University of Southern California Dissertations and Theses

On the ultrastructure and morphogenesis of a marine chonotrichous ciliateprotozoan, Lobochona prorates

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On the ultrastructure and morphogenesis of a marine chonotrichous ciliateprotozoan, Lobochona prorates

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Content ON THE ULTRASTRUCTURE AND MORPHOGENESIS OF A MARINE
CHONOTRICHOUS ClLIATE PROTOZOAN, Lobochona prorates
by
Hitoshi Matsudo
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biology)
June 1967
UNIVERSITY O F S O U T H E R N CALIFORNIA
T H E GRA DUATE SC H O O L
UN IV ER SITY PARK
LO S A N G ELES, C A L IFO R N IA 9 0 0 0 7
This dissertation, written by
HITOSHI f^TSUDO
under the direction of hXS....Dissertation C om
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R O F P H I L O S O P H Y
Dean
Date................June 8^ 1967
DISSERTATION COM M ITTEE
Chairman
TABLE OF CONTENTS
Page
LIST OF TABLES.................................... iii
LIST OF FIGURES.................................. iv
INTRODUCTION .................................... 1
Acknowledgements ........................... 2
HISTORICAL REVIEW OF THE ORDER CHONOTRICHIDA . . 4
MATERIALS AND METHODS ........................... 7
OBSERVATIONS .................................... 15
General Morphology of Lobochona prorates . . 15
Pellicle and Associated Structures ........ 16
Buccal Apparatus ............................ 21
Nuclear Apparatus ..................... 25
Cytoplasmic Matrix and Inclusions .......... 27
Morphogenesis .............................. 31
Morphogenesis of the Bud................... 34
Diminutive Budding ..................... 36
Conjugation................................ 38
DISCUSSION...................................... 42
SUMMARY ........................................ 74
LITERATURE C I T E D ................................ 77
EXPLANATION OF FIGURES......................... 89
ii
LIST OF TABLES
Table Page
I. Osmium Fixatives ........................ 8
II. Aldehyde Fixatives ...................... 9
iii
LIST OF FIGURES
Figure Page
1. Drawing of mature Lobochona prorates . . 90
2. Electron micrograph of mature L. prorates 92
3-28. Pellicle and associated structures ... 94
29-47. Buccal apparatus ....................... 110
48-51. Nuclear apparatus ........ . 132
52-63. Cytoplasmic matrix and inclusions .... 136
64-84. Morphogenesis ........................... 144
85-88. Morphogenesis of the b u d ................ 164
89-94. Diminutive budding ..................... 172
95-99. Conjugation ............................. 182
iv
"There is nothing so minute, or inconsiderable, that I
would not rather know it than not."
Samuel Johnson 1709-84
INTRODUCTION
Anthony van Leeuwenhoek's observations on the free-
living Protozoa began with his discovery of certain "very
little animalcules" which he saw in the year 1674 with his
"best microscope" that he kept "for himself alone" and by
his "particular manner of observing very small creatures"
for "best observation" (Dobell, 1932). Since then, the
development of optical lenses and construction of the
compound microscope and many researchers have contributed
to the knowledge of these animalcules. Their contributions
have resulted in an abundance of literature about Protozoa.
The observations of the "classical" workers and the present
state of perfection of the electron microscope and con
comitant development of techniques for the preparation of
biological specimens for electron microscopy together make
these organisms especially well suited for ultrastructural
investigation.
Most ultrastructural studies of protozoans have been
2
limited to specific organelles (e.g. cilia) or to the
ultrastructure of an organism. These studies, however,
have shown that the basic organization and structure of
organelles in Protozoa — cilia, mitochondria, endoplasmic
reticulum, Golgi — parallel closely the structure of the
same organelles found in other organisms but also that
great variation occurs among Protozoa.
The object of this investigation is to study the
ultrastructure and morphogenesis of Lobochona prorates,
a chonotrichous ciliate living on the abdominal appendages
of a crustacean wood borer, Limnoria tripunctata, in order
to better understand its biology and to get a better notion
as to its affinities with groups which are reported to be
closely related.
ACKNOWLEDGEMENTS
The author wishes~-to acknowledge his indebtedness and
sincere gratitude to Professor John L. Mohr, Chairman of
the Dissertation Committee, to Dr. Robert F. Bils, Co-
Chairman, for instruction and use of the electron micro
scope, to Dr. Russel L. Zimmer for helpful suggestions, and
to Dr. John S. Garth, Dr. Thomas R. Pray, and Dr. Donn S.
Gorsline, members of the Dissertation Committee.
The writer also wishes to acknowledge his indebtedness
to the research programs supported by the Office of Naval
Research's Biology and Geography Branches (NONR 228(19),
NR 307-270) and the National Science Foundation (GB-4254)
to Dr. John L. Mohr through which an enlarged possibility
of materials were made available. Various other facilities
were provided by the Allan Hancock Foundation of the
University of Southern California. Earlier work of Mr.
J. A. LeVeque, who found the organism and studied its
natural history (Master's thesis), provided many leads.
HISTORICAL REVIEW OF THE ORDER CHONOTRICHIDA
The Order Chonotrichida is a relatively small and
little known group of ciliates which generally occur as
epizoans on crustaceans. The original species, Spirochona
qemmipara, was described by Stein (1851) from Tharand,
near Dresden, Germany, from the gills of Gammarus pulex —
a fresh-water gammaridean amphipod widespread throughout
Europe. Because of its spiral apical funnel, Stein (1854)
put £>. qemmipara in a new family (Spirochonina) beside
the vorticellids, and later erected the Order Peritricha
to hold both Spirochonina and vorticellids. Clapar&de and
Lachmann (1858) observed that the affinities of Spirochona
were uncertain and placed them in an appendix to the
vorticellids. The Peritricha was divided by Delage and
Herouard (1896) into dexiotrichs (right spiralled) and
scaiotrichs (left spiralled) and spirochonines. Eismond
(1890, 1895) studied the peristome of Spirochona critical
ly; however, it remained for Wallengren (1895) to separate
and establish the "Sektion" (= Order) Chonotricha with two
families — Chilodochonina and Spirochonina. Mohr (1948),
further divided the order into three families on the basis
4
of funnel structure — Chilodochonidae (with "lips" rather
than a funnel, Spirochonidae (with spiralled funnel), and
Stylochonidae (with funnel not spiralled).
Recently, the taxonomic position of the Chonotrichida
has been questioned. Faur&-Fremiet and disciples
(Guilcher, 1951; Corliss, 1956, 1961) would place the
chonotrichs as an independent order near the suborder
Cyrtophorina of the Order Gymnostomatida. Dobrzanska-
Kaczanowska (1963) and Raabe (1964) treat the chonotrichs
as a suborder of the gymnostomes and suggest that possibly
it should be considered only a family in the cyrtophorine
hypostomes. However, Cheissin and Poljansky (1963) con
sider the chonotrichs as one of the subclasses of Ciliata.
Mohr (1966), in considering the age of the chonotrichs, is
inclined to agree with the position of Cheissin and
Poljansky.
Cytological studies have been made by Hertwig (1877),
Plate (1886), Balbiani (1895), Stein (1859), Doflein
(1897), and Swarczewsky (1928)* Recently, Guilcher (1951)
made a comparative study of Chilodochona quennerstedti and
Spirochona qemmipara, and Tuffrau (1953), a cytological
study of conjugation in ,S. qemmipara. Dobrzanska-
Kaczanowska (1963) compared morphogenesis of Heliochona
acheutenii with that of the gymnostomes, Chilodonella
uncinata and Allosphaerium paraconvexa. Matsudo (1966)
studied the life cycle of Lobochona prorates and also
included a few electron micrographs. There has been but
one other electron microscopic observation of a chonotrich-
ous ciliate — that by Faure-Fremiet, Rouiller, and
Gauchery (1956) on the attachment organelle of Chilodochona
guennerstedti.
The genus Lobochona was described and curiously
figured by Dons (1940). LeVeque (thesis) and Matsudo
(1966), however, were able to confirm the curious features
of the genus.
MATERIALS AND METHODS
Over a period of three years, Limnoria tripunctata
were collected and fixed instantly in various fixatives
(Tables I and II) at the California Yacht Club Anchorage,
San Pedro, California.
Mitochondria are considered to be sensitive organelles*
thus indicative of good or poor fixation (Pease, 1960).
The electron micrographs of ciliate mitochondria in the
literature generally appear as dense and compact organelles.
However, the fixatives used by other investigators for
Protozoa (also used for Metazoa) produced empty, swollen
mitochondria for Lobochona prorates. The writer has also
assumed that the dense, compact mitochondria are indicative
of good fixation and representative of the most nearly
true state of the organelle attainable with fixation tech
niques employed at present. Fifty combinations of
principal component, buffer, temperature, pH, and plastic
were tested before mitochondria of L. prorates appeared
reasonably "normal".
Glutaraldehyde proved to be superior to osmium
tetroxide for fixing mitochondria of L. prorates. The
"1 'VIMNINT EIR CENT BUFFER pH
■ ' >: 1 j" ' t i t I i X 1 d i . P.lladf 1 Veronal acetate B. 3
u s m l u i m c* troxi Zetter4vist 1 Veronal acetate 7 .1
Gsmium tetroxide 1 Sea water 8.6
Osmium tetroxide Caufleld 1 Veronal acetate 8.2
Osmium tstroxids Palade 1 Veronal acetate 7.4
OSRUum-chrooste Dslton 1 7.1
Osmium tstroxids + MgCl 1 Veronal acetate 7.4
Osmium tstroxids 1 Sea water 8,6
Osmium tstroxids vapor 2
Osmium tstroxids vapor 2
Osmlnm tstroxids vapor 2
Osmium tstrumids vapor 2
Osmlsm tstrsmids 1 Sea water 8.6
Osmium tstrumids 1 Sea water 8.6
Osmium tstrumids 1 Sea water 8.6
Oamlm tstrumids 1 Sea water 7.3
Osmium tstrsmids 1 2*1* Sea water 8.4
Osmium tstrsmids 1 Veronal acetate 7.4
OsmiiM tstrumids 1 2*1* Sea water 8.6
Osmium tstrumids 1 lodioa bicarbonate 6.4
Osmium tstroxids 1 Veronal acetate 6.4
Osmium tstroxids 2 2"X" Sea water 6.9
os ml um tstroxids 2 Sodiua bicarbonate 6.9
Osmium tstroxids ♦ HAc 2 2"X" Sea water 3 . f c
TEMPERATURE TIME EMBEDDING MEDIUM
Ice 2 hrs Me thacr ylate
Ice 2 hrs Methacrylate
Ice 2 hrs Methacrylate
Ice 2 hr* Methacrylate
Ice 2 hrs Methacrylate
Ice 2 hr* Methacrylate
Ice 15 coins Vestopal
Ice 15 nine Veatopal
Ice 2 nine Vestopal
Ice 5 nine Veatopal
Air 2 nine Veatopal
Air 5 aina Veatopal
Air 10 aina Veatopal
Air 10 aina Methacrylate
Air 30 aina Veatopal
Ice 1 hr Veatopal
Ice 30 aina Veatopal
Ice 1 hr Veatopal
Ice 1 hr Epon
Ice 1 hr Epon
Ice 1 hr Epon
Ice 1 hr Epon
Ice 1 hr Epon
Ice 1 hr Epon
00
TABLE
\l component PER CENT BUFFER
CluL.ii .ildi hyde
Glutjr.ildehyde
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde + NaCl
Glutaraldehyde Manton
Glutaraldehyde
Glutaraldehyde + NaCl
Glutaraldehyde
Glutaraldehyde
Glutaraldehyde + NaCl
Glutaraldehyde + NaCl
Glutaraldehyde + NaCl
Glutaraldehyde + NaCl
Glutaraldehyde + NaCl
Hydroxyadipaldehyde
Hydroxyadipaldehyde
Hydr oxyadipaldehyde
Hydroxyadipaldehyde
Hydroxyadipaldehyde
Hydroxyadipaldehyde
Paraformaldehyde
Paraformaldehyde/Glutaraldehyde
Acrolein
Permanganate t,uft
Permanganate Luft
5 Sea water 6.1
5 Phosphate 7.4
4 Phosphate 7.4
4 Phosphate 7.4
4 Phosphate 7.4
6,5 Cacodylate 7.2
5 Sea water 7,1
5 2"X" Sea water 7.2
5 Cacodylate 7.0
5 2"X" Sea water 7.0
5 Sea water 6.S
5 Sea water 4.2
5 Sea water 6.25
5 Sea water 6.2
5 Sea water 6.1
12.5 Cacodylate 7.1
12.5 Veronal acetate 7.0
12.5 2"X" Sea water 7.4
12.5 2"X" Sea water 7.5
12.5 2“X" Sea water 7.5
12.5 Cacodylate 6.4
4.0 2"X" Sea water 7.2
4/4 2"X" Sea water 7.2
10.0 Sea water 6.8
0.5
0.5
TEMPIRATURI TIME i MM EE INC MINIUM
Ice
Ice
Ice
Alt
Ice
Ice
Ice
Ice
Ice
Ice
32 5C
32°C
32 °C
32°C
32°C
Ice
Ice
Ice
Ice
Ice
Ice
Ice
Ice
Ice
Ice
Icc
2 hrs Mttr t v 1 a tt
2 hrs Methacryljti
2 hrs Vestopal
2 hrs Vestopal
2 hrs Vestopal
2 hrs Vestopal
2 hrs Vestopal
2 hrs Epon
2 hrs Epon
2 hrs Epon
2 hrs Epon
>
2 hrs Epon w
F
2 hrs Epon w
2 hrs Epon H
H
2 hrs Epon
2 hrs Vestopal
2 hrs Vestopal
2 hrs Vestopal
2 hrs Vestopal
2 hrs Epon
2 hrs Epon
2 hrs Epon
2 hrs Epon
2 hrs Vestopal
1 nun Methacrylate
21 nuns Methacrylate
10
10
ciliates were originally fixed as recommended by Palade
(in Pease, 1960), using his fixative at high pH (8.4 for
hydrated tissues and protozoans) and embedded in pre
polymerized methyl-butyl methacrylate. The result of
fixation was not readily apparent because of polymerization
damage caused by methacrylate. The use of Vestopal W
reduced polymerization damage and the swollen mitochondria
became apparent. Osmium tetroxide fixation followed by
embeddment in Epon 812 (provided by Dr. R. L. Zimmer)
essentially eliminated polymerization damage? however,
mitochondria still appeared swollen. Addition of sucrose,
magnesium chloride or calcium chloride to the fixative
proved useless. Better results were obtained when sodium
chloride was added to an equal (v/v) mixture of a 2%
aqueous solution of osmium tetroxide and sea water to bring
the total sodium chloride concentration to approximately
that of sea water. Finally, in an attempt to equalize
tonicity of fixative with that of sea water, the sea water
was allowed to evaporate to half its original volume
(= "2X" sea water) and this was mixed with an equal volume
of 2 or 4% aqueous solution of osmium tetroxide to make a
1 or 2% osmium fixative. This fixative, however, did not
appreciably improve the fixation of mitochondria.
11
For glutaraldehyde fixatives, the stock 25% glutar
aldehyde solution was diluted to 5% with sea water and
sufficient sodium chloride was added to bring the sodium
chloride concentration of the fixative to approximately
that of sea water.
Slow penetration rate of osmium became the prime
suspect as cause of poor mitochondrial fixation. The
pellicle of L. prorates is thinner within the funnel than
at the level of the cytosome. Mitochondria in the funnel
appeared more compact than those within the cytosome. A
review of cytological methods for mitochondria by light
microscopy by earlier workers showed fixatives to be acidic
and that penetration rates of aldehydes in general were
about twice that of osmium tetroxide. To increase the
rate of penetration, the temperature of the osmium fixative
was raised to 32° C. and pH lowered to 3.6; however, mito
chondria were still swollen in the cytosome. Lowering of
pH and raising temperature of the glutaraldehyde fixative
proved most fruitful. Mitochondria appeared best after
exposure to pH 6.1 to 6.25. At pH 4.2 the cytoplasm of
the ciliate appeared somewhat flocculent. Glutaraldehyde-
fixed specimens were postfixed in 1% or 2% osmium tetroxide
(a mordant for subsequent lead staining) buffered with sea
12
water.
Acid and alkaline phosphatase reactions were carried
out according to the methods of Gomori (1952); adenosine
triphosphatase activity was localized according to the
method of Tice and Barrnett (1962). Silver impregnation —
both Bodian (1937) and Chatton-Lwoff (after Corliss, 1953)
techniques — was carried out as described for light micro
scopy. Ribose nucleic acid was extracted according to the
method of Erickson (in Pearse, 1954).
After fixation the chonotrich-bearing gribbles were
dehydrated in a graded series of ethanol. The pleopods
were removed in a mixture of solvent and plastic and only
those infested with chonotrichs were embedded in meth
acrylate, Vestopal W, or Epon 812. Specimens were
sectioned with a diamond knife on a Porter-Blum microtome
at 500 to 600A. The thin sections were collected on
collodion coated and carbon backed copper grids and stained
with lead ammonium acetate (Bjftrkman and Hellstr&m, 1965).
Lead hydroxide (Watson, 1958), lead citrate (Reynolds,
1963), and lead oxide (Karnovsky, 1961) stains were also
tested, but they produced more contamination than lead
ammonium acetate; their use was therefore discontinued.
Grids were examined with an RCA EMU3F electron microscope
13
at 50KV.
The writer discovered accidentally that aldehyde-
fixed specimens not postfixed in osmium tetroxide were not
stainable with lead. Grids with sections of glutaraldehyde
-fixed chonotrichs not postfixed with osmium tetroxide
were floated face down on a few drops of 1% osmium
tetroxide solution for about 30 minutes. Specimens treated
in this manner were stainable with lead. Apparently*
osmium tetroxide acts as a mordant for the lead stain.
Another interesting phenomenon is the effect of the
copper grid on staining of the sections. Sections lying
next to the copper meshwork of the grid were not stained
or were not stained as intensely with lead as others.
Possibly the copper ions interfered with the staining
properties of lead. The resolution of this problem was
not sought* although, a seemingly simple solution would
be the use of grids made with other metals (e.g. nickel*
stainless steel, etc.).
Mitochondria, at least those of L. prorates, are
indeed sensitive to different fixatives and to variations
of the fixative. With techniques now employed for fixation
— the very fact that the organism is "fixed" — and ultra-
structural observations, there is reasonable doubt that
electron micrographs of mitochondria retain the micro
structure of the living state. One may, however, hold the
position that the possibility of fixation artifact may be
assumed to be of limited significance by seeking refuge
in the adage that beautiful preparations are properly
fixed preparations.
OBSERVATIONS
General Morphology of Lobochona prorates
The mature L. prorates (Figs. 1/ 2) have an urn-like
appearance — with a funnel# a cytosome# and an attachment
organelle — all characteristic of the collar ciliates
generally. The total length of L. prorates is about 65
microns and the width about 23 microns. Within the
moderately flared funnel are two fields of cilia# a
diagonal field of about 21 rows and a horizontal field of
about 8 rows. Cilia are restricted to the inner surface
of the funnel.
The pellicle of L. prorates increases in thickness
more or less gradually from the rim of the funnel to the
attachment organelle with which the pellicle appears to be
continuous.
A cytostome opens at the base of the funnel and a
cytopharynx continues to about the middle point of the
cytosome. The cytoplasm is finely granular? there is no
apparent ectoplasm nor endoplasm. There is no contractile
vacuole.
The nuclear apparatus consists of a single# usually
15
16
two-zoned macronucleus and usually three micronuclei. At
the base of the cytosome is the vesicle of the attachment
organelle and posterior to it is the attachment disc which
adheres to the marginal bristle of the host's pleopod
(Mohr, LeVeque, and Matsudo, 1963).
Pellicle and Associated Structures
The external surface of L. prorates is covered by a
plasma membrane as found almost without exception on Proto
zoa that have been studied (Pitelka, 1963). In section,
the plasma membrane of L. prorates appear as two dark lines
(each representing a unit membrane) separated by a light
line. The dark lines are each about 5 millimicrons thick
and the light line about 15 millimicrons thick; the thick
ness of the whole is about 25 to 30 millimicrons (Pigs. 3,
7) .
The plasma membrane overlies the pellicle which
extends from the cytostome within the funnel to the
juncture of the attachment organelle. The thickness of
the pellicle varies from area to area of the cytosome. It
is thinnest within the funnel (about 60 millimicrons) and
increases in thickness gradually (about 210 to 300 milli
microns midway) to the thickest point (about 600 to 750
17
millimicrons) at the attachment organelle {Pig. 2).
In longitudinal section, the pellicle has a ribbed,
comb-like appearance and appears to be composed of two
layers (Fig. 3). The external surface is smooth (except at
the neck region (Fig. 8) where the pellicle is irregularly
thickened and appears wrinkled), whereas the inner surface
has digitations about 250 to 320 millimicrons long, 25 to
40 millimicrons wide, and with a space of about 35 to 40
millimicrons between each projection. A layer which is
more electron dense interdigitates with the outer portion
(Figs. 3, 4, 5). In a fortuitous section through a dead
individual, the cytoplasm and inner electron dense portion
have disappeared and only the portion with digitations
remains (Figs. 16, 17, 18). In cross sections, the
pellicle appears as a homogeneous structure with no
projections (Fig. 8). In specimens treated to show
alkaline phosphatase activity, cobalt is deposited in the
spaces between the digitations (Fig. 14).
Curious structures of the pellicle are the pellicular
pores (about 125 to 500 millimicrons in diameter) which
are aligned as longitudinal rows and occur throughout the
cytosome and funnel (Fig. 11). They are closed by the
limiting membrane externally and usually have a vesicle
18
internally (Figs. 5, 6). Dense granules 10 to 250 milli
microns or less dense spherical (19 by 26 millimicrons)
structures may occur within the vesicle (Figs. 5, 7). In
some sections, rough endoplasmic reticulum or smooth endo
plasmic reticulum is apposed to the vesicle while other
vesicles are associated with a complex structure which is
apparently attached to or is smooth endoplasmic reticulum
(Figs. 6, 7, 13). These pores receive a deposition of
silver in the Chatton-Lwoff technic but not in the Bodian
technic (Figs, 9, 10). Localization of adenosine tri
phosphatase activity in the vesicle adjacent to the pore
is suggestive of energy requiring functions of the vesicle
apposed to the pore (Fig. 15).
Immediately beneath the pellicle, but not in contact
with it (a space of about 10 to 15 millimicrons), are fine
tubular fibrils or filaments — called subpellicular
fibrils here. There appear to be two fibrils in longi
tudinal section (each about 25 millimicrons) and 3 in
tangential and cross sections (Figs. 3, 8, 13). There is
a space of about 10 to 15 millimicrons between each bundle
cf 3 fibrils in cross section. These fibrils are aligned
with the longitudinal axis of the cytosome; there are 4 to
6 groups of 3 fibrils between the pores of the pellicle
19
{Figs. 8, 13). In longitudinal section, these fibrils are
seen to arise at the ciliary field (kinetodesmata), pass
along the pellicle, and insert upon the attachment organ
elle (Figs. 19, 23, 26). Some subpellicular fibrils appear
to form a fibrous ring-like structure in the neck region
(Figs. 41, 42, 44). Near the ciliary field, these fibrils
appear intertwined and separate to form a "Y" configuration
(Fig. 12). There appears to be no connection of these
fibrils to the pores nor are they impregnable with silver.
Light microscopic observations show the attachment
organelle to have four prominent structures: 1) a membrane-
lined, spherical vesicle, 2) an attachment mechanism -- a
crown-like, discoid structure below the vesicle, 3) an
adhesive disc which adheres to the marginal bristle of the
host's pleopod, and 4) a stem which extends from the sphere
through the discoid structure to the adhesive disc (Mohr,
LeVeque, and Matsudo, 1963).
Electron micrographs show that the sphere is enclosed
by a limiting membrane. However, there occurs beneath this
membrane another membrane system about 10 millimicrons
thick with outpockets into the sphere which appear as
"donuts" or a tube within a tube in cross section and as
three dark lines in longitudinal section. Portions of this
inner membrane form vesicles 225 to 275 millimicrons that
penetrate into the cytoplasm through the outer membrane.
The outer portion of the crown-like attachment mechanism
appears to be composed of a sponge-like homogeneous
material — presumably protein. Subpellicular fibrils
appear to be attached to this structure. Within this
sponge-like region is a portion not unlike the pellicle in
structure and composition. Within this portion is a
vesicle with 16 extensions into the pellicle-like portion.
The vesicle communicates with the sphere and appears to be
enclosed by the same membrane. The stem, which originates
in the sphere, continues through the disc-like portion into
the 16 extensions of the vesicle becoming the core of the
attachment disc. The attachment disc is composed of what
appears to be the same material and is also lined by the
same membrane enclosing the sphere. The walls of the
attachment disc are from 125 to 200 millimicrons thick;
fibrils 30 to 35 millimicrons in diameter from the stem —
probably of the same material — are attached to the wall.
The attachment disc is presumably "cemented" or "glued" to
the pleopodal bristle; there are no apparent "roots" enter
ing the host (Figs. 18-25).
In some individuals, presumably older ones, the
21
tubules within the sphere appear to disintegrate (Fig. 27).
In certain sections the fibrils from the sponge-like
portion of the attachment mechanism appear to be arranged
in rings of about 185 millimicrons (Fig. 28).
Buccal Apparatus
The adoral ciliary field is easily recognizable from
the horizontal field in sections. Between each row of
cilia of the adoral field is a fold about 200 millimicrons
wide and 500 millimicrons high. It is covered by the
plasma membrane and also includes the thin pellicle (35 to
45 millimicrons) and cytoplasm. There is no fold between
rows of cilia of the horizontal field (Fig. 29). The folds
may possibly function in directing the current and food
particles toward the cytostome.
Cilia of L. prorates, about 3 to 4 microns long and
325 millimicrons in diameter, are like those generally
described for Protozoa and Metazoa with typical "9 plus 2"
structure (Fawcett, 1961; Sleigh, 1962; Pitelka, 1963).
An outer ciliary membrane 4 millimicrons thick which is
continuous with the plasma membrane, surrounds the ciliary
shaft and matrix. Nine peripheral pairs of fibrils, each
fibril about 25 millimicrons in diameter, and a central
22
two, each about 30 millimicrons in diameter, are embedded
in the matrix. Radial links (10 millimicrons thick) occur
between the central fibrils and outer fibrils. Secondary
fibrils (20 millimicrons thick) occur on the radial links.
The ciliary shaft is constricted at the transition zone;
the central fibrils terminate at the level of the cell
surface on the spherical axosome (120 millimicrons) which
is hollow and rests on a slightly curved basal plate. The
outer fibrils continue below the level of the cell surface.
At this level, the outer fibrils are still paired. About
20 millimicrons below the level of the basal plate,
transitional filaments connect the paired fibrils to the
cell membrane. Also at this level, a third fibril occurs
with each of the paired fibrils to make triplets. Dense
granules about 6 millimicrons in diameter occur within the
kinetosomes. They appear to be aligned in longitudinal
rows of about 8 and each row is associated with the
triplet (Figs. 30-40).
The infraciliature of L. prorates is not like that
described for other ciliates. Each row of kinetosomes
overlies a band of five fibrils (each fibril about 25
millimicrons in diameter and the entire fibrillar band
about 200 millimicrons wide) which probably corresponds to
23
the kinetodesma of other ciliates. Two fine fibrils (about
6.5 millimicrons thick), one from each side of the proximal
end of the kinetosome, appear to connect the kinetosome
to the fibrillar band (Fig. 30). Each kinetosome has in
turn three fibrils about 15 millimicrons thick, two from
one side and one from the other, which are attached to
three closest kinetosomes in the next row and perpendicular
to the fibrillar band (Fig. 12). In longitudinal section,
beginning with the row of cilia nearest the cytostome, the
three fibrils originate at about the level where the
triplets of the kinetosome begin and are inserted at the
proximal end of the kinetosome(s) in the next row and
possibly to the fibrillar band (Fig. 30). In apical view,
the three fibrils of one kinetosome in the row nearest the
cytostome are attached to three adjacent kinetosomes in
the next row. It appears, therefore, that all kinetosomes
are interconnected. Some subpellicular fibrils join the
fibrillar band (Fig. 33) while others appear to pass to
the pellicle beneath alveoli which occur between cilia
(Fig. 32).
The cytostome (about 3.5 microns in diameter) opens
centrally at the base of the funnel (Fig. 43) and continues
as a cytopharynx to about the lower level of the macro-
nucleus. Twenty eight fibrils (each 25 millimicrons by
450 millimicrons) extend into the cytopharynx (Pigs. 41-
47). Each fibril is composed of about 16 subfibrils 10
millimicrons by 25 millimicrons which may possibly be
interpreted as one folded sheet. Below the cell surface,
the cytopharynx appears to be partially filled with cyto
plasm (Figs. 42, 44, 45, 46). Fine fibrils about 15 milli
microns thick and tubules 50 millimicrons in diameter
surround the cytopharynx between the cytostome and the
ring-like structure in the neck region (Fig. 41). Ribo
somes are present in this region, however, they are not as
abundant as they are elsewhere nor do they appear to be
attached to endoplasmic reticulum. Small vacuoles 75 to
150 millimicrons in diameter are scattered throughout the
region. The ring-like structure of the neck region appears
to be composed of subpellicular fibrils and possibly from
pellicular fibrils. Mitochondria are lacking in this
region.
The tubular cytopharynx continues to one side through
the neck region to the posterior level of the macronucleus
(Fig. 46). At the level of the macronucleus, the cytoplasm
within the cytopharynx contains many vesicles 75 to 175
millimicrons in diameter. Some smaller vesicles contain
25
particles 15 to 35 millimicrons in diameter while a larger
one contains a bacterium (Figs. 46, 47). The cytopharynx
is expansible, as shown by the bulge containing a large
vacuole with a bacterium (Fig. 46).
The transition zone between the cytoplasm within the
cytopharynx and the cytosome is filled with vesicles or
vacuoles 150 to 500 millimicrons in diameter. The inner
diameter of the cytopharynx here is about 4.5 microns
(Fig. 47).
Nuclear Apparatus
Light microscopic observations show that the single,
interkinetic macronucleus of L. prorates is an eliptical,
usually two-zoned heteromere 7 to 9 microns wide and 8 to
10 microns long. The chromophilic zone is coarsely
vesicular, stains very darkly, and contains nucleoli within
the vesicles. The anterior part of this zone is split off
from the larger portion by an achromatic nuclear cleft
("fente nucleaire" of Faure-Fremiet, 1957). The chromo
philic portion is separated from the chromophobic zone by
a homogeneously darkly staining border. Between the darkly
staining border of the chromophilic zone and the chromo
phobic zone is another achromatic area. The remainder of
26
the chromophobic zone is also vesicular but stains less
intensely. Nucleoli occur within the vesicles.
The micronuclei are spherical, 1 to 2 microns in
diameter. Each is surrounded by a delicate membrane and
contains siderophilic granules (Matsudo, 1966).
Electron micrographs of the macronucleus show two
membranes (= nuclear envelope) 10 to 15 millimicrons thick
(Figs. 48, 49). Each membrane is about 3 to 5 millimicrons
and the light zone between, about 5 millimicrons. Pore
like structures 5 to 10 millimicrons wide occur on the
membrane. Granules about 20 millimicrons in diameter,
presumably ribosomes, occur on the surface of the outer
membrane. The anterior portion of the chromophilic zone
consists of granules of two sizes as does the adjacent
portion. The Feulgen-positive portions are compact,
composed of smaller granules (5 to 7 millimicrons) while
granules of the nucleoli are larger (15 to 20 millimicrons),
more diffuse, and less electron-dense. Fine granules 5 to
10 millimicrons occur in the achromatic nuclear cleft and
in the vesicles. The homogeneously darkly staining border
appears to be a concentration of deoxyribonucleic acid
granules. The chromophobic portion also contains two types
of granules; less dense granules of nucleoli (7 to 12
27
millimicrons) and granules of deoxyribonucleic acid (5 to
10 millimicrons). The achromatic area also has 5 to 10
millimicrons granules like those in the matrix. It is
interesting to note that the chromatin material does not
lie next to the nuclear membrane but about 15 millimicrons
within it.
The micronucleus is limited by two membranes each
about 10 millimicrons thick with a space of about 10 milli
microns between (Figs. 48, 50, 51). Pore-like structures
5 to 10 millimicrons in diameter occur on the micronuclear
membrane. Most of the granules of the micronucleus are
7 to 10 millimicrons in diameter. Fine filaments 7 to 10
millimicrons thick also occur in the micronucleus. Ribo
somes 10 to 20 millimicrons in diameter occur on the outer
membrane.
The Cytoplasmic Matrix and Inclusions
The cytoplasmic matrix appears homogeneous or finely
granular — difficult at times to distinguish from the
graininess of the photographic emulsion or embedding
medium — and has low electron density.
Mitochondria, ribosomes, endoplasmic reticulum, and
food vacuoles are prominent and easily identifiable inclu-
28
sions of the cytoplasmic matrix. Others, such as Golgi
elements, are more difficult to identify.
The mitochondria of L. prorates are like those
described for other ciliates (Pitelka, 1963). They are
oval to elongate (450 to 900 millimicrons in greatest
dimensions) and limited by two membranes 5 to 7 milli
microns thick with a light zone of 7 to 10 millimicrons
lying between. Some mitochondria are as long as 1800
millimicrons (Fig. 26). Cristae are microtubular, 40 to
50 millimicrons in diameter, and appear to be continuous
with the inner limiting membrane. The mitochondria occur
throughout the organism except in the tubular region of
the neck. They appear to be concentrated beneath the
ciliary fields, below the ring-like structure in the neck,
and peripherally in the cytoplasm beneath the pellicle
(Fig. 52). The mitochondria do not appear to have
definite orientation but lie at random in the cytoplasm.
Ribosomes occur throughout the cytoplasm both as free
granules and along membranes. The granules range in size
from 10 to 20 millimicrons. Ribosomes or ribosome-like
granules 10 to 20 millimicrons across occur on the nuclear
membranes and on endoplasmic reticulum (= rough endoplasmic
reticulum).
29
The endoplasmic reticulum (ergastoplasm of earlier
workers) extends as a system of tubules, canaliculi, and
sacs throughout the cytoplasm. The tubules are about 35
to 50 millimicrons in diameter.
The Golgi apparatus (or structure identified as such)
of L. prorates appears to be associated with a membrane
lined with ribosomes (Fig. 50). The entire complex is
about 1000 millimicrons long and 175 millimicrons wide.
Generally, there are three cisternae with small vesicles
at the ends of each cisterna. In non-budding individuals,
Golgi structures are most abundant around the muclei;
however, they may be found elsewhere in the cytoplasm.
The large food vacuoles of L. prorates presumably form
by the fusion of the numerous smaller- vacuoles in the
transition zone at the terminus of the cytopharynx (Fig.
53). The new food vacuoles are characterized by the smooth
outline and undisrupted condition of food (bacteria) in the
vacuole (Fig. 54). A later stage shows a partially
digested bacterium and dense material within the vacuole
(Fig. 55). A still later stage shows outpocketing of the
vacuole (Fig. 56). The dense material appears to become
entrapped in the small vacuoles formed by the outpockets.
Older vacuoles appear empty and the membrane of the vacuole
3 0
apparently collapses and dissolves — sometimes leaving
a vacuole without a membrane (Fig. 57). Many small
vacuoles with dense material occur around the older empty
vacuoles (Figs. 57, 58). Myelin figures appear in some
food vacuoles (Fig. 58), however, their significance is
not known.
In an attempt to identify lysosomes or organelles with
lysosome-like activity on food vacuoles, specimens were
tested for acid phosphatase activity. A specific organelle
with acid phosphatase activity could not be identified.
However, deposition of cobalt (indicating activity) is
correlated with the digestive events described above (Fig.
59) .
An unidentified body (found only in specimens
incubated in sodium beta glycerophosphate) 550 to 650
millimicrons in diameter with a dense body (about 250 by
400 millimicrons) and with lamellar or lattice-like
structures within occurs in the cytoplasm near food
vacuoles (Fig. 60). The dense bodies within this organ
elle do not appear to show cobalt deposition. It is
interesting to note, however, that these bodies appear to
be associated with the food vacuoles and that they seem
to become disorganized internally with advancing stages
31
of digestion of the food vacuole (Figs. 61-63).
A very perplexing problem is the occurrence of
bacteria in vacuoles not lined with a membrane.
Morphogenesis
The first indications of budding are increase in width
of cytosomei increase in number of food vacuoles, appear
ance *>f a karyosome in the jnacronucleus, the migration of
micronuclei from its posterior position toward the macro
nucleus, and appearance of kinetosomes at the site of bud
formation.
Buds of L. prorates usually rise from the anterior
third or half of the cytosome and about ninety degrees
away from the adoral ciliary field (Figs. 79-84). As the
bud grows, the parental cytosome is deflected at the fore
end so that the bud appears to grow from a very shallow
pouch (Matsudo, 1966).
The electron micrographs show that a pouch, "gener
ative pouch", has already been formed at the site of bud
formation before the kinetosomes appear ( the pouch is not
readily discernible with the light microscope except under
ideal conditions). Subpellicular fibrils, free ribosomes,
and rough endoplasmic reticulum appear near the inner wall
32
of the pouch. Pores occur on the walls of the pouch which
is apparently dedifferentiated and is thinner (from 50 to
100 millimicrons) pellicle (Figs. 66, 67). Presumably the
subpellicular fibrils are the precursors of the kineto
somes but specimens showing the actual transition from
fibril to kinetosomes have not been found (Fig. 68).
Individuals of L. prorates of a slightly later budding
stage impregnated with silver protein by the Bodian technic
show deposition of silver on the recognizable kinetosomes
but not on any other structure in the area (Fig. 69). A
section through another individual in a similar stage of
budding but at a different level shows concentrations of
fibrils — each bundle approximately the same width as a
kinetosome and aligned and spaced in a pattern and position
where kinetosomes would probably occur. These fibrils and
the subpellicular fibrils are not impregnable with silver
by the Bodian nor by the Chatton-Lwoff technics (Fig. 70),
The primordium of the attachment organelle appears to
be an outpocketing of the same pouch (Figs. 71, 73). It
apparently develops at about the same time as the kineto
somes (Fig. 68). The portion which becomes the disc-like
attachment mechanism is continuous with the subpellicular
fibrils. The cytoplasmic granules (Figs. 51, 64, 65) which
33
occur near the micronuclei migrate with the micronuclei and
become concentrated about the primordium of the attachment
organelle. Many vesicles 50 to 200 millimicrons in dia
meter and canaliculi 50 by 3 50 millimicrons as well as
ribosomes surround the sphere of the attachment organelle
primordium (Pig. 71). The number of cilia increases as
the bud increases in size. Dividing kinetosomes and "pre-
kinetosomes", however, have not been observed (Fig. 71).
The cytopharynx develops from the same pouch anterior
to the primordium of the attachment organelle (Fig. 72).
Lipid bodies (900 to 1000 millimicrons in diameter)
occur in the cytoplasm of the developing bud (Figs. 75,
76) .
The 16 vesicles of the adhesive disc produce canal
iculi (about 78 to 92 millimicrons in diameter and about
5 microns long) around and beyond the sphere of the
primordium of the attachment organelle — to give the
appearance of finger-like projections surrounding a sphere
(Fig. 92). The cytoplasmic granules are disposed in a
"floral" pattern around the canaliculi (Figs. 77, 91).
The enlarged pouch with developing ciliary field, cyto
pharynx, and primordium of the attachment organelle remains
in communication with the exterior environment (Fig, 74).
34
Morphogenesis of the Bud
After budding, the individual apparently attaches to
the host's pleopodal bristle by wrapping its posterior
portion around the bristle, thereby enabling the bud to
attach temporarily to the bristle. The attachment disc is
secreted subterminally within the area of contact between
the bud and the bristle (Fig. 85). Neither the mechanism
of secretion nor the chemical composition of the adhesive
material is known other than that it is protein. It is
believed that the granules in the cytoplasm secrete the
attachment disc or are precursors of the protein stalk.
Unfortunately the solitary specimen, available of the
stage immediately post budding, had been stored in 95%
ethanol for a few weeks before impregnation with the
Chatton-Lwoff silver technique and the alcohol soluble
components of the cell had been extracted. However,
preservation appears adequate for identification of ribo
somes and membranes and to show an interesting phenomenon
which occurs at this stage. In the budding process, mito
chondria from the parent's cytosome in the region of bud
formation are incorporated into the bud. At this young
stage immediately after release from the parent, a few
dense, rounded mitochondria of varying sizes occur in
35
groups throughout the cytosome with apparently degenerating
mitochondria suggesting formation of new mitochondria in
the bud. The dense mitochondria are not like those seen
in other stages of the life cycle (Fig. 86).
In a slightly later stage of development, the pre
viously oval outline of the bud becomes elongate. The
pouch (funnel) with cytostome which was open to the side
is now open at the anterior end of the organism. The
attachment organelle remains subterminal, however, a
smaller part of the cytosome is around the bristle of the
pleopod (Figs. 87, 88).
The cytoplasmic granules that presumably secreted the
attachment disc are fewer and are smaller (210 to 310
millimicrons), and the finger-like projections of the
attachment mechanism are shorter. The rounded, dense mito
chondria of earlier stages have now taken the more charac
teristic elongate profile (400 by 800 millimicrons) and
are not as dense. There appear to be more mitochondria
at this stage than at maturity. The cytoplasm appears
about as compact as that of a bud. Lipid bodies are still
present; there appear to be as many free ribosomes, rough
endoplasmic reticulum, and small vesicles (150 to 200
millimicrons) with dense contents (from food vacuoles of
36
the parent) as in younger individuals. Large, dense,
amorphous bodies (about 500 by 1200 millimicrons) appear
in the chromophobic zone of the macronucleus. Similar
bodies have been observed in the macronucleus of a budding
individual.
The ring-like structure in the neck of an adult is
not apparent at this stage.
Diminutive Budding
In addition to the more usual asexual budding process,
there occurs in L. prorates another pattern in which the
individuals appear to begin budding in the same manner, but
there is a diminution of the parent's cytosome accompanying
the formation of a bulge at the posterior third of the
cytosome opposite the bud. The lappets of the collar dis
appear and there is a decrease in the size of the parent as
the bud increases in size. When the bud is fully formed,
the size of the parent has been reduced to about 9 microns.
After budding, the parent has little resemblance to the
"normal" adult individual. The cytosome is compressed
along the longitudinal axis and there is only a slight
protrusion at the anterior end. The bud appears normal
(Matsudo, 1966).
37
Because relatively few diminutive budding individuals
were among the Lobochona collected, only few individuals
have been observed with the electron microscope. The
electron micrographs show a reduction in number of cilia
in the funnel and an apparent thickening and wrinkling of
the pellicle (Fig. 89). Portions of the pellicle of the
parent appear mottled. The cytoplasm in the funnel
appears fibrous and bodies (400 by 1200 millimicrons)
composed of many 10 to 20 millimicrons granules is present
(Fig. 89).
The cytosomal cytoplasm also appears fibrous and
compact and most mitochondria appear to be degenerating.
Many ribosomes persist, however, the amount of smooth
endoplasmic reticulum and rough endoplasmic reticulum
appears to be less. The old food vacuoles are still
present as are the cytoplasmic granules. Structures
identifiable as lysosomes have not been observed (Fig. 90).
Electron micrographs of the bud of a diminutive parent
show the cytoplasm to be normal (Fig. 91).
The parental pellicle, after budding, appears rough
on the outer surface — particularly the upper portion
(Fig. 92). The cytoplasm appears even more fibrous; mito
chondria and ribosomes are scarce. A section through the
38
above individual at a different level shows many bacteria
in the cytoplasm. The bacteria do not appear to be in
membrane-lined vacuoles (Fig. 93).
The pellicle of a diminutive parent at the last stage
available appears thinner and smoother. The remnant of
the collar remains as a plug-like protuberance. There are
few mitochondria in the cytoplasm which is no longer dense
but rather appears vacuolated (Fig. 94).
Conjugation
Conjugation among L. prorates is not like the process
generally described for other ciliates (e.g. Paramecium)
— a temporary union of two individuals for the exchange
of nuclear material. Copulation, a complete fusion of two
gametes, may be a more appropriate description of sexual
reproduction for L. prorates. However, in view of the fact
that the term conjugation has been used for sexual repro
duction among the Chonotrichida — although a highly
specialized form of conjugation — and that copulation is
not an entirely satisfactory term, the writer chooses to
refer to sexual reproduction among L. prorates as conju
gation .
Conjugation occurs between adjacent individuals on the
39
same pleopod. The conjugants appear morphologically
similar to juvenile forms. They are smaller than non
conjugating adults (25 to 35 microns as opposed to 45 to 69
microns long), and possess a macronucleus similar to that
in juvenile forms. The lappets of the collar also resemble
those of juveniles.
One of the pair, the migrating conjugant, is smaller
(10 microns) than the stationary one and has a collar with
minimal lappets. The two animals bend toward each other
at the neck; the migrating conjugant enters the cytostome
of the other, detaching itself from its adhesive disc.
The anterior part of the migrating conjugant is more or
less pointed and does not possess a collar. The macro
nucleus and micronuclei of the migrant (presumably the
entire animal) enter the stationary conjugant. The
posterior part of the migrating conjugant remains as a
cylindrical "plug" about 7 microns long and 4 microns in
diameter in the cytostome of the stationary individual.
Conjugants in later stages do not possess this "plug".
Whether the "plug" is completely absorbed or degenerates
i
and is ejected has not been observed (Matsudo, 1966).
Unfortunately, only two conjugating pairs, both in a
similar stage were among L. prorates collected and
4 0
embedded in plastic. The external surface of the pellicles
of both conjugants is rough and a pouch (1.2 by 4.2
microns) is present in the stationary conjugant. The
ring-like structure is not apparent and a vacuole (about
1.8 microns) is present in the neck region of the station
ary conjugant. The outline of the macronucleus is not like
that of a normal individual but is constricted between the
anterior and posterior portion of the chromophilic zone
and between the chromophilic and chromophobic zones. The
anterior portion of the chromophilic zone is not vesicular
as in an interkinetic macronucleus. It contains numerous
homogeneous granules (60 to 90 millimicrons) and larger
bodies which are probably nucleoli (600 to 750 millimicrons
composed of granules 30 to 40 millimicrons). The posterior
portion of the chromophilic zone appears vesicular.
Nucleoli occur within the vesicles. The dense portion is
composed of granules 60 to 90 millimicrons and the
nucleoli of granules 30 to 40 millimicrons. The chromo
phobic zone is like the anterior portion of the chromo
philic zone with granules (60 to 90 millimicrons) and
nucleoli (600 to 800 millimicrons). The cytoplasm of the
stationary conjugant contains many ribosomes and more
smooth endoplasmic reticulum than do non-conjugating
41
individuals. Lipid bodies occur in the neck region (Figs,
95, 96).
The macronucleus of the migrating conjugant is also
constricted. Micronuclei appear to be undergoing meioses.
Chromosomes appear as dense areas (100 to 150 milli
microns) in the micronuclei. Pore-like structures occur
in the intact micronuclear membrane which is not lost
during meiosis (Figs. 97, 99).
Lipid bodies and cytoplasmic granules occur in the
neck region of the migrating conjugant (Fig. 97). Some
food vacuoles and small vesicles containing dense material
occur in the posterior portion of the migrating conjugant.
Cisternae 25 by 200 millimicrons occur in groups of 3 to 4
and some in rows of 9 to 14 occur in the anterior portion
of the migrating conjugant (Fig. 98). Similar arrays
(25 by 200 millimicrons) also occur in the stationary
conjugant (Fig. 96).
The pellicle apparently fuses at the point of contact
(Fig. 98). Kinetosomes are not evident in the pointed
anterior portion of the migrating conjugant. All of its
structures anterior to the ring-like structure have
disappeared (Figs. 97, 98).
DISCUSSION
The pellicle of L. prorates is covered by a plasma
membrane like that which is also found on other Protozoa
(Beams and Anderson, 1961; Pitelka, 1963) and universally
among all types of cells (Sjdstrand, 1959; Robertson, 1960).
Little attention has been given to pellicular struc
ture of Protozoa generally. The familiar polygonal cortex
of Paramecium was interpreted by Ehret and Powers (1959)
and Parducz (1958); however, their interest was focused on
argentophilic structure of the pellicular and fibrillar
systems and not on the ultrastructure of the pellicle
itself. The pellicle complex of Euglena gracilis has been
studied by Sommer (1965) and mucocysts and pellicle of
Tetrahymena pyriformis by Tokuyasu and Scherbaum (1965),
but neither has any resemblance to the pellicle of L.
prorates. The pellicle of Hyalophysa chattoni (Bradbury,
1966), a foettingeriid apostome, is most like that of
Id- Prorates. It is formed of 3 layers: an outer layer 20
millimicrons thick; a dense, homogeneous middle layer 30
millimicrons thick; and an inner layer 50 millimicrons
thick. The inner layer has regularly spaced fibrils which
42
are perpendicular to the pellicle and reminescent of the
projections of the pellicle of L. prorates. Bradbury
(1966) suggests for the pellicle of Hyalophvsa that the
projections may be an adaptation for growth. The trophont
which may triple its volume in minutes does not have the
ordered pellicle of the tomite. "Perhaps the tomite's
inner pellicular layer with its oriented rods may be
easily expanded to provide new membrane, so that synthesis
would not interfere with ingestion" (Bradbury, 1966, p.
603). The function attributed to the pellicle of Hyalo-
physa probably would not apply to L. prorates as the
pellicular structure remains constant throughout its life
cycle.
The chemical composition of the pellicle of L.
prorates is not known. Mucopolysaccharide has been
suggested as the predominant component of the cuticle of
Ephelota while the epiplasm has been reported to be protein
(Rouiller, Faure-Fremiet, and Gauchery, 1956). The
fortuitous finding of the pellicle of a dead L. prorates
suggests that the pellicle may not be part of living
protoplasm; however, the role of portions of the pellicle
in morphogenesis and during conjugation suggests a contrary
view. The pellicle stains lightly with Heidenhain's iron
44
haematoxylin but is stained much more intensely by Brom-
phenol blue, a specific stain for protein (Mazia, Brewer,
and Alfert, 1953). The deposition of cobalt in the elec
tron dense portion of the pellicle between the projections
(after incubation in glycerophosphate to show alkaline
phosphatase activity) suggests a number of functions for
the structure. Mucopolysaccharide, which may be a com
ponent of the pellicle, is reported to occur at sites of
high alkaline phosphatase activity (Moog and Wenger, 1952).
Hinsch and Buxbaum (1965) report the occurrence of alkaline
phosphatase at sites of absorption and a role in histo
genesis and metabolism (transfer of molecules) of the cells
in developing chick oesophagus and trachea. It is possible
that absorption could take place through the pellicle of
L. prorates, and there is also a possibility of a role of
alkaline phosphatase in morphogenesis of L. prorates.
However, there is another possibility that the deposition
of cobalt may be an artifact, that the cobalt could not
diffuse through the pellicle and was deposited between the
projections. It appears, however, that the pellicle is
living: possibly as a gelated portion of the cytoplasm of
L. prorates.
The pores in the pellicle of chonotrichs (Chilodochona,
Spirochona, Heliochona. Trichochona) have been reported to
be kinetosomes of somatic cilia that have been lost (Guil-
cher, 1951). Electron microscopic observations of the
pellicular pores of L. prorates and Heliochona psychra
show no resemblance of the pellicular pores to kinetosomes
The pores are impregnable with silver by the Chatton-Lwoff
technic but not by Bodian's technic which appears to be
more specific and consistent for impregnation of kineto
somes provided one has a good batch of protein silver.
Light microscopic observations of L. prorates impregnated
with silver by the Chatton-Lwoff technique show the pores
to be aligned in parallel rows along the longitudinal axis
of the organism which is suggestive of kinetosomes.
Similar observations have been made for certain suctorians
(Chatton, Lwoff, Lwoff, and Tellier, 1929) and peritrichs
(Chatton, 1936). Pottage (1959) has shown with electron
micrographs that the argentophilic granules of another
suctorian, Discophrva piriformis, are indeed kinetosomes
in the ectoplasm. However, Faure-Fremiet, Favard, and
Carasso (1962) have shown that the argentophilic granules
of a peritrich, Epistvlis anastatica, are pellicular pores
The derivation and the function of the pores are not
known. For peritrichs, a group that has been studied more
46
completely# the argentophilic structures were believed to
be the relic of infraciliature (Chatton, 1936; Klein,
1928) or pellicular sculpturing (Kofoid and Rosenberg,
1940). Grass6 and Mugard (1961) suggest for the pores of
Qpisthonecta — a peritrich which forms cysts — a role in
formation of cysts. Bradbury (1965) suggests that the
pores may represent parasomal sacs whose associated
kinetosomes have vanished or may facilitate diffusion of
oxygen through the pellicle. The pellicular pores pictured
by Bradbury (1965) and Faur^-Premiet, Favard, and Carasso
(1962), however, appear smaller (about 0.1 by 0.2 micron)
and not like the pores of L. prorates. The juxtaposition
of the vesicle to rough endoplasmic reticulum and adenosine
triphosphatase activity in the vesicle suggests a more
dynamic function — at least for those of L. prorates —
than diffusion or a parasomal sac. The pellicular pores
of L. prorates will be discussed further along with the
buccal apparatus.
Fibrous structures occur widely among protozoans
(particularly ciliates) in the structure of organelles.
A large number of fibrils and fibrous structures have been
described by the electron microscopists and have been
categorized by Pitelka (1963) into 3 groups: 1) banded
47
fibrils exhibiting a period of 30 to 60 millimicrons and
varying greatly in width — possibly made up of bundles of
periodic filaments in phase; 2) cylindrical fibrils
described as tubular with dense peripheries and low-
density centers and ranging from 12 to 30 millimicrons in
diameter; 3) fine filaments, less than 15 millimicrons in
diameter and often in bundles or sheets.
Subpellicular fibrils and fibrils associated with the
ciliary fields (kinetodesmata) of L. prorates fall into the
second category in size and appearance. However, a
comparison of the fibrillar structures of L. prorates with
that of other ciliates is difficult because of the high
degree of structural specialization among the examples
that have been studied and lack of sufficient study on
ciliates which are reported to be closely related to the
chonotrichs.
Pitelka (1963) chooses tetrahymenids as the represent
ative group to describe cortical morphology and infra-
ciliature because ciliates (gymnostomes) considered to be
most primitive have not been studied in detail by electron
microscopy. A schematic diagram of the somatic infra-
ciliature of Colpidium (Pitelka, 1961) shows that the
parasomal sac lies anterior to the kinetosome. In such
48
units of organization# fibrils (which are striated) from
the kinetosome passing anteriad and outward toward the
pellicle alongside the parasomal sac are called kineto-
desmal fibrils while those going in the opposite direction
are called post-ciliary fibrils. Each kinetodesmal fibril
merge into a longitudinal fibril. Transverse fibrils lead
from the kinetosome to the ciliate's left and to the
observer's right. Fibrils do not connect somatic kineto
somes; however# kinetosomes in the buccal region are inter
connected by fine filaments. In tetrahymenids "each
kinetosome of the somatic infraciliature is the point of
origin of three diverging fibrous structures: the striated
kinetodesmal fibril from its right anterior margins# the
transverse fibril band from its left lateral margin# and
the post-ciliary fibrils from its right posterior margin"
(Pitelka, 1963). There are also three fibrils originating
from the kinetosome of L. prorates, however# they do not
appear to correspond to the fibrils of tetrahymenids. The
fibrils also interconnect with other kinetosomes and not
with kinetodesmata. Each row of kinetosomes of L. prorates
is associated with one band of fibrils (kinetodesma)# how
ever, the fibrillar bands lie neither to the right nor left
but directly beneath the kinetosomes and therefore do not
observe the rule of desmodexy (Chatton and Lwoff, 1935a).
Dumont (1961) found that the rule of desmodexy did not
apply to Dileptus anser, a tracheliid gymnostome. In D.
anser, small fibrils are attached to kinetosomes of the
feeding cilia on both the right and left sides of a larger
fibril bundle. That the rule of desmodexy applies to all
ciliates as implied has been questioned by other workers
(Corliss, 1956; Pitelka, 1963; Mohr, pers. comm.). An
attempt to make comparisons and homologies of the infra-
ciliature at this time would be conjectural.
The buccal apparatus of L. pr- • .£S is relatively
simple by comparison with other ciliates. The cytopharynx
opens directly into the funnel as a cytostome. In Chlamy-
dodon pedarius, a gymnostomatous ciliate, thus a member of
a group which is believed to be closely related to the
chonotrichs, the oral apparatus is complex. A membranous
system outlines the cytostome to form an oral cavity, and
trichites line the cytopharynx (Kaneda, 1962). Trichites
or protein rods occur in both rhabdophorine and cyrto-
phorine gymnostomes and in certain spirotrichs. There is
no similar structure in L. prorates. In Tetrahymena pyri-
formis, a “valve1 ' is present which opens and closes the
opening of the cytopharynx (Elliot and Clemmons, 1966).
5 0
The pharyngeal tube of Chlamvdodon pedarius, also
filled with cytoplasm, is not very unlike that of L. pro
rates, The laterally compressed tube of about 100 lamellae,
each about 30 millimicrons thick and spaced about 40 milli
microns apart, resembles that of L, prorates. At the open
ing of the cytopharynx, the lamellae of C. pedarius are
about 350 millimicrons wide but are twice that deeper in
the organism. The micrographs, not of superior quality,
do not show the "very fine, short fibrils randomly arranged
... in longitudinal sections" (Kaneda, 1962, p. 191). The
increase in width of lamellae of C. pedarius compares
interestingly with that of L. prorates which decreases in
width toward the inner end.
Neither the function nor significance of the fine
fibrils and tubules surrounding the cytopharynx of L. pro
rates is clear. In Tetrahymena pyriformis, the oral ribs
are underlain by thick-walled microtubules (arranged in a
regular pattern) which are believed to support the buccal
cavity wall (Elliot and Clemmons, 1966); similar tubules
in many Protozoa are reported to have a supportive function
(Pitelka, 1963). The tubules of L. prorates, however, are
not regularly arranged. Lack of mitochondria and presence
of few ribosomes (which do not appear to be along membranes
51
in the region) do suggest support as their function. How
ever, in consideration of the fact that L. prorates has no
apparent osmoregulatory device, it follows, accordingly,
that some mechanism or structure subserving osmoregulation
may be present. In a study of contractile vacuoles of
Paramecium aurelia and P. caudatum, Schneider (1960) found
a network of nephridial tubules (15 to 20 millimicrons)
which are connected to the endoplasmic reticulum and larger
50 millimicrons tubules around the periphery of the
nephridial tubules. Mitochondria are not among the tubules.
In the above and other studies, "... the cytoplasmic zone
in which segregation of water and resorption of solutes
almost certainly takes place is seen to be occupied by
tubules or vesicles providing a large, and in the more
extreme cases, enormous, membrane surface area. Linkage of
these tubules with endoplasmic reticulum may be significant
if the latter system can be demonstrated to function in
water transport elsewhere in the cell" (Pitelka, 1963, p.
31). Only few tubules and canaliculi in the neck region
of L. prorates may be seen passing through the neck to the
cytosome. These may be continuous with endoplasmic retic
ulum or are possibly endoplasmic reticulum. These tubules
may, conceivably, remove water or salts taken in with
52
ingestion of food and transport water or salts to other
parts of the cell as suggested by Pitelka. If the tubules
are continuous with endoplasmic reticulum, water or salts
may possibly be removed where endoplasmic reticulum is
apposed to the vesicular structure of a pore. Adenosine
triphosphatase activity at the pores and also at the cyto
pharynx suggest that some means of active regulation may
exist.
The ring-like structure in the neck will be discussed
below with conjugation.
Electron micrographs of ciliate macronuclei show a
porous double nuclear envelope that encloses a fine fibrous
and granular matrix with dense, fibrous or granular irreg
ularly shaped bodies (Pitelka, 1963).
The macronucleus of a juvenile L. prorates is more or
less spherical (Matsudo, 1966) as is that of Tokophrya
infusionum, whereas the macronuclei of older T. infusionum
(Rudzinska, 1961b) and L. prorates are larger and have an
irregular outline.
Dense, fibrous or granular bodies from 100 to 200
milliirucrons in the macronucleus of Paramecium bursaria
(Ehret and Powers, 1955), P. caudaturn (Tsujita, Watanabe,
and Tsuda, 1954), tetrahymenids (Pitelka, 1963), and
53
Colpoda maupasi (Rudzinska, Jackson, and Tuffrau, 1966)
are believed to be the Feulgen positive chromatin material
seen in the same forms while larger dense bodies are
believed to be nucleoli. Jurand, Beale, and Young (1962)
studying the macronucleus of P_. aurelia with ribonuclease,
deoxyribonuclease, and "silver-Feulgen" treatment observed
that the numerous 0.5 micron diameter structures in the
macronucleus which are thought to be nucleoli consist of
an outer ribonucleic acid containing portion and an inner
deoxyribonucleic acid containing portion and that the
smaller bodies have neither ribonucleic acid nor deoxyribo
nucleic acid. They suggest that the 0.5 micron bodies are
the "most likely candidates" for the postulated "sub
nuclei" of Sonneborn (1947). For Blepharisma intermedium,
Seshachar (1964) also describes two types of bodies: small
irregular bodies 0.05 to 0.2 micron diameter and large 0.4
to 0.6 micron diameter bodies. The smaller bodies are
construed to be cross sections of long branching filaments
that cross the macronucleus in all directions and which
$
correspond to deoxyribonucleic acid filaments centrifuged
out of the macronucleus and composed of 15 millimicrons
fibrils. The larger bodies, also fibrillar, contain 10 to
80 millimicrons granules and correspond to nucleoli.
The macronuclei discussed above, however, are home-
omerous while that of L. prorates is heteromerous (Faure-
Fremiit, 1957; Matsudo, 1966). The chromophilic portion
of the Lobochona macronucleus shown to be Feulgen positive
and not extractable with ribonuclease in a light micro
scopic study is composed of 10 millimicrons fibrils and
fine 5 millimicrons granules which appear flocculent.
Structures identified as nucleoli and extractable with
ribonuclease are less electron dense and composed of 15 to
20 millimicrons granules. They compare favorably with
structures identified as nucleoli of other ciliates.
Hollow nucleoli have been reported for P. caudaturn
(Vivier, 1961), however, they do not appear to be the
spherical structures usually called nucleoli. Only one
hollow nucleolus-like structure has been observed in
macronuclei of L. prorates.
Young (1939) observed spherical structures in the
macronucleus of Blepharisma undulans and found that they
occurred at a certain time during the nuclear cycle.
Kennedy (1965), on basis of cytochemical tests, suggested
that they are a carbohydrate-protein complex storage
product used by the macronucleus in preparation for divi
sion.
55
An inclusion in the chromophobic portion of the
Lobochona macronucleus has been observed in a young
individual and in an individual beginning to bud. Its
electron density and fine granularity are not unlike those
of the cytoplasmic granules which are believed to secrete
the attachment organelle; however, chemical composition
and function are not known.
Unlike the homogeneously granular micronuclei of L.
prorates, the electron micrographs of other ciliate micro
nuclei (Blepharisma intermedium, Seshachar, 1964; B.
undulans, Kennedy, 1965; P. aurelia, Jurand, Beale, and
Young, 1962; Colpoda maupasi, Rudzinska, Jackson, and
Tuffrau, 1966) show a dense chromatin network like the
Feulgen positive portion of the chromophilic zone in the
macronucleus of L. prorates. Micronuclear pores have not
been reported for P. aurelia (Jurand, Beale, and Young,
1962) nor Blepharisma intermedium (Seshachar, 1964).
Kennedy (1965), however, pictures pores in the micronucleus
of B. undulans. Pores also occur in the micronuclear
envelope of L. prorates and probably occur in those of
other ciliates.
The reorganization band of Euplotes has been studied
with the electron microscope (E. patella, Roth, 1957;
5 6
E. eurvstomus, Faure-Fremiet, Rouiller, and Gauchery, 1957;
and cytochemically (Gall, 1959). The reorganization band
of Stvlonvchia has been studied by Roth (1960). In the
above studies, coarse granules were found to appear after
the passage of the reorganization band. Similar obser
vations have been made for Urostyla grandis (Inaba and
Suganuma, 1966) in the early interphase macronucleus.
Flickinger (1965) however, found no "impressive change" in
the interphase macronucleus of Tetrahymena pyriformis and
could not correlate ultrastructural changes with deoxy
ribonucleic acid synthesis. Cytochemical studies of
ciliate macronuclei have shown that deoxyribonucleic acid
is doubled with the passage of the reorganization band
(McDonald, 1958; Prescott, 1966; Gall, 1959). The passage
of a reorganization band has been described for Spirochona
qemmipara (Tuffrau, 1953) and Heliochona scheutenii (Dobr-
zanska-Kaczanowska, 1963) with light microscopic obser
vations. A reorganization band-like structure also occurs
in the macronucleus of L. prorates but only fixed and stain
ed Lobochona were observed (Matsudo, 1966). Electron
micrographs of the L. prorates macronucleus show no struc
ture resembling the passage of a reorganization band as in
the other ciliates that have been studied. It is possible
57
however# that the manifestation of the reorganization band
of L. prorates is not like that of other ciliates or that
there is no passage of a reorganization band at all.
Mitochondria of L. prorates have the ultrastructural
characteristic of those of most Protozoa; however, similar
microtubular mitochondria have been observed in cells of
Metazoa (Sedar and Rudzinska, 1956; Rouiller, 1960;
Novikoff, 1961).
In Tokophrya infusionum, mitochondria of young and old
organisms look similar, however, the mitochondria of older
organisms appear to have reduced internal structure
(Rudzinska, 1961a) . Mitochondria of young T. infusionum
are distributed randomly while in older organisms there are
fewer mitochondria and they are concentrated near the per
iphery of the cell. The concentration of mitochondria
peripherally has also been reported for Tetrahvmena
(Pitelka, 1961) and in peripheral endoplasm of astomes
(dePuytorac, 1960). The mitochondria of mature L. prorates
are also concentrated peripherally. In the buds that are
being formed, mitochondria (maternal) appear also at the
periphery of the cytoplasm of the bud. However, mito
chondria of a bud that has recently been attached to its
host occur in groups throughout the cytoplasm and are
58
spherical and dense. These appear to be new mitochondria;
the maternal mitochondria apparently degenerate.
Three prominent theories have been proposed for the
origin of mitochondria: 1) mitochondria are like chromo
somes (carry genes) and new ones are formed by preexisting
mitochondria (Novikoff, 1961; Rouiller, 1960); 2) de novo
synthesis (Harvey, 1946; Novikoff, 1961); and 3) formation
from a non-mitochondrial structure (endoplasmic reticulum
or plasma membrane).
Mitochondria of Pelomvxa have been reported to arise
from the nucleus (Brandt and Pappas, 1959) and in Para
mecium from small, undifferentiated, membrane-lined, pre
cursors arising de novo (Wohlfarth-Bottermann, 1958; Ehret
and Powers, 1955). For L. prorates, scarcity of material
(only one specimen) and its poor condition preclude a
systematic study of the origin of mitochondria at this
time. The occurrence of mitochondria in groups suggests
division of new mitochondria from certain maternal mito
chondria that do not degenerate or from other precursors,
as dense granules that appear near the mitochondria. Some
mitochondria, however, appear to be continuous with mem
branes of the endoplasmic reticulum.
The problems encountered by the writer for "proper"
59
fixation of mitochondria for L. prorates make him hesitant
to make categorical statements. Many variations in pattern
of cristae or microtubules have been reported for mito
chondria, but neither the significance of occurrence nor
function have been explained (Pitelka, 1963). "Abnormal"
mitochondria have not been observed for L. prorates.
Two types of endoplasmic reticulum have been des
cribed: 1) rough endoplasmic reticulum — endoplasmic
reticulum lined with ribosomes, and 2) smooth endoplasmic
reticulum — endoplasmic reticulum without ribosomes.
Endoplasmic reticulum is reported to occur as a system of
tubules, canaliculi, and sacs throughout the cytoplasm and
which presumably separate the protoplasm from the membrane
enclosed spaces; the enclosed spaces are believed to comm
unicate with each other and with the environment outside.
The spaces between the membranes may be filled with
material of varying electron density. In certain cells
actively engaged in "protein synthesis", the space between
membranes of the endoplasmic reticulum appear distended
(Pitelka, 1963; DeRobertis, Saez, and Nowinski, 1965).
In L. prorates, rough endoplasmic reticulum is most abun
dant around the nuclei and in regions where protein syn
thesis is probably taking place, that is in budding
6 0
individuals and in young buds. At times, rough endoplasmic
reticulum is seen in close association with mitochondria,
and as mentioned above, with pellicular pores. Canaliculi
occur next to the posterior portion of the macronucleus.
The food vacuoles of protozoans have been studied
extensively by light microscopy and electron microscopy
(Mast, 1942; Kitching, 1956; Jurand, 1961; Schneider, 1964;
Rudzinska, Jackson, and Tuffrau, 1966) and more recently
by electron histochemistry (Mtlller, Rblich, Toth, and Tftro,
1963; Carasso, Favard, and Goldfischer, 1964; Elliot and
Clemmons, 1966).
Although the method of ingestion differs among various
groups of ciliates because of the structural differences of
the oral apparatus, the sequence of events following in
gestion appear to be consistent.
Small vesicles in the vicinity of the cytopharynx are
believed to fuse to form larger food vacuoles in Tetra-
hvmena (Elliot and Clemmons, 1966; Miller and Stone, 1963)
as is the case also in L. prorates. Electron micrographs
of young food vacuoles show a relatively smooth outline
(Jurand, 1961, Paramecium aurelia; Elliot and Clemmons,
1966, Tetrahvmena pyriformis; Rudzinska, Jackson, and
Tuffrau, 1966, Colpoda maupasi) with few small vesicles
6 1
close by. The presence of acid phosphatase activity in the
small vesicles (Carasso, Favard, and Goldfischer, 1964;
Goldfischer, Carasso, and Favard, 1963; Mtlller, Rftlich,
Toth, and Tttro, 1963; Elliot and Clemmons, 1966) suggests
fusion of the small vesicles with food vacuoles to intro
duce enzymes (Rudzinska, Jackson, and Tuffrau, 1966).
Kitching (1956) observed the occurrence of a short alkaline
phase preceding the acid phase and Rudzinska and co-workers
(1966) suggest, accordingly, that the originally alkaline
food vacuole is made acidic by the contents of the small
vacuoles.
The interpretations of this writer for L. prorates are
contrary to that of Rudzinska and co-workers (1966). The
small vesicles are not associated with those food vacuoles
which appear to be newly formed and, if present, are not
apposed to the food vacuole. Acid phosphatase activity
has not been found in the small vesicles and the dense
material found in older food vacuoles appears to be the
same material as in the small vesicles. Finally, in
developing buds, there are many small vesicles with dense
material but there are no food vacuoles. It is, therefore,
the opinion of the writer that the small vacuoles are
vesicles with digested food that may be transported to
various areas of the organism; in L. prorates food vacuoles
occur only in the posterior part of the cytosome.
The structure identified as a lysosome in Tetrahvmena
(Elliot and Clemmons, 1966) is not like the "unidentified
body" (see p. 30) found in L. prorates. In Tetrahvmena,
acid phosphatase activity was found in the lysosomes.
A related phenomenon is egestion of waste products by
L. prorates. A cytopyge occurs in certain ciliates; a
similar structure has not been reported for chonotrichs nor
was a corresponding structure or process observed for L,
prorates during this study (the dense granules in the
pellicular pores may possibly be waste products).
Two theories, "kinetosome" and "pattern", have been
proposed for ciliate morphogenesis. Lwoff (1950) and Weisz
(1951, 1954) propose that the kinetosomes are the basic
morphological units in the cortex of the ciliate and that
the self-replication and differentiation of these kineto
somes, influenced by the microenvironment, determine the
structure and arrangement of cortical organelles that are
generally characteristic of a ciliate. Tartar (1941,
1961), Sonneborn (1963), Beisson and Sonneborn (1965), and
Uhlig (1960) have treated fully the pattern theory which
states that interaction of underlying metabolic fields
6 3
determine the site, initiation, and development of the
apparent cortical structures.
Evidence for the kinetosome theory based on silver
impregnation of infraciliature came from the laboratory of
Chatton, Lwoff, Lwoff, and Tellier (1929); later by Chatton
and Lwoff (19 35b) and expounded recently in this country
by Corliss (in Evans and Corliss, 1964). Experimental and
descriptive observations on Stentor (Tartar, 1941; Uhlig,
1960) and Paramecium (Sonneborn, 1963, 1965) and others on
Blepharisma and Spirostomum have provided evidence for the
pattern theory.
Whether kinetosomes have genetic continuity or not has
been debated (Lwoff, 1950; Gall, 1961). That silver
impregnation provides sufficient evidence that kinetosomes
are self-replicating has been questioned (Ehret and Powers,
1959; Grimstone, 1961; Randall and Hopkins, 1963; Sonneborn,
1963; Evans and Corliss, 1964). It is the opinion of this
writer that use of silver impregnation at the light micro
scopic level is a probably futile effort in attempting to
observe kinetosome replication. Kinetosomes of L. prorates
impregnated with silver and observed with the electron
microscope show a haphazard deposition of silver about the
kinetosomes. It also seems that a dividing silver-impreg-
nated kinetosome could not be resolved with the light
microscope. Furthermore, even at the electron microscopic
level it may not be possible to identify developing kineto
somes of de novo origin, which seems to be the origin in
L. prorates. If, as the writer sees it, subpellicular
fibrils are giving rise to kinetosomes, the "prekineto-
somes" are not impregnable with silver. Possibly the
property that makes a kinetosome stainable with silver does
not exist in a "prekinetosome" but appears only with
maturation of the kinetosome.
Roth (1960) observed for dividing Stylonychia, groups
of ciliary fibrils in the cytoplasm not close to existing
cilia at a period during which kinetosomes were expected
to be developing. A similar observation has been made for
L. prorates. Groups of fibrils in the cytoplasm where
kinetosomes later appear, suggested the development of
kinetosomes (de novo) from the fibrils. Noirot-Timoth^e
(1960) also presents, in electron micrographs of Ophrvo-
scolecidae, evidence for de novo origin of kinetosomes. A
light microscopic study of the gullet organelles of
Paramecium by Ehret and Powers (1959) showed that kineto
somes arise de novo, not near preexisting kinetosomes.
Wise (1965), also by light microscopy, found kinetosomes
65
of Euplotes eurvstomus arising in cortical regions apart
from visible kinetosomal structures and, interestingly for
our findings here, from an invagination of the cortex.
Events leading to origin of kinetosomes of L. prorates
lend support to the pattern theory of morphogenesis. As
mentioned previously, a pouch is formed at the site of bud
formation before kinetosomes appear. The appearance of the
pellicle lining the pouch also suggests that some dediffer
entiation of the pellicle occurs before the pouch is
formed.
The pouch appears to be the presumptive primordial
site for L. prorates which corresponds to the region that
forms the oral apparatus of Stentor (Tartar, 1961). The
pouch of L. prorates, however, also gives rise to the
attachment organelle primordium, a structure not present
in Stentor. Miller and Stone (1963) and Williams and
Anderson (1960) have shown for Tetrahvmena patula that the
"oral ribs" are formed from the inner pellicular layer and
descend to the cytopharynx. A layer of fine fibrils inter
connects the oral ribs with the fibrous network of the
undulating membrane of T. patula. Miller and Stone suggest
that an interchange of stimuli could occur between the
kinetosomes of the undulating membrane and the ribs,
6 6
However, the role of the kinetosomes in formation of the
oral ribs during stomatogenesis of T. patula is not known.
The relationship of oral ribs of T. patula to fibrils
lining the cytopharynx of L. prorates is also not known.
Similarities exist in the cytostomal region of L. prorates
and oral ribs of T. patula. The fibrils lining the cyto
pharynx of L. prorates also appear to arise from the
pellicle. There are also fine fibrils surrounding the
cytopharynx of L. prorates; however, they do not appear to
be connected with kinetosomes and probably have a function
of support rather than of the transmission of stimuli.
The primordium of the attachment organelle is also an
outpocketing of the generative pouch. Mohr (1948, p. 8)
pointed out for Trichochona lecvthoides, "that the peri
stomal ciliary mechanism and the fibrillar portion of the
stalk develop from a common anlage and that this rises de
novo in the cytoplasm of the embryo". In an electron
microscopic study of apparently only mature Chilodochona
quennerstedti, Faure-Fremiet, Rouiller, and Gauchery
(1956) interpret the origin of the peduncle as a secretion
of intracytoplasmic glandular ampullae. Matsudo (1966) in
a preliminary electron microscopic study of L. prorates,
also found the attachment organelle primordium associated
6 7
with developing ciliary fields and fibrils of a budding
individual and suggested that the origin of the attachment
organelle is involved with that of cilia or basal granules,
although somewhat different from those reported for the
Peritrichida. Further observations of L. prorates have
shown that a basal granule(s) is(are) probably not involved
in the origin of the attachment organelle primordium
although subpellicular fibrils are involved. The plasma
membrane, dedifferentiated pellicle, and subpellicular
fibrils contribute to the formation of the attachment
organelle primordium. The spherical portion of the attach
ment organelle primordium originally faces away from the
developing ciliary field. Late in bud formation (through
growth or torsion or both) the primordium rotates 180
degrees so that the sphere faces the anterior end of the
bud. As the bud attaches temporarily to the host's
bristle, it is believed that the vesicular portion of the
primordium and the pellicle fuse. Presumably an opening
appears at the point of contact (between the vesicle and
pellicle) through which the adhesive disc is secreted.
The stem and the adhesive disc, which are continuous, are
the only new structures of newly budded and mature
individuals not present in buds still associated with
68
their parents. Decrease in number of cytoplasmic granules i
j
in newly budded and attached forms# reduction in length of i
the finger-like extensions of the vesicle and deposition
of the dense adhesive disc material suggest that the cyto- !
plasmic granules secrete the adhesive disc as in Chilodo-
chona cruennerstedti (Faure-Fremiet, Rouiller, and Gauchery,
1956). A process similar to but simpler than that in
Chilodochona has been reported for some Dysteriidae and
compared with chonotrichs. "On peut conclure de ces
comparaisons que le filament fin et souple qui assure la '
fixation de Hartmannula acrobates ou de Trochilioides
filans est l'homologue de 1 *une des fibres styliennes de
Chilodochona. les premiers possedant a la base du pied une
glandule simple, et le second possidant e i la base du
p^doncule une glandule composee" (Faure-Fremiet, Rouiller,
and Gauchery, 1956, p. 193). It is the opinion of this
writer that sufficient observations have not been made to
warrant conclusions about homologies.
Observations of diminutive budding individuals have
been too limited to gain an understanding of ultrastruc-
tural changes that take place. The fibrous nature of the
cytoplasm is of interest, however, in that subpellicular
fibrils have been reported to be contractile (Randall and
69
I
Jackson, 1958; Yagiu and Shigenaka, 1963; Dembitzer and j
Hirschfield, 1966). If the subpellicular fibrils of L.
I
prorates are indeed contractile, they do not change their
width with contraction as they remain about 25 millimicrons:
I
in diameter.
It also occurred to the writer that the diminutive
parent may possibly be able to encyst. However, there are
no mucocysts of the sort characteristic of encysting
ciliates nor are food reserves apparent in the cytoplasm.
Conjugation at the electron microscopic level has been
studied for Tetrahvmena pyriformis (Elliot and Tremor,
1958), Paramecium bursaria (Ehret and Powers, 1959), and
Paramecium caudaturn (Vivier and Andre, 1961; Vivier, 1962).
Elliot and Tremor (1958) found the pellicle of conjugating
individuals of Tetrahvmena pyriformis closely apposed and
cytoplasmic "bridges" occurring between the conjugants
through pores in the pellicle. Alveoli, cilia, and kineto
somes are not present in the area of contact. For Para
mecium bursaria (Ehret and Powers, 1959), and Paramecium
caudaturn (Vivier and Andre, 1961) the method of attachment
differs from that of Tetrahvmena. Vivier and Andre (1961)
show that the cytoplasmic communication occur at the
"ridges of the periciliary depressions" and that kineto-
70 i
!
i
somes are present while cilia and trichocysts are not.
Porter (1960) suggests, from light microscopic silver j
impregnation studies, that the infraciliature of Parameciumi
i
aurelia dedifferentiates in the area of contact.
(
In L. prorates, all organelles anterior to the ring- ;
like structure in the neck of the migrating conjugant are
apparently dedifferentiated and lost, whereas the funnel
and cilia of the stationary conjugant remain intact. The
ring-like structure of the migrating conjugant, however,
is not apparent.
Conjugation of L. prorates and Paramecium or Tetra-
hymena would be difficult to compare because of the
ultimate fate of the conjugants. Whereas the several ex-
con jugants of Paramecium and Tetrahvmena separate, only
one Lobochona remains after conjugation. Among Tricho-
nvmpha (Cleveland, 1956), the male gamete enters the female
gamete in a manner similar to that of L. prorates. The
extranuclear organelles of the male gamete are digested by
the cytoplasm of the female gamete, only a remnant of the
organelles of the male gamete remaining by the time the
pronuclei fuse. Although only one stage of conjugation
for L. prorates has been observed with electron microscopy,
entrance of the entire migrating conjugant into the
71
stationary conjugant (as also described with light micro
scopy by Matsudo, 1966) probably does not occur. The
pellicle at the point of contact appears to be fused and
it appears likely that only the nuclei and cytoplasm of
the migrating conjugant enter the stationary conjugant,
presumably the pellicle of the migrating conjugant is
ejected. The "plug" that remains in the funnel of the
stationary conjugant probably is analagous to a diminutive
parent (I.e. a diminution of the pellicle of the migrating
conjugant).
There is a pouch in the stationary conjugant; this
suggests that the stationary conjugant will undergo
division soon after conjugation is completed.
The cisternae that occur in the conjugants are
especially interesting because of their Golgi-like appear
ance, although the writer has tentatively identified other
structures around muclei as Golgi material. For ciliates,
however, Golgi material has been observed in but two
groups, the Ophryoscolecidae (Noirot-Timoth^e, 1957) and
Blepharisma (Kennedy, 1965). For Tetrahymena pyriformis.
Elliot, Zieg, and Hunt (1966) found that starved ciliates
possess Golgi-like structures and that when the starved
ciliates conjugate, the Golgi-like structures are
72
concentrated near the region of contact between mates.
The cisternae or saccules of L. prorates also occur near <
I
|
the region of contact; the phenomenon may be similar to
that of T. pyriformis. Elliot, Zieg, and Hunt suggest j
that since these structures occur only in starving and
conjugating cells, they may be related either to starvation
or to the sexual process or both. That the conjugating
i
L. prorates were starving appears doubtful because food
vacuoles are present in the conjugants.
A number of problems that require further investiga
tion have appeared during the course of this study. Two
prominent problems are the origin of kinetosomes and the
origin of mitochondria. L. prorates appears to be an
excellent organism for studying the origin of kinetosomes.
Many budding individuals occur during the warmer months
of the year and the developmental stages are easily
recognizable. Another interesting problem is the apparent
degeneration of maternal mitochondria and origin of new
mitochondria in the newly budded individual. There is,
however, a difficulty in obtaining individuals at this
particular stage in development. Apparently the newly
budded individual passes through this phase of development
rapidly. Among the many L. prorates observed, very few
73
Newly budded individuals have been found. Both problems
are controversial and the method of formation of kineto
somes and mitochondria for L. prorates may be peculiar to
the Chonotrichida and not in accord with other organisms.
The greatest difficulty in studying L. prorates is
the inability of the organism to survive in the laboratory.
Most individuals become moribund if brought back to the
laboratory from San Pedro — in less than an hour. The
writer has not yet attempted to culture L. prorates.
SUMMARY
The ultrastructure of Lobochona prorates shows the
basic organization and structure of organelles — cilia,
mitochondria, endoplasmic reticulum, ribosomes, food
vacuoles — found in other Protozoa. The infraciliature
of L. prorates departs from the plan of most ciliates,
however, in that the "rule of desmodexy" is not followed.
The kinetodesmata of L. prorates lie directly beneath the
rows of kinetosomes rather than to the right as in most
ciliates that have been studied.
Ultrastructural changes during morphogenesis were
studied and correlated with a previous study at the light
microscopic level. The presumptive primordial site for
L. prorates is the "generative pouch" which forms at
maturity as an invagination of the pellicle. Cilia, cyto-
pharynx, and attachment organelle are derived from this
"generative pouch". Kinetosomes appear to develop de novo
from subpellicular fibrils at the innermost surface of the
pouch. Sections of L. prorates impregnated with silver
by the Bodian technic and observed with the electron
microscope show deposition of silver about the kinetosomes
but not about bundles of subpellicular fibrils which are
approximately the same size as kinetosomes and are aligned
and spaced in a pattern and position where kinetosomes
would probably occur. It is possible that the property
that makes a kinetosome "stainable" with silver does not
exist in a "prekinetosome" but appears only with maturation
of the kinetosome. The attachment organelle primordium
arises as an outpocketing of the "generative pouch" and is
also associated with the subpellicular fibrils. The
attachment disc is presumably secreted by the cytoplasmic
granules. The cytopharynx appears to be an extension of
the wall of the pouch (pellicle) into the cytosome.
In the budding process, mitochondria from the parent's
cytosome in the region of bud formation are incorporated
into the bud. In newly budded individuals, however, dense,
rounded mitochondria of varying sizes occur in groups
throughout the cytosome with apparently degenerating
mitochondria suggesting formation of new mitochondria in
the bud. The precursor, if any, or site of formation of
mitochondria has not been elucidated.
Few gymnostomatous ciliates have been studied with the
electron microscope; the affinities of L. prorates with
these ciliates which are reported to be closely related
remain obscure.
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Blepharisma undulans. J. Morphol., 64: 297-347.
Fig. 1. Drawing of mature Lobochona prorates showing
pellicle (Pe), adoral ciliary field (Ca), horizontal
ciliary field (Ch), cytostome (Cs), cytopharynx (Cp),
macronucleus (Ma), micronucleus (Mi), and attachment
organelle (Ao).
89
90
£&V'Ca
1
I
1
i
i
i
»
i
i
j
Fig. 2. Longitudinal section through mature L. prorates.
Pe - pellicle, C - cilia, Cs - cytostome, M - mitochondria,
Ma - macronucleus, Mi - micronucleus, Cg - cytoplasmic
granules, Ao - attachment organelle, B - marginal bristle
of host’s pleopod. 3240X.
91
92
Pigs. 3, 4, 5. The pellicle (Pe) of L. prorates showing
the plasma membrane (Pm), the external portion with pro
jections (0) and inner dense portion (D). Subpellicular
fibrils (Sf) and endoplasmic reticulum (Er) occur beneath
the pellicle. Po - pellicular pore, Pog - pellicular pore
with granules. 20,000X.
Fig. 6. Pellicular pore with apposed rough endoplasmic
reticulum (REr). 32,OOOX.
Pig. 7. Pellicular pore with spherical structure within
vesicle (Pos) and another vesicle with apposed endoplasmic
reticulum (Er). M - mitochondrion. 32,000X.
Fig. 8. Cross section through neck region showing wrinkled
external appearance and no indication of projections. Pe -
pellicle, Po - pore, Sf - subpellicular fibrils. 32,000X.
Figs. 9, 10. Pellicle of individuals impregnated with
silver by the Chatton-Lwoff technique showing silver (Ag)
deposited in the pellicular pores (Po). 32,000X.
93
94
^ ' v;V „>
•V- Ji'j 'j
^T, * r-
« ^ - V .v * .-,
f /
V . V ’.
Fig. 11. Tangential section through pellicle showing the
alternating light and dark portions and longitudinally
aligned pellicular pores (Po) with dense granules.
32,000X.
Fig. 12. Tangential section through pellicle of the
funnel showing the diverging subpellicular fibrils (Sf)
and kinetosomes (K) with dense granules (Kg) and kineto-
somal fibrils (Kf). 32,000X.
95

Fig. 13. Tangential section through pellicle showing
subpellicular fibrils (Sf) and vesicles (Ve) with complex
structure and attached endoplasmic reticulum (Er).
9,750X.
Fig. 14. Longitudinal section through pellicle showing
deposition of cobalt (arrow) indicating alkaline phos
phatase activity between projections of outer portion of
the pellicle. 20,000X.
Fig. 15. Tangential section through pellicle showing
adenosine triphosphatase activity in vesicles (arrows) of
pellicular pores. 20,000X.
97
98
% & & & it &*'■
I
I
i
Fig. 16. Longitudinal section through dead (degenerating)
L. prorates. Pe - pellicle# Ao - attachment organelle.
3,240X.
Fig. 17. Longitudinal section through pellicle of dead
individual showing lighter outer pellicle (PeO) and
remains of the dense portion (PeD). 20,000X.
Fig. 18. Longitudinal section through attachment organelle
of dead individual. 20,000X.
99
100
Fig. 19. Longitudinal section through attachment organ
elle. As - sphere of the attachment organelle with
tubules which appear as a tube (arrow) in cross section
and vesicular (Asv) outpocketing. Asp - outer sponge-like
portion of the attachment mechanism with attached sub-
pellicular fibrils (Sf). Ap - pellicle-like portion
surrounding the stem (Ast) and vesicles of the attachment
mechanism (Amv). Ad - attachment disc with fibrils within,
Cg - cytoplasmic granules, M - mitochondria, Ba - bacteria.
20,000X.
101

i
i
Figs. 20, 21, 22, 23. Cross sections through successively
lower levels of the attachment organelle. Ast - stem of
the attachment organelle, As - sphere of the attachment
organelle with enclosed tubules, Amv - vesicle of the
attachment mechanism, Ap - pellicle-like portion of the
attachment mechanism, Ad - attachment disc, M - mito
chondrion. 20,000X.
103
104
S&43&W
m
R m i
Fig. 24. Cross section through the attachment disc and
marginal bristle (B) of the host's pleopod. Fibrils
radiate from the stem (Ast) to the wall of the attachment
disc. Bacteria (Ba) appear to be embedded in some portions
of the attachment disc. 9,750X.
Fig. 25. A higher magnification micrograph showing the
relationship of attachment disc (Ad) to the bristle (B).
20,000X.
105
106
Fig. 26. Longitudinal section through attachment organelle
showing sponge-like portion (Asp) of the attachment
mechanism with attached subpellicular fibrils (Sf).
20.000X.
Fig. 27. Sphere of the attachment organelle (As) with
tubules that are apparently degenerating. 20,000X.
Fig. 28. Ring-like organization of subpellicular fibrils
(arrows). As - sphere of the attachment organelle.
20,000X.
107
108
Fig. 29. Cross section through funnel showing horizontal
ciliary field (Ch) and adoral ciliary field (Ca) with
folds (Caf) between rows of cilia. Arrows indicate gaps
between adoral and horizontal fields. 9,750X.

Fig. 30. Longitudinal section through horizontal ciliary
field showing absence of fold between rows of cilia. Cross
sections through ciliary shaft shows ciliary membrane (Cpm)
and enclosed matrix with typical "9 +2" fibrillar
arrangement. Alveoli (Cal) occur between rows of cilia.
Cax - axosome, Cb - basal plate, Cp - parasomal sac, K -
kinetosome, Kg - dense granules within kinetosome, Kf -
kinetosomal fibrils, Kr - fibrils from proximal end of
kinetosome, Kd - kinetodesma, M - mitochondrion. 32,OOOX.
Fig. 31. Cross section through cilia and kinetosomes of
adoral field at successively deeper levels from right to
left. Caf - fold between rows of cilia, Cp - parasomal
sac, Kf - kinetosomal fibril, Kd - kinetodesma, Sf - sub-
pellicular fibril, M - mitochondrion. 20,000X.
Ill
112
0
-
(0
Pig. 32. Longitudinal section through horizontal field
showing subpellicular fibrils (Sf) passing to the pellicle |
i
between cilia. 20f000X.
j
Fig. 33. Subpellicular fibrils (Sf) appear to join j
fibrillar band (kinetodesma = Kd). 20,000X.
Fig. 34. Cross section through adoral field showing
deposition of silver (Ag) in the parasomal sacs. Chatton-
Lwoff technic. 20,000X.
Fig. 35. Longitudinal section through horizontal field
showing deposition of silver (Ag) in parasomal sacs.
Chatton-Lwoff technic. 20,000X.
Fig. 36. Cross section through adoral field showing
adenosine triphosphatase activity (dense granules at
arrows) on the surface of fibrils. 32#OOOX.
Fig. 37. Longitudinal section through cilia and kineto-
somes showing adenosine triphosphatase activity. 32,000X.
113
114
Fig. 38. A composite drawing of a longitudinal section
through a cilium and kinetosome. Cax - axosome, Cb - basal
plate, Kr - fibrils frcm proximal end of kinetosome, Kd -
kinetodesma, Cp - parasomal sac, arrows indicate transi
tional filaments.
Fig. 39. Cross sectional drawings through different
levels of cilium and kinetosome. Letters {a, b, c)
indicate corresponding levels in Fig. 38. Arrow -
secondary fibril on radial link.
Fig. 40. Diagramatic drawing of kinetosomes and fibrils.
The cytostome opens to the left (arrow) of the figure.
K - kinetosome, Kf - kinetosomal fibril, Kd - kinetodesma,
Kr - fibrils from proximal end of kinetosome.
115
116
Ca
Cp
38
CO
< * » CP
39
Kf
K
Kd
40
Fig. 41. Longitudinal section through neck region showing
cytostome (Cs) and fibrils lining the cytopharynx (Cp).
Fine fibrils (F) and tubules (Tn) surround the cytopharynx.
Mitochondria (M) and rough endoplasmic reticulum (REr)
occur below the ring-like structure (Rn). Tubules appear
to pass through the ring-like structure (arrow). Dense
granules occur in the pellicular pores (Po). Sf - sub
pellicular fibrils. 20,000X.
117
118
Fig. 42. Longitudinal section through neck region showing
cytopharynx (Cp) filled with cytoplasm and fine fibrils (F)
and tubules (Tn) and vesicles (Ve) anterior to the ring
like structure (Rn). Canaliculi (arrows) pass through the
ring-like structure. M - mitochondria. 20,000X.
Unstained.
120
Fig. 43. Cross section through cytostome. Fibrils of the
cytopharynx (arrow) appear continuous with the pellicle.
Ba - bacterium, C - cilia. 32#OOOX.
121
122
c - >*
, T i x 1
/
5
m
f t
Fig. 44. Cross section through neck region and through
ring-like structure (Rn) showing differences in cytoplasm.
Ribosomes (R) and rough endoplasmic reticulum (REr) occur i
below the ring. Cp - cytopharynx, Po - pellicular pore
with dense granules, Sf - subpellicular fibrils. 20,000x.
123

Fig. 45. Cross section through neck region showing
cytoplasm in the cytopharynx (Cp) and mitochondria (M) in
cytoplasm below the ring-like structure (Rn). Sf - sub-
pellicular fibrils, Po - pellicular pore with dense
granules. 20,000X.
125

Fig. 46. Longitudinal section through cytopharynx (Cp)
with bacterium (Ba) within. The transition zone between
the cytoplasm within the cytopharynx and the cytoplasm of
the cytosome is filled with vesicles (Ve) and vacuoles
(Va). 20,000X.
128
Fig. 47. Oblique section through transition zone of the
cytopharynx (Cp) and the cytoplasm showing vesicles with
dense particles (arrows). 32f000X.
129
130
H
Ml
r * r t* %
% » * ^
¥ S +
1
Fig. 48. Oblique section through macronucleus (Ma) and
micronuclei (Mi) of a young individual. Pore-like
structures (arrows) occur on the nuclear envelope which
appears smoother than in mature individuals. Ch -
chromatin/ Nu - nucleolus. 9,750X.
Fig. 49. Longitudinal section through macronucleus of
mature individual. Nucleoli (Nu) occur in vesicles of the
anterior portion of the chromophilic zone (Mpa) and in
vesicles of the posterior portion of the chromophilic
zone (Mp). Nucleoli (Nu) also occur in the chromophobic
zone (Mb). A homogeneously dense layer (arrow) lies
between the chromophilic and chromophobic zones. Rough
endoplasmic reticulum (REr) and canaliculus occur by the
macronucleus. Mac - nuclear cleft, M - mitochondrion,
G - Golgi material. 20,000X.
131

Fig. 50. Section through micronuclei (Mi) showing pore
like structure (Mpo) on the nuclear envelope and Golgi
material (arrows) around micronuclei. Bacteria (Ba) occur
in vacuoles not lined by membranes. M - mitochondrion.
20,000X.
Fig. 51. Micronucleus (Mi) showing outpocketing with
unidentified dense body (arrow). M - mitochondrion, Cg -
cytoplasmic granules. 20,000X.
133

Fig. 52. Cross section through cytosome below level of
macronucleus showing peripheral distribution of mitochon
dria (M) and rough endoplasmic reticulum (REr) beneath
pellicle. Po - pellicular pore, Ba - bacterium. 20,000X.
136
52
Fig. 53. Oblique section through terminus of cytopharynx j
showing many small vesicles (Ve) and two large food
vacuoles (Fv). The vacuole at the right (Fvi) is newer
than that at the left (FV2)• Note outpocketing (arrow)
and small vesicles (Ve) with dense contents. Ma - macro
nucleus, M - mitochondrion. 20,000X.
Fig. 54. Section through young food vacuole (Fv) showing
smooth outline and undisrupted condition of food (Ba).
20,000X.
Fig. 55. Section through a food vacuole (Fv) in a later
stage showing partially digested bacterium (Ba) and dense
material (arrows). 32,000X.
137
138
t
I
Fig. 56. Section through a still later stage shows out-
pocketing (arrows) of the food vacuole and entrapment of
the dense material into the vesicles. 20,000X.
Fig. 57. Section through older vacuole (Fvi) which appears
empty and surrounded by small vesicles (Ve) with dense
material. Other vacuoles (FV2) appear collapsed, leaving
portions of the vacuole without a membrane (arrows).
Bacteria (Ba) occur in the cytoplasm and in vacuoles not
lined by a membrane. M - mitochondrion. 20,000X.
Fig. 58. Old vacuole (Fv^) surrounded by many vesicles
(Ve) with dense contents. A myelin figure appears in
some food vacuoles (FV2). M - mitochondrion. 20,000X.
139
• ?■ : v
x
»
J
i
Fig. 59. Section through food vacuoles showing absence of i
acid phosphatase activity in a new vacuole (Fv^) and
activity (dense granules) in older vacuoles (FV2). Note
absence of activity in small vesicles with dense contents
(arrows). U - unidentified body* M - mitochondrion.
20* 000X.
Fig. 60. Unidentified body (U) with dense bodies and
lamellar structure. 20,000x.
Fig. 61. The unidentified body (U) closely apposed to a
rather new food vacuole (Fv). 20,000X.
Fig. 62. The unidentified body (U) apposed to an older
food vacuole (Fv) appears to be degenerating. 20,000X.
Fig. 63. The apparent remains of the unidentified body
(U) occurs by old food vacuoles (Fv). 20,000X.
141
142
Fig. 64. Oblique section through dividing micronuclei (Mi)
showing tubular spindle fibers (S), dense areas which are
apparently chromosomes (Chr) and pore-like structures
(arrow) in the persistent micronuclear membrane. Rough
endoplasmic reticulum (REr) is closely associated with
mitochondria (M). Cg - cytoplasmic granules, Fv - food
vacuole, G - Golgi material. 20,000X.
143
144
? v 3 S . r . * ; f c ' . " f '
V/a^>s* * s
i
I
ft
%
K.
f t
*
Fig. 65. Longitudinal section through dividing micronuclei
(Mi) showing tubular spindle fibers (S) and pore-like
structures (Po) on persistent micronuclear membrane.
Ribosomes occur on the nuclear membrane (arrows). Many
ribosomes and rough endoplasmic reticulum occur in the
cytoplasm. M - mitochondrion, Cg - cytoplasmic granules.
20,000X.
145
146
e
9
m u .
> -W /
X J & -
h
L# 9' ? *
v^L , : r
* 7* , *•*+. - ►
” r J; • , t' *
I
Fig. 66. Longitudinal section through generative pouch
showing concentration of subpellicular fibrils (Sf) and
many ribosomes (R) and rough endoplasmic reticulum (REr).
M - mitochondrion. 20,000X.
Fig. 67. Cross section through generative pouch showing
closely associated subpellicular fibrils (Sf) and rough
endoplasmic reticulum (REr). 20,000x.
Fig. 68. Longitudinal section through generative pouch
of an individual at a later stage of budding. The tubules
of the attachment organelle primordium (Ao) and kineto-
somes (K) and cilia (C) are apparent. Ma - macronucleus.
20,000X.
147

Fi9. 69. Section through generative pouch showing
deposits of silver (Ag) on kinetosomes after treatment
with the Bodian technic. Ma - macronucleus, M - mito
chondrion. 20,000X.
Fig. 70. Section through another individual in a similar
stage of budding but at a different level shows concen
trations of fibrils (arrows) aligned and spaced in a
pattern and position where kinetosomes would probably
occur. Ao - attachment organelle primordium, M - mito
chondrion, Ma - macronucleus. 20,000x.
149

Fig. 71. Longitudinal section through developing ciliary
field (C) and attachment organelle primordium with sphere
toward posterior of developing bud. Many vesicles (Ve)
and canaliculi (Cn) and ribosomes (R) surround the sphere
of the attachment organelle primordium. Ma - macro
nucleus, M - mitochondrion, Cg - cytoplasmic granules,
K - kinetosomes, Kd - kinetodesma. 20,000x, (After
Matsudo, 1966).
151
152
Pig. 72. Section through generative pouch showing
developing cytopharynx (Cp). C - cilia, M - mitochondrion,
Ma - macronucleus. 20,000x. (Ribonucleic acid extracted).
Pig. 73. Section through generative pouch showing
development of attachment organelle primordium (Ao) by
outpocketing of pouch. C - cilia, Cg - cytoplasmic
granules. 20,000X, (Ribonucleic acid extracted).
153
154
vV'/
r"*anr
Fig. 74. Longitudinal section through generative pouch
showing communication with the exterior (arrow). M - mito
chondrion, K - kinetosomes. 20,000X. (Ribonucleic acid
extracted).
Fig. 75. Lipid bodies (L) occur in the cytoplasm of the
developing bud. 20,000X.
Fig. 76. Section of developing bud showing lipid body
(L) and many small vesicles with dense contents (arrow).
20,OOOX.
155

I
Fig, 77. Cross section through canaliculi (Cn)
attachment disc with cytoplasmic granules (Cg).
macronucleus, M - mitochondrion. 32#000X.
of the
Ma -
157
158
• »
c j y * t
> * { * .
#
■ * V * J V i
< * ; V - ' ’ ?.»• » ‘J - ’ •
O
Fig. 78. Longitudinal section through developing bud
showing compact cytoplasm with many small vesicles with
dense contents (Ve), rough endoplasmic reticulum (REr),
ribosomes, and Golgi material (G). M - mitochondrion,
L - lipid body, Ba - bacterium. 20,000X.
159
160
* * y * *
. ' > '••.-■ V « * v .
• '"'J%
v-' ■ • ''••/
?v i ^ j - . f T,i '
*?
f
Figs. 78-83. Partially diagramatic drawings showing
development of generative pouch, kinetosomes, attachment
organelle primordium, and cytopharynx.
161
162
4
Fig. 85. Oblique section through posterior portion of
cytosome showing subterminal attachment organelle and
cytosome "pinching" (arrow) marginal bristle (B). M -
mitochondrion, Mi - micronucleus. 6750X.
163
164
85
I
Fig. 86. Rounded mitochondria (M) of varying sizes occur
in groups in cytosome with apparently degenerating mito
chondria (Mg). Some mitochondria appear to be continuous
with membranes of the endoplasmic reticulum (arrows) while
others appear to be of homogeneous composition (H).
Ma - macronucleus. 20f000X.
165
166
*
m
i
i
i
|
i
i
|
i
i
Fig. 87. Longitudinal section through recently budded
individual showing compact cytoplasm and subterminal
attachment organelle (Ao). Dense bodies (arrows) occur
in the macronucleus. Mitochondria (M) appear elongate.
Food vacuoles (Fv) and few cytoplasmic granules (Cg) are
present, 4,750X.
167
168
87
Fig. 88. Longitudinal section through attachment organelle
(Ao) showing a smaller part of the cytosome around the
bristle (B). Many small vesicles with dense contents (Ve)
occur in the cytoplasm. Mitochondria (M) appear elongate.
Cg - cytoplasmic granules. $750X.
170
Fig. 89. Oblique section through funnel of diminutive
budding individual showing reduced number of cilia (C).
The pellicle (Pe) appears thickened and wrinkled, some
portions appear mottled. The cytoplasm appears fibrous,
granular bodies (arrows) are present. M - mitochondrion.
20,000X,
171
172
Pig. 90. Oblique section through cytosome of diminutive
budding individual. Cytoplasm appears fibrous and compact,
mitochondria (M) appear to be degenerating. Fv - food
vacuole, Cg - cytoplasmic granule, Po - pellicular pore
with associated vesicle and endoplasmic reticulum.
20,000X.
173
174
Fig. 91. Section through bud of diminutive budding
individual appears "normal". M - mitochondrion, Cn -
canaliculus of attachment mechanism, Cg - cytoplasmic
granule, As - sphere of the attachment organelle with
tubules, arrow indicates outpocketing from sphere.
20,000X.
175
M .
■ # • ' * • • . . . ■ v ' . .;.vW'-'» ' ' ■
::"v i ' \ ■ M ' .:*■
9Z.T
Fig. 92. Longitudinal section through diminutive budding
individual. Parental pellicle appears thick and wrinkled
and cytoplasm fibrous. The bud appears normal. The sphere
of the attachment organelle primordium (As) faces the
anterior end of the bud. Ao - attachment organelle, B -
marginal bristle of pleopod. 4,750X.
177
178
Fig. 93. Section through diminutive parent showing rough
outer surface of the pellicle (Pe) and bacteria (Ba) in
vacuoles not lined by membranes. About 12#000X.
Fig. 94. Section through diminutive parent showing
smoother pellicle (Pe) and remnant of collar (arrow). Few
mitochondria (M) occur in the vacuolated cytoplasm. About
10,000X.
179
180
Fig. 95. Longitudinal section through stationary conjugant
and portion of migrating conjugant. The surface of the
pellicle appears rough and a pouch (arrow) is present in
the stationary conjugant. A vacuole (Va) occurs in the
neck region. Ma - macronucleus, Mpa - anterior portion
of the chromophilic zone, Mb - chromophobic zone, Mi -
micronucleus, Cp - cytopharynx. 3,240X.
181

Fig. 96. Section through neck region of stationary
conjugant showing large vacuole (Va). Er - endoplasmic
reticulum, L - lipid body, Ma - macronucleus, arrows
indicate cisternae. 20,000X.
183
m^mmz
Fig. 97. Longitudinal section through migrating conjugant
showing absence of all structures anterior to the ring-like
structure (Rn) in the neck region and funnel of stationary
conjugant. Fv - food vacuole. Mi - micronucleus, Ma -
macronucleus. 4,750X.
185

Fig. 98. Longitudinal section through point of contact
between migrating (upper) and stationary (lower) conjugants.
Rows of cisternae (arrows) occur in the migrating conju
gant. Structures anterior to the ring-like structure (Rn)
have disappeared. A lipid body (L) occurs in the funnel
of the stationary conjugant. 20,OOOX.
187
188
\ V » ' ■ v w & H E ^*s ' ' ■
'•W XBflRSSw ' • • • ’ •.
'*vV.V-J> '•• • * # •
' % y ^ > -V .
' V > ; . # A - ' - t(* . J
7^ ^ .: "A.< • , ; , , v f i / ! -
1 V-.*
^ v,.-
Fig. 99. Longitudinal section through migrating conjugant
showing micronuclei (Mi) and macronucleus (Ma). Pore-like
structures (arrow) occur on the micronuclear envelope.
Chromosomes (