcalf, Ph. D., 

Professor of Zoology, Oberliu, 0., U. S. A. 
With 15 Plates und 9 Text-Figures. 



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Opaliiia. 

Its Anatomy and Reproduction, with a Description of Infection 
Experiments and a Chronological Review of the Literature. 

By 

Maynard M. Metcalf, Ph. D. 

Professor of Zoology, Oberlin 0., U. S. A. 

From the Zoological Institute, Wiirzburg. 
(With Plate XIV— XXVIII and 17 Textfigures.) 

Table of contents. 

page 

Acknowledgements 197 

Material and methods 198 

Occurrence of the species 207 

The structure of Opalina and the phenomena of mitosis 210 

Cilia 210 

Pellicula 211 

Ectosarc 211 

Subpellicular layer 211 

Alveolar layer 212 

Endosarc 215 

Endosarc spherules 216 

Excretory organs 222 

Nucleus and mitosis 224 

"Resting nucleus" 226 

Achromatic foam 226 

Nucleolus 227 

Chromatin 282 

Prophases of mitosis 283 

Centrosomes 234 

Equatorial plate stage . 234 

Anai)liases 234 

Telophases 2^7 



196 M. M. Metcalf 

pape 

Spireme 238 

Resting nucleus 238 

Division of the body 239 

Time of appearance of the new nucleolus 242 

Differences between the two nuclei 242 

Chromatin spherules 243 

Origin of the ectosarc spherules 246 

Splitting of the chromosomes 247 

Nuclear conditions in other species compared with those of 0. intestinalis 247 

Enlarged individuals of 0. caudata and of other species 250 

General considerations in connection with the structure of Opalina and the 

phenomena of mitosis .... 252 

Ectosarc and endosarc 252 

Excretory organs 254 

Anterior end of the body 254 

Absence of centrosomes 254 

The spindle 255 

The mechanism of mitosis 256 

The polarity of the nucleus and the planes of division of the nucleus and 

of the body 258 

Time relations in the division of the body and of the nucleus .... 260 

Splitting of the chromosomes 260 

An alternative explanation of the mitosis 264 

The evolution of mitosis 266 

Kuclear condition and cytoplasmic movements 269 

Aualogies of the chromatin spherules 269 

Phylogeny of the nuclei of Ciliafa 272 

Compound nature of the Ciliata 276 

The phenomena in the spring which preceede and accompany copulation . . 276 

Phenomena in 0. intestinalis 276 

Decrease in the number of chromosomes 277 

The last division before encystment 277 

Extrusion of vegetative chromatin 278 

Encystment 281 

Formation of the gametes 284 

Copulation 289 

Chromatin spherules in the gametes and zygotes 294 

Nucleoli in the gametes and zygotes 294 

Can the heterogamous copulation described be abnormal? .... 295 

Encystment following copulation? 295 

Phenomena in other species 298 

Opdlina caudata 298 

Opalina dimidiata 298 

Opalina ranarum 300 

Further general considerations 300 

Vegetative chromidia 300 

Reduction 301 

Relationships of Opalina . ." 303 



Opalina. 197 

page 

Abnormalities 309 

Infection exiteriments 314 

Description of O. zelleri 316 

Chronological review of the literature of Opalina 319 

Appendix. Table showing the staining reactions of the different parts 

of the body of Opalina 346 

Literature index 348 

Explanation of plates 354 



Acknowledgements. 

The work upon which this paper is based has been done in 
the Zoological Institute in Wiirzburg. Professor Boveri directed 
my attention to the favorable character of Opalina caudata for cyto- 
logical study, and suggested also that it would be interesting to 
compare the reproductive processes in this binucleated species with 
the phenomena of reproduction which Nere^-heimer had then 
recently described for 0. ranarum and 0. dimidiata, both multi- 
nucleated forms. I cannot adequately express my thanks to Professor 
Boveri for these suggestions and for the constant advice he has 
given during the course of the work. I am especially indebted 
to him for suggestions in regard to the comparative cytological part 
of this paper. 

Professor Spem ANN kindly shared with me larvae of Bana esculenta 
which he had reared for experimental study. To Dr. Lo Bianco 
and the Director of the Zoological Station in Naples I am much 
indebted for material preserved and sent me. Dr. J. Wilhelmi gave 
me material of Gimda segmentafa infected with Hoplitophrya uncinaia 
which I desired to compare with its reputed relative Opalina. He 
also most kindly lent me many of his series of sections of this 
Turbellarian, that 1 might study the parasites. Dr. F. Balzer 
preserved material of Opalina for me during two months in the 
early spring when I was away from Wiirzburg, and, during my 
absence in the summer, Mr. E. Schneiut did me a similar service. 
Mr. \V. B. von Baehr kindly lent me a fine preparation of sections 
of 0. ranarum in the rectum of a tadpole of liana temporaria, the 
only preparation I have had showing zygotes of this species. To 
all of these, who have so greatly helped me in obtaining material 
for studv, I wish to express my most hearty thanks. 

14* 



198 M. M. Metcalf 

I desire to thank Geheimratli Prof. Dr. F. E. Schulze, and 
especially Dr. Max Hartmann for assistance in obtaining numerous 
books, I also wish to thank most cordially the authorities of the 
Smithsonian Institution, through whose kindness I was enabled to 
spend two months at the Zoological Station in Naples. During this 
time I did but little upon Opalina, but had the opportunity to study 
HopUtophrya, which I was very glad to compare with Opalina. It 
is a pleasure to express my appreciation of the assistance and many 
courtesies received from the Director and Staff of the Zoological 
Station. 

In the spring of 1900 Mr. Ernst Teichmann, at Prof. Boveri's 
suggestion, began a study of the cytology and reproduction of Opalina 
caudata. The study, however, was never completed. Most of his 
preparations are mislaid and cannot be found, but I have had the 
use of one series of his sections, and more recently Prof. Buveri 
obtained from Mr. Teichmann his drawings and lent them to me. 
I found in these drawings interesting observations most of which 
my study had already confirmed, but, as Mr. Teichmann's results 
were never published, I cannot well refer to them, since, in attempting 
to do so, I would be in danger of falsely interpreting his drawings. 



Material and methods. 

In my study of the cytology of Opalina I have used chiefly the 
binucleated species 0. intestinalis and 0. caudata, both of which are 
found in the recta of Bomhinator pachypus and B. igneus. The nuclei 
in these species are much larger and more satisfactory for study 
than are those of the multinucleated species. Opalina intestinalis 
is especially good, its nuclei being a little larger than those of 
0. caudata. I have also studied 0. ranarum, 0. oltrigona, 0. dimidiata 
and 0. selleri, using all the methods that were applied to the binucleated 
species. 

For the study of the processes of reproduction I have used 
0. intestinalis, 0. caudata and 0. dimidiata, that is — two binucleated 
and one multinucleated species. 

Infection experiments were made with the cysts of these three 
Opalinas. 

The study of the living animals has given valuable results, 
confirming almost in detail results obtained from the study of 



Opalina. 199 

pi'eserved material. These parasitic animals do not live long- outside 
the host. In water they live usually about one day; in water 
containing- some of the rectal contents and part of the rectum of the 
host they may live two or three times as long-. In 0.6 "/o Sodium 
chloride solution they live generally about two days. If part of the 
rectum of the host and a little of tlie rectal contents be added to 
the salt solution the animals live longer, from three to nine days. 
Locke's fluid ') semes about as favorable a medium as phj'siological 
salt solution.-) Opalina oUrigona lived longest in my cultures. 
Opalina caudata seemed generally the most delicate, though I have 
several times kept it seven days. Occasional!}^ I have had all the 
animals in a culture die in less than a day, some chang-e in the 
rectal contents doubtless occurring which poisoned the OpaJinae. 
Often some individuals in a culture will live after many others 
have died. Generally, for a day or two before the Opalinae in a 
culture die, i\\%y will show gradually slower and slower movements. 
Abnormal nuclear conditions are found in these dying animals, as 
will be described in the chapter on abnormalities. 

It is interesting to note that keeping the animals outside the 
host tends to cause division, perhaps through the unfavorable environ- 
mental conditions. 

Large watch-glasses were used to contain the cultures of adult 
Opalinae, these glasses bring covered to prevent evaporation. Attempts 
to rear isolated adults in microscopic aquaria (hollow-ground slides) 
were not made; such attempts with the gametes and zygotes were 
unsuccessful. These are more delicate than the large forms, so 
that very likely the latter could be kept alive a couple of days or 
so in such microscopic aquaria. 

For the study of living gametes and other minute forms from 
the tadpoles, slide cultures were used. The intestine of the tadpole 
would be placed upon a slide with a drop or two of 0.6 % NaCl 

') Calcium chloride (anhydrous) 07% 

Potassium chloride 0.01 % 

fiodium chloride 0.06 "/o 

Sodium bicarbonate 0.01—0.03%. 

From Journ. of the Boston Soc. of Med. Sc. 1896. 

^) Putter (1905) says that the best culture medium for Opalina is made of 

sodium chloride 0.8% 100 parts 

sodium and potassium tartrate 307o- • • • 5 „ 

distilled water 400 „ 

and that in this fluid, ivhen it contains no free, oxygen, Opalina, if fed, will live 
three weeks. I have not tried this fluid, nor used any oxygen-free culture media. 



200 M. M. Metcalf 

solution, or Lockf/s fluid, and be opened under a Zeiss binocular 
dissecting microscope (magnification fifty diameters) and rapid 
observation be made of the forms found, all especially interesting 
phenomena bring noted. This preliminary survey is important for 
comparison with later appearances which may be suspected of being 
abnormal. The intestinal w^all and contents would then be separated 
from the OpaUnae by pushing the former to one side wdth dissecting 
needles. The OpaUnae would then be covered with a thin cover-glass 
and, after a few moments waiting to allow the edges to dry, the 
culture would be sealed with Cheeseborough Manufacturing Company's 
white vaseline. Wax is not a satisfactory sealing for slide cultures 
which are to be studied with an immersion lens, as pressure upon 
the cover-glass tends to cause leaks in the w^ax sealing. These 
slide cultures live sometimes as much as two days, but often die 
within eight to twelve hours. Similar slide cultures were often used 
for studying the adult OpaUnae. the cover being supported by a 
couple of very fine hairs. The slide cultures of adult OpaUnae may 
live three days, though more die the first or second day. 

For studying the finer structure of living OpaUna the binucleated 
species are, as already said, by far the better, but not all individuals, 
even of 0. intestinalis, are equally clear. Sometimes one finds nuclei 
in which while alive one can observe with remarkable clearness the 
chromosomes, the spindle fibres, and the achromatic granules. It is 
certain that the structures described in the dividing nuclei are not 
artifacts, for they have been observed not only in preserved material, 
but in the living animal as well. Probably no one really gives 
much weight to the sw^eeping objections that have been made to 
cytological studies as dealing largely with artifacts, yet many reagents 
do undoubtedly produce artifacts w^hich are likely to be misleading; 
it is therefore a satisfaction to be confident that one is describing 
natural structures and not things that have been produced by mani- 
pulation. 

Not only does one find many living individuals which do not 
show their nuclear structure clearly; occasionally one is even unable 
to distinguish the nuclei at all. One must usually carefully observe 
a good many individuals before finding one in wiiich the nuclear 
structures are very clearly seen. It is interesting to note that the 
posterior nucleus is often clearer than the anterior. At first I 
thought this was due to the fact that the protoplasm of the anterior 
end of the body is more dense than that of the rest of the body, 
but there is a further and even more important reason for this 



Opalina. 201 

difference in clearness in the two nuclei. In individuals in which 
the system of excretory vacuoles ') is well developed, one sees that 
these vacuoles usually lie close along one side of the posterior 
nucleus (Fio-. 1, PI. XIV, 248. PI. XXVI). They may extend also 
alongside of the anterior nucleus, though this is less usual. When 
such a vacuole is large and lies above the nucleus under observation, 
one is likely to see the nuclear structures vei-y clearly. If, as 
sometimes occurs, the sj'stem of vacuoles divides, sending also a 
branch along the opposite side of the posterior nucleus, one has his 
best opportunity to observe this nucleus, if only the animal is so 
oriented that one vacuole lies above and the other below the nucleus. 
In this case the refractive bodies in the cytoplasm (to be discribed 
later) lie so far above or below the focal plane of the objective of 
the microscope that they distort the image but little. It is therefore 
well to search for the most favorable individuals before settling 
down to careful study of the nucleus. The anterior part of the 
excretory organ is seldom well seen in the living animal. It is 
chiefly through the study of stained preparations that one reaches 
this explanation of the remarkable clearness of some living nuclei. 

In studying the reproductive processes in the spring, it is often 
valuable first to use living animals and later to treat the same 
animals with acetic acid or acetic carmine. For example, one can 
thus allow copulation to proceed to a particular point, and can 
then confirm his observations of nuclear and other phenomena in 
the living animals by studying the same animals treated with one 
of these reagents. It is often well in such cases to use first acetic 
acid and, after study, to follow with acetic carmine. Some structures, 
the nucleolus for example, show far better with acetic acid than 
with acetic carmine. Often the whole structure of cytoplasm and 
nucleus comes out with remarkable clearness with acetic acid. 

Acetic carmine used ui)on fresh material is very satisfactory 
for the study of the outlines of the excretory organs and is invaluable 
in the study of the minute forms in the spring, for. while it is not 
a sharply definitive stain and while its results, even in the same 
slide, are often very uneven, yet it is so simple in its application 
and so prompt in giving its results, that with it one can examine 
a very great amount of material, and this is essential to the proper 
understanding of the reproductive processes. 

Intra vitam staining with all the usual dyes was tried upon all 



') Metcalf. 1907 h and c. 



202 M. M. Metcalf 

the species at my disposal execpt 0. seUeri. The results will be 
given in the proper connections. They are also shown in a table 
in the appendix. 

For fixing- 1 used chiefly Schaudinn's alcoholic- corosive-sublimate, 
corosive-sublimate-acetic acid, picro-acetic acid, Flemming's fluid, 
formol, and absolute alcohol. Of these corosive-sublimate-acetic acid 
(20 minutes to 86 hours) gave the best results and in the later work 
was chiefly used. 

For staining- in toto I used principally Grenacher's borax 
carmine, Mayer's paracarmine, Mayer's haeraalum, and Delafield's 
haematoxylin. Paracarmine did not give very good results. Borax 
carmine gives a good general stain, but does not show the finest 
details with sufficient clearness. If a thin sheet of green gelatine 
be placed on the table of the microscope beneath the slide, the 
definition of detail is much improved,'] but even then the borax 
carmine preparations are not the best. No satisfactory stains were 
obtained with Mayer's haemalum, except for protoplasmic structure. 
Delafield's haematoxylin far outclasses all the other stains used 
for total objets. It is best to stain as darkly as possible (12 to 24 
hours in ^3 strength, ^2 strength, or even full strength stain) and 
then to decolorize with exceedingly dilute hydrochloric acid. The 
decolorization should be watched under the microscope and when it 
has reached the right point it can at once be stopped by adding a 
drop of weak ammonium hydrate. The decolorization should be 
carried to a point that seems extreme, for the objects become much 
darker upon adding the ammonia. A little experience enables one 
to regulate the stain very accurately. If upon adding the ammonia 
the objects are found to be too dark, most of the liquid can be 
drawn off" and acid again added, the decolorization being carried to 
the desired point. It should, however, be noted that, upon adding 
acid after ammonia has been used, the decolorization is much more 
rapid than before the objects were treated with ammonia. With 
this stain used in this way preparations of total objects can be 
obtained which rival for clearness the best sections. 

The animals when stained were run through graded alcohols to 
cedar oil and w^ere mounted in balsam. As Delafield's haematoxylin 
is exceedingly sensitive to the presence of the least acid, readily 
fading when in balsam, if this be in the least degree acid, it is 

') For suggesting this method, which is a very useful one, I am indebted 
to Mr. W. Fkeytag of Wiirzburg. 



Opalina. 203 

well before covering- to hold tlie slide, with the animals in cedar oil 
upon it, upside down for a few moments over the top of an ammo- 
nium hydrate bottle, and to do the same with the balsam on the 
cover-glass. My preparations so treated have not faded in ten 
months except near the edges of the cover-glass. x4pparently the 
carbon dioxide of the atmosphere causes decolorization of the objects 
near the edge of the cover-glass. The objects can be kept fiom 
running out from the center toward the edge of the cover-glass by 
the simple but effective device of placing the balsam before covering 
in a complete circle just inside the outer edge of the cover. 

For sectioning single individuals in predetermined planes 
Yatsu's (1904) Viva leaf method was used. For imbedding large 
numbers of animals together I used either Boveri's method of wrap- 
ping the animals in a bit of the sloughed skin of a large amphibian 
{Cryptobranchus) , or a method which combines suggestions from 
Lefevre (1903) and from Paul Mayer (1907). In the latter method 
the animals are carried up to absolute alcohol in ordinary embryo 
glasses. After dehydration all but a few drops of the alcohol is 
drawn off. Then with a iine pipette the remaining alcohol, with 
the animals, is removed and placed in a small gelatine capsule 
(20 mm by 5 mm) which is set upright in a hole in a pasteboard 
box (Text Fig. I, A). The ends of the box should be removed so that 
one can look through and see the objects in the bottom of the 
capsule. After the animals have settled to the bottom of the capsule, 
the supernatant alcohol is drawn off" and xylol added. It is well 
to change the xylol once or twice to remove all trace of alcohol. 
After sufficient time, the xylol is removed, melted paraffin in added, 
and the capsule is set into the warm chamber. The paraffin must 
be changed to remove all xylol. With care this may be done with 
a warm pipette, but I find it much easier to effect the change in 
another way. After the animals have become well infiltrated with 
the paraffin, the capsule may be removed from its supporting box 
and its contained paraffin cooled by placing the capsule in cold 
water. After a few minutes the gelatine capsule will be softened 
and swollen by the water and the cylinder of paraffin can be easily 
removed. A second capsule should then be nearly filled with melted 
paraffin and the tip of the cooled paraffin cylinder, with the con- 
tained objects, be cut off and placed in the top of the capsule of 
melted paraffin and the capsule placed in the warm chamber. As 
the paraffin cylinder tip melts, the objects sink through the whole 
length of the capsule, losing en route whatever xylol they may 



204 



M. M. Metcalf 



still have had. This capsule may now be taken out of the warm 
chamber and its contents be cooled in water as before. 

A paraffin cylinder with rounded tip is not easy to cut. This 
difficulty can readily be avoided by using a Lefevee watch glass for 
reimbedding (Text Fig, I, C and D). The tip of the cylinder, con- 
taining all the objects, is cut oif and is placed in the center of the 
groove of an unwarmed Leeevre watch glass, which has previously 
been lightly smeared with glycerine. With a hot pipette, melted 
paraffin in added on each side of the cool paraffin block, care being 
taken to leave this block with its contained objects still in the 
center of the groove. The watch-glass is now placed in the warm 
chamber until all is melted. It is then removed, without jarring, 
and placed in water, or alcohol (Lefevre), to cool. The resulting 
block of paraffin is of a shape convenient for sectioning (Text Fig. I, B). 





A 



B 





C B 

Text Fig. I. Illustrating the method of imbedding small objects. A, a box con- 
taining three gelatine capsules; i?, the block of paraffin taken from a Lefevre 
watch-glass; C and D, sections of a Lefevre watch glass. {B, C and D from 

Mayer after Lefevre.) 

Since the objects to be sectioned are all in the center of the pro- 
jecting ridge, the ends of the ridge may be cut away and a com- 
pact series of sections be obtained. This method is not tedious. 
It requires no watching. ^) 

^) I am greatly indebted to Professor Paul Mayer for suggesting the use 
of gelatine capsules in imbedding. His further suggestion that they might well 



Opalina. 206 

To obtain sections of tlie g-ametes, wliole recta of infected tad- 
poles were cut. The dirt in the rectum usually prevents cutting 
sections thinner than 3, 4, or 5 micra, but these suffice. 

Sections were stained with Delafield's haematoxylin, Heiden- 
hain's iron - haematoxylin , Gkenacher's borax carmine, safranin, 
safranin and light green (Liclifgri'm), thionin, gentian violet, methyl 
violet, methylen blue, methyl green, eosin, dahlia, orange G, fuchsin, 
magenta, Kemschivar-, Bioxdi-Ehrlich-Heiuenhain's mixture, Ehr- 
Licirs triacid mixture and Ehrlich's indulin-aurantia-eosin mixture. 
All gave results of some value and the comparison of the results 
obtained from diiferent stains was important. esi)ecially in studying 
the refractive si)herules. Delaeield's haematoxylin and Heiden- 
hain's iron-haematoxylin were the most generally useful stains. The 
most diflFerential stain was obtained with safranin and light green 
(safranin 12 to 24 hours, light green in 95 " o alcohol ' .2 to ^^ of a 
minute). The results with all of the stains used are shown in a 
table in the appendix. 

For illumination the light from a Welsbach gas mantel was 
used, daylight not being strong enough. 

The degree of accuracy of the figures is told in each instance 
in the explanation of plates, any figures, or parts of figures, schemati- 
cally drawn being so indicated. 

Since writing the major part of tliis paper I have found that 
smear preparations of undiluted rectal contents of Rana tenqwraria 
give fine results with cysts and free swimming individuals of 
O. ranarum and I do not doubt that equally good results would be 
obtained with other species. The method should be of especial 
value with the minute forms in the recta of tadpoles. The smear 
preparations should not be allowed to dry, but should at once, while 
moist, be fixed a moment in a hot fixing fluid and then be trans- 



be used for storing small objects in alcohol has also proven veiy useful. Much 
of my Opalina material has been kept in gelatine capsules in a jar of 95% Alcohol. 
{Alcohol weaker than 80 °o softens the capsules.) A further suggestion, from Dr. 
R. DoHRN and Dr. Gast, that the lower half of the capsule be sealed with celloidin 
before covering with the lid is important, since with very small objects there is 
danger that some may get between the capsule and its lid and be crushed. Be- 
fore adding the film of celloidin, it is well, as Doctors Dohrn and Gast suggested, 
to puncture the capsule in several places just below its upper edge with a needle, 
so that the celloidin film will hold firmly. Minute objects so stored transport 
without danger of breakage and the capsules require much less room than the 
glass tubes ordinarily used. Furthermore there is no cotton plug in which any 
of the objects may be lost. 



206 



M. M. Metcalf 



fered to cold fluid of the same sort. The heat is valuable since it 
makes caagulation instantaneous and the objects hold firmly to the 
cover-glass upon which they are spread. 



o 



o 



o 




baXuxno-Vva xuUsUucsixt) cau.(L<iXCk 



macrowucleala 




O 



\(L^ctoVaXcL. (i\m\<i\a\a 




idUtv 



lOTl(^Q. 






RCLYCL 



IclVcl 



xauaTcxLTcv 





e0tCi.C0l(k5.CL 



■115.C: 



q\>XxV(^oyiq. 



Textfig. II. Outline drawings of surface views and cross sections of the known 
species of Opalina. The nuclei are indicated only for those forms that have few 
nuclei. The relative size of the different species is not shown. In each case the 
anterior end is above and the bend of the anterior end of the longitudinal axis is 

toward the right. 



Opaliua. 



207 



Occurrence of the species of Opalina. 

We now know thirteen species of Ojmlina, twelve parasitic in 
tailless Batrachia (one of these also in Triton) and one in a Me- 
diterranean fish, Box hoops. Their occurrence is as follows (cf. Text- 
fig. II): 

Species with two nuclei, bodies circular in cross section. 
O. sahirnalis, Leger & Duboscq, in Box boops, Laur.* ^) 



0. intestinalis, Stein (Bloch?) 



0. caudata, Zeller 



O. macronucleata. Bezzenb. 



Bomhinator pachypus, Bon.* 
Bomhinator igneus, Laur.*) 
Discoglossus pictus, Otth. ^) 
Pelobates fnscus, Wagl. 
Bana esculenia, L. •^) 
Triton tacniatus, Schn. ^) 
Bomhinator pachypus, Bon.* 
Bomhinator igneus, Laur.* ^) 
Bufo variahilis, Pall. *) 
Bufo melanostichus, Schn. •^) 



Species with four to five nuclei, body circular in cross section. 
0. lanceoJata, Bezzenb. in Eana esculenta, L., var. cJiinensis, Osb. ^) 

Species with many nuclei, body circular or broadly oval 
in cross section. 



O. dimidiata. Stein 



O. zelleri, Neresheimer 
0. longa, Bezzenb, 



in cross section. 

in Ba)ia esculenta, L.* ") 

Bufo vulgaiis, Laur (cinereus, L.). 

Bufo variahilis, Pall. 
„ Bana esculenta,* ^) 
„ L'ana limnocharis, Wigm. ^) 



Species with many nuclei, body flattened. 

O. flava, Stokes in Scaphiopus holhrooMi, Harlan. ^) 

„ Rana limnocharis, Wigm. ^) 



O. lata, Bezzenb. 
O. ranarum, Ehrb. 



Bana temporaria, L.* (fusca Rosel). 
Btifo vulgaris, Laur. 
Bufo variahilis, Pall.* 



208 M. M. Metcalf 

0. coracoidea, Bezzenb. in Rana cyanophlydis, ^) 

0. ohtrigona, SxEm „ Hyla arborea, L.* ^) {viridis Laur.). 

*) Coufirmed by my own observations. 

1) LtQEB. & DUBOSCQ (19046). 

-) Stein (lf^67), Zeli-er (1877), very rare in this host. 

2) CoNTE & Vaney (1902). 

*) T found in Naples one toad of this species whose rectum contained a few 
dozen Opalinae caudatae. 

^) Bezzenberger (1904). 

•) Zeller (1877), Neresheimer (1907), Metcalf (1907 a). 

') Stokes (1884). 

») CoHN (1904). 

») Zeller (1877). 

All the species of Opalina which I have studied live chiefly at 
the upper end of the rectum of the host. A few individuals may 
be found scattered throug^h the contents of the whole upper half of 
the rectum (this is especially true of 0. ohtrigona in Hyla arborea), 
but generally the parasites lie in one or more masses between the 
rectal contents on the one hand and rectal wall on the other. In 
frogs or toads which have been dead for some hours, the Opalinae 
are often found also in the lower part of the intestine, and occasion- 
ally, in frogs that were evidently diseased, I have found the 
posterior part of the intestine to contain some Opalinae. Several 
species of Opalina have been reported from the intestines, as well 
as the recta, of their hosts. It is possible that these reports are based 
on observations upon diseased frogs and toads, or upon those that 
were dead some time before they were examined. Legek & Duboscq 
(1904 &) report their new Opalina saturnalis as occuiTing in the whole 
intestine of Box boops. It would be of some interest to know if 
the whole intestine of normal, freshly killed fish of this species con- 
tains the parasites. 

Opalina caudata and 0. intestinalis are rarely, if ever, found in 
the same individual host. In the two instances in which I have 
found 0. zel'eri, 0. dimidiata Avas also present. Zeller also found 
these two forms together. Neresheimer (1907), the only other student 
who has recorded the occurrence of 0. zelleri, does not say whether 
he found it with 0. dimidiata or not. There is a little doubt of the 
independence of the two species. 

The frequency of infection of the several hosts by the several 
species is shown for, the animals I examined in the following table. ^) 

^) Records were not kept of a number of the hosts which were killed early 
in the fall of 1906, or of most killed in Naples. 



Opaliiia. 



209 





fa 


Xuruber coiitainin<>: Ofxdinae 








Bomhinator pachypus 


105 


0. caudata 61 

0. intcsfiimlis 34 

Botli 0. caudata and 0. intestinalis 1 ^) 

Uncertain, either 0. caudata or 0. in- 

testinahs 
No Opalinae 8 -) 


Bomhinator igneus 


63 


0. caudata 25 
0. iniestinalis 15 
No Opalinae 23 ») 


Hyla arhorea 


49 


0. oUrigona 21 
No Opalinae 28 


Rana escnJenia 


77 


0. dimidiata 59 

0. zelleri and 6>. dimidiata 2 

No Opalinae 16 


Rana temporaria 


15 


0. ranarum 10 

No Opalinae 5 (One had been starved 
a long time) 


Btifo variahilis 


4 


0. ranarum 1 
0. caudata 1 
No Opalinae 2 


Bufo vulgaris 


1 


No Opalinae 1 



1) This frog was sick, the stomach being- greatly distended by a very acid 
fluid, and the whole intestine being full of gas. The Opalinae in the rectum 
were shrunken, twisted and distorted, and in consequence the identification of the 
two species of parasites is not reliable. Zeller does not say whither he found 
these two species of parasites in the same individual host, though he describes 
them both from Bomhinator igneus. 

'-) Two of these were unusually large individuals, obtained in Berlin in the 
spring of 1908. In one the spots on the ventral surface were of the orange color 
typical of B. igneus. In the other the color was paler orange, more nearly 
approaching the lemon yellow characteristic of B. parhi/pns. The size and the 
character of the dorsal surface showed that these animals belonged to the species 
B. pachypus. 

') Some of these Bomhinator had been kept several months in the laboratory 
and were very thin. 



210 M. M. Metcalf 



The structure of Opalina and the phenomena of mitosis. 

In the description of the structure of Opalina, I will begin for 
each organ with the conditions in 0. intestinalis and will then com- 
pare with the other species studied. 

Cilia. 

I have little to add to or to modify in Maier's (1903) dis- 
cription of the cilia in Opalina ranarum. In all species studied the 
conditions of the cilia are similar. It is easy to cause the dis- 
integration of specimens of 0. intestinalis and 0. caudata by pressing 
intermittently upon the cover-glass above them. One then sees many 
of the cilia, with their basal granules attached, floating freely in the 
salt solution. Occasionally such isolated cilia may show a few 
faint contractions after their complete separation from the body. 
Similar phenomena have been observed in ciliated cells by Klebs (1883), 
BtJTSCHLi (1885 a), Fischer (1895) and Peter (1899), and for the iso- 
lated tails of spermatozoa by numerous students [cf. Meves 1899 h). 

In tangential sections of the body of any species of Opalina, 
one sees a network of fibres beneath the pellicula (Fig. 2). The 
spiral-longitudinal rows of basal granules lie just below the larger 
fibrils, the course of the two exactly coinciding. The much more 
delicate transverse fibrils stretch between the longitudinal fibrils, 
each end coinciding in position with a basal granule. In this regard 
it is seen that my studj^ confirms Tonniges (1898) rather than 
Maier (1903). The observation of contraction in isolated cilia shows 
that their movements are automatic, as Maier claims in opposition 
to Tonniges, but it seems probable that the coordination of the 
movements of the cilia may be connected with the presence of this 
network. The transverse fibrils of this network lie beneath and not 
in the pellicula, as accurate focussing clearly shows. The longitudinal 
fibrils are a little more superficial, lying apparently at the level of 
the outer ends of the basal granules. 

Nether 0. intestinalis nor 0. caudata have any tuft of longer cilia 
at the anterior end of the body, such as Legee & Duboscq (1904 h) 
discribe for 0. saturnaUs. In 0. saturnalis the anterior tuft of cilia 
is not very distinct from the adjacent cilia. 

There is no posterior zone, as in 0. saturnalis, from which the 
cilia are absent. 



Opalina. 211 



Pellicula. 



All species studied have a pellicula of appreciable thickness 
which is quite distinct from the subjacent cytoplasm (Figs. 3, 6, 7, 8, 
PI. XIV). With many stains it colors dilfei'ently from the ecto- 
plasm. Some of these differentiating stains are gentian violet, methyl 
violet, methylen blue, thionin, fuchsin, dahlia, Ehklich's triacid mix- 
ture, and Delafield's haematoxylin. With methylen blue the whole 
ectoplasm is stained green while the pellicula is pale blue (Fig. 18, 
PI. XV); with methyl violet the pellicula has a lighter shade than the 
ectoplasm; with thionin the pellicula is unstained while the ectosarc 
is green ; with fuchsin the light red pellicula is readily distinguished 
from the more faintly colored ectoplasm. Dahlia gives perhaps 
the clearest picture, the pellicula being a very faint purplish gray, 
while the ectoplasma is purple. The contrast between the green 
pellicula and the blue ectoplasma after staining with Ehrlich's 
triacid mixture is also very marked. With all stains used the pelli- 
cula seems homogeneous. 

I have not been able to demonstrate with entire clearness the 
minute longitudinal ridges which Maiee describes as present on the 
outer surface of the pellicula. The longitudinal striae are very clear 
in surface views of tangential sections, but none of my cross sections 
give satisfactory views of the ridges. In some sections stained with 
Delafield's haematox3'lin they are faintly seem. One cannot, how- 
ever, "doubt the accuracy of Maiee's description, for his work is 
clearly very careful. 

Zellee figures the outer surface of the body as breaking into 
thin narrow strips after treatement with acetic acid. He called 
these strips muscle threads. From my own preparations it seems 
probable that what he described were strands of pellicula which had 
separated along the course of the lines of cilia (cf. Maiee, 1903, p. 80). 

Ectosarc. 
Siih-pellicular layer. 

Immediately beneath the pellicula, between it and the usually 
large alveoles which fill the greater part of the ectoplasm, there is 
a thin layer of finely alveolar protoplasm (Fig. 8, PI. XIV). In the 
outer part of this layer lie the basal granules of the cilia. Other 
granules, similar in size, lie more internally in the same layer. They 
resemble in size the granules that lie at the nodes and along the 



212 M. M. Metcalf 

course of the films of the endoplasmic web, but in their staining 
reactions they resemble more the ectoplasmic spherules soon to be 
described. 

Alveolar layer ^) (Figs. 4, 7, 8, PL XIV). 

Beneath the sub-pellicular layer the ectoplasma shows many 
alveoles always larger, usually very much larger, than those of 
either the sub-pellicular layer, or the endoplasma. Metlijd violet 
most sharply defines all the structures of the alveolar layer (Fig. 8j. 
In sections stained with this reagent one sees that there is usually 
an outer irregular row of moderately large alveoles and that within 
this is a second irregular row of huge alveoles. It is not difficult 
to understand how Maiee failed to observe these alveoles in 0. ra- 
narum. if he used only iron-haematoxj'lin in staining his sections, 
for this reagent often gives very unsatisfactory pictures of the 
structure in this region (cf. Fig. 5). The walls of all these alveoles 
are delicate films along which lie scattered granules. Each alveolus, 
whether large or small, contains a body which, following the 
nomenclature of my preliminary paper (Metcalf 1907a) may be 
called an ectoplasmic spherule. Sections stained with most reagents 
fail to show a fact which methyl violet clearly demonstrates, namely, 
that each alveolus, whether large or small, contains only one spherule. 

The spherules as seen in sections vary greatly in size. In 
some cases, even when an alveolus is of great size, the spherule 
may nearly fill it. In general, the size of the spherule is roughly 
proportional to the size of its alveolus. The spherules are more 
or less irregular in form. Often, especially in sections of animals 
which are a little shrunken by reagents or by the heat used in. 
imbedding, one sees the spherules showing a shape that irresistably 
suggests that they have been coagulated and shrunken from a more 
fluid substance which previously filled the alveoles. The finely 
granular character of these irregular spherules is not inconsistant 
with such an interpretation. 

The ectoplasmic spherules are often clearly seen in the living 
animals. They have usually a distinct yellow tinge. This is empha- 
sized by acetic acid. In many acetic-carmine preparations their yellow 
color is sharply contrasted with the red of the spherules in the 
endoplasm (Fig. 31 «, PI. XV). 



^) I make no attempt in any part of this paper to distinguish in terminology 
between the minutest alveoles and the larger spaces found within the protoplasm, 
which may arise by the enlargement or fusion of minute alveoles. 



Opalina. 213 

These spherules or drops of liquid in the ectosarc stain intra 
ntam with neutral red (darkly stained alter half an hour), methylen> 
blue (deep stain), toluidin blue (deep stain) (Fig. 20, PL XV). They 
tlo not stain iuira vitam with Congo red, indigo-carmine, methyl 
violet, dahlia, Bismarck brown, gentian violet, thionin or eosin. 
With methylen blue and toluidin blue the anterior end of the body 
remains almost entirely unstained, showing either that the ectosarc 
spherules are wanting there, or are in a different condition. The 
study of sections shows that the ectosarc spherules are very small 
in that region. After intra vitam staining with toluidin blue (Fig. 20, 
I'l. XV) one finds a few bodies, larger than the ordinary ectosarc 
spherules, stained a much darker blue. These are evidently in a 
different condition, if they be not of a wholly different nature. It 
is probable that these larger, darkly staining bodies lie in the outer 
part of the endosarc. Compare the results, obtained by staining 
with iodine and with Fischee's glycogen stain (page 218). 

The statements in the last paragraph apply only to true intra 
vitam staining, the animals remaining alive and active. As the 
animals become inactive and die, the ectoplasmic spherules com- 
mence to stain with methyl violet, though later they again fade. 
If one sections animals which have been fixed in corrosive subli- 
mate-acetic acid and stains the sections with the same dyes 
that were used for intra vitam staining, somewhat different re- 
sults are obtained. Methylen blue stains the ectosarc spherules 
green, not blue as in life; methyl violet colors them violet like the 
protoplasm; dahlia stains them purple; gentian violet stains them 
pale violet; thionin stains them green; Bismarck brown colors them 
brown; though the last five reagents left these spherules unstained 
in the living animal. In the table in the appendix the color re- 
actions of the different parts of the body to all the stains used are 
given. I would call attention to the fact that with safranin and 
light green the ectoplasmic spherules are all colored green, though 
with safranin when used alone they stain a good red. 

The ectosarc spherules show little structure with most stains 
(Figs. 4, 7, 8, 9, 14, PI. XlVj. With dahlia some of the larger, 
more faintly stained ones show granules which seem to be peripheral 
(Fig. 7j. Iron-haematoxylin when insufficiently extracted shows the 
spherules apparently homogeneous (Figs. 4, 9), but after longer de- 
colorization one often finds them showing clearly the presence of 
peripheral granules and even apparently alveolar structure (Fig. 5). 
When found, these indications consist merely of faint dark lines 

15* 



214 M. M. Metcalp 

stretching across the interior of the spherule and connecting certain 
of the deeply stained granules which lie at the periphery of the 
spherule with smaller granules in the interior. I have never seen 
ectosarc spherules showing indication of division. 

The ectosarc spherules do not stain at all with potassium iodide, 
so they cannot be composed of glycogen. They do not stain at all 
with a solution of iodine in aqueous potassium iodide. They do 
stain strongly with safranin in sections of animals fixed in absolute 
alcohol and treated, after sectioning, with tannin and potassium 
bichromate. Fischer (1905) regards this staining after such treat- 
ment as indicative of glycogen, but the entire absence of any re- 
action to iodine on the part of these spherules seems to indicate 
that they are not glycogen and probably are not of a substance 
closelj^ related to glycogen. 

It is difficult to form an idea of the function of the ectoplasmic 
spherules. The internal structure demonstrated by iron-haematoxylin, 
and less well by certain other stains, seems to argue against their 
being wholly secreted bodies. On the other hand, their position 
within the alveoli rather than upon the alveolar walls, would suggest 
that they are a product rather than a constituent part of the proto- 
plasm. The fact that with certain dyes they readily stain intra 
vitam casts further doubt upon their interpretation as living con- 
stituents of the cell, though these dyes, neutral red, methylen blue, 
and toluidin blue, are well known to stain some kinds of living 
tissue in Metazoa. Leger & Duboscq (1904 h) think that the similar 
bodies in 0. saturnalis are probably connected with nutrition and 
may be of a nature similar to lecithin. In the species I have 
studied, however, they are not soluble in warm alcohol and ether, 
so that they cannot be composed of lecithin. I know of nothing to 
indicate that they are excretory. They have no connection with the 
system of excretory vacuoles I have described (Metcalf 1907 6 and c). 

The ectosarc spherules are present in all species I have studied, 
though they are very small in some forms of 0. dimidiata. In 
0. caudata they resemble closely those of 0. intestinalis. In 0. ra- 
narum and 0. ohtrigona they are smaller, but otherwise similar. In 
0. selleri they are still smaller, but are clearly recognised. In 
0. dimidiata, in all but the minute forms in the spring and the 
young forms in the tadpole, one almost fails to find them with cer- 
tainty, for, they are little if any larger than the largest of the 
ordinary granules of the ectosarc. On the other hand, yellow ecto- 
sarc spherules of laige size are abundant in this species in the 



Opaliiia. 215 

macrogametes and other small forms from the tadpole, except possibly 
the microgametes. and also in the smallest forms found in the spring 
in the rectum of the frog. I regret that my notes do not say as to 
the presence of the ectosarc spherules in the microgametes of 
(). dimidiafa and I do not remember with certainty, though I think 
they are present and of good size in this species as in the micro- 
gametes of 0. iniesiinalis and 0. caiidaia. 

It is not easy to be certain who of the students of the Opalinae 
have seen the ectosarc spherules, for, excepting by Leger & Duboscq 
(1904 h) for 0. saturnalis, they have not been figured or clearly 
described. Tonniges (1898) describes for 0. ranariim certain greenish 
granules, disc-shaped, elongated, or of irregular form, and varying 
in size. He says that in most individuals they lie exclusively in 
the endoplasm and goes on to describe at considerable length their 
minute structure. In the main his description applies surely to 
what I have called endosarc spherules (Metcalf 1907 a), but the 
greenish color he ascribes to these is characteristic of the ectosarc 
spherules. Tonniges has not distinguished between the two kinds 
of spherules and it may be that the greenish color of the outer 
ones has been ascribed by him to them all. Conte & Vanet (1902) 
have evidently not distinguished the ectosarc spherules. Neres- 
HEiMER (1906 and 1907) describes remarkable phenomena connected 
with certain disc-shaped spherules. I am not sure I understand 
correctl}' his description, but it seems to apply to the larger ecto- 
plasmic spherules and not to the smaller sort of spherules which lie 
in the endoplasma. He says that these disc-shaped bodies change 
into spherical or ovoid spherules into which the reproductive chro- 
midia migrate, each spherule with its chromidia constituting a new 
reproductive nucleus in which the spherule furnishes the achromatic 
portion and the chromidia the chromatic portion of the new nucleus. 
The conditions in 0. intestinalis previous to and during the spring 
sexual reproduction preclude any such interpretation, useless 0. ra- 
narum. upon which Neresheimer worked, is fundamentally different 
from the binucleated species. Opalina ranarmn is a far less favorable 
species than the binucleate forms for the study of the ectosarc 
spherules, for in 0. ranarum they are not only proportionally but 
actually much smaller. 

Endosarc. 

The endosarc of all the species of Opalina I have studied shows 
usually a finely granular and fibrous appearance in which the real 



•216 M. M. Metcalf 

foam-like alveolar hiatiire can hardl3^ be discerned. Occasionally 
one finds indiyidiials whose endosarc clearly shows the alveoles, 
especially when stained in tofo with Delafield's haematoxylin 
(Figs. 15, PL XIV, 87. PI. XX), or in section by Ehelich's triacid 
mixture. Matee's haemalum after Flemming's fluid shows the same 
structure but less clearly. The clearest pictures of the alveoles of 
the endosarc are found in very small individuals of 0. dimidiata 
and 0. ohtrigona. The structure in 0. mtestincdis is the same, but 
is a little less clearly seen. 

The endoplasma diifers markedly from the ectoplasma in the 
very much smaller size of its alveoles, which are very minute. The 
nodal granules of the endosarc (Fig. 15, PL XIV) resemble in size 
the granules which lie at the nodes and along the films of the ecto- 
sarc foam. The endoplasmic alveoli are usually so minute that one 
is unable to see if any granules lie along their walls at other points 
than the nodes of the foam. When however one finds an animal 
in whose endoplasma some of the alveoles are enlarged, the walls 
of the alveoles are seen to bear frequent granules. 

The endoplasma in 0. intestinalis is more dense in the anterior 
end of the body, in front of and near the anterior nucleus. In all 
other species studied a similar greater density of the endoplasm in 
the anterior end of the body is observed, though it is less noticeable 
in the flattened species, especially in 0. ranarum. 

Endosave spherules (Figs. 4, 5, 6, 10, 11, 12, 13, 16, PL XIV). 

In the endosarc are many refractive bodies which I have called 
endosarc spherules (Metcalf 1907 a). They have been observed 
by most of those who have studied Opalina. Zeller describes little 
refractive bodies in the "parenchyma" of each of the five species of 
which he treats. These he says are slightly flattened and disc- 
shaped, and show a central dark spot which may be due to the 
presence of a central cavity or to a hollow in each face of the disc. 
He says their diameter is about 0'004 mm. 

Baefueth (1885) describes for 0. ranarum certain masses of 
"glycogen" which stain brown with iodine, and near them many 
light yellow strongly refractive drops of another substance ("fat?"). 
The latter were probably the refractive spherules. 

ToNxiGES (1898) describes minutely the structure of the spherules 
as they appear in sections of 0. ranarum stained with iron-haema- 
toxylin. I can confirm his statements that they are disc-shaped. 



Opaliua. 217 

elonpited. or irre.o-nlar in form and are of various sizes; that they 
lie usually [in this species] in a regular direction in the whole body, 
the flat side of the disc [when they are disc-shaped] being parallel 
to the surface of the body, so that in sections parallel to the flattened 
surfaces of the body one sees them almost all circular, while in other 
sections they appear almost all rod-shaped; that they appear homo- 
geneous when strongly stained with [most] aniline dyes; but not so 
with [well extracted] ii-on-haematoxjiin ; that they show an alveolar (?) 
structure; that one often finds them constricted in the middle like 
a dumb bell; that they are insoluble in alcohol, alcohol and ether, 
strong acetic acid, or wTak mineral acids; that they are soluble in 
concentrated mineral acids. I can add that they are but slightly 
colored by osmic acid; that they are insoluble in cold water; that 
the}' are insoluble in tannic acid ; that after boiling with hydrochloric 
acid or after digesting them with diastase, the solution gives no 
sugar reaction with Fehling's solution (not a very delicate test); 
that they do not stain at all wdth potassium iodide; and that for 
the most part they do not stain with iodine dissolved in a water 
solution of potassium iodide, though occasionally some of them in 
some part of the body stain a good brown with the iodine solution.') 

It is of interest that, when treated with this iodine solution, 
most of the individuals of 0. ranarum used for experiment did not 
stain at all; a few showed brown color in some large irregular 
masses which seemed to be on one side or the other of the boundary 
between ectosarc and endosarc, probably in the endosarc; many 
showed a diffuse brown stain in the endosarc in one or more regions 
of the bodj^ this diffuse stain usually not affecting the spherules, 
though in a few instances the spherules in these stained areas were 
themselves tinged with brown. Heat does not change the stain; 
adding strong sulphuric acid darkens it but slightly and does not 
give a red or violet tone. 

In 0. caudata there is no color reaction to potassium iodide; 
the reaction to iodine dissolved in a water solution of potassium 
iodide is similar to that described in 0. ranarum, except that I found 
that the whole endosarc in all individuals stained strongly and a 
more reddish brown, the ectosare, like that of 0. ranarum showing 
only a faint yellow tinge. Addition of strong sulphuric acid darkens 

') Glycogen is said to stain reddish brown with potassium iodide or with 
iodine, the color disappearing- upon heating. If sulphuric acid be added to the 
stained glycogen the color is said to become redder or show a violet tone. (See 
especially Barfurth 1885.) 



218 M. M. Metcalf 

the color of the endosarc stain but does not seem to make it more 
red or to give it a violet tone. The substance in the endosarc 
which stains seems for the most part to be in solution, though here 
and there dense irregular masses are seen which seem as if 
coagulated. The endosarc spherules also stain, but less strongly than 
the protoplasm. 

Individuals of Nyctotherus and Balantidium on the same slides 
with the 0. ranarum and 0. caudaia show no reaction to potassium 
iodide. With iodine dissolved in a water solution of potassium iodide 
they show dark brown bodies in the endoplasma, the endoplasma 
itself, like the ectoplasma, being merely tinged with yellow. 

Fischer (1905) has described a method of treatment which he 
says gives a distinctive stain for glycogen. The tissue containing 
the glycogen is fixed in absolute alcohol, sections are made by the 
paraffin method, these are brought through graded alcohols into a 
ten percent solution of tannin (to precipitate the glycogen) and are 
then placed in potassium bichromate to render this precipitate 
insoluble in water. Ofter washing the sections they are stained in 
safranin, only the glycogen bodies becoming red, the cytoplasm and 
nuclei being hindered from staining by the treatment with tannin. 
I tried this stain upon sections of 0. dimidiata. The rectum of a 
Bana esculenta was opened and the contained Opalinas and Balantidiums 
were diyided into two parts, one lot being fixed at once and stained 
according to Fischee's directions; the other lot was kept two days 
in a solution of sodium chloride until the infusoria were mostly 
inactive, then they were fixed and stained by the same method. In 
the sections of the first lot of Opalinas, killed before starving, the 
ectosarc spherules were a bright red. In most individuals the 
endosarc was wholly unstained: in other individuals the endosarc 
was strongly stained, great irregular red masses, of what appeared 
like coagulated material, completely filling it; in still other individuals 
but little color and few masses of coagulum were seen in the endosarc. 
The endosarc spherules were usually unstained but in some animals 
with well stained endosarc and coagulum the endosarc spherules 
were also stained, but showed a fainter red than the protoplasm. 

In the sections of Opalinae which were starved forty-eight hours 
before killing and staining, the ectosarc spherules were stained as 
strong a red as in the other sections. In almost all individuals the 
endosarc was wholly unstained; a few individuals, on the other 
hand, showed red masses of coagulum in the endosarc, the whole 
endosarc being stained. In all the Opalinas on this second lot of 



Opaliua. 219 

slides the eiidosarc spherules were present in their usual abundance 
but were unstained. 

In the Balantidia on both lots of slides there were abundant 
spherules in tlie endosarc which took the safranin well, each 
spherule showing a red peripheral layer and an unstained or faintly 
stained core. 

The natural interpretation of all these microchemical tests and 
digestion experiments seems to be: 1) that the endosarc of Opalina 
contains a nutrient substance abundant in freshly taken 0. caudata 
and in many individuals of 0. dimidiata and in parts of the body 
of many individuals of 0. ranarum; in starved individuals its pre- 
sence is infrequent. This nutrient material seems to be usually in 
solution in the endoplasma, though even in living animals it may 
form some irregular semi-solid masses. In some cases the endosarc 
spherules as well as the endoplasma seem to be permeated by the 
nutrient fluid, but usually they are not so. 2) The nutrient sub- 
stance is not true glycogen, but seems to be related to glycogen. 
The term paraglycogen which BtJTSCHLi has suggested seems to be 
appropriate. 3) The endosarc spherules are not oil for they do not 
stain with osmic acid or dissolve with alcohol or ether or xylol. 
4) The endosarc spherules are not lecithin for they do not dissolve 
when warmed for four hours with a mixture of equal parts of ab- 
solute alcohol and ether. 5) The ectosarc spherules for the same 
reasons are neither oil nor lecithin. 6) Probably neither sort of 
spherules contains true glycogen ; whether they contain any related 
substance is uncertain. I have failed to get a sugar reaction with 
Feeling's solution after diastatic digestion, but this test is not a 
very sensitive one. 7) The spherules of the endoplasma of Nydo- 
tlierus and Balanfidium, seem to be composed of paraglycogen. It 
is, of course, natural to suppose that those of Opalina are of a 
somewhat similar nature, but they are not exactly similar chemi- 
cally, as is shown by the difference in their reaction to iodine and 
to Fischer's glycogen stain. 

The whole subject of the nutrient fluids and refractive bodies 
in the cytoplasm of the Protozoa needs more successful study than 
it has yet received. For valuable papers upon the subject see 
Certes (1880), Maupas (1885, 1886), Barfurth (1885), Bijtschli 
(18856, 1880—1889 and 1906 i)), Stolc (1900) and Bott (1907). 

^) In some way I overlooked this valuable paper of Butschli's upon the 
paramylon bodies of Euglena, and I have not yet had opportunity to study the 
spherules in Opalina in the light of Bijtschli's vv'ork. There is much divergence 



220 M. M. Metcalf 

Contrary to Tonniges (1898) I do not find the endosarc spherules 
much, if any, more numerous near the periphery of the body even 
in 0. ranarum. Tonniges says that these spherules very frequently 
divide. According to his description they become first dumbbell- 
shaped, then still more constricted, the connecting portion becoming- 
a mere thread and then breaking, the two halves separating. He 
says that, before division, a spherule becomes smaller and more 
dense, loosing its visible alveolar structure, doubtless by exuding 
the liquid in the alveoles, and that in this condition they stain more 
strongly. In my preparations, the dumbbell-shaped spherules are not 
on the whole smaller than the others, nor do they show less internal 
organisation (Figs. 11 and 10, last two spherules, PI. XIV). 

KuNSTLEE & GiNESTE (1905) describe the endosarc spherules of 
0. dimidiata as containing a central granule. They say the spherules 
divide by constriction, the central granules first dividing. This I 
cannot confirm. 

I am unable to convince myself that the endosarc spherules 
divide. The dumbbell-shaped forms are not infrequent, but after 
long search I have not found a single spherule in which the con- 
necting portion is very slender as if ready to part. By for the 
most constricted one I have seen is shown in Fig. 11. This point 
is an important one, aftecting the question of the nature of these 
bodies, so I have studied it with care. At the time I wrote my 
preliminary paper (Metcalf 1907 a) I assumed that the frequent 
dumbbell shape indicated division, but I now think that these 
bodies do not divide any more than do the ectosarc spherules. One 
never finds two of either sort of spherule in one alveole or any 
other indication of division in them. 

The endosarc spherules are more numerous in the anterior part 
of the body, where the endosarc itself is denser (Fig. 1). 

In 0. oUrigona certain strands of minutely alveolar protoplasm 
stretch out from the endosarc and, passing between the large alveoles 
of the ectosarc, join the subcuticular layer (rather poorly shown in 
Fig. 6). Along these strands, and in the subcuticular layer near the 
outer ends of the strands, one finds endosarc spherules. In no other 
species have I seen the endosarc spherules outside the limits of the 
endosarc proper. 



even among the Ciliophora as to the character of their refractive spherules, and 
it is probable that the spherules in Opalina are more or less different from those 
in Etiglena. 



Opalina. 221 

The endosarc spherules stain well intra vitam with neutral red, 
methyl violet, dahlia, and gentian violet. This fact makes it doubtful 
if they are living constituents of the cell. 

The si)herules of the endosarc are so similar, in all the species 
I have studied, that no distinctions of size, structure or reaction to 
stains can be described. The questions of the origin, nature and 
function of these spherules will be further discussed after the description 
of the nucleus has been given. 

One must agree with Tonniges (1898) that there is no indication 
that the endosarc spherules are either excretory or parasitic. To 
his interpretation of them as a diifuse macronucleus we will refer 
again. 

CoNTE & Vaney (1902) believe that the spherules arise in the 
nucleus from chromatin and wander out into the cytoplasm through 
the nuclear membrane. They think that they are similar to zymogen 
granules in gland cells and to yolk nuclei. To this we will return 
again. 

Maier (1903) fails to confirm Tonniges' description of internal 
structure in the endosarc spherules, saying that they are homogeneous. 
It must be that in the sections upon which this statement is based 
the haematox^iin (Haidenhain's) was insufficiently extracted {cf. 
Fig. 4). Sufficient extraction of the stain always shows the internal 
structure. 

Leger &: DuBoscQ (19046) figure certain apparently similar 
bodies in what seems to be a microgamete of 0. saturnalis (my 
Text Fig. XVII, page 338), and their Fig. 3, representing an optical 
longitudinal section of a full grown form of this species, shows in 
the endoplasma deeply staining bodies of the right size and form to 
represent endoplasmic spherules, yet these authors say that the 
endoplasma is without particular inclusions, though showing here 
and there small spherical vacuoles with very sharp contours. This 
appearance of vacuoles is exactly what is seen after staining with 
borax-carmine, Mater's or Delafielu's haematoxylin, or any of the 
numerous dyes which do not color the endoplasmic spherules. 

KuNSTLER & Gineste (1905) interpret the endosarc spherules as 
a "secretory apparatus". 

Nekksheimer (1907) did not see any alveolar structure or any 
division stages in the endoplasmic spherules. He suggests that 
C'onte & Vaxey's description of the origin of the spherules from 
the nucleus may indicate that they saw the formation of reproductive 
chromidia, a process which Neresheimer describes at length, I feel 



222 M. M. Metcalf 

confident, however, that Conte & Vanet, who worked on 0. intesfinalts, 
refer to certain very evident chromatin spherules in the nucleus, 
which will soon be described. 



JExcretory ortfans 

(Figs. 1, 17, PI. XIV; 97, PL XXI; 248—250, PI. XXVI). 

A system of excretory vacuoles is present in the axis of the 
body. I have published a description of these organs (Metcalf 
1907 & and c) for the full grown forms of 0. iniestinalis, 0. caudata 
and 0. oUrigona and for the small spring individuals and the 
macrogametes (not the microgametes) of 0. infestinalis, 0. caudata and 
0. dimidiata. Eeference must be made to this very primitive excretory 
organ when we discuss the relationships of Opalina, so I include here 
a few figures showing its character, and summarize the chief points 
in the published description. 

In 0. intestinalis (Fig. 1) the excretory organ, when highly 
developed, consists of an axial series of more or less irregular fused 
vacuoles, opening to the exterior by a transient aperture at the 
posterior end of the body, and stretching forward usually as far as 
the posterior nucleus, or often nearly to the anterior end of the body. 
In its course, as it passes the posterior nucleus, it lies close against 
the nuclear membrane, usually bending spirally around it. It often 
has a similar relation to the anterior nucleus. Frequently the series 
of fused vacuoles branches behind the posterior nucleus, the branch 
running along another side of the posterior nucleus, which is thus 
almost enveloped by the excretory organ. I have recently found full 
grown 0. intestinalis and 0. caudata in which the excretory organ 
stretches as far forward as the anterior nucleus. Heretofore I had 
seen the elongated excretory organs only in small forms in the spring. 

Generally the posterior end of the organ in 0. intestinalis shows 
one or more enlargements of considerable size surrounded by un- 
usually large granules in their walls (Fig. 97, PI. XXI, also cf. 
Metcalf 1907 b). Usually one sees a mass of such larger granules 
in the cavity of the posterior chamber. These granules stain some- 
what differently from the ordinary endoplasmic granules with most 
stains. They are from time to time extruded from the excretory 
aperture at the posterior end of the body and are cast away. One 
often sees individuals dragging after them a mass of these extruded 
granules (Figs. 248, PI. XXVI, 147, 153, PI. XXII). 



Opaliua. 223 

In 0. candaia the conditions are very nearly the same, but the 
posterior end of the organ is usually branched, one or two shorter 
branches being seen in addition to the chief branch which runs 
forward along: the axis of the body. The relation to the nuclei is 
like that in 0. intcsiinalis and the excretory granules are similar. 

In 0. dimidiata (Fig. 17) the conditions resemble those in 
0. infest inaJis;. In this s{)ecies there are many nuclei. One often 
sees that most, if not all, of these nuclei are surrounded by narrow 
perinuclear vacuoles. Many of the posterior nuclei are enveloped 
by the excretory organ. Probably by no means all of the perinuclear 
vacuoles have any direct connection with the excretory organ, but 
their contained excreta probably reach the excretory vacuoles by 
dialysis through the intermediate alveoles of the endoplasm. In 
Fig. 17. PI. XIV, which represents the posterior end of an unusually 
slender but nearly full grown 0. dimidiata, the axial series of excretory 
vacuoles is very clearly seen to consist merely of enlarged and 
irregularly fused alveoles of the endoplasm. The most posterior 
nucleus, in mitosis, is shown entirely enveloped by the excretory 
vacuoles. One often sees individuals of 0. dimidiata, especially 
small forms in the spring, dragging behind them masses of extruded 
excretory granules. 

The three species already mentioned are circular in cross section 
and have the excretory vacuoles along the axis of the body. 
(>. obtrigona and 0. ranarmn are very flat. It is possibly because 
of this flattening that their excretory organs are so much less deve- 
loped. In 0. ranarum 1 have found no trace of any excretory organ, 
though one often finds perinuclear vacuoles present. In 0. oUrigona 
there is present only a slight rudiment of the posterior end of the 
excretor}^ organ in the form of a small elipsoidal or semilunar 
vacuole at the extreme posterior tip of the body. This vacuole 
occasionally contracts and one sees the shrunken, depressed contour 
where the vacuole previously was. No excretory granules have been 
found in this vacuole, nor have I seen the living animals dragging 
a mass of extruded granules after them, as is so frequent in the 
three spindle shaped species above described. 

Excretory organs have not been seen before in Opalina, the 
genus being always described as unique among the Ciliata in having 
no excretory vacuoles. 

The condition of the excretory canals in the new Ciliate Pycnothrix 
monoajstoides, described by 8chubotz (1908) is very interesting 
in comparison with the cylindrical Opalinae. This simple axial 



224 M. M. Metcalf 

system of irregular, branching canals is more developed than the 
excretory organ of Opalina in having 1) a definite limiting membrane, 
2) a permanent external aperture, and 3) cilia lining the outer 
portion of its duct, and also in being evidently a permanent organ 
of the cell. 

In my first paper on the excretory organs of Opalina (Metcalf, 
1907 &) I wrote: "Under pressure from a cover-glass, in gradually 
drying preparations, oil globules are generally protruded from the 
body at different points on the periphery. The largest of these oil 
globules is generally found at the posterior end of the body" [in 
connection with the excretory pore] "and is usually the first to 
appear, in spite of the fact that the posterior end of the body is 
the most slender part and must be the last to feel the pressure." 
Further observation shows that I was mistaken in describing any 
special connection between the excretory pore and an especially 
large drop of this exuded liquid. Often a drop is found here and 
it may be large, but observation of a much larger number of Opalinae 
under pressure shows that it was an error to emphasize the size 
and early appearance of this drop. The exuded liquid is not oil, 
but is either the protoplasm itself, or is derived from the protoplasm. 
It cannot be derived from the ectosarc spherules, for similar exuded 
drops are found in Ciliaia which have no ectosarc spherules {cf. also 
KoLSCH 1902). 

Nucleus, and mitosis. 

Few if any known nuclei among the protozoa are clearer and 
better for study than those of 0. intestinalis and 0. caudata; the 
nuclei are large and the chromatin is small in amount and does not 
obscure the achromatic structures; the chromosomes are few in 
number, eight in 0. intestinalis and six in 0. caudata; all the structures 
usually found in typical nuclei, including a plasmosome nucleolus, 
are present and stain readily and distinctively; and, as already 
mentioned, all the structurec in the nucleus are sometimes very 
clearly seen in the living animal. I have therefore given chief 
attention to the nuclear phenomena, especially those of mitosis. 

Opalina intestinalis has usually two ovoid nuclei lying in the 
anterior half of the body, sometimes in the anterior third (Fig. 1 
and PL XVII, Fig. 38). The ends of the nuclei which are turned 
towards each other are generally more or less pointed. Commonly 
these pointed ends are connected by a more or less elongated delicate 
strand consisting of the attenuated nuclear membrane, which was 



Opalina. 225 

constricted in the middle at the hist division and diawn out to a 
thread (PL XVI, Figs. 34, 35, 37). This thread persists for a long 
time, disappearing, generally', as the two nuclei are entering upon 
the next division. Frequently the connecting thread is much bent 
or even coiled, being far longer than the shortest line between the 
two nuclei (Figs. 35 and 37), a condition which suggests that the 
thread elongates by its own growth. 

The two nuclei divide at the same time, becoming first spindle- 
shaped, then dumbbell-shaped, and finally separating into two 
daughter nuclei which are still for a long time united by the thread 
wliich indicates their common origin. While the division of the two 
nuclei is occurring, the body divides (PI. XVII). This is usually during 
the anaphases, but one often finds the body still but partially divided 
when the nuclei are entering on the telophases. One often sees a 
daughter cell with only a single nucleus, but this, if normal, is always 
in an anaphase or early telophase stage of division (Figs. 32, PI. XVI; 
43, PI. XVII; 54, PI. XVIII). It is during the early telophase that the 
nucleus constricts into two (Figs. 32, PI. XVI; 60—65, PI. XIX). 

The nuclear membrane is very definite and clear, not thick, 
but very firm and strong. This is indicated by the fact that the 
connecting strand between the daughter nuclei persists for so long 
a time. It is seen even more clearly when the living animals are 
crushed by pressure upon the cover-glass, causing the nuclei to come 
out into the surrounding salt solution. Such isolated nuclei, even 
when connected by very slender threads, one seldom succeeds in 
causing to break apart by the most violent currents one can produce 
by pressing upon the cover-glass. Often one of the two united 
nuclei will be held in place by its connection with the broken body, 
while the other nucleus projects into the clear liquid. One can then 
make it jerk about and tug violently upon the thread that holds it, 
yet without breaking the thread. I kept one such pair of nuclei 
for three days, trying several times daily to break the thread by 
violent currents, but even the third day it held as firmly as ever. 
It is very evident that under this severe treatment the thread con- 
necting the nuclei does not stretch. It seems not to be at all elastic. 

CoNTE & Vaney describe the endosarc spherules as arising from 
the nucleus, from which they emerge through an opening in 
the membrane. We wdll return again to this point. It is well 
here merely to emphasize the remarkable toughness of the nuclear 
membrane, which could be penetrated only with the greatest difficulty, 
unless it were weakened (chemically?) at some point. 



226 M. M. Metcalf 

The nuclear membrane never disappears even during mitosis. 

The nuclear membrane shows no structure. Under all conditions, 
whether living, or after treatment with acetic acid, silver nitrate, 
or fixing agents without staining, after all sorts of staining in total 
preparations or in sections, one finds it always appearing homogeneous 
and uninterrupted. There are no indications that the achromatic 
structures in the nucleus are in any way continuous through the 
nuclear membrane with the structures of the cytoplasm. Of course 
in each division the membrane is ultimately broken at the point of 
constriction, but this break occurs in the slender connecting thread 
at a distance from the cavities of the daughter nuclei and there are 
no wounds at the surface of either nucleus. 

The nucleus lies in the cytoplasm, as it were in a great alveolus, 
being suspended and held in place by the films of the cytoplasmic 
foam. Fig. 205, PI. XXIV, gives a clear picture of this condition in 
the case of a male pronucleus in a zygote of 0. intestinalis. 



The resting nucleus (PI. XX, Figs. 76 and 77). 

The phrase "resting nucleus" of course does not imply that the 
nucleus is inactive physiologically, but only that it is not engaged 
in the movements which constitute or accompany mitosis. One can 
hardly speak with propriety of such a resting stage in the nuclei 
of 0. intestinalis, for there seems to be no time throughout the year 
when changes in their visible structure are not constantly occurring. 
The divisions of the nuclei and of the body never cease, and every 
nucleus seen is either in actual division or is preparing for or 
recovering from division. There is no condition in which the nuclei 
seem to pause for any prolonged period. The stage which corresponds 
to the ordinary resting nucleus of metazoan cells is probably that 
in which the chromatic network is most branched and diflfuse. I will 
begin the description with the stage just prececding the formation 
of the mitotic spindle. 

Achromatic foam. 

The whole space within the nuclear membrane is seen to be 
filled with alveolar protoplasm, the alveoles in many places being 
fused to form vacuoles of different sizes (Fig. 77. PL XX). In many 
other nuclei the alveoles are not fused but are of fairly uniform 
size (Fig. 69, PL XIX). Granules of varying sizes and irregular 



OiKilina. 227 

shape lie at tlie nodes of the foam. 'J'hese are higlily refractive in 
the living nucleus (PL XVI). Their reaction to stains shows them 
to be of achromatic, not chromatic, material. The granules not only 
differ in size in the same nucleus, their average size in different 
nuclei vai'ies perceptably. They seem largest in nuclei in which 
the spindle is forming preparatory to mitosis (Fig. 47, PI. XVIII). 
The lines uniting these granules (optical sections of the walls of 
the alveoli) generally show very clearly in well-stained nuclei both 
in preparations of total objects and in sections. The lines are 
not discernable in living nuclei, at least with the illumination I 
have used. 



Nucleolus (Figs. 18, 19, 22, 25, 27, 29, PI. XV; 55, 56, PL XVIII; 

Fig. 73, PL XIX). 

The always spherical, or nearly spherical, nucleolus belongs to 
the achromatic group of nuclear structures. It is always present 
in fully formed nuclei and lies near the axis of the nucleus, never 
at the surface. It is held in an alveolus of the achromatic foam 
(Fig. 55, PL XVIII), completely filling this alveolus, so that the 
films of the foam are seen radiating from its surface. Where these 
strands touch the surface of the nucleolus they are seen to enlarge 
to form typical nodal granules, triangular in optical section, as are 
many of the other nodal granules. 

The nucleolus stains strongly with plasma stains. One does 
not find it in total preparations stained with borax-carmine, but in 
many Delafield haematoxylin preparations it shows very distinctly 
and is sharply distinguished from the chromatin by its fainter color 
and browner tone. In other Delafield haematoxjiin preparations, 
which are not so well decolorized, one often cannot distinguish the 
nucleolus from the chromatin. The most selective and distinctive 
stain for the nuclear structures is safranin followed by light green 
(Lichfgriin). The chromatic elements take the safranin strongly 
while the achromatic elements are green. With this stain the 
nucleolus is a clear bright green and is a very conspicuous object, 
for it is large (Figs. 22, 25, 27, 29, PL XVj. Often with light 
green, and still better with Delafield's haematoxylin, one sees that 
the nucleolus is not homogeneous, from one to ten or more circular 
lighter areas being visible within it (Figs. 55, 56, PL XVIII; 73, 
PL XIX). Generally the more central light spot appears the larger. 
These vacuoles (?) are generally of different sizes in the same 



228- M. M. Metcalf 

nucleolus and their averag-e size may be different in different 
nucleoli. As a rule the size of the vacuoles is in inverse proportion 
to their number. 

Sections stained with methylen blue often show an interesting' 
condition in many nucleoli (Figs. 18 and 19, PI. XV). The nucleolus 
proper is stained a bluish green. This portion is spherical. In one 
or two regions on its periphery, it bears cap-like structures which 
are stained a clear blue darker than the pale blue of the nucleolus 
proper. This is no accidental condition, for it is present in almost 
all nuclei seen upon these slides which were made from two diffe- 
rent lots of Opalinas. Vacuoles are not seen in the nucleoli which 
show these blue caps, though in other nucleoli upon the same slides 
vacuoles are found. The history of these peculiar nucleoli has not 
been followed, so nothing can be said as to the meaning of the 
conditions found. 

The behaviour of the nucleolus in dividing nuclei is interesting. 
Zellee observed that in dividing nuclei the nucleolus did not divide 
but remained intact in one of the daughter nuclei, the nucleolus of 
the other daughter nucleus being a new structure. I can fully con- 
firm this for 0. intestinalis and 0. caudata. The nucleolus is less 
easy to see in the smaller nuclei of the multinucleated species, and 
as my material of the multinucleated forms shows comparatively 
few nuclei in division I have not taken the considerable time re- 
quired to study the nucleoli carefully in them. 

To Zeller's interesting observation I would add the further 
facts: — first, that in 0. intestinalis the old nucleolus remains in 
the posterior of the two daughter nuclei (Figs. 70—72, PL XIX), 
and second, that in 0. caudata this relation is usually reversed, the 
old nucleolus remaining generally in the anterior daughter nucleus 
(Fig. 82, PI. XX). In the many hundreds of nuclei of 0. intestinalis 
examined I have found but a single exception to this rule (Fig. 74, 
PL XIX). In this young daughter cell whose nucleus is still in the 
dumbbell stage of division, the nucleolus was found in the anterior 
part of the nucleus near the constriction. The narrow tube connec- 
ting the daughter nuclei was not too small to allow the nucleolus 
to pass through it and reach its usual position in the posterior 
daughter nucleus, but that the nucleolus would have done so does 
not seem very probable. 

In a large majority of cases, in dividing 0. caudata the old 
nucleolus remains with the anterior daughter nucleus, yet one occassio- 
naUy finds these relations reversed. 



Opaliua. 229 

One of course must inquire as to tlie meaning of this puzzling 
ditference between the two species. I can suggest no adequate ex- 
planation. 'I'he nuclei lie much further forward in 0. iniestinalis 
than in 0. caudata (compare Fig. 38, PI. XVII, with Pig. 81, PI. XX). 
It suggests itself that the nucleolus in both species may remain in 
the nucleus which is nearer to the center of the body, or rather 
nearer to the protO))lasmic rather than the geometric center of the 
body. The geometric and protoplasmic centres are not the same, 
for the protoplasm in the anterior part of the body is more dense 
than that further back. This suggestion fits the conditions in 
0. intestinnlis and, for the most part, also, the conditions in 0. cau- 
data. If one takes into account the greater density of the anterior 
part of the body, it is true that in 0. caudata in the cases in which 
the old nucleolus remains in the anterior daughter nucleus it is 
nearer the center of the protoplasm. I have looked through many 
preparations, comprising many hundreds of 0. caudata, to see if in 
cases in which the old nucleolus remained in the posterior daughter 
nucleus the nuclei were unusually far forward, so that the proto- 
plasmic center might in these cases be nearer to the posterior than 
to the anterior nucleus. In the majority of instances of this sort 
it was found that the nuclei were unusually far forward, almost as 
much so as in 0. intestinalis, but, unfortunately for my suggestion, 
no less than six instances were found in which the old nucleolus 
remained in the posterior daughter nucleus, although this was placed 
not only not exceptionally far forward but even unusually far back. 
I believe, therefore, that there is probably no worth in the suggestion 
made. 

The new nucleolus in the daughter nucleus of 0. intestinalis 
arises always at the pointed end of the nucleus near the thread 
which connects it with its sister nucleus (Fig. 72, PI. XIX). At 
first it is very small. It grows rather slowiy, but by the time the 
nucleus is ready for its next division the nucleolus is again of 
full size. 

I have little suggestion to make as to the nature or function 
of the nucleolus. I would merely emphasize 1) that it is wholly 
distinct from the chromatin elements and never at any time has 
any discernable genetic relation to them; 2) that a nucleolus once 
formed persists throughout the year and until the nucleus con- 
taining it is ready to throw off its vegetative chromidia and enter 
upon sexual reproduction (processes which will be described later). 
I have not found nucleoli in the nuclei of any forms between the 

16* 



230 M. M, Metcalp 

time of extrusion of the veg-etative chromidia and copulation, 
though I have stained sections of all of these forms with safranin 
and light green, which gives such clear pictures of the nucleolus. 
In at least some zygotes which have grown a little since copulation, 
the nucleoli are seen. Its apparent absence from those nuclei which 
have recently cast oif vegetative chromidia, and are probably but 
slightly active in nutrition, seems of much interest, though I would 
not venture to suggest what may be the real meaning of this 
relation. In the case of large dividing individuals in the summer, 
fall and winter, the new nucleolus appears in the anterior daughter 
nucleus at the time when this nucleus is forming the chromatin 
spherules, which seem to be essentially vegetative chromidia. These 
phenomena will soon be described. It seems probable that the 
nucleolus has some connection, not necessarily causal, with the 
nutritive activities of the nucleus. 3) In the third place I would 
emphasize that the new nucleolus, when it arises, seems to come 
from some substance in liquid form in the nucleus, and not from 
the immediate transformation of any previously visible structures. 
4) The old nucleolus does not grow beyond a certain size, though 
it may persist for nearly a year. The new nucleolus, after each 
division, grows to the same size as the old and then stops its 
growth. This suggests that there is some balance between the 
nucleolus and the other structures of the nucleus (or cytoplasm?) 
which requires the presence in an ordinary fully-formed nucleus of 
a nucleolus of a given size. The diminution of the vegetative 
chromatin, preceeding and during the period of conjugation, seems 
to do away with the necessity of a nucleolus during that time, or 
to remove something which if present would have caused a nucleolus 
to form. 

The various stages in the growth of the new nucleolus are of 
great assistance in determining the sequence of phenomena in the 
telophases of mitosis. Until this criterion was found it was almost 
impossible to be certain of the relative order in mitosis of several 
of the stages observed. 

This description of the condition and behavior of the nucleolus 
in 0. intestinaUs is based upon series of preparations of animals from 
many ditferent hosts. I have since studied a series of preparations 
of apparently normal Opalinas from an apparently normal Bombinator^ 
in which the nucleolar relations are quite ditferent. These Opalinas 
were killed at once upon opening the rectum of the host so that 
the divergent condition of the nucleolus is not due to degenerative 



Opalina. 231 

changes caused by living- in cultures. In all of these animals, when 
binucleated, one nucleus contains a nucleolus and the other does not. 
Generally the nucleolus is in the posterior nucleus, but in S^o of 
the binucleated forms the nucleolus is in the anterior nucleus. All 
of the forms found with the nucleolus in the anterior nucleus were 
antei-ior daughter cells formed by transverse division indicating 
probably that about 16% of the divisions are transverse. 

The conditions in these Opalinas show that the old nucleolus 
does not persist. It diminishes and disappears just before, or during, 
or some times just after, the spireme stage, which in these animals 
is less marked than usual. It reappears generally at about the 
time the spindle is forming for the next division, or before. 
Occasionally it does not appear until the spindle is formed. We 
find therefore some animals with no nucleolus in either nucleus 
(spireme stage), some (8 %) with a nucleolus in the anterior nucleus 
only, and the rest with a nucleolus in the posterior nucleus only. 

The discrepancy between the different series of preparations as 
to the condition of the nucleoli necessitates more careful study of 
this subject. My notes upon the several sets of preparations do not 
say whether the hosts had been starved, for a time or not. Until 
this relation has been carefully, observed one cannot be certain which 
of the nucleolar phenomena are normal and which abnormal. Possibly 
all are normal, varying with the conditions of nutrition. It is but 
a surmise that the conditions of nutrition explain the divergent 
conditions of the nucleoli, but it seems the most probable explanation 
Opalina must be very sensitive to surrounding conditions, and it is 
possible that in this genus we have an opportunity to study, with 
unusual hope of some success, the problem of the function of the 
plasmosome nucleolus. 

Zellek saw and clearly described and figured nucleoli in all 
stages from the cysts to the full grown forms, in all five of the 
species he studied. He even figures a central dot in the nucleolus, 
which he calls a central vacuole. It is barely possible that in the 
cysts and in certain small forms he has mistaken certain chromatin 
masses for the true nucleoli, but there is no doubt that, at least in 
the case of full grown forms, he has described the true plasmosome 
nucleolus. This is easily demonstrated with acetic acid, which is the 
reagent he chiefly used. 

Since Zellee, no student of the Opalinae has observed the true 
nucleolus. Pfitzner (1886j, Tonniges (1899) and Leger & Duboscq 
(19046) refer to certain chromatin masses as nucleoli, but make no 



232 M. M. Metcalf 

reference to the plasmosome nucleolus. Neeesheimer (1901) says 
lie has not seen a true nucleolus, not accepting the name nucleolus 
as applicable to the masses of chromatin described by Peitzner, 
ToNNiGES, and Legee & Duboscq. Neeesheimer is surely right in 
not applying- the name nucleolus to the chromatin masses so charac- 
teristic of the nuclei of Opalina. They seem so entirely unrelated to 
the true plasmosome nucleoli that no one term should be applied to 
both. One must admit, however, that in some other animals it is not 
easy to disting-uish clearly between plasmosome nucleoli and bodies 
related to chromatin, so that in general the word nucleolus must be 
allowed to have the broader meaning. 



Chromatin. 

The chromatic material of the nucleus in 0. intestinalis, and all 
other species studied, lies near the surface of the nucleus just beneath 
the membrane (Fig. 59, PI. XVIII).^) This seems to be true of all con- 
ditions of the nucleus except just before, during, and after encystment, 
when, in the multinucleated species, the chromatin contracts toward 
the centre of the nucleus. One sees in the resting nucleus that 
there are irregular masses of chromatin, of larger and smaller sizes, 
scattered here and there beneath the nuclear membrane (Figs. 23, 27, 
PL XV; 45—48, PI. XVIII; 76, 77, PI. XX). These chromatin masses 
are drawn out into numerous points each of which connects with a 
fiber of chromatin which runs over the surface of the nucleus beneath 
the membrane. These fibres branch and the branches anastomose 
with one another and with the branches of similar fibres from other 
chromatin masses (Figs. 27, PI. XV; 46, PL XVIII). In other words, there 
is just beneath the nuclear membrane a network of chromatin fibrils, 
the chromatin masses described lying upon and being in connection 
with the network. One could say that the fibres seem like delicate 
reticulate pseudopodia from the chromatin masses. 

The fibres of chromatin are sometimes quite even (Fig. 46, PL XVIII) ; 
again one finds them considerably enlarged at the nodes (Fig. 27, PL XV) ; 
in other nuclei one sees them as rows of different sized granules strung 
on a thread {cf. Fig. 50, PL XVIII, a nucleus in mitosis). These diffe- 
rences cannot be wholly due to differences in staining, but represent 
real divergent conditions of the chromatin threads during the so-called 
resting stage. The chromatic structures of the nucleus never seem 



1) Cf. ToNNiGES (1899), BovERi (1900). 



Opalina. 233 

to be arranged in the form of an alveolated foam, but are in the 
form of masses and fibrils. It is difficult Avith most dyes to distinguish 
the fliromatic fibrils from the achromatic, but doul)le staining with 
safraniu and light green gives a very clear demonstration of the 
distinctness of the two sorts of fibrils, the chromatin being red, the 
linin green. Care must, however, be taken not to extract the light 
green too much. The slides must be left in absolute alcohol but a 
moment, else the light green ma}^ be extracted from the nucleus 
and the whole endoplasma as well, remaining only in the ectoplasma. 

The chromatin masses are not homogeneous, but contain many 
granules which after staining with safraniu (Fig. 31, PL XV) iron- 
haematoxylin (0. caudata. Figs. 83, 84, 86, PI. XX), or Delafield's 
haematoxylin (Fig. 09, PI. XIX), decolorize more slowly than the rest 
of the chromatin mass. The character of these granules can best 
be discussed in connection with the later stages of mitosis. 

In the preliminary notice of this work (Metcalf 1907 «) I wrote 
"The chromatin net in the 'resting' nucleus consists of large and 
small chromatin masses and their branching anastomosing pseudo- 
podia-like processes. In certain conditions of the nucleus no such 
processes are found"'. After further study, it seems that the last 
statement is mistaken and that more or less of a network of chromatin 
is always present, though in some conditions of the nucleus it may 
be very delicate and difficult to distinguish from the achromatic 
foam even with differential stains. 



ProjyJmses of wltosls. 

One sees from the study of total preparations and of sections 
that, as the nucleus prepares for mitosis, the longitudinal fibres of the 
chromatin net become emphasized and the transverse fibrils become 
fainter (Figs. 45—52, 57, PI. XVIII). One imagines that the latter'are 
drawn in and that their substance is added to the longitudinal fibres. 
At this time the nodes that lie along the longitudinal fibres are 
especially emphasized (Fig. 50). While the chromatic spindle is thus 
forming, the longitudinal films of the achromatic foam thicken and 
the transverse films become fainter (left side of Fig. 47, PI. XVIII), 
the whole nucleus at the same time becoming elongated. 

The spindle is never regular and is hardly well enough formed 
to be comparable to the spindle in the mitosis of metazoan cells. 
Figs. 49 to 52, Plate XVIII, show it in its fullest development. The 
thicker fibres in the whole nucleus are seen to have an irregularly 



234 M. M. Metcalf 

longitudinal direction, yet ihey are always connected by transverse 
fibrils (Fig. 50). The general form of the group of fibres is spindle- 
shaped, it being thickest at the equator of the nucleus, where it 
bulges out almost or quite to the nuclear membrane. As the chief 
chromatic fibres converge toward the two poles of the nucleus they 
frequently, one can say usually, bend inward toward the axis of 
the nucleus, presenting a peculiar and very characteristic appearance 
of a spindle with acuminate ends. 

Centrosomes. 

There are no centrosomes visible either inside or outside the 
nucleus.^) The thick chromatic fibres extend to and are in contact 
with the nuclear membrane at the poles of the nucleus (Figs. 51, 55, 
PL XVIII). Usually these fibres are somewhat swollen at or near 
their ends, forming granules of quite noticeable size (Figs. 51, 57, 
PI. XVIII). There are no special aggregations of achromatic material, 
either granular or fibrous, at the poles of the nucleus. None of the 
structures found can be interpreted as a centrosome. 

Equatorial lylate stage. 

These is no well defined equatorial plate stage in the mitosis. 
The chromatin masses make an irregular group scattered through 
the whole equatorial third of the nucleus (Figs. 45—48, PI. XVIII). 
There is at this time no indication of any longitudinal splitting of 
the chromosome masses. If it occurs at all, it has occurred previous 
to this stage. 

Anaphases, 

This very irregular and imperfect equatorial plate stage soon 
passes into an early anaphase condition in which one sees the 
chromatin masses arranged in two transverse rows (Figs. 49 — 52, 
PL XVIII). These masses are still united one to another by the 
longitudinal fibrils and one often finds this connection so definite as 
to suggest that the masses so united in pairs are products of a 
transverse division of the chromatin masses of an earlier stage. 
Probably some have recently divided, but others divide at a con- 
siderably earlier stage, before the spindle is formed. Some of the 



1) Cf. Pfitzner (1886), Tonniges (1899), L^ger & Duboscq (1904 &) aud 
Nekesheimer (1907). 



Opalina. 235 

masses of chromatin may be slower than the rest in coming to 
the center of the nucleus and in taking their place in this double 
transverse row, but in the end all do so. 

Each chi-omatin mass is connected with the pole of the nucleus 
by one, or sometimes by two, of the thicker chromatin fibres (Fig. 50). 
The chromatin masses of the two groups are also connected with 
one another by thick fibres which cross the equator of the nucleus. 
Occasionally one sees a fibre start from a chromatin mass, cross 
the equator of the nucleus and pass on directly to the opposite pole 
without connecting on the way with a second chromatin mass (Fig. 50). 
This, however, is not very general. One may say that the chro- 
matic spindle is composed of fibres which in general stretch from 
pole to pole of the nucleus and connect in their course with one or 
two chromatin masses. The fibres may branch and unite in a more 
or less irregular way. 

The chromatin masses now begin to migrate toward the poles 
of the nucleus (Figs. 53 — 55, 58, PI. XVIII). During these migration 
stages, one often sees that the chromatin fibres connecting the chro- 
matin masses with the poles are thicker, while the fibres stretching 
across the equator are fainter (Fig. 54, PI. XVIII; 0. candata, Fig. 81, 
PI. XX). During this migration the chromosome masses assume more 
definite shape, becoming in general more elongated (Fig. 58). They 
lie side by side and because of their regular arrangement and com- 
pact form are best studied at this stage. They seem to be true 
chromosomes. They keep their regular parallel position during the 
whole migration, but, as thy approach the pole, first some and then 
others may divide into two (Fig. 54), and at about the same time 
they send out thin broad plates of chromatin which unite them 
together {cf. the upper end of the nucleus in Fig. 58). The stage, 
then, when the chromosomes can be counted and studied to best 
advantage, is but a brief one during the middle portion of the 
migration. As some chromosomes maj^ be late in coming into the 
double transverse equatorial plate, and as others may divide pre- 
cociously during the migration, one finds, in certain nuclei, conditions 
that are very confusing. I have, however, carefully counted the 
chromosomes in more than a hundred favorable nuclei in the middle 
anophase condition and find their number to be eight for 0. in- 
testinalis (Figs. 32, 33, 36, PI. XVI; 58, PI. XVIII; 65, PI. XIX; 
119, PI. XXII, anterior nuclei of 201-203, PI. XXIV). The apparent 
exceptions are due, I believe, wholly to precocious division of the 
chromosomes, or to their precocious fusion by means of the band- 



236 M. M. Metcalf 

sliaped pseudopodia. In some dumbbell-shaped nuclei one sees, in 
one end, the chromosomes already thus fragmented or fused, while 
in the other end of the same nucleus the eight chromosomes are 
compact and distinct (Fig. 34, PI. XVI; 63, PI. XIX). 

The chromosomes, even in their most compact condition, give 
off numerous threads which connect with the general chromatic net- 
work. Unless the staining is satisfactory the threads themselves 
are sometimes difficult to see, but one rarely fails to notice upon 
the surface of the chromosomes the pointed protrusions with whicli 
the threads connect. 

The chromosomes differ in size and form and in the number of 
granules they contain. After much study I must confess that I am 
not sure whether these differences are constant. The granules are 
so small and, especially in this stage, so difficult to see, that the 
margin of error in the count in a single chromosome is greater than 
the difference betwen the numbers in different chromosomes. In the 
first attempts to count in different nuclei the granules in the chromo- 
some which is the first to divide during the telophase, the numbers 
so nearly agreed as to give hope that evidence from this source 
would prove valuable, but further study has rendered the whole 
matter so doubtful that it is best to say nothing further of it. 
Similarly, after prolonged study of the form and size of the chromo- 
somes, I feel that it would be unsafe to express an opinion as to 
the constancy of these characters. It is true that there is usually 
a remarkable degree of resemblance between the chromosomes of 
the two ends of the same nucleus in their size and form and in 
the time and manner of their division or fusion in the early telo- 
phase. It is also true that one finds nuclei in different animals 
whose chromosomes show equally remarkable resemblance in these 
regards. Yet the whole series of phenomena is so often confused 
by the early appearance of division or fusion in the telophase and 
the precocious appearance of the characteristic differences between 
the anterior and posterior nuclei, that is seems unsafe to conclude 
from the resemblance referred to that the chromosomes have con- 
stant and characteristic differences from one another, I incline to 
that belief, but cannot quite convince myself. It would be easy to 
give rather convincing drawings, if only the most favorable nuclei 
were selected, but the study of hundreds of nuclei shows the 
conditions to be too various for satisfactory solution of the 
question. 



Opalina. 237 

Teloiyhases. 

When tlie chromosomes liave passer! almost to the poles of the 
nucleus, they stop their migi^ation and enter upon the chanj^es of 
the telophase. These changes affect both the chromosomes them- 
selves and the fibrillar portion of the chromatin, as well as the 
achromatic films. The chromatin fibres in the equatorial area are 
alread.y, or soon become, more slender and more branched. The 
libi-es uniting the chromosomes to the poles of the nucleus somewhat 
more tardily undergo the same change. The longitudinal films of 
the achromatic foam similarly become less emphasized until one sees 
no distinction between the longitudinal and transverse films. The 
nucleus has now wholly lost its longitudinally striated appearance. 

Two sets of changes occur in the chromosomes, they constrict 
transversely, and they fuse. The transverse constriction of the 
chromosomes is very frequently seen during the telophases (Figs. 53, 
54, 58, PI. XVIII). They do not all constrict transversely at this 
time for one never finds a nucleus in the condition which would thus 
result. In many cases no transverse constriction occurs until later. 
In one of the chromosomes, the first to so constrict, the division is 
very unequal, the larger moiety lying toward the equator and the 
smaller toward the pole (Figs. 53, ^) 54, PI. XVIII). The two parts 
are generally clearly seen to be united by a distinct thread resembling 
one of the thick fibres of the chromatin spindle. Another of the 
chromosomes divides more nearly equallj^, the slightly smaller moiety 
being toward the pole of the nucleus. As already, said this trans- 
verse constriction of the chromosomes does not always occur before 
their fusion. One cannot say which is the proper and which the 
divergent time relation between these two sets of phenomena, trans- 
verse constriction and fusion. 

The chromosomes unite by sending out thin plates of chromatin 
which pass from one chromosome to the next (Figs. 58, PL XVIII; 
60—63, PI. XIX). At first perhaps but a single pair will unite 
(Fig. 58), then others will become connected. There may thus arise 
a very irregular complete ring of chromatin just beneath the nuclear 
membrane '^) {cf. 0. caudaia, Fig. 82, PI. XX). This fusion may begin 

^) This figure shows the only exception I have found to the rule that the 
smaller moiety of the chromosome first to constrict lies toward the pole. In the 
posterior end of the anterior nucleus the smaller moiety of the divided chromosome 
is nearer the equator. 

^) LtGER & Duboscq's Fig. 19 is interesting in this connection {cf. Text 
Fig. IV H, page 249). 



238 M. M. Metcalf 

during the early telophases, before the elongated nucleus has become 
dumbbell-shaped (Fig. 58), or may not have occurred by the time 
the daughter nuclei are quite distinct (Fig. 65, PI. XIX). 



Spire^ne. 

Ultimatly a condition is reached in which the chromosomes are 
completely united to form a long ribbon coiled irregularly over the 
surface of the nucleus, beneath the nuclear membrane (Fig. 34, lower 
half, PI. XVI; 67, PI. XIX). Along the course of this ribbon, from 
very many points, threads run out to join the chromatin network. 
In the preliminary notice of this paper (Metcalf, 1907 a). I referred 
to the chromatin ribbon as a more or less compact mass. The more 
compact condition is found in animals which have been kept for a 
time in cultures, and is probably slightly abnormal. 

Resting nucleus. 

Later the ribbon breaks up into band-shaped portions of un- 
equal size, varying in number from six to sixteen (Figs. 68, 70, 
PI. XIX; 76 — 79, PI. XXj. Either at first, or at some time before 
the next division of the nucleus, the chromatin masses become 
sixteen in number (Figs. 35, 37, PI. XVI), for we find sixteen of 
them, arranged in two rows of eight each during the next anaphase, 
as described (Fig. 36, PI. XVI). Often, in nuclei in which the 
spindle is beginning to form, sixteen chromatin masses (chromosomes) 
can be counted (Fig. 35). In other similar nuclei fewer chromatin 
masses are found, the division of some of them evidently being 
retarded (Figs. 46—48, PI. XVIII). 

The constriction of the nucleus into two daughter nuclei con- 
nected by a thread occurs before the complete fusion of the chromo- 
somes into a ribbon. By the time this ribbon has broken into its 
smaller portions the two nuclei are far separated in the cell, being 
connected only by a very slender thread consisting of the attenuated 
nuclear membrane. 

After the separation of the chromatin ribbon into its several 
portions, the spindle for the next division begins to form as already 
described. 

The nuclear membrane has remained intact during this whole 
mitotic cycle. 



Opalina. 239 

J) iris ion of the body. 

The division of the bod}" beg:ins while the two parent nuclei 
are in a late anaphase of mitosis (Fig'. 38, PI. XVII), and the 
separation of the daughter cells, in normal vigorous animals, is com- 
plete during- the latest anaphase (Fig. 43), or less often during the 
early telojjhase, when the daughter nucleus is dumbell-shaped (Fig-. 42). 
In division one daughter cell receives the anterior nucleus of the 
parent, the other daughter cell receives the posterior nucleus, both 
nuclei soon completing their division and becoming double. The 
division of the body, after it beg-ins, occupies, in the fall and winter, 
about one day. As this begins and ends generally during the 
anaphases, it is evident that the whole mitotic cycle must occupy 
many days. Less vigorous animals, weakened by being kept too 
long in unnatural conditions outside the host, may take two or three 
days for division, or may even fail to complete the division. 

Oecasionally one sees individuals fresh from division, one side 
of whose body is drawn out into irregular strands (Fig. 43, PI. XVII). 
This appearance is explained when one observes the last stages of 
the division itself and finds the two daughter animals united by 
such strands that have been drawn out by the efforts of the animals 
to pull away from one another. Zeller described these conditions. 

Division of the body in 0. indestinalis is usually longitudinal. 
In one series of preparation of individuals which were probably 
slightly abnormal, only one of the two nuclei in each individual having 
a nucleolus, I found that the conditions of the nucleolus gave a 
criterion enabling one to estimate the relative frequency of trans- 
verse division. In individuals resulting from transverse division, 
the posterior daughter cell, when its nucleus completed its division, 
showed the nucleolus in the posterior of its two nuclei; the anterior 
daughter cell, in a corresponding stage, showed the nucleolus in the 
anterior of its two nuclei. Only young anterior and posterior daughter 
cells can with certainty be distinguished by their form and general 
appearance. In these preparations of abnormal individuals the 
nucleolar relations were, without exception, as described, in the case 
of the young daughter cells, and doubtless held good for the older 
cells. In the case of longitudinal division of the body each daughter 
cell, when its nucleus divides, shows the nucleolus in the posterior 
nucleus. Eight per-cent of the individuals on these slides show the 
nucleolus in the anterior nucleus. We can therefore estimate that 
sixteen per-cent of the divisions were transverse. Probably normal 



240 M. M. Metcalf 

individuals would show a similar proportion of transverse divisions. 
Figs. 44, PI. XVII, and 20, PI. XV show individuals in transverse 
division. Zellek describes and figures transverse divisions for 
0. iniestinalis, but does not speak definitely as to their relative fre- 
quency', though implying that they are numerous. 

In 0. caudata transverse division is of the same character and 
about as frequent as in 0. intestinalis. The longitudinal divisions 
are exactly similar in the two species. In 0. dimidiata longitudinal 
division resembles that of the binucleated species, except that in 
this multinucleated form there is no apparent connection between 
nuclear division and the division of the body. I have not observed 
transverse division in this species, but Zeller's observations show 
clearly that it occurs. I have once seen longitudinal division of the 
body in 0. zelleri: it resembled that of 0. dimidiata. In 0. oh- 
trigona and 0. ranarum one finds longitudinal, transverse and 
irregular divisions, the latter in the spring when division is very 
rapid (Text Fig. III). In all species the longitudinal divisions follow 
the main axis of the body. As this is bent, ^) the really longitudinal 
divisions, especially in the flattened species, appear to be oblique, 
as Zeller has described them. Cohn (1904) and Schouteden (1905) 
have shown that the so-called oblique division of 0. ranarum is 
morphologically longitudinal. I have studied 0. ranarum but little, 
but from observation of 0. oUrigona I doubt if the longitudinal and 
transverse divisions are so definite in their sequence as Zeller 
describes them. Reference to the figures of irregular division in 
0. ohtrigona (Text Fig. Ill) shows that in the spring one may find 
almost any sort of irregularily, even two or three entirely irregular 



^) Ciliata and Flagellata have either the body form asymmetrical, or the 
organs of locomotion asymmetrically arranged, or both, so that the animals rotate 
on their main axes as they swim, producing spiral progression, Opalina is no 
exception to this rule. The spiral motion in Opalina is caused by two factors, 
first by the bend in the anterior end of the body, second by the spiral arrangement 
of the rows of cilia. The latter is not a result of the former, for, if one should 
straighten out the bend in the body of an Opalina, the rows of cilia would still 
be spiral. Dale (1901), Wallengben (1903), quoted by Jennings (1906), em- 
phasize also the direction of the beat of the individual cilia in 0. ranarum in 
producing spiral progression, rows of cilia along the right side of the anterior 
end being said to beat forward and to the left, while the others beat backward 
(Text Fig. XVI, page 335). It seems to me these authors have failed to emphasize 
that the broad anterior end of the body in this species is bent "to the right'' as 
is so evident in other more slender species, so that the morphological anterior end 
is not the actual anterior end. The cilia in all species seem to beat nearly if not 
quite in the morphologically posterior direction. 



Opalina. 



241 















Text Fig. III. Outline drawings of 0. obtrigona showing the irregular divisions 
in the spring, previous to the formation of the infection cysts. In each figure the 
anterior end is uppermost. Transverse division A — H and M—P. Oblique 
division Q. Longitudinal division beginning as a perforation of the central part 
of the body Y. Longitudinal division beginning at the posterior end of the 
body X Three divisions occurring at the same time 0, Q. 



242 ^^- M. Metcalf 

divisions occurriug at once {Q}. Sometimes the division furroM^ begins 
in the middle of the body, instead of at the edge, and spreads to 
the edge (F). Tonniges (1899) has described exactly parallel phe- 
nomena in the irregular divisions of 0. ranarum in the spring. 

Were the divisions of the body always as regular as Zellee 
describes, one would be tempted to compare them with the regular 
divisions of an ^g^, in which each division plane has a definite and 
predictable direction. Vegetative division in most CUiata is trans- 
verse ; in the Flayellata it is longitudinal ; in Opalina it is generally 
longitudinal, sometimes transverse, and, in the multinucleated flattened 
species, sometimes irregularly oblique. 



Thne of aiypearance of the new nucleolus. 

We have already seen that the nucleolus in the parent nucleus 
does not divide, but remains in one of the daughter nuclei, the other 
daughter nucleus acquiring a new nucleolus. This new nucleolus 
appears near the pointed end of the nucleus, where it narrows to 
the thread which connects it with its sister nucleus (Fig. 72, PL XIX). 
The new nucleolus does not appear in the daughter nucleus until 
the chromatin ribbon is ready to break up, or has already broken 
up, into separate chromatin masses. It increases and is of full size 
by the time the new spindle for the next division begins to form. 

Differences between the two nuclei. 

Very often the two nuclei are not in exactly the same stage 
of mitosis. Frequently one half of the dividing nucleus will have 
its chromosomes all distinct, while the other half shows them be- 
ginniDg to unite by means of band-shaped pseudopodia (Fig. 58, 
PI. XVIII; 34, PL XVI); or one nucleus may show a complete 
chromatin ribbon while the other has its chromatin ribbon already 
broken into a number of pieces {cf. Fig. 37, PL XVI, in which one 
nucleus shows fourteen (?) chromatin masses, while the chromatin 
ribbon of the other nucleus has not yet completely divided). In 
veiy many cases in nuclei which are forming and casting off the 
chromatin spherules, soon to be described, one sees these spherules 
larger in one nucleus than in the other, or already separated from 
the chromatin masses in one nucleus but not in the other. In ab- 
normal nuclei of animals kept too long outside the host, there are 
often differences between the two nuclei (Fig. 95, PL XXI). Gener- 



Opalina. 243 

ally, but by no means always, it is the anterior nucleus, in 0. 
iniestinalis, which is in the more advanced condition of the two, if 
they be different, althouirh in this nucleus the restoration of fully 
typical structure after mitosis is somewhat delayed because of the 
late appearance of the new nucleolus. One would naturally expect 
the chromatin of this nucleus to be in the less advanced condition 
of the two. if there is to be a difference. 



Chromatin sph eruJes, 

In this description of mitosis no reference has been made to 
one of the most interesting series of phenomena, i. e. the diminution 
of the chromatin by the throwing- off' and dissolving- of a large pro- 
portion of the material of the chromatin masses into which the 
chromatin ribbon constricts as described. 

Mention has been made of the granules present in the chromo- 
somes at all times. These are small, usually no larger than the 
achromatic granules of the nucleus. In addition to these, in 0. in- 
testinalis, many larger spherical granules appear during the late 
spireme stage, or as soon as the chromatin ribbon is broken into 
separate masses (Figs. 71—73, PI. XIX; 21, 22, 28-31, PL XV). 
These spherules ^) are formed at the surface of the chromatin masses 
and protrude bej'ond their contour. They are of different sizes, 
not only in the same nucleus, but upon the same chromatin mass. 
They are compact and seem homogeneous with all stains used. 
Before the spindle for the next mitosis forms, the chromatin sphe- 
rules break away from the now divided chromatin ribbon and come 
to lie free in the nucleus (Fig. 72, PI. XIX). At first they stain 
very strongly, but. by the time the spindle for the next mitosis is 
formed, they stain much more faintly (Fig. 75, PL XX, also Figs. 23, 
PL XV; 65, PL XIX; 79, PL XX). By the time the anaphase stage 
is reached they usually can no longer be recognised, though occas- 
ionally they can be faintly discerned even in the early telophases.^) 

The formation of chromatin spherules was not seen in the 
gametes or zygotes or in the gamete mother -cells. I did not 



') 111 the preliminary notice of this paper I called these structures sometimes 
spherules and sometimes spheres. It seeme best to call them chromatin spherules 
and to reserve the term chromatin spheres for certain much larger bodies formed 
in and extruded from the nuclei in the spring before the sexual phenomena occur. 

"-) Fig. 1)4, Pi. XXI. shows a pair of abnormal nuclei in the anterior of which 
are granular bodies which may be dissolving chromatin spherules. 



244 M. M. Metcalf 

observe the chromatin spherules in the early part of my study of 
Opalina in the fall and early winter.^) They were abundant in the 
late winter and early spring. I have since found them in material 
preserved in the fall. As I have not yet studied material from tad- 
poles preserved in the summer, I cannot say for how long- a period 
their formation is interrupted, though it seems probable that they 
are absent from the nuclei which have cast oif their vegetative 
chromatin (a process to be described later) and in v»^hich the ordinary 
proportions of vegetative and reproductive chromatin, characteristic 
of the vegetative phase of the life cycle, have not been restored 
by subsequent growth. 

Their fate is a little doubtful. They seem to go into solutiou. 
It is apparently these chromatin spherules which Conte & Vakey 
(1902) have desciibed as passing through the nuclear membrane into 
the cell-body and there giving rise by division to the refractive 
spherules in the cytoplasm. It seems as if they do occasionally 
pass undissolved through the ends of the nucleus at the place where 
the nuclear membrane broke in a former division. In several dozen 
instances I have found the end of a nucleus drawn out into an 
irregular protrusion and one (rarely more) body resembling a chro- 
matin spherule lying in this protrusion, apparently ready to pass 
out into the cell-body (Figs. 78 — 80, PI. XX). In some instances 
the membrane bounding the protrusion is noticeably more delicate 
than that of the rest of the nucleus. The chromatin spherules in 
these protrusions generally stain but faintly, having already some- 
what changed their character. On the other hand there were 
several times seen, in these protrusions from the nucleus, spherules 
which with iron-haematoxylin were deeply stained. Fig. 5, PL XIV, 
shows one such nucleus. The slide was well decolorized and the 
endoplasmic spherules are quite light colored, only their granules 
being dark. Even the chromosomes are lighter than usual, but the 
chromatin spherule in the nuclear protrusion, and two other sphe- 
rules at the base of the protrusion, are heavily stained. Apparently 
they stain almost as strongly as if newly formed, though this nucleus 
is in a late anaphase. Their persistence in this condition to so late 
a stage in very exceptional. 

There are from twenty to one hundred or more, of the chromatin 
spherules in one nucleus (cf. PI. XV: Figs. 21 and 22 show one 
nucleus. Figs. 28 — 31 another). If they all passed undissolved through 



^) Because my attention was not then directed to them. 



Opalina. 245 

the nuclear iiiembraue, one would surely see more frequent evidence 
of their doing so. It seems certain that only a very small proportion, 
if any. pass as solid bodies out of the nucleus. In the great majority 
of nuclei apparently none do so. 

It seems, however, not improbable that the chromatin spherules 
of the nucleus and the endoplasmic spherules may be somewhat 
related. Their staining reactions suggest this. Stained with Dela- 
field's haematoxylin the newly formed chromatin spherules are very 
dark blue; the older chromatin spherules stain less and less, and 
finally are not stained at all. The endosarc spherules are entirely 
unstained with this reagent. Similarly with safranin and light green 
the chromatin spherules, if newly formed, stain deep red; older 
chromatin spherules stain more faintly, and in nuclei in which the 
new spindle has appeared they are either very faint or have already 
disappeared. With the same dye the endoplasmic spherules are 
colored a very faint pink, resembling the almost dissolved chromatin 
spherules within the nucleus. As the chromatin spherules loose 
their staining capacity one sees that many are growing smaller; 
some, on the other hand, in the same nuclei often seem to be 
enlarged and more diifuse. Not infrequently one finds faintly staining 
irregular masses which look like dissolved chromatin spherules filling 
several alveoli of the nuclear foam, and I believe this is the proper 
interpretation {cf. Fig. 58, PI. XVIII, in which near the centre of the 
nucleus are such faintly stained areas). Is seems well-nigh certain that 
the chromatin spherules dissolve, and it is probable that they pass in 
liquid form through the nuclear membrane into the cytoplasm. It is not 
improbable that, having reached the cytoplasm, this material reforms 
in the endoplasmic spherules. Very likely, however, material from the 
cytoplasm as well is used in the formation of the cytoplasmic spherules. 

The origin of the endoplasmic spherules from the chromatin 
spherules is by no means assured. The granules that with proper 
staining are always seen in the endoplasmic spherules, and especially 
the lines occasionally seen within them, connecting their granules, 
suggest that they are formed elements of considerable complexity. 
If they really divided, as Tonniges (1898) and Kunstler & Gineste 
(1905) describe, their interpretation as living constituents of the 
cell would seem unavoidable. I do not, however, find evidence of 
their division, the constricted portions of the frequent dumbbell- 
shaped spherules never being very slender as if about to part. 

If the two sorts of spherules be related as suggested, probably 
the material from one chromatin spherule is enough to form or aid 

17* 



246 M. M. Metcalf 

in the formation of many endoplasmic spherules; otherwise it would 
be difficult to explain the great number of the latter present in the 
body at all times. The whole body divides during each mitosis, so 
that the number of endoplasmic spherules is reduced to half. The 
chromatin spherules are formed during each mitotic cycle, but there 
are rarely, if ever, more than one hundred and twenty in one 
nucleus, while there are many hundreds of the endoplasmic spherules 
in an oi'dinary sized Opalina mtestinalis. Kunstlee & Gineste 
estimate eight thousand for an ordinary sized 0. dimidiata, a much 
larger species than 0. intestinalis and having many more endoplasmic 
spherules. If the endoplasmic spherules are in any way derived 
from the chromatin spherules, and if they do not increase by division, 
there seems no escape from the conclusion that the material of one 
chromatin spherule suffices for many endoplasmic spherules. The 
endoplasmic spherules are larger than the chromatin spherules. New 
chromatin spherules do not continue to form in the nucleus while 
the earlier formed ones are dissolving, for one does not find them 
showing all varieties of staining in the same nucleus. They stain 
all about alike. The period during which the chromatin spherules 
form is indeed a rather brief one, extending from the end of the 
spireme (chromatin ribbon) stage to the beginning of the formation 
of the spindle. 

