The Neuromotor System of Euplotes patella
during Binary Fission and Conjugation.
By
Datus M. Hammond,
Department of Zoology, University of California, Berkeley.
With Plates 21-22 and 11 Text-figures.
CONTENTS.
INTRODUCTION
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PAGE
507
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METHODS
508
Cultures, p . 508. Observation of Living Organisms, p . 509. Demonstration of the Peripheral Polygonal System, p . 510. Demonstration of the Neuromotor System, p . 511.
STRUCTURE OF THE NEUROMOTOR SYSTEM
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512
The Neuromotorium, p. 515. Cirri and Membranelles, p . 515. Cytopharynx, p . 520. Peripheral Polygonal System, p. 521. Basal
Apparatus of t h e Bristles, p . 522.
ASEXUAL REPRODUCTION
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526
Peristome, p . 528. Development of Cirri, p . 529. Resorption of
Cirri, p . 532. Differentiation of Fibrils, p . 532. Multiplication
of Bristles, p . 533. Peripheral Polygonal System, p . 537.
SEXUAL REPRODUCTION
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541
Reorganization of t h e Gamete, p . 542. Reorganization of t h e
Zygote, p . 545.
DISCUSSION
546
SUMMARY .
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552
REFERENCES
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553
INTRODUCTION.
THERE is a life-cycle in the Protozoa, which is comparable to
that of the Metazoa (see Kofoid, 1923). This life-cycle begins
with the zygote, which undergoes successively periods of
cleavage, differentiation, asexual reproduction, senescence, and
finally death. Although the nuclear aspects of the life-cycle of
several ciliates have been worked out, there is little detailed
knowledge concerning plasmic structures during the different
NO. 316
L 1
508
DATUS M. HAMMOND
stages of the cycle. Stein (1859) discovered that there is a more
or less complete loss of cytoplasmic structure during asexual
reproduction in the complex ciliates in the order Hypotricha,
and Wallengren (1901) worked this out more completely.
Engelmann (1862) showed that a similar change occurs during
sexual reproduction in this group, and Maupas (1889) confirmed
and extended his observations. However, our knowledge concerning the extent and manner of these cytoplasmic changes in
different stages of the life-cycle, and their relationship to the
nuclear phenomena, is still incomplete.
The hypotrichous ciliate E u p l o t e s p a t e l l a Ehrenberg
provides admirable material for the study of this problem. It
has a highly differentiated body, with a well-developed neuromotor system, consisting of complex locomotor organelles, coordinated by an integrated system of fibrils, as shown by Yocom
(1918) and Taylor (1920). Also, the nuclear phenomena during
the entire life-cycle have been thoroughly studied (Turner,
1930). The same variety as that studied by Yocom and by
Turner was used as the subject of this investigation. This
variety is easily recognized by the deep peristomial groove with
its wide extension under the frontal field, and by the deep indentation in the macronucleus near the left anterior side (Text-fig. 2).
The purpose of this investigation was to determine to what
extent and in what manner the structure of the neuromotor
system changes during the different stages of the life-cycle of
the organism, and how these changes are related to the basic
phases of the life-cycle, such as asexual reproduction, and the
haploid and diploid conditions at the time of sexual reproduction.
The writer wishes to express appreciation to Dr. C. A. Kofoid
for the use of his library, and for his many helpful suggestions
during the course of the investigation; also to Dr. Harold Kirby
for his helpful criticisms of the work.
METHODS.
Cultures.
The organism was cultured by a modification of Turner's
(1930) method. Timothy hay and wheat grains in standard
solution (Chalkley, 1930) provided the most satisfactory nutrient
EUPLOTBS PATELLA
509
medium. Five grammes of timothy hay and 3 grains of wheat
were added to 50 c.c. of this solution at boiling-point, and
allowed to boil for one minute. When cool, this was added to
250 c.c. of the solution in a round refrigerator jar. After standing for one day the culture was started by inoculation with
E u p l o t e s from a previous culture. Cultures made from grains
of wheat also provided a good growth of the organisms. In
these cultures, 10 grains of wheat were substituted for the
timothy hay and wheat in the procedure already given. The
E u p l o t e s were obtained originally from Thornhill Pond, a
freshwater pool in the vicinity of East Oakland. Various
organisms were used as food for the E u p l o t e s . Chilom o n a s provided the most abundant growth, but A s p i d i s c a
gave better results, for the E u p l o t e s feeding on it were more
free from food masses which obscure cytological details.
Since specimens undergoing binary fission were of especial
interest in this study, cultures of from three to ten days in age
provided most of the material. In cultures of this age a large
percentage of the organisms were undergoing fission. For the
study of conjugation cultures three weeks to one month in
age were used. The organisms were very abundant in the
cultures at the time epidemics of conjugation began.
O b s e r v a t i o n of L i v i n g O r g a n i s m s .
Organisms were studied in hanging drops over depression
slides, and under cover slips supported by vaseline rings. Since
E u p l o t e s p a t e l l a is extremely active, study of the living
organisms entails some means of retarding movement. This was
accomplished by reducing the amount of water in the hanging
drop to such an extent that surface tension held the organisms in
place. With the organisms under cover slips movement was
retarded by several methods, of which those suggested by Lund
(1935), namely, the use of sodium amytal as a narcotic agent,
and keeping the organisms stationary by applying the cover
slip closely to the slide were found most useful. Gum arabic as
a means of increasing the viscosity of the solution and thus
retarding movement was found to be unsuccessful, because the
organisms became abnormal in appearance.
510
DATUS M. HAMMOND
Several vital dyes were used, including cresyl blue, Janus
green B, Bismarck brown, acid fuchsin, methylene blue, toluidin
blue, and neutral red. Acid fuchsin in a 1 per cent, solution, as
suggested by Cole (1934), was useful for studying the structure
and movement of the ciliary apparatus of the cytopharynx.
Neutral red in a dilution of 1-10,000 was the best vital stain for
study of the rodlets at the bases of the bristles, membranelles,
and cirri.
One per cent. NiSO4 and Ni(N03)2were employed as suggested
by Gelei (1935) in order to differentiate the parts of the cytopharynx, but the results were unsatisfactory.
In order to ascertain the mechanics of feeding and to study
the path of food particles in the endoplasm, organisms were
observed in a medium containing finely ground indigo and
carmine. The particles of indigo were taken in readily by the
organisms, while carmine was rejected. As an aid in observation
of the process of ingestion the action of the organelles was
retarded with sodium amytal.
Microdissection methods were employed in an attempt to
determine experimentally whether the so-called 'sensory
bristles' are really sensitive to tactile stimuli, but this was
found to be too difficult on account of the small dimensions of
these bristles.
D e m o n s t r a t i o n of t h e P e r i p h e r a l P o l y g o n a l
system.
The silver impregnation methods of Klein (1926), Yabroff
(1928), Gelei (1934 a), Lund (1933), and Turner (1933) were
used. The silver-osmic acid-formalin method of Gelei (1928)
gave the best results in differentiating the rodlets at the base of
the bristles. These could not be demonstrated by the osmic
acid-toluidin blue process of Gelei (1926-7). A modification of
the mercuric chloride-Golgi solution-silver method of Gelei
(1934 a) was the most advantageous in demonstrating the polygonal network. This method as used consists of the following
steps:
(1) Fixation for one minute in concentrated aqueous mercuric
chloride, to which are added, just before use, two drops of a
EUPLOTBS PATELLA
511
3 per cent, potassium-bichromate solution, and one drop of a
1 per cent, osmic acid solution per c.c. of mercuric chloride.
(2) Wash twice in standard solution.
(3) Place in a 2 per cent, silver-nitrate solution and expose
to bright sunlight until the organisms reach the proper stage of
darkening. This must be ascertained under the microscope.
Uniform exposure of the organisms is accomplished by circulating air through the solution as suggested by Lund (1933).
(4) Wash several times in distilled water.
(5) Dehydrate rapidly, clear in xylol, and mount in balsam.
The organisms were handled in centrifuge tubes. This method
produces uniformly good results, and has the advantage over
Klein's method that the organisms are not distorted by drying.
D e m o n s t r a t i o n of t h e N e u r o m o t o r S y s t e m .
For whole mounts and sections Schaudinn's, Bouin's, Flemming's, Yocom's, Champy's, and Zenker's fixing fluids were used.
Champy's and Flemming's fluids were used cold, and the others
hot. Schaudinn's fluid gave the best results for differentiation
of fibrils, while Champy's fluid was the most useful in fixation
of material for study of the basal apparatus of the bristles.
The organisms were fixed in centrifuge tubes, then for whole
mounts were mounted on slides from 70 per cent, alcohol after
fixation in Schaudinn's, Bouin's, Yocom's, or Zenker's fluids.
In this way the distortion and uneven results of fixation on
cover slips was avoided. The material fixed in Flemming's and
Champy's fluids was handled in centrifuge tubes throughout,
because of the difficulty which was experienced in making the
organisms adhere to the slides.
Stains used were Heidenhain's iron alum-haematoxylin,
Mallory's triple connective tissue stain, and toluidin blue.
Heidenhain's haematoxylin gave the best results with the short
method, using the mordant 6-12 hours and the stain half an
hour, both cold. Alcoholic haematoxylin as recommended by
Dobell (1914), except that he used haematein instead of haematoxylin, gave excellent results. This method differentiated the
basal structures of the locomotor organelles to better advantage
than that using aqueous haematoxylin.
512
DATUS M. HAMMOND
For sectioning the individual organism method used by J. P.
Christenson (unpublished paper) was employed. This consists
of staining the organisms with eosin in 95 per cent, alcohol, and
impregnating in paraffin in a Syracuse watch glass. Only a
small number of organisms were placed in each dish, and these
were uniformly distributed over the bottom of the dish. After
cooling of the paraffin and removal from the dish, the organisms
may be seen under a dissecting microscope and the blocks of
paraffin cut so that each block contains only one or a few
organisms, whose orientation is known. This method involves
more precision and less waste than mass methods of sectioning.
Kolatchev's osmic acid method, as given by Bowen (1928),
was employed for the demonstration of mitochondria and Golgi
material.
STRUCTURE OF THE NEUROMOTOR SYSTEM.
In order to understand the changes in structure of the neuromotor system during the different stages in the life-cycle, its
typical structure must first be considered.
The essential features of the neuromotor apparatus, as
described by Yocom (1918), are five fibrils running anteriorly
from the bases of the anal cirri and converging near the anterior
end of the body, where the resulting single fibril is attached to
a motorium. Another fibril, the membranelle fibril, runs anteriorly from the motorium along one side of the adoral zone of
membranelles and connects with a lattice-work structure in
the anterior lip. Yocom interpreted this fibrillar system as
functioning in the co-ordination of the anal cirri with the
membranelles. He also described fine fibrils attached to the
bases of all the remaining cirri. These fibrils were found in
from 1-3 groups of from 4-6 fibrils each, radiating out in different
directions from the bases of each of the frontal and ventral cirri.
Only one group was seen to be attached to each of the marginal
cirri. These fibrils were considered as a dissociated part of the
neuromotor apparatus which functions in conveying stimuli to
the cirri, but which has not yet developed the function of coordination. Yocom noticed that the cirri are composed of cilia,
the basal granules of which are arranged irregularly in round
513
EUPLOTES PATELLA
X
mot
ad. memh
TEXT-FIG. 1.
Ventral view of E u p l o t e s p a t e l l a showing structure of neuromotor system. Fixed in Schaudinn's fluid and stained in alcoholic
haematoxylin. X 890. ana.cir.fib., fibril of anal cirrus; ad.memb.,
membranelles of anterior adoral zone; end.cil., endoral cilia;
memb.jib., membranelle fibril; mot., motorium; postphar.fib., postpharyngeal fibril; postphar.mem., postpharyngeal membrane;
rad.fib., radiating fibrils; sub.memb., suboral membranelles;
vewt.menib., membranelles of ventral adoral zone.
514
DATUS M. HAMMOND
basal plates. The membranelles were seen to be composed of
two parallel rows of fused cilia.
Taylor (1920) confirmed Yocom's interpretation of the function of the neuromotor apparatus by experimental methods.
Also, he discovered the presence of fibre plates beneath the basal
plates of the cirri and membranelles, to which the fibril or fibrils
are attached.
Klein (1926) in an undetermined species of E u p l o t e s
described a superficial network of lines to which he gave the
name ' Silberliniensystem', because of its selectivity for silver
in the silver impregnation method. This was found to be
arranged in a pattern of regular polygons on the dorsal surface,
and of irregular polygons on the ventral surface. For this reason
the term peripheral polygonal system will be used to designate
these structures. The lines were found to connect the bases of
the bristles, cirri, and membranelles. Klein supposed this network to be the means of co-ordination, rather than the neuromotor system of Yocom, which he considered to be supporting in
nature. However, more recently, Klein (1932) modified this
viewpoint to include the neuromotor system with the system
of co-ordination as a conductor of internal impulses to the locomotor organelles, while the polygonal system receives external
stimuli and effects the co-ordinated working of these organelles.
Jacobsen (1931) concluded that both the silverline system and
the neuromotor apparatus are supporting in function.
Gelei (1929 a) with his osmic acid-toluidin blue and silverosmic acid-formalin methods described an ectoplasmic 'neuroplasm' which he considered to be a stimulus-conducting structure. Gelei (1929 b) supposed that the neuroplasm together
with the silverline system provides for co-ordination between
the locomotor organelles, with the assistance of the neuromotor
apparatus. At first (1929 a) he regarded the 'silverline system'
as being of a skeletal nature, but he later (1934 V) concluded
that it may be also neuroid.
Turner (1933) demonstrated the polygonal system in E u p l o t e s p a t e l l a . He came to the conclusion that this system
conducts sensory stimuli received from the bristles to the neuromotor system, which acts as a motor conductor system. Turner
EUPLOTES PATELLA
515
was unable to demonstrate the motorium, but otherwise confirmed Yocom's description of the neuromotor system.
In the present study the neuromotor system was found to
follow the description of Yocom (1918) in its essential features.
However, the location of the motorium and the finer structure
of the cirri, membranelles, and cytopharynx was found to differ
in some respects from his account.
The N e u r o m o t o r i u m .
The motorium is located differently than Avas described by
Yocom (1918). The fibril formed by the convergence of the anal
fibrils continues its straight course anteriorly beyond the place
at which the motorium is located according to Yocom. This
point is immediately to the left of the most anterior frontal cirrus.
The fibril passes just to the left of the base of this cirrus and
continues anteriorly to the edge of the frontal area.
The beginning of the membranelle zone is immediately dorsal
to the anterior margin of this horizontal frontal area, on which
the cirri are located. In favourably stained preparations in side
view a vertically placed bilobed body is present in the region
between the anterior margin of the frontal area and the membranelle zone (Text-fig. 1). The fibril from the anal cirri is connected to this body on its ventral side and the membranelle
fibril is attached on the dorsal side. This is probably the
motorium described by Yocom (1918). Its vertical position and
its location at the edge of the frontal area where destaining is
relatively rapid makes the demonstration of the motorium very
difficult. However, there is not a direct connexion between the
fibril from the anal cirri and the membranelle fibril as described
by Turner (1933). In side view the bilobed body is seen to
intervene between these two fibrils.
Cirri a n d M e m b r a n e l l e s .
The arrangement of the eighteen cirri into seven frontal, two
ventral, five anal, and four marginal cirri as given by Taylor
(1920) will be followed. For convenience in description those of
each group will be numbered from left to right as indicated in
Text-fig. 2. The basal plates of the anal cirri have a rectangular
516
DATUS M. HAMMOND
micmemb.
md
B V ; J
cuirec-
vent.br—uentar.—
ci/rrodsuhmemb. -
cytophaz—
ana. c/r
rn ac •
mat cit
TEXT-FIG. 2.
Ventral view, showing arrangement of cirri and relationship of the
rodlets to the cirri and membranelles. Silver-osmio acid-formalin
method, supplemented by observations on living organisms stained
with neutral red. Ventral view. X 700. ana.cir., anal cirri; dr.rod.,
group of rodlets at base of cirrus; cytophar., cytopharynx; cyt.rec,
cytostomal recess; fr.cir., frontal cirrus; mac, macronucleus;
mar.cir., marginal cirrus; intmb.rod., rodlets at base of membranelles; mtc, micronucleus; ros.vent.br., rosettes of ventral row
of bristles; sub.memb., suboral membranelles; vent.cir., ventral
cirrus.
shape as indicated by Taylor (1920). However, the basal plates
of the frontal and ventral cirri are rhomboidal to square in
shape rather than round (Text-fig. 1). At least two and most
often three sides of the basal plates of these cirri are straight.
BUPLOTES PATELLA
517
The bases of the marginal cirri are very difficult to observe
because of their slanting position on specimens lying flat on
the slide. These cirri have irregular square to oval bases.
The basal granules of the cirri are arranged in definite straight
rows, contrary to what was found by Yocom. This arrangement
consists of primary rows and secondary rows approximately
perpendicular to the primary rows, resulting in the appearance
of a grating. The primary rows are oriented in a longitudinal
direction in the anal cirri and in an oblique direction in the
frontal and ventral cirri, with the anterior ends of the rows
pointing toward the middle of the body (Text-fig. 1). In the
marginal cirri the primary rows of basal granules are in general
perpendicular to the margin of the body. There are nine to ten
rows of basal granules in the anal cirri and six to seven in the
other cirri. A similar arrangement of the basal granules in
straight rows was described in E u p l o t e s w o r c e s t e r i by
Griffin (1910 a). He found only three to four rows in each cirrus,
with a maximum of fourteen granules in a row according to his
fig. 8, p. 300. The number of basal granules in the rows in
E u p l o t e s p a t e l l a could not be determined, but it is much
greater than Griffin found in E u p l o t e s w o r c e s t e r i .
Studied in relationship to the rows of basal granules of the
cirri the arrangement of the fibrils of the cirri is given added
significance. These fibrils have a definite orientation with respect
to the direction of the rows of basal granules of the cirrus to
which they are attached. In the left ventral cirrus, for example,
three groups of fibrils extend out in different directions from the
base of the cirrus (Text-fig. 1). One group proceeds anteriorly,
another posteriorly, and the third in a transverse direction
toward the left side of the body. The fibrils of the anterior and
posterior groups are parallel with the primary rows of basal
granules of the cirrus, while the transverse fibrils are parallel
with the secondary rows of these granules. This close correlation in direction of fibrils with the rows of basal granules does
not hold true for all cirri. In some cases the fibrils can be seen
to attach to the ends of rows of basal granules. Usually three to
five fibrils make up each group. For some cirri there are two
groups of fibrils and for the marginal cirri only one group.
518
DATUS M. HAMMOND
According to Yocom's description the anal cirri are supplied
with only the main longitudinal fibrils, one for each cirrus, but
in critically stained preparations transverse fibrils can also be
mewb,
£v- - mac.
-anpeif
mic.
an. fit
citf
-on. lit
marxiii
-h-anaa'r.
fern,
mac.
- anri.
maf-cif.
TEXT-ITO. 3.
Reorganization of zygote. Schaudinn's, alcoholic haematoxylin.
Ventral view. X 700. ana.cir., anal cirri; an.fr.cir., rudiment of
frontal cirrus; an.lft.mar.cir., rudiment of left marginal cirrus;
an.per., rudiment of posterior portion of peristome; an.rt.mar.dr.,
rudiment of right marginal cirrus; mac, macronucleus; memb.,
membranelles; mic., micronucleus; rem.mac, remnant of old
macronucleus.
seen. These extend laterally from each anal cirrus, although
those attached to the first cirrus are most noticeable.
These fibrils of the frontal, ventral, and marginal cirri extend
much farther from the bases of the cirri to which they are
EUPLOTES PATELLA
519
attached than was described by Yocom. No connexion could
be demonstrated at the distal ends except in one case, where the
frontal cirri one and two are connected by these fibrils.
The membranelles consist of three rows of cilia rather than
two, as has hitherto been described. In the part of the zone of
membranelles which is on the ventral side of the body, this third
row is incomplete in that it extends only part of the way across
the width of the zone. In that part of this region which lies in
a frontal plane the third row consists of about 7-10 cilia (fig. 10,
PI. 21; Text-fig. 1). The membranelles which extend across the
anterior end of the body have three complete rows. The transition between these membranelles and those with an incomplete
thircj»row is abrupt, and occurs at the place where the zone of
membranelles turns around the left anterior end of the body.
This third row was first seen in material prepared by the silverosmic acid-formalin method, but it can be plainly distinguished
in the living organism as well.
Kahl (1932) found a difference in number of lamellae in the
membranelles in different parts of the adoral zone in S t e i n i a.
In this species nine to ten of the membranelles near the cytopharynx have two lamellae, while the remainder have three.
The arrangement of the basal granules of both the cirri and
the membranelles in rows, and the relationship of the radiating
fibrils to these, may be explained by supposing that the complex
protozoan E u p 1 o t e s with its composite organelles arose from
a simple form in which the cilia were distributed in slightly
spiral longitudinal rows over the entire surface of the body.
According to this interpretation, the rows of cilia in the cirri are
regarded as being derived from the longitudinal rows of the
once evenly distributed cilia. Griffin (1910 a) regarded each
cirrus of E u p l o t e s w o r c e s t e r i as having been derived
from several rows of cilia of a simpler form. The oblique direction of the rows in the frontal and ventral cirri is indicative of
a fundamental spiral organization, which is also expressed in
the spiral arrangement of the adoral zone of membranelles.
Following this idea farther, the radiating fibrils are considered
as derivatives of the interciliary fibrils of the cilia of the simple
ancestor. In each cirrus those fibrils in the groups parallel with
520
DATUS M. HAMMOND
the primary rows of basal granules are derived from fibrils
connecting the cilia of the same rows, while the fibrils parallel
with the secondary rows of basal granules are derived from the
transverse fibrils. Since these fibrils are not so numerous as are
the rows of basal granules of the cirrus, either loss or fusion of
fibrils may have occurred, resulting in a reduction in number. In
several cases groups of two to three fibrils are fused into a single
fibril at their distal ends, indicating how such a reduction in
number may have occurred.
Although this interpretation is entirely speculative it supplies
an explanation of the arrangement of the basal granules in
uniform rows and the constant relationship of the fibrils to
these rows.
Cytopharynx.
The cytopharynx of E u p l o t e s p a t e l l a has not been
adequately described. Yocom (1918) noted that the adoral zone
of membranelles extends along the left wall of the cytopharynx.
He also noted that the right wall of the cytopharynx is ciliated,
but did not describe the arrangement of the cilia.
In the present study the cytopharynx was found to correspond
in structure with the description given for E u p l o t e s worc e s t e r i by Griffin (1910 a). The cytopharynx of E u p l o t e s
p a t e l l a is much shorter and less curved than is the case in
E u p l o t e s w o r c e s t e r i , but the ciliary apparatus is similar.
This consists of the adoral zone of membranelles on the leftposterior wall, several oblique rows of endoral cilia on the dorsal
wall, and suboral membranelles on the right wall of the cytopharynx (Text-fig. 1). An undulating membrane or ridge of
protoplasm on the right wall of the cytopharynx was seen in
living specimens stained with acid fuchsin. This structure is
evidently located along one side of the suboral membranelles.
The suboral membranelles are connected by a longitudinal fibril
with the membranelle fibril. Another fibril extends into the
endoplasm from the end of the membranelle fibril. There is a
slight enlargement where these three fibrils are joined.
The endoplasm in a circular area immediately adjacent to the
cytopharynx is sharply demarcated from the surrounding cyto-
EUPLOTES PATELLA
521
plasm. This area appears to be delimited by a circular fibril
attached at one end to the cytopharynx. In sections this is seen
to be a continuous membrane lying close to the ectoplasm of
the ventral surface. This membrane has an incomplete dorsal
boundary, and thus is in the shape of a partially enclosed sac.
In the feeding experiments the food particles were seen to
follow along the deep groove at the right side of the peristomial
field. Fairly large particles up to 15 microns were ingested. As
soon as a large particle entered the endoplasm, streaming movements carried it away from the cytopharynx along a definite
course. This course followed the circumference of the membranous sac in an anti-clockwise direction. The particles were
seen to make from one-fourth to one or more revolutions along
this course. Plasmosis occurred only at the time of ingestion
of particles and only in the area within the membrane. No
definite food vacuoles were seen.
The food masses are received into the membrane as they are
first ingested. Processes of digestion are evidently started in
this partially enclosed sac. The large dorsal opening allows the
food particles to leave the sac and circulate freely in the endoplasm during the later stages of digestion.
Peripheral Polygonal System.
The arrangement of the polygonal system agrees in general
with the description given by Turner (1933). The polygonal
lines, however, do not come into direct contact with the basal
granules of the bristles, as will be described later, nor is there
any uniform connexion with the basal plates of the cirri. The
membranelles are, however, directly connected with the polygonal lines at the ends of the membranelle plates (Text-fig. 1).
The median membranelle fibril described by Turner (1938) is
coincident with the central row of rodlets of the membranelle
zone.
The polygonal lines in E u p l o t e s p a t e l l a are pellicular
structures rather than subpellicular as described by Turner
(1933). The position of the lines in the pellicle was demonstrated
in the modification of the wet silver method, in the silver-osmic
acid-formalin method, and in preparations of material fixed in
522
DATUS M. HAMMOND
Schaudinn's fluid and stained with the alcoholic haematoxylin
method. The latter preparations show the polygonal lines only
when the organisms have been allowed to become partly dry in
mounting on the slides after fixation. The clearness of the lines
is proportional to the degree of drying which the organisms
underwent in mounting. In the organisms which became more
dry the lines show up as definite ridges on the surface of the
organism. This supports the interpretation that the polygonal
system is pellicular in position.
The diagram of Turner (fig. 2, p. 56, 1933) supports this view
if the structureless superficial layer labelled ectoplasm is interpreted as the pellicle, and the deeper layer labelled endoplasm
as the ectoplasm.
B a s a l A p p a r a t u s of t h e B r i s t l e s .
The so-called sensory bristles occur in longitudinal rows along
the surface of the body. There are nine rows of these on the
dorsal surface and one on the ventral surface, near the left
margin of the body. The bases of these bristles are surrounded
by elongate granules arranged in rosettes (fig. 7, PL 22). These
rows of bristles are coincident with the sides of ridges along the
surface, which may or may not be evident, and thus the number
of rows is the same as the number of ridges. The bristles are not
rigid, nor are they vibratile, but they do show slight movements,
which may be passive.
In the silver preparations these bristles are seen to be connected directly by longitudinal fibrils and indirectly by cross
fibrils (Text-fig. 5), as described by Klein in E u p l o t e s
h a r p a (1926) and by Turner in E u p l o t e s p a t e l l a (1933).
Each bristle arises from a basal granule which forms part of
a complex basal apparatus. In E u p l o t e s p a t e l l a this
consists of a depression in the ectoplasm in the form of an inverted cone, of which the basal granule forms the apex (Textfig. 7). Toward the surface the membrane lining the depression
ends in a ring, which connects on either side with the longitudinal fibril of the polygonal network (Text-figs. 5 and 7). Surrounding the depression is a group of rod-like bodies arranged
in rosette fashion. There are usually six of these for each basal
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TEXT-FIGS.
4-11.
All figures, except 7, 9, 10, and 11, x 700.
Fig. 4.—Rudiment (Anlage) of right marginal cirrus for anterior
daughter developing in row of bristles in region of multiplication.
Champy's fluid, Heidenhain's haematoxylin.
Fig. 5.—Portion of peripheral polygonal system of dorsal surface.
Bach ring surrounds a depression in which the basal granule of a
bristle lies. Wet silver method.
Fig. 6.—Sketch showing the relation of the peripheral polygonal
system to the membranelle zone. Wet silver method.
Fig. 7.—Diagrammatic representation of the structure of the basal
apparatus of a bristle. The basal granule is at the bottom of a
depression surrounded by rodlets, two of which are shown. The
ring which encircles the depression is connected on either side with
the peripheral polygonal system.
Fig. 8.—Sketch of a portion of the ventral surface of an organism in
an early stage of binary fission, showing the networks of the new
peripheral polygonal system and their relation to the old polygonal
system and rudiments of the cirri.
Figs. 9, 10, 11.—Diagrams illustrating successive stages in the
fission of a basal granule of a bristle.—Bristle omitted.
NO. 316
Mm
528
524
DATUS M. HAMMOND
granule, although there may be more or less. These are tilted
at an angle toward the surface, so that the outer end of each is
pointed toward the surface and toward the centre of the depression. Thus the appearance of the group is similar to that of the
poles of a wigwam. The outer ends of the rodlets are located
at the boundary between ectoplasm and pellicle, but each one
makes a tiny bulge on the surface. This accounts for an early
description of them (Stokes, 1885) as minute prominences
arranged in a stellate cluster. These rodlets can be demonstrated
in the living organism with or without vital dyes and are well
differentiated with Gelei's silver-osmic acid-formalin method.
They are not mitochondria or Golgi material because they do
not stain with methods for differentiation of these.
This agrees with the description of the basal apparatus of
the bristle as given by Gelei (1933) for an undetermined species
of E u p l o t e s , except that the depression is cone-shaped
instead of cylindrical and is not filled with protoplasmic
material. Also, the neuroplasm connecting the bases of the
bristles in the same longitudinal row was not seen. Gelei (1929 a)
has named the depression ' Sensucylinder' and the rodlets
' Sensucysten', but since these terms imply a function which has
not been demonstrated they will not be used here. Jacobsen
(1931) saw that the bristle arises from a depression in the
surface of the body. She considered that the edge of the pellicle
surrounding this depression is coincident with the silverline ring
described by Klein. Turner (1933) described the bristles and
the rosettes surrounding the bases of these, but did not see the
depression or ring.
Basal structures without bristles occur on the ventral surface
in connexion with the membranelles and cirri (Text-fig. 2).
Their occurrence on the ventral surface has been described by
Gelei (1929 a) for an undetermined species of E u p l o t e s , and
Griffin (1910a) in E u p l o t e s w o r c e s t e r i saw the bristles
as well as the rosettes in these locations. Each basal apparatus
present at the bases of the cirri and membranelles in E u p l o t e s
p a t e l l a appears to consist only of the rodlets, ring, and the
central depression. There are two to three groups of rodlets
at the base of each cirrus. They always have a definite location
EUPLOTES PATELLA
525
with regard to the cirrus at whose base they lie. Where there are
only two groups of rodlets they are placed on opposite sides of
the cirrus. They are always placed near the corners of the basal
plates of the cirri and along the side of the primary rows of
granules. Thus, they have an antero-posterior relationship with
regard to the frontal cirri one, two, four, five, and seven, an
oblique position with regard to frontal cirri three and six, and
a lateral position at the base of the ventral cirri. They are
placed near the anterior and posterior ends of the anal cirri.
The groups of rodlets at the bases of the marginal cirri are
parallel with the margin of the body.
There are three rows of groups of rodlets along the adoral
zone of membranelles. This results from the location of one
group near the centre and one near either end of each membranelle plate. For those membranelle plates occurring on the
ventral surface these groups occur just posterior to each plate.
These continue anteriorly along the frontal portion of the zone
(figs. 8 and 9, PI. 22), and posteriorly into the pharyngeal portion of the zone. There is also a group of rodlets at the anterior
end of the suboral membranelles.
These rosettes have been seen by many investigators. Eees
(1881) observed rows of very small vesicle-like structures along
the dorsal ridges of E u p l o t e s l o n g i p e s . He suggested
these might be of the nature of contractile vacuoles. Stokes
(1885) saw the rodlets in E u p l o t e s c a r i n a t u s and
E u p l o t e s p l u m i p e s and described them as variable minute
dots or oblong elevations marking the surface of the body.
Minkiewicz (1901) considered the rosettes in E u p l o t e s
v a n n u s to be crystalline in nature. Griffin (1910a) applied
microchemical tests to the structures in E u p l o t e s worc e s t e r i and on the basis of these determined that they were
of the nature of reserve fatty food substances. Gelei (1929 a,
1934&) confirmed the fatty nature of the bodies but supposed
that they are used in amplifying the sensory stimuli received
by the bristles. Jacobsen (1931) follows this interpretation of
the significance of the rosettes. Gelei further suggests that the
groups of rodlets at the bases of the cirri receive their stimuli
from the cirri and are thus rheoceptive sensory organs. This
526
DATUS M. HAMMOND
view is not supported by the position of the rodlets with relation
to the cirri, because the rodlets are not in the direction of stroke
in the case of the ventral cirri, frontal cirri three and six, and some
of the marginal cirri.
The function of the rosettes is obscure. That they amplify
the stimuli which might be received by the bristles is improbable
because they do not come into direct contact with the basal
granule or bristle. They are apparently connected in some way
with the function of the bristle.
The function of the bristles is also obscure. It has generally
been assumed that they possess a sensory function (Griffin,
1910 o; Gelei, 1929 a; Turner, 1933), since they are not used for
propulsion. Until experimental evidence is obtained the view
that the bristles are sensory in nature seems most plausible.
The bristles are generally considered to have arisen by a
transformation of functional cilia. This view has been suggested
by Johnson (1893), Wallengren (1901), and Klein (1932). Gelei
(1933) has advanced a different interpretation. He supposes
that the bristles are not derived from cilia, but are outgrowths
from the end of a sensory neuroid structure, similar to the
Nebenkorn in P a r a m e c i u m. This view cannot be supported
by a consideration of the structure in E u p l o t e s p a t e l l a ,
since no fibrillar continuation of the basal granule of the bristle
has been demonstrated. Furthermore, the structure of the membranelles, cirri, and bristles is comparable as regards the
possession of basal granules and basal groups of rodlets. The
cirri and bristles are associated in ontogeny, as will be brought
out later. Therefore, the weight of evidence is in favour of the
derivation of the bristles by a modification of cilia.
The body of E u p l o t e s p a t e l l a is thus seen to be relatively
complicated. There are several kinds of ciliary organelles which
are co-ordinated in their action by the fibrillar system. The
behaviour of these differentiations during binary fission will
next be considered.
ASEXUAL BEPKODUCTION.
It has long been known that the common method of reproduction in ciliates is binary fission or asexual reproduction.
EUPLOTES PATELLA
527
Stein (1859) observed one stage in fission of E u p l o t e s
p a t e l l a and several stages in E u p l o t e s c h a r o n . He was
the first investigator to note the interesting phenomenon of
complete resorption of the entire set of old cirri and its replacement by two sets of new cirri, during binary fission. Mobius
(1887) described the process of binary fission in E u p l o t e s
h a r p a. He supposed that the set of old cirri of the parent is
halved, and only enough new cirri are formed to make up the
deficiency in the two daughters. Schuberg (1899) investigated
binary fission in E u p l o t e s p a t e l l a , but observed only a few
stages and consequently was not able to give a complete
description of the process. Wallengren (1901) made a thorough
study of binary fission in E u p l o t e s h a r p a , and Griffin
(1910) gave a detailed account of the process in E u p l o t e s
w o r c e s t e r i . Yocom (1918) briefly described binary fission in
E u p l o t e s p a t e l l a , and Turner (1930) made a detailed study
of the nuclear phenomena in asexual and sexual reproduction
in this species.
It has been shown by Turner (1930) that the first evidence
of approaching fission is the appearance of bands composed of
lightly and darkly staining areas on the ends of the macronucleus. These bands gradually move along the macronucleus
until they meet at the centre. This process Avas interpreted by
Turner (1930) as a reorganization of the material in the macronucleus, resulting in a rejuvenation of the organism. In later
stages the micronucleus undergoes a mitotic division and the
condensed macronucleus is split into two parts. Each daughter
receives one macronucleus and one micronucleus.
The dedifferentiation and redifferentiation of the nuclei is
followed by similar processes in the cytoplasm, as will be shown
later. Before the parent organism divides into the two daughters
there are extensive changes in the cytoplasmic structure, involving the appearance of a new peristome for the posterior
daughter; the differentiation of two complete sets of new cirri,
one for each daughter, and the dedifferentiation of the old set;
the development of a number of new bristles in each row; and
the reorganization of the polygonal system over almost the
entire surface of the body.
528
DATUS M. HAMMOND
Peristome.
The first cytoplasmic change to occur during binary fission
is the appearance of the rudiment of a new peristome as a depression in the ectoplasm immediately posterior and to the left of
the old peristome (fig. 11, PL 22). This becomes visible soon
after the reorganization of the macronucleus begins. A patch
of cilia appears in this depression, and these cilia become
arranged in rows, which are the beginning of the membranelles.
The depression deepens into a pocket, which extends first
posteriorly, then anteriorly, and the membranelles develop along
the floor and side of this pocket. The development of the peristome has been described by Griffin (1910b) and, since the
development in E u p l o t e s p a t e l l a agrees with this description, it will not be described in detail here. As soon as the
structure of the individual membranelles can be made out it
is seen that the organization of these is similar to that in the
fully developed organism. The third row is complete in the
anterior one-third of the zone, and incomplete in the remaining
portion.
There is apparently no change in structure in the old peristome
during binary fission. This agrees with Yocom's (1918) and
Turner's (1930) observations. There is likewise no visible
reorganization in the old peristome during binary fission in
E u p l o t e s w o r c e s t e r i (Griffin, 1910b), or in S t e n t o r
(Hetherington, 1932; Schwartz, 1935).
On the other hand, Wallengren (1901) noted a gradual replacement of the old adoral zone in E u p l o t e s h a r p a , and
a similar process has recently been observed during binary
fission in U r o n y c h i a (Taylor, 1928) and D i o p h r y s
(Summers, 1935). A reorganization of the adoral zone during
binary fission has also been reported in several heterotrichous
ciliates including B l e p h a r i s m a u n d u l a n s (Calkins, 1912),
and B u r s a r i a t r u n c a t e l l a (Schmahl, 1926).
It is possible that a gradual reorganization of the adoral zone
might occur before or after binary fission in the forms in which
the old peristome is retained without visible change of structure
by the anterior daughter.
EUPLOTES PATELLA
529
D e v e l o p m e n t of C i r r i .
Although the development of the cirri has been thoroughly
described for E u p l o t e s h a r p a by Wallengren (1901) and
for E u p l o t e s w o r c e s t e r i by Griffin (19106), there has been
no adequate study of this process in E u p l o t e s p a t e l l a .
Maupas (1889) gave a brief description of the origin of the new
cirri during conjugation, while Schuberg (1899) and Yocom
(1918) have given general accounts of this process during binary
fission.
The first indication of the new cirri can be made out soon
after the appearance of the rudiment (Anlage) of the new peristome as a series of ten slit-like depressions along the middle of
the ventral surface. These depressions are clearer in appearance
than the surrounding ectoplasm and they result from a resorption of the pellicle and a change in the appearance of the
ectoplasm at these points.
The depressions are arranged in two approximately transverse
rows which consist of five depressions each. One row, which
is directly anterior to the other five, gives rise to cirri which
become a part of the anterior daughter, while the posterior row
gives rise to cirri which become a part of the posterior daughter
(fig. 11, PI. 22). The depressions do not appear simultaneously,
as in E u p l o t e s w o r c e s t e r i , but those on the left appear
first, as in E u p l o t e s h a r p a .
These depressions, which are at first narrow slits, become
broader and longer. Beginnings of cirri appear along the floor
of each. In each of the three depressions to the left three cirri
arise, and in each of the remaining two depressions two rudiments (Anlagen) appear, making a total of thirteen rudiments
of cirri for each daughter (fig. 4, PL 22; Text-fig. 8). According
to Yocom (1918) and Turner (1930) only the last depression to
the right has two rudiments and all the rest have three rudiments,
making a total of fourteen cirri arising out of the five depressions, as is the case in E u p l o t e s h a r p a (Wallengren, 1901)
and E u p l o t e s w o r c e s t e r i (Griffin, 19106). This account
is incorrect for there are only nine anteroventral cirri in
E u p l o t e s p a t e l l a as compared with ten in the same group
530
DATUS M. HAMMOND
in E u p l o t e s h a r p a and E u p l o t e s w o r c e s t e r i , and
this difference develops from the absence of the third rudiment
in the second depression from the right in E u p l o t e s p a t e l l a .
The posterior rudiment in each depression becomes an anal
cirrus. The anterior rudiments in the two depressions to the
right develop into ventral cirri, and all the remaining rudiments become frontal cirri. Of these the two anterior rudiments
in the middle develop into frontal cirri three and six; the two
anterior rudiments in the second depression from the left give
rise to frontal cirri two and five; and the two anterior rudiments
in the first depression to the left develop into frontal cirri one
and seven. As development occurs the cirri migrate to their
definitive positions, and at about the time these definitive locations are reached the old cirri are resorbed. Since the migration
of the cirri agrees with that described for E u p l o t e s h a r p a
by Wallengren (1901) the details of this process will not be
considered here.
Soon after the two rows of depressions become visible a
rudiment appears on the median wall of the pocket in which the
new peristome is developing. A corresponding rudiment of a
cirrus arises on the median wall of the old peristomial field in
the region of frontal cirrus seven. These rudiments develop
into frontal cirrus four for the anterior and posterior daughter
respectively. This cirrus arises in the peristomial field rather
than on the frontal field as in E u p l o t e s w o r c e s t e r i
(Griffin, 19106). Wallengren (1901) describes this cirrus as
arising in E u p l o t e s h a r p a 'auf dem protoplasmatischen
Theil der Unterlippe des alten Peristome'.
The left marginal cirri arise in two depressions to the left of
the peristome (fig. 5, PI. 21). These were seen by Schuberg
(1899), but he was unable to determine how many rudiments of
cirri were present in each depression, and he did not follow their
development. Wallengren (1901) found in E u p l o t e s h a r p a
two rudiments in each depression and saw that the posterior pair
give rise to the left marginal cirri of the posterior daughter,
while the anterior pair form the left marginal cirri of the anterior
daughter. The development of the left marginal cirri in
E u p l o t e s p a t e l l a agrees with this description. The rudi-
EUPLOTES PATELLA
531
ments of these cirri appear slightly later than the groups of
depressions already described.
The origin of the right marginal cirri has not been described
for E u p l o t e s p a t e l l a , and only incompletely for E u p l o t e s w o r c e s t e r i and E u p l o t e s h a r p a . In both of
the latter species the right marginal cirri were described as
originating upon or near the margins of the daughters' bodies
near their definitive location at the time of constriction of the
parent into the two daughters.
By using material fixed in Champy's or Plemming's fluids the
writer was able to trace the origin of these cirri to a much
earlier stage in binary fission. In organisms in which the rudiments of the cirri have just appeared and before there is any
sign of the constriction between the two daughters it is possible
to identify the rudiments of the right marginal cirri. These have
a remarkable origin, in that they arise on the dorsal surface
along the rows of bristles, and only at about the time of separation of the daughters do they assume their definitive ventral
positions. The rudiments of the right marginal cirri for the
anterior daughter arise near the centre of each of the last two
rows of bristles to the right (fig. 3, PI. 21), and those for the
posterior daughter appear in the same two rows near the
posterior end.
The first indications of these rudiments appear as minute
deeply staining areas immediately posterior or anterior to a
basal granule of a bristle (Text-fig. 4). These increase in size,
and, during constriction, the plane of division passes immediately posterior to the right marginal cirri for the anterior
daughter. By this process these cirri reach their definitive
positions on the ventral surface. The right marginal cirri for
the posterior daughter reach their ventral positions by a migration around the posterior margin of the body. This movement
is closely associated with the replacement of the polygonal
system as will be shown later.
The dorsal location of the rudiments of the right marginal
cirri may account for their being overlooked by the earlier
investigators. The differentiation of these rudiments along the
row of bristles indicates a possible kinship between the cirri
532
DATUS M. HAMMOND
and bristles. This will be further discussed in connexion with
the multiplication of the bristles.
K e s o r p t i o n of C i r r i .
The dedifferentiation of the cirri was studied in organisms in
the living condition. Since the marginal cirri project beyond the
margin of the body, and thus have the greatest visibility, they
were found most suitable for this study. In the cases observed
the two laterally placed marginal cirri on either side were the
first of these to be resorbed. This occurred about a half-hour
before separation of the two daughters. The median marginal
cirri were resorbed later. Usually the left one of these disappeared at about the time of separation of the daughters, while
the right one persisted five to thirty minutes after separation.
Thus, the posterior daughter can be recognized for some time
after fission by the presence of a supernumerary marginal cirrus.
The resorption of a cirrus is preceded by sudden cessation of
movement of this cirrus. Immediately after it stops beating
a decrease in size occurs. The fimbriation which Taylor (1928)
noted in the dedifferentiation of cirri in U r o n y c h i a , and
Wallengren (1901) observed in E u p l o t e s h a r p a , was not
seen. The cirrus decreases in diameter as well as in length.
This process occurs rapidly and is completed with the disappearance of the cirrus within two minutes of the time it
becomes motionless. The cirrus is drawn in the direction of the
fibrils at its base as dedifferentiation occurs. Thus, it is pulled
out of its former position, which the corresponding new cirrus
then occupies.
D i f f e r e n t i a t i o n of F i b r i l s .
The fibrils of the anal cirri are first visible as heavier lines
directly beneath the basal plates and parallel with the rows of
basal granules of the cirri to which they are attached. They
appear first beneath the second and third cirri from the left side,
followed by the first, then the fourth, and finally the fifth. The
fibrils begin to develop at about the same time in the anterior
and posterior daughters.
In a later stage in binary fission, the fibrils are extended
EUPLOTES PATELLA
588
anteriorly from the basal plates and are seen to run superficially
to the old fibrils. They are thus ectoplasmic in origin. This
extension continues until the fibrils from the anterior set of cirri
reach the anterior end of the organism. At this time the old
set of fibrils is resorbed and the membranelle fibril becomes
connected with the new set.
The fibrils from the posterior set of anal cirri are compressed
into a single fibril at the time of constriction. At this time the
connexion with the membranelle fibril is effected, completing
the integration of the neuromotor apparatus of the posterior
daughter. The origin of the motorium was not determined.
The fibrils of the frontal and ventral cirri also develop from
the basal plates of the cirri. When they first appear, these fibrils
are more closely in line with the rows of basal granules of the
cirri to which they are attached than in the fully developed
organism.
M u l t i p l i c a t i o n of B r i s t l e s .
Until recent times there has been no detailed knowledge concerning the manner in which the bristles are increased in number
during binary fission. Sterki (1878) suggested that at least a part
of the bristles are formed anew during binary fission, since the
daughters have as many bristles as the parent. Wallengren
(1901) studied the origin of the dorsal bristles in U r o n y c h i a .
He found that, in a stage in which the new ventral and marginal
cirri have begun to develop, clear lines appear on the dorsal
surface to the left of the longitudinal furrows in which the old
bristles are located. Soon after, small, closely set, cilia-like,
actively moving structures appear in this line. He supposed
the old bristles to be resorbed, but did not follow this process.
Gelei (1929 a, 1933,1984 b) has conducted intensive investigations on the subject of the origin, structure, and function of the
bristles. He studied the multiplication of these in an undetermined species of E u p l o t e s , and found that only the bristles
in the central part of each row take part in multiplication, and
that from the basal structure of each of these several new
bristles arise.
Klein (1932) supposed that the new bristles are derived from
534
DATUS M. HAMMOND
the polygonal system during binary fission. Gelei (1934&) has
shown that this is not ordinarily the case in the species of
E u p 1 o t e s with which he was working.
The two methods used by Gelei, and in addition material
fixed in Champy's and in Flemming's fluids and stained in
Heidenhain's haematoxylin, were used by the writer in studying
the multiplication of the bristles. The results obtained agree in
general with those of Gelei.
In E u p l o t e s p a t e l l a only the central bristles in each row
are involved in the process of multiplication. This begins soon
after the rudiments of the left marginal cirri have appeared on
the ventral surface. The basal granules of about the three
central bristles in each row divide, so that two granules instead
of one can be plainly seen at the bases of each of these bristles
(Text-fig. 9). The adjacent basal granules anteriorly and posteriorly are then involved until five to eight basal granules in
each row are undergoing fission (figs. 1 and 2, PI. 21). This
fission continues until up to as many as six new basal granules
are produced from each old basal granule involved (Text-figs.
10 and 11). Those basal granules on the anterior and posterior
margins of the area of multiplication give rise to fewer new
granules, sometimes as few as one, while those in the centre of
this area may give rise to as many as six new ones. Thus there
is a gradient of regeneration, with the highest point at the centre
of the organism, and decreasing both anteriorly and posteriorly.
In the process of fission of a basal granule there are tubular
outgrowths extending anteriorly and posteriorly from the basal
apparatus of each bristle involved (Text-figs. 10 and 11). These
develop soon after the first fission of the basal granule has taken
place. The daughter granules move out within this tubular
membranous process and, since the interior of these outgrowths
appears relatively clear with the stains used, the new granules
can be followed through all the stages of their multiplication.
The new granules give rise to the new bristles and it is probable
that the accessory parts of the basal apparatus such as the cone
are derived from the membranous process (fig. 3, PI. 21). The
origin of the rodlets was not determined. They were seen to be
EUPLOTES PATELLA
535
present around the bases of the new basal granules at about
the time that these gave rise to the new bristles.
Gelei (1934 b) did not see the division of the basal granules
and supposed that the new granules arise by differentiation from
the membranous outgrowths. By the methods he used these
processes appeared solid, and thus masked the origin of the new
basal granules.
In the form studied by Gelei an average of seven basal
apparatuses in each row participated in multiplication, and four
to five new basal granules arose from each of these. In E u p l o t e s p a t e l l a fewer basal structures take part, but each
one produces more new basal granules.
There is no dedifferentiation of bristles during binary fission.
This conclusion is supported by the observation of different
stages of the process from the earliest indication of multiplication until the new bristles are fully formed. The old bristles
were seen to be present throughout this process until they
could no longer be distinguished from the new bristles interpolated between them. Also, the bristles outside the zone of
multiplication showed no evidence of resorption or replacement.
Another line of evidence which supports the assumption that
the bristles are not resorbed is supplied by a numerical study
of the new bristles as compared to those in the completed
organism. There are about as many new bristles produced in
each row as were present in the row before fission. Thus, if the
number is to be kept uniform, resorption must not occur. Gelei
(1934 b) described dedifferentiation of the bristles in the region
of multiplication in the species of E u p l o t e s with which he
worked, but the writer found no evidence of this process.
The bristles anterior and posterior to the region of multiplication are received without any change in structure by the anterior
and posterior daughters respectively. There is a redistribution
of the new bristles along the rows so that a more or less uniform
spacing results.
Indications of division of individual bristles independently of
the remainder have been noted. In several organisms at a stage
in which the rudiment of a new peristome was just detectable,
536
DATUS M. HAMMOND
and long before the beginning of multiplication of bristles as
described above, two bristles so closely placed as to be almost
contiguous were seen.
This might be interpreted as a process of reorganization and,
if so, would indicate how the reorganization of the bristles
occurs. At any rate, a reorganization of the bristles might take
place during stages which have not been observed.
The origin of the right marginal cirri along the rows of bristles
is worthy of further notice here. The anterior set of rudiments
of the cirri occur, as previously mentioned, along the last two
rows of bristles to the right and near the centre of the region of
multiplication. In the early stages of the multiplication of the
bristles no indication of the rudiments of the cirri can be seen. The
basal granules of the bristles in the rows in which rudiments
will appear undergo fission, as do the basal granules in the other
rows. The first indication of a rudiment is a more deeply
staining area which is usually immediately posterior to an old
basal granule, and just anterior to the centre of the region of
multiplication (Text-fig. 4). This darkened area has at first the
same dimensions as the membranous outgrowth from the basal
apparatus and is possibly derived from it, or from the products
of division of the basal granule. Later, the area becomes greater
in diameter, and sinks beneath the level of the dividing basal
granules. All the new basal granules which occupied the area
between the two old basal granules wherein the rudiment arose
disappear.
The rudiments of the right marginal cirri for the posterior
daughter arise slightly earlier than those for the anterior
daughter. They appear just posterior to the last bristles in the
same two rows wherein the rudiments for the anterior daughter
develop. As this location is outside the region of multiplication the appearance of the rudiments is somewhat different than
is the case for the more anterior rudiments. However, here also,
the first indication of the rudiment is a deeply staining splinterlike area immediately posterior to the bristle, and in a direct line
with the row of bristles. The development is similar to that
already described for the rudiments of the anterior marginal
cirri. It is possible that the rudiment arises as an outgrowth
EUPLOTES PATELLA
537
from the basal granules of the last bristle in the row involved,
but this could not be determined.
The behaviour of the bristles during asexual reproduction is
very different from that of the cirri. While two sets of cirri are
formed entirely anew and the old set of cirri is dedifferentiated,
the new bristles are formed from multiplication of the previous
structures, and there is apparently no dedifferentiation of the
bristles. This difference may be associated with the fact that
the bristles are not vibratile and thus would not become worn
out or injured so readily as the actively beating cirri. However,
the bristles may also be replaced during stages of the life-cycle
which have not come under observation during this study.
Peripheral Polygonal System.
Klein (1928) studied a stage in binary fission of an undetermined species of E u p l o t e s with especial reference to the
so-called silverline or polygonal system, but confined his observations to the development of the peristome. According to Klein,
the polygonal lines have the power to reproduce themselves, to
grow, and to form new locomotor organelles. Gelei (19846) has
described in detail the behaviour of the polygonal system on
the dorsal surface of an undetermined species of E u p l o t e s
during binary fission. According to this description a new
system of polygons arises in connexion with the multiplication
of the bristles. A row of meshes appears on either side of each
row of developing bristles and spreads out laterally until the
meshes of adjacent rows meet, entirely replacing the old polygonal system in the middle region of the body. These new
meshes grow out from the already differentiated basal apparatuses
or from the longitudinal connexions between these. This is
directly opposite to Klein's view of the origin of these structures.
The results of Gelei on the origin of the polygonal system on
the dorsal surface were in general confirmed by the present
study. Klein's view that the polygonal system produces the
basal granules and thus indirectly the bristles cannot be supported because the basal granules arise by fission from previous
basal granules. The new polygonal system arises after fission
of the granules is completed. The exact sources and points of
538
DATUS M. HAMMOND
origin of this system have not been determined. The new meshes
arise along the rows of developing bristles and spread laterally
from these, as Gelei (1934 b) has described.
In E u p l o t e s p a t e l l a replacement of the polygonal
system occurs over the entire dorsal surface, and is not limited
to the area of multiplication of the bristles, as indicated by
Gelei. The process goes on differently outside this region. In
the extreme anterior and posterior parts of the body, each
bristle acts as a centre for development of a unit of the new
system. The development in these areas proceeds later than
that in the middle of the body, and is not noticeable until after
separation of the daughters. In such a stage each bristle toward
the posterior end of a posterior daughter is surrounded by a
circle with a longitudinal partition, in the centre of which is the
bristle. This circle widens during development until it comes
in contact with those anterior and posterior to it. A similar
replacement occurs in the anterior end of the anterior daughter.
When this process is completed, an entirely new dorsal polygonal system results.
The networks are completely formed in miniature at the time
when they can first be distinguished, and development consists merely in enlargement of the meshes and integration of the
units. The old system is resorbed as the new networks spread
out. The integration of the meshes of adjacent rows of granules
involves a fusion of the lateral walls of the approaching meshes,
since only a single line can be seen at these points in the completed system. Since the new polygonal system originates in so
many independent loci a delicate co-ordination, both as to time
and space, is involved during its development.
Other than Klein's description of a stage in the development
of the new peristome, no work has been done on the behaviour
of the polygonal system on the ventral surface of E u p l o t e s
during binary fission. This process is of considerable interest,
in that it throws light on the differentiation of the cirri.
The first indication of a new polygonal system on the ventral
surface appears as a fine network on the floor of the depression
in the ectoplasm which will develop into the new peristome.
According to Klein (1928) the appearance of the rudiment of
EUPLOTES PATELLA
539
the new peristome is the first indication of approaching binary
fission. Turner (1930) pointed out that the first of the changes
which constitute binary fission is the appearance of the reorganization bands in the macronucleus. This latter viewpoint
is upheld in the writer's study of many dividing organisms, in
which the reorganization bands invariably appeared before the
rudiment of the peristome.
The rudiment of the zone of membranelles appears along the
side of the depression. It is visible, however, as early as the fine
network, and thus there is no reason to follow the belief of Klein
that the membranelles arise from the network.
The network covering the walls and floor of the depression
spreads out over the ventral surface surrounding the opening
of the depression. In this way the frontal cirrus four, which
arises within the depression, is carried to its definitive position
on the frontal field.
Other parts of the new polygonal system on the ventral surface appear as networks of fine lines in each of the depressions
wherein the rudiments of the cirri arise (figs. 5 and 6, PI. 21;
Text-fig. 8). Including the depressions in which the left marginal
cirri arise, there are twelve separate areas covered by these fine
meshes. These become visible at about the time when the rudiments of the cirri can first be made out. Delicate networks can
be seen covering the entire surface of the depressions except the
areas occupied by the rudiments of the cirri.
As the rudiments of the cirri develop, the originally small
areas covered by the networks of fine lines increase in extent,
especially in a longitudinal direction. The networks are apparently completely formed, except for size, when they first
become visible, and development consists only in enlargement
of the meshes as the areas which they cover expand. As this
spreading occurs, there is a dedifferentiation of the old polygonal system at the edges of the areas, so that the surface
occupied by the new system increases at the expense of that
occupied by the old. This process continues until there is a
meeting of adjacent new networks, including the one arising
in the depression in which the new peristome develops, which
then form an integrated new system covering the entire ventral
NO.
316
N
n
540
DATUS M. HAMMOND
surface except the old peristomial field, and connecting with the
new system of the dorsal surface.
This throws new light on the process of ^differentiation. The
depressions in which the rudiments of the cirri arise are formed
by a dedifferentiation of the pellicle and ectoplasm at these
points. Differentiation then occurs over this area, forming
rudiments of cirri. The appearance of the fine networks in the
depressions around the rudiments indicates that a new pellicle,
and new ectoplasm or superficial layer of ectoplasm, are also
differentiated at these loci.
As these redifferentiated areas gradually expand, dedifferentiation of the old ectoplasm and pellicle with its polygonal
system occurs at the edges of the areas. That expansion of the
redifferentiated areas takes place by intussusception of new
material over the whole surface of the differentiated area, rather
than by differentiation at the margin, is shown by the fact that
the polygons are completely formed in miniature over the area
before expansion occurs. So far as could be determined, no new
meshes were added at the edges. The growth in size of meshes
can be explained only by assuming that the area is stretched or
that intussusception of new material occurs, increasing the surface without alteration of the pattern.
Since the groups of cirri arise in the centre of the expanding
areas, each cirrus is carried to its definitive position by the
spreading of those areas. A very precise co-ordination is required
both as to time and space in the development of the independent
rudiments of the polygonal system into an integrated uniform
pattern. The degree of perfection of this co-ordination is shown
by the lack of any indication of multiple origin in the completed
system. There are some indications, however, that the polygonal
lines are labile structures, thus allowing for adjustment as the
separate meshworks come together.
If, as was indicated above, the polygonal system is located in
the pellicle, the origin and development of this system shows
that the pellicle is not a lifeless secretion, but undergoes the
processes of dedifferentiation and redifferentiation characteristic of such active structures as the cirri. Also, the development by a process of spreading out, while retaining the
EUPLOTES PATELLA
541
pattern of structure, would not be possible with a secreted
covering.
It is evident that during asexual reproduction there must be
a doubling of organelles if each daughter is to have a complete
set. This doubling occurs differently in the different kinds of
cytoplasmic structures. By a series of fissions a limited number
of basal granules of bristles produce enough new bristles so that
each daughter will be provided with a complete set. One new
peristome is formed independently of the old peristome, which
is retained by the anterior daughter. In both of these cases
no apparent dedifferentiation of the structures of the parent
organism occurs. Two new complete sets of cirri and fibrillar
systems are formed independently of the old set, which undergoes resorption. The peripheral polygonal system is likewise
formed independently of the old system, which is replaced over
the entire surface pf the body with the exception of the old
peristomial field. The development of the polygonal system in
connexion with the parts of the neuromotor system indicates
a close relationship between these two systems.
SEXUAL EEPEODUCTION.
The occurrence of reorganization of the cirri and membranelles
was briefly described in E u p l o t e s c h a r o n by Engelmann
(1862). He also noted that the exconjugants possess no cytostome until two to three days after separation. Maupas (1889)
found a similar behaviour during conjugation in E u p l o t e s
p a t e l l a . According to his description the exconjugants, in
addition to having no cytostome and posterior part of the
adoral zone of membranelles, lacked one frontal cirrus of having
a complete set. This missing cirrus was found to arise at the
same time as the cytostome and remaining part of the membranelle zone, about eighty-five hours after separation of the
conjugants at twenty-two degrees. The cytostome and its
ciliary apparatus were seen to arise in a region separate from the
membranelles already present.
Turner (1930) confirmed Maupas's description of the reorganization of the cirri and membranelles during conjugation
in E u p l o t e s p a t e l l a , but did not notice the incomplete
542
DATUS M. HAMMOND
condition of the set of cirri of the exconjugant. Also, he supposed that the cytostome and missing membranelles are
gradually formed without having a separate origin as described
byMaupas(1889).
Since the nuclear phenomena of conjugation were thoroughly
investigated by Turner, that aspect of conjugation was not
considered in this study. In order to relate the cytoplasmic
processes to the nuclear behaviour, a brief summary of this will
be given as worked out by Turner (1930).
The micronucleus undergoes a preliminary division and two
maturation divisions, forming eight nuclei, of which two undergo
a post-meiotic division. Of the four products of this division
two become pronuclei and two degenerate. Eeduction of the
eight chromosomes occurs in the second maturation division,
and the migrating haploid pronuclei, with four chromosomes
each, are mutually exchanged. The zygote nucleus divides
twice and, of the four products of these divisions, one becomes
the micronucleus, one develops into the macronucleus, and two
degenerate. The macronuclear rudiment joins with a reorganized
portion of the old macronucleus to form the new macronucleus.
In the present study it was found that in addition to the
reorganization during conjugation described by Maupas (1889)
and Turner (1930) there is another reorganization which takes
place after the conjugants have separated. Thus there are two
reorganizations of the neuromotor system during sexual reproduction. The first, or that of the gamete, begins while the
conjugants are together, but before the pronuclei are exchanged;
the second, or that of the zygote, occurs in the exconjugants
during the development of the nuclei. Each of these reorganizations is similar to that which occurs during binary fission, except
that only one neuromotor system is redifferentiated instead of
two and that the peristome is replaced. This renewal of the
peristome occurs partly in the reorganization of the gamete and
partly in the reorganization of the zygote, so that in this respect
the two reorganizations are complementary.
K e o r g a n i z a t i o n of t h e G a m e t e or C o n j u g a n t .
Two organisms come together in conjugation in such a way
EUPLOTBS PATELLA
543
that the ventral surfaces of each are contiguous. A fusion of
the two bodies occurs in a small triangular area in the left
anterior region of the peristomial field of each conjugant (fig. 13,
PI. 22). At the edges of the area the peripheral polygonal lines
of the two organisms become joined, making the polygonal
systems of the two conjugants continuous with one another in
this area. Klein (1929) observed a similar coalescence of the
so-called silver-lines in an undetermined species of E u p l o t e s .
This behaviour further supports the conception of the pellicle
as a living substance.
Soon after the conjugants come together the posterior part
of the peristome is resorbed, leaving intact only the anterior
portion. This resorption is peculiar in that the cytostome with
its membranelles is first drawn into the endoplasm, where the
membranelles may remain visible for as long as two days
(fig. 12, PL 22). This phenomenon was noted by Maupas (1889).
It is probable that the fusion of the bodies of the two conjugants
in the regions of the membranelles prevents their resorption
in the manner which is usual for the cirri.
The rudiment of the anterior portion of the new peristome
arises as a depression in the ventral surface. Lining the floor
of this depression is the rudiment of the anterior portion of the
zone of membranelles. The development of this rudiment agrees
in general with the description of Turner (1930), except that the
rudiment gives rise to only the anterior portion of the new peristome instead of the whole organelle as was stated by him.
After separation of the conjugants the rudiment increases in
size, moves anteriorly, and assumes a more transverse position.
The replacement of the remnant of the old anterior portion of
the peristome by the new one occurs about two hours after
separation of the conjugants. The old membranelles are resorbed by dedifferentiation of the anterior lip region, which
starts on the sides and progresses towards the middle. The
membranelles beat irregularly until the zone has disappeared.
After replacement is completed the posterior portion of the
peristome, including the cytostome, is still lacking.
The structure of the new portion of the zone of membranelles
is similar to that of the fully developed organism, with the
544
DATUS M. HAMMOND
membranelles across the anterior end each composed of three
rows of cilia, and those on the ventral surface having each two
complete and one incomplete rows.
The new cirri become visible during the second maturation
division. Their appearance is usually, but not always, preceded
by the development of reorganization bands in the macronucleus. The new cirri arise in exactly the same manner as in
binary fission, except that only one set develops instead of two,
and the frontal cirrus four, which develops in the peristomial
field during binary fission, does not appear. This is associated
with the fact that the portion of the peristome in which the
cirrus ordinarily arises has not been redifferentiated at this
stage. As a result of this cirrus not developing the exconjugant
has only seventeen cirri instead of eighteen after the old set of
cirri has been resorbed. This agrees with the observation of
Maupas (1889). The incomplete condition of the cirri was not
noticed by Turner (1930), due to his incorrect observation of
the numbers of cirri arising in the rows of depressions on the
ventral surface. Both the left and the right marginal cirri arise
in the same localities as do those for the posterior daughter in
binary fission.
The development of the cirri and peristome is usually, but
not always, at the same stage in the two conjugants. Sometimes the rudiments are noticeably further developed in one conjugant than in the other. In these cases the reorganization bands
are farther along in the conjugant in which the cytoplasmic
organelles are further developed.
Eesorption of the cirri begins while the conjugants are still
together, and is completed within one hour after their separation
occurs.
Although no preparations by silver impregnation method of
late stages of conjugants were obtained, the replacement of
the ventral polygonal network during conjugation was indicated
by the alcoholic haematoxylin preparations.
The bristles undergo no visible change in structure during
conjugation. That the bristles are not included in the reorganization process during conjugation may be correlated with the fact
that they are not reorganized during binary fission, but merely
EUPLOTES PATELLA
545
undergo a multiplication process so that there will be a sufficient
number for both daughters.
R e o r g a n i z a t i o n of t h e Z y g o t e .
When replacement of the cirri and anterior portion of the
peristome is completed, the exconjugants are lacking frontal
cirrus four and the posterior part of the peristome. About two
to three days after separation of the conjugants, the macronuclear rudiment, which until that time has a spherical shape,
begins to elongate in a lateral direction. At this time the rudiment of the posterior part of the peristome appears as a depression immediately posterior to the end of the incomplete zone of
membranelles (fig. 14, PI. 22; Text-fig. 3). On the floor of this
depression a series of rudiments of membranelles appear, the
most anterior of which is contiguous with the most posterior of
the membranelles already present. This rudiment of the posterior part of the zone of membranelles has nearly the same
width as the fully formed anterior part of the zone, but the
bases of the membranelles are crowded very close together. The
pocket gradually extends backward, lengthening the interval
between membranelles, and at the same time the cytopharynx
and its ciliary apparatus are formed.
This agrees in general with Maupas's account of the origin of
the posterior portion of the peristome, except that he described
the rudiment as arising more posteriorly in the middle of the
ventral surface.
Soon after the appearance of the posterior portion of the
peristome, the rudiments of an entire set of cirri appear in the
same manner as in the reorganization of the gamete (Text-fig. 3).
The frontal cirrus four arises under the right margin of the
developing peristome, so that, when replacement is complete,
there results an organism with an entire peristome and a full
set of cirri. So far as could be determined the bristles do not
undergo any change of structure during this period. Immediately after the completion of this reorganization, and very
often before the macronucleus has assumed its usual shape, the
organism undergoes a binary fission during which cytoplasmic
reorganization proceeds as usual.
546
DATUS M. HAMMOND
Maupas (1889) observed only a portion of the reorganization
of the zygote in E u p l o t e s p a t e l l a . The replacement of
the cirri escaped his attention and he supposed that cirrus four,
missing in the organization of the gamete, arises separately
either as a new formation, or from a division of a cirrus already
present. However, the entire process of reorganization of the
zygote, including replacement of the cirri, was described by
Maupas (1889) in O n y c h o d r o m u s g r a n d i s , S t y l o n y c h i a p u s t u l a t a , and O x y t r i c h a f a l l a x . He interpreted
the reorganization during conjugation as a simple process of
regeneration with the function of replacing the organelles, which
might have been injured in the course of conjugation. The
second reorganization as found in O n y c h o d r o m u s , &c,
was regarded as associated with the particular arrangement of
the ciliary apparatus of the species in question.
In the present study it has been shown that the two periods
of reorganization during sexual reproduction have a definite
relationship to the phases of the life-cycle. The first reorganization takes place in the gamete, and is associated with the
haploid condition of the chromosomes. The second reorganization occurs in the zygote, and is associated with the restoration
of the diploid condition resulting from fertilization.
Each phase of the life-cycle has its own organization. Some
of the structures, such as the bristles, are apparently continuous
from one organization to the next, but for the most part the
differentiated structures in the organization are resorbed at the
end of each phase. This periodic loss of differentiation may be
a factor in preventing senescence of the organism.
DISCUSSION.
It has been shown that profound changes in protoplasmic
organization are correlated with both asexual and sexual reproduction in E u p l o t e s p a t e l l a . During each of these periods
in the life-cycle there is a dedifferentiation and a ^differentiation
of certain cytoplasmic structures, including the cirri, the fibrils
attached to the bases of these, and the peripheral polygonal
system. This process occurs twice during sexual reproduction:
once in the gamete, and once in the zygote. The peristome
EUPLOTES PATELLA
547
undergoes this process of renewal during sexual reproduction,
but there is no evidence of dedifferentiation in this structure
during asexual reproduction. The more complete reorganization
of the neuromotor system during sexual reproduction is associated with the more profound nuclear changes at this time.
Dedifferentiation of the bristles was not seen at any time.
Unless some kind of renewal occurs, the peristome and bristles
must be worn out or broken off in the course of time. The old
peristome is similarly retained during binary fission in several
ciliates, notably S t e n t o r (Hetherington, 1932; Schwartz,
1935). Schwartz (1935) described a periodic reorganization in
S t e n t o r, during which there is a reorganization of the nuclei
and a replacement of only the oral portion of the membranelle
zone. Thus the aboral portion of the membranelle zone is
retained apparently unchanged both in the periodic reorganization and during binary fission. It has been shown that when
S t e n t o r is placed in a 0-5 per cent, solution of NaCl the
membranelles are thrown off, but the basal structures are retained, and new membranelles grow forth from these (Prowazek,
1904). Schwartz (1935) suggests that this may occur normally
when the membranelles are in need of replacement. This sort
of renewal might also take place in the membranelles and bristles
of E u p l o t e s .
Eeorganization of protoplasmic structure during asexual
reproduction has been described in many other Protozoa. Taylor
(1928) confirmed Wallengren's (1901) description of the replacement of the entire set of locomotor organelles in U r o n y c h i a .
Summers (1935) noted a more or less complete cytoplasmic
reorganization in D i o p h r y s , A s p i d i s c a l y n c e u s , and
S t y l o n y c h i a . Poljansky (1934) noted a complete renewal
of cytoplasmic organelles in B u r s a r i a
truncatella.
MacDougall (1925) found that the cilia and trichites are entirely
renewed in C h i l o d o n u n c i n a t u s during binary fission.
Lucas (1932) described a complete reorganization of the ciliary
apparatus in C y a t h o d i n i u m p i r i f o r m e .
Reorganization of cytoplasmic structure during conjugation
has also been described in many ciliates. In C h i l o d o n
u n c i n a t u s (MacDougall, 1925)a reorganization of the pharyn-
548
DATUS M. HAMMOND
geal basket and some of the cilia takes place at this time. Two
reorganizations occurring during sexual reproduction have been
noted only in O n y c h o d r o m u s g r a n d i s , S t y l o n y c h i a
p u s t u l a t a , and O x y t r i c h a f a l l a x (Maupas, 1889),
although it is probable that other cases would be found if a
thorough study of other hypotrichous ciliates were made.
Keorganization of cytoplasmic structure may also occur at
other periods during the life-cycle. Although encystment has
not been observed in E u p l o t e s p a t e l l a , Garnjobst (1928)
showed that a complete dedifferentiation of external organelles,
accompanied by nuclear reorganization, occurs during the process of encystment in E u p l o t e s t a y l o r i .
It is known that a reorganization, similar to that which occurs
during binary fission, takes place if the organism is mutilated.
This has been proven for E u p l o t e s p a t e l l a (Taylor, 1923;
Reynolds, 1932), E u p l o t e s v a n n u s , E u p l o t e s c h a r o n ,
D i o p h r y s , A m p h i s i a (Dembowska, 1926), and U r o n y c h i a (Calkins, 1911; Young, 1922; Taylor, 1928).
A periodic reorganization of certain of the structures in
S t e n t o r (Schwartz, 1935) and C y a t h o d i n i u m p i r i f o r m e
(Lucas, 1932) may occur without reference to any basic phase
of the life-cycle.
The manner of reorganization is different in the various forms
described. In some cases certain cytoplasmic structures persist
through the stage of dedifferentiation and give rise to the new
ciliary apparatus during redifferentiation. Chatton and his
collaborators have described the formation of the new ciliary
apparatus by division of the basal bodies of the cilia of the parent
in C h i l o d o n (1931 a) and other ciliates. A process of multiple
fission of the basal granules of the cilia on either side of the plane
of division has been described i n P a r a m e c i u m (Gelei, 1934d),
which closely corresponds to the method of multiplication of the
bristles in E u p l o t e s p a t e l l a .
Klein (1928, 1932) supposed that the new ciliary organelles
are formed in s i t u from the so-called silver-line system. As
pointed out above, the rudiments of the ciliary organelles in
E u p l o t e s p a t e l l a are not connected with the old polygonal
lines, and they arise either before or at the same time as the
EUPLOTES PATELLA
549
new polygonal lines. Therefore the ciliary apparatus is not
derived from the polygonal system. However, the differentiation
of the polygonal system is closely associated with that of the
neuromotor system, which is indicative of a relationship between
them.
MacLennan (1935) found a continuity in structure of the
neuromotor system through the stage of dedifferentiation in
I c h t h y o p h t h i r i u s . During redifferentiation the new ciliary
apparatus was found to develop from fibrils of the neuromotor
system.
All the above cases involve some degree of visible cytoplasmic structure persisting through the stage of dedifferentiation and giving rise to the new parts of the ciliary apparatus
during redifferentiation. In E u p l o t e s p a t e l l a the bristles
are the only structures derived from similar structures in the
previous organization.
In another group of organisms, especially the hypotrichous
ciliates, the new ciliary apparatus is derived from apparently
undifferentiated cytoplasm. This group includes E u p l o t e s
h a r p a (Wallengren, 1901), E u p l o t e s w o r c e s t e r i (Griffin,
1910b), and U r o n y c h i a u n c i n a t a (Taylor, 1928). The
redifferentiation of the peristome, polygonal system, and cirri
in E u p l o t e s p a t e l l a belongs to this category. In all these
forms the ciliary organelles arise in the ectoplasm, but an endoplasmic origin of the ciliature has been described in C y a t h o d i n i u m p i r i f o r m e (Lucas, 1932).
The regenerative activities are localized in a region at the
future plane of constriction in E u p l o t e s p a t e l l a . This is
shown by the appearance within this region of all the rudiments
of the various parts of the neuromotor system except the right
marginal cirri, and the extreme posterior and anterior parts of
the peripheral polygonal system. This localization is best shown
in connexion with the multiplication of the bristles, which is
most active at the future plane of constriction and decreases
both anteriorly and posteriorly from this region.
That these reorganization processes occur at very definite
times in the life-cycle of the organism may be correlated with
the physiological condition of the organism at these times.
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DATUS M. HAMMOND
Calkins (1911) and Young (1922) have shown in U r o n y c h i a
that the power to regenerate is at a maximum at the time of
binary fission, and at a minimum after division is completed.
This was explained by suggesting that during its condensation
the macronucleus gives off a substance into the cytoplasm which
influences the development of new organelles (Calkins, 1911), or
that a substance dependent upon the presence of the micronucleus accumulates in the protoplasm up to the time of binary
fission, when it is used in the regeneration of organelles (Young,
1922). That the macronucleus might give off an activating
substance is supported by the fact that the progressive reorganization of the macronucleus always precedes the changes
in the visible structure of the cytoplasm i n E u p l o t e s p a t e l l a .
Child (1916) advanced the view that the processes of reorganization which accompany binary fission in the Protozoa
result in rejuvenescence. This tends to counteract senescence,
which consists in a decrease in the metabolic rate and a progressive accumulation of the more stable components of the
substratum of the protoplasm during growth, development, and
differentiation. Calkins (1933) has developed this viewpoint,
and has shown that there are evidences of reorganization during
division in all the groups of the Protozoa. Taylor (1928)
suggested how rejuvenation might be brought about by dispersion of the molecular components of the formed structures
during dedifferentiation and recombination of these during
redifferentiation, in which process the products which tend to
decrease the metabolic rate may be eliminated.
More recently, Taylor (1935) has proposed the view that there
is a comparable return to a condition of minimal differentiation
in binary fission, conjugation, encystment, and regeneration.
This does not hold true for E u p l o t e s p a t e l l a , since reorganization in binary fission differs from that during conjugation in having no replacement of the peristome. Thus Taylor's
conclusion that binary fission, as well as each of the other stages
in the life-cycle in which reorganization occurs, is comparable
to the end of an old life-cycle, and the beginning of a new one
in the Metazoa is not supported by the facts in E u p l o t e s
patella.
EUPLOTES PATELLA
551
The structural changes in the protoplasm are closely related
to the basic phases of the life-cycle. The zygote is formed at
the time of fertilization and inherits the body of the gamete
with its cytoplasmic structures. The zygote then undergoes
nuclear divisions, in which four products of the fertilization
nucleus are formed, followed by differentiation of nuclear
material into macronucleus and micronucleus. Coincident with
the differentiation of the nuclei there is a change in cytoplasmic
structure of the body, during which the organization of the
gamete is replaced by an organization developed under the
influence of the new combination of genetic material. Any
hereditary changes in the structure of the organism should be
expressed at this time.
The zygote then undergoes a period of asexual reproductions,
during which no new nuclear material is introduced. Some of
the cytoplasmic changes during binary fission may be explained
by the need of the doubling of organelles. This would include
the multiplication of bristles and origin of the new peristome.
Changes such as the development of two sets of cirri may be
explained only partly on this basis. Wallengren (1901) supposed
that two entirely new sets of cirri were developed during binary
fission in order to produce a matched set, and to replace the
old worn-out cirri. This explanation would not hold in E u p l o t e s p a t e l l a where the old membranelle zone is retained,
resulting in an unmatched condition of the locomotor organelles.
The dedifferentiation of structure during asexual reproduction
may serve the function of rejuvenation.
This periodic rejuvenation during asexual reproduction might
help to explain why certain species of Protozoa may continue
to thrive in culture for an indefinite period of time without the
occurrence of sexual reproduction. A somewhat similar protoplasmic reorganization occurs in the cells of Metazoa during
the process of mitosis, as pointed out by Taylor (1928). The
rejuvenation which may accompany this reorganization might
help to explain why tissues can be cultured indefinitely in the
proper medium.
Under certain conditions not fully known the organism may
undergo sexual reproduction. During this process there is a
552
DATUS M. HAMMOND
maturation of the micronuclei involving the reduction of
chromosomes from the diploid to the haploid number. A reorganization of cytoplasmic structure is associated with this
change in nuclear condition so that the haploid gamete acquires
a new cytoplasmic organization. By the process of fertilization
the diploid condition is restored. The resulting zygote receives
a new set of cytoplasmic structures by means of the reorganization following fertilization.
The problem of endomixis was not touched in this study.
Investigation of the protoplasmic reorganization during endomixis is needed to complete the problem.
SUMMARY.
1. The neuromotor system of E u p l o t e s p a t e l l a Ehrenberg was studied in different stages of the life-cycle, including
the adult, and asexual and sexual reproduction.
2. In the adult the basal granules of all the cirri are arranged
in straight primary and secondary rows. The fibrils from the
bases of the cirri are in general parallel with either the primary
or secondary rows of basal granules of the cirri to which they
are attached. This is interpreted as an indication of the evolution of the organism from an ancestor with a simple neuromotor
system in which the cilia were uniformly distributed over the
surface of the body in longitudinal, slightly spiral rows.
3. The basal apparatus of a bristle consists of a basal granule
at the bottom of a depression in the ectoplasm which is surrounded by a group of rodlets and ends at the surface in a ring
connected with the polygonal system. The polygonal or silverline system is pellicular in position.
4. During asexual reproduction the entire set of cirri is
resorbed and replaced by two new sets of cirri, one for each
daughter. One frontal cirrus for the anterior daughter develops
in the old peristomial field of the parent, while the corresponding
cirrus for the posterior daughter develops in the new peristome.
The right marginal cirri arise on the dorsal surface along the
rows of bristles and later move to the ventral surface. The
fibrils develop outward from the bases of the cirri.
5. During binary fission a new peristome arises in a depression
EUPLOTES PATELLA
553
in the ectoplasm independently of the old peristome. The old
peristome remains in the anterior daughter without at the time
undergoing any visible change in structure.
6. The multiplication of the bristles is limited to a zone on
either side of the future plane of constriction. Multiplication
occurs by a series of fissions of the basal granules in this area.
Dedifferentiation of bristles was not seen.
7. The polygonal system is replaced over the entire surface
of the body with the exception of the old peristomial field. The
new system originates in many separate loci in connexion with
the bristles on the dorsal surface, and with the rudiments
(Anlagen) of the cirri and peristome on the ventral surface.
8. During conjugation there are two successive reorganizations of the neuromotor system, that of the 'gamete' (conjugant)
and that of the zygote.
9. The first reorganization, that of the gamete, begins during
the maturation divisions preparatory to fertilization. During
this reorganization the posterior portion of the peristome is
resorbed and the remaining anterior portion is later replaced.
The eighteen cirri are replaced by a set containing only seventeen
cirri.
10. The second reorganization, that of the zygote, occurs
during the differentiation of the nuclei in the exconjugants. It
involves the completion of the peristome by differentiation of
the missing posterior portion, and the replacement of the set
of seventeen cirri by a complete set of eighteen cirri.
11. These protoplasmic reorganizations, invariably associated
with both asexual and sexual reproduction, involve a structural
and presumably a physiological rejuvenation resulting in the
possibility of an indefinitely continued existence of the individual
in the Protozoa. It may be that sexual reproduction is not
necessary to bring about this result, but further work on the
relationship of endomixis to the problem is needed.
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EXPLANATION OF PLATES 21 AND 22.
All figures are microphotographs taken by Mr. J. E. Gullberg of the
Cinematographical Laboratory of the University of California.
PLATE 21.
Kg. 1.—Dorsal surface of E u p l o t e s p a t e l l a in stage of binary
fission, showing process of multiplication of bristles. Specimen fixed in
Champy's fluid, stained in Heidenhain's haematoxylin. x 1,300.
Fig. 2.—Another specimen in about the same stage as that in fig. 1.
The basal granules of the six central bristles are undergoing fission.
Champy's, Heidenhain's haematoxylin. X 1,300.
BUPLOTES PATELLA
557
Fig. 3.—A later stage in binary fission. The new basal granules have
given rise to new bristles. In the centre of the row of bristles is the rudiment of one of the marginal cirri (right) of the anterior daughter. Chanipy 's,
Heidenhain's haematoxylin. x 1,300.
Fig. 4.—Ventral surface of organism in stage of binary fission. Cytopharynx near lower right (of organism). A portion of the peripheral polygonal system is visible posteriorly and to left of cytopharynx. In the two
rows of five depressions, the rudiments of the new cirri can be seen. Wet
silver method, x 650.
Fig. 5.—A later stage in binary fission. New networks of the peripheral
polygonal system are visible in each depression. The rudiments of the cirri
are more widely separated. The rudiments of the left marginal cirri for
the anterior daughter appear to the (organism's) left of cytopharynx. Wet
silver method, x 650.
Fig. 6.—Same aa fig. 4, at a higher magnification, showing detail of one
of the networks of the new polygonal system. X 1,650.
PLATE 22.
Fig. 7.—Dorsal surface o f E u p l o t e s p a t e l l a , showing rodlets which
surround the depression wherein the basal granule of the bristle lies.
Silver-osmic acid-formalin method. X 650.
Fig. 8.—Dorsal surface, showing groups of rodlets at the ends of the
membranelle plates in the anterior portion of the adoral zone. Silver-osmic
acid-formalin method. X 650.
Fig. 9.—Same asfig.8, at a higher magnification, to show detail of groups
of rodlets. X 1,650.
Fig. 10.—Ventral surface, showing basal structure of membranelles.
Incomplete row of cilia is visible at the (organism's) right side of each
membranelle. Silver-osmic acid-formalin method. X 1,650.
Fig. 11.—Ventral surface of organism in stage of binary fission. Rudiment of new peristome is posterior and to the (organism's) left of the old
cytopharynx. Two depressions of each transverse row of five are visible,
and three rudiments of cirri appear in each of these. Champy's, Heidenhain's haematoxylin. X 650.
Fig. 12.—Organism during conjugation, showing posterior portion of
zone of membranelles in a rolled-up position inside the body. Macronucleus appears anteriorly. Schaudinn's, alcoholic haematoxylin. X 1,300.
Fig. 13.—Conjugants, showing triangular area of fusion of the two
bodies. Membranelle zones, visible posterior to the area of fusion, have not
yet been taken into the endoplasm. Wet silver method. X 1,300.
Fig. 14.—Reorganization of zygote. Macronuclear rudiment appears as
a round, darldy staining body. The rudiment of the posterior portion of
the peristome is visible as a crescentic structure anterior to remnant of old
macronucleus. Rudiments of new cirri, and fibrils of the old anal cirri appear at (organism's) left side. Schaudinn's, alcoholic haematoxylin. X 1,300.
. Journ. Micr. Sci
D. M. Hammond
Vol. 79, N. S., PI 21
Quart. Journ. Micr. Sci. Vol. 79, N. S., PI 22
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D. M. Hammond