It is, of course, possible that the chromatin spherules are but 
by products of the metabolism of the chromosomes and that they 
have little significance. Yet their staining reactions, with all stains 
used, seem to indicate that they are composed of chromatin, and the 
gradations in their staining, as they dissolve, seem to connect them 
with the refractive spherules of the endosarc. 

In 0. caudata the condition of the chromatin spherules and 
endoplasmic spherules is like that in 0. intestinalis. The multi- 
nucleated species, 0. dimidiata, 0. ^elleri, 0. ranarum and 0. ohtri- 
gona, have endoplasmic spherules of the same character. Their nuclei 
are so small that it is not easy to study in them the formation of 
the chromatin spherules, and I have not attempted it. 



Orif/in of the eetosarc spherules, 

I have seen nothing to indicate any genetic connection between 
the spherules of the endosarc and those of the eetosarc, except that 
with iron-haematoxylin, when very thoroughly extracted, one some- 
times finds, in the eetosarc, spherules showing an internally granular 



Oi)alina. 247 

and fibrous appearance exactly similar to that seen in the endosarc 
spherules. I am somewhat puzzled by this observation. Under all 
other conditions the two sorts of spherules seem very distinct. To 
most stains they react in an utterly different way (cf the table in 
the appendix). It is difficult to belive that the bodies in the ectosarc, 
which with iron-haematoxylin show this structure, are really the 
ordinary ectosarc spherules. They are not endosarc spherules which 
have been misplaced by the microtome knife, and they cannot be 
endosarc spherules which have wandered unmodified into the ectosarc, 
for in sections stained with differential stains one never sees endosarc 
spherules in the ectosarc. These conditions sometimes seen in sections 
stained with iron-haematoxj^in are not enough to indicate that the 
ectosarc spherules arise from the endosarc spherules. They ai'e far 
more probably formed in situ. 

Splitting of the chroniosotnes. 

PriTZNER (1886) is the only student who has described splitting 
of the chromosomes in Opalina, and all more recent workers agree 
that he was mistaken in his description. The chromosomes do not 
form into a definite equatorial plate and then split, as he described 
for 0. ranarum. 1 find no convincing evidence of splitting of the 
chromosomes at any stage of the mitosis, but in sections of 0. intestinalis 
and 0. raudafa, stained with iron-haematoxylin, one often sees, in 
the early telophases, a condition that suggests that the chromosomes 
may possibly be splitting (Fig. 86, PI. XX). The chromosomes, when 
seen in side view, have a lighter axis and darker edges. Close 
observation shows that the darker appearance of the edges is due 
to the presence there of deeply staining granules which are absent 
from the axis of the chromosome. It would seem very simple to 
find cross sections of chromosomes in this condition and to determine 
definitely if we do have here a true splitting involving a division of 
the granules, but, so far, I have failed to obtain convincing pictures. 

Leger & DuBOSCQ (1904 ft) describe for 0. satiirnaUs the for- 
mation of an equatorial ring and its division which they interpret 
as equivalent to the ordinary splitting of the chromosomes (Text 
Fig. IV, B, C, D). To this we will return. 

Nuclear conditions in other species compared ivith those 
in O. intestinalis* 

The nuclear conditions in 0. caudata are so similar to those in 
0. intestinalis that but one point needs mention, namely, that the 



248 M. M. Metcalp 

number of the chromosomes is six instead of eight (Fig. 81, 82, 
PI. XX). In the multinucleated species the nuclei are much smaller 
and less favorable for study and I have given them much less 
attention. The nuclear membrane persists through the whole mi- 
tosis; there are no indications of centrosomes; the spindle is similar 
to that of 0. intesfinaJis; the chromatin lies just beneath the nuclear 
membrane. 

The number of the chromosomes in 0. ranarum and 0. dimidiafa, 
as Neresheimee has said, seems to be twelve. The nucleus of 
O. dimidiata shown in Fig. 17, PI. XIV, is unusually clear, bring sur- 
rounded by vacuoles of the excretory organ, and in this instance 
there seems little doubt that there are twelve rows of superficial 
granules, each row probably corresponding to a chromosome. The 
chromosomes in these multinucleated forms are more granular and 
less compact than in the binucleated species. The relations of the 
chromatin spherules are difficult to make out. The resting nuclei 
show a very characteristic appearance with a superficial chromatin 
network Avith enlarged nodes, and one to four disc-shaped chromatin 
masses closely applied to the nuclear membrane (Figs. 99 — 101, 
PI. XXI; Text Fig. X, a and b). I have not yet attempted to follow 
the course of the mitosis, nor have I studied the nucleolus carefully. 
The chromosomes in multinucleated Opalinae seem to be more gra- 
nular and less compact than those in 0. infestinalis and 0. caudata. 
They often appear merely as rows of granules. The chromosomes 
of 0. caudata and 0. intestinalis are always granular as described, 
but the granules instead of being in linear aggregates are scattered 
through a mass of less darkly staining chromatin, this mass with 
its granules composing the chromosome. 

Leger & DuBOscQ (19046) have described mitosis in 0. satur- 
nalis in a way that is somewhat difficult to reconcile with ray 
description of the phenomena in 0. intestinalis and 0. caudata. 
Text Fig. IV shows eight of their figures: ^ is a resting nucleus; 
B shows the characteristic gathering of a part of the chromatin 
into an interrupted band around the equator of the nucleus; in C 
this band is shown dividing ; in D the two parts are seen migrating 
toward the poles of the nucleus; E and F show how the daughter 
bands break into numerous parts which join the lines of granules 
("chromosomes") and move with them to the poles; G and H show 
telophase stages in the reconstitution of the daughter nuclei. 

Leger & Dfboscq suggest that the division of the equatorial band 
of chromatin is equivalent to the ordinary splitting of the chromo- 



Opal ill a. 



249 



somes in metazoan nuclei. It seems more probable that the equa- 
torial chromatin ring* in 0. saturnaUs is comparable to a chromatin 
nucleolus. It seems impossible that its division can be equivalent 
to the splitting- of the chromosomes in Mciasoa. I have not yet 
examined nuclei of 0. sainrnalis and cannot interpret the nuclear 
conditions in this species in comparison with those described for 
(). hitesiinalis and 0. caudafa. It may be that the equatorial band 



'ftm 






AM 



B (12) 



CH 



D (15) 




I^lre) 




F(i7) 



(it 

G(J8) 



sm 







H(19> 



Text Fi^. IV. Mitosis in 0. saturnaUs. After LfiGER & Duboscq. The numbers 
in parentheses are their figure numbers. X 1^00 diameters. 

of chromatin in 0. saturnaUs is homologous with the irregular ring- 
of chromatin in daughter nuclei of 0. intcstinalis and 0. caudata 
formed by the fusion of the chromosomes, as desci'ibed, preceeding 
the formation of the chromatin ribbon (spireme). In multinucleated 
species and in 0. macronudeata (Bezzenberger 1904) much of the 
chromatin often gathers in one or more large masses beneath the 
nuclear membrane (Figs. 99—101, PI. XXI, and Text Fig. V, a). It 
is possible that these superficial chromatin masses correspond to the 
equatorial band in 0. saturnaUs. 

Bezzenbeeger has described for the binucleated 0. ntacronucleata 



250 



M. M. Metcalf 



a type of mitosis that resembles that of the multinucleated species 
much more than that of 0. intestinalis and 0. caudaia (Text Fig. V, 
a—f). The resting- nucleus is like that of the multinuclear species; 
the chromosomes are numerous and are linear. In his Text Fig. XV 
(my Text Fig. V), a shows superficial chromatin masses like those in 



f 










a 



d 







cf h j k I 

Text Fig. V, Bezzenberger's figures of mitosis, a — /' in 0. macronacleata ; g — I iu 
0. lanceolata : a resting nucleus ; b—f stages in division : a — / X 2000 diameters ; 

g — / X 350 diameters. 

a resting nucleus of 0. ranarum ; h shows the chromatin net without 
such larger masses; c is a spireme stage with the chromatin thread 
apparently ready to fragment to form the many chromosomes; 
d, e and f show anaphases. Bezzenbeegee gives also five figures 
of nuclei of 0. lanceolata, whose mitosis seems to resemble somewhat 
that of 0. saturnalis (Text Fig. V, g — /). 



Enlarged individuals of Ojyalina caudata and of other species. 

In both species of BomUnator one finds frequently, especially 
in the spring, certain very thick individuals of 0. caudata (Fig. 88, 



Opalina. 251 

PI. XX). These are generally associated with other normal forms, 
but rarelj' may be the only sort present in the rectum. I have 
never seen these very lai-ge individuals in division. That they are 
not a distinct species but are really Opalinae caudaiae is proven by 
numerous transitional stages between the two forms. The chromatin 
in their nuclei is often, through not always, aggregated into larger 
masses than is the case in normal nuclei of ordinary forms, and one 
suspects that the animals are not entirely normal, yet they are as 
active as other forms and are frequently found in large numbers 
in freshly taken material. 

It is chieHy the finding of these broad individuals of 0. caudata 
that makes one a little doubtful as to the status of 0. zelleri as a 
true species rather than as a condition of 0. dimidiata. I have 
never seen similar enlarged forms of 0. intesiinalis or 0. ohtrigona. 
In one lot of 0. ranarum from the rectum of an apparently normal 
liana temporaria, there were, among a large number of oidinary forms, 
a few (about ten) individuals wiiich were very much thicker than 
usual, being almost cylindrical. Their length was twice their width 
and their width half again as great as their thickness. I have not 
sectioned these thick individuals, but stained total preparations show 
nothing unusual in their appearance except the unusual thickness 
of the endosarc, the nuclei being a little less closely set in the endo- 
sarc than in individuals of ordinary thickness. 

Leger & DuBOSCQ (1904 h) describe certain individuals of 
0. saturnalis as very broad and thick in comparison with their 
length. In these individuals the increased thickness is due to the 
greatly increased thickness of the ectosarc, in which the alveoles 
and spherules are of remarkable size. In 0. caudata and 0. zelleri 
and in the few thick individuals of 0. ranarum seen, the increased 
thickness of the body is due to the unusual development of the 
endosarc. Legek & Duboscq suspected that the broad individuals 
of 0. salurnaUs might be products of transverse division, but it is 
difficult to see wiiat suggests this interpretation. 

It is well known (Zellee 1877, Neresheimee 1907) that in the 
spring, when division becomes very rapid and most of the Opalinae 
become very small, some individuals in all species remain almost 
of full size, apparently not dividing any more rapidly than during 
the rest of the year. These large individuals do not encyst, but 
remain in the host and secure a continuance of its infection. It is 
possible that the great enlargement of some individuals is related 
in some way to the retardation of division. It is possible that 



252 M. M. Metcalf 

0. zeUeri may be but a similar enlarged form of 0. dimidiaia. 
I have found 0. selleri only twice and then in the spring, and the 
thick forms of 0. caudata are rather rare, except in the spring at 
a time when many individuals have already become small through 
repeated division. Both Zeller and I have found 0. selleri and 
0. dimidiata together in the same individual host. Neresheimer 
describes these two species as from Rana esniJenta, but does not 
say if both occur in the same individual host. In no other instance, 
except one very doubtful one in Bomhinator, have I ever found two 
species of Opcdina in one rectum. Bezzexberger describes 0. Jaia 
and 0. Jonga as occurring in liana Umnochans, but does not say if 
the two species are found together parasitic in the same individual. 
It seems to be verj' unusual to find two species of Opalina together 
in the same host. The presence of individuals of 0. dimidiata with 
those of 0. selleri in the same host casts some doubt upon the 
status of 0. seUeri as an independent species. Until, however, we have 
more evidence of its connection with 0. dimidiata, we must, as Neres- 
HEiiviER has done, treat it as independent. Zeller, the discoverer 
of this form, expressed doubt as to its connection with 0. dimidiata. 
The chromatin spherules which are formed and dissolved, or 
extruded from the nuclei, during the course of each mitosis, seem 
to be especially connected with nutrition and growth. It is not im- 
possible that careful study of the chromatin spherules in these large 
individuals of 0. caudata and 0. ranartim and in 0. selleri, might 
throw some light on their- origin, but as jet I have found nothing 
of special import in this direction. I have not enough preparations 
of nuclei of any of these thick forms, in the right stage of mitosis, 
to allow me to study the point with sufficient care. 



General considerations in connection with the structure 
of Opalina and of the phenomena of mitosis. 

Ectosarc and endosarc. 

The ectosarc and endosarc of Opalina are quite sharply distinct, 
both the protoplasmic granules and films and the refractive spherules 
of the two regions staining very diiferently with many stains. It 
is difficult to suggest to what this may be due. Is the primary 



Opalina. 253 

ditference in the spherules or in the })rotoplasm itself? If the 
refractive spherules are products of the cytoplasm then, of course 
the priniar}' ditference between the ectosarc and endosarc must lie 
in the protoplasm itself. It seems, however not unlikely that the 
endosarc spherules are derived in part at least from the chromatin 
of the nucleus. On the other hand, the spherules of the ectosarc 
probably are formed by the ectoplasma itself. In 0. obtrigona a few 
of the smaller spherules in the ectosarc are merely endosarc sphe- 
rules that have wandered toward the periphery (Fig. 6, PI. XIVj. 
In other species studied, the endosarc spherules seldom, if ever, leave 
the endosarc. Even in 0. oUrifjona the migrated endosarc spherules 
lie in endosarc-like tissue which has protruded in strands between 
the alveoles of the ectosarc. In any case one can ignore these dis- 
placed endosarc spherules in inquiring as to tlie ditferences between 
endosarc and ectosarc. 

That the peculiar staining reactions of the ectosarc are probably 
not due to the presence of the ectosarc spherules is shown by the 
fact that in the anterior end of the body, where only a few very 
small ectosarc spherules are present (Fig. 1), the ectosarc stain is 
just as divergent from that of the endosarc as it is in the rest of 
the bod}'. That the difference in staining is not due to the absence 
of endosarc spherules from the ectosarc is shown by the fact that in 
0. ohtrigona the ectosarc shows the same peculiar staining reactions 
as in other species, although in this species the endosarc spherules 
migrate into the ectosarc between the large alveoles, even reaching 
the sub-pellicular layer. 

Apparently we can safely emphasize two points, first that there 
is very decided structural difference between the two regions, and 
second that there is an equally marked chemical and phj^siological 
difference, as indicated in the staining reactions and by the divergent 
character of the refractive spherules of the respective regions.^) 

One might suspect that the ectosarc spherules are excretory 
and that one of the chief functions of the ectoplasm itself is excretion, 
were there not present in the body, in several species, such a well 
developed system of excretory vacuoles. As these vacuoles lie in the 
endosarc and have no discernable connection with the ectosarc, we 
seem debarred from attributing any special excretory function to the 
ectosarc. We must rest for the present with the mere statement 



') ScHUBOTZ (1908) finds, in Pycnothrix monocystoides, that when stained by 
VAN Gibson's method the ectosarc is yellow, the endosarc red. 



254 M. M. Metcalp 

of the fact of a chemical and physiological difference between the 
ectosarc and endosarc, leaving unexplained the nature of this difference. 

Excretory organs. 

The special connection of the excretory organs with the nuclei 
is worth emphasis, though just what its physiological meaning may be 
is unexplained. 

It is also of interest that the granules massed in the posterior 
end of the system of excretory vacuoles, which are from time to time 
extruded from the body, seem to be derived from granules of the 
cytoplasm, as indicated by their size and their exact resemblance 
to the granules of the cytoplasm bounding the posterior vacuole of 
the excretory system. In the processes of excretion certain of the 
cytoplasmic granules seem to be thrown away bodily. 

The very primitive character of the excretory organs in Opalina 
has been emphasized in a previsions paper (Metcalf 1907 c}. 

Anterior end of the body. 

The divergent character of the anterior end of the body also 
deserves special note. One sees that in this region the granules of the 
endosarc are more numerous, and the endosarc spherules much more 
abundant (Fig. 1), while in the ectosarc the very large alveoli and 
the large ectosarc spherules are wanting. As division of the body is 
constantly going on, growth must be constant, and one naturally thinks 
that the denser character of the anterior end may be related to 
special activity in this growth, yet this is not easy to prove. There 
are no definite points in the body which can be taken as landmarks 
in estimating the relative growth of different regions. The nuclei 
move within the plasma and so cannot be used as a fixed point for 
reference in studying the relative growth of different regions. That 
they so move is shown by the fact that one daughter cell, in each 
division, receives the posterior nucleus from the parent and that in 
a short time this comes to lie as near, or almost as near, to the 
anterior end of the body as does the nucleus in the other daughter 
cell (PI. XVII). 

Absence of centrosomes. 

The absence of centrosomes in the mitosis is of interest. Centro- 
somes are well known among the Protozoa (e. g. in the Sporozoa). 
Among Flagellata and Foraminifera and in certain Ciliata, structures 



Opal ilia. 255 

which seem clearl}^ to be rehited to true centrosomes are found 
both inside and outside the nuclei. The absence of centrosomes in 
Opalina is apparently not a piimitive cliaracter. 

In so for as the function of the centrosome is a mechanical 
one. furnishing a point of resistence in the movements attending 
mitosis, it is not needed in the mitosis of Opalina, for the ends of 
the spindle are attached to the nuclear membrane and this membrane 
can furnish the necessary resistence points, if any such be really 
needed. At each constriction of a nucleus in division, both the 
chromatic and tlie achromatic fibrils at the equator of the nucleus 
are pinched and held by the constricted nuclear membrane, and 
apparently the attachment of at least the chromatic fibrils to each 
end of the nuclear membrane persists even during the resting stage. 

The spindle. 

The mitotic spindle in Opalina is interesting in its simplicity, 
being formed merely by the enlarging of those fibrils and films which 
run lengthwise in the oval nucleus and by the concomitant diminishing 
in size of the transverse fibrils and films, the latter, apparently, foi* the 
most part, being drawn in like pseudopodia. There is nothing that can 
be interpreted as an outgrowth of fibres from any formative center, as 
seems to occur in connection with the centrosomes in many mitoses.^) 

The mitotic spindle in Opalina is also interesting in the fact 
that it is formed from both chromatic and achromatic material. In 
the resting nucleus the achromatic foam fills the whole nucleus, a 
network of chromatin fibrils being also pi-esent over the surface of 
the nucleus just beneath the nuclear membrane. The appearance 
of longitudinal striation in the dividing nucleus is due to the 
emphasizing of the longitudinal strands of the chromatin net and 
the longitudinal films of the achromatic foam. The spindle, therefore, 
is composed of a central achromatic portion and a superficial chromatic 
portion. To what extent the two are connected in either the resting 
or dividing nuclei it is difficult to say.^) 

There seems to be little true resemblance between the condition 
in Opalina, with an outer spindle composed of chromatin and an 
inner spindle of achromatic substance, and the condition in many 

') Very similar conditions have been desfribed by Boveri (1887 6, p. 21) in 
the formation of the spindle in the maturation divisions of Asraris meyalocephala. 

'■'j Wilson i1895, 1^00) believes that the linin which gives rise to the mitotic 
spindle in sea-urchin &gg& arises from the chromatin. 



256 M. M. Metcalf 

metazoan mitoses in which one distinguishes a central and an outer 
portion in the achromatic spindle. It seems somewhat doubtful if 
the mitotic spindle in Opalina is really comparable at all to the 
mitotic spindle of a metazoan cell, though the presence of perfect 
spindles of nearly, if not exactly, the metazoan type in Sporosoa^ 
and the occurrence in other Protozoa of spindles of intermediate 
character, seem to justify our regarding the structure in Opalina as 
a true, through very lowly developed, spindle. Its achromatic portion 
is evidently more nearly related to the inner than to the outer 
spindle of Metazoa, being as Boveri (1900) has shown, a "netrum"i 



The mechanism of mitosis. 

The machanism of mitosis in Opalina seems as difficult, in some 
regards, to understand as it is in other forms. One sees nothing 
in the cytoplasm which seems to be acting upon the nucleus. So 
far as one can judge, the nucleus is automatic in its movements, for 
even the separation of the daughter nuclei cannot be due to the 
pull of the cytoplasm as the body elongates, since the thread 
connecting the daughter nuclei is often long and coiled, indicating 
that it has itself been in rapid growth. The whole nucleus wanders 
forward in that daughter cell wiiich receives the posterior nucleus 
of the parent, and some sort of contraction in the cytoplasm seems 
necessary to explain this migration, but the changes of form in the 
nucleus itself seem due to its own activity. 

In the changes of shape and in the movements accompanying 
mitosis are certain portions of the nucleus active and others passive? 
Does the nuclear membrane elongate and become spindle-shaped 
because it is pushed upon by the fibres of the spindle forming 
within, or is the nuclear membrane the active agent, itself elongating 
by growth at the same time that it serves to supply points of 
resistence to the pull of the spindle fibers? Do the chromosomes 
migrate of their own accord along the chromatin fibres of the 
spindle; or do the latter contract and pull the chromosomes towards 
the poles of the nucleus; or do those portions of the spindle-fibres 
between the chromosomes elongate and push the chromosomes apart ? 
Are both the chromatic fibres and achromatic films in the spindle 
active, or is one set of structures active and the other passive? 
A little evidence upon some of these points can be found. 

The change in form of the nucleus from oval to elongated 
eliptical or spindle-shaped seems to be due to growth of the nuclear 



Olialiiui. 257 

iiieinbiaiR'. The spindle usually does not fill the whole nucleus, the 
uucleiir membrane apparently growing more rapidly than the rest 
of the nuclear structures. During the whole mitotic cycle the nuclei 
are in constant growth, as is indicated by their increase in size. 
That the nuclear membrane shares in this active growth is shown 
not only by the fact just mentioned that the spindle does not fill 
the nucleus, but also by the fact that in the late telophases the 
thread connecting the tw^o daughter nuclei grows even to unnecessary 
length and becomes coiled. 

The usual peculiar form of the spindle, with acuminate ends, 
is instructive. It does not seem as if the chromatin fibres uniting 
the chromosomes to the poles of the nucleus can be contracting, 
for they are much, and quite irregularly, bent. They are not 
taut, as if pulling upon the chromosomes, ^'et it is barely possible 
that the minute transverse fibrils connecting the longitudinal 
fibres of the spindle are drawing these together with sufficient force 
to bend them into the irregular bows which are seen. Naturall}'^, 
at the narrowed ends of the nucleus, these transverse fibrils are 
more numerous in a given area than they are near the equatoi-, 
where the nucleus has nearly its original diameter. The first 
impressions one receives from such a nucleus as that shown in 
Figs. 49 — 52, PL XVIIT, may be that the spindle is elongating and 
pushing the nuclear membrane in front of it; yet the whole character 
of the chromatin fibres of the spindle is such as to suggest that 
they are pliable and not so stiff as any such hypothesis of the force 
of their elongation would imply. The fibres are usually quite irre- 
gular and curved, and it seems impossible to think of their pushing 
with any appreciable force. Such irregular fibres may exert some 
pull, but that they can effectirely push is unbelievable.') 

The migration of the chromosomes is accompanied by a perceptable 
thickening of the chromatin fibres connecting them to the poles of 
the nucleus, suggesting that the chromosomes are pulled toward the 
poles by the shortening of these fibres. As the chromosomes 
approach the pole they become less branched, less irregular in form 
and larger. On their way to the pole they seem to absorb most of 
the substance of the fibres of the chromatin spindle, drawing in the 
transverse strands of the chromatin net and taking up the substance 
of the longitudinal fibres and adding it to their mass. Thus, during 
the late anaphase, a very large proportion of all the chromatin in 

') Prandtl (1905, 1906) believes that the equatorial portions of the spindle- 
fibres in Didinium elongate and push the chromosomes apart. 



258 M. M. Metcalp 

the nucleus is in the chromosomes. Soon the chromosomes begin 
again to send out processes and the chromatin network of the resting 
nucleus is formed. The whole migration of the chromosomes and 
the reformation of the chromatin network suggests comparison with 
the movements of a reticulate foraminiferan. All the chromatin 
seems to be active in this movement. 

No explanation of the migration of the daughter chromosomes 
as dependant upon some repulsion between them, connected with their 
splitting, can apply here, for no such splitting occurs in the equatorial 
plate stage or immediately preceeding it. The splitting, if it occurs 
at all, is found in the telophases of mitosis, after, not before, the 
migration of the daughter chromosomes. 

A chromosome cannot crawl upon nothing any more than can an 
animal. There must be some resistant substance upon which the 
moving chromosomes can advance. The resistant substratum in this 
case seems to be the alveolar achromatic material which fills the 
center of the nucleus, and whose alveoles with their delicate walls 
and contained liquid seem to furnish the necessary resistence for the 
movements of the chromosomes and their pseudopodia (chromatin 
fibrils). The attachment of the spindle fibres to the nuclear mem- 
brane also, of course, aids in these movements. 

What causes the chromosomes to arrange themselves at first in 
the equatorial third of the nucleus and later to crawl to the poles 
of the nucleus, and what causes the changes in the form of the 
nucleus, are questions whose answer the conditions in Opalina do 
not help us to approach. 

Such a mitotic division as we see in Opalina, in which all parts 
of the nucleus seem to be active, membrane, chromatin and achromatin 
all sharing by active growth and movement, seems not only less 
specialized than the mitosis of higher forms, but also less removed, 
at least mechanically, from amitotic division, in which also probably 
all parts of the nucleus share by active movements. The presence 
of centiosomes in a cell allows the nuclear membrane and the chro- 
matin and, in the most highly developed mitoses, the achromatic 
foam of the nucleus also, to be less active. 



The polarity of the nuclei aud the planes of division of the 
nuclei aud of the body. 

The nuclei of the binucleated Opalinae are always somewhat 
elongated, their two poles being always clearly recognisable. This 



Opaliiia. 259 

polarity is seen not only in the shape of the nucleus but also in 
the fact that the chromatin network is attached to each pole of 
the nucleus. This attachment is very clearly seen during mitosis 
and apparently persists through the "resting period". The orientation 
of the nuclei is constant and unchanging, their long axis being 
about parallel with, usually coincident with, the long axis of the 
body. The nuclei never rotate, except possibly upon their long axes, 
which would be without significance in this connection. This con- 
stancy of orientation may be due in part to the fact that the two 
nuclei are generally connected by a thread. The longitudinal axis 
of a daughter nucleus always remains in the same position as that 
of the parent nucleus. This enables us to clearly see the interesting 
fact that the nuclei of the binucleated species of Opalina always 
divide in the same direction and that they do so whether the ac- 
companying division of the body is longitudinal or transverse. 

In Metazoa and plant cells and in most Protozoa in which the 
relations are clearly discerned, the plane of division of the cell-body 
is parallel to the plane of division of the nucleus, both being per- 
pendicular to the long axis of the mitotic spindle. The binucleated 
OpaUnae show the same relation in the case of the unusual trans- 
vei'se divisions, but in the more fi"equent longitudinal divisions the 
plane of division of the body is parallel with the long axis of the 
nuclear spindle. A similar discrepancy between the directions of 
the division of the body and of the nucleus is seen in the Trypano- 
somes. but there the nuclear relations are not so clear, there being, 
especially, no certain indication that the orientation of the parent 
and daughter nuclei remains constant generation after generation. 
The constancy in the direction of the division of the nuclei and the 
variability in the direction of the division of the body in Opalina 
show that there is a lack of coordination between the direction of 
division of the nucleus and of the body. This lack of coordination 
is much more marked in the multinucleated species. 

The conditions are interesting to consider from the point of view 
of phylogeny. If the anterior end of Opalina is homologous with 
the anterior end of a Flagellate, and doubtless it is so, we see that 
the two agree in dividing longitudinally. In most Flagellata we have 
only longitudinal divisions. ^) Probably in Oimlina the longitudinal 
<livision is primitive and the unusual transverse division secondarj^ 
The latter is probably comi)arable to the transverse division char- 



') Oxyris marina is said to divide transversely. 



260 M. M. Metcalp 

acteristic of the higher Ciliata. Opalma shows, therefore, a condition 
intermediate between that of Flagellata and that of the higher Ci- 
liata, since, while retaining the more primitive type of division, it 
shows occasional divisions of the secondary type. The flattened 
multinucleated species of Opalina show more frequent transverse di- 
visions than do the probably more primitive cylindrical binucleated 
species. 

The phylogenetic significance of these phenomena is further 
considered on page 274 in connection with the discussion of the 
relationshipes of Opalina and the origin of distinct micro- and macro- 
nuclei in higher Ciliata. 



Time relations in the division of the body and of the nucleus. 

The two sets of daughter chromosomes in each nucleus of the 
binucleated Opalinae remain for one cell-generation in the same 
daughter cell, though soon separating into different nuclei; that is, 
division of the body lags one step behind the division of the nuclei. 
At the second division of the body the daughter chromosomes of the 
preceeding division become distributed to separate cells. Probably 
originally the binucleated condition was brought about by the de- 
lay of one division of the body, the temporary binucleated con- 
dition thus secured persisting until the body itself finally divides. ^) 
The multinucleated condition of other species seems due to the 
further "suppression of other divisions of the body, nuclear division 
and division of the body in them being still more loosely related. 

Splitting of the chromosomes. 

In the mitosis of Opalina we do not find any longitudinal split- 
ting of the chromosomes in connection with the imperfect equatorial 
plate stage. The chromosomes are then already present in nearly 
or fully the double number and, wiiile one or two chromatin masses 
may constrict transversely during the equatorial plate stage, the 
majority merely rearrange themselves in two transverse rows pre- 
paratory to migration to the poles. Careful observation of the chro- 
mosomes in both ends of many nuclei during the anaphases shows 
that generally each chromosome in one end of the nucleus corre- 



^) I find that Boveri (1900, p. 189) has similarly interpreted the binucleated 
condition of Opalina as due to delay in the division of the cell-body. 



OpaliiKi. 261 

spouds more or less closely in size and form and in nnmber of con- 
tained granules to one of the chromosomes at the other end. These 
corresponding- chromosomes are opposite to one another. This all 
suggests very strongly that the daughter chromosomes in a dividing 
nucleus of Opalina are paired, just as truly as the}^ are in a meta- 
zoan nucleus. 

We have already seen in the early telophase a condition which 
suggests that the chromosomes may be splitting longitudinally. The 
fate of the two halves that may be so formed is well-nigh impos- 
sible to follow, for the chromosomes almost at once unite to form a 
continuous ribbon. I have never found sixteen chromosomes at each 
end or even at one end, of a nucleus in the telophase, nor have I 
seen evidence that the chromatin ribbon is double. This, very in- 
teresting stage in the mitosis, needs further study, though I have 
little hope of obtaining conclusive results. At present we can only 
say that splitting of the chromosomes does not occur at the equa- 
torial plate stage, that it may occur in the telophases, and that in 
the anaphases the daughter chromosomes seem to be paired as in 
metazoan mitoses. 

GoNDER believes that splitting of the chromosomes may often 
occur during the anaphases or telophases of maturation mitoses, but 
1 know of no description of other mitoses in which the splitting of 
the chromosomes occurs after their distribution to the daughter 
nuclei instead of in the equatorial plate or in the prophases. 

If there be true splitting of the chromosomes in the telophase, 
one cannot be certain whether each granule in a chromosome divi- 
des (Fig. 86, PI. XX). Some of the granules at the edges of the 
chromosome, seem spherical, others eliptical, others elongated rod- 
shaped, it is possible that adjacent granules often lie in contact, or 
even fuse, and so give an appearance of an elongated rod. In com- 
paring the two rows of granules at opposite edges of the apparently 
split chromosome, one sees a general resemblance but often the 
fusion or the contact of the granules in one line does not corre- 
spond exactly to that in the other line, and the two rows are not 
alike. Very likely, of course, even if the granules were all perfectly 
distinct, the two rows would not be found to be alike. 

Of course, if, in general in mitosis, chromosomes retain their in- 
dividuality during the spireme stage, is makes no real difference 
whether splitting of the chromosomes occurs before or during or 
after the spireme stage, so that mitosis in Opalina is not funda- 
mentally different from that in most Meta^oa, if splitting of the 

18* 



262 M. M. Metcalf 

chromosomes really occurs during- the telophases. If, on the other 
hand, the chromosomes and their contained granules do not divide 
into equivalent parts in each division of the nucleus, it seems that 
nuclear division in Opalina must be much simpler than mitotic di- 
vision in higher forms. 

I at first inclined toward the first hypothesis in interpreting the 
mitosis in Opalina and tried to imagine how these daugther chro- 
mosomes, formed in the telophase, could pass into the spireme and 
reappear later in the next double equatorial plate. Three salient 
facts are seen: 1) the fusion of the chromosomes in the late telo- 
phase is not a union end to end, but is instead an irregular lateral 
union, more or less broad plates of chromatin passing from the side 
of one chromosome to the side of the next, until they all become 
united to form the chromatin ribbon, 2) the chromosomes when 
they again become distinct, previous to the next mitosis, arise by 
transverse constriction of the chromatin ribbon and 3) the chromo- 
somes remain permanentle, attached at each end to the poles of the 
nucleus by means of fibres some at least of which do not split. 
The transverse constriction of some of the chromosomes in the early 
telophases is a transient phenomenon, all the unconstricted chromo- 
somes and the parts of the constricted chromosomes soon completely 
fusing in the spireme. 

If the eight chromosomes split longitudinally in the telophase 
and then unite laterally to form the chromatin ribbon of the spireme 
stage, this ribbon might consist of the sixteen daughter chromosomes 
lying side by side in a single row. When now the spireme con- 
stricts transversely to form the sixteen chromosomes of the next 
mitosis, these might be the original sixteen daughter chromosomes 
from the longitudinal division in the last telophase. On the basis 
of such a schema, the relations of the granules in the chromo- 
somes would not be very difficult to bring into ha.rmony with the 
usual conception of the chromosomes as consisting of a linear aggre- 
gate of chromioles which retain their individuality and which, in 
division, give one half of their substance to each daughter chromo- 
some. But the chromosomes which in the telophase show appearance 
of splitting do not, as already noted, show their granules arranged 
in pairs, so that we have, even on the basis of this schema, no 
satisfactory indication that equivalent daughter chromioles are 
distributed in the daughter chromosomes to each daughter nucleus; 
and furthermore, the attachment of each end of each parent chromo- 
some to the corresponding pole of the nucleus by means of a per- 



Opalina. 263 

sisteiit fibre Avhicli, at least in some instances, does not itself split 
seems to introduce insuperable difficulty into the schema. 

Calkins & Cull (1907) describe splitting of the chromosomes in 
the maturation divisions of Paramaecium, with a clearness Avhich 
leaves no doubt that this type of mitosis occurs in at least some 
of its divisions, and so is known among the Protozoa. 

Professor Boveri directed my attention to conditions in Ascaris 
megalocephala which seem similar to those in the telophases of mi- 
tosis in Opalina and which make it seem likely that the appearance 
of splitting in the chromosomes of Opalina during the telophases is 
not significant. In Ascaris the chromosomes are seen to be granular 
duiing the telophases, and the granules lie more or less distinctly 
in two rows at the edges of each chromosome, leaving the axes of 
the chromosomes lighter, as in Opalina. Van Beneden (1883, p. 343; 
1887. Plate V, Fig. 8) has referred to these conditions in Ascaris as 
showing a second longitudinal division of the chromosomes in the 
telophases, one having already occurred in the equatorial plate stage 
of mitosis. Heidenhain, at the Anatomical Congress in Wiirzburg, 
1907, described an appearance of splitting in the chromosomes in the 
telophases of mitosis in cells of the skin of salamanders. Boveki, 
during the discussion of Heidenhain's paper, suggested that, inas- 
much as this appearence, when seen, is found in all parts of all the 
chromosomes, whatever their position may be, it cannot indicate a 
splitting of the chromosomes, but probably shows that the axes of 
the chromosomes in the cells of the salamander skin stain at this 
stage less deeply than the periphery. Chromosomes with unstained 
or faintl}' stained axes have since been interpreted by Haidenhain 
(1907j as having an axial linin fibril. The lighter axis of the chro- 
mosomes of Opalina during the telophases seems not to be due to 
the presence of an axial fibril of linin, but to the absence of gra- 
nules at the axis and their presence at the edges of the chromosomes. 

The appeai-ance of granular edges and a lighter axis in the 
chromosomes of Ascaris is said by Boveri to be transient, disap- 
pearing when the chromosomes branch to form the network of the 
resting nucleus, and not showing in the chromosomes when these 
reappear from the resting nucleus preparatory to the next mitosis. 
There is no reason to believe that the splitting of the chromosomes 
in the new mitosis is a reappearance of a double condition in the 
previous telophase. The appearances of doubling of the chromo- 
somes in the telophases seem quite similar in Ascaris and Opalina, 
and the condition in the cells of the salamander skin is somewhat 



264 M. M. Metcalf 

comparable. In Ascaris and the salamander the condition in the 
telophase seems to be unrelated to the true splitting of the chromo- 
somes, which occurs during- the next mitotic cycle, a resting stage 
intervening. This comparison with Ascaris and the salamander adds 
more doubt to the already very doubtful interpretation of the ap- 
pearance in the telophases of Opalina as indicating a splitting of the 
chromosomes. 

Calkins & Cull '(1907) found that splitting of the chromosomes 
occurs in the two maturiation divisions of Paramaecium, but not in 
the third division by which the conjugating nuclei are formed. The 
ordinary vegetative divisions of Paramaecium have not been so stu- 
died as to show whether splitting of the chromosomes occurs in 
them or not. Neither Hamburger (1904) nor Calkins & Cull show its 
presence in the nuclear divisions immediately following conjugation. 
These conditions suggest the possibility that in Opalina the matu- 
ration divisions may differ from the vegetative divisions and that 
splitting of the chromosomes may be found in the maturation di- 
visions. I have used for the most part, with the minute individuals 
of Opalina in the spring, methods whicli do not show the finest de- 
tails. My study of carefully stained sections has not yet shown in 
detail the phenomena of maturation. Another spring's work will 
propably be necessary to determine this interesting point. 

An alternative explanation of the mitosis. 

The alternative interpretation of the nuclear division in Opalina 
as a very primitive mitosis in which there is no longitudinal splitting 
of the chromosomes needs further development. We have seen that 
the chromatin masses (properly called chromosomes only in certain 
conditions) are always branched, their branches being connected to 
form a network just beneath the nuclear membrane; and we have 
also seen that some of the fibres of the network are attached to the 
nuclear membrane at each of the two poles of the nucleus. This 
attachment of the fibres is very clearly seen when the spindle is 
well developed. The manner in which the nucleus constricts in each 
mitosis until the membrane at the equator of the nucleus pinches 
and holds the fibres of the spindle, explains the fact that these 
fibres are attached to the membrane only at the two poles. One 
night conceive each longitudinal chromatin fibre of the mitotic 
spindle in Opalina, with all its branches and with the two chro- 
matin masses upon it, as forming one unit, the units being in con- 



Opaliua. 



265 



iiection by means of tlieir united branches (Text Fig. VI, B). When 
the division of the nucleus is completed, one would find in each 
daughter nucleus eiiiiit dauojiter units each consistinp: of a single 
mass of chromatin with numy branches which unite with branches 






Text Fig. VI. 
Diagrams of mitosis in 0. intestinalis, only 
two chromatin units, instead of eight, being 
shown : A = an anterior nucleus in the irre- 
gular "equatorial plate" stage, four daughter 
chromosumes being present; ^ = an early 
anaphase; C = a later anaphase; Z) = an 
early telophase, the chromosomes being con- 
nected by their broad lateral outgrowths. 



Text Fig. VII. 
Diagrams of mitosis in O. intestinalis, only 
two chromatin units being shown. E ^ 9. late 
telophase (spireme). The chromosomes are unit- 
ed into a ribbon which, if all the chromosomes 
were shown, would extend over nearly the 
whole nucleus. The undivided nucleolus is 
shown in the posterior daughter nucleus. 
F = an anterior daughter nucleus in an early 
prophase; the chromatin ribbon is breaking up 
again into chromosomes; the new nucleolus 
is forming near the more pointed end of the 
nucleus. (? = an anterior nucleus in a late pro- 
phase; chromatin spherules have been formed. 



266 M. M. Metcalf 

of the neighboring chromatin masses to form the nuclear net which 
is always recognisable at all stages of mitosis, however faint the 
transverse fibrils may become. The chromatin masses of the neigh- 
boring units send out also broad plates of chromatin (Text Fig. VI, D) 
which soon completely unite them into a chromatin ribbon (Text 
Fig. VI, E). Preparatory to the next mitosis the chromatin masses 
of neighboring units again separate (Text Fig. VII, F and G) and 
each constricts into two thus producing sixteen chromatin masses, 
whose branches are all interconnected, each unit of course having 
two chromatin masses (Text Fig. VI, A). The eight units now 
draw in most of the substance of their lateral branches and reassume 
their position side by side in eight more distinct parallel lines 
stretching from pole to pole of the nucleus, each line having upon 
to two chromatin masses (Text Fig. VI, B). The cycle then repeats 
itself. 

Upon this interpretation it would be seen that the division is 
not a highly developed mitosis, but that, still, by means of the per- 
sistence of the longitudinal chromatin fibres in all stages, even in 
the resting nucleus, and through their retention of their connection 
with the two poles of the ovoid nuclear membrane, the chromatin 
masses, after they divide, are able to send one of their daughter 
masses to each pole of the nucleus, securing thus a result somewhat 
similar to that obtained in the fully developed mitoses of higher 
animals. There is no means in this division to secure the distribution 
of one half of each granule of each chromosome to the daughter 
nucleus, but each daughter nucleus does receive about half of the 
mass of each chromosome of the parent nucleus. In this case, we 
see that the emphasis is upon the chromosomes and not upon the 
chromioles. 

The eyolution of mitosis. 

Is this type of mitosis in Opalina aberrant or does it correspond 
to a stage in the phylogeny of the more highly developed mitoses 
of Metasoa? The nuclei of many, probably of all, Plasmodroma are 
centronuclei (Boveri 1900) each containing, in addition to the chro- 
matin elements and indifferent plastin, a differentiated karyosom 
which functions as a more or less perfect centrosome. Compare 
Euglena (Keuten 1895), Trypanosoma (Schaudinn 1904, v. Peowazek 
1905, Salvin Moore & Breinl 1907), Amoeba (Schaudinn 1894, 
Haetmann & V. Prowazek 1907). It is somewhat doubtful whether 
the simplest nuclei have differentiated karyosomes, but at any rate, 



Opalina. 267 

we may probably assume the former existence of such simi)le nuclei, 
even though they may not exist to-day. 

One naturally conceives a series of stages in which both the 
plastin and the chromatin constituents of the nucleus are becoming 
more highly developed. On the one hand the phylogenetic develop- 
ment of the chromatic structures probably showed at one time a 
stage with the chromatin in the form of diffuse granules not grouped 
into chromosomes, and this may have been succeeded by a stage 
such as we now seem to have in Trypanosomes, in which we have 
diffuse granules irregularly arranged during the vegetative mitosis 
(Salyin Mooee & Beeinl), but true chromosomes during some of 
the divisions preceeding conjugation (Schaudinn, v. Pkowazek). A 
further evolution gives definite chromosomes persisting throughout 
the whole life of the cell. Ultimately the chromosomes show morpho- 
logical differences corresponding to their differences in function. On 
the other hand we conceive the plastin elements of the nucleus as 
giving rise to an intranuclear centrosome ^), which soon becomes 
developed to the point of containing a centriole. The final develop- 
ment, showing a spindle and astral rays, is best seen when the 
centrosome becomes extra nuclear. The original centronucleus has 
thus evolved into an elaborate double set of structures, one con- 
sisting of the chromatin elements associated with some indifferent 
plastin, and the other being the kinetic plastin in the form of the 
centrosome, whose structure in some phases of mitosis becomes 
elaborately developed. 

The conditions in Opalina seem to throw light upon this phylo- 
geny, though its own nuclear structure seems aberrant and not to 
correspond to any stage in the phylogeny of the mitosis of higher 
forms. It seems to have substituted another and simpler mechanical 
device for the centi'osome ; has developed its mitosis to a c'ertain 
point and has stopped there, unable to go further because of the 
absence of developed centrosomes. Its method of mitosis is simple 
and is efficient to a degree, but is incapable of producing the 
remarkably perfect results reached by those cells which kept and 
further evolved their centrosomes. 

The indication that in the mitosis of Opalina the emphasis seems to 
be placed upon the chromosomes and not upon the chromioles is 



^) The karyosom of Plasmodroma seems to cousist of both plastin and chro- 
matin and to be therefore more than a centrosome. It contains tlie centrosome. 
These relations are shown with especial clearness in an as yet unpnblished paper 
by Habtmann upon Amoeba tetragena. See also Haktmann & v. Prowazek 1907. 



268 M. M. Metcalp 

not without sig-nilicance. May it be that the longitudinal splitting 
of the chromosomes has been overemphasized, and is not so funda- 
mental as is often thought, having been evolved from a less definite 
simpler method of division in which the chromosomes but not their 
granules divide into equal halves? Probably the vegetative divisions 
of the micronuclei of Paramaecium are of this type. Has this simpler 
type of division itself been evolved from lowly mitoses, like those 
in many Plasmodroma, in which there is no method of securing so 
equal a distribution of the mass of each chromosome to the daughter 
nuclei, no distinct and constant chromosomes, indeed, appearing to 
be present? In such divisions as these in the Plasmodroma the 
masses of chromatin in the two daughter nuclei may be about equal. 
Possibly amitotic division stands as still more primitive. It is 
difficult to distinguish the two in some cases. Has the individuality 
of the chromosomes and perhaps of the chromioles been developed 
pari passu with the elaboration of the process of division? Are the 
conditions in Opalina intermediate between ordinary amitotic division 
and highly developed mitoses, Opalina having a method of preserving 
the distinctness of the several chromosomes generation after genera- 
tion, but not having a perfect method for securing such an exact 
equality of the daughter chromosomes as results from the longitudinal 
splitting in highly developed mitoses, in which one half of each 
chromiole goes to each daughter nucleus? The latter is secured 
only in mitoses in which the chromosomes split longitudinally and this, 
it seems, is not the case in Opalina, in which the persistent attach- 
ment of the two ends of each chromosome (chromatin unit) to the 
two poles of the nucleus is the means of securing the separation 
of the daughter chromosomes and the independence of the several 
sister chromosomes. The conditions in the binucleated Opalinae seem 
to favor such a general interpretation as that here developed, but 
mitosis among the Protozoa must be better understood before one 
can accept as sufficient the evidence in favor of such a phylogeny 
of mitosis. 

Opalina seems to have the chromosomes not only distinct but 
somewhat different from one another, as is indicated by differences 
in size and form. Of course differences between the chromosomes, 
once established, could readily persist if the schema of division 
suggested for Opalina is correct, and it seems to be so, provided 
one assumption is correct, namely, the assumption that when the 
chromatin ribbon constricts to form the chromosomes for the new 
mitosis, constrictions occur at points where the eight chromosomes 



Opalina. 269 

of the previous telophase united to form the ribbon. Fundamentally 
the same assumption is involved in the belief in the individuality 
of the chromosomes in any animal. In the binucleated Opalinae the 
chromatin is less diffuse in the ''resting nuclei" than it is in most 
animals, so that it is easier to conceive of the chromatin masses 
which appear before the new mitosis as being merely the old chro- 
mosomes of the previous telophase which have again become distinct. 
Each chromosome itself soon constricts into two, this being appa- 
rently the true division of the chromosomes. Division of some of 
the chromosomes may occur at the time when the chromatin ribbon 
is constricting to reform the chromosomes, so that the number of 
masses coming out of the chromatin ribbon may be more than eight, 
but this, of course, does not alfect the interpretation of the phenomena. 

Nuclear condition and cytoplasmic movements. 

Attention has been called to the fact that the two nuclei in 
the binucleated Opalinae are often in slightly different condition. 
This divergence is never great. In the multinucleated species the 
conditions of the numerous nuclei are very different. No disturbance 
of the movements of the cell arises from this diversity in condition 
of the different nuclei, which tends to confirm the general belief 
that the chromatin of the nucleus is not directly concerned in the 
control of protoplasmic movements. Meves (1899) showed that 
secretion in the cells of the kidney in salamander larvae is inter- 
rupted during mitosis, the chemical activities of the cytoplasm being 
influenced by the condition of the chromatin in the nucleus. 

Analogies of the chromatin spherules. 

Some light may be thrown upon the problem of the nature of 
the chromatin spherules and cytoplasmic spherules by comparison 
with the conditions in other ciliate Infusoria. The macronucleus of 
most Ciliata consists of gran-ules aggregated into a more or less 
compact group. This group breaks up under certain conditions, the 
granules scattering through the whole endoplasm and soon disap- 
pearing by solution. When a new macronucleus is formed, it arises 
from one (or more?) of the micronuclei. The macronucleus of the 
Ciliata is generally regarded as especially connected with nutrition. 

The chromatin spherules in Opalina are derived from the chro- 
matin of the chromosomes and by their origin from chromatin and 
their solution and disappearance remind one of the macronuclear 



270 M. M. Metcalf 

granules of other Ciliata. But one notes a decided difference between 
the two. In Opalina the chromatin spherules are formed and dis- 
solved in the course of every cell division, while in most of those 
Ciliata which have been most carefully studied the macronucleus 
dissolves and is reformed only during or after conjugation, or under 
conditions of nutrition which often induce conjugation. In Hopli- 
tophrya imcinata, a probable near relative of Opalina, the macro- 
nucleus is very often found fragmented into granules or groups of 
granules which are scattered through the cell (Text Fig. VIII, C). 
One cannot believe that this fragmentation is usually connected 
with conjugation. I have not followed the fate of the scattered 
granules in HoplHophrya uncinata, nor have I as yet had time to 
study carefully the abundant refractive spherules in the endoplasm 
of this species. They seem to be formed in the macronucleus and 
also in the scattered fragments of the macronucleus when this 
breaks to pieces (Text Fig. VIII). They do not react to intra vitam 
stains exactly as do the endoplasmic spherules of Opalina, yet they 
are probabl}^ of a generally similar nature. The apparent connection 
of refractive spherules and macronucleus in Hoplitophrija uncinata, 
and the frequent fragmentation of the macronucleus, make this 
species a peculiarly favorable one for the study of the refractive 
spherules and their relation to the macronucleus. I hope soon to 
give the matter further study. The scattered macronuclei of Loxodes 
rostrum (Joseph 1907) are perhaps comparable to the scattered groups 
of macronuclear granules seen in Hoplitophrya. 

The relation of the refractive spherules in the endosarc to the 
chromatin spherules in Opalina is not fully established. The resem- 
blance between the two in their staining reactions is not a demon- 
stration of their relation, but it does suggest that the substance of 
the chromatin spherules may find its way into the endoplasmic 
spherules. This, is rendered still more probable by the fact mentioned 
that in Hoplitophrya apparently similar refractive spherules arise in 
the macronucleus and in the scattered groups of macronuclear gra- 
nules when the macronucleus fragments, 

Eefractive spherules somewhat comparable to those in the endo- 
sarc of Opalina are not rare among the Ciliata, Nydotherus and 
several species of Salantidium, present in the same hosts with 
Opalina, have many such refractive spherules in their endoplasma, 
which, however, seem alw^ays to stain darkly wdth iodine. They 
are not composed of true glycogen but are apparently paraglycogen 
of a somewhat different nature from that in the spherules of Opalina. 



Opalina. 



271 



It seems probable that some of the refractive bodies found in Fla- 
gellata and Foraminifera are of the same general nature. There 
seems to be something the same doubt as to the origin of the 






A 

Text Fig. Vin. 
Sections of Hoplitophrya uncinafa : 
A, stained with Mayer's haemalnm, 
shows the unstained refractive sphe- 
rules in the tnacronuclens ; B, stained 
with iron-haematoxylin shows the 
refractive spherules in the macro- 
nucleus darkl}' stained, the micro- 
nucleus and a bit of the cj'toplasmic 
foam being also shown; C, stained 
with Apathy's haematein I A and 
orange G, shows unstained refrac- 
tive spherules both in the dividing 
macronucleus and in some of its 
scattered fragments. The light area 
in the micronucleus is not a refrac- 
tive spherule. X lOlO diameters. 

refractive bodies in Pelomijxa that there is as to their origin in 
Opalina. Greef (1874) and Goldschmidt (1905) describe them as 
arising from the nucleus, Gould (1893) says that they divide by 



272 M. M. Metcalf 

constriction. Stolc (1900) and Bott (1907), on the other hand, are 
certain that they arise in the cytoplasm and that they do not in- 
crease by division, Stolc describes them as consisting of two parts, 
an outer envelope and an inner substance, the latter glycogen, the 
former a carbohydrate. Bott confirms Stolc, agreeing that the re- 
fractive bodies are probably reserve nutritive material. The mor- 
phological structure of the endosarc spherules in Opalina, with their 
outer layer of granules and more lightly staining central-portion, 
resembles the structure of the refractive bodies of Pelomyxa and their 
interpretation as reserve nutrient material seems altogether probable, 
though they are not true glycogen. As already shown, the chro- 
matin spherules of Opalina and the macronuclear granules of other 
Ciliata may be comparable. If, then, the endoplasmic spherules of 
Opalina are derived from the chromatin spherules, our series of com- 
parisons would include the macronuclear granules of most Ciliata, 
the chromatin spherules in the nucleus of Opalina, the endoplasmic 
spherules of Opalina, and the refractive bodies in Ilagellata and 
Foraminifera. There is a general resemblance also between the re- 
fractive spherules of Protozoa and the pyrinoids of plant cells. Both 
seem to be a reserve food supply and both are handed down from 
parent to child when the cells divide. We know nothing, however, 
to indicate any special connection between the pyrinoids and the 
substances in the nuclei of plant cells. 

Phylogeny of tlie nuclei of Ciliata. 

The question as to what in Opalina is the full homolog of the 
macronucleus in higher Ciliata can best be approached through a 
discussion of the evolution of the condition with two functionally 
diverse nuclei. The macronucleus of higher Ciliata arises by the 
metamorphosis of a nucleus which has itself arisen by division from 
the micronucleus. ^) It is therefore phylogenetically a complete 
nucleus and not a mere mass of granules extruded from a nucleus 
and gathered into a group. The macronucleus seems to be speciali- 
zed in connection with the nutrition of the cell. It is able to di- 
vide, as does the micronucleus, in the vegetative divisions of the 
cell, but it takes no part in the special phenomena, interpreted as 
maturation, which preceede conjugation. The micronucleus, apparently 



*) Nekesheimer (1908) does not describe the origin of the macronucleus in 
Ichthyophthirius (species?). The remarkable phenomena which he does describe, 
if correctly described, make it improbable that the macronucleus in this species 
arises by metamorphosis from a micronucleus. 



Oimlina. 273 

holds ill abeyance the t'unctioiis connected with the nutrition of the 
cell, but the potentiality of these functions must be present, since 
daughter nuclei from the micronuclei are able to transform into 
macronuclei. Probably each type of nucleus is a complete nucleus, 
the nucleus especially connected with conjugation being slightly 
specialized by diminution of some of its functions and probably of 
the chromatic material upon which these functions rest. The macro- 
nucleus is specialized by the great development of its nutritive activi- 
ties and a corresponding great increase in the amount of chromatin 
especially associated with these functions. The specialization and 
hypertrophy of the macronucleus seems to have gone so far that it 
is difficult to secure conjugation of the macronuclei, and so, partly 
as a consequence, the macronuclei degenerate. The germinal (un- 
specialized) chromatin is so overbalanced by the nutritive (speciali- 
zed) cbromatin in the macronuclei that it is unable to assert itself 
and bring about conjugation of these nuclei, and, without occasional 
readjustment such as is secured through conjugation, ultimate de- 
generation seems unavoidable. 

Before copulation or conjugation there seems to be quite gene- 
ralh^, among the Protozoa at least, a process of reestablishing the 
proper balance of the nutritive and other chromatin in the nucleus. 
It is apparently the nutritive chromatin which especially increases 
in amount during growth and ordinary vegetative divisions and the 
excess of this nutritive chromatin is gotten rid of before conjugation 
by the formation of chromidia, either the excess of vegetative chro- 
matin leaving the nucleus, or the excess of this specialized chro- 
matin being left in the nucleus, the ordinai-y chromatin going out 
into the cytoplasm and there reforming into a new nucleus or new 
nuclei, or, as in Chromidina (Gondek 1905), all the chromatin passing 
into the cytoplasm where after a time part degenerates and the rest 
forms the generative nuclei. We doubtless do not know the full 
significance of these phenomena, but this much seems probable, that 
there is division of labor between different parts of the chromatin 
and consequent hypertrophy of some parts during their periods of 
special activity. The specialization and hypertrophy of chromatin in 
connection with nutrition has gone so far in the macronucleus of 
Ciliata that it is simpler to secure a new macronucleus than to re- 
establish in the old macronucleus such a balance of the respective 
parts as will allow it to share in conjugation. 

What was the phylogenetic origin of the condition with two 
nuclei, one of which is highly developed for nutrition while the other 



274 M. M. Metcalf 

remains minute and hardly shares in the activities of growth. The 
divergence must have occured in a binucleated (or multinucleated) 
condition. We have in Opalina such a binucleated (or multinucleated) 
form. In what way could its condition with similar nuclei be chan- 
ged into a condition with dissimilar nuclei? 

First let us note again the fact that the nuclei of the bi- 
nucleated Opalinae are often slightly dissimilar in regard to mitosis, 
one being often in a slightly more advanced condition than the other. 
There is a similar divergence in regard to the formation of the prob- 
ably nutritive chromatin spherules, one nucleus showing these in 
a more advanced stage of formation. The exact balance of the two 
nuclei seems already somewhat disturbed in Opalina. 

May we conceive this divergence as going further, the nutritive 
chromatin becoming hypertrophied in one nucleus and not in the 
other, the second nucleus ultimately giving up almost all its con- 
nection with nutrition and becoming, much smaller, giving us ulti- 
mately the condition seen in higher Ciliata with very divergent 
micro- and macronuclei? 

One thing seems to stand in the way of such an interpretation 
so far as Opalina is concerned: in the division oi Opalina one whole 
nucleus, and not two half nuclei, is given to each daughter cell. 
The condition in Opalina is not a true binucleated condition. We 
have merely a delayed division of the body, which causes two 
daughter nuclei to lie for a long time in one cell, indeed even until 
they have entered upon the next mitosis. Division of the cell when 
it does occur is not associated with the mitosis in the nuclei which 
is taking place at the same time, but is really the delayed cell- 
divion that belonged with the last nuclear mitosis. Division of the 
cell-body lags one step behind the division of the nuclei. To get a 
proper understanding of the real meaning of this division we must 
bring together that division of the body and that division of the 
nucleus which really belong together. 

In attempting to do this we see at once that the direction of 
division of either nucleus or body must be changed. At present 
the long axes of nuclei and body coincide and remain constantly in 
this relation. The nucleus divides transversely and the body gen- 
erally longitudinally. ^) Can we find a plausible scheme which will 
get around this difficulty? 

') Lougitiulinal division of the body is characteristic of Flagellata and is 
dovibtless, primitive for Opalma. Many Flagellata show nnclei which when dividin;? 
elongate at right angles to the plane of division of the body and then divide 



Opalina. 275 

The present coiulitiou in Opalina, with an apparent but not a 
true binucleated state, could be changed into a true binucleated state 
comparable to that of Paramaecinm, if cell-division should change 
from longitudinal to transverse and at the same time should bisect 
each of the two nuclei. Instead of the condition shown in Text 
Fig. IX. A, as now, we would have that illustrated in Text Fig. 
IX. C, each cell recieving two daughter nuclei instead of one whole 
nucleus. From this condition, that of Paramaecium could be reached 
by functional and accompanying structural divergence of the nuclei, 
as suggested above. 'J'lie ordinary infrequent transverse divisions 
of the binucleated OpaUnae, do not help us in this schema, for they 
do not bisect the nuclei (Text Fig. IX, B). The false binucle- 





A B C 

Text Fig-. IX. Illustrating the development of the truly binucleated condition of 

the higher Ciliata from a pseudobinucleated form like Opalina. A and B are 

drawings of actual conditions found in 0. caudafa; C shows a hypothetical 

transverse division which bisects the two nuclei. 

ated condition of Opalina can be changed to a real binucleated con- 
dition only by the complete suppression, not the mere delay, of one 
division of the body. Were this to occur, then a transverse division 
of the body, such as we occasionally find, would bissect the two 
nuclei, being not a delaged division belonging to the last mitosis, 
but the division which properly belongs with the present mitosis of 
the two nuclei (Text Fig. IX, C). We can conceive the same re- 
sult as following still longer delay in the postponed division of the 



across the equator, their plane of division coinciding with that of the body. In 
Trypanosonies as in Opalina the division of the body and that of the nucleus 
are not synchronous and the planes of division of nucleus and body do not coin- 
cide. One cannot say which is the more primitive condition, that which does or 
that which does not show coordination between the division of the nucleus and 
the division of the cytoplasm. 



276 M. M. Metcalf 

body, it not occuring until the daughter nuclei are separated to a 
considerable distance, so that the division of the body (transverse 
in this case) could easily pass between the daughter nuclei, produ- 
cing thus in each daughter cell a truly binucleated condition. 

It seems to me quite probable that such has been the history 
of the evolution of the binucleated condition in higher Ciliata: first 
delay in division of the body, establishing a temporary binucleated 
condition; then complete suppression of this delayed division of the 
cell-body, establishing a true binucleated condition, each nucleus, as 
apparently now in Paramaecium, belonging to a potentially, but not 
actually, independent individual. 

The remarkable phenomena which Neeesheimee (1908) descri- 
bes for Ichthyophthirius probably cannot be brought into harmony 
with the interpretation of the nuclear conditions in the Ciliata here 
suggested. There are gaps in Neeesheimee's work, and an absence 
of detail in both figures and description, which make it desirable 
that this genus be further studied. 

Compound nature of the Ciliata. 

The truly binucleated forms, as well as the falsely binucleated 
Opalinae are really potentially double individuals; and similarly the 
multinucleated Opalinae, arising by further temporary suppression of 
divisions of the body, are highly compound forms composed of many 
potential individuals. These individuals all become ultimately distinct 
before or in connection with copulation, even in the multinucleated 
Opalinae, the temporarily suppressed divisions of the body finally 
appearing rapidly in the spring and producing unicellular gametes.^) 



The phenomena in the spring which preceede and 

accompany copulation. 

Phenomena in Ojtalina intestinalis* 

As the breeding season of the host approaches most of the 
Opalinae in the rectum increase the rapidity of their division, be- 



^) The fact that the macrogametes are often still biuucleated at the time of 
conjugation does not indicate that they are really binucleated forms, but merely 
that conjugation may occur before the complete separation of the definitive gametes, 
as if fertilization in a Metazoan should occur before completion of the maturation 
of the egg. Compare pages 285 and 290. 



Opalina. 277 

coming: very minute, a few individuals retain nearly their full size and 
do not encyst, but remain in the lectum of the frog-, apparently con- 
tinuing the infection of the host. The minute individuals encyst, 
pass into the Avater with the foeces of the host, and are eaten by 
tadpoles, in whose alimentary canals the little Opalinae work their 
way ont of the cysts and divide, forming micro- and macrogametes 
which copulate. ^) 

Decrease in the number of chromosomes. 

In the later mitoses before encystment one finds but four 
(Fig. 120. PI. XXII) instead of eight (Fig. 119, PI. XXII) chromo- 
somes. This change in the number of the chromosomes takes place 
in animals from four to eight times as large as the individuals which 
enter the cysts. It occurs before the vegetative chromatin is thrown 
out of the nucleus, the latter process, under normal conditions, 
taking place just before encystment and in the cysts or in the 
rectum of the tadpole. The decrease in the number of the chromo- 
somes might be due either to their union in pairs (synapsis of 
Montgomery) or to an actual "reduction division" at this stage. 
The chromatin ribbon breaks into eight instead of sixteen parts, 
and these do not seem to be unusually large. Possibly counting 
the granules in these chromosomes would show whether they are 
double or not. I have not yet done this, such work being necessa- 
rily slow, and my material needing restaining before any such counts 
can be made. It will probably be necessary to wait until another 
spring in order to have sufficient favorable material for studying 
this interesting point. The reduced number of chromosomes persists 
until copulation occurs (cf. Fig. U8— 152, PI. XXII; 168, 173, 
PI. XXIII; 183, 185, 187—191, PI. XXIV). 

The last division before encystment. 

One sees very clearly that in the last (?) division by which uni- 
nucleated animals ready for encystment are formed no mitosis of the 
ordinary type occurs (Figs. 121, 122, PI. XXII). The nuclei seem 
not to be in division at all, but rather are occupied in getting rid 
of a part of their chromatin, a process which will soon be described. 



') It seems necessary to accept the german use of the word copulation to 
denote fusion of two gametes to form one zygote, and of the word conjugation to 
denote the mutual fertilization of two gametes each by the other, as in higher 
Ciliata. The natural English use of these words would be the exact reverse. 

19* 



278 M. M. Metcalf 

The uninucleated condition is reached by suppressing one nuclear 
division while the body divides. There is at no time any such 
degeneration of nuclei as Neresheimer has described for 0. ranarum 
and 0. dimidiata, nor is there any formation of new nuclei from 
chromidia in the cytoplasm. The old nuclei discharge a portion of 
their chromatin and themselves persist as the reproductive nuclei. 

Extrusion of yegetative chromatin. 

In living nuclei, at this stage, which are getting rid of a part 
of their chromatin in this peculiar manner, one observes two large 
balls or discs which by staining are clearly shown to be composed 
of chromatin (Figs. 121—139, PL XXII; 236. PI. XXV). Occasionally 
instead of two such chromatin spheres one finds three (Figs. 132, 
134, PI. XXII), one or two of these being smaller. In other cases 
but one sphere is found, but in these cases another may have been 
present and have been extruded. The rest of the contents of the 
nucleus lies in the form of granules, generally in an hour-glass- 
shaped group, transversely, between the two chromatin spheres when 
two are present (Fig. 121). In the nuclei of the cysts one finds 
sometimes one (Figs. 131, 135, 138, 139, PI. XXII; 236, PI. XXV), 
sometimes tw^o (Figs. 136, 137, PI. XXII), sometimes three (Figs. 132, 
134, PI. XXII) such chromatin spheres. In the animals hatched 
from the cysts one finds usually but one such compact sphere of 
chromatin, or often none, the granules remaining in the nucleus 
being often also gathered into a spherical group, which however in 
both the living animals and in acetic carmine preparations can be 
distinguished from the denser sphere. When stained with Dela- 
field's haematoxylin the difference between the two is very clearly 
seen (Figs. 136, 137, PL XXII). By the time the gametes are ready 
for copulation, the dense chromatin spheres have entirely disappeared 
from their nuclei (Figs. 148—152, PL XXII; 168, 173, PL XXHI, 
also PL XXIV). 

These compact spheres of chromatin are extruded from the 
nucleus into the cj^toplasm and there degenerate. I have studied 
but little the minute animals in the rectum of the frog, before their 
encystment, and have but twice found in them the extrusion of the 
first chromatin sphere (Fig. 124, PL XXII; 279 \0. dimidiata], 
PL XXVII); I think, though, this usually occurs at this stage, as 
Neresheimre has said. In the cysts (Fig. 253, PL XXVI, 0. caudata), 
and in young forms hatched from the cysts in the rectum of the 
tadpole (Fig. 143, PL XXII, and 0. dimidiata, PL XXVIII, Figs. 306, 



Opaliua. 279 

308, o09), I have often seen one or two compact chromatin spheres 
already extruded and lying in the cytoplasm, or in the process of 
being extruded (Fig. 302, PI. XXVIII, 0. dimidiata). In animals which, 
without being encysted, liav(^ been ingested by the tadpoles and have 
passed unencysted through the whole alimentary canal to the rectum, 
one often finds the extrusion of great masses of chromatin from the 
nuclei. These masses, sometimes before, but generally just after 
their extrusion, become reticulated, with lighter areas in the meshes 
of the heavily stained chromatin net (Figs. 237—247, PI. XXVI). 
In some cases the nuclei from which the chromidia have been ex- 
truded show very distinct cliromosomes (Fig. 238, 239, 241). In 
other cases irregular chromatin masses are left in the nuclei 
(Fig. 240). In still other instances the chromatin left in the nucleus 
is very finely granular (Fig. 245). In Fig. 241, in the posterior 
end of the body, is shown a hollow sphere of net-like chromatin 
surrounding an unstained central sphere. In many degenerating 
nuclei of 0. oUrigona exactly similar structures were found (Figs. 104 
— 109, 111 — 115, PI. XXI). In a few instances, when the staining 
was exactly right, I have seen that the chromatin spheres in the cysts 
were composed of a net-like, darkly staining envelope surrounding a 
central sphere (Fig. 133, PL XXII: the central sphere was present 
but is not indicated in the drawing which shows the reticulated sur- 
face of the sphere). I have not yet attempted to test microchemi- 
cally the nature of these central spheres. The unencysted ingested 
animals just described showed very clearly that the chromatin spheres 
fragment and scatter through the cell, there disappearing. It is 
not quite certain that always two chromatin spheres are extruded, 
but that there are usually two appears certain from the phenomena 
observed. It seems altogether probable that we have in these pheno- 
mena a throwing away of nutritive chromatin similar to that describ- 
ed by Hektwig, Schaudinn and others for numerous Plasmodroma. 
I have described the formation of chromatin spherules during 
the course of each mitosis throughout the year (except perhaps just 
before and after copulation) and have suggested that these chro- 
matin spherules are nutritive — comparable to the granules of the 
macron ucleus of higher Ciliata. Their formation and extrusion is 
positively useful, being probably connected with nutrition and per- 
haps with the formation of the refractive spherules of the endosarc. 
The formation and extrusion of the large chromatin spheres before 
copulation is apparently negatively useful, leaving the nuclei in the 
right condition for copulation. It seems likely that the chromatin 



280 



M. M. Metcalf 



spheres are composed of nutritive chromatin essentially similar to 
that seen in the numerous small chromatin spherules throughout 
the year. 

LowENTHAL (1904) describes the presence and manner of for- 
mation of one or more large dense chromatin spheres in the nuclei 
of encysting 0. ranarum (Text Fig. X). He did not observe their 
extrusion into the cytoplasm. He interprets them as homologous 
with micronuclei, whereas they are probably more comparable with 



(■:■■ ' 



tt 









dL 



-^Tv^ 




y 






n 



J 




fe 



Text Fig. X. Loewenthal's figures of the formation of a "micronucleus-like body" 
in the nuclei of 0. ranarum: a, a nucleus with chromatin net and nodal thi- 
ckenings; fc, a nucleus with the chromatin gathered mostly into large masses at 
the periphery; c. shows the chromatin masses again fragmented; d and e, show 
the gathering of the chromatin at the center of the nucleus; f—li, show the 
separation of a compact darkly-staining chromatin sphere from the central mass, 
and its wandering to the periphery and suggest |its possible extrusion from the 
nucleus: the material remaining in the central mass fragments and scatters 
through the nucleus; j, shows two chromatin masses at the periphery of the 
nucleus ; A-, a nucleus figured but not described by Loewenthal. X 2250 diameters. 

macronuclei, being probably composed of nutritive chromatin. I have 
not studied the process of formation of these chromatin spheres with 
sufficient care to justify me in commenting upon Lowenthal's de- 
scription of the manner of their formation. I would only suggest 
that it must be difficult to be certain of the sequence, of the iso- 
lated phenomena observed. 

Neeesheimer (1906 and 1907) gives an account of these chro- 
matin spheres essentially similar to that I have given above. He 
emphasises the comparison of the two spheres with the two polar- 
bodies of Metasoa (cf. p. 302). 



Opalina. 281 

It is intfrestin^ to note that at least some of the individuals 
which pass unencysted through the alimentary canal of the tadpole 
to the rectum ') form and extrude chromatin spheres but not ([uite 
in the noinial manner. I have not observed that they encyst if liv- 
ing in the natural species of tadpole. I have, however, had such 
individuals of 0. intestinalis and 0. caudata encyst in the recta of 
large tadpoles of Baua esculcnta four days after the infection was 
secured, and have successfully infected tadpoles of Bufo vulgaris 
with these cysts from the Eana esculenta tadpoles. In two series 
of sections of the rectum of an tadpole of Bomhinator padiypus in- 
fected six days with 0. intestinalis, I find numerous large individuals 
with eight chromosomes, and numerous smaller forms with four 
chromosomes, extruding their chromatin. Perhaps in the rectum of 
the frog extrnsion of the chromatin may occur either before or 
after the reduction in the number of the chromosomes, though usu- 
ally, if not always, under normal conditions the extrusion occurs 
after the number of chromosomes has been reduced. 



EiicijstiiieHt. 

The number of nuclei in the cysts varies with the species and 
within the same species. In one series of preparations of 0. ranarum 
out of 146 cysts tabulated 1 had no nucleus (abnormal), 25 had 
1 nucleus of the ordinary size, 70 had 2 nuclei, 32 had 3 nuclei, 
16 had 4 nuclei, 1 had 5 nuclei and 1 had 6 nuclei. According to 
Neresheimek (1907) three to five nuclei are most frequent. Six to 
twelve or more nuclei in the cj^sts are described by Zeller, Tonniges 
(1899), Loewenthal and Neresheimer (1906 and 1907), though these 
larger numbers are infrequent. In 0. ohtrigona (Zeller) and 0. dimi- 
diata (Zkller, Neresheimer, Metcalf 1907 «) conditions are the same 
(Figs. 285—288, PI XXVII). In the binucleated species 0. saturnalis 
(Leger & Duboscq), 0. intestinalis (Zeller, Metcalf) and 0. caudata 
(Zellek, Metcalf) the cysts have generally one nucleus (0. intestinalis. 
Figs. 130—136, 140—143, PI. XXII; 236, PL XXV; 0. caudata, 
Figs. 137—139, PI. XXII; 252, 253, PI. XXVI) though I have often 
found two nuclei in the cj'sts of the latter two species (Figs. 254, 
255, PI. XXVI). Occasionally one finds the binucleated encysted 
animal in the process of division (Fig. 255), though I do not think 
the division can often be completed until the animal emerges from 



^) Compare the chapter on infection experiments, page 314. 



282 M. M. Metcalf 

the cjat, the free action of the cilia apparently being of great 
assistance in all divisions at all times of year, helping to separate 
the daughter cells. I have, however, found, two cysts of 0. caudata 
each containing two individuals either entirely distinct or so nearly 
so that the connection between them could not be observed (Fig. 
256, PI. XXVI). Zeller and Loewenthal describe cysts of 0. ranarum 
containing individuals in division, and Doflein quotes Puzesmecki 
as having seen the animals in the cysts divide into several offspring. 
I have seen nothing of this multiple division in the cysts. 

The cysts do not need to lie in water in order to produce suc- 
cessful infection. 

Tadpoles eat, often eagerly, the foeces of frogs and toads, so 
that it is easy to infect them. Offer feeding cysts to the tadpoles, 
the cysts will be found throughout the whole alimentary canal in- 
cluding the rectum. The little Opalinae leave the cysts usually in 
the rectum of the tadpole but occasionally they are found in the 
small intestine. Wherever hatched the little Opalinae collect at 
the upper end of the rectum of the tadpole, just as the larger forms 
do in the rectum of the frog or toad. They mostly keep together, 
lying between the foecal mass and the rectal wall, a few individuals 
only being found scattered through the foecal mass. 

I have studied the spring reproduction in 0. mfestinalis, 0. caudata 
and 0. dimidiata, and will describe the phenomena for all three species, 
beginning with 0. intestinalis. 

The minute forms of 0. intestinalis ready for encystment do not 
have the body form characteristic of larger individuals, but look 
more like Amoeba Umax (Figs. 121 — 129, PI. XXII). They are ciliated, 
but the cilia are unusually delicate, being distroyed by acetic car- 
mine, while the cilia of larger individuals in the same preparations 
are only considerably injured. The narrow posterior end of the 
body often shows a peculiar minutely lobulated appearance similar 
to what one often sees at the posterior end of an actively moving 
Amoeba proteus (Figs. 122, 123, PI. XXII). Ectosarc and endosarc 
are distinct and each contains the usual spherules. The last division 
before encj^stment is almost always longitudinal (Figs. 121, 122, 
PL XXII) but possibly may sometimes be transverse (Fig. 123, 
PI. XXII). I have not had the good fortune to see the whole 
process of encystment in any species. Zeller describes the process 
for 0. ranarum as follows (p. 359). — "i>^e Tierchen scliivimmen zwar 
noch eine Zeitlang mit grofler LebhaftigJceit umher, dann aber werden 
sie zuseliends langsamer in ihren Bewegungen, zielien sicli Jcugelformig 



Opalina. 283 

zusammen und scheideti, indcni sic sich dabei schneUer oder langsamer 
drchen. eine farUose, glasheUe Cijste um sich ab." 

The cysts vary considerably in size. They are mostly spherical 
or nearly so. some are oval (Fi^. 130, PI. XXII), and a few some- 
what irregular in form. One frequently sees encysted animals many 
of whose ectosarc spherules are at the extreme outer edge of the 
ectosarc (Fig-. 134, PL XXII), and in a few instances, I have seen 
cysts in which, between the cyst wall and the ectosarc, there were 
numerous refractive globules which seemed to be extruded ectosarc 
sperules (Fig. 135, PI. XXII). It is easy to see in some cases a 
mass of granular debris left behind, in the cysts which the animals 
are leaving (Fig. 140, PI. XXII). Other cysts are left entirely 
empty when the animal hatches, no such debris being found (Figs. 
142, 143, PI. XXII). It seems as if some individuals, during en- 
cystment, got rid of an excess, through generally not of all, of the 
ectosarc spherules. 

Cysts of 0. ranarum stained with Mooee & Bkeinl's lithium- 
iron-haematoxylin often show the presence of endosarc spherules. 
On the other hand many cysts contain no endosarc spherules. In 
minute individuals ready for encystment and in slightly larger forms, 
similar diverse conditions are seen. In the binucleated species and 
in 0. dimidiata most if not all of the animals hatching from the 
cysts contain endosarc spherules. The presence or absence of the 
spherules in 0. ranarum is probably dependent on nutrition. 

It happens that none of my sketches of 0. intestinalis show 
binucleated cysts, though I have seen many. Fig. 124, PI. XXII, 
shows a binucleated individual in the process of encystment. 

Before hatching from the cysts the little Opalinae become very 
active, the speed of their revolutions increasing until one becomes 
fairly dizzy as he watches them. Cysts in the duodenum of the 
tadpole may contain these very active animals, indicating that hatch- 
ing may take place soon after ingestion. Much more frequently 
hatching occurs in the posterior part of the intestine or in the 
rectum. The cyst wall weakens at some point and the little Opalina 
squezes through, sometimes slowly, sometimes rapidly, and swims 
off with a very rapid motion quite different from the motion of other 
individuals. The newly hatched individuals have well developed, 
cilia, longer in proportion to the size of the body than are the cilia 
of larger forms Figs. 140—144, PI. XXII). The cysts begin to 
hatch within three hours of the time of their ingestion, pro- 
bably even earlier. 



284 M. M. Metcalf 

Legek & DuBOscQ (1904 a) describe a second type of "endo- 
genous cysts" for 0. ranarum. In an ordinary multinucleated indi- 
vidual, a bit of protoplasm containing one to four nuclei is said to 
isolate itself from the rest of the protoplasm and acquire a cyst wall. 
The cyst so formed is extruded from the body of the parent. This 
description, which is very brief and unillustrated, needs confirmation 
before it can be accepted. No other student of Opalina has seen 
anything of this sort. 



The formation of the gametes. 

Longitudinal division is observed almost as soon as one finds 
the animals hatching from the cysts, (Figs. 146, 147, 153, PI. XXII). 
I have found no way of determining how many divisions take place 
before the gametes are formed. The minute animals do not live long 
enough outside the body of the tadpole to allow one to directly 
follow the phenomena from the time of hatching from the cyst to 
conjugation. Size is not a safe criterion, for the cysts and the ani- 
mals that hatch from them are of various sizes; so also are both 
the micro- and macrogametes. Time relations have failed to deter- 
mine the point, for one cannot be certain how long a time is re- 
quired for one division, and the time when one observes copulation 
is different in different infections. 

In animals removed from the tadpole and placed in salt solution 
with a bit of the rectal wall and some of the rectal contents, di- 
visions just begun before removal from the host require generally 
from two to twelve hours to complete even under the most favo- 
rable conditions; in many other cases division is not completed at 
all, even pairs which upon removal from the host were almost di- 
stinct remaining unseparated after more than twelve hours. It is 
not improbable that division under natural conditions in the rectum 
may be more rapid than in even the most favorable artificial cul- 
tures. In one series of infections both micro- and macrogametes 
were abundant after forty-two hours and frequent instances of co- 
pulation were observed. Generally copulating pairs are abundant 
fifty to eighty hours after ingestion of the cysts. In material five 
and a half days after feeding the cysts I have found many zygotes 
but not copulating pairs. The average time required between in- 
gestion of the cysts and copulation is therefore uncertain, as is also 
the number of divisions that intervene, if indeed the number be 
constant. In the multinucleated Opalinae the number of nuclei in the 



Opalina. 285 

cysts varies from one to twelve, so probably the number of divisions 
intei'venin;? between hatching- from the cyst and the formation of 
definitive g-ametes is not constant. We have already seen that the 
condition of tlie nuclei in different cysts is different, indicating that 
the time required after hatching to produce the ripe gametes may 
be different in different cases. 

As a result of the divisions following emergence from the cysts, 
two sorts of gametes are formed 1) macrogametes which do not mark- 
edly differ from the individuals of the asexual generation ') and 
2) microgametes of very minute size and peculiar form. The 
macrogametes differ from the asexual forms in being smaller and in 
having relatinly longer cilia (Figs. 144, 148, PI. XXII; 164, PL XXIII). 
They have one (Figs. 164—168, PI. XXIII; 183, 185, PL XXIV; 210, 
PL XXV) or two (Figs. 170—180, PL XXIII) nuclei. Probably the 
typical fully mature gamete would be uninucleate, but copulation 
may occur before the final division which produces the uninucleate 
condition. The nuclear phenomena following copulation will soon 
be described. The macrogametes have large excretory organs, often 
very clearly seen in these small bodied forms {cf. Metcalf 1908 &). 
In the Opalinas, at all times of year, the excreta, which are for a 
time dragged behind the body, are sticky. They are no less so in 
the macrogametes (Figs. 147, 153, PL XXII), but this stickiness of 
their excreta is not a phenomenon comparable in any way to the 
stickiness of Paramaecinm when ready for conjugation (Calkins 1906) 
and has no relation to copulation. 

The microgametes are much smaller (Fig. 163, PL XXIII). Their 
cilia are few in number and are long and weak. They are absent 
from the posterior end of the body, which is drawn out into a long 
aud slender tail wiiich is bent, generally at right angles, near its base, 
at a point usually about midway in the whole length of the body. 
This bend in the tail probably aids in securing spiral movement in 
swimming. Near the tip of the tail is usually a small swelling. 
Perhaps this is always present in functional microgametes. The tail 
appears homogemous and transparent. It seems to be composed of 
ectosarc alone. It is very sticky. One imagines that the little 
swelling near the tip is a special accumulation of the sticky material, 
but the animals are so small that it is not easy to find conclusive 
evidence of this point. The stickiness of the microgametes is 



• Opalina has uo true alternation of generations, so that this term whil 
convenient to use, is not strictly accurate. 



286 ^I- M. Metcalf 

clearly an adaptation to copulation and is probablj^ comparable to 
the stickiness of the isogametes in Paramaecium. 

The microganietes usually swim tail foremost, though sometimes 
one finds them swimming in the opposite way. Doubtless the habit 
of swimming with the sticky end of the body foremost is an adap- 
tation to copulation, helping the gametes to become attached. I think 
that the microgametes wiiich an found swimming with the tail 
behind lack the ball of sticky material near the tip of the tail. In 
every case in which this point was noticed it was found to be so, 
but the observations are too few to make one certain that mature 
and immature microgametes can always be distinguislied by their 
mode of swimming. The microgametes contain but one nucleus. This 
is usually difficult to see in the living animal, though sometimes it 
shows clearly. No excretory organs or extruded excretory granules 
are seen in the microgametes, but the extruded granules are some- 
times found in the mother-cells from which the microgametes arise 
and in individuals of the preceeding generation. Endosarc spherules 
are present in the microgametes (Fig. 161, 163, PI. XXIII). Ectosarc 
spherules are found in the microgametes of 0. caudata (Fig. 259, 
PL XXVI) and 0. dimidiata, and are doubtless present in the micro- 
gametes of 0. intestinalis also, though I find nothing in my notes 
upon this point. 

The gametes arise by longitudinal division in every case which 
I have observed (Figs. 146, 147, 143, XXII, 159, PI. XXIII). Appa- 
rently transverse division does not occur between the time of hatch- 
ing from the cyst and copulation, though it might be about as 
frequent as in the asexual generation and still very likely not be 
observed. There is nothing of special note in the divisions which 
result in the formation of the macrogametes. Division begins al- 
most always at the anterior end (Fig, 146, 153, PI. XXII) rarely at 
the posterior end (Figs. 207, 209, PI. XXIV) sometimes at both ends 
at the same time (Fig. 147, PI. XXII). The strand of tissue, which 
ultimately is all that is left connecting the two daughters, may lie 
at any level in the posterior two-thirds of the bodies; usually it is 
quite near to the posterior end. 

In the formation of the microgametes the divisions for at least 
two generations do show some divergence from the ordinary di- 
visions. The daughter cells are more slender than usual and they 
seem to have unusual difficulty in pulling apart. The division be- 
gins apparently always at the anterior end of the body and moves 
bockwards until the daughter cells are connected only by the extreme 



Opaliua. 287 

posterior tips of the bodies (F\g. 159, PL XXIII). The dauf?hter 
cells, by attempting to swim in opposite directions, draw the poste- 
rior ends of tlieir bodies out into slender points before tliey finally 
separate (cf. 0. dhnkUata, Figs. 307—309, PI. XXVIII). The very 
pointed animals whicli thus arise (Figs. 154, 155, 160, PL XXIII) can 
by this feature be distinguished from the macrogametes and the cells 
from wliicli the latter arise. This division required in some ob- 
served instances from two to four hours, reckoning from the first 
appearance of bifurcation of the anterior end of the body until the 
complete separation. 

A second division of the same type follows. It may begin in 
one of the daugliter cells before the last division is complete {cf. 
0. dimidiata. Figs. 308, PL XXVIII). I have never seen both 
daughter cells so dividing again before their separation. One cannot 
say how many divisions of this sort occur before the definitive mi- 
crogametes result. In the final division which forms the microgametes 
a very long and very slender thread is drawn out between the two 
daughter cells. It seems as if the animals become more sticky at 
their posterior ends and so have more difficulty in separating. One 
can watch the elongation of this thread until it becomes finer than 
one of the cilia of the body. It may reach a length more than 
twice as great as that of the body proper of one of the animals. 
Generally, on this thread, the point of original contact of the two 
bodies is indicated by a few granules resembling excretory granules 
with sometimes a little debris. One can thus determine that the 
elongation of the two bodies is frequently unequal, even as much as 
two-thirds of the thread coming from one daughter-cell. It seems 
probable that the tail of the raicrogamete is derived from this thread. 
In sevei'al cases in which I saw one daughter-cell dividing before 
it had completely separated from its fellow, the dividing cell had 
by far the longer tail. In each instance the undivided cell was 
somewhat pointed posteriorly, but had practically no tail {cf. 0. 
dimidiata, Fig. 308, PL XXVI IIj. It is possible that the gametes 
are formed by diiferential division, the two sorts diverging at least 
one generation before the formation of the definitive gametes. This 
is, however, merely a suggestion with very insufficient evidence in 
its favor. 

There are two sorts of tailed forms 1) larger ones with short 
straight tails (Figs. 154, 155, PL XXIII, cf. 0. dimidiata, PL XXV III, 
Figs. 307, 308) and 2) smaller forms with much longer tails usually 
bent at a right angle (Fig. 163, PL XXII). Only the latter have 



288 M. M. Metcalf 

the ball of sticky material near the tip of the tail. These are the 
true microgametes. I have seen individuals of the first type in a 
late stage of division and have followed the division to its com- 
pletion seeing two forms with very long tails arise from one short- 
tailed form (Figs. 159, 160, PI. XXIII). I have never seen the 
transformation of such daughter-cells with long straight tails into 
typical microgametes with bent tails bearing a ball of sticky matter 
near their tips. This transformation does not occur immediately 
after the division. It seems to be the tailed microgamete mother- 
cells which Neresheimer has described as the gametes of 0. dimi- 
diata. I do not find either these or the true gametes of either type 
^^gans plaW\ as he describes them. All are generally circular in 
cross section, though they may be broadly oval. The microgametes 
vary in size ; so do the short-tailed forms from which they arise. 
The largest of the true microgametes are nearly or quite as large 
as the smallest of the short-tailed forms. 

In my preliminary notice (Metcalf 1907) I wrote "The tailed 
gametes are of two sizes, one about twice as large as the other, the 
smaller being found from the larger by longitudinal division". This 
was probably an error. There are larger and smaller microgametes, 
the largest being fully twice as large as the smallest, but it is 
doubtful if the latter arise from the former, both probably arising 
by division of the short-tailed forms, as described. In one case, I 
have seen, in 0. dimidiata, a microgamete mother-cell, in process of 
division, attached by its unusually long straight tail to the center 
of the body of a uninucleated macrogamete no larger than the 
microgamete mother-cell (Fig. 313. PI. XXVIII). The attachment 
was a firm one, lasting over three quarters of an hour while the 
animals were Actively swimming. During this time no change oc- 
curred. The individuals were then lost. This seems to have been 
probably an abnormal attempt to copulate on the part of a micro- 
gamete mother-cell. I doubt if it would have been successful or if 
it indicates that fully formed and functional microgametes are ac- 
customed to divide. 

Both sorts of gametes are often numerous in the rectum of the 
tadpole. The forms resembling raacrogametes are far the more nu- 
merous, probably in part because one cannot distinguish the defini- 
tive macrogametes from forms destined to divide further. The lar- 
gest number of microgametes seen in'one rectum was seventy eight; 
in the same rectum the number of pairs was forty-two; and the 
number of the larger, tailed forms (parent-cells requiring probably 



Opaliiia. 289 

but one more division to form definitive microgametes) was twenty- 
two. Similar proportions, but with smaller numbers, were frequent. 



Copulation, 

In copulation a micro^-amete fuses completely with a macro- 
gamete (Figs. 210—217, PI. XXV, also 164—182, PL XXIII). The 
first contact is purely accidental, there being no evidence of any 
attraction of the gametes for one another. Nekesheimer describes 
the sliort-straight-tailed forms in 0. dimidiata, wliich he interpreted 
as gametes, as circling around each other, as if under the influence 
of some mutual attraction. May this not have been merely the usual 
spiral movement seen in all Ciliata and Flagellata, appearing circular 
in this case because confined by the slide and cover-glass almost 
to one plane? The microgametes cling to anything which they touch 
with their sticky tails, though they seem never to cling to one 
another. Indeed I believe I have never happened to see two of 
them in contact even for a moment. 

The animals in the rectum gather chiefly in a single group 
(sections of the recta show this clearly). They have the same 
habit of gathering in groups in the slide cultures, collecting usually 
about some bit of foecal matter. In such a group thei-e are often 
thirty to one hundred forms which look like macrogametes and per- 
haps a dozen microgametes. The latter are constantly striking the 
macrogametes, clinging to their cilia and again breaking away, 
either because of the active swimming movements of the macro- 
gametes, or because of violent contact with other individuals. Even 
true copulating pairs may be torn apart by other individuals 
swimming between them. In two such instances copulation between 
the intruder and the microgamete immediately followed. Frequently 
two (0. caudata, Fig. 271, PI. XXVII) and once three microgametes 
were seen attached to the cilia of one macrogamete. Microgametes 
were seen attached to individuals in an advanced stage of division 
to form two macrogametes (0. caudata, Figs. 269, 270, PI. XXVII). 
They seem readily to attach to any of the individuals of the macro- 
gamete type. 

The loose attachment of the microgamete to the cilia of the 
macrogamete changes to the closer union and ultimate fusion of co- 
pulation (Figs. 210—217, PI. XXV, various, stages on PL XXIII, 
cf. 0. caudata, PL XXVII). The tail of the male fuses at its tip 
with the body of the female: then the tail of the male becomes 



290 M. M. Metcalp 

shorter and thicker and, after from half an hour to an hour, the 
two bodies are almost completely fused. Frequently one can distin- 
guish for quite a time upon the zyg-ote a slight protuberance bearing 
a few weak cilia of the microgamete type, thus marking very 
clearly the point of fusion, which may be in any region of the body 
(Fig. 182, PI. XXIII, cf. 0. caudata, Figs. 265, 268, PI. XXVII). 
I have seen all possible stages in the copulation, have three times 
followed the whole process from the first contact (observed) to the 
complete fusion, and have many times followed the process through 
the earlier or later or middle period and have then killed and stain- 
ed the animals in order to observe more closely the nuclear phe- 
nomena. 

The macrogametes may have either one or two nuclei (PL XXIII). 
The microgamete has always but one. When the macrogamete is 
uninucleated the nucleus may be in the resting stage (Fig. 164, PI. 
XXIII) or in mitosis (Figs. 167, 169, 170, 182, PL XXIII); similarly 
the two nuclei in the binucleated females may be either resting 
(Figs. 171—173, 178. PL XXIII) or in mitosis (Fig. 180, PL XXIII). 
I have never seen the nucleus of the microgamete in mitosis before 
the complete fusion of the two bodies. 

When the macrogamete has but one nucleus this unites ob- 
liquely end to end with the nucleus from the microgamete (Figs. 
187 — 196, PL XXIV). The two nuclei apply themselves closely, and 
ultimately the double membrane between them breaks down (Figs. 
191 — 193), sometimes first in the middle sometimes first at one edge. 

If the macrogamete has two nuclei, conditions become more 
complicated. If the two female nuclei are in the resting condition, 
the male nucleus may fuse with either one. In one instance of 
copulation of this type I have watched the entrance of the male 
nucleus and have seen it fuse with the posterior nucleus of the 
macrogamete, the resultant fusion nucleus being considerable larger 
than the other nucleus in the same zygote (Figs. 174 — 177, PL XXIII). 
I had a single acetic-carmine preparation which suggested that the 
male nucleus had entered and had passed by the posterior nucleus 
of the binucleated macrogamete and was just uniting with its anterior 
nucleus (Fig. 186, PL XXIV). Sometimes one sees the male nucleus 
lying between the two female nuclei, genej'ally nearer to the anterior 
one (Figs. 197—200, PL XXIV). In one lot of fine material from 
an eighty-eight hour infection I found that over fifty of the bi- 
nucleated forms had one nucleus (almost always the anterior one) 
much the larger. In some of these larger nuclei it was easy to see 



Opalina. 291 

eight chromatin masses which were doubtless eiglit chromosomes 
(Figs. 201—208, PI. XXIV), showing that the nuclei were syncaria 
resulting from copulation. 

Copulation may occur while the single nucleus of the macro- 
gamete is in division. In tiiis case the male nucleus waits until 
the division is complete and then fuses with one of them, the 
anterior one in the best case I have observed (Fig. 200, PI. XXIV). 
Both nuclei of the macrogamete may be in division when cop- 
ulation takes place. One preparation showing this condition was 
very clear (Fig. 204, PI. XXIV). The male nucleus, by its longitudinal 
striation and the position of its chromosomes in two polar groups, 
showed that it also was dividing. A second preparation shows a 
zygote in division with eacli of the femali nuclei already divided, 
giving four daiigther nuclei and the male nucleus, in an early stage 
of mitosis, lying beside them (Fig. 208, PI. XXIV). Under perfectly 
normal conditions division of the cell-body of this animal should 
have occurred before the complete division of the nuclei. Another 
preparation of a daugther cell ^) just from division and whose nucleus 
was in a little later telophase than the female nuclei in the next 
to the last case, showed a male nucleus lying against the constricted 
portion of the dumbbell-shaped female nucleus (Fig. 206, PI. XXIV). 
This male nucleus was in a very early anaphase stage of division. 
It must have entered before the division of the macrogamete arid 
have passed to one of the daughter cells {cf. Fig. 204, PI. XXIV). 
I have seen two instances of another condition which may pos- 
sibly stand as the next term in this series of copulation conditions. 
These individuals seemed each to show four nuclei. In one instance 
the four nuclei were in an oblique row (Fig. 207, PI. XXIV). As 
the posterior end of the body showed a slight division-furrow, it 
is somewhat doubtful if this was a zj^gote. It may possibly have 
been a dividing binuclear macrogamete in which the dumbbell- 
shaped nuclei overlapped, somewhat more than in Fig. 204, PL XXIV, 
producing in edge view the appearance of four nuclei. The pre- 
paration, stained with acetic-carmine was not entirely clear. The 
other instance was of a living animal, a dividing macrogamete, 
with a considerable division fuirow at its i)osterior end (Fig. 209, 
PI. XXIV). It seemed to show four unusually small nuclei side by 
side in pairs, but the picture was not very clear. Both of these 

') That it was a daughter cell fresh from division was indicated by the 
irregular contour of one side, the side by which it had been attached to its 
sister cell. 



292 M. M. Mktcalf 

cases are so doubtful that they can hardly be taken into account 
in endeavoring to understand copulation. They may also have been 
abnormal, division of the body being- delayed be3'0nd the proper 
time, giving- nuclear relations not found in perfectly normal cells. 
In all other cases described the phenomena were clear. 

It is uncertain what would have been the further history of 
each type of zygote described. When the male nucleus unites with 
one of the nuclei of a binucleated macrogamete, it seems probable 
that the next division would separate the syncarion from the smaller 
nucleus, one going to each of the daughter cells, but I have not 
observed the division of these forms. If the binucleated zygotes do 
divide in this way, one sees no reason why the daughter cell w^hich 
receives the nucleus with four chromosomes might not itself fuse 
with another microgamete to form a uninucleated zygote, just as 
a polar body may be fertilized, but I have made no observations 
bearing upon the point. If it does not do sd, it would seem to 
indicate that changed cytoplasmic conditions due to the entrance of 
the microgamete stand as a bar to further copulation. Professor 
BovEEi tells me he has never succeeded in fertilizing a nonnucleated 
fragment of an already fertilized sea-urchin egg, though it is easy 
to fertilize fragments of unfertilized eggs. Sufficient study of old 
infections in Opalina would probably determine this interesting 
point. The comparison of these binucleated macrogametes with an 
egg. wliich has not yet completed its maturation seems a correct 
comparison, for doubtless the typical fully-formed macrogamete in 
Opalina would be uninucleated. Entrance of the male cell in Opalina 
seems to occur either after or during the maturation divisions, as 
in Metazoa. 

The later history is much more doubtful in those cases in which 
the male nucleus divides before fusing with any of the female nuclei 
{cf. Fig. 204 and Fig. 206, PI. XXIV). Fig. 206 seems to interpret 
Fig. 204 to the extent of showing that the dividing male nucleus 
all goes to one of the daughter cells. Apparently there it must so 
behave as to give four of its eight daughter chromosomes to each 
of the daughters of the female nucleus, but just how this is effected 
we cannot say. It might be either by fusing while the female 
nucleus is still incompletely divided, or by waiting until both nuclei 
are completely divided and then the four daughter nuclei conjugating 
in pairs. I hope to find in the sections of infected recta of the 
tadpoles answers to some of the questions still unsettled, but the 
preparation of the sections is difficult because of the dirt in the 



Opalina. 293 

rectum and tlieir stiuly witli an immersion lens necessarily takes 
much time. I am. therefore, not delaying- the publication of this 
paper until all of these sections have been made and studied. If 
from this study further results of interest are obtained, they will 
be communicated later. 

In the material from the older infections one finds very many 
peculiar nuclei of huge size, much more pointed than usual and 
with peculiar spindles and chromatin (Figs. 222—227, PI. XXV). 
It seems probable that these are copulated nuclei (syncaria). This 
is strongly suggested by such a condition as we see in Fig. 188, 
PI. XXIV, in which we see the very pointed distal ends of the 
nuclei developed even before the two nuclei have fused at their 
apposed surfaces. I have not yet found such a spindle form in 
each nucleus in binucleated individuals, or in one nucleus while the 
other nucleus is in division according to the regular "asexual genera- 
tion" type. I have however often found uninucleated gametes in 
a late stage of nuclear division in which the forming daughter 
nuclei were both of the peculiar type just described (Figs. 226, 227, 
PL XXV). It is doubtful whether the spindle form of these nuclei 
indicates division. The syncaria in many species of Protozoa are 
spindle shaped when not in division. ^) It is probable that such of 
my material as is carefully preserved does not include old enough 
infections to determine fully the phenomena following the copulation 
of the nuclei. 

CoHN (1904) has described fundamentally diftereut phenomena 
in the conjugation of 0. intcstinalis. It is evident that the animals 
seen apparentl}' in conjugation could not have been Opalinae. The 
character of the nuclei, as well as the manner of the conjugation 
shows this. The brief unillustrated description of conjugation in 
0. ranarum, given by Leger & Duboscq (1904 a) is also fundamentally 
difterent and is probably erroneous. They say that two OpaU'nae 
"resembling those of the ordinary cysts" come together by their 
anterior ends, lie for a long time rubbing against each other and 
turning, and then from a cyst which contains the two, each animal 
occupying half of the cyst. These phenomena are so divergent from 
those observed by other students that the description can hardly be 
accepted without contirmation. It is possible that Leger & Duboscq 
mistook the encj^stment of a dividing individual for conjugation, 
though with such experienced observers this seems improbable. 

') Somewhat similar spindle-shaped nuclei of peculiar appearance are found 
in degenerating 0. obtriyona. 

20* 




294 M. M. Metcalf 

Neeesheimer figoe, 1907) saw both microgametes and macro- 
gametes and saw them in copulation (Compare his Fig. B, on p. 26. 
One of his drawings from this figure is reproduced here in Text 
Fig. XI), but he failed to recognise this as copulation, interpreting 
it as abnormal budding. The forms which he describes as isogametes 
seem from his figures to have been microgamete mother-cells. He 
once saw two of these come together by their anterior ends and 
gradually fuse, closing together like the blades of a pair of shears. 
The nuclear phenomena in this case were not followed. This he 
took as showing the presence of isogamous copulation. Doubtless it 

was abnormal. In one instance I have found 
in an acetic carmine preparation two short- 
tailed forms of 0. caudata. apparently micro- 
gamete mother-cells, attached, the tail of one 
being united to the side of the other (Fig. 
276, PI. XXVII). This instance, with the 
Text Fig. XI. One of single instance which Neresheimer describes, 
Neresheimer's figures of seems to indicate that very rarely micro- 
"abnorinal division in the ^^mete mother-cells of the same size may 

formation of the gametes .^ ^-i • •. • j^ ■ , ^■ 

■ ri 1- -7/ " rru- „„ unite. I have in one instance m 0. mtesti- 

m O. (mmdinta . i his was 

probably the attempted co- nalis seen a microgamete attached by the 
pulation of three micro- Avhole anterior half of the body to a macro- 
gametes with one macro- gamete, the tail of the microgamete, being 
gametes. ^-^.g^ ^^-^^ ^^^ ^ pj xXillj. Neither the 

previous nor the subsequent stages of this copulation were seen. 
It was of course abnormal in the sense of being a departure from 
the regular method of copulation, but it may not have been patho- 
logic. It is similar to the instance that Neresheimer describes 
in that the male was not attached by its tail as in every other 
of the several hundred instances of copulation I have seen. 

Chromatin spherules in the f/a/inetes and zygotes. 

In the mature nuclei, after the formation and extrusion of the 
chromatin spheres, I have not observed the formation of chromatin 
spherules, nor have I seen their formation in the syncaria soon after 
copulation. I cannot say positively that there is an interval when 
they are not formed, but this seems to be the case. 

Nucleoli in the f/anietes and zygotes. 

I have failed to find nucleoli in the nuclei of the gametes and 
young zygotes. In older zygotes they are present. 



Opalina. 295 

Cant/ieheterof/ainous copnJation described heahnornial? 

When I first saw lieterogamous coi)ulation in Opalina, it seemed 
possible that it might be abnormal, for several reasons: — 1) be- 
cause it was observed in mateiial that had been about three hours 
in a slide culture; 2) because the cysts from which the infection 
was secured had not lain in water before being fed to the tadpoles; 
3) because there were present in the culture individuals of different 
sizes which jhad passed unencysted through the alimentary canal 
of the tadpole to its rectum. All doubt however was later removed. 
One of my best series of infections was secured from cysts which 
lay 36 hours in water until all the unencysted Opalinae with them 
were killed. When opening, under the microscope, the recta of the 
tadpoles infected from these cysts, I very often immediately found 
typical heterogaraous copulating pairs in different stages of copu- 
lation. Indeed nearly half of the drawings of gametes and zygotes 
of 0. mtestinalis on the accompanying plates were made from this 
series of infections. Other good infections were made in the same 
manner, giving similar results. Six instances of heterogamous co- 
pulation have since been found in a series of sections of the rectum 
of a tadpole infected sixty hours with 0. intestinalis (Figs. 183 — 185, a, 
PI. XXIV). 

Encystment follow ing copulation ? 

Neresheimer has described encysted individuals which he regards 
as zygotes. He believes that encystment normally follows copulation. 
The zygote cysts, according to his description, are of the same size 
as the infection cysts, but are distinguishable by their usually 
spindle-shaped nuclei, by the fact that the animals more nearly fill 
the cysts, and by the faint concentric striation of the contained body, 
Neresheimer saw individuals: containing two spindle-shaped nuclei 
become quiescent, change to oval form and throw off part of their 
cilia, and he interprets these changes as the early phenomena of 
encystment. Engelmann had already described for 0. ranarum cysts 
with large nuclei in the rectum of the tadpole. Zeller described, 
for the multinucleated Opalinae, multinucleated cysts in the rectum 
of the frog and uninucleated cysts with large nuclei from the rectum 
of the tadpole. Influenced by the observations of these three stu- 
dents, 1 fully expected to find encysted zygotes in the recta of the 
tadpoles, but I have not seen them for any of the three species 
studied. 



2'96 M. M. Metcalf 

It seems to me that the eiicystment, which I have often seen 
in dying zj^gotes and in dying- individuals of other sorts, is abnor- 
mal. In material from infected tadpoles, which has been kept too 
long in slide cultures, one sees very many individuals of all sorts 
rapidly change the form of the body, becoming first oval, then 
spheroidal. Following this change of form, they throw off most or 
all of their cilia with many of their basal granules, and extrude 
part of their protoplasm in the manner so characteristic of Opalina 
when under pressure (Figs. 219—221, PI. XXV). The pellicula 
remains as a very delicate cyst. Within this the body becomes 
very transparent and the nuclei and their contained chromatin be- 
come very clearly visible. Observe especially the granular chromo- 
somes in figures 219 and 220 which show such pseudoencysted in- 
dividuals drawn from ilfe, or rather death {cf. 0. caudata, Fig. 275, 
PI. XXVII). Figs. 210—218, PL XXV, show successive conditions 
in copulation and pseudoencystment in one pair of gametes from 
material of 0. intestinalis forty-two hours after infection. In this 
case, after complete fusion of the two bodies, the male nucleus broke 
down, entirely disappearing in the cytoplasm of the zygote. The 
female nucleus remained long intact, showing considerable changes 
in the character and arrangement of its contents. Fig. 220 shows 
the condition in another pseudocyst of 0. intestinalis, formed after 
the union of a binucleate macrogamete and a microgamete. The 
four granular chromosomes of each female nucleus were very clear. 
The male nucleus had but a single chromatin mass in one side of 
which an elongated bod}", probably a group of contiguous granules, 
was clearly seen. The excretory vacuole, with its contained excre- 
tory granules in Brownian movement, was also clearly seen. It lies 
uppermost in the figure. Fig. 219, is from a macrogamete of 
0. intestinalis which was found in copulation and was followed through 
a similar series of changes. In this case the nucleus of the macro- 
gamete was in mitosis. Before copulation was complete the animals 
separated again and each formed a pseudocyst in the typical manner. 
There seems no doubt that these instances of encystment and all 
others observed were all abnormal and pathologic. I have never 
found encj^sted zygotes in material from old infections and I believe 
encystment after copulation does not normally occur. 

I have occasionally seen infection cysts of 0. dimidiata, with 
oval nuclei or with one of their nuclei oval while the rest appeared 
spherical (Fig. 286, PL XXVII). I doubt if the oval form of the 
nuclei can be taken as evidence that cysts are copulation cysts 



Opal ilia. 297 

{cf. Figs. loG. loH, 139, PI. XXII). Many of the infection cysts are 
almost completely filled by the little animals within them (Fig. 130, 
PI. XXII). A faint appearance of stiiation. which is usually spiral 
but may be concentric, is often found in tlie infection cysts, being 
due to the rows of cilia and their basal granules, the direction of 
the course of the lines being determined by the position of the ani- 
mal within the cyst. These three features, which Neresheimek gives 
as distinctive criteria between the copulation cysts and infection 
cysts, seem insufficient to establish the presence of copulation cysts. 
Neeesheimek's description may possibly apply in part to the pheno- 
mena of abnormal encystment. 

On the other hand, the description by Engelmann and by 
Zeller. for multinucleated OpaUnae, of uninucleated cysts with large 
nuclei, in the recta of the tadpoles, seems to indicate the presence 
of a second type of cysts, which might well be copulation cysts. If, 
however, there really are such copulation, cysts, I do not understand 
why I have not found them. The ease with which cross infections 
are secured in aquaria in wiiich adults and tadpoles of different 
species of frogs are kept (see the chapter on infection experiments, 
p. 314) suggests that possibl}" some cysts of 0. intestinalis or 
0. caudata might have been present in Engelmann's and 
Zeller's tadpoles, the infection cysts in tliese species having usu- 
ally a single nucleus. I have had collected tadpoles of Rana tem- 
poraria, li. esadenta and Biifo vidgaris become infected with both 
0. intestinalis and 0. candata by leaving them an hour in a jar with 
adult Bomhinator pachtjirus, while on the way from the field to the 
laboratory. Some of these tadpoles w^ere already infected with Opa- 
linas of the natural species, so that, upon opening them, three spe- 
cies of Opalina were found living together. As the cysts of Opa- 
lina live in w'ater for quite a time without injury, of course such 
cross infection could be secured by placing tadpoles in aquaria in 
which the Bomhinator adults had been placed, perhaps only for a 
short time, many, days before. Encystment after copulation seems 
to me doubtful. The matter needs further study. 

Legeb & Dtjboscq (1904 a) describe copulation cysts of an 
utterly diff'erent type in 0. ranarum. These cysts are said to en- 
close two distinct gametes which lie side by side, each occupying 
one hemisphere of the cyst. I can suggest no satisfactory expla- 
nation of the phenomena, which Avere evidently not true copulation. 



298 ^I- M. Metcalf 

Phenomena in other species. 

Opalina caudata. 

In 0. caudata all the phenomena are like those described in 
0. intestinalis, except that the reduced number of chromosomes in 
the gametes is three instead of four (Figs. 249, 250, PI. XXVIj, 
as one would expect from the fact that in the nuclei of full-grown 
individuals there are six chromosomes. Compare Fig. 248, PI. XXVI, 
in which each nucleus is in an early stage of mitosis showing 
twelve chromosomes ready to arrange themselves in double rows 
preparatory to migration to the poles. Compare also Figs, 81 and 82, 
PL XX, which represent anaphases and a telophase in mitosis, the 
number of chromosomes being clearly six. 

The cysts have either one or two nuclei (Figs. 252— 255, PI. XXVI), 
and these show the characteristic chromatin spheres which are 
thrown off in the process of getting rid of the vegetative chromatin 
(Figs. 252 — 254). The gametes and the manner of their formation, 
and the external and nuclear phenomena of copulation are similar 
to what has been described. (See Figs. 257, PI. XXVI, to 276, 
PI. XXVII. Observe especially the number of the chromosomes in 
Figs. 273—276.) I have not, however, followed the nuclear pheno- 
mena in the zygotes to quite so late a stage as in 0. intestinalis. 
Fig. 276, PL XXVII, shows the only instance I have seen of the 
apparent copulation of equal gametes. The drawing is from an 
acetic-carmine preparation and I do not know what would have been 
the later behaviour of the animals. 

Large chromatin spheres, ready for extrusion from the nuclei, 
are shown in Fig. 251, PL XXVI, in a rather small individual from 
the rectum of an adult Bombinator pachypus. 

Opalina diniidiata. 

In 0. diniidiata, which is a multinucleated species, the earlier 
phenomena are somewhat different. The nuclei are not so easy to 
study, for they are small and the number of chromosomes is greater, 
apparently twelve in the full-grown forms. The chromatin is gener- 
ally arranged in numerous granules just beneath the nuclear mem- 
brane, giving a very characteristic appearance (Figs. 277—280, 
PL XXVII). I find no evidence of degeneration of nuclei, or of 
formation of new nuclei from reproductive chromidia, as Neresheimer 



Opalina. 299 

describes. This species shows phenomena exactly similar to those 
described for 0. intesiinalis and 0. cmidata in tlie extrusion of the 
vegetative chromatin, the original nuclei peisisting as reproductive 
nuclei (Fig. 285, PI. XXVII; 299-306, PI. XXVIII). 

It is evident that the number of the chromatin masses in the 
nuclei of the minute Opalinae dimidiatae in the recta of the tad- 
poles is much less than in the larger animals in the frog's rectum. The 
number of chromosomes seems to be either five or six (Figs. 307 — 309, 
PI. XXVIII, indicating ten or twelve chromosomes in the nuclei of 
the full grown forms. I neglected to study this point carefully in 
the nuclei of the living animals and in the acetic-carmine prepora- 
tions last spring, and have not yet worked through my preserved 
material of the gametes of this species, so that the statement of 
the reduced number of chromosomes is based on sketches made 
without that thought especially in mind. There is, however, no 
doubt that the number of chromosomes is reduced. The only doubt 
is as to the exact number present. 

The cysts have from one to seven or more (Neresheimee 
twelve) nuclei (Figs. 285—288, PI. XXVII), and the animals which 
hatch from the cyst have as many (Figs. 289—292, PI. XXVII; 
299—303, PI. XXVIII). On this account, as well as because the 
nuclei are less clear, this species is not so favorable for study. 
Macro- and microgametes arise as in the binucleated forms. 
The microgametes are simular to those described for other species, 
their tails sometimes being of very great length (Fig. 310, PI. XXVIII). 
The true long-tailed microgametes arise by longitudinal division from 
short-tailed forms, as in other species (Fig. 308, PI. XXVIII). The 
macrogametes, like the full grown individuals in the asexual genera- 
tion, are often rather pointed posteriorly. On this account they 
frequently somewhat resemble the short-tailed mother- cells of the 
microgametes, but the latter can be distinguished by their fewer, 
longer and weaker cilia. 

Copulation occours between uninulecated microgametes and macro- 
gametes which have one or two (or possibly more?) nuclei (Fig. 312, 
PI. XXVIII). The compound nuclei resulting from fusion are unusually 
large, and they have a characteristic elongated spindle form which 
may indicate division; it is less pointed, however than that seen in 
the division of syncaria in the zygotes of the binucleated species. 
In this species the nuclei are more independent of one another than 
in the binucleated species, not dividing simultaneously but usually 
one dividing while the other remains quiescent (Figs. 315, 316, 



300 M. M Metcalf 

318 — 821, PL XXVIIIj. This fact renders the species unfavorable 
for the study of the nuclear phenomena following copulation. 

Neeesheimer's Text Fig. B. (page 26) is very interesting. That 
which he interprets as budding was probably copulation. The second 
figure seems to' indicate the copulation of three microgametes with 
one macrogamete (my Text Fig. XI, Page 294). I have often seen 
two males attached to one female, but in every case the attachment 
was a loose one and not true copulation {cf. 0. caudata, Fig. 271, 
PI. XXVII). In the instance figured by Neresheimer the union 
seems to be an intimate one indicating true conjugation. It is to 
be regretted that Neresheimer does not figure the nuclei. If the 
macrogamete had several nuclei there seems no reason why several 
male nuclei might not enter at the same time and fuse with them. 

Opalina ranaruni, 

I have not seen tlie gametes of 0. ranarum. I have found 
the multinucleated cysts as described by other students (Fig. 326, 
PL XXVIII). The zygotes show large spindle-shaped nuclei, re- 
sembling those of 0. dimidiata, 0. intestinalis and 0. caudata (Fig. 827, 
PL XXVIII). 

Further general considerations. 

Vegetative cJironiidia, 

The extrusion into the cytoplasm of a portion of the chromatin, 
preceeding sexual reproduction, seems in all probability to be a 
throwing oif of vegetative chromatin ^) comparable to that described 
by E. Hertwig, Schaudinn and others in so many Plasmodroma. 
The disappearance of the macronucleus in conjugating CiUata is prop- 
ably a similar phenomonen. The solution, and osmotic expulsion 
from the nucleus, of the chromatin spherules during each mitosis in 
the asexual generation is probably also a somewhat related phe- 
nomenon, the chromatin spherules being probabl}^ nutritive, and their 
extrusion into the cytoplasm probably having to do with nutrition, 



^) It seems to me altogether probable that there is no fundamental distinction 
between generative and vegetative chromatin, the latter being derived from the 
former. Generative chromatin is probably complete perfect slightly specialized 
chromatin, vegetative chromatin being secondary modified chromatin. If the 
modification is too great the vegetative chromatin is unable to share in the pro- 
cesses of conjugation and so degenerates. (Compare Hertwig 1907.) 



Opalina. 301 

In the latter case the "purpose" of the extrusion is positive, to 
aid in nutrition : in the former it is more negative, to free the nuclei 
from that which must in some way be a hinderance to reproduction. 
Before comparisons can confidently be made between Metazoa 
and Protozoa as to the phenomena preceeding fertilization, the former 
must be carefully studied in the light of recent work upon the 
freeing of the generative nuclei in Protozoa from nutritive chromatin 
before sexual union, and the Protozoa must be studied more success- 
fully with reference to reduction division. Boveei (1887 a, 1892, 1899) 
has shown the extrusion from the nucleus and subsequent degeneration 
of a definite part of each chromosome in those blastomeres of Ascaris 
which are to develop into soma cells, but we have no satisfactory 
understanding of the formation of vegetative chromidia in the germ 
cells of Metazoa. Yolk nuclei are well known. They seem to be 
somewhat comparable to vegetative chromidia, but their formation 
seems generally to have a positive value comparable to the formation 
of zymogen granules in metazoan gland cells, and of the chromatin 
spherules in Opalina, all bring connected with the manufacture of 
nutritive substances, in the cytoplasm. On the other hand, the 
degenerating residual chromatin in the germinal vesicles of many 
Metazoa seems more strictly comparable to vegetative chromidia. 
There is in Protozoa a somewhat similar formation of vegetative 
chromidia which degenerate before sexual union (chromatin spheres 
of Opalina, vegetative chromidia in many Plasmodroma), the result 
of the process being to free the generative nuclei from the nutritive 
chromatin which seems in some way to be an obstruction to the 
sexual process. 

Ileductio)i, 

In the Metazoa the ripening divisions in ovogenesis and sper- 
matogenesis are accompanied by a reduction of the number of the 
chromosomes. Phenomena of this sort have been described in maturing 
Protozoan cells; by Schaudinn (1904) and v. Pkowazek (1905) for 
Trypanosomes and by Peandtl (1905 and 1906) for Didinium. The 
phenomena in Paramaeciimi are not quite clear, but Calkins & Cull 
(1907) interpret them as involving reduction. The accuracy of the 
observations of Schaudinn and v. Pkowazek on Trypanosomes has 
recently been questioned by Salvin-Moore c^- Beeinl (1907), though 
it is difficult to see upon what grounds, since they did not study 
the species or stages in which the phenomena were described as 
occuring. Dr. M. Hartmann tells me that Schaudinn's further work, 



302 M. M. Mbtcalf 

soon to be published by v. Peowazek, leaves no doubt of the 
existence of reduction in Trypanosomes. Several as yet unpublished 
papers by Haetmann and his students show reduction division in 
Amoeba and Flagellata. In Opalina we find clearly a reduced number 
of chromosomes in the gametes, and it is easy to see that the usual 
full number is restored by copulation. I have not found how the 
reduction is effected, but that it occurs is beyond doubt. 

It is interesting to note that the diminished number of chromosomes 
appears long before copulation, in animals from four to eight times 
too large for encystment. Four chromosomes are found in the nuclei 
for at least four generations before copulation, and probably for a still 
longer period. It is impossible to say how many times the animals 
divide after leaving the cysts before they are ready for copulation. 
It is possible that for a time the appearance of fewer chromosomes 
is due to their bivalence.^) If the smaller number is due from the 
first to true reduction division, and apparently this is true, we have 
the interesting fact that the condition with a reduced number of 
chromosomes persists for several generations. In Metasoa fertilization, 
immediately follows the maturation divisions. 

Neresheimee (1907) has compared the extrusion of the two 
chromatin spheres in Opalina to the formation of the two polar bodies 
in Metasoa, a comparison which seems wholly unjustified.'^) The 
chromatin spheres neither of them consist of whole chromosomes, 
but, like the chromatin spherules, are formed from chromatin given 
off from all the chromosomes. In some nuclei in which the chromatin 
spheres are present one sees eight chromosomes remaining in the 
nucleus, in other cases four chromosomes are found, showing that the 
formation of these spheres may occur before the number, of chromo- 
somes is diminished and that it is a process distinct from that by 
which the diminished number of chromosomes is brought about. As 
a further objection to Neeesheimer's interpretation of the chromatin 
spheres, which he seems to have based only upon their number and 



^) In the preliminary notice of this work (Metcalf 1907 a) I wrote "These 
chromosomes [of reduced number], as seen in the living animals, show about half 
as many grannies as do the chromosomes of the full grown individuals". Further 
study of carefully stained material in all stages of the spring phenomena is 
necessary before further treatment of this interesting point will be profitable. 

^) Neresheimeb had already described a series of phenomena which would 
necessitate the nuclei at this stage being interpreted as purely generative nuclei 
formed from generative chromidia, which prevented his interpreting the chromatin 
spheres as vegetative chromidia. 



Opulina. 303 

their extrusion from the nuclei, is tlie fact that we often find three 
of these spheres, and that very likely but one is formed in some 
cases. When only one is seen, it may be. of course, that another 
was present but has already been extruded. 

It may be well to note, before leaving- this subject, that, in 
OpaJina, each nuclear division is associated which a division of the 
cell. The phenomena in Paramaecium seem less primitive, two cell- 
divisions being suppressed dui'ing- maturation, and as a consequence 
three nuclei in each g-amete degenerating:. In Opalina all the cells 
produced by the divisions preceeding copulation are functional 
gametes as in the spermatogenesis of Mctasoa. Doubtless the con- 
ditions in the maturation of the eggs of Metasoa and in Paramaecium 
are secondary. 

Relationshiiys of Ox>alina, 

There seems no ground for doubting the close relationship of 
the several species of Opalina. Possibly the binucleated species 
should be placed in one genus and the multinucleated species in 
another genus. The character of the nuclei, as well as their num- 
ber, is somewhat different in the two groups. It seems, however, 
better to retain the genus as at present constituted and to recog- 
nise the binucleated species as a rather distinct subgenus. 

The connection of Opalina with the Ciliata has recently been 
questioned by Neresheimee (1906 and 1907) who concludes, on the 
basis of the method of reproduction by gametes, that Opalina is 
more nearly related to the Plasmodroma than to the Ciliopliora. I 
find myself unable to agree with this suggestion. The cilia with 
their basal granules, and the manner of the arrangement of the 
cilia in spiral rows, so exactly agree with what we see in Ciliata, 
and these organs are so highly developed, that their independent 
origin in two distantly related groups should not be assumed without 
convincing evidence. The resemblance between the exci-etory or- 
gans in Opalina and Hopliiophrya suggests relationship, and the excre- 
tory organ of Pycnothrix monocystoides (Schubotz 1908) shows still 
closer resemblance. The presence of a macronucleus containing re- 
fractive spherules in Hopliiophrya makes this form a good transition 
between Opalina and other Ciliata. The frequent fragmentation of 
the macronucleus in Hoplitophrya^ without connection with conjugation, 
is again a chai-acter somewhat intermediate between higher Ciliata 
and Opalina in which chromatin spherules are formed in the nucleus 
during each mitosis and are dissolved and extruded into the cytoplasm. 



304 M. M. Metcalf 

On the other hand the restriction of sexual phenomena in Opa- 
lina to one period of the year is quite different from what we find 
in most Ciliata. This is probably associated in some way with its 
parasitic habit. 

The gametes are true ciliates with characteristic basal granules 
upon their cilia. They are not formed during encystment, or in 
great numbers simultaneously from one individual, as is so usual 
among Plasmodroma. They result from ordinary", though very rapid, 
division. They differ in no very decided manner from the indivi- 
duals of the asexual generation, though the microgametes do show 
special adaptation in the naked sticky tail. The posterior end of 
0. saturnalis in the asexual generation is naked, so this character 
in the microgametes is not so much of a departure from the usual 
conditions. 

The formation of large numbers of minute individuals in the 
spring, and their encystment and extrusion from the rectum of the 
host into the water, is doubtless an adaptation to the fact that the 
most favorable time for infecting new hosts is a brief one in the 
spring while the adult frogs and toads are found almost exclusively 
in the water, and while the vegetarian tadpoles are browsing over 
the bottom of the ponds where the cysts lie. As my experiments 
have shown, adult frogs can be infected by Opalina cysts, but the 
feeding habits of the adult are such that infection of this sort must 
be very infrequent. Infection of the tadpoles, on the other hand, is 
very easy. 

The period of very rapid division in Opalina is followed by 
sexual union, as is usual among Ciliata, the restriction of copulation 
to the spring being probably due to the occurrence of such rapid 
divisions only in the spring. 

The formation of microgametes is by no means unknown among 
the Ciliata — witness the Vorticellas. In the Vorticellas w^e also 
find complete fusion of the gametes. 

The resemblance between the reproductive processes in Opalina 
and those in Plasmodroma seems superficial, the gametes in Opalina 
being true ciliates and the chief peculiarity being the restriction, 
for ecologic reasons, of the period of rapid division and copulation 
to a brief time in the spring, and the consequent false appearance of 
distinct asexual and sexual generations. There is no true alternation 
of generations in Opalina, any more than there is in Paramaecium. 

The question as to whether Opalina is a primitive or a highly 
modified genus of the Ciliata also needs considering. Its parasitic 



Opaliiia. 305 

Imbit does not argue against its primitive nature. TJie secluded 
liabitat of parasitic life, like that of deep sea life, has enabled manj- 
species to retain lowly character without extermination. The question 
of the primitive or secondary character of Opalina must be studied 
tlierefore chiefly through an examination of the structure of the 
animal itself in comparison with other forms. 

In its cilia Opalina in as highly developed as most holotrichous 
Ciliata. The absence of a gullet seems probably secondary, an 
adaptation to parasitism, since it uses predigested dialyzable food 
from the rectum of its host and has no need of ingesting solid food. 
Even Mastigophora, which are doubtless more primitive than Opalina. 
have a gullet which is functionally comparable with that of Ciliata. 
Those species of parasitic Ciliata which retain the mouth have prob- 
ably adopted the parasitic habit more recently than Opalina. 
Chromidina elegans, which in certain conditions of its nuclei resembles 
the multinucleated Opalinae, has what has been interpreted as a 
vestigial mouth Gonder (1905). So also has Anoplophryn hrasili 
(Leger & DuBOSCQ 19046). Lang (1901) classes Opalina with the 
Hijmenostomidae among the Ciliata Jiolotricha, describing the suborder 
Hijmenostomidae as possessing an undulating membrane and as having 
the mouth always open. Of course Opalina has neither undulating 
membrane nor mouth, yet Lang's phrase, "mouth always open", 
suggests a very interesting interpretation, which, however, he doubt- 
less did not intend, namely, that the mouth (ingestion tube) of 
Opalina has disappeared by becoming shallower until finally it has 
joined the even contour of the outer surface of the body, being no 
longer distinguishable from the latter. This seems not at all un- 
likely in view of Gonder's description of the mouth of Chromidina 
as a shallow pit over whose walls run rows of cilia continuous 
with and similar to those on the surface of the bodj^, and also in 
view of Leger & Duboscq's description of a vestigial mouth in 
Anoplophrya hrasili. 

The presence of functional vegetative and reproductive chro- 
matin in the same nucleus in Opalina seems surely more primitive 
than their segregation in sepai-ate nuclei in higher Ciliata. In 
Opalina, spherules of vegetative chromatin are formed in the nucleus, 
are then dissolved and pass into the cytoplasm, where very likely 
they take part in forming the refractive spherules. In Hoplitophrya 
tlie functional vegetative chromatin is in a macronucleus distinct 
from the micronucleus. In this macronucleus refractive spherules 
are formed. (This process has not yet been described, Text Fig. VIII, 



306 M. M. Metcalf 

page 270, shows the refractive spherules in the macronucleus and 
in the groups of granules into whicli it fragments.) In higher 
Ciliata distinct vegetative and reproductive nuclei are present, but 
there are but few indications that the refractive spherules in the 
cytoplasm, are formed hy the macronucleus. Each nucleus of Opalina 
is functionally comparably to both micronucleus and macronucleus 
of higher Ciliata, and is in much more primitive condition. 

The formation of chromatin spheres in Opalina and their ex- 
trusion from the nucleus before copulation seems comparable to the 
formation of vegetative chromidia in Plasmodroma and is probably 
primitive. The degeneration of the macronucleus in higher Ciliata 
is probably somewhat comparable. 

The conditions of maturation in Opalina seem simpler than 
those in higher Ciliata. In the maturation of higher Ciliata the 
nuclear divisions unaccompanied by cell-division have generally been 
discussed from the standpoint of cytopbysiology or of the mechanism 
of heredity. ^) The conditions in Opalina seem to indicate that the 
degeneration of the nuclei in maturing Paramaecium and other 
higher Ciliata is secondary and is connected with the suppression of two 
divisions of the cell body. In Opalina there is no such suppression 
of divison of the cell body and there is no degeneration of nuclei 
or cells in connection with maturation. In the division of the 
macrogamete mother-cells in Opalina both daughter cells become 
functional gametes; similar relations obtain in the formation of 
Opalina's microgametes. In Paramaecium three of the four nuclei 
(that is three of the four daughter cells) resulting from the matura- 
tion divisions in the similar gametes degenerate. In Metazoa sper- 
matogenesis follows the type found in Opalina, maturation producing 
four functional gametes, but in the maturation of the eggs of 
Ildasoa usually one functional gamete and three degenerate gametes 
(polar bodies) arise. The fact that the polar bodies are capable of 
fertilization and development {cf. Feancotte 1897) seems to show 
beyond doubt that the usual interpretation of the polar bodies as 
degenerate gametes is correct. 

Complete fusion of the gametes as in Opalina and the Vorticellas 
is doubtless more piimitive than such temporary partial union, with 
exchange of parts of the nuclear material, as we see in Paramaecium. 



^) BovEEi (1892 &) is the only one, so far as I know, who has discussed the 
phenomena of maturation in Ciliata from the comparative morphological point of 
view and has recognised that the two matuiation divisions here, as in the matura- 
tion of the eggs of Metazoa, give rise to rudimentary individuals. 



Opaliiia. 307 

Usually in hip:lier Ciliata tlie conjug-atirifr individuals are nearly 
or exactly equal in size. It niijiiit seem that Opaliuu. having o-ametes 
of unequal size, is in this regard less primitive, but the fact that 
in the Vorticellas. whose gametes completely fuse, the gametes 
are often very unequal in size, and also the conditions in (ipalina, 
argue in favor of a belief that anisogametes are for the Ciliata 
more primitive than isogametes. Calkins Sc Cull reach a similar 
conclusion. They say (1907, p. 405). "There is little evidence to 
indicate the lines of evolution that have been followed in the deve- 
k)i)ment of the participants in conjugation. The view that is usually 
adopted, without supporting evidence, is that the isogamous type 
like that of Paramaeciiim, was piimitive and has developed into 
an anisogamous type with sexually differentiated gametes (e. g. 
Hartog 1906). It is our belief that the reverse has been the case 
and that the Paramaecium type of conjugation has arisen from a 
type with sexuall}' differentiated gametes, with intermediate stages 
in forms like the Vorticellidac. where the size diiference is great in 
Lagcnophnjs ampulla^ less marked in Episfijlis, and still less in Vorti- 
cella; and in Trachylinidae, where in Lionotus fasciotas the two or- 
ganisms are alike save for a slight diiference in size (Calkins 1902). 
In the Vorticellidae the macrogamete fuses with the microgamete and 
there is no mutual fertilization, but in Paramaecium and probably 
in Lionotus. mutual fertilization takes place. The case of Lionotus 
is to be interpreted as a reminiscence of anisogani}^, and we would 
expect in this case, that the smaller conjugant, if fertilized, would 
have a reduced vitality. In Paramaecium, finally, there is no mor- 
phological evidence of the relation to an earlier anisogamous con- 
dition, but there is well-marked physiological evidence in the lesser 
vitality of one of the ex-conjugants, apparent in 72*'/o of all con- 
jugations in which the history of both was followed (Cull 1907)." 
^^'e can, I think, at least say that its anisogamous and slightly 
heterogamous copulation does not argue strongly, if at all, against 
the comparatively primitive character of Opalina. 'j 

Finally the chai-acter of the excretory organs in Opalina — 
merely enlarged and confluent vacuoles of the ordinary cytoplasmic 



^) The very interesting Pycnothrix monoeystoides described by Schubotz 
(1908), which is apparently a holotrochoiis Ciliate, seems to have unequal gametes, 
but the exact systematic position of this remarkable form cannot be determined 
until its life history is more fully known, so that we cannot now say whither its 
condition argues in favor of the primitive nature of anisogamy for the Ciliatn, 
or not. 



308 M. M. Metcalp 

foam — is very lowly. As parasitic life does not seem in general 
to produce degeneration of the excretorj^ organs, the lowly character 
of the excretory organs of Opalina is probably primitive. Their re- 
semblance to the excretory organs of Hoplitophrya argues for the 
relationship of the two genera. 

It seems, therefore that Opalina is a member of the Ciliata and 
that in many i-espects it is quite primitive. 

Leger and Duboscq (1904 h) suggest that, because of its habi- 
tat in a marine fish, 0. saturnalis is probably the most primitive 
Opalina known, all others being parasitic in terrestrial or fresh- 
water forms. I have suggested above that the restriction of the 
rapid division and subsequent conjugation in Opalina to a brief time 
in the spring is an adaption to the habits of its hosts. It would 
be interesting to know of 0. saturnalis 1) if it shows a similar re- 
stricted period of rapid division followed by conjugation, and 2) if 
the habits of Box hoops are such as to make such a restriction in 
the rapid reproduction period of its parasite a decided advantage. 
If the habits of Box hoops are not such as to render infection much 
more easy at one period, and if, in spite of this fact, 0. saturnalis 
still has a restricted period of rapid reproduction, then probably 
the adoption of Box hoops as a host is recent, 0. saturnalis showing 
a peculiarity in its reproduction, which was originally adopted in 
adaptation to conditions such as we find among the Amphihia. I do 
not know the habits of Box hoops. Leger & Dubosq found the cysts 
of 0. saturnalis in the month of September, but do not say whether 
they are found only at that season. We must, therefore, leave un- 
answered this interesting question as to the comparatively recent 
adoption of Box hoops as a host for Opalina. 

It seems probable that the Opalinas were originally uninucleated, 
that the binucleated condition was brought about by the suppresion 
of one division of the body when the nucleus divided, and that the 
multinucleated condition is still more secondary bring due to further 
suppression of divisions of the body. In Text Fig. 11, page 206, I 
have grouped the several species of Opalina in three groups which 
seem to me to indicate their probable relationship: group 1) bi- 
nucleated species, all circular in cross section; group 2) multi- 
nucleated species, circular in cross section; group 3j multinucleated 
flattened species. Compare also the list of species on page 207 in which 
the same arrangement is followed. Opalina ranarum, the form which 
has been most studied, is probably one of the most highly modified 
species. 



Opalina. H09 

Lkger & DuBosci,) (1904 a) separate the family Opalinae, iiiclu- 
dino: tlie genera Opalina, Opalin(ypsis, and Foefiingcria, from the Ano- 
phphrijinac including the genera Anoplophrya and HoplitopJmja ; a 
classification which seems reasonable. Instead, however, of believing 
with Lkgkr c<: Duboscq that the resemblance between these two 
families is a superficial one due to convergence caused by parasitism, 
I think that the two families show real, though not close relationship, 
the Anoplophryinac being the nearest relatives of the Opalinklae, 
unless ScHUBOTz' new Pycnothrix monocystoides stands still nearer. 
The difference between the two groups, is, however, probably too 
great to allow them with propriety to be placed in the same family, 
Opoliuidae, as is usual. The absence of a distinct macronucleus in 
the Opalininae is the chief distinction between this family and other 
CiHata; but, as I have shown, the distinction is not a fundamental 
one, the macronuclear elements being present in the nuclei of Opalina. 



Abnormalities. 

Some very interesting abnormal phenomena have been observed 
in the course of the work described in the previous pages. Brief 
reference should be made to some of these. 

Under unfavorable conditions the animals enter on changes which 
in some respects resemble the phenomena proceeding or accompany- 
ing sexual reproduction. We have already noted that rearing Opa- 
linae outside the host tends to make them divide. Even in the fall, 
when division is very infrequent in freshly taken material, it is 
found quite readily among animals that have been kept from one 
to three days in cultures. This recalls the fact that in the spring, 
when the period of sexual reproduction is approaching, division be- 
comes much more rapid. 

The major part of the chromatin in the nuclei of animals kept 
long in cultures tends to aggregate into compact masses (PI. XXI, 
Fig. 94, lower nucleus of Fig. 95). This drawing together of the 
chromatin may go so far as to form a single ball. These pheno- 
mena recall the gathering of the vegetative chromatin into two 
masses before encystment, previous to their extrusicm into the cyto- 
plasm. R. Hkrtwjg (1898) has shown that in starved individuals 
of Adinosphaerium the chromatin condenses into a single mass, while 

21* 



310 M. M. Metcalf 

in richly fed animals it is divided into fine granules scattered through 
the nucleus. In one case I found in the rectum of a Hyla viridis 
only six individuals of 0. obtrigona all of which were abnormal, 
showing in their degenerating nuclei preliminary phenomena which 
very closely parallel the phenomena accompanying extrusion of the 
vegetative chromatin in normal animals at the time of formation of 
the infection cysts (Figs. 99—118, PI. XXIj. 

Although keeping Opalinae outside the host tends to instigate 
division, life under the unfavorable conditions in the cultures tends 
to so weaken the animals that they generally do not succeed in com- 
pleting the divisions. Division seems normally to be aided by the 
cilia, the swimming movements of the daughter animals tending to 
draw them apart. The cilia beat less vigorously in the animals 
weakened by life in the cultures, and, perhaps chiefly on this account, 
division is rarely completed. In animals which have thus failed 
to complete their division, the nuclei are very often almost completely 
divided so that four daughter nuclei are present, bound together in 
pairs by their connecting threads. Frequently one sees but one of 
the daughter nuclei of the anterior parent nucleus in one cell, the 
other daughter of the anterior nucleus remaining in the other cell 
along with the two daughters of the posterior nucleus, so that this 
cell contains three nuclei, two united to each other by a thread and 
a third united by a thread to the single nucleus in the other cell 
(Fig. 98, PI. XXI). In nine such instances I have seen the odd 
nucleus in the trinucleated daughter cell either already fused with or 
in the process of fusing with the anterior of the two nuclei which pro- 
perly belong to the cell. This recalls the fusion of nuclei in the zygote. 

In material of 0. intestinalis and 0. caudata one finds, rarely 
during the fall and eaily winter, and a little more frequently in 
early spring, individuals with single nuclei of very large size (Figs. 
92, PI. XX; 96, PI. XXI). In the character of their mitotic spindle 
and in the distribution of their chromatin these huge nuclei very 
closely resemble tlie large dividing syncaria in the zygotes in the 
tadpole {cf. Figs. 222, 224, PI. XXV). The apparently abnormal uni- 
nucleated forms are found in freshly taken material. I have no 
evidence as to the mode of their origin or their relation to normal, 
nuclei. They may possibly be compound nuclei resulting from the 
union of two nuclei, or from the failure of a nucleus to divide. 

The very thick, stocky individuals of 0. caudata often seen in 
the spring, have been described ^) (Fig. 88, PL XX). They may be 

') page 250. 



Opalina. 311 

abnormal. tliouo:li there is little to indicate that they are so. With 
them are often found clearly abnormal forms. Fi^. 89 shows one 
such very stocky individual of 0. caudata which had four nuclei 
each in a telophase stage of division. Two of these are seen in the 
figure in end view and so do not show that they are in mitosis. 
Fig. 90 shows an individual whose two nuclei have almost completely 
degenerated. I have found one individual of 0. intestinalis with ab- 
solutely no trace of a nucleus. I'hese wTre clearly pathologic 
forms and not in any way comparable to the non-nucleated indivi- 
duals of Actinosphacrimn, which Hertwig (1899) describes as reforming 
their nuclei from chromidia. Fig. 91 shows an 0. caudata whose two 
nuclei are in a condition characteristic of multinucleated Opalinae, 
but very rare and [ think abnormal in 0. caudata. 1 have never 
found nuclei of this sort in 0. intestinalis. 

In one lot of material of 0. intestinalis from a fifty-four hour 
infection there were many individuals showing abnormal divisions 
similar to what Cohn has described for the same species as budding ^) 
(Figs. 228—235, PI. XXV). When first seen these animals falsely 
seem to be zygotes in which fusion of the gametes is not yet com- 
plete. All the animals in this culture died within an hour. Fig. 229 
shows an individual just hatched from the cyst which lies collapsed 
near by. The little spine-like tip of the body shown in Fig. 230 
suggests that this individual was an imperfect microgamete (cf. Figs. 
163. PI. XXIII; 261, PI. XXVI). 

The abnormal nuclei in the six degenerating individuals of 
0. ohtrifiona, referred to above, were ver}' interesting and deserve 
further description. Figs. 99—101 , PI. XXI, recall some of 
Lowenthal's figures of nuclei of cysts of 0. ranarum (Text 
Fig. X, b. page 280). Similai- nuclei are quite usual in the 
full-grown foi'nis of the multinucleated species, as well as in their 
cysts. The chromatin is found in two conditions 1) in a fine super- 
ficial net which slight nodal thickenings, this is hardly distinguish- 
able from the acrhomatic foam; and 2) in from two to six, or more, 
large disc-shaped or hemispherical masses pressed close to the nuclear 
membrane. Figs. 102 and 103 show somewhat similar superficial 
chromatin masses in which the chromatin is in the form of a darkly 
stained netwoik with much lighter meshes. The difference in the 
appearance in the two sorts of chromatin discs in probably not 
wholly due to difference in staining. Very heavy staining and long 

') Cohn 1904, Figs. 14 and lo. 



312 M- M. Metcalf 

extraction of the stain seems always to bring out the netted appear- 
ance in some of the discs (generally the larger and thinner ones) 
and not in others which seem more nearly normal. In Fig. 102 and 
103 each nucleus is seen to contain a central mass of granules 
whose later behaviour indicates that they are probably chiefly 
achromatic. Fig. 105 shows a radiate ari-angement of these achroma- 
tic granules, the chromatin having gathered into a sphere which 
shows the characteristic netted appearance. Figs. 106—109 show 
that there may be one or two of these spheres. When two are in 
the same nucleus one may be much smaller (Fig. 107, a). In many 
of the figures, especially in Figs. 106 and 107, one sees that the 
center of each chromatin sphere is filled with a refractive body 
which does not stain with Delafield's haematoxylin. The chromatin 
net lies like a cap partially, or almost completely, enclosing this 
central body. The presence of these referactive bodies in such 
intimate association with the aggregated chromatin recalls the for- 
mation of refractive spherules in the macronucleus or its fragments in 
HopUtophrya and the possibly similar phenomena in Loxodes rostrum 
(Joseph 1907), and gives a little more probability to the suggestion 
that in 0. intestinaUs and 0. caudata the chromatin spherules after 
they leave the nucleus and reach the cytoplasm, may aid in the 
formation of the refractive spherules of the endosarc. Boveki (1907, 
see his plate XXIII) has described in degenerating nuclei of disperm 
Echinoderm larvae compound chromatin and refractive bodies very 
closely resembling those here described in the degenerating nuclei 
of 0. obtrigona. 

Figs. 110 to 118 show a very interesting arrangement of the 
achromatic granules in the form of two polar groups with lines of 
granules connecting them and often with a more or less evident 
radiate arrangement of the granules around the polar groups. One 
or two of the netted chromatin spheres lie on the outer side of the 
granular spindle-fibres, at the equator of the spindle. Fig. 110 shows 
the chromatin sphere still adhering to the nuclear membrane. In 
Fig. Ill we see the two chromatin spheres withdrawn from the 
nuclear membrane and lying upon the spindle. The polar groups of 
granules, and the lines of granules composing the spindle, at first 
sight suggest comparison with a mitotic spindle with large polar 
centrosomes. It seems to me not improbable that they are essentially 
similar to the structures in some protozoan nuclei wiiich have been 
regarded as spindle and centrosomes. (Compare R. Hertwig 1899, 
Paramaeciuni micronucleus ; Schaudinn 1894, Amoeba crystalligera ■ 



Opalina. 313 

Keuten 1895, Euglena viridis; Calkins 1901, Clepsidrina; Hartmann 
A: V. Phowazek 1907, numerous Plasmodroma) 

These are not functional mitotic si)indles in these degenerating 
nuclei of 0. ohtri(jo)i(t. The nuclei, though they become elliptical, 
do not divide, but soon go to pieces, leaving spaces in the cj'toplasm 
where they lay (Fig. 118). louring the process of degeneration the 
nuclear membrane becomes fainter and fainter and ultimately entirely 
disappears. Sometime the chromatin sphere, including both the 
chromatin net and the central refractive bodj^, is extruded into the 
cytoplasm, leaving within the degenerating nucleus only one or 
more masses of debris representing the achromatic structures (Figs. 
116 and 117). In other cases one finds the degenerate chromatin 
sphere lying in the space from which the nucleus has dissappeared. 

The chromatin sphere itself resembles those of the cysts {cf. 
Fig. 133, PL XXII), and its extrusion is propably comparable to that 
of the latter. That is, under unfavorable conditions, the degene- 
rating nuclei undertake a part of the activities which usually 
preceede copulation, I have not found a perfectly clear spindle-like 
arrangement of the achromatic granules in the nuclei of the cysts 
or of the minute individuals in the spring which were preparing 
to extrude the vegetative chromatin, but most of my material of 
these forms was stained with acetic-carmine which does not give 
sharp pictures of the finest details. In sections of cysts stained 
with Delafield's haematoxylin, one sees in the center of the nuclei 
groups of granules (Figs. 134, 136—139. PI. XXII) resembling those 
in the earlier stages of degeneration in the nuclei of 0. obtriyona 
(Figs. 105—109, PI. XXI). Up to this point the two sets of phe- 
nomena, normal and abnormal, seem quite comparable. The granular 
spindles and polar masses do not seem to be paralleled in the 
normal nuclei at any stage of the life cycle. Their resemblance, 
however, to what is found normally in some Plasmodroma. e. g. 
Amoeba eristaUiffera, is such as to suggest that these abnormal phe- 
nomena in degenerating nuclei of 0. obtrigona are a reminiscence of 
archaic normal conditions. 

The abnormal phenomena described in this chapter are probably 
due to unfavorable conditions of life. Their resemblance to some of 
the phenomena usually preceeding copulation suggests comparison 
with the well-known fact that many animals, e. g. BoHfera. Cladocera, 
which reproduce asexually under favorable conditions, are induced 
by unfavorable conditions to introduce sexual phenomena. 



314 M. M. Metcalf 



Infection Experiments. 

Under natural conditions the several species of Gpalina are found 
only in certain definite hosts, as noted in the table on page 207. 
In the hope of reaching- a better understanding of this restricted 
distribution, many artificial infections were made with the cysts of 
0. intestinalis, 0. caudata and 0. dimidiaia upon the larvae of liana 
esculenta, Bufo imlgaris and BomUnator pachypus and upon the adults 
of several species of frogs and toads and Triton cristatus. Attempts 
were made also to infect the same larvae and some of the same 
adults with adult Opalinae of four species, 0. intesiinalis, 0. caudata^ 
0. dimidiaia and 0. oUrigona. 

Opalina intesiinalis cysts cause infection of the tadpoles of Bona 
escidenta and Bufo vulgaris as readily as of the tadpoles of BomU- 
nator pachypus. Under natural conditions Piana esculenta only very 
rarely contains this parasite. It has never been reported from 
Bufo vulgaris. In both of these hosts the Opalinae form normal 
gametes which copulate. After four weeks the infection appeared 
normal when studied from living material. Preserved material from 
older infections has not get been examined. 

Adult Hyla arhorea and Rana temporaria. as well as tadpoles 
of the latter species, are also readily infected if forcibly fed with 
the cysts, the young Opalinae in the rectum being apparently entirely 
normal. The later historj' of these infections was not followed to 
see if copulation occurred. 0. intesiinalis has never been reported 
from Hyla arhorea or Bana temporaria. 

Tadpoles of Bombinator pachypus, Bufo vidgaris, and Bana esculenta, 
when placed with foeces of either species of Bombinator containing 
adult 0. intesiinalis, ingest many of the parasites with the foeces. 
Many others of the parasites pass into the nostrils with the respira- 
tory current. Many of these ingested adult Opalinae are digested 
by the tadpoles of Bufo vidgaris, but some pass uninjured through 
the whole alimentary canals to the recta and there establish thriving 
colonies. T3idi)o]eii of Bombincdor pachypus digest a smaller proportion, 
and tadpoles of Bana esculenta digest almost none of the adult 
Opalinae, their recta becoming very richly infected with large 
Opalinae. There is no subsequent degeneration of the Opalinae in 
any of these infections, at least within four weeks. Adult Hyla 
viridis and adult Bana temporaria are also readily infected if forcibl,y 
fed with adult 0. intesiinalis. 



Opalina. 315 

Attempts to infect two adult individuals of Triton cristutus with 
cysts and adults of 0. intestinalis failed, possibly because the newts 
did not swallow the Opaliuac The negative result cannot be trusted 
to show that such infection is difficult to secure. Conte & Vaney 
report Opalina intestinalis from Triton taeniatus. 

Opalina caudata gave exactly similar results with only the 
additional fact that adult Jlana esculenta are abundantly infected 
from the cysts. Doubtless 0. intestinalis cysts would infect adult 
Jiana esculenta, but no such experiment was made. Opalina caudata 
has never been repoi'ted from Rana esculenta, nor from Rana tempo- 
raria, Bufo viilyaris, nor Jlyla viridis. 

Opalina dimidiata cysts cause abundant infection of the tadpoles 
of Bufo vulgaris, Rana temporaria and Bomhinator pacliypus. Adult 
0. dimidiata cause abundant infection in tadpoles of the same species 
and in tadpoles of Rana esculenta. Rana esculenta is the usual host 
for 0. dimidiata. Bufo vulgaris is also frequently infected with this 
species, but it has never been reported from Rana temporaria. 

Adult Opalina ohtrigona cause abundant infection of tadpoles of 
Bufo vulgaris, Rana escidenta and Bomhinator pachypus, though this 
parasite has never been reported from these species. 



Table showing results of infection experiments, 

I Asterisks indicate infections different from those known to occur 

in nature.] 

O. intestinalis cj'sts cause infection of Bomhinator pachypus tadpoles. 

Bufo vulgaris tadpoles.* 

„ „ metamorphosing tadpoles.* 

Rana esculenta tadpoles. 
Rana temporaria tadpoles.'' 

„ „ adult.* 

Hyla viridis adult. * 
„ „ adults* „ „ „ the same tadpoles and adults. 

0. caudata cysts „ „ „ Bomhinator pachypus tadpoles. 

Bufo vulgaris tadpoles. "*• 

„ „ metamorphosing tadpoles. * 

Rana esculenta tadpoles.* 

„ „ adult.* 

Rana temporaria tadpoles.* 

„ ,, adult. * 

Hyla viridis adult.* 
„ ., adults* „ „ ,. the same tadpoles as the cysts. 



316 M. M. Metcalf 

O. dimidiata cysts cause iutection of Ra7ia esculenta tadpoles. 

Bufo vulgaris tadpoles. 
Rana temporaria tadpoles.* 
Bombinator imchypus tadpoles.* 
„ „ adults* ,, „ „ the same tadpoles as the cysts. 

O. ohtrigona adults* „ „ „ Btifo vulgaris tadpoles.* 

Rana esculenta tadpoles. * 
Bombinator pachypiis tadpoles.* 

In the preliminary notice of this work there were two errors: 
1) adult Rana esculenta were infected by cysts of 0. caudata, not 
of 0. intestinalis as there stated; 2) the Bufo vulgaris called "young 
toads" in that paper were not adult, but were metamorphosing" tad- 
poles with fully formed leg-s, with the tails only beginning to 
diminish, and with mouths of the larval type. 

In the light of the results of these infection experiments, the 
restricted distribution of the parasites in the several hosts is very 
difficult to understand. It seems probable that any species of frog 
or toad can be infected hj cysts or adults of any species of Opalina 
(except, of course. 0. saiurnalis). Why, then, is the distribution of 
the parasites so restricted? Why, for example, do not 0. dimidiata, 
0. intestinalis and 0. caudata all naturally occur in both Rana 
esculenta and Bombinator imchypus? The tadpoles of Rana esculenta 
and Bombinator pachypus live together in the same ponds and streams. 
Why does one species become infected only with 0. dimidiata and 
the other species only with 0. intestinalis and 0. caudata, when all 
three kinds of cysts are present in the same ponds at the same 
time of year and must doubtless often be ingested by both species 
of tadpoles? The question deserves more attention than I had time 
to give it last spring. If, upon my return to America, I find con- 
ditions there favorable for experiment upon this point, I shall studj^ 
it further. I hope also the matter will be further studied upon 
Europaean forms, the species mentioned above being especially favor- 
able for study. 



A Description of Opalina ^elleri, Neeesheimer. 

In his fine paper upon the Opalinas, published in 1877, Zeller 
describes finding, along with 0. dimidiata, in the rectum of Rana 
escidenta, certain individuals much more stocky than the ordinary 
0. dimidiata. They were especially characterized by having the 
body folded posteriorly, with deep furrows between the folds, ordinary 



Opalina. 317 

Opalwac dimidiaiae liavinp: tlie posterior end of tlie body pointed. 
Zeli.er was uncei'tain wlietlier to i-eg-ard these stocky individuals 
as Opalinae dimidiatae of a peculiar form, or as belonging to a new 
species. 

Delage & Herouaed (1896), without having seen these Opalinas, 
interpreted Zellee's figure as indicating the presence of vestigial 
excietory organs, an interpretation which T have shown to be 
mistaken (Metcalf 1908//). 

Nebesheimke (1907j again found these peculiar Opalinae in 
liana escidenfa, and, without adding to Zeller's description, gave 
them the name 0. .seUeri, believing them to belong to a distinct 
species. 

In the same year I independently (but later) gave them the 
same name, it being only natural to name them after their discoverer. 

I have seen these forms but twice, Zeller apparently saw 
them several times, though he does not say definitely. In both 
instances wdien I saw them they were with Opalinae which un- 
doubtly belonged to the species dimidiata. Zeller reports the 
two forms as occuring together. Neresheimer does not say how 
often he saw these peculiar forms, or whether 0. dimidiata was 
present with them. All tlie Opalinae zelleri I have seen were 
large, all the small individuals present with them, as well as many 
of the lai'ge ones, being typical 0. dimidiata. The fact that very 
much swollen and stocky individuals of 0. caudata are frequent in 
the late winter and in the spring, and the fact that I once found 
a few very thick individuals of 0. ranarum, make one suspect that 
the forms called zelleri may be merely similar stocky individuals of 
0. dimidiata. Until this question can be definitely settled, it is 
convenient, and is apparently justifiable, to give these forms a 
specific name. 

Opalina zelleri (Text Fig. II, p. 206) is the largest Opalina known, 
when we consider its breadth and thickness as well as its length. 
A large example has a length of 0.25 mm and a breadth of 0.13 mm. 
In cross section the animal is circular except that there are present 
upon the body four to eight longitudinal ridges with intervening 
furrows, which show, of course, in cross section. The anterior end oi 
the body is bent to one side, as in all other Opalinas. The animal is, 
however, so stocky that the bend is not quite so noticeable as in 
slenderer forms. In 0. ranarum the corresponding bend in the body 
is present, but the decided flattening of the body, and its consequent 
great breadtii. somewhat obscure the bend. The anterior end of 



318 M. M. Metcalf 

0. zelleri is slightly compressed to the form of a very thick wedge. 
If this compression could be carried much further until the whole 
body was thin and flat, its anterior end, and indeed its whole form, 
would resemble that of 0. ranarum, for frequently 0. ranarum has 
the contour of the posterior end of the body concave. 

At the posterior end of the body the longitudinal ridges show 
rounded ends, there being a terminal depression of considerable 
depth between their posterior ends. 0. cUmidiaia is pointed posteriorly, 
often very sharply pointed and slendei*, differing most markedly 
from 0. seJleri. It was the superficial furi'ows between the ridges at 
the posterior end of the body, rather poorly drawn by Zeller, which 
Delage & Herouaed interpreted as internal canals, remnants of a 
system of water canals. 

The broad rounded longitudinal ridges are usually five or six 
in number, though one finds individuals showing four or eight ridges. 
They are constant, not changing as the animal swims. They are 
slightly spiral, following the same general direction as the spiral 
lines of cilia. 

Opalinae of any cylindrical species may often, when living under 
adverse conditions, show a decided spiral twisting of the body, which 
is then raised into ridges. These ridges are constant, not changing 
as the animals swim. They may possibly be, in a general may, com- 
parable to those of 0. zelleri. 

Always at least one and often two of the longitudinal ridges 
show at the posterior end a rounded protrusion, giving a more 
pointed appearance than that of the other ridges. It is possible that 
this rounded point marks the morphological posterior end of the 
body and that when two such points are present they indicate nascent 
longitudinal division of the body. These points remind one slightly 
of the short sharp protrusions from the posterior ends of very stockj^ 
Opalinae caudatae (Fig. 88, PI. XX), though I have never seen two 
points upon an animal of the latter species. 

The minute structure of 0. zelleri, as seen in sections, agrees 
so exactly with that of 0. dimidiata, as to need no description. The 
anterior end of the body, as in all other species of Opalina, has 
denser endoplasma and more numerous endosarc spherules than the 
rest of the body. 

This form deserves further study, but it is rare. I found it 
only twice, once on the 22*^ of June and again on an unrecorded 
date at about the same time. No cysts were present with the 
Opalinas in the rectum. Zeller describes its reproduction as like 



Opalina. 



319 



that uf 0. dimidiata, but it is difticult to see how he knew tliat the 
reproductive stages studied belong-ed to 0. zellcri and not to 
0. dnnidiafa which was living' in the same host. I have once seen 
an individual of 0. zeJJeri in longitudinal divison, the phenomena 
being as in 0. dimidiata. 

I have no constant opinion as to the distinctness of 0. zelleri 
from (). dimidiata. 





A 



B 



Chronological Review of the Literature of Opalina.^) 

Opalina was first mentioned 
by Leecwexhokk in 1685. In his 
Opera omnia (1722) he quotes the 
earlier record of finding innumer- 
able animalcidae of various sizes 
and forms in the foeces of the 
frog. One of these figured seems 
in all probability to have been 
0. raminim (Text Fig. XII, B). An- 
other may have been 0. dimidiata 
(Text Fig. XII, A) [not 0. intestinalis 
as Kent (1881—1882) supposed].-) 

One hundred years later than Leeuwenhoek, Bloch (1782, p. 36, 
Taf. XXIII) described and figured two forms from the alimentary 
canal of the frog, which he called Hirudo intestinalis and Chaos in- 
testinalis cordiformis. The former was probabl}' 0. dimidiata or 



Text Fig. XII. 

Leeuwenhoek's figures of animalculae 

from the rectum of frogs : A may be 

0. dimidiata^ B is almost surely 

0. ranarum. 



') Only papers and books which include observations upon Opalina, or dis- 
cussions based definitely upon conditions in Opalina, are included in this review. 
General discussions which do not especially mention Opalina are omitted, so also 
are most text books which refer but briefly to observations upon Opalina made 
by others than the authors of the books in question. I have endeavored, with 
these exceptions, to make the review as complete as possible, but doubtless I have 
failed to find numerous references to the genus. I should cordially appreciate the 
kindness of any one who would direct my attention to references to Opalina not 
mentioned in this review. 

^) Throughout this review of the literature my own comments are included 
within brackets. 



320 M. M. Mbtcalf 

0. intestinalis (Text Fig. XIII, A). A late stage of division was 
observed in Bloch's preparations by his friend '•''Herr Oberprediger 
Hekbst" and was interpreted by Bloch as copulation (upper two 
animals of Text Fig. XIII, A). As the animals were united by their 
pointed ends, these were regarded as posterior and it was assumed 
that the mouths must be at the opposite broader ends. The second 
form, Chaos, may possibly have been 0. ranarum (Text Fig. XIII, i? 
and C). Block's artist saw many small particles come out of the 
posterior (?) end of a quiet individual, which soon died, phenomena 
which Block interpreted as the birth of young. [Doubtless the 
animal was going to pieces.] 





ABC 

Text Fig. Xlir. 
Block's figures of Opaliva (?). A, three of his nine figures of "Hiriido intestinalis 
{der Eingeweideblntigeiy\ The upper pair [in a late stage of division] show what 
he interpreted as copulation. B, two of his seven figures of ^^ Chaos intestinalis 
cordiformis {das herzfdrmige Infusionstierchen)". C, an animal of the same species 
"giving birth to young". B and C, probably represent Balantidium and not Opalina. 

GozE (1782, p. 429-433, Taf. 34), in the same year, makes a 
more important contribution to our knowledge of Opalina. He says 
Chaos includes several sorts of intestinal worms which are found in 
the '' Landfrosclf \Rana temporaria], the ^' Wasserfrosch" [R. escidenta], 
the ^'Mittelfi'osch'^ [Bomhinaior pachypus?], in "Landkroten" [Bufo 
cinereus?] and '■' Wasserkroten" [Pelobates fuscus?]. He says they live 
naturally in the recta of these amphibia and have not merely 
wandered in from the water because: 1) they are never found in 
water; 2) they are found in land-toads as w^ell as in water-toads; 

3) they are found only in the anterior end of the rectum, just 
behind the constriction which separates it from the small intestine, 
never in the small intestine or in the back part of the rectum; 

4) one finds three or four species always of constant shape; 5) if 
frogs are kept a quarter of a year in water the rectum becomes 
entirely empty of the animals, and still none of these animals are 
found in the water, though many other Infusoria are present. He 
says that one finds frogs or toads with either very few or none of 
the Chaos in the recta, that the little animals are more abundant 



Opalina. 



321 



in Ai)iil and .May; that they decrease in the hot summer months; 
tliat in December and January tliey are entirely absent. On March 22* 
numerous •'Flinnucnvalzen'" [appai-ently 0. intesfinalis], were found in 
the rectum of the " MiUelfrosch" during the winter sleep, but they 
were not so large as in summer. The ''■Flimmerivalzen" (Text 
Fig. XIV, jB) are found only in the "Mitlelfrosch", never in the 
" LandfroscJi" or ^^Wasserfrosck". When magnified 370 diameters they 
appear 1 inch long and Vjo of an inch broad. On p. 311 he gives 
the name Leucophra to these forms. Certain much larger "Flimtner- 
qnadrafe'^ [probably 0. rananim] are mentioned and figured (Text 
Fig. XIV, .1). 
























B 

Text Fig. XIV. 

Goeze's figures of Opalina (?). A, three oi his eight Hgmes of "Flimmerquadrate'' 

[apparently 0. ranaruni]\ B, a group of '' Flimmenvalzen {Leucophra^ [probably 

0. intestinalis or 0. caudata] from the rectum of the '■^Mittelfrosch". 

[0. F. MiJLLER's (1786) Leucoiihra globuUfera thought by Eheen- 
BEEG (1821) to hav£ been an Opalina, seems clearly not to have belonged 
to this genus]. 

ScHRANK (1803) gives a brief description (p. 68) of Paramaecmm 
incubus, which he regards as perhaps the same as Block's Hirudo 
intestinalis, which was probably Opalina intestinalis or 0. dimidiata. 
jHis description however seems to apply to Balantidium entozoon and 
not to an Opalina.] 

[BoRY DE St. Vincent's (1824) reference to Leucophra globuUfera, 
thought by Ehrenrerg to apply to Bursaria [Opalina] rananmi, is 
really to a different form, as 0. F. MiIller's (1786) original description 
of Leucophra globuUfera shows.] 

Ehrenrerg (1831) (p. 110) describes very briefly Bursaria 



322 M. M. Metcalf 

fOpalina] ranarum, and (p. Ill) briefly describes Bursaria intestinahs, 
[which according' to his description cannot be an Opalina]. 

Ehrenbeeq (1835, p. 164) in a discussion of the "male gland" 
of Protozoa, says that that of Bursaria [Opalina] ranarum is band- 
shaped or has a '•'■Seidenschmirfornf. 

PuKKiNJE & Valentin (1835) mention a form which they believed 
may perhaps be the same as Eheenberg's Bursaria ranarum. To 
this form the}^ give the name Opalina ranarum, the first use of the 
name Opalina. ^^Propter rolorum superficei splendorum et varietatem sub 
sole pleno adparentem Opalinam earn vocavimusr 

Von Siebold, in 1835, mentions the occurrence in the spring 
in Bana temporaria of a great number of completely ciliated, light 
gray animalcula, and refers to the regularly undulating stripes 
over the whole body, due, as he [correctly] says, to the^ serial wave- 
-like movement of the cilia. [The reference is clearly to 0. ranarum] 

Ehrenbeeg (1838) gives drawings of his Bursaria intestinalis ^) 
and B. ranarum, which bej^ond doubt are respectively of 0. iniesti- 
nalis and 0. ranarum. His description of the former species shows 
that he confused with it 0. dimidiata. He says it is abundant in 
Februar}^, near Bei'lin, in Bnfo cinereus, Bana temporaria [incorrect] 
and B. esculenia. The nucleus is called a male gland, and a mouth 
is [of course erroneously] described as present at the pointed [posterior] 
end of the body. Numerous digestive vacuoles [probably nuclei of 
0. dimidiata] are described. The same interpretation is given to the 
nuclei of Bursaria [Opalina] ranarum. The abundant refractive sphe- 
rules of both species are called egg-granules. Transverse division of 
O. intestinalis is mentioned and figured. Bursaria [Opalina] ranarum 
is decribed as large, flat, with 32—33 longitudinal rows of cilia; a 
mouth is described at the pointed anterior end, and an anus at 
the broad posterior end; a small curved male gland is also described. 
Neither species was found to ingest pigment granules given to it, 
so the position of the egestion opening was only doubtfully identified. 
[Of course Opalina has neither mouth nor anus.] The dimensions 
of B. intestinalis are given as: length ^340 — Viao of an inch, diameter 
of the eggs V4noo of an inch; of B. ranarum, length V256 — V72 of 
an inch. Eheenberg's figures give the first entirely certain identi- 
fication of any species of Opalina, there being no doubt as to the 
species from which they were made, but 0. intestinalis, though well 



^) He makes no reference to his previous description of Bursaria intestinalis 
(1831) which does not apply to any Opalina. 



Opalina. 323 

figured, is not distiiioiiislied in the description from 0. dimidiata 
[which has the same shape but contains many nuclei]. 

DujAKDix (1841) makes very brief reference to the Opalinas, 
giving an unrecognisable figure [probably not of an Opalina]. 

Max Schultze (1851) says (p. 68) that it seems to him very 
probable that the Opalinas form no true independent genus, but 
are rather developmmital stages or nurses {'"'■ Entivieklunysstiifcn oder 
Ammen") of other animals. [The word Ammen is difficult to under- 
stand in this connection.] He notes (p. 69) the absence of a con- 
tractile vacuole in 0. ranarum. 

Perty (1852) correctly describes the form of 0. ranarum from 
the alimentary canal of Bana temporaria. He says that the mouth 
and body cavity aie scarcely recognisable; that the whole surface 
in life seems evenly ciliated, and that the longitudinal striation in 
dead individuals is due to delicate folds, not to cilia. The appearance 
of the waves of motion of the cilia is aptly compared to that of 
the waves which pass acioss a wheat field in the wind. No figures 
are given. Bursaria [Opalina] intestinalis is mentioned but not 
described. 

Stein (1856) mentions (p. 56) 0. rananmi and also suggests that 
Bursaria intestinalis may be an Opalina. On p. 37 he diagnoses the 
genus Opalina (in the broader sense) saying that the cilia are in 
rows over the whole surface of the body, that these animals are 
distinct from all other Infusoria in having no mouth and therefore 
taking their nourishment in liquid form through the whole surface 
of the body; in having for the most part no contractile water 
vacuole; and in being often without nucleus. He considei's it doubtful 
whether they are to be regarded as true Infusoria, or as developmental 
stages of endoparasitic worms. They are said to reproduce almost 
always by transverse division. 0. ranarum is said to be most diver- 
gent from other ciliate Infusoria since it has no nucleus or con- 
tractile vacuole and has never been seen in division. 

Leydig (1857) doubts the position of the Opalinas as Infusoria 
arguing fi-om the many nuclei of the multinucleated forms and from 
the -'beautifully cellular structure" of the outer plasma of 0. intesti- 
nalis that they may be multicellular foims. 

Pagenstecher (1857) regards Opalina as probably a stage in 
the development of a Trematode. He figures a form which seems 
to be 0. ranarum. 

KiJHNE (1859, p. 823) stimulated Opalina (species not mentioned) 
and other Ciliata with strong induction electric currents and saw 



324 M. M. Metcalp 

vigorous movements arise, followed by protiusions from the body as 
if it were broken, and Anally the body became completely flat. 
"With moderate currents, at the first stimulation the animals drew 
back strongly, then lay entirely quiet in a sort of tetanus of all 
the muscles, if they were not carried forward by the action of the 
cilia, upon which the current seemed to have absolutely no influence." 
If the stimulus was strengthened, constrictions soon appeared and 
then breaks at different places at the edge of the body. In this 
condition many animals swam about for a considerable time if the 
stimulus was removed. After long stimulation with verj^ strong 
currents the whole animal liquefied, forming a shapless flat mass 
{^^unformigen Bret") in which for a long time the cilia here and 
there would continue moving. 

Stein (1859) mentions (p. 72) Opalina as belonging to the 
holotrichous Infusoria; he again refers (p. 75j to the divergence of 
this genus from the rest of the Infusoria in its lack of mouth and 
anus and in absorbing liquid food through the surface of the body; 
he describes (p. 91) the presence of many vacuoles with definite 
contour, filled with liquid containing granules. [This reference is 
apparently to the nuclei.] Division or budding had not up to that 
time been observed in 0. ranarum (p. 94). He says he had sought 
in vain for a nucleus in 0. ranarum (p. 94). 

Stein (1860, p. 54) again emphasises the divergence of the 
Opalinas from the other holotrichous Infusoria. He says the former 
genus Opalina divides itself into several genera : Discophrya includ- 
ing the forms with a sucking disc at the anterior end of the body; 
Hoplitophrija including the forms bearing horny hooks at the anterior 
end of the body; AnopJophrya including forms, nearly related to 
HopUtophrya but without discs or hooks, which have simple nuclei 
in the axis of the body and contractile vacuoles of different forms; 
and Opalina including 0. ranarum, the form longest known, and 
0. dimidiata, ^) a nearly related, slenderer, more elongated form, 
also living in the alimentary canal of the frog. These two species 
have no contractile vacuoles and no ordinary nuclei, but instead 
have numerous small nucleus-like structures scattered through the 
whole parenchyma. 

Peitchard (1861) describes at considerable length the Opalinidae, 
giving however but little attention to 0. ranarum the only true 
Opalina he recognises. He says of 0. ranarum that the mouth 



^) I have not found Stein's original description of 0. dimidiata. 



Opaliua. 325 

described bj' Eiikenberg is no true mouth but a mere fold of the 
surface as may be seen after the body has been distended by 
adding- a little dilute solution of iodine, alcohol, or acetic acid (Stein) ; 
tliat no nucleus was found by iS-PKiN; that contractile vacuoles are 
wanting'; tliat tlie cilia ai'C disposed in longitudinal lines; that the 
species is common in the intestine and bladder of frog-s; that "the 
absence of a mouth affords evidence of the merely transitive nature 
of Opali)weci\ that "'these simple beings are not independent but 
the mere embryonie or transitional phases of other animals", that 
"they are probably larvae of various worms", "consequently this 
group of beings is at best but provisional, serving only the purposes 
of definition and nomenclature"; that „neither the intimate structure 
nor the developmental history of the Opalinoea is sufficiently well 
understood for them to be arranged in well-defined genera; that 
0. iritonis (Perty) "is very like 0. ranariim and requires further 
examination"; that 0. nucleus, 0. entosoon and 0. intestinaHs „. . . are 
nothing more than different phases of growth and development of 
Opalina rcouirmn''. Two unnamed and unrecognisable figures are 
given. 

KoLLiKER (1864, p. 24) recognises the many nuclei of 0. raimrum 
as true nuclei and for the first time mentions the cysts, which he 
describes as multinucleated. He regards the cysts as eggs, and 
thinks that the fact that Opalina develops from eggs confirms 
Max Schultze's view that they are developmental stages of metazoa. 

QuENKERSTEDT (1865) discusses the organization of the Infusoria, 
making but brief reference to Opalina. In his description of species 
he treats 0. rananim, giving fairly good figures. 

Stein (1867) opposes (p. 10) Leydig's belief that the Opalinas 
are multicellular, saying that 0. intestinalis is clearly not so, and 
that the numerous clear vesicles of 0. ranarum, 0. dimidiaia and 
0. obtrigona, which are demonstrated with acetic or chromic acid, 
are not nuclei, but are vacuoles of liquid containing granules. The 
structure of the Opalinae therefore, does not confirm belief in the 
multicellular nature of the Froiosoa. The binucleated Opalinas are 
classed as numbers of his genus Anoplophrya. On p. 311 brief 
reference to the synonomy and occurrence of these forms is made, 

Clapahede & Lachmann (1868, p. 373) class the Opalinae, with 
many of the other forms now placed as members of the family 
Opalinidae, as an appendage to the ciliate Ivfusoria. 

Lankester (1870) excludes from the genus the forms now called 
Opalina, reserving this name for the species "so frequently found in 

2iJ* 



326 M- M. Metcalf 

both marine and frisli-water Annelids". He says "the simple struc- 
tureless body of these first named parasites lias really very little 
in common with Opalina, properly so-called — an abundance of highly 
refringent granules being the only differentiated portions of its 
substance, no trace of the nucleus and contracted vesicles, nor of 
the furrowed cuticle of true Opalina being observable. It is not 
impossible that these swimming flakes of sarcode — for they are 
nothing more — may undergo subsequent metamorphosis of the most 
extreme character." 

Engelmann published in Dutch (1875) and in German (1876) the 
first account of the growth of the young Opalinas in the rectum 
of the tadpole. He reared tadpoles from the e^g in glass dishes. 
He does not say how they were fed or how they became infected, 
though he mentions remnants of plants as present in their alimentaiy 
canals. [Probably the tadpoles were naturally infected from the 
material in the dishes in which they were kept, else Engelmann 
would have mentioned aitificially infecting them from the material 
in the recta of the frogs.] He found uninucleated cysts in the recta 
of the tadpoles and he also describes stages in the development 
of the little Opalinas from the uninucleated to the multinucleated 
condition. [His figures suggest tha the may have seen both micro- and 
macrogametes, though he did not recognise them as such, or observe 
copulation. Engelmann says he studied 0. ranarum from tadpoles of 
Rana esculenta. As this parasite has never before or since been 
reported from this host it seems possible that cross infected material 
was studied. The minute Opalinas figured seem clearly to be 
0. ranarum, for many of them are not slender enough for the 
corresponding stages of 0. dimidiata '), while they resemble Zellee's 
figures of the minute 0. ranarum in the tadpoles. His observation 
of uninucleated cysts suggests either that possibly Engelmann saw 
encysted zygotes, as Neeesheimek thinks, or that infection cysts of 
0. intestinalis or 0. caudaia may have been present also, for uni- 
nucleated infection cysts are common only in these two of the 
Europaean Opalinae though they are not rare in 0. ranarum [cf. 
p. 281). As the tadpoles were reared from eggs, there can be little 
doubt of their correct identification, since the time of year wiien 
the eggs were found, and their size and color, as well as the size 
and color of the tadpoles themselves, would distinguish them from 



') Neeesheimek (1907) thinks that they were 0. dimidiata, but I have not 
found the minute individuals of this species presenting this appearance. 



Opalina. 327 

those of Ixtma iotiporaria. In the chapter on infection experiments, 
page 314. I have shown that cross infections are verj^ easily secured. 
In view of this fact it seems hardly safe to accept Engklmann's 
i-eport of O. ranarum from liana esculenta as surely establishing that 
that species naturally occurs in this liost.] Engelmann's work 
definitely settled the fact that the nuclei of the multinucleated 
Opaliuac are true nuclei. He regards Anoplophrya and IIopHtophrya 
as transitional forms between Opalina and other Infusoria. He 
sought unsuccessfully to find the manner of origin of the cysts 
in the rectum of the frog. 

Zeller. the following year (1877) published a very accurate 
paper describing for five species (0- ranarum, 0. ohtrigona, 0. dimi- 
diata, 0. intesHnalis and 0. caudata, n. s.) 1) the rapid transverse 
and oblique [really longitudinal] divisions in the spring, within the 
frog's rectum, by which the Opalinas become minute; 2) the cysts 
in the rectum of the frog and the process of encystment ; 3) the cysts 
in the rectum of the tadpole; 4) the character of the animals hatched 
from the cysts; 5) their growth to adult chai-acter. Good descriptions 
of the form and structure of the adults are given. Zeller is the 
first to describe the nucleoli and their behaviour in mitosis and 
gives also the first good description of the disc-shaped refractive 
spherules. In studying the multinucleated species of Opalina he 
found the cysts in the rectum of the frog 
to be multinucleated (usually 4 nuclei); in 
the recta of the tadpoles he found both multi- 
nucleated and uninucleated cysts, the latter 
with large nuclei, as Engelmann described. 
He figures a minute "abnormal" individual Text Fig. XV. 

of 0. ranarum from the rectum of the tad- ^kller'.s figure of a minute 

. (). ranarum irom a tadpole 

pole [wliich was probably a microgamete or ^^ ^^,^„ tanporaria. It 
a microgamete mother-cell (Text Fig. XV), waspossibly a microgamete 
but he did not so interpret it, nor did he or a microgamete mother- 
observe copulation]. He figured and briefly ^e"- though the tail bears 
described a large form occurring with 0. 

dimidiaia in Rana esculenta, which he said might be either a new 
species or a form of 0. dimidiata. [Neresheimek has since named 
this form 0. zeUeri?^ This, the finest of all the papers upon Opalina 
will long serve as the starting point with all students of the genus. 

Certes (1880) discusses the presence of glycogen in the Infusoria, 
either in the form of refractive spherules or in solution in the endo- 




l^^S M. M. Metcalf 

plasm. Opalina is referred to as ag-i-eeing- with the otlier Infusoria 
in never having* the glycogen granules in the nuclei. 

Balbiani (1881) makes brief I'eference to observations of Engel- 
MANN and of Zellee upon the division of the nucleus in Opalina. 

Kent (1881 — 1882) gives good diagnoses of the five species of 
Opalina then known. He quotes ENfiELMANN's and Zeller's descrip- 
tions of the phenomena of encystment and the growth of the young 
animals in the tadpoles. Many of Zeller's and Engelmann's figures 
are well copied. The hosts of 0. intestinalis are given as Pelobates 
fuscus and Eana esculenta [in both of which it is very rare, its 
usual hosts being Bomhinaior pachypus and B. igneus]. 

Krukenberg (1882) in a discussion of digestion refers to Opalina 
among the animals which do not ingest solid food. 

De Lannesan (1882) gives a very brief description of Opalina and 
says that the Opalinae are doubtless descended from non-parasitic 
forms which became parasitic and gradually lost mouth and anus. 
Brief reference is made to the cysts and to the reproduction. 

Stokes (1884) describes 0. flava n. s., from Scaphiopus holbrooMi, 
the Hermit spade-foot Toad of eastern North America. 

NussBAUM (1884) a preliminary notice of Nussbaum 1886. 

Barfcrth (1885) includes 0. ranarmn in a general discussion 
of glycogen in the bodies of animals. He showed that, when trea- 
ted with '^Jodgunitni" or with iodine glycerine, some individuals re- 
mained merely yellow, others showed in certain restricted areas of 
the body a red brown color "characteristic of glycogen", the color 
sometimes being diff"use, sometimes following the lines of the cilia. 
Under higher magnification irregular masses of glycogen were found 
staining brown, while near them were many light yellow strongly 
refractive drops of another substance ("fat") [probably the refractive 
spherules of the endosarc]. 

Von Kolliker (1885) says (p. 23) that division in multinucleate 
forms like Opalina, which takes place without cooperation of the 
nuclei, is not comparable to true division among the protozoa, for 
the fragments do not grow to the size of the parent, but continue 
their division until they become very minute particles comparable 
to spores. The process may be described as cell-formation without 
division of the nucleus. [The distinction is superficial, not funda- 
mental. Nuclear division, of course, occurs, but the properly 
concomitant divisions of the body are for a time suppressed, to 
appear later, in the spring, when the animals are preparing for 
encystment.] 



Opalina. 329 

Grubek (1885 a) in a discussion of multinucleate protozoa, refers 
briefly to Opalina, saying that the frao:nientation of the multinucleate 
Opalinas in the spring-, by which a generally uninucleate condition 
is reached before encystment, is not comparable to ordinaiy binary 
fission. [The encepted forms usually have two to four nuclei.] 

Grubeu (1885i) in a discussion of artificial division in infusoria, 
refers briefly to Nussbaum's (1884) work, mentioning- the fact that 
Opalina frag-ments into dissimilar pieces which later by regeneration 
reach normal form. 

BiJTSCHLi (1886) describes and figures the alveolar structure of 
the endoplasma of 0. ranamm and says that the ectoplasma shows 
similar structure. 

Pfitzner (1886) gives a [too schematic] account of the division of 
the nuclei in 0. ranarum. He interprets the refractive spherules as 
algae. He showed clearly that the nuclei of 0. ranarum divide mi- 
totically. The true nucleolus was not seen. The absence of centro- 
somes and the persistence of the nuclear membrane were observed. 
Splitting of the chromosomes was [mistakenly] said to occur during 
a typical equatorial plate stage. 

NussBAUM (1886j gives a brief outline of the life history of 
0. ranarum after Engelmann and Zeller. He says the least 
particle of foecal matter causes a culture of Opalina quickly to die. 
[Others, myself included, have found that cellures with foecal matter 
live longer than those without.] He describes form, mode of 
swimming, and structure. He says that division apparently stops 
during the winter sleep of the host: that animals ready for encyst- 
meut have four or more nuclei. [I find frequently one, two, or three, 
as well ar four or more] : division is described at considerable length ; 
the products of division are not always alike; sometimes there is 
division into three pieces; at the temperature of the room division 
occupies forty to forty-five minutes; division of the body is in- 
dependent of the division of the nuclei, but division of the nucleus 
does not occur during division of the body. [I have not found the 
last statement correct for my preparations]; the direction of the 
mitotic spindle with reference to the planes of the body is very 
various; the nuclei show no interrelation in their divisions, dividing 
at different times without reference to one another: he found multi- 
nucleated and uninucleated cepts in the recta of tadpoles and 
thought it probable that the latter are derived from the former by 
fusion of nuclei: the nuclei of the young Opalinae in the tadpoles 
divide mitotically: he confirms Zeller that some Opalinae in the 



330 M. M. Metcalf 

frog's rectum remain large in the spring and do not rapidly divide : 
the cysts in the tadpole hatch in either the intestine or the rectum, 
some within an hour after ingestion, some remainiug unhatched as 
much as seven days : the cysts never hatch in water but will hatch 
in the aqueous humor of the frog: experiments in artificial division 
were unsuccessful for the pieces, like the whole animals, died. 

Balbiani (1887) refers at some length to the Opalinas; naming 
the hosts of the five species then known [Bomhinator should have 
been included as a host of 0. intestinalis {cf Zeller 1877)]; diag- 
nosing the genus and describing the shape of all the species except 
0. intestinalis; brief citations are made of the work of Zeller, Engel- 
MANN, and NussBAUM, upon the rapid division in the spring and en- 
cystment; he mentions having, frequently himself seen, in 0. rana- 
rum, conjugation preceeding the rapid multiplication in the spring 
[doubtless it was oblique division already correctly described by 
Zeller]. 

Entz (1888) [whose paper I have been unable to obtain] is 
quoted as saying that Opalina upon a partly shaded slide will swim 
out of the lighted area into the shaded area [a result not confirmed 
by other students]. 

BiJTscHLi (1887—1889) in his great work upon the Protozoa in 
Bronn's Klassen und Ordnungen des Thierreichs, gives [not quite com- 
plete] literature references to that date. He describes the genus, 
giving figures of 0. ranarum, 0. dimidiaia and 0. intestinalis. He 
[mistakenly] suggests that the oblique division described by Zeller 
was propably conjugation. The figures of mitosis, apparently taken 
from PriTZNER, are inaccurate. He [mistakenly] says (p. 1500) that 
the nuclei lie close under the cuticle, irregulary distributed in a 
single layer. He says the nuclei are comparable to micronuclei, not 
raacronuclei [They are probably comparable to both, each nucleus 
being both nutritive and generative]. 

Fabre-Domergue (1888) mentions with approval Bijtschli's (1886) 
"first" ') description of alveolar protoplasm in Ciliata, observed in the 
ectosarc [Butschli says endosarc] of 0. ranarum. 

Verworn (1889) found that Opalina [probably 0. ranarum] gave 
no reaction to stimulation by light. The center of a drop of water 
was brilliantly illuminated while the rest remained dark. There 
was no difference in the behaviour of the Opalinas in the two areas 



') Leydig (1857) had already mentioned the "beautifully cellular structure" 
of the ectosarc of Opalina. 



Opalina. 331 

and tliey were eciiially abundant in the two regions. Light causes 
no iniury to OpaUua. A frog was opened in faint light and the 
Opalinas in the rectum were placed in two cultures, one remaining- 
in the dark, the other being placed in bright daylight. No sub- 
sequent difference between the two cultures was observed. He found 
that Opalinas in a culture swim indifferently toward the warmer 
or toward the cooler area. 

In a second paper (1890) Vekworn showed that 0. ranarum, 
stimulated by an electric current, swims towai-d the anode. 

Parker (1891 and subsequent editions) describes briefly the 
structure and life history [so far as then known] of 0. ranarium. 
[The only error is the statement (after Engelmann) that] the little 
OpaUnac, which hatch from the infection cysts in the alimentary 
canal of the tadpole are uninucleated. 

Perrier (1893) refers to the difference between ectosarc and 
endosarc in 0. ranarum, to the presence of paraplasmatic bodies [re- 
fractive spherules] in the cytoplasm, to the process of encystment, 
to the fact that nuclei and cytoplasm divide independently, to the 
process of mitosis which is described according to Pfitzner's abser- 
vations [and therefore inaccuratelj^]. 

Verworn (1896) mentions again the anodic galvanotropism of 
0. ranarum and says further that under stimulation from a strong 
current the side of the body toward the kathode becomes clearer 
and more strongly refractive; that the granules of protoplasm and 
the nuclei withdraw^ more and more from that edge of the body; 
that small hj^aline vesicles soon appear there; that the cilia of that 
side are then destroyed; and that the contour of that part of the body 
then becomes uneven. There follows immediately a granular disin- 
tegration of the side of the body toward the kathode. Verworn 
believes that the anodic galvanotropism of 0. ranarum is due to a 
contractile irritation of the kathodic side of the body [later shown by 
Dale (1901) and Wallengren (1903) to be a mistaken interi)retation]. 

Delage & Herouard (1896) [mistakenly] interpret Zeller's 
figure of the form [which Neresheimer has named] 0. selleri, as in- 
dicating the presence of remnants of an excretory organ. 

LoEB ct BuDGETT (1897) quote Verworn's description of the 
fragmentation of the kathodic side of the body of 0. ranarum under 
strong electric stimulation, ascribing this to the action of acid; they 
believe that this anodic reaction of Opalina is due to the fact that 
it is always studied in physiological sodium chloride solution. [Pijtter, 
1900, has shown the error of this assumption.] 



332 M. M. Metcalf 

Paeker & Has WELL (1897) say "in Opalina numerous nuclear 
bodies are present which divide by mitosis, and therefore resemble 
micronuclei: if they are to be considered as such, this genus must 
be held to differ from the other Ciliaia in the total absence of a 
meganucleus". They refer to the processes of reproduction in the 
spring as described by Zellee and give figures from Zelller. Kent 
and Pfitzner (mitosis). 

Von Pkowazek (1898) says that the protoplasm of 0. ranarum 
stains intra vitam rosy red with neutral red, the unstained nuclei 
then being more evident. [I have not found a diffuse protoplasmic 
stain with very weak solutions of this reagent.] 

ToNNiGES (1898) recognised the alveolar structure of the proto- 
plasm of 0. ranarum and distinguished the ectosarc and endosarc. 
He describes the cilia as perforating the pellicle [vs. Bijtschli 1887 
— 1889 p. 1325. The cilia propably consist of a prolongation of 
the pellicula containing an axial fibril which extends at its base 
through the pellicula, to the basal granule. Both BiJTSCHLi's and 
ToNNiGEs' statements seem to be correct but incomplete.] and as ari- 
sing from the nodal points of a network of very delicate fibrils lying 
beneath the cuticle, ascribing the motion of the cilia to the contraction 
of these fibrils. He describes well the alveolar structure of the re- 
fractive spherules of the endoplasma. He says that they divide by 
constriction [probably an error]; that they are not excretory and 
are probably not parasitic, but are to be interpreted as a diffuse 
macronucleus. 

In a further communication the following year (1899), in regard 
to 0. ranarum, he notes that the division of the body has no dis- 
cernable relation to the division of the nuclei; he describes irre- 
gular divisions of the body ; he says that the several nuclei within 
the multinucleated cysts fuse into one [not confirmed by later stu- 
dents]. The nuclear membrane is described as showing alveolar 
structure [not confirmed by my study]. Amitotic division is said to 
occur side by side with mitotic division in the fullgrown forms [not 
confirmed by later students]. The true nucleolus was not observed. 
Pfitznee's work was confirmed in that no centrosomes were found 
and the nuclear membrane was seen to persist during the mitosis. 
The chromatin is described as superficial, lying just beneath the 
nuclear membrane. No longitudinal splitting of the chromosomes 
was seen. 

BiEUKorr (1899) discusses Veewoen's observations upon positive 
galvanotropism in 0. ranarum. 



Opalina. 333 

BovEKi (1900) discusses the evolution of centrosomes and mito- 
tic spindle, illustrating- from the nucleus of 0. caudafa one stap:e in 
this hypothetical evolution. He notes (p. 185) that the division of 
the nuclei and the division of the cytoplasm are relatively inde- 
pendent phenomena in the multinucleated Opalinac, and (p. 187) that, 
in the binucleated Opalinae, the binucleated condition is apparent, 
not real, being- due to delay in division of the body after the division 
of the nuclei. 

PiJTTER (1900) opposes Loeb & Budgett's (1897) statement that 
the anodic I'eaction of Opalina. differing: from all other Ciliata, is 
due to its al\va3'S being experimented upon in sodium chloride so- 
lution. He found that when Opalina and Balaniidium from the same 
host were experimented upon together in the same culture, Opalina 
showed anodic reaction while the reaction of Balantidium was ka- 
thodic. 

Lang (1901) classes Opalina in the suborder Hymenostomidae 
among the Ciliata holotricha. In the same suborder, which he cha- 
racterizes as having the mouth always open and as possessing an 
undulating membrane, he places Colpoda, Colpidium, Urocentrum. Para- 
maecimn. Anoplophrya, Frontonia, Leucophrys, Ophryoglcna, and Pleuro- 
nema. [Opalina of course has no undulating membrane and ho 
mouth.] 

DoFLEiN (1901) gives a suscinct account of the structure and 
development of Opalina. [as liable to mislead , may be noted the 
statements thatj the ectoplasma is homogeneous and that the endo- 
plasma is granulated; and, [as a probable error] quoted from Przes- 
MYCKi, the statement that the animals in the cysts often divide into 
several offspring. 

Dale (1901) refers to Verworn's observation of positive galvano- 
taxis in 0. ranarum; he notes that this species is found in the anterior 
end of the rectum of the frog; he describes experiments upon its 
chemotaxis and galvanotaxis which he summarizes as follows — 

Alkalinated Neutralized Acidified 

Chemotaxis Attraction to acid Attraction to acid Attraction to alkali 

Repulsion from alkali Repulsion from alkali Repulsion from acid 

Galvanotaxis Collects at anode Collects at anode Collects at kathode; 

he describes the normal movements of the cilia and their reaction 
to chemical and electrical stimuli; he describes the modification of 
galvanotaxis by changes in concentration of the media; and finally 
considers theoretically the phenomena. 



334 M- M. Metcalp 

CoNTE & Vaney (1902) mention the occurrence of 0. infestinalis 
in Triton taeniatus, the first report of Opalina from a tailed 
Batrachian. They say the refractive spherules arise in the nucleus 
and wander out throug'h the nuclear membrane into the cytoplasm. 
There are regarded as comparable to zymogen granules and yolk nuclei. 

KuNSTLER & GiNESTE (1902) give some anatomical notes upon 
0. dimidiata emphasizing especially the presence in the endoplasma 
of certain "vesicles" which contain each a central granule, which 
reproduce by division and which therefore have an individuality of 
their own. [Instead of one granule, these bodies, the endosarc 
spherules, contain many. They apparently do not divide. Instead of 
being constituent parts of the living protoplasm they seem to be 
nutritive material, paraglycogen, so that Kunstler & Gineste's con- 
clusions in regard to them seem inadmissible.] 

KoLscH (1902) studies minutely the drops of liquid which are 
extruded from 0. ranariim and 0. dimidiata when under pressure. 
He believes that the pressure causes partial liquefaction of the pellicle, 
that the culture flind (sodium chloride solution) is thus allowed to 
enter the body, and that this fluid unites chemically with the 
protoplasm forming "paramylin". He confirms Verwohn's description 
of anodic galvanotropism in 0. ranarum and describes the peculiar 
but constant curve through which it swims slowly toward the anode. 

HicKsoN (1903) makes numerous [inaccurate] references to Opalina. 
He says (p. 364) "The mouthless Opalina found in the bladder [!] of 
frogs may owe its many peculiarities of form to its entozoic habits"; 
Opalina is included with other Ciliata in the [mistaken because in- 
complete] statement (p. 368), quoted from BiJTSCHLi, that the cilia 
spring from the pellicula and are continuous with it; reference 
is made to the nuclei of Opalina as follows (p. 378): "If the current 
views concerning the nuclei of Opalina are trustworthy, this genus 
should no longer be regarded as a member of the Heterocaryota 
[Ciliata]. Opalina possesses, according to Pfitzner and others, a large 
number of meganuclei, but no micron uclei. [Pfitzner (p. 466) regards 
the nuclei of Opalina as homologous with the micronuclei of Para- 
maecium.] Thin sections of Opalina that are suitably stained show, in 
addition to the numerous macronuclei, a large number of small 
bodies containing chromatin. They are probably micronuclei. [The 
accompanying figure shows them to be refractive spherules of the 
endosarc] The meganuclei divide sometimes amitotically [probably 
not true], and it is probable that they always do so [mistaken]. 
The mitotic figures discovered by Pfitzner are clearly seen in a 



Opalina. 



335 



larf^e miniber of sections examined, but they are smaller than the 
mef^anuclei [no] whicli. as in other forms, increase considerably [ver}^ 
slightly] in size before division." [Each nucleus of Opalina seems 
to be functionally comparable to both micro- and macronucleus of 
higher CiliaiiL but to be houiologous with each, both the nuclei of 
Ciliata being phylogenetically complete nuclei.] 

Wallengkkn (1903) desci'ibes the form of the body of 0. rananim 
(Text Fig. XVI) [which has its very broad anterior end bent "to the 
rig-ht"]; the arrangement of its cilia; the normal movement of the 
cilia in "which the cilia that lie along the "anterior half of the 
right side" [corresponding to the morphological anterior end] beat 
forward [morphologically backward and toward the left] while all 
the others beat backward, the animal thus turning over to the 
right as it swims; he describes and analyzes the reactions of the 
cilia under electric stimulation, which cause the animal with a weak 
current to swim forward tot\'ard 
the anode, with a stronger current 
to swim forward towai-d the 
kathode, with a still stronger 
cun-ent to swim backward or 
sidways toward the anode. 

Maiek (1903) describes care- 
fully for 0. ranarum the pellicula, 
and the cilia and their basal 
granules. He denies the connec- 
tion of the cilia with a network 
of subcuticular fibrils such as 
ToNNioEs describes, ascribing the 
appearance of the transverse lines 
observed to ridges in the pellicle. 
[My study confirms Tonniges as 
to the presence of a sub-pellicular 
network in connection with the 
cilia, though I would not ascribe 
the movement of the cilia to the 
contraction of the fibrils of the 
network. The network seems 
more likely to be useful for the 
coordination of the movements of 
the cilia.l Maiek opposes ToiNNKiEs' 
description of the alveolar struc- 




Text Fig. XVI. 

VVallengren's figure of 0. ranarum, 
illustrating the direction of the motion 
of the cilia waves (plain arrows) and 
the direction in which the animal turns 
(feathered arrow). The* dotted lines 
across the hody do not indicate the 
lines of insertion of the cilia but the 
waves of contraction of the cilia. What 
I interpret as the morphological anterior 
end stretches form -j- to -[- and is in- 
dicated by the dotted index line. 



336 ^1- ^I Metcalf 

ture of the refractive spherules, saying tliey are homogeneous. 
[Bezzenberger and I confirm Tonniges.] Maier opposes Tonniges' 
description of the ectoplasma as containing large alveoles, saying 
that its alveoles are as small as those of the endoplasma. [My work 
confirms Tonniges.] 

Veneziani (1904) divided cultures of 0. ranarum into two 
exactly equal parts and placed a tube containing Vj„ gram of active 
radium bromide in one dish and none in the other. In ten experi- 
ments when the culture medium was 0.5 '% or 0,6 % sodium chloride 
solution, and in four experiments when the animals very kept in 
ordinary water (not distilled), the Opalinas in the culture containing 
the tube of radium bromide remained active longer than in the 
corresponding unstimulated cultures. The author says it is doubtful 
whether the longer continued activity of the Opalinas is due to the 
direct etfect of the radium upon their protoplasm, or to same 
modification of the density or chemical composition of the culture 
media. 

Statkewitsch (1904) finds that 0. ranarum does not react at all 
to weak constant and induction electric currents (0.5—1 MA), with 
stronger currents (3-4 MA) they generally swim slowly toward the 
kathode. With rather strong currents (2.3 or 4 MA) they often 
swim slowly toward the anode; sometimes they first approacli the 
anode and later turn and approach the kathode. Often with a weak cur- 
rent they start toward the kathode, but, without reaching it, turn 
back and swim in various directions through the culture. In the 
latter cases they probably become accustomed to the stimulation. 
The character of the reaction depends on the strength of the current. 

In another paper (1905) Statkewitsch mentions 0. ranarum in 
a discussion of the reactions of cilia in the Ciliaia to electric cur- 
rents. 

Bezzenberger (1904) describes five new species of Opalina from 
Asiatic frogs and toads (0. macronudeata, 0. lanceolata, 0. coracoidea, 
0. lata and 0. longa), giving many anatomical details. He figures 
mitosis in 0. macronudeata and 0. lanceolata (cf. Text Fig. V, page 250). 
For 0. longa he describes very peculiar elongated rod-shaped basal 
granules of.,the cilia, reaching from the pellicula through the whole 
ectoplasma and as far again into the endojdasma. The ectoplasma 
is described as having a zone entirely without demonstrable struc- 
ture. [His figure was evidently drawn from very poorly preserved 
material in which it was probably not possible to recognise the real 
structure of pellicula, cilia, basal granules, or ectoplasma.] The proto- 



Opaliiia. 337 

l)lasm of OpaUiia is spoken of as containing- intestinal contents. [It 
is, I think, always free from food particles or foecal matter, as Stein 
had already- shown.] He confirms Tonniges' statement that the re- 
fractive spherules contain granule.*?, but finds no sig^n of alveolar 
stiuctuie in them. [My work confirms T(')nniges in the latter re- 
gard.] He saw no indications of division of the refractive spherules. 
The longitudinal striae, between the rows of cilia, he describes as 
compesed of lows of granules. [My study indicates that this always 
liazy appearance of granules (Fig. 2, PI. XIV) may be an optical etfect 
produced where the longitudinal ridges (MaiekI of the pellicula pass 
above the transverse fibrillae (Tonniges) of the subpellicular net- 
work which is connected with the basal granules of the cilia.] 

LowENTH.4L (1904) describes for 0. ranarum the formation of 
the chromatin spheres in the nuclei before encystment (Text Fig. X. 
l)age 280). He distinguishes the more strongly staining sphere from 
the less deeply staining [and, according to my own observations, gra- 
nular] mass, saying that the former arises from the latter. The 
deepl}' staining compact sphere he regards as homologous to a micro- 
nucleus (sexual), the weaklj^ staining residue to a macronucleus (nu- 
tritive). His figures show wiiat he believes to be the sequence of 
phenomena. [The darkly staining spheres are extruded fiom the 
nucleus, as Neeesheimeu and I have shown, and go to pieces in the 
cytoplasm. They are probably composed of nutritive chiomatin.] 

CoHN (1904) gives an account of 0. intesiinalis, which is either 
inaccurate in most points, or is based wholly or in part on abnormal 
animals or on some other form or forms. Some of the features 
described [which do not fit normal 0. hitesfinalis] are: that the body 
is often triangular and flattened; that the refractive spherules 
disappear after twenty-four hours if the animals be kept without 
food in a hanging drop in a moist chamber; that the alveoles of the 
cytoplasmic foam grow smaller from the center of the body toward 
the periphery; that small forms are never binucleated and laige 
forms never uninucleated. The individuals figured in conjugation 
are evidently not Opalinas. The budding desciibed appears, to have 
been pseudoencystment following fragmentation. 

Leger & DuBoscQ (1904 a and h) give a fine account of the 
structure of an interesting new species, 0. saturnalis, which is found 
in the rectum of Box hoops, a fish from the Mediterranean Sea. This 
is the only Opalina which is reported from a host which is not an 
Amphibian. Tiie authors describe elongated and stocky foi-ms [as 
in 0. cat(data]; "lecithin (?)" bodies [my "ectosarc spherules"] are 



338 



M. M. Metcalp 



described in the outer, coarsely alveolated layer of the body; the 
endosarc is said to show no special inclusions [but bodies apparently 
resembling- the ordinary refractive spherules of the endosarc are 
figured]; the phenomena of mitosis are described with very clear 
figures {cf. Text Fig-. IV, page 249); longitudinal division is descri- 
bed and figured; uninucleated infection cysts are shown; true copu- 
lation was not observed; an individual resembling a microgamete or 
microgamete mother-cell is described and figured (Text Fig. XVII) 
without being recognised as a gamete; 0. saturnalis. on the ground 
of its occurrence in a marine fish, is regarded as the most primitive 
of the Opalinae. The authors say the family Opalininae (including 
the genus Opalina^ Opalinopsis and Foettingeria) should be sharply 
distinguished from the Anoplophryinae (including Anoplophrya and 
Hoplitophrya, which they would unite to form one genus Herpetophrya) 

and that the two families should not be 
regarded as closely related, the resemblance 
between them being a superficial one due 
to convergence caused by parasitism. The 
authors describe for 0. ranarum three sorts 
of cysts 1) the well known infection cysts, 
"exogenous", 2) "endogenous" cysts — in an 
ordinay large individual a bit of the proto- 
plasm containing one to four nuclei separates 
itself from the rest of the protoplasm and 
forms a cyst around itself, being then ex- 
truded from the body — , 3) conjugation 
cysts — two individuals like those of the 
ordinary cysts come together by their anterior 
ends, lie for a long time rubbing against 
each other and turning, and then form a 
cyst enclosing them both, each animal occu- 
pying half of the cyst. [These remarkable 
phenomena of the formation of the second 
and third kinds of cysts have not been 
observed by other students. Leger & Duboscq's very brief un- 
illustrated description can hardly be accepted without confirmation.] 
Fauee-Feemiet (1904), in a brief discussion of the structure of 
protoplasm, refers to Kunstlee's [Kunstlee & Gineste's] idea of 
the vesicular structure of protoplasm. He says that the "vesicles" 
of KuNsi LEE [in part refractive spherules of the endosarc in 0. dimidiata 
and other Protosoa] are not inclusions, are not reserve food, are not 




Text Fig. XVII. 
An individual of 0. satur- 
naliH figured by LSgee & 
DuBOSCQ. Doubtless it was 
a microgamete or a micro- 
gamete mother-cell. 
X 600 diameters. 



Opalina. 339 

excretory vesicles. [Several diifei'ent unrelated structures seem to 
be included under the term "vesicles" as here used. The endosarc 
spherules of Opalina seem to be composed, chiefly at least, of para- 
globuliu and to be a reserve food supply. The often reiterated 
conceptions of Kunstler & Gineste and Faurk-Fkemiet seem there- 
fore to be founded on an insecure foundation.] 

KuKSTLiR & Gineste (1905) discuss the refractive spherules of 
the endoplasma in 0. dimidiata; they say that they divide by con- 
striction, a central granule in each dividing fiist [not confirmed by 
Neresheimer or myself]^ They estimate eight thousand of these 
spherules to be present in an 0. dimidiata 112 '/.^ f.i long by 87^2 i^* 
broad. These spherules are regarded as a secretory apparatus. 
[They are apparently paraglj'cogen.] 

Schouteden (1905) in a brief note reports finding longitudinal 
division (Zeller's oblique division) in 0. ranarum very frequent in 
the spring. Division took fiom 50 to 90 minutes. In isolated in- 
dividuals he saw the longitudinal division begin and complete itself, 
thus confirming Zeller's description of the division (as Cohn had 
done before) and refuting Butschli's suggestion that Zeller had 
probably mistaken conjugation for division. 

PtJTTER (1905) finds that after an hour in sodium chloride 
solution 0. ranarum begins to show signs of injury, cilia movements 
becoming slower. The animals are at first clear and transparent, as 
they become abnormal they get darker. The abnormal condition 
and final death may be caused by the unnatural environment, or 
may be due to the noxious etfect of free oxygen in the cultures. Opalinae 
in culture media containing no free oxygen live longer than those in 
conti'ol cultures in which free oxj^gen is present. A better culture 
medium than sodium chloride solution is a solution made of 

sodium chloride 0.8 7o 100 parts 

sodium and potassium tartrate 30 "/o 5 „ 

distilled water 400 „ 

In this fluid, free from oxygen, Opalinae, if fed, live up to three 
weeks. Without food they live from one to seven days, showing 
how long they can live upon the energy already stored in their 
bodies, for there is in the fluid no source of ener-gy for the Opalinae. 
The stored eneigy in the bodies of the OimUnae is not in the form 
of "Folysachariden", for with iodnie we do not get tiie characteristic 
color reaction. [This statement needs modification Compare Bakfurth 
(1885) and my statement of the reaction to iodine — page 216.] The 
stored energy is not in the foini of fat, so probably it must be 



340 M. M. Metcalf 

some form of proteid. The appearance of myelin after solution 
(Kolsch) would favor the presence of Lecithin. [Nutrition in a form 
similar to glycogen seems to be present.] The Opalinae live longer 
in this ox3'gen-free culture fluid if certain substances be added. 
Addition of egg albumin, maltose (?), and uric acid especially a mixture 
of uric acid and dextrin, produce this result. It is not certain 
whether in the case of egg albumin the Opalinae form an extra- 
cellular enzyme which digests it, or whether the egg albumin is 
acted upon by anaerobic bacteria and is changed to a liquid 
dialyzable form. In the case of uric acid it is doubtful if the effect 
is an indirect one, or if the acid is used as food. PiJTTER says 
that after a few days in the cultures rather numerous instances of 
conjugation [doubtless really longitudinal division] and of transverse 
division appear. 

Faube-Feemiet (1905 a and b, 1906 a and b discusses the re- 
fractive spherules in the Protozoa, referring with approval to Kunstler's 
[KuNSTLER & Gineste's] interpretation of these bodies in Opalina as 
a secretory apparatus. He distinguishes [upon grounds that are not 
clear] between "spheroplasts of internal secretion" and "spheroplasts 
of external secretion". The spherules of Opalina are said to belong 
to the latter group. They are regarded as fundamental elements of the 
cell, comparable to the "leucites" of plants and to the nucleus, in 
this regard. 

Hartog (1906) notes Opalina's positive galvanotaxis and refers 
to Dale's experiments which show that the direction of motion varies 
with the nature and concentration of the medium; "It would tlius 
be a reaction to the ion liberated in contact with the one or the 
other extremity of the being". Opalina is classed among the Fla- 
gellata in the group Protomastigaceae. The author says "The numer- 
ous similar long flagella of the Trichomjmphidae afford, a transition 
in the genus Pyrsonympha to the short abundant cilia of Opalina, 
usually referred to the Ciliate Infusoria" ; and again "The Opalinidae 
have also an investment of cilia, which are short and give the 
aspect of a Ciliate to the animal. But despite the outward resem- 
blance, the nuclei, of which there may be as many as 200, are all 
similar, and consequently this group cannot be placed among the In- 
fusoria at all". 

Ktjntstler & GiNESTE (1906 a and c) describe for 0. climidiata, a 
mouth and spiral oesophagus, a cup-shaped depression, in front of 
the mouth, into which opens an excretory tube whose branches ra- 
mify through the body, and retractile papillae at the posterior end 



Opaliiia. 341 

of the body in tlie midst of wliich is an anal aperture leading from 
a short rectal tube. The position of the mouth indicates the true 
ventral surface, the animals having bilateral symmetry. [In careful 
study of living animals, and of prepaiations of total objects and 
sections, of 0. dimidiata and other species, I have found no trace of 
any of the organs mentioned, and cannot believe them to be present.] 

KuNSTLEE & GiNESTE (1906 b) describe the protoplasm of ab- 
normal 0. [dimidiata?] (kept too long in pui'e water, or from frog 
kept too long in captivity [?]) as resembling a continuous gelatinous 
substance, the appearance of a net, seen in the protoplasm of normal 
animals, being no longer discernable. 

Schneider (1906j. after studying iron-haematoxylin sections of 
0. ranariim, quotes with approval (p. 48) Maier's statement that the 
ectosarc is homogeneous [apparently referring to only what I have 
called the subcuticular layer, since he later mentions the presence 
of large spaces filled with a thick substance, which were doubtless 
the large alveoles of the ectosarc]. He describes (p. 49. Fig. 14 a — c) 
the appearance of threads, coarser and finer, wliich one sees in iron- 
haematoxylin preparations, distinguishing coarser branching fibres, 
connected with the basal gi-anules of the cilia, from more delicate 
ones forming a network not connected with the cilia. At the nodal 
points of this delicate network one sees thickenings. The coarser 
threads lie chiefly in the ectosarc, but extend also into the endo- 
sarc, the finer threads lie throughout both ectosarc and endosarc. 
Lying upon the latter [error] are found the disc-shaped granules 
[refractive spherules] described by Zellee. Schmeidke strongly 
opposes BuTscHLi's conception of the cytoplasm of the Infusoria as 
alveolar, saying that the threads described form clearly a frame- 
work within the cytoplasm, such as is present in the ciliated cells 
of Metasoa. [Had Schneider studied sections of Opalina, especially 
0. intesiinaUs or 0. caudata, which had been stained with Ehrlich's 
triacid mixture and others stained with meth3'l violet, he could 
hardly doubt the alveolar nature of both ectosarc and endosarc. The 
threads he describes seem to me chiefly optical sections of the walls 
of the alveoles.] Schneider describes (p. 50) and figures (his 
Fig. 14 c and d) the appearance [described by T()nni(;es, Maier and 
Bezzknberger. Cf. my Plate I, Fig. 2] of longitudinal markings 
between the rows of cilia, and other similar transverse maikings at 
a more internal level. He says the latter have no connection with 
the cilia. [Tonniges, and, in the present paper, I also, describe the 
transverse fibrils as uniting the bases of the cilia.] Schneider 

23* 



342 M. M. Metcalf 

describes (p. 62) the drops of liquid which exude from the ectosarc 
of Opalina when pressed; the fact that they do not mix with the 
water; and the further fact that on relieving the animal from 
pressure the drops are again absorbed by the body. These he 
regards as drops of hyaloplasma. Schneidee says (p. 71) that 
the protoplasmic currents, so general in the Infusoria, are entirely 
wanting in Opalina. His discussion of the hyalo plasma (p. 83 — 111) 
is based in part upon his studies of Opalina. He strongly opposes 
BuTSCHLi's belief in the prevalence of alveolar structure in living 
protoplasm. [It seems to me that few objects could be found more 
satisfactory^ than 0. intestinalis and 0. caudata for demonstrating 
the alveolar structure of protoplasm.] 

Jennings (1906) describes the "avoiding reaction" in 0. ranarum, 
and its behaviour with reference to acid and alkaline media showing 
that Opalina's reactions to stimuli, like those of other Protozoa, are 
always negative or null, never positive; he also quotes, with figures, 
Wallengren's analysis of the reaction of the cilia of this species 
to electric stimuli of different intensities. 

Neresheimee (1906 preliminary notice, and 1907) is the fir.st 
to recognise the presence of gametes in Opalina and is the first to 
describe the extrusion from the nuclei of the chromatin spheres 
previously described as micronuclei by Loewenthal. He saw no 
splitting of the chromosomes, confirming Toxniges and Bezzenbeegf.e 
against Pfitzner. He found the chromosomes in the asexual forms 
of 0. ranarum and 0. dinndiaia to be apparently 12 in number. 
He describes in detail phenomena preceeding encystment in the 
rectum of the frog; the process of encystment and the character of 
the infection cysts; the hatching of the cysts in the rectum of the 
tadpole; the formation of isogametes and their copulation; encystment 
following copulation; a spindle-like shape of the male and female 
pronuclei in the copulation cysts, and of the large syncaiia of the 
zygotes. He gives the name 0. zelleri to the large stocky form 
which Zellee found with 0. diimdiata in Rana esctilenia, having 
himself seen the same form in the same host. On the basis of its 
manner of reproduction, he regards Opalina as related to the Plasmo- 
droma rather than to the Ciliophora. [In many points the results 
of mj" stud}^ are opposed to Neee^^heimee. Some of his statements 
and beliefs to which I am unable to subscribe are : — his detailed 
description of the formation of generative chromidia, the degenera- 
tion of the old nuclei, and the formation of new sexual nuclei from 
the generative chromidia and refractive spherules, all of which is 



Opaliiia. 343 

said to preceede the formation of tlie infection cysts in the rectum 
of the froii": the complete disap{)earance of the refractive spherules 
before the formation of the infection C3'sts; the presence of always 
two and only two chromatin spheres in the nuclei before the forma- 
tion of the infection cysts, and their extrusion, one before encyst- 
ment, and one afterwards in the water or in the rectum of the 
tadpole (The definitiness of these phenomena seems to me uncertain); 
the presence of a double number of chromosomes in the sexual 
nuclei. 24 instead of 12 for 0. ranarum and 0.dimidiata\ isogamous 
copulation ; encystment following copulation (This seems to me 
doubtful); his interpretation of certain phenomena as abnormal 
budding (probably heterogamous copulation); his statement that the 
refractive spherules are homogenous; his belief that OpctUna is related 
to the Plasniadroma rather than the Ciliophora\ and his belief that 
all healthy frogs contain Opalinas.] 

DoBELL (1907) observed 0. ranarum and says he "confirms 
fully" Neresheimer's description of the nuclear phenomena preceeding 
encystment, i. e., "(1) formation of chromidia, (2) synthesis of fresh, 
nuclei from these chromidia, (3) reduction of chromatin and (4) en- 
cystment" [(1) and (2) I have not succeeded in finding in 0. ranarum, 
or in any multinucleated Opalina; they do not occur in 0. intestinalis 
or 0. caudata: (3) seems to have no connection with true "reduction"]. 
He observed and interprets as degenerative 1) the irregular divi- 
sions preceeding encystment [described by Tomniges for 0. ranarum ; 
see my Text Fig. Ill, page 241]: 2) the loss of cilia from animals 
kept "some days" outside the host; 3) the presence of refractive 
spherules in the cytoplasm, which "ultimately run together forming 
large masses lying in the cells" [the refractive spherules are, of 
course, present in normal Opalinas; I have never seen them fused 
to form large masses]; 4) nuclear degeneration accompanied by amitotic 
division, equal or unequal: the degenerating nuclei are said to 
extrude their chromatin in the form of chromidia and entirely dis- 
appear; "as a rule most of this chromatin is cast out of the organism, 
which then dies and breaks up, but occassionally only a part of the 
chromatin in cast out and perishes, the remaining granules" running 
together to form "two nuclei, consisting of solid chromatin", which 
"then approach one another and fuse". 

Metcale (1907 a) a preliminary notice of the present paper, 
Hartmaxn (1907) accepts Nehesheimer's (1907) conclusion that 
Opalina should be removed from the Ciliophora to the Flasmodroma, 
but does not assign it a definite position in the latter group. 



344 M. M. Metcalf 

Metcalf (1907 h and c) describes large excretory org-ans in 
0. intestinalis. 0. caudafa and 0. dimidiata, and a very rudimentary 
excretory organ in 0. oUrigona. No excretory organs were found in 
0. ranarum or 0. seUeri. that which Delage &Herouaed interpreted 
as remnants of excretor}^ canals in the latter species not being so. 
The excretory organ is very simple, being merely a series of enlarged 
and connected alveoles of the ordinary cytoplasmic foam. The pri- 
mitive character of the excretory organs and their resemblance to 
those of Hoplitophrya is emphasized. 

Loewexthal (1908) ^) found cysts of 0. ranarum in young partly 
grown Banae temporariae [showing that sexual maturity of the frog 
and the approach of its bi'eeding season is not a necessary stimulus 
and probably does not furnish any stimulus to cause encystment 
of the parasites.] Cysts are sometimes found containing two Opalinas. 
This condition does not surely indicate division within the cyst, but 
in one observed instance arose by a fusion of at first independent 
cysts. This is regarded as very likely pathologic. Neeksheimer's 
(1907 and 1908) description of the extrusion of chromatin spheres 
from the nuclei of encysted Opalinas is confirmed. Loewenthal gives 
up his former (1904) interpretation of these bodies as micronuclei 
and accepts Neeesheimee's comparison of the phenomena with the 
formation of polar bodies [This I have opposed]. Stained with 
Giejmsa's solution the spheres ') are clear blue [a reaction usually 
thought to indicate achromatic nature] while the smaller granules 
are red. If the nuclei are stained with methyl green in weak acetic 
acid, only the spheres are green [a reaction generally accepted as 
almost definitive for chromatin]. Treated with acetic acid the spheres 
become more highly refractive, if ammonia be added they become 
invisible but are not dissolved, both reactions indicating that the 
spheres are chromatin [?; the nucleoli show similar reactions]. On 
the basis of the reaction to Giemsa's stain the substance of the 
spheres is called cyanochromatin and the finer granules in the 
nucleus erythrochroraatin, the two sorts of chromatin being regarded 
as functionall}' different, corresponding the cyanochromatin to 
Schaudinn's somatic chromatin, the erythrochromatin to his sexual 



') Through the kindness of Dr. Haetmann I have been able to read the 
manuscript of this interesting paper, sent to him for publication, and to include 
a reference to it here. 

') Loewenthal cells them " KiJrperchen" . 



Opalina. 345 

chromatin. In "Paramaecia" [probably Balantidinm] from the rectum 
of the frog, and in other Ciliafa, Giemsa's staining- colors the 
micronucleus red, the macroniicleus blue. [The finer nuclear granules 
in Opalina I have regarded as chiefly achromatic, being influenced 
largely be their behaviour in degenerating nuclei of 0. oUrigona, 
and by the fact that after staining with safranin and light green 
they are green, not red.] 



346 



M. M. Metcalf 









Appendix. 


Staining reaction 






. 


Basal 


Ectosarc 


Ectosarc 
spherules 


Stains and Reagents 


Cilia 


Pellicula 


granules of 
Cilia 


films and 
granules 


Intra Titam. 












1 Neutral red 


O 


O 


O 


O 


dark red 


2 Methylen blue 


O 


O 


O 


o 


blue 


3 Toluidin blue 


O 


O 


O 


o 


blue 


4 Congo red 





O 





o 


O 


5 Indigo-carmine 


O 


O 


O 


o 


O 


6 Methyl violet 


O 


O 


O 


o 





7 Dahlia 





O 


O 


o 


O 


8 Bismarck brown 


O 


O 


O 





O 


9 Gentian violet 





O 


O 


o 


O 


10 Thionin 


O 


O 


O 





O 


After fixation with 












corosive-sublimate- 












acetic acid. 












11 Methylen blue 


O 


faint blue 


green 


green 


green 


12 Methyl violet 


violet 


very pale 
violet 


violet 


violet 


pale violet 


13 Gentian violet 


pale violet 


pale violet 


dark violet 


violet 


violet, the 

smaller are 

darker. 


14 Thionin 








green 


green 


green 


15 Dahlia 


very faint 
purple 


very faint 
purplish 


purple 


purple 


purple 


16 Fuchsin 


pale red 


gray, 
good red 


pale red 


films faint 
pink, granu- 
les yellow 
with faint 
pink flush. 


yellow with 

faint pink 

flush 


17 Rubin S. 


pale red 


red 


red 


red 


pale red 


18 Orange G. 


yellow 


yellow 


yellow 


yellow 


yellow 


19 Bismarck brown 


brown 


brown 


brown 


brown 


brown 


20 Kernschwarz 


brown 


brown 


brown 


brown 


brown 


21 Safranin 


red 


red 


red 


red 


red 


22 Safranin and light green 


green 


green 


green 


green 


green 


23 Ehrlich's triacid mixture 


? very faint 


green 


blue 


blue 





24 Biondi-Ehrlich-Heiden- 












haiu's mixture 


? very faint 


green 


blue 


blue 


O 


25 Borax carmine 


faint red 


? very faint 


red 


red 


O 


26 Paracarmine 


faint red 


? very faint 


red 


red 


O 


27 Delafield's haematoxylin 


blue 


blue gray 


blue 


blue 


muddy 

yellow, i. e. 

unstained. 


28 Heidenhain's iron -haema- 


O 


O 


black 


black 


larger, gray ; 


toxylin 










smaller, black. 


29 Ehrlich's indulin-aurantia- 


blue 


blue 




very faint 


O 


eosin 








blue,' almost 
unstained 





Opalina. 



347 



Opal hilt. (O = unstained.) 



Endosarc 
films and 
Sfiannles 



Endosarc 
spherules 



EndusaiT, 

Granules in 

posterior end 

of Excretory 

ors:an 



Nuclear 
Membrane 



Nucleus, 

Achromatic 

films and 

granules 



Nucleolus 



Nucleus, 

Chromatin 

including 

fibres 



blue 
violet 
violet 



dark red 

O 

O 

O 

O 

stain strongly 

stain w^ell 

O 

violet 

O 



O 



O 



blue O 

i 

purple O 

faint pink good red 



pale red 
yellow 
brown 
brown 
red 
green 
blue 

blue 
red 
red 
blue 



black 
blue 



good red 

yellow 

brown 

brown 

fainter red 

very pale red 

red purple 

red 
O 
O 

o 



black 
red 



good red 

yellow 

brown 

brown 

red 



blue 
dark red 

red 

dirty dark 

dull blue 

black 



very faint very faint 
blue blue, almost 



faint blue U 



dark violet violet 



violet 



violet 



dark blue faint blue, 
almost un- 
stained, 
purple ! faint purple 



unstained, 
very faint . red violet 12 
blue, almost I 
unstained. 

O violet IB 



blue 14 



red 



red 
yellow 
brown 
brown 

red 
green 

blue 

blue 

red 

red 

dark blue 



black 
blue 



films pink, 
granules red 



red 

yellow 

brown 

dark brown 

red 
green 

blue 

blue 

red 

red 

dark blue 



black 
blue 



red 



red 
yellow 
brown 
brown 

green 



blue 
O 
O 

light grayish 
blue 

black 



purple 15 
pink 16 



red 17 

yellow 18 

brown 19 

dark brown 20 

red 21 

red 22 

purple 28 

blue 24 

red 25 

red 2f> 

very dark 9- 

blue ^' 

black 28 

red 29 



348 M. M. Metcalf 



Literature Index. 

Balbiani (1881): Les organismes imicellulaires. Les protozoaires. in: Journ. de 
Micrographie T. 5 p. 357. 

— (1887): Evolution des microorganismes animals et vegetatives parasites, in: 

Journ. de Micrographie T. XI p. 293. 
Barfurth (1885): Vergleicheud-histochemische Untersuchungen iiber das Glycogen. 

in: Arch. f. mikr. Anat. Bd. 25. 
Van Beneden (1883): Recherches snr la maturation de I'oeuf, la fecondation et la 

division cellulaire. Gand (Leipzig und Paris). 
Van Beneden & Neyt (1887) : Nouvelles recherches sur la fecondation et la division 

raitosique chez I'ascaride megalocephale. Bruxelles. 
Bezzenberger (1904) : tjber Infusorien aus asiatischen Anuren. in : Arch. f. Pro- 

tistenkunde Bd. 3. 
BiRUKOFF (1899): Untersuchungen iiber Galvauotaxis. in: Arch. f. d. ges. Physiol. 

Bd. 77 1899. 
Bloch (1782): Abhandlungen iiber die Erzeugung der Eingeweidewiirmer. Berlin 

1782. 
BoRY de St. Vincent (1824) : Encyclopedie methodique. Paris. 
BoTT (1907): tJber die Fortpflanzung von Pelomyxa palustris. in: Arch. f. Pro- 

tistenkunde Bd. 8. 
BovERi (1887 a): Uber Differenzierung der Zellkerne wahrend der Befruchtung des 

Eies von Ascaris megalocephala. in : Anat. Auz. Bd. II. 
- (1887 6): Zellen-Studien. Heft I. Die Bildung der Richtungskorper bei Ascaris 

megalocephala und Ascaris lumbricoides. Jena. 

— (1892 a): Uber die Entstehung des Gegensatzes zwischen den Geschlechtszellen 

und den soniatischen Zellen bei Ascaris megalocephala. in: Sitz.-Ber. 
d. Ges. f. Morphol. u. Physiol, in Miincheu Bd. VIII. 

— (1892?*): Befruchtung. in: Ergebn. d. Anat. u. Entwicklungsgesch. Bd. I. 

— (1899): Die Entwicklung von Ascaris megalocephala mit besonderer Riicksicht 

auf die Kernverhaltnisse. in : Festschr. z. 70. Geburtstag von C. v. Kupffer. 
Jena. 

— (1900): Zellen-Studien. Heft 4. Uber die Natur der Centrosomen. Jena 1900. 

— (1907): Zellen-Studien. Heft 6. Die Entwicklung di.spermer Seeigel-Eier. Ein 

Beitrag zur Befruchtungslehre und zur Theorie des Kernes. Jena. 
BtJTSCHLi (1880—89): Bronn's Klasseu u Ordnungen d. Tierreichs. Bd. I. Protozoa. 
Abt. I (1880—82): Sarkodina und Sporozoa. Abt. II (1883—87): Mastigo- 
phora. Abt. Ill (1887 — 89) : Infusoria und System der Radiolarien. Leipzig. 

— (1885 a): Einige Bemerkungen iiber gewisse Organisationsverhaltnisse der sog. 

Cilioflagellaten und der Noctiluca. in : Morph. Jahrb. Bd. X. 

— (18856): Bemerkungen iiber einen dem Glycogen verwandten Korper in den 

Gregarinen. in: Zeitschr. f. Biol. Bd. 21. 

— (1886): Kleine Beitrage zur Kenntuis einiger mariner Rhizopoden. in: Morpb. 

Jahrb. Bd. XI. 

— (1906): Beitrage zur Kenntnis des Paramylons. in: Arch. f. Protistenk. Bd. 7. 
Calkins (1899): Mitosis in Noctiluca railiaris and its bearing on the nuclear relations 

of Protozoa and Metazoa. in : Journ. of Morphol. Vol. 15. 

— (1901) : The Protozoa. New York. 



Opalina. 349 

Calkins (1902): Miuino Protozoa from Wood's Hole, in: II. S. Fish Oommiss. Reports 
1901. 

— (1906): The Protozoan Life Cycle, in: Biol. Bull. Vol. XI No. 5. 

Calkins & Cull (1907): The coujugation of Paramaecium anrelia (caudatum). in: 

Arch. f. Protistenk. Bd. X. 
Certes (1880): Siir la glycogen^se chez les infnsoires. in: Compt. rend. Acad. Sc. 

Paris T. 90. 
CLAi'ARfeDE & Lachmann (1868): Etudes sur les lufusoires et les Rhizopodes. Geneva 

et Bale (Extiait des tomes V, VI et VII des Memoires de I'lnstitut 

Genevois 1858—60). 
CoHN (1904): Zwei parasitische Infusorien aus Discoglossus pictus. in: Arch. f. 

Protistenk. Bd. 4. 
CoNTE & Vaney (1902): Sur des emissions nucleaires observees chez des protozoaires. 

in : Compt. rend. Acad. Sc. Paris T. 135. 
Cull (1907): Rejuvenescence as the Result of Conjug-ation. in: Journ. Exper. 

Zool. Vol. 4. 
Dale (1901): Galvanotaxis and cheraotaxis of ciliate infusorians. Part. I. in: Journ. 

of Physiol. XXVI. 
Delage & Herouard (1896): Traite de zoologie concrete. T. 1. La cellule et les 

protozoaires. Paris. 
DoBELL (1907): Physiological degeneration in Opalina. in: Quart. Journ. Microsc. 

Science Vol. 51. 
DoFLEiN (1900): Studieu zur Naturgeschichte der Protozoen. IV. Zur Morphologie 

und Physiologie der Kern- und Zellteilung. in : Zool. Jahrb., Abt. f. 

Anat. u. Ontog. Bd. XIV. 

— (1901): Die Protozoen als Parasiten und Krankheitserreger. Jena. 
DujARDiN (1841): Histoire iiaturelle des zoophytes iufusoires Paris. 
Ehrenberg (1831): tjber die Entwicklung und Lebensdauer der Infusionsthiere, 

nebst ferneren Beitragen zu einer Vergleichuiig ihrer organischen Systeme. 
in: Abb. d. kgl. Akad. d. Wiss. zu Berlin Jahrg. 1881. 

— (1835) : Zusatze zur Erkenntnis groCer organischer Ausbildung in den kleinsten 

thierischen Organisinen. in: Idem 1835. 

— (1838): Die Infusionsthierchen. Leipzig. 

Engelmann (1875): Over ontwikkeling en voortplanting van Infusoria: I. Ont- 
wikkeling van Opalina ranarum binnen bet darmkanal van den kikvorsch. 
in: Onderznek. pliysiol. Laborat. Utrecht Hoogeschool. dirde Recke Bd. III. 

— (1876): IJber Entwicklung und Fortpflanzung von Infusorien. I. Entwicklung 

und Fortpflanzung von Opalina ranarum innerhalb des Darmkanals von 
Rana esculenta. in: Morph. Jahrb. Bd. 1. 

Entz (1888): Studien iiber Protisten. in: Auftr. d. k. Ung. Naturw. Ges. Budapest. 

Fabre-Domergue (1888): Recherches anatomiques et physiologiques sur les iu- 
fusoires cilies. in : Annales des sciences naturelles Zoologie T. V. 

FAURfi-FREMiET (1904): Sur la structure des protoplasma chez les infusoires cilies. 
in: Compt. rend. Soc. Biol. Paris. 

— (1905a): La structure intime du protoplasma chez les Protozoaires. in: Compt. 

rend. Soc. Biol. Paris T. LIX. 

— (1905ft): Sur la structure du protoplasma chez les Protozoaires. Idem p. 197. 

— (1906 «): Sur la structure intime du ))rotoplasma chez les Protozoaires. in: 

Compt. rend. Acad. Sc. Paris T. 142. 



350 M. M. Metcalp 

FAURfe-FEEMiET (1906 &): A propos de la structure du protoplasma chez les Proto- 

zoaires. in: Compt. rend. Soc. Biol. Paris. 
Fischer (1895): Untersuchungen iiber Bakterien. in: Jahrb. f. wiss. Botauik Bd. 27. 

— (1905): Fine neue Glycogen-Fiirbung-. Aiiat. Anz. Bd. XXVI. 

Francotte, p. (1897): Recherches sur la maturation, la fecondatiou et la segmentation 

chez les polyclades. Bruxelles. Published by the Royal Acad, of Sciences 

of Belgium. 
GozE (1782): Versuch einer Naturgeschichte der Eingeweidewiirmer thierischer 

Korper. Blankenberg. 
GOLDSCHMIDT (1905): Die Chromidien der Protozoen. in: Arch. f. Protistenk. Bd. V 

Heft 4. 
GoNDER (1905) : Beitrage zur Kenntnis der Kernverhaltnisse bei den in Cephalo- 

poden schmarotzenden Infusorien. in: Arch. f. Protistenk. Bd. V. 
Gould (1893): Notes on the minute structure of Pelomyxa palustris Greef. in: 

Quart. Journ. of Micr. Science Vol. 36. 
Greef (1874): Pelomyxa palustris, ein amobenartiger Organismus. in: Arch. f. 

mikr. Anat. Bd. 10. 
Hamburger (1904): Die Conjugation von Paramaecium bursaria Focke. in: Arch. 

f. Protistenk. Bd. 4. 
Hartmann (1907): Das System der Protozoen. in: Arch. f. Protistenk. Bd. 10. 
Habtmann & V. Prowazek (lt07): Blepharoplast, Caryosom und Centrosom. in: 

Arch. f. Protistenk. Bd. X. 
Hartog (1906): Protozoa, in: Cambridge Natural History Vol.1. London. 
Heidenhain (1907): Plasma und Zelle. Jena. 
Hertwig, R. (1889) : Uber die Conjugation der Infusorien. in : Abh. d. bayr. Akad. 

d. Wiss. II. CI. Bd, XVII. 

— (1898): Kernteilung, Richtungskorperbildung und Befruchtung von Actino- 

sphaerium. in: Abh. d. k. bayer. Akad. Wiss. XIX, 2. 

— (1899): Was veranlaCt die Befruchtung der Protozoen? in: Sitz.-Ber. d. Ges. 

f. Morphol. u. Physiol, in Miincheu. 
— . (1907) : tjber den Chromidialapparat und den Dualismus der Kernsubstanzen. 

in: Sitz.-Ber. d. Ges. f. Morphol. u. Phy.siol. in Munchen. 
HiCKsoN (1903) : Protozoa, Infusoria, in : A Treatise on Zoology, edited by E. Ray 

Lankester Vol. I. London. 
Ischikawa (1899): Further observations on the nuclear division of Noctiluca. in: 

Jouru. Coll. Science Tokyo Vol. 6. 
Jennings (1899) : Studies on reactions to stimuti in unicellular organismes. II. The 

mechanism of the motor reactions of Paramaecium. in : Amer. Journ. 

Physiol. II p. 311. 

— (1906) : Behaviour of the lower organisms. Nevr York 1906. 

Joseph (1907): Beobachtungen iiber die Kernverhaltnisse von Loxodes rostrum 

0. F. M. in: Arch. f. Protistenk. Bd. 8. 

Kent (1881—1882) : A Manual of the Infusoria. Vol. II. London. 

Keuten (1895): Die Kernteilung von Euglena viridis. in: Zeitschr. f. wiss. Zool. 

Bd. LX. 
Klebs (1883): Uber die Organisation einiger Flagellatengruppen. Botan. Inst. 

Tiibingen I, 1. 
KoLLiKER (l864j: Icones histologicae, oder Atlas der vergleichenden Gewebelehre. 

1. Bd. Der feinere Bau der Protozoen. Leipzig. 



Opalina. 351 

Koi.MKER (1885): Die Bedeutuiiff der Zellkeriie fur die Vorgange der Vererbung. 

in: Zeitsclir. f. wiss. Zool. P.d XLII ji. 23. 
KoLSCH (1902|: rjntersutlnintren iiber die ZertlieGnngserscheinnngen der ciliaten 

Iiifusorien (nebst Benierkunj>'en iiber Protoplasniastrulitur, Protopiasma- 

bewegungen und Vitalfiirbungen). iu: Zool. Jalirb. , Abt. f. Auat. u. 

Antol. Bd. 16. 
Krukenberg (1882): Grundziige einer vergleichenden Physiologle der Verdaming. 

Heidelberg. 
KuHNE (1859): Untersucbnngen iiber Bewegungen und Veriiiiderungen der con- 

traotileu Substanzeii. IV. Die Verandenuigen der contractilen Sub.stauz 

nacb dem Tode. in: Arch. f. Aiiat. u. Pbysiol. Jabrg 1859. 
KiTNSTLER & GiNKSTE (1902): Notice preliminaire sur I'Opaline dimidiate, in: 

Bibliographie anatomiqne T. 10. 
(1905) Les spbernles trophoplasmique des infusoires cilies. in: Compt. rend. 

Acad. Sc. Paris T. 141. 

— — (1906 rt): Contribution a la morphologic generale des Protozoaires superieurs. 

iu: Compt. rend. Acad. Sc. Paris T. 142. 
(1906 b) : Les cultures de Protozoaires et ces variations de la matiere vivante. 

in : Compt rend. Acad. Sc. Paris T. 142. 
(1906 f;): L'orientation du corps des Opalines, in: Compt. rend. Soc. Biol. 

Paris T. 61. 
Lang (1901): Lebrbuch der vergleichendeu Anatomic der wirbellosen Tiere. 2. Aufl. 

2. Liefg. Protozoa. 
Lankester (1870): Remarks on Opalina and its contractile vesicles, in: Quart. 

Journ. Micr. Sci. n. s. Vol. X. 
DE Lannesan (1882): Traite de Zoologie. Paris. 
Leeuwenhoek (1685): Outledingen en Ontdekkingeu. 

— (1722j: Opera omnia, seu Arcana naturae ope exactissimorum microscopiorum 

detecta, experimentis variis compiobata. Lugdnni Batavorura. 
Lefevre (19U3): A new metbod of embedding small objects, in: Juurn. Applied 

Microscopy Vol. V p. 21180-2081. 
Lf:GEE & DuBOCQ (1904o): Notes sur les iufnsoires endoparasites. L Les Astomata 

represent-ils un groupe naturel? in: Arch, de Zool. exper. et gen., Notes 

et revue, 4^ serie T. 2. 

— — (1904 b): Notes sur les infusoires endoparasites. IL Anoplophrya brasili L. & D. 

in. Opalina saturnalis L. & D. in: Arch. Zool. exper. et gen. 4'' serie T. 2. 
Leydig (1857): Lebrbuch der Histologie des Menscben und der Tiere. Frankfurt. 
LoEB & BuDGETT (1897): Zur Theorie des Galvanotropismus. in: Arch. f. Anat. u. 

Physiol. Bd.65. 
LoEWENTHAL (1904) : Das Auftreten eines Micronucleus-artigen Gebildes bei Opalina 

ranarum. in: Arch. f. Proti.stenk. Bd. Ill li04. 

— (1908): Notizen iiber Opalina ranarnm nebst Beinerkuiigen iiber die Untersnchung 

von Erythro- und Cyanochromatin. in: Arch. f. Protistenk. Bd. XIII. 
Macparland (1897): Cellulare Siudieu an Mollusken-Eier. in: Zool. Jahrb., Abt. 

f. Anat. u. Ontog. Bd. X. 
Maier (1903j: Uber den feinereu Ban der Wimperapparate der Infusorien. in: 

Arch. f. Protistenk. Bd. II 
Macpas (1885): Sur le glycogeue chez les infusoires cilies. in: Comjit. rend. Acad. 

Sc. Paris T. 101. 



352 M. M. Metcalf 

Mattpas (1886): Sur les granules amylaries du cytosorae des Gregarines. in: 

Compt rend. Acad. Sc. Paris T. 102. 
Mayer (1907): Uber die Einbettnng kleiner Objekte zum Schneiden. in: Zeitschr. 

f. wiss. Mikrosk. u. niikr. Technik Bd. XXIV. 
Metcalf (1907a): Studies on Opalina (Preliminary jS'otice). in: Zool. Anz. Bd. 32 

Nr. 3/4. 

— (1907 ft and c): The Excretory Organs of Opalina. Parts I and II. in: Arch. 

f. Protistenk. Bd. X. 
Meves (1899a): Uber den EinfluC der Zellteilung auf den Sekretionsvorgang, nach 
Beobacbtungen an der Niere der Salamanderlarve. in: Festschrift zum 
70. Geburtstag von Carl v. Kupffer. Jena. 

— (1899 b) : Uber Struktur uud Histogenese der Samenfaden des Meerschvveinchens. 

in: Arch. f. mikr. Anat. u. Entwickluugsgesch. Bd. 54. 
MiJLLER, 0. F. (1786): Animalcula infusoria fluviat. et marina. Leipzig. 
Neresheimer (1906): Der Zeugungskreis von Opalina. in: Sitz.-Ber. d. Ges. f. 

Morphol. u. Physiol. Miinchen Bd. 22, also in: Mlinch. med. Wochenschr. 

Nr. 36 4. Sept. 1906. 

— (1907): Die Fortpflanzung der Opalinen. in: Arch. f. Protistenk., Suppl. I 1907. 

— (1908) : Der Zeugungskreis des Ichthyophthirius. in : Ber. d. k. bayr. Biol. Ver- 

suchsstation in Miinchen Bd. I. 
NussBAUM (1884): Uberspontane u. kiinstliche Zellteilung. in: Sitz.-Ber. d. Niederrh. 
Ges. f. Natur- u. Heilkunde Bonn 1884 p. 259. 

— (1886): Uber die Teilarbeit der lebendigenMaterie. I. Mitteilung. Die spontaneund 

kiinstliche Teilung der Infusorieu. in: Arch. f. mikr. Anat. Bd. 26 p. 487. 
Pagenstecker (1857): Trematodenlarveu und Trematoden. Heidelberg. 
Parkek (1891 and subsequent editions): Elementary Biology. 
Parker & Haswell (1897): A Text Book of Zoology. London. 
Perrier (1893): Traite de Zoologie. Paris. 
Perty (1852): Zur Kenntnis kleinster Lebensformen. Bern. 
Peter (1899): Das Centrum fiir die Flimmer- uud GeiCelbewegung. in: Anat. 

Anz. XV 14/15. 
Pfitzner (1886): Zur Kenntnis der Kernteilung bei den Protozoen. in: Morphol. 

Jahrb Bd. U. 
Prandtl (1905) : Reduktion und Caryogamie bei Infusorieu. in: Biol. Centralbl. Bd. 25. 

— (19U6) : Die Conjugation von Didinium nasutum 0. F. M. in : Arch. f. Protistenk. 

Bd. VIL 
Pritchard (1861): A History of Infusoria. London, p. 267, 569. Plate XXVI 

Figs. 28, 29. 
VON Prowazek (1898): Vitalfarbungen mit Neutralrot an Protozoen. in: Zeitschr. 

f. wiss. Zool. Bd. 63. 

— (1905): Studien iiber Saugetiertrypauosomen. in: Arb. a. d. kais. Gesuudheits- 

amte Bd. XXII. 
PuRKiNjE & Valentin (1835): De phenomeno generali et fundamentali motus 

vibratorii. Vratislaviae. 
PtJTTER (1900): Studien iiber Thigmotaxis bei Protisten. in: Arch. f. Anat. u. 

Phys., Physiol. Abt., Suppl -Bd. IIOO. 

— (1905): Die Atmung der Protozoen. in: Zeitschr. f. allgem. Physiol. Bd. V. 
Qoennerstedt (1865): Bidrag til sveriges infusoriefauna. in: Acta universit. 

Lundensis T. II. 



Opalina. 353 

Salvin-Moouk & Breinl (1007): Tlie C'ytolofjy of the Trypaiiosoines. in: Annals 

of Tropical Medicine and Parasitology Vol. I. 
ScHAUDiNN (1894): Uber Keniteilung: niit nacbfolgender Korperteihmg bei Amoeba 

ciystalligeia Gruuek. in: Sitz.-Ber. d. Akad. d. Wiss. zu Berlin. 

— (18y6a): Uber das Centralkorn der Heliozoen. Ein Beitrag zur Oentrosomen- 

frage. in: Verb. d. dentsch. Zool. Ges. 

— (18966): ilber den Zeiigungskreis von Paramoeba eilhardi. in: Sitz.-Ber. d. 

Akad. d. Wiss. Berlin 18% I. 

— (19(M): Generations- und Wirtswechsel bei Trypanosoma und Spirocbaete. in: 

Arb. a. d. kais. Gesnndbeitsamte Bd. XX. 
Schneider (1906): Plasmastruktnr nnd -Bewegnng bei Protozoen und Pflanzenzellen. 

in: Arb. a. d. Zool. In.st. der Univ. Wien Bd. XVI. 
ScHOUTEDEN (1905): Liingsteilnug bei Opalina ranarum. in: Zool. Anz. Bd. 28. 
ScHRANK (180^): Fauna boiea. Ill, 2. Landshut. 
ScHUBOTZ (1908) : Pyenothrix monocystrides nov. gen., uov. spec, in : Denkschr. d. 

med -naturw. Ges. Jena Bd. XIII. 
ScHULTZE, Max (1851): Beitrage zur Natnrgeschichte der Tnrbellarien. Greifswald. 
VON SiEBOLD (1835) : Helminthologische Beitrage. in : Arch. f. Xaturgesch. Bd. I. 
Statkewitsch (190-4): Galvanotropi-smus und Galvanotaxis der Ciliaten. I. Mit- 

teilung. in : Zeitschr. f. allgem. Physiol. Bd. IV. 

— (1905): Idem. II. Mitteilung. Reaktion der Wimpern — die Grunderscheinung 

des Galvanotropismus der Protisten. in: Zeitschr. f. allgem. Physiol. Bd. V. 
Stein (read 1856, published 1859): tJber die ihm bis jetzt bekannt gewordenen 
und von ihm genauer erforschteu Infu.sorien, welche im Innern von 
audereu Tieren eine parasitische Lebensweise flihren. in : Abh. k. bohm. 
Ges. Bd. X. 

— (1859): Der Organismus der Infnslonstiere nach eigenen Forschungen in syste- 

matischer Reihenfolge bearbeitet. I. Abt. : Die hypotrichen Infusionsthiere. 
Leipzig. 

— (1860): Uber die Eiuteilung der holotrichen Infusionstiere: neue Gattungen und 

Arten dieser Ordnung. in: Sitz.-Ber. der k. bohm. Ges. d. Wiss. in Prag 
Jabrg. 1860 p. 56. 

— (1867): Der Organismus der Infusionstiere. Bd. II. Leipzig. 

Stokes (1884): Notice of some new parasitic Infusoria, in: Amer. Naturalist. 

Vol. XVIII p. 1081-86. 
Stolc (1900): Beobachtungen und Versuche iiber die Verdauung und Bildung der 

Kohlehydrate in einem amobenartigen Organismus, Pelorayxa palustris 

Greef. Zeitschr. f. wiss. Zool. Bd. 68. 
ToNNiGES (1898): Die feineren Bauverhaltnisse von Opalina ranarum. in: Sitz.-Ber. 

d. Ges. z. Beford. d. ges. Naturw. zu Marburg Jahrg. 1898. 

— (1899j : Nachtrag zu den Untersuchungen iiber die feineren Bauverhaltnisse von 

Opalina ranarum. Ibid. Jahrg. 1899. 

Veneziani (1904): Uber die physiologische Einwirkung des Radiums auf die Opa- 
lina ranarum. in: Centralbl. f. Physiol. Bd. 18 1904. 

Verworn (1889): Psycho-physiologische Protisten-Stndien. Jena. 

— (1890): Die polare Erregung der Protisten durch den galvanischen Strom, in: 

Arch. f. d. ges. Physiol. Bd. 46. 

— (1896j: Untersuchungen iiber die polare Erregung der lebendigeu Substanz durch 

den konstanten Strom. III. Mitteilung. in : Arch. f. d. ges. Physiol. Bd. 62. 



354 M. M. Metcalf 

Wallengren (1903): Znr Keuutnis der Galvanotaxie. I. Die anodische Galvano- 

taxis. in: Zeitschr. f. allg. Physiol. Bd. II. 
Wilson, E. B. (1895): Archoplasm, Centrosome and Chromatin in the Sea Urchin 

Egg-, in : Journ. of Morphol. XI. 
— (1900): The Cell in Development and Inheritance. New York. 
Yatsu (1901): On the Use of "Sea Lettuce" (Ulva). in: Orienting Small Objects 

for Sectioning, in: Jonrn. of Applied Microscopy Vol. VI No. 12. 
Zeller (1877): Untersuchnngen liber die Fortpflanzung und Entwicklung der in 

unseren Batrachiern schmarotzenden Opalinen. in: Zeitschr. f. wiss. Zool. 

Bd. XXIX 1877. 



Explanation of Plates. 

All figures are camera drawings unless otherwise indicated. The degree of 
accuracy of all figures is told. When nothing is said as to accuracy, everything 
shown is carefully drawn with the camera; omissions are not always noted. Draw- 
ings from acetic-carmine preparations cannot show fine details for the stain does 
not bring out the finer structure. 

Plate XIV. 
Fig. 1. A schematic drawing of an optical longitudinal section of 0. intestinalis, 
showing cilia, basal granules of cilia, ectosarc with ectosarc spherules (gray), eudo- 
sarc with endosarc spherules (black), axial excretory organ, two nuclei (connected 
by a thread) each containing eight chromosomes. In the posterior nucleus is seen 
the vacuolated nucleolus. 

Fig. 2. Part of a tangential section (superficial) of 0. ranarum. Two rows 
of basal granules of the cilia are shown. Above each of these rows is a fibril 
which probably connects the outer ends of the basal granules. Transverse fibrils, 
at a little lower level than the last, run between the basal granules. The two 
longitudinal striae between the rows of basal granules are probably ridges in the 
pellicula. Where they cross over the transverse fibrils one sees a misleading 
hazy appearance of granules which do not exist. Coros. subl. -acetic acid, iron 
haeraatoxylin. X 410*3 diameters. 

Fig. 3. Part of an oblique section of 0. intestinalis, showing cilia (diagram- 
matically drawn), pellicula, basal granules of cilia, five alveoles of tlie ectosarc, 
two ectosarc spherules with contained granules, and two endosarc spherules with 
contained granules. Coros. subl.-acetic acid, iron haematoxylin. X 2000 diameters. 

Fig. 4. A cross section of 0. intestinalis, showing the endosarc spherules 
(black) and the ectosarc spherules (gray). No attempt to indicate protoplasmic 
structure is made, merely the large alveoles of the ectosarc being drawn. Coros. 
subl.-acetic acid, iron haematoxylin (but little extracted). X 14^5 diameters. 

Fig. 5. Part of a longitudinal section of 0. intestinalis, showing cilia 
(slightly diagrammatic), basal granules of cilia, granular spherules in ectosarc 
and endosarc, cytoplasmic granules, nucleus with three large granular masses of 
chromatin and granular achromatic foam, also three chromatin spherules seeming 
about to be extruded from the nucleus. The small faintly stained spherical body 
in the nucleus is probably a partly dissolved chromatin spherule. The arrows in- 



Oralina. 355 

dicate the boundary between ectosarc and eudosarc. Coros. subl. -acetic acid, iron 
haenmtoxyliu (well extracted). X 2000 diameters. 

F\g. 6. Part of a cro.ss section of 0. obirinonn, showing cilia (semi-dia- 
graniniatic). pellicula, rows of basal granules of cilia, larye alveoles of ectosarc 
containing finely granular ectosarc spherules (gray), granular endosarc spherules 
(more darkly stained) each iu an alveole (some of these endosarc spherules lie in 
strands of endoplasma which have pushed out into the ectosarc), three nuclei in 
two of which one sees masses of chromatin lying against the nuclear membrane, 
while all show the superficial network of chromatin with nodal thickenings. The 
achromatic structures in the nuclei are not drawn, and the fine-meshed cytoplasm 
is but conventionally shown as granular. Some of the rows of basal granules and 
cilia are double because the rows are dividing where the body broadens, so as to 
cover the broader body surface. Coros. subl. -acetic acid, iron haematoxylin (w^ell 
extracted). X ^000 diameters. 

Fig. 7. Part of an oblique section of 0. intentinaUs, showing pellicula, well 
stained ectosarc spherules (some granular), unstained endosarc spherules looking 
like vacuoles, and nucleus. The boundaries between the different alveoles of the 
ectosarc were not well shown, nor was the structure of the endosarc clear. Coros. 
subl.-acetic acid, dahlia. X -^""0 diameters. 

Fig. 8. Part of an oblique section of 0. intestinalis, showing pellicula (its 
ridges are drawn at the left of the figure) basal granules of cilia, sub-pellicular 
layer of ectosarc, and the alveolar layer of ectosarc, each alveole containing one 
ectosarc spherule. Coros. subl.-acetic acid, methyl violet, x 2000 diameters. 

Fig. 9. Three ectosarc spherules from a section of 0. intestinalis. Coros. 
subl.-acetic acid, iron haematoxylin (not much extracted). X 2000 diameters. 

Fig. 10. Ten endosarc spherules from the same animal as in Fig. 9. The 
last two spherules show the not infrequent dumb bell shape. The last spherule 
shows superficial granules. Coros. subl.-acetic acid, iron haematoxylin (not much 
extracted). X '-000 diameters. 

Fig. 11. An eudosarc spherule from a section of 0. obtrigona. This slender 
dumbbell-shaped spherule shows the nearest appi'oach to division 1 have found in 
the endosarc spherules of Opalina. Coros. subl.-acetic acid, iron haematoxylin 
(not well extracted). X -^'^O diameters. 

Fig. 12. Five endosarc spherules from a section of 0. ranarum, showing 
internal (alveolar?) structure. In each instance the left side of the figure represeuta 
the side of the spherule toward the outer surface of the body. Coros. subl.-acetic 
acid, iron haemato.xylin (long extracted). X 28f)0 diameters. 

Fig. 13. Five endosarc spherules from a section of 0. intestinalis. All are 
from the same animal, yet one sees that they are differently stained, indicating 
difference of condition. Coros. subl.-acetic acid, iron haematoxylin (not much 
extracted). X 2000 diameters. 

Fig. 14. Six ectosarc spherules from the same animal as iu Fig. 13. This 
animal was considerably shrunken and the spherules of the ecto.sarc and endosarc 
weie also. Coros. subl.-acetic acid, iron haematoxylin (not much extracted). X 2000 
diameters. 

Fiy. 15. A bit of unusually coarse-meshed endoplasmic foam from a section 
of 0. caudata. Coros. subl.-acetic acid, Delafield's haematoxylin. X l"^'^^ diameters. 



356 M. M. Metcalf 

Fig-. 16. A bit of ejidosarc from a section of O. intesfinalis, showing that 
each endosarc spherule lies in an alveolus. The structure of the endoplasma is not 
well shown. Coros. subl. -acetic acid, Ehrlich's triacid mixture. X 2000 diameters. 

Fig. 17. An optical section of the posterior end of a slender 0. dimidiata, 
showing- part of the excretory organ. One nucleus, in niito.sis, lies surrounded by 
the excretory vacuoles. Only the upper half of this nucleus is drawn. Ten (?) or 
twelve rows of chromatin granules (chromosomes) were present. The general cyto- 
plasmic foam is not shown. Coros. subl.-acetic acid, Delafield's haematoxylin. 
X 1^-0 diameters. 

Plate XV. 
Opalina intestinalis (Figs. 18 — 31) and 0. dimidiata (Fig. 31 a). 

Fig. 18. Part of a section stained with methylen blue. Pellicula pale blue, 
ectosarc films and spherules green, endoplasma blue (structure very obscure and 
not shown), endosarc spherules unstained appearing like vacuoles, nuclear membrane 
blue, nuclear contents not shown, except the nucleolus (pale greenish blue with a 
dark blue vacuolated cap). Coros. subl.-acetic acid, methylen blue. X 2(J00 diameters. 

Fig. 19. Another nucleolus from the same preparation as Fig. 18, but with 
two dark blue unvacuolated caps. X 2000 diameters. 

Fig. 20. An individual stained intra-vitam with toluidin blue. The ectosarc 
spherules are stained, the endosarc spherules imstained. The band of ectosarc 
spherules shown crossing the cell in front of the posterior nucleus is superficial 
and is half of a complete ring. Very darkly stained bodies of a problematic 
nature are shown. The nuclei were entirely unstained until after all motion of 
the cilia ceased. Then certain masses (of chromatin?) began to stain as indicated. 
This individual was in an early stage of transverse division. X 500 diameters. 

Figs. 21 and 22. Two sections showing the whole of one nucleus, stained 
with safranln and light green. The heavily stained red spherules are chromatin 
spherules. The chromosomes are paler red and granular. (The granules were not 
drawn with the camera. They should be one-half larger and one-third less 
numerous.) The achromatic granules and the large nucleolus are green. Neither 
the chromatin network nor the achromatic films were clearly enough seen to draw. 
The former was composed of extremely delicate threads. Coros. subl.-acetic acid. 
X 2000 diameters. 

Fig. 23. Part of a section stained with safranin and light green, showing 
cilia, outer contour of pellicula, basal granules of cilia, ectosarc with coarser and 
finer fibrillae or films (green, semi-diagrammatic) and green-stained spherules, 
endosarc with pink-stained spherules and coarser and finer fibrillae and films (These 
are stained green in this preparation. If left a moment longer in absolute alcohol 
they would be red. It was impossible to draw these with entire accuracy), nucleus 
with chromatin masses and threads stained red, and the achromatic structures 
green. The three faint red bodies in the nucleus are probably dissolving chromatin 
spherules. All the nuclear structures shown (accurately drawn) lie near the upper, 
uncut, surface of the nucleus. The nucleolus is not in this section. Coros. subl.- 
acetic acid. X 2000 diameters. 

Fig. 24. A longitudinal section of a nucleus in a late telophase of mitosis. 
Only a portion of the achromatic granules present are drawn. The nucleolus is 
not in this section. Coros. subl.-acetic acid, safranin and light green. X 2000 
diameters. 



Opalina. ;-J57 

Fig. 25. A section of another nucleus similarly preserved and stained. The 
chief lines of achromatic granules at the upper surface of the section are accurately 
shown; the other granules are drawn free hand. X -00(* diameters. 

Figs. 26 and 21. Sections of other nuclei in which the fibrillar structures 
were more clearly seen. In Fig. 27 the achromatic granules and films lying 
heneath the chromatin net in the center of the nucleus are not drawn. Coros. 
subl -acetic acid, safranin and light green. X 2C00 diameters. 

Figs. 28 — 31. Four sections showing the whole of one nucleus and, in 
Fig. 29, part of the cytoplasm. The chroniatin spherules are numerous; the 
nucleolus is vacuolated. Except the very delicate chromatin net, Avhich was 
difficult to see, all the chromatin structures present are drawn. In Fig. 29 the 
thickness of the pellicula (green) is uncertain, for the section was a little oblique. 
The interruption of the pellicula, as shown, was undoubted. Ectosarc films, granules, 
tilirils (?) and spherules, green; endosarc spherules pink; endosarc granules, films 
and fibrils (V). green. X -0'^^ diameters. 

Fig. 31 rt. On optical section of a macrogamete or macrogamete parent cell 
of 0. diwidinfa from a tadpole of Bufo adijaris, infected 19 '/> hours. The ectosarc 
spherules are yellow, the endosarc spherules red. The nuclei are not drawn. 
Acetic-carmine. X 1010 diameters. 

Plate XVI. 
Licing Opalina intestiiialis. 

Except in Fig. 34, the anterior end of each nucleus in toward the top of 
the plate. 

Fig. 32. The anterior end of a daughter cell whose nucleus is in a telophase 
of mito.sis. Only the nuclear structure is shown. This was the posterior nucleus 
of the parent cell, as is indicated by its pointed anterior end. Eight chromosomes 
are seen in the anterior end of the nucleus; in the posterior end but seven can 
be seen, the eighth lying in the lower half of the nucleus below the right hand 
chromosome of the upper group of four. All the structures in the nucleus were 
remarkably clear, as clear as in the best stained preparations. The achromatic 
granules of only the upper half of the nucleus are shown, except that in the 
auterior end of the nucleus two rows of granules at a lower level are also drawn. 
X 10 10 diameters. 

Fig. 3.'5. A nucleus drawn five to forty-five minutes after active swimming 
movements of the animal ceased, the cilia being then but slightly active. The 
granules of only the upper third of the nucleus are drawn. A few endosarc 
spherules and large granules of the cytoplasm are drawn for comparison. Eight 
chromosomes are seen in the anterior nucleus; three of those in the posterior 
nucleus are hidden. This nucleus was later almost completely isolated from the 
body, being held at one end by part of the broken body. It withstood uninjured 
the most violent currents produced by intermittent pressure on the cover-glass 
and after three days was entirely unchanged, except that the chromo.somes seemed 
more nearly spherical and the achromatic granules were lies refractive, some 
having disappeared. X l-'So diameters. 

Fig. 34. A pair of nuclei from an animal with active cilia, which was held 
immovable by pres.sure between a hair and the cover-glass. Eighteen hours after 
all movements of the cilia ceased the nuclei were in the same condition. Eight 
(or ninej distinct chromatin masses are in the anterior nucleus (one chromosome 

24* 



358 M. M. Metcalf 

has constricted or is constricting into two); in the other nucleus the chromosomes 
are already united into a ribbon. The difference in condition in the two nuclei 
is greater than usual, but is apparently not abnormal. The achromatic granules, 
in this case, were not carefully counted or drawn with entire accuracy. X ^010 
diameters. 

Fig. 35. A pair of nuclei connected by a bent and somewhat spiral thread. 
In the anterior nucleus seventeen chromatin (?) masses (one may he the nucleolus) 
are seen, also several spindle fibers which were remarkably clear. This is an 
early prophase of mitosis. X ^010 diameters. 

Fig. 36. The posterior nucleus of a binucleated individual, in a late anaphase 
of mitosis. Eight chromosomes are in each end. But few of the spindle fibres 
were clear enough to draw and even these showed only faintly. The achromatic 
granules of only the upper half of the nucleus are shown. These were unusually 
large and were irregular in shape, a fact difficult to show in a drawing at this 
scale. X 14S5 diameters. 

Fig. 37. A pair of nuclei connected by a very long and irregularly bent 
thread. Fourteen to sixteen chromatin masses are shown in the anterior nucleus, 
in the posterior nucleus the chromatin ribbon is still incompletely fragmented. 
Almost none of the achromatic granules in the anterior nucleus are drawn. In 
the posterior nucleus only the larger achromatic granules are shown. X 1-010 
diameters. 

Plate XVII. 
Opn Una in tcstinalis. 

Longitudinal division Figs. 38 — 43, transverse division Fig. 44. 

In drawings at this scale the nuclear phenomena cannot be accurately shown. 

Fig. 38. A very early stage of division, the anterior end of the body showing 
merely a slight indentation. The nuclei are in a late anaphase of mitosis. Coros. 
si;hl.-acetic acid, borax carmine. X 4t-0 diameters. 

Fig. 39. A little later stage of division. The nuclei are in a late anaphase 
of mitosis. Coros. subl. -acetic acid, borax carmine. X 460 diameters. 

Fig. 40. A still later stage of division. The nuclei are in a late anaphase of 
mitosis. Both anterior and posterior ends of the body are dividing, Coros. subl.- 
acetic acid, borax carmine. X 460 diameters. 

Fig. 41 A dividing, individual, slightly abnormal, having been kept four 
days in Lock's fluid before killing. The nuclei are in a late telophase of mitosis. 
The body should before this have completely divided. The genoal form of the 
body, however, well represents the usual normal manner of division. Coros. subl.- 
acetic acid, Delafield's haematoxylin. X 460 diameters. 

Fig. 42. An almost completely divided individual. The nuclei are in an 
early telophase. This individual had been kept three days in sodium chloride 
solution before killing. Probably under normal conditions division would have 
been completed before the nuclei reached this .stage. The general form is exactly 
similar to what is found in entirely normal animals in a late stage of divLsiou. 
Coros. subl.-acetic acid, Delafield's haematoxylin. X 460 diameters. 

Fig. 43. A daughter cell fresh from division, as is shown by the irregular 
contour of one side of the body where it was connected with its sister cell (com- 
pare Fig. 40) . The nucleus is in a rather late anaphase, as is usual in such 
young daughter cells. This cell has received the posterior nucleus from the parent, 



Upaliiia. 359 

as is shown by its position (compare Fi^s. 40 — 42). Coros, snM.-afotic acid, borax 
carmine. X '160 diameters. 

Fig. 44. An individual in transverse division. The nuclei are in an anaphase 
of mitosis. Coros. subl.-acetic acid. Dei.ai'iei.d's baematoxylin. X "160 diameters. 

Plate XVIII. 

Opalina intesfinalis. 

All figures are reduced one-fifth, to the magnification indicated. 

Fig. 45. An individual with two nuclei each in a pro])liase of mitosis. 
Coros. subl.-acetic acid, Dei.ai-iki.d's liaematoxylin. Drawn at 1010 diameters, 
reduced to 808 diameters. 

Figs. 46—48. Three optical sections, through the upper, middle and lower 
thirds respectirely, of the posterior nucleus shown in Fig. 45. In Fig. 46 most 
of the fibrils shown belong to tlie chromatin network. In Fig. 47 the varicose 
fibrils in the center of the figure are optical .sections of the films of the achromatic 
foam. In Fig. 48 only the chromatin masses and a few of the chromatin fibres 
are shown. X ^^00 diameters. 

Fig. 49. An individual with two nnclei in an early stage of mitosis. Espe- 
cially in the posterior nucleus, one sees that the chromosomes are arranging them- 
selves in two adequatorial rows preparatory to migration to the poles of the 
nucleus. The distinct and rather coarse fibers are fibers of the chromatin spindle. 
Most of the achromatic structures and the finer chromatin threads are omitted. 
Coros. subl.-acetic acid, Delafield's baematoxylin. X ^^^ diameters. 

Figs. 50 — 52. Three optical sections, through the upper, middle and lower 
thirds respectirely of the posterior nucleus shown in Fig. 49. The coarse varicose 
longitudinal chromatin fibres of the spindle are well seen in Fig. 50. Fig. 51 
shows the emarginate form of the ends of the chromatin spindle (compare Fig. 49). 
Fig. 52 shows that not all of the chromosomes have yet been formed from the 
chromatin ribbon (spireme). The achromatic structures lying in the deeper layer 
of the upper third of the nucleus are omitted from Fig 50. In Figs. 50 and 51 
one sees that the chief longitudinal fibres of the chromatin spindle are attached 
to each pole of the nuclear membrane. The nucleolus lies in the lower part of 
Fig. 52. It is darkly shaded, not because it was heavily stained, but to make it 
seem to lie near the top of the section. X 1600 diameters. 

Fig. 53. An individual with its nuclei each in an anaphase of mitosis. 
Eight chromosomes were present in each end of each nucleus. Most of the achro- 
matic structures and the finer chromatin fibres are omitted in each nucleus. Coros. 
subl.-acetic acid, Delafield's baematoxylin. X 808 diameters. 

Fig. 54. A daughter cell with its single nucleus in a late anaphase of 
mitosis. Some of the chromosomes are constricted transversely into two unequal 
portions (compare Fig. 53). All structures are omitted except the chromosomes 
and the thickest chromatin fibres. Coros. subl.-acetic acid, Delafield's baema- 
toxylin. X 808 diameters. 

Fig. 55. A nucleus, in an anaphase of mitosis, in which the fibres of the 
chromatin spindle were more delicate, more numerous and less distinctly longi- 
tudinal than usual. Except in the center of the nucleus, the achromatic structures 
are omitted. Here some of the chromatin fibres are omitted, leaving a "window" 
through which the vacuolated nucleolus and the films of the achromatic foam are 
seen. The nucleolus is seen to lie in an enlarged alveolus of the achromatic foam, 



360 M. M. Metcalf 

whose fibres radiate from the surface of tlie nucleolus. Where the}' touch the 
nucleolus triangular nodal thickenings of the films are seen. The chromosomes 
are granular. Coros. subl -acetic acid, Delafie^d's haematoxylin. X 1600 diameters. 

Fig. 56. A nucleolus -with central vacuole and minute peripheral vacuoles. 
Coros. subl. -acetic acid, Delafield's haematoxylin. X 1' 00 diameters. 

Fig. 57. A nucleus showing characteristic enlargement of the ends of the 
longitudinal fibres of the chromatin spindle, only a small part of the structures 
in the nucleus are drawn. Coros. subl.-acetic acid, Delafield's haematoxylin. 
X If-OO diameters. 

Fig 58. A nucleus iu an earlj' telophase of mitosis. In one end of the 
nucleus tw'o of the chromosomes have fused by sending out a broad band of 
chromatin which unites them. There are ei:;ht chromosomes in this end. At the 
other end two of the chromosomes are constricted each into two. None of these 
chromosomes are yet united as in the opposite end of the nucleus. At the centre 
of the nucleus are a few alveoles of the achromatic foam which stain more deeply 
than the rest and are probably filled with dissolved chromatin from the "chromatin 
spherules". With the exception of these alveoles, none of the achromatic structures 
in this nucleus are shown, and only the larger fibres of the chromatin net are 
drawn. Coros. subl.-acetic acid, Delafield's haematoxylin. X I'^OO diameters. 

Fig. 59. A very thin transverse section of a nucleus, showing the chromo- 
somes just beneath the nuclear membrane, also lines of achromatic granules, and 
four partially dissolved masses of chromatin ("'chromatin spherules"). The finer 
details of the achromatic structures were not clear enough to draw. Coros. subl.- 
acetic acid, borax carmine. X 1188 diameters. 

Plate XIX. 

Opalina intestinalis. 

All figures are reduced one-fifth, to the magnification indicated. 

Fig. 60. A daughter cell with the nucleus in a late telophase of n;itosis. 
Coros. subl.-acetic acid, Delafield's haematoxylin. X 808 diameters. 

Figs. 61 — 62. Two optical .sections, through the upper and lower halves 
respectively, of the nucleus shown in Fig. 60. The chromosomes are seen to be 
united together by broad thin bands of chromatin. The transverse line near the 
upper end of Fig. 61 is a fold in the nuclear membrane. The finer chromatin 
fibres are not drawn nor are the fibres of the achromatic foam. Only a few of 
the achromatic granules are shown. The nucleolus is not drawn. Coros. subl.- 
acetic acid. Delafield's haematoxylin. X 1600 diameters. 

Fig. 63. An older daughter cell whose nucleus is nearly separated into two 
daugther nuclei, one of which shows eight distinct chromosomes, while in the 
other the chromosomes are mostly united as in Fig. 61. The drawing shows the 
chromosomes, the polar fibres connecting one set of these with the pole, and 
several faintly stained bodies, nearer the constricted center of the nucleus, which 
seem to be dissolving "chromatin spherules". Coros. subl.-acetic acid, Delafield's 
haematoxylin. X 808 diameters 

Fig. 64. Outlines of the nucleus and anterior end of the body of another 
individual. X '10"! diameters. 

Fig. 65. The same nucleus as that shown in Fig. 64. It is in a late telo- 
phase of mito.sis, the daughter nuclei being nearly distinct. Eight chromosomes 
were present in each daughter nucleus, as was shown when the animal was 



Opaliua. 361 

sli«;htly I'olled. These are all distinct. A few dissolving- elironiatin spherules are 
shown near the constricted end of each daughter nucleus. The nucleolus is not 
drawn. Coros. snhl. -acetic acid, Delafield's haematoxylin. X 1^00 diameters. 

Fig. 66. Outlines of the nuclei and anterior end of the body of another 
individual. X -^^^^ diameters. 

Fig'. (i7. The posteiior of the two nuclei shown in Fig-. 66. Only the granular 
chromatin rihhon and a little of the achromatic foam is shown. Coros. subl.-acetic 
acid. Delakielu's haematoxylin. X 1*^00 diameters. 

Fig. 68. The anterior end of a binucleated individual, showing in each nucleus 
the chromatin ribbon beginning to break up into the chromosomes preparatory to 
the next mitosis. The achromatic granules are conventionally drawn. The nucleolus 
is not shown. Coros. subl.-acetic acid, Dei.afikld's haematoxylin. X ^08 diameters. 

Fig. 69. A thin optical section through the posterior nucleus shown in 
Fig. 68. All the granules in the chromosomes and the achromatic granules and 
film-lines are accurately drawn, except that the level of some of the achromatic 
granules is not correctly shown. X 1^00 diameters. 

Fig. 70. The anterior end of an individual in whose nuclei the chromatin 
ribbon has apparently already constricted to form separate chromatin masses, 
although, from the short thread connecting them, the nuclei seem to be young. 
The chromatin masses of only the upper half of the nuclei are drawn. The spheri- 
cal nucleolus is seen in the posterior nucleus. Coros. subl.-acetic acid, Delafield's 
haematoxylin. X 808 diameters. 

Fig. 71. The anterior end of an individual with two nuclei. The chromatin 
masses of only the upper half of the nuclei are drawn. The spherical nucleolus 
is seen in the posterior nucleus. Chromatin spherules are forming from the chro- 
matin masses. Coros. subl.-acetic acid. Delafield's haematoxylin. X ^08 diameters. 

Fig. 72. The anterior end of an individual with two nuclei in which the 
chromatin spherules are for the most part already separated from the chromatin 
masses. The old nucleolus is seen in the posterior nucleus. A new nucleolus is 
forming near the pointed end of the anterior nucleus. Coros. subl.-acetic acid, 
Delafield's haematoxylin. X 808 diameters. 

Fig. 73. The old nucleolus and a single chromatin mass from a nucleus in 
a condition slightly earlier than that shown in Fig. 72. The chromatin mass is 
granular, as the chromosomes always are. The chromatin spherules, much larger 
than the chromatin granules, are about to be cast off. Coros. subl.-acetic acid, 
Delafield's haematoxylin. X 1600 diameters. 

Fig. 74. An injured daughter cell (posterior end broken) whose nucleus shows 
the only exception I have found to the rule that in this .'species the old nucleolus 
remains in the posterior daughter nucleus. In this case it is in the anterior daughter 
nucleus. No attempt is made in this small scale drawing to represent all the 
ihromosomes. Coros. subl.-acetic acid, Delafield's haematoxylin. X '"^68 diameters. 

Plate XX. 

Opalina intesllnalis and O. caudata. 

All figures are reduced one-fifth, to the magnification indicated. 

Figs. 75 — 80. Opalina hitestinalis. 

Fig. 75. The posterior nucleus of a binucleated individual, entering upon 

mitosis. Numerous faintly stained partially dissolved chromatin spherules are 



362 ^- ^I- Metcalf 

shown. The achromatic structures (except the nucleolus), the tinerthreads of the 
chromatin net, and the details of form of the chromosomes are omitted from the 
figure. Cores, subl.-acetic acid, Delafield's haematoxylin. X 1600 diameters. 

Fig-. 76. An individual with its nuclei in what may perhaps be called the 
resting condition. Only the chromatin masses and the larger vacuoles of the 
achromatic foam are shown. Coros. subl.-acetic acid. Delafield's haematoxylin. 
X 808 diameters. 

Fig. 77. The posterior nucleus shown in Fig. 76. Only the achromatic foam 
and the chromatin masses are shown, the nucleolus and the chromatin net being- 
omitted. X IfiOO diameters. 

Fig. 78. An anterior nucleus drawn to show a weakly stained chromatin 
spherule in a tubular process from the anterior end of the nucleus. The chromatin 
masses also are drawn. Coros. subl.-acetic acid, Delafield's haematoxylin. X 1^00 
diameters. 

Fig. 79. A nucleus similar to that drawn in Fig. 78, showing several par- 
tially dissolved (weakly stained) chromatin spherules, one of which lies in a pro- 
jection from the anterior end of the nucleus. All achromatic structures and the 
chromatin net are omitted from the drawing. Coros. subl.-acetic acid, Delafield's 
haematoxylin. X 1600 diameters. 

Fig. 80. A dumbbell-shaped nucleus of a daughter cell. A few partially 
dissolved chromatin spherules are seen in each end of the nucleus. The chromatin 
net, the nucleolus, and all the other achromatic structures except a few granules 
are omitted from the drawing. Coros. subl.-acetic acid, Delafield's haematoxylin. 
X 1600 diameters. 

Figs. 81 — 92. Opalina caudafa. 

Fig. 81. An individual with each of its nuclei in a late anaphase of mitosis. 
Six chromosomes are seen in each end of each nucleus. The spherical bodies at 
the centers of the nuclei are the nucleoli. The achromatic foam and the finer 
fibres of the chromatin net are omitted from the figure. Coros. subl.-acetic acid, 
borax carmine. X 246 diameters. 

Fig. 82. A daughter cell showing the nucleolus in the anterior daughter 
nucleus, and six chromosomes in each nucleus; some of these are already united 
preparatory to forming the chromatin ribbon. Coros. subl.-acetic acid, borax car- 
mine. X 262 diameters. 

Fig. 83. A section of a nucleus showing granular chromatin masses, some 
fibres of the chromatin network with darkly stained nodal thickenings, and some 
films and granules (lightly stained) of the achromatic foam. Observe that the 
granules in the chromatin masses are of various sizes and shapes. Coros. subl.- 
acetic acid, iron haematoxylin. X H^^S diameters. 

Fig. 84. Part of a section of a nucleus, showing one granular chromatin 
mass, and a few other granules probably achromatic. Near the nucleus is shown 
a single granular endosarc spherule. Coros. subl.-acetic acid, iron haematoxylin. 
X 1188 diameters. 

Fig. 85. Part of a longitudinal section, showing one nucleus and a little of 
the adjacent cytoplasm. This nucleus was in an anaphase of mitosis. In order 
to show more clearly the chromatin granules upon the chromatin net these are 
drawn much darker than the chromosomes and the fibres of the chromatin net. 



Upaliiia. 363 

In reality all were stained e(|Ually dark. Coros. subl. -acetic acid, iron liaeinatoxylin. 
X 808 diameters. 

Fig. 86. A longitudinal section of a nucleus in a telophase of mitosis. Four 
of the chromosomes are seen to have at their edges rows of more or less elongated 
grannies. These granules are not arranged in pairs. Most of the hmgitudinal 
fibres of chromatin are seen to be very granular. The liner chromatin threads 
and most of the achromatic structures are omitted. In this nucleus it was im- 
possible to sharply distinguish between chromatin granules and achromatic granules. 
Cores, subl. -acetic acid, iron haematoxylin. X 1600 diameters. 

Fig. 87. An abnormal individual showing a great lateral swelling in which 
the endosarc was evenly granular, and the ectosarc ap])arently structureless. There 
were no endosarc spherules in the swollen area. They were abundant in the 
rest of the body, but are not drawn. The finer structures of the nuclei were not 
clear enough to draw. Ooros. subl.-acetic acid, Delafield's haematoxylin. X '^04 
diameters. 

Fig. 88. A very stocky individual. In the nuclei only the chromosomes 
and chief chromatin fibres of the upper surface are shown. Coros. subl.-acetic 
acid, Delafield's haematoxylin. X ^56 diameters. 

Fig. 89. A very stocky and abnormal individual, with four nuclei each in 
a telophase of mitosis. Two of these, seen in end view, do not show their mitotic 
condition. In the right hand nucleus the chromatin is abnormally compact. In 
the nucleus in the middle most of the granules drawn are probably achromatic. 
In the other two nuclei only the chromosomes and the chief fibres of the chromatic 
spindle are drawn. Coros. subl.-acetic acid, Delafield's haematoxylin. X ^56 
diameters. 

Fig. 90. An individual with almost completely degenerated nuclei. Coros.- 
subl.-acetic acid, Delafield's haematoxylin. X ■^OO diameters. 

Fig. 91. A probably abnormal individual with nuclei in a condition charac- 
teristic of the multi-nucleated Opalinae, but never, I think, found in normal in- 
dividuals of 0. caxidata. Coros. subl.-acetic acid. Delafield's haematoxylin. 
X 808 diameters. 

Fig. 92. An individual with abnormal nucleus. Coros. subl.-acetic acid, 
borax carmine. X "10^ diameters. 

Plate XXI. 

Abnormalities. Opalina intestinalis and O. ohtrigona. 

All figures are reduced one-fifth, to the magnification indicated. 

Figs. 93—98. Opalina intestinalis. 

Fig. 98. Outlines of the nuclei and the anterior end of a young individual. 

X -^04 diameters. 

Fig. 94. The same nuclei as those shown in Fig. 98. The aggregation of 
the chromatin into large irregular masses is abnormal, although the animal was 
killed immediately after removal from the host. Coros. subl.-acetic acid, Delafield's 
haematoxylin. X 1600 diameters. 

Fig. 95. The anterior end of an individual where posterior nucleus is ab- 
normal, as is shown by the aggregation of the chromatin into a few dense compact 
masses. This was first observed upon staining with acetic-carmine, after which 
the animal was stained with Delafield's haematoxylin and drawn. In the anterior 



364 M. M. Metcalf 

nucleus only the chromatin masses are drawn. Kept alive three days in 0,6% 
NaCl solution, acetic-carmine four hours, 0,6 °/o NaCl solution V2 ^^J, coros. subl.- 
acetic acid, Delafield's haematoxylin. X '^^'^ diameters 

Fii>-. 96. A small individual with a single abnormal nucleus. (Compare Plate XX 
Fig'. 92.) Flemming's stronger fluid. Mayer's haemalum. X 368 diameters. 

Fig. 97. An individual kept alive three days in 0,6% NaCl solution. The 
shape of the body and the position of the nuclei are unusiial and may be abnormal. 
In the nuclei, which were in a late anaphase of mitosis, the chromatin masses 
are not carefully drawn. At the posterior end of the body one sees a depression 
which marks the position of the excretory aperture, an ovoid mass of the excretory 
granules, and the enlarged posterior vesicle of the excretory organ. The method of 
preparation was the same as for the individual shown in Fig. 95. X -^68 diameters. 

Fig. 98. A dividing individual, still active after living S'/a days in 0,6 7o 
NaCl solution. Division began the third day and the form of the body reached 
the condition shown upon that day; the animal remained apparently unchanged 
two days more and was then killed, stained and drawn. Three nuclei are in one 
daughter cell. The anterior daughter of one nuclear pair and the posterior daughter 
of the other are about to fuse. (In other instances more advanced stages of this 
fusion were found.) The four daughter nuclei seem normal ; each has eight chromo- 
somes; each posterior daughter nucleus contains a nucleolus. Coros. subl.-acetic 
acid, Delafield's haematoxjiin. X 368 diameters. 

Figs. 99 — 118. Abnormal Opalina obtrigona. 

(Coros. subl.-acetic acid, Delafield's haematoxylin. All figures X ^600 dia- 
meters, except Fig. Ill which is a free hand sketch on a slightly smaller scale.) 

Figs. 99—101. Nuclei seeming almost if not quite normal. The network 
with thickened nodes is superficial and is probably chromatin, but the achromatic 
foam (not drawn), filling the whole nucleus, presents much the same appearance. 
Each nucleus shows from two to six discoid masses of chromatin upon the nuclear 
membrane. The bodies which are lightly shaded are chromatin discs on the far 
side of the nucleus. 

Figs. 102 and 103. Nuclei in which the chromatin plates upon the nuclear 
membrane are reticulate. In the center of each nucleus is a mass of granules 
probably chiefly achromatic. A little of the superficial, chromatin (?) net is shown, 
and a little of the achromatic foam is drawn in Fig. 103. It is hardly distinguish- 
able from the superficial net. except by its more central position and the smaller 
size of its meshes. 

Fig. 104. A nucleus in which the chromatin in chiefly in two reticulate 
masses, in one of which a central refractive body is seen. The reticulation of the 
lower mass is not shown. 

Figs. 105 — 109. Optical sections through nuclei showing similar conditions. 
In each is a central mass of granules probably chiefly achromatic and one or two 
bodies consisting of a central refractive sphere surrounded by a layer of chromatin 
which shows a denser net with more faintly staining interspaces. Bits of the 
achromatic foam are indicated in some of the figures. 

Fig. 110. A nucleus in which the achromatic granules are in two masses, 
connected by lines of similar granules. One sphere, with its chromatin either more 
compact or not well differentiated by the stain, is shown, as is also the achro- 
matic foam. 



Oimlina. 365 

Figs. Ill — 115. Optical sections (if other nuclei in similar conditions. Note 
tliat in Yig. Ill there are two clironiatiii-covered spheres upon the pseudo- 
spindle. 

Figs. IIG and 117. Nuclei which have almost completely degenerated, .showing- 
only one or two masses of debris within the still intact nuclear membrane. 

Fig. 118. An optical section through a part of the body showing two nuclei 
and the remains of eight or nine others. In some cases merely an empty space 
marks the former jiosition of a nucleus; in other cases one sees small spheres, the 
remains of the chromatin-coverod spheres: in other ca.ses there are left merely 
masses of debris representing the achromatic structures. 



Plate XXII. 

Opalina intcstinnlis. Figs. 137 — 139 0. caudata. 

All figures reduced one-third, to the magnification indicated. 

Fig. 119. A small individual with eight chromosomes, from a tadpole of 
Bomhinator pachypns. infected 5 days. This Opalina passed unencysted through 
the alimentary canal of the tadjiole. Acetic-carmine. X 673 diameters. 

Fig. 120. A small individual with four chromosomes, from the same tadpole. 
It passed unencysted through the alimentary canal of the tadpole. The reduced 
number of chromosomes is seen in animals of this size and smaller. Acetic-carmine. 
X 673 diameters. 

Figs. 121 — 128. Optical sections of minute individuals about ready for en- 
cystment. from the rectum of an adult Bomhinator pacJiypiis. Figs. 121 and 122 
show respectirely an early and a late stage of the last division before encystment. 
Observe that the nuclei are not in mitosis. In Figs. 122 and 123 the posterior 
end of the body shows a very delicate pellicula and numerous slight lobulations. 
In each nucleus the chromatin spheres are shown, and in most of them the group 
of achromatic granules is drawn. The cytoplasmic structure is either omitted or 
conventionally drawn. The ectosarc spherules are shown in Figs. 123 and 128. 
In Figs. 121. 126 and 127 probably the larger spherules belong to the ectosarc. 
the smaller to the endosarc. Acetic-carmine. X 673 diameters. 

Fig. 129. An optical section of an individual which had begun to encyst. 
Endosarc spherules (shaded, more abundant in the anterior part of the body) and 
a few ectosarc spherules (unshaded, in the posterior end of the body) are shown. 
The nuclear and cytoplasmic structure is not carefully drawn. Acetic-carmine. 
X 673 diameters. 

Figs. 130 — 132. Optical sections of cysts from the rectum of an adult Bom- 
binator pacltypus. In Fig. 130 the unusually small endosarc spherules are drawn. 
Fig. 131 shows one chromatin sphere in the nucleus. Fig. 132 shows three. Acetic- 
carmine. X 673 diameters. 

Fig. 133. The nucleus of another cyst from the same preparation, showing 
only the reticulate character of the superficial chromatin in the chromatin sphere. 
Acetic-carmine. X 1010 diameters. 

Fig. 134. An optical section of a newly formed cyst with very delicate 
wall, from the same preparation. There are three chromatin spheres in the nucleus. 
The ectosarc spherules are at the outermost edge of the ectosarc. Acetic-carmine. 
X 673 diameters. 



366 M. M. Metcalp 

Fig'. 135. An optical section of another cyst from the same preparation. 
Most, perhaps all, of the ectosarc sphernles have been extruded and lie between 
the body and the wall of the cyst. Acetic-carmine. X ^^^ diameters. 

Fig. 136. An optical section of another cyst from the same preparation, 
showing in the nucleus one large and one small chromatin sphere and a central 
mass of granules. Between the cell body and the cyst wall is a mass of debris 
probably derived from degenerated ectosarc spherules. Acetic -carmine. X ^'^•^ 
diameters. 

Figs. 137—139. Oimlina candata. Sections (2 fi) of cysts from the rectum 
of an adult Bombinator j)achy2)ns. All of the nuclear structures present are 
accurately drawn. In Figs. 137 and 138 all of the chromatin seems to be gathered 
into the chromatin spheres. In Fig. lo9, in addition to the chromatin sphere, ten 
small chromatin masses are seen. Others were present in the adjacent sections. 
Coros. subl.-acetic acid, Delafield's haematoxylin. X l'^34 diameters. 

Figs. 140 — 143. Animals hatching from the cysts in the rectum of a tadpole 
of Bombinator pachypus five hours or less after ingestion of the cysts. Ectosarc 
spherules are shown (shaded) in Fig. 140. In the same figure granular debris is 
seen in the cyst. The boundary between ectosarc and endosarc is indicated by 
dotted lines. Most of the cilia were distroyed by the acetic acid and the currents 
caused in making the preparation; all which were seen are drawn. The details 
of nuclear structure were not well shown. Acetic-carmine. X 6T3 diameters. 

Figs. 144 and 145. Macrogametes or macrogamete parent-cells ') from the 
rectum of a tadpole of Bombinator pncltypus^ infected 6 days. Few cilia were 
drawn in the original sketch for Fig. 144, the rest having been filled in later. 
The endosarc spherules are shown in Fig. 145. No nucleus was visible in this 
darkly stained animal. Possibly it had degenerated. Acetic-carmine. X ^^^ di^^^^^ters. 

Fig. 146. A living dividing individual from a tadpole of Bufo vulgaris, in- 
fected 60 hours. Endosarc spherules (shaded) and ectosarc spherules (unshaded) 
are shown. The nuclei were not clear. A second, more anterior, seemed to be 
present at a lower level, but I could not be sure of it. The long and sparce 
cilia make it probable that (after not less than two divisions) this individual 
would have given rise to microgametes. X 673 diameters. 

Fig. 147. A free-hand sketch of a very active, dividing macrogamete mother- 
cell (it may be a i)areut-cell) from a tadpole of Bufo vulgaris, infected 36 hours. 
The two daughter cells are of the same size. They were slightly flattened, one 
being seen more in edge view. Extruded excretory granules were seen dragging 
behind one daughter cell. 

Figs. 148 and 149. Optical sections of macrogametes or macrogamete parent- 
cells from a tadpole of Bombinator pachypus, infected 136 hours. But few cilia 
were in the original sketch for Fig. 148, the rest having been supplied later. 
Each nucleus shows four chromosomes. Acetic-carmine. X 673 diameters. 

Fig. 150. An optical section of a macrogamete from a tadpole of Bombinator 
pachypus, infected 43 hours. Acetic-carmine. X 6^3 diameters. 



^) I use the word parent-cell to indicate a cell which after one or more divisions 
will produce gametes. The word mother-cell is used for a cell whose next division 
will give rise to gametes. I realize that the phraseology is not satisfactory, but 
with the understanding that parent is entended to include grand parent or still 
earlier generations, it may do for the purposes of the present paper. 



Olialiiia. 367 

Fig", lol. An optical section of a niacrog'amete from a tadpole oi Bomhinator 
pachypus. infected 6 days. Acetic-carmine. X 673 diameters. 

Fig-. 152. A section (4 ii) of a macroganiete or niacroganiete parent-cell in 
the rectum of a tadpole of Bonib'uiator pacJii/jtns, infected 24 hour.s. Coros. subl.- 
jicetic acid, DKi.AviKr.n's liaematoxylin. X ^'^'^•i diameters. 

Fig 153. A living dividing gamete parent-cell from a tadpole of Bufo 
ndgarifi, infected 42 hours. Extruded excretory granules are seen at the posterior 
end of the body. The long and sparce cilia make it probable that (after not less 
than two divisions) this cell will give rise to microgametes. X 673 diameters. 

Plate XXIII. 

Opnl'ma inteatinalis. 
All figures are reduced one-third, to the magnification indicated. 

Fig. 154. A niicrogamete parent-cell from a tadpole of Bombinator pachypus, 
infected 70 hours. The cilia are taken from a sketch of the living animal; the 
body fnrm and nucleus were drawn after treatment with acetic-carmine. X 673 
diameters. 

Figs. 155 158. Microgamete parent -cells from a tadpole of Bombinator 
pachypus, infected 70 hours. In Fig. 155 parts of the excretory organ are seen. 
Acetic-carmine. X 673 diameters. 

Fig 159. A living microgamete parent-cell in division, from a tadpole of 
Bombinatiir pachypus, infected 70 hours. The cilia are too short. X ^010 diameters. 

Fig. 160. A living cell ready to metamorphose into a niicrogamete, from a 
tad])ole of Bontbhiator pacltypris, infected 42 hours. I suspect that the posterior 
two or three cilia were attached further forward than is shown, since the naked 
end of the tail is longer in the fully formed microgamete than in this animal as 
here drawn. X 673 diameters. 

Figs. 161 and 162. Microgametes (the first mature, the second probably 
not so) whose tails are contracted by acetic-carmine; from tadpoles of Bombinator 
pachypus, infected 91 hours (Fig. 161) and 70 hours (Fig. 162). In Fig. 161 the 
eudosarc spherules are drawn. X 673 diameters. 

Fig. 163. A living microgamete from a tadpole of Bufo vulgaris, infected 
42 hours. Accurately drawn, except that the nuclear structure, which was not 
clear, is omitted. The endosarc spherules are shown. X 673 diameters. 

Fig. 164. An early stage of copulation. From life. The animals were too 
active for drawing with the camera. The record of the infection from which these 
animals were obtained is lost. Some cilia have been added to the microgamete 
in the original sketch, which represented rather too thin an optical section. 
(Cf. Metcai.p 1907 «, Fig 3.) 

Figs. 165—167 Later stnges of copulation in different individuals from a 
tadpole of Bombiunfor pachypus, infected 88 hours. Free hand drawings fron) life. 

Fig. 168. A copulating pair from a tadpole of Bombinator pachypus infected 
88 hours. The nucleus of the male seemed to contain two chromatin masses of 
uneijnal size, but the staining was not sufficiently clear to determine accurately 
the structure. Probably the larger mass was composed of three chromosomes 
lying close together. Acetic-carmine. X 673 diameters. 

Fig. 169. An early stage of copulation, from a tadpole of BoiitbiHator pachypu%. 
infected 70 hours. Acetii -carmine. X 693 diameters. 



368 ^I- M. Metcalf 

Fig. 170. A copulating pair from a tadpole ol Bomhinator pachypus, infected 
88 hours. 

Fig. 171. A copulating pair from a tadpole of Bufo vulgaris, infected 45 hours. 
The microgamete was the smallest I have seen of this species. Its nucleus was 
not visible. Drawn from life. 

Fig. 172. A copulating pair from a tadpole of Bnfo vulgaris, infected 60 hours. 
Acetic-carmine. Magnification not recorded. 

Fig. 173. A copalatinff pair from a tadpole oi Bomhinator pachypus. infected 
7 days. The macrogamete is much larger than usual. Acetic acid. X 673 diameters. 

Figs. 174 — 177. Successive stages of copulation, drawn from life: Fig. 174 
at ri" P.M., Fig. 175 at 1" P.M., Fig. 176 at 1'^ P. M , Fig. 177 at l*^ P.M. 
From a tadpole of Bomhinator pachypus, infected 70 honrs. 

Fig. 178. A copulating pair from a tadpole oi Bomhinator pachypus infected 
70 hours. Acetic-carmine. X 673 diameters. 

Fias. 179 and 180. Two stages in the attempted copulation of a pair of 
gametes from a tadpole of Bomhinator pachypus, infected 70 hours. The micro- 
gamete was unusixally large. Later it separated from the macrogamete and formed 
a pseudocyst. Drawn from life. 

Fig. 181. A copulating pair from a tadpole oi Bomhinator pachypus, infected 
91 hours. The microgamete is attached by the whole anterior half of its body, 
the tail remaining free. This is the only instance 'n wliich such a condition was 
seen. The preparation was inadvertentlj' stained before I learned if the conjugation 
would become complete. From life. X ^'^^ diameters. 

Fig. 182. A copulating pair from a tadpole of Bomhinator pachypus, infected 
7 days. There are four chromosomes in the nucleus of the male and four in one 
end of the dividing nucleus of the female. In the other end of the nucleus of 
the female only two chromosomes were visible. Acetic-carmine, magnification not 
recorded. 

Plate XXIV. 

Opalina intestinnlis. 

All figures are reduced one-third to the magnification indicated. 

Figs. 183— 185a. Copulating pairs in sections of the rectum of a tadpole 
of Bomhinator pachypus, infected 60 hours. Figs. 185 and 185 a, are from .successive 
sections. The macrogamete shown in Fig. 183 was broken as indicated. Coros. 
subl.-acetic acid, Delafield's haematoxylin. X 9^0 diameters. 

Fig. 186. A copulating pair from a tadpole of Bombinator pai-hypus, infected 
88 hours. One of the two anterior nuclei may be from the male If so it passed 
by the posterior nucleus to reach its present position. Acetic-carmine. X 673 
diameters. 

Fig. 187. A zygote, with the nuclei still unfused, from a tadpole of Bom- 
hinator pachypus, infected 7'/2 days. Acetic acid. X 673 diameters. 

Fig. 188. A zygote, with the nuclei ready to fuse, fri^m a tadpole of Bom- 
hinator pachypus, infected 7 days. Acetic acid. X 673 diameters. 

Fig. 189. A similar zygote from a tadpole oi Bomhinator pachypus, infected 
91 hours. Acetic-carmine. X ^^0 diameters. 

Fig. 190. A section of a zysj-ote from a tadpole of Bomhinator pjachypus 
infected 7 days. The two nuclei are still unfused. Coros. subl.-acetic acid, Dela- 
field's haematoxylin. X 673 diameters. 



Opalina. 369 

Fig. 191. A section of a zygote from a tadpole of lioiiihinator pacJiypus, 
infected 64 hoars. The inembiaiie between the two nuclei is broken down in the 
middle. Coros. subl. -acetic acid, Delakield's haematoxylin. X ^90 diameters. 

Fig. 192. A zygote, with nuclei marly fnsed, from a tadpole of Bomhinnfor 
paclnjpus. infected 7/., days. The nuclear structure was not clearly seen. Acetic 
acid. X 673 diameters. 

Fig. 193. A zygote from a tadpole of Bombinatov pachypus, infected l^j., days. 
Acetic acid. X 673 diameters. 

Figs. 194—196. Zygotes from tadpoles of Bombinator pachypus, infected 
respectirely 474 days, 5 days, 4^4 days. Each nucleus (syncarion) shows eight 
chromosomes. In the nucleus shown in Fig. 196 were two structures, of different 
refractive quality from the chromosomes, which may have been nucleoli or vacuoles, 
probably the latter. Figs. 194 and 196 acetic acid; Fig. 195 acetic-carmine. 
X 910 diameters. 

Figs. 197—200. Zygotes formed by the fertilization of binucleated macro- 
gametes. In each case the nucleus from the male lies between the two macro- 
gamete nuclei. In Figs. 198—200 it is evident that the nucleus from the male 
will fuse, or is fusing (Fig. 2( 0), with the anterior of the macrogamete nuclei. 
The first is an acetic acid preparation, the others are acetic-carmine preparation, 
from tadpoles of Bomb'uvitor pachypm, infected respectirely 7 days, 136 houis, 
88 hours, 88 hours. Figs. 197, 199, 200 X ^73 diameters; magnification of Fig. 198 
not recorded. 

Figs. 201—203. Binucleated zygotes, the anterior nuclei all being syncaria 
as is shown by their size and the number of their chromosomes. From tadpoles 
(if Boinblnator pachypu!<, infected 7 days. Acetic acid. X •'"'•'^ diameters. 

Fig. 204. A zygote formed by the union of a male with a binucleated female 
whose nuclei were in a telophase of mitosis. The nucleus from the male is also 
in mitosis. The radiations at its two ends are merely films of the cytoplasmic 
foam (compare the next figure). From an acetic-carmine preparation; this animal, 
however, lay in a part of the slide where only the acetic acid, and not the carmine, 
had taken effect. The preparation was very clear. From a tadpole of Bontbinafor 
pachypux, infected 10(i hours. X 673 diameters. 

Fig. 205. An optical section through the same animal, showing on a larger 
scale the nucleus from the male and the cytoplasm surrounding it. The nucleus 
lies in a vacuole of the cytoplasm, which it completely fills, its membrane heing 
covered by a film of cytoplasm from which radiate other films. Where the radiating 
films join the film covering the nuclear membrane, granules aie seen similar to 
tho>e in the rest of the cytoplasm. The drawing — an ink copy of the original 
pencil drawing — is inaccurate, for these grannies upon the nuclear contour should 
be shown as lying wholly outside (though abutting upon) the membrane. X '•^'-'^ 
diameters. 

Fig. 206. A daughter cell from the division of a zygote similar to that 
shown in Fig. 204; from a tadpole of Bombinator jjar//?//;i(S, infected 7 days. 
Acetic acid. X 673 diameters. 

Fig. 207. An unclear acetic-carmine preparation from a tadpole of Bom- 
binator pachypus, infected 91 hours. X 673 diameters. 

Fig. 208. An acetic-a<id preparation from a tadpole of Bombinator pachypus, 
infected 7 days. A spindle-shaped microgamete nucleus lay near what seemed to 
be four daughter nuclei. X 673 diameters. 



370 M. M. Metcalf 

Fig. 209. A living animal from a tadpole of Bomhinator pachypuft, infected 
88 hours. The nuclei were not perfectly clear. In the original free-hand sketch 
they are annotated with a question mark. 

Plate XXV. 

Opalina infestinalis. 

All figures are reduced one-third, to the magnification indicated. 

Figs. 210 — 218. Successive stages of copulation and pseudoencystment of a 
pair of gametes from a tadpole of Bufo vulgaris. Fig. 210 is a free-hand sketch 
from memory made immediately after the male hecome attached; the other figures 
X 673 diameters. From life. 

Figs. 219 — 221. Pseudocysts from tadpoles of Bombinnior pachypus (Figs. 
219 and 221) and Biifo vulgaris (Fig. 220). In Fig. 219 four granular chromo- 
somes are seen in each end of the dividing nucleus. The granules apparently 
were not arranged in pairs, their number in two of the chromosomes being uneven. 
In Fig. 220 each macrogamete nucleus had four chromosomes the uppermost two 
•of which in each case showed distinct granules, so arranged however as not to be 
readily counted; the granules of the lower two chromosomes were less distinctly 
seen. The nucleus from the microgamete showed a single compact chromatin 
mass which contained a group of granules at one side. The excretory vacuole 
with faintly visible granules (in Brownian movement) is shown near the top of 
the figure. Fig. 219 from life, X 990 diameters; Fig. 220 from life, X 673 dia- 
meters; Fig. 221 acetic-carmine, X 673 diameters. 

Figs. 222 — 22b. Zygotes with peculiar spindle-shaped syncaria, from tadpoles 
of Bomhinator pacJiypus, infected respectively 7 % days, 88 hours, 7 days, 113 hours. 
In Fig. 224 a vacuole of the excretory organ is shown in dotted outline. In Fig. 
225 the large disc in the nucleus was probably not a nucleolus but a mass of 
chromatin. Figs. 222 and 224 acetic acid; Fig. 223 acetic-carmine; Fig. 225 from 
life. X 673 diameters. 

Figs. 226 and 227. Zygotes whose daughter nuclei somewhat resemble the 
nuclei shown in Figs. 222 and 224; from a tadpole of Bomhinator pachypus, in- 
fected 7 '/a days. Acetic acid. X 673 diameters. 

Figs. 228 — 235. Abnormal individuals from tadpoles of Bomhinator pachypus, 
infected 43 hours (Fig. 228), 54 hours (Figs. 229—233), 91 hours (Fig. 234), and 
114 hours (Fig. 235). These tadpoles Avere infected from two different lots of 
cysts, Figs. 229 — 233 being from one series of infections, Figs. 228, 234 and 235 
from another. From life. Figs. 234 and 235 free-hand sketches; the rest camera 
draAvings X 673 diameters. 

Fig. 236. A section of an infection cyst from a tadpole oi Bomhinator pacJiypus. 
€oros. subl.-acetic acid, Delafield's haematoxyliu. Xo record was made of the 
infection or of the magnification of the drawing. 

Plate XXVI. 

Opalina intestinalis and 0. caudata. 

All figures are reduced one-third, to the magnification indicated. 

Figs. 237 — 247. Opalina intestinalis. 

Figs. 237 — 239. Three sections of a small individual which passed unencysted 

through the alimentary canal into the rectum of a tadpole oi Bomhinator jjacliy pus. 



Opaliiia. 371 

Larije reticulate masses of chromatin and some smaller frajL^ments are seen extruded 
from the nucleus and lying in the cytoplasm. Apparently the full number of 
chromosomes (8) were in these nuclei. Vacuoles around some of these masses are 
indicated by dotted contours. From a (i day infection. Coros. subl.-acetic acid, 
Delafikld's baeniatoxylin. Restaining- with iron haematoxylin gave the same 
results. X 673 diameters. 

Fig. 240. The anterior end of another individual from the same preparations, 
showing a reticulate mass of chromatin slightly disi)laced, probably by the micro- 
tome knife, from its position in the nucleus. X ^'^H diameters. 

Fig. 241. A combination of three drawings from sections of an individual 
which had passed unencysted through the alimentary canal and had been six 
days iu the rectum of a tadpole of Bombinator pachypus. Vacuoles, probably of 
the excretory system, are shown in dotted outline. Six masses of chromatin are 
seen lying in the cytoplasm. One of these is in the form of a reticulate layer 
partly surrounding a central refractive sphere {cf. the figures on Plate XXI). The 
nuclei contained the reduced number of chiomosomos (4). Coros. subl.-acetic acid, 
Delafield's haematoxylin. X 673 diameters. 

Figs. 242 — 244. Successive sections from a small individual from a tadpole 
of Bombinator pachypns, infected 6 days. Extruded masses of chromatin are seen 
iu the cytoplasm. The animal may or may not have come from a cyst so for as 
its size would indicate. It seems probable, however, that animals with this type 
of chromidia passed unencysted through the alimentary cansil. Four chromosomes 
are in each nucleus. Coros. subl.-acetic acid, Delafield's haematoxylin. X 673 
diameters. 

Figs. 245 — 247. The three central sections from a series of five through an 
individual from a tadpole of Bombinator pachy pun, infected 6 days. Extruded masses 
of chromatin are seen in the cytoplasm near each of the two nuclei. The other 
two sections showed no such chromidia. Coros. subl.-acetic acid, Delafield's 
haematoxylin. X 673 diameters. 

Figs. 248 — 262. Opalina caudata. 

Figs. 248—250. Individuals which passed unencysted through the alimentary 
canal into the rectum of a tadpole of Bombinator pachypxis; 118 hours infection. 
Excretory vacuoles are seen in each. In Fig. 248 the boundary between ectosarc 
and eiidosarc is indicated by a dotted line. In this figure there are shown twelve 
chromatin masses (chromosomes) in each nucleus which must be ready to enter 
upon mitosis. The other animals show the reduced number of chromosomes (3). 
Acetic-carmine. X 673 diameters 

Fig. 251. A section through a small individual from the rectum of an adult 
Bombinator pachyjnis which contained many infection cysts. In each nucleus are 
two chromatin spheres whose presence at this early stage is unusual. Coros. subl.- 
acetic acid, Delafield's haematoxylin. X 673 diameters. 

Figs. 252 — 256. Infection cysts from aquaria in which Bombinator pachypus 
was kept (Fig. 254 shows a cyst from the rectum of one of these adult Bombinator). 
Fig. 252 shows one chromatin sphere in the nucleus; Fig. 253 shows two chromatin 
spheres extruded into the cytoplasm and none in the nucleus; Fig. 254 shows a 
binucleated cyst with one chromatin sjdiere in one nucleus and none in the other; 
Fig. 255 shows a binucleated animal which encysted while in division; in Fig. 256 
two animals are shown inside one cyst (one cannot be certain that they are entirely 



372 M. M. Metcalf 

separate; they may be connected by snch delicate strands as are found uniting' 
the daughter cells in late stages of division). Figs. 252 — 254 acetic-carmine; 
Figs. 255 and 256 from life. X 440 diameters. 

Figs. 257 and 258. Macrogametes (Fig. 257 possibly a macrogamete parent- 
cell) from a tadpole of Bombinator pachypus (?), infected 22 hoars. The first 
figure shows the u.sual size, the second the smallest size found. The ectosarc 
spherules are indicated. Acetic-carmine. X 6T3 diameters. 

Fig. 259. A microgamete from the same tadpole of Bombinator pachypus (?), 
infected 22 hours. Ectosarc spherules (unshaded) and endosarc spherules (shaded) 
are shown. In no other series of infections have I found apparently mature gametes 
before 42 hours after the beginning of infection. Acetic-carmine. X '-'90 diameters. 

Fig. 260. A macrogamete (or macrogamete parent-cell?) from the same tad- 
pole of Bombinator pachypus (?), infected 22 hours. The ectosarc spherules are 
shown. Acetic-carmine. X ^^3 diameters. 

Fig. 2rtl. A living but almost quiescent microgamete. X 673 diameters. 
Infection data were not noted. 

Fig. 262. A free-hand drawing of a living copulating pair from a tadpole 
of Buf'o vulgaris, infected 66 hours. 

Plate XXVII 

Opalina caudata and Opalina dimidiata. 

All figures are reduced one-third, to the magnification indicated. 

Figs. 263 — 276. Opalina caudata. 

Fig. 263. A copulating pair from a tadpole of Bufo vulgaris infected 66 hours. 
The macrogamete is in division. Free-hand drawing. 

Figs. 264—271. Examples of copulation in living animals from tadpoles of 
Bombinator jmcJiyjms, infected 60 hours. Fig. 265 shows a late stage of copulation; 
Figs. 269 and 270 show copulation while the macrogamete is in division; Fig. 271 
shows two males attached to one female. Fig. 264 X 438 diameters; the other 
figures are free-hand drawings. 

Figs. 272 — 274. Copulating pairs from a tadpole of Bombinator pachypus 
infected 60 hours. The endosarc spherules are drawn in Figs. 272 and 273. Each 
nucleus in Figs. 273 and 274 shows three chromosomes. The nature of the body 
just outside the microgamete nucleus in Fig 273 is uncertain. Acetic-carmine. 
X 673 diameters. 

Fig. 275. A pseudocy.st (macrogamete?) from a tadpole of Rana csculmta, 
infected with both 0. caudata and 0. intestinalis by being placed for an hour in 
a jar with adult Bombinator pachypus. Cast off cilia and extruded globules lie 
around the pseudocyst. Each nucleus has three chromosomes. X 673 diameters. 

Fig. 276. Abnormal copulation of two individuals of nearly similar size, the 
smaller of which showed a nucleus in division. Acetic-carmine. Infection data 
and the magnification were not noted. 

Figs. 277 — 298. Opalina dimidiata. 

Figs. 277 — 281. Minute individuals from a culture in 0,6% NaCl .solution, 

kept three days after removal of the animals from the rectum of an adult Bana 

esculenta. Division continued during this time and many of the animals became 

minute, as shown, and were apparently ready for encystment. Fig. 277 shows 



Opaliua. 373 

some of the emlosarc sjilieniles present; in the cytoplasm only the larg'er granules 
are carefully drawn ; the living animal was treated directly with Ehrlich's acid 
haematoxylin. Figs. 278—280 are acetic-carmine preparations; Fig. 279 shows 
apparently a chromatin sphere being extruted from the anterior nucleus. Fig. 281, 
Ehrlich's acid haematoxylin. All figures X ^^-^ diameters. 

Figs. 282 — 284. Spheroidal individuals, not true cysts, from a tadpole of 
Ixana esculcnta, infected 24 hours. From these apparently pseudocysts some of 
the endosarc spherules are being extruded. My notes on these drawings, as also 
my memory of them, are insufficient to explain the conditions shown. Acetic- 
carmine. X 678 diameters. 

Figs. 285—288. Infection cysts from tadpoles of Bafo vulgaris, naturally in- 
fected. The size of the cysts, their multinucleated condition, and the presence of 
free-swimming minute Opalmae dumdiatae in the same recta, show these to be 
cysts of 0. dimidinfa. Fig. 285, from a living cyst; the other figures from 
acetic-carmine preparations. X ^"^ diameters. 

Figs. 289 291. Peculiarly shrunken individuals which passed unencysted 
through the alimentary canal and were lying in the rectum of a tadpole of Bana 
esculenta, infected 24 hours. Endosarc spherules (shaded) and ectosarc spherules 
(unshaded) are shown. Acetic-carmine. X ^^^ diameters. 

Fig. 292. An individual from a large tadpole of Bana esculenta, infected 
24 hours. Acetic-carmine. X 673 diameters. 

Fig. 293. A living gamete parent-cell from a tadpole of Bufo vulgaris, in- 
fected 6 hours. The endosarc spherules are shown. X 673 diameters. 

Fig. 294. A living gamete parent-cell in division; from a tadpole of Bufo 
vulgans, infected 36 hours. The endosarc spherules are shown. The granules 
shown as black were highly refractive. The condition of the nuclei is of interest. 
X 673 diameters. 

Figs. 295—298. Macrogametes from a tadpole of Bava esculenta. X 673 
diameters. 

Plate XXVIII. 

Opalina dimidiata and Opalina rananim. 

All figures are reduced one-third, to the magnification indicated. 

Figs. 299 — 324. Opalina dimidiata. 

Figs. 299 — 302. Gamete parent-cells from tadpoles of Bana esculenta, infected 
respectively 32 hours, time?, 9772 hours, 32 hours. Fig. 302 shows the eudosarc 
spherules and also the extrusion of chromatin spheres from each nucleus. Acetic- 
carmine. X 673 diameters. 

Fig. 3i)3. An individual from a tadpole (of a species not noted) infected 
8 days. The structures in the elongated nuclei were not clearly seen. It is 
doubtful whether the animal was a gamete parent-cell with both nuclei in mitosis, 
or a zygote with its syncarion already divided into two. Acetic-carmine. X 673 
diameters. 

Fig. 304. An individual from a tadpole of Bana esculenta, infected 80'/2 hours. 
Its nucleus was in mitosis. The nature of the bodies at the two ends of the 
spindle was not clear; they were probably chromatin spheres similar to those 
shown extruded from the larger nucleus in Fig. 309. The animal was probably a 
microgamete mother cell. Acetic-carmine. X 673 diameters. 

25* 



374 M. M. Metcalf 

Figs. 305 and 306. Macrog-ametes from a tadpole of Rana esculenfa, infected 
97 '/z hours. In the second figure endosarc spherules are shown, and also a chro- 
matin sphere extruded from the nucleus into the cytoplasm. Acetic -carmine. 
X 673 diameters. 

Figs. 307 309. Divisions in the formation of the microgametes; from a tad- 
pole of Rana esculenta, infected 80'/.^ hours. The number of chromosomes seemed 
in some nuclei five, in others six. Probably six is the correct reduced number of 
chromosomes for 0. dimidiata. Some of the nuclei were not sufficiently clear to 
draw and are left empty. In Fig. 309 the division may be abnormal, but in this 
species the nuclei are so independent of one another that the lack of synchronism 
in their division in this case may not indicate abnormal condition. The endosarc 
spherules are shown in all figures. From life. X 673 diameters. 

Fig. 310. A living raicrogamete, with unusually long tail, from a tadpole 
of Rana esculenia, infected 8 days. There was no swelling at the tip of the tail 
such as is usually seen in microgametes in this and other species. X 673 diameters. 

Fig. 311. An individual which seems to be a microgamete mother -cell, 
from a tadpole of Bufo vulgaris, naturally infected for at least three weeks. The 
tail is some what contracted by acetic carmine. Copulation was found among the 
Opalinas in this tadpole, this being by far the oldest infection in which copulation 
or microgametes were seen. Acetic-carmine. X 673 diameters. 

Fig. 312. A living copulating pair from a tadpole of Rana esculenta. infected 
98 hours. X 673 diameters. 

Fig. 313. A pair of living individuals from a tadpole of Rana esculenta, 
infected 80 V2 hours, showing a dividing microgamete mother-cell attached to 
another cell of about the same size. The manner of the attachment and the 
condition of the nuclei indicate that this was either a chance connection or an 
abnormal attempted coj)ulation. There was no change after three-quarters of an 
hour. Free-hand drawing. 

Figs. 314 — 318. Zygotes from tadpoles of unnoted species (probably Rarui 
esculenta. otherevise the fact would have been noted), infected 7 days, except the 
host for the animal shown in Fig. 117, which was infected 97 '/z hours. The nuclei 
were not sufficiently well stained to allow accurate drawing of the chromosomes 
or chromatin masses, which were so numerous as to obscure one another. The 
posterior nuclei shown in Figs. 315 and 316 are the syncaria. Possibly all the 
nuclei shown in Fig. 318 have come from the syncarion, the po.-<terior daughter 
having completed a second division with which the anterior nucleus is still engaged ; 
or the macrogamete may have contained originally three nuclei. The smaller size 
of the posterior nuclei makes the first interpretation much the more probable. 
The details of nuclear structure are not adequately shown. The chromatin masses 
were so numerous as to obscure one another. Acetic-carmine. X 673 diameters. 

Fig. 319. An individual from the same preparation as Fig. 318. The anterior 
nucleus from its shape would seem not to be a syncarion. It may be abnormal. 
The chromatin is not accurately drawn. Acetic-carmine. X 673 diameters. 

Fig. 320. Another individual from the same preparation, in which the anterior 
nucleus is the syncarion with the spindle form which persists until after at least 
one division. The chromatin could not be accurately drawn. Acetic-carmine. 
X 673 diameters. 

Figs. 321 — 324. Young individuals from tadpoles of Bufo vulgaris, naturally 
infected for an unknown period. Still larger individuals, with twice as many 



Opalina. 375 

imclei irregularly arranged instead of in an axial row, were present in the same 
tadpoles. Acetic-carmine. X 6"^^ diameters. 

Figs. 325 — 327. Opalina ranarum. 

Fig. 325. A minute individual ready for encystment, from the rectum of an 
adult Rana teniporaria. The endosarc spherules (unshaded) are shown; cilia are 
not drawn because injured. Tlie method of preparation was not noted, but the 
injury to the cilia makes it probable that acetic-carmine was used. X 673 diameters. 

Fig. 326. A cyst from the same host. Cilia were present within the cyst 
but were too confused to draw. The method of preparation was not noted. X 673 
diameters. 

Fig. 327. A section of a zygote in the rectum of a tadpole of Rana tenqio- 
raria. The nucleus is carefully drawn, as are also the endosarc spherules. Coros. 
subl.-acetic acid, iron haematoxylin. X 990 diameters. 



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Dr. Whitm an-Chicago, U.S.A., hag. vou Prof. Dr. F. Keibel, Freiburg i. Br. 

1. Normentafel zur Entwicklimgsgeschichte des Schweines (Siis scrofa 

domesticus). Herausgegeben vou Prot. Dr. F. Keibel. 1897. Preis : 'JO Mark. 

II. Norinentafel zur Eutwieliluiigsgeschiclite des Huhues (Oallus domes- 
ticus). Herausgegeben von Prof. Dr. F. Keibel und cand. med. Karl Abraham. 

Mit -6 lithographischen Tafeln. 1900. Preis: '20 Mark. 

III. Normentafel zur Entwickluugsgeschichte des Ceratodus forsteri. 

tierausgegelien von Prof. Dr. Richard Semoii. Mit 3 Tafeln und 17 Figuren 
im Text. 1901. Preis: 9 Mark. 

IV. Normeutafel zur Entwicklungsgeschichte der Zauneidechse (Lacerta 

agilis). Herausgesjeben vou Karl Peter in Breslau (jetzt in Wiirzburg). Mit 
4 Tafeln und 14 Figuren ira Text. 1904. Preis: 25 Mark. 

V. Normal Plates of the development of the Rabbit (Lepus cuniculus L.). 

By Charles S. Miiiot and Ewiug Taylor, Harvard Medical School Boston, Mass. 
With 3 plates and 21 figures in the Text. 1905. Preis : 20 Mark. 

VI. Normentafel zur Eutwicklungsgeschichte des Rehes (Cervus capre- 

olus). Von Dr. Tsunejiro Sakurai, Fuknoka (Japan), z. Z. Freiburg i. Br. 
Mit eineni Vorwort von Prof. Dr. F. Keibel. Mit 3 lithographischen Tafeln und 
1 Figur im Text. 1906. Preis: 20 Mark. 

VII. Norraentafeln zurEutwicklungsgeschichte desKoboldmaki (Tarsins 

spectrum) und des Plumplori (Jiycticebus tardigradus). Von A. VV. Hu- 
brecht, Utrecht und Franz Keibel, Freiburg i. B. Mit einem Vorwort von 
Franz Keibel. Mit 4 Tafeln und 88 Textfiguren. 1907. Preis: 20 Mark. 

VIII. Xormentafeln zur Eutwicklungsgeschichte des Menschen. Vou 

Franz Keibel, Freiburg i. Br. und Curt Elze, Halle a. S. Mit Beitragen von 
Prof. Broman-Lund: Prof. Hammar-Upsala und Prof. Tandler-Wien. Mit 

6 Tafeln und 44 Figuren im Text. 1908. Preis: 36 Mark. 

Ueber die Bastarde you Helix Horteiisis Miiller und 

Helix XeillOralis L. ■'^^"^ Untersuchung zur experimentellen Ver- 

'- 1 erbungslehre von Arnold Lang, o. Professor 

der Zoologie und vergleichenden Anatomic an der Universitat und am eid- 
geuossischen Polytechnikum in Ziirich. Mit 4 lithographischen Tafeln. 1908. 
Preis: 1.5 Mark. 

Vorlesuiii»eii iiber Deszeiideiiztlieorieii mit besonderer Beruck. 

2 . sichtigung der botaui- 

schen Seite der Frage gehalteu an der Keichsuniversitiit zu Leiden. Von Dr. 
J. P. Lotsy. Erster Teil. Mit 2 Tafeln u. 124 Textfiguren. Preis 8 Mark, 
geb. 9 Mark. — Z welter Teil. Mit 13 Tafeln und 101 Textfiguren. 1908. 
Preis: 12 Mark, geb. 13 Mark. 



Veriag vou GriistcLv Fischer* in Jena. . 

Die Fauna Siidwest-AUStralieUS. Ergebnisse der Hamburger sUdwest- 

australischen Forschungsreise 190o. 

Heransgegeben vou Prof. ^\. Michaelsen uud Dr. R. Hartmeyer. 

Band I, Lieferuug 1. Reisebericht vou Prof. W. Micbaelseu iu 
Hamburg und Dr. R. Hartmeyer in Berlin. Preis: 4 Mk. 

Lief. 2. Oligochaeta vou Prof. W. Micbaelseu, Hamburg. Mit 2 litbo- 
grapb. Tafeln. 1 Karteuskizze uud 34 Abbildungen im Text. 1907. Preis: 

5 Mark. 

Lief. 3—5. Copeognatha vou Dr. Giiutber Enderlein, Stettin. Mit 

6 Abbildungen im Text. Ophiuroidea par Prof. R. Koebler, Lyon. Avec 
10 figures dans le texte. Panorpata imd Flanipeunia von Dr. H. W. van 

der AVeele, Leideu. Mit 1 Abbilduug im Text. Preis: 1 Mark 50 Pf. 

Lief. 6 — 7. Lief. 6. Apidae von J. D. Alftken, Bremen. Lief. 7. Formi- 
cidae von Prof. A. For el, Chigny. Preis: 2 Mark 50 Pf. 

Lief. 8-13. 1908. Preis : 6,50 Mk. Lief. 8. Dysticidae, Hydropbilidae et 
Gyriuidae, von M. Regimbart, Evreux Lief.9. Braconidae und Ichuenmo- 

uidae von Gy. Szepligeti, Budapest. Mit Figur 4 uud 2 auf Tafel III u. 2 Ab- 
bildungen im Text. Lief. lU. Tencbrionidae vou Hans Gebien. Hamburg. 
Mit Figur 3—8 auf Tafel III und 4 Abbilduugen im Text. Lief. 11. Alleculidae 
von H. Borcbmann, Hamburg. Mit Figur 9—14 auf Tafel III und 4 Ab- 
bilduugen im Text. Lief. 12. Arancae, Ire partie, vou Eugene Simon. 
Paris. Mit 1 Karteuskizze und 14 Abbildungen im Text. Lief. 13. Fossores 
von W. A. Schulz, Genf. Mit 3 Abbildg. im Text. 

Band II, Lieferuug 1 — 4. Chrysoraelidae und Coccinellidae, vou 
J. Weise, Berlin. Staphylinidae, von Dr. Max Bernbauer, Griinburg. 
O.-Oe. TrichopteiM und Ephemeridae, von Georg Ulmer, Hamburg. Mit 
44 Abbildiiugen im Text. Tbysanura, per F. Silvestri. Portici. Con. Tab. 
I— X. 1907/08. Preis: 12 Mark. 

Lief. 5—8. Dermaptera by Malcolm Burr, Eastry, Kent. Rotatoria, 
Tardigrada und andere Moosbewohner von Prof. F. Richters, Frank- 
furt a. M. Scorpiones von Prof. Dr. K. K r a e p e 1 i n , Hamburg. Scolopou- 
dridae von Prof. Dr. K. Kraepeliu, Hamburg. Mit 2 Tafeln und 3 Abbil- 
dungen im Text. 1908. Preis: 5 Mark. 

Untersiicliiiiigeii ziir yergieicliendeii Muskellehre der 

Wii'hpltiPVP I^ie Musculi Serrati Postici der Saugetiere uud ihre 
TTllPJClLACic. phyiogenese. Von Dr. F. Maurer, o. Professor der 
Anatomie und Direktor der Anatomiscben Anstalt in Jena. Mit 4 Tafeln und 
28 Figuren im Text. 1905. Preis : 20 Mark. 

Soeben erscbien: 

AlloPinAiiiP PliA'Oolno'iP Eiu GruudriO der Lehre Tom Leben. 
All^emeilie ril,) MUlU^ie. ^.^^ ^^^ Verworn, Dr. med. et pbil., Prof. 

der Pbvsiologie und Direktor des pbysiologiscben Instituts der UniversitJit 
Gotringeu. Mit 319 Abbildungen. Funf te, vollstandig neu bearbeitete Auflage. 
Preis: 16 Mark, geb. 18 Mark. 

Ergebuisse mid Fortscliritte der Zoologie. I^'^^^ei/pr^f* 

der Zoologie in GieCeu. Erster Band. Erstes Heft. Inhalt: Valentin 
Haecker: Die Chromosouien als angeuommeue Yererbungstrager. Mit 
43 Abbildungen. — Richard Heymons: Die verschiedenen Formen der 
Insectenmetamorpbose uud ihre Bedeutung im Vergleich zur aietamor- 
phose auderer Artliropodeu. Mit 7 Abbildungen. — Otto Maas: Die 
Scyphomedusen. — Erster Band. Zweites Heft. — Inhalt: M. F. 
N i'e r s t r a s z , Die Amphiueuren. Mit 22 Abbildungen. — U 1 r i c b G e r h a r d t . 
Der gegeuwartige Stand der Keuntnisse von den Copulatiousorganen der 
Wirbeltiere, insbesondere der Amnioten. Mit 16 Abbildungen. 

.Jabrlicb erscheint eiu Band in zwanglosen Heften im Gesamtumfang von etwa 
40 Druckbogen. Preis des Baudes: 20 Mark. 



Lippert & Co. (G. Patz'sche Buchdr.), Kaumburg a. S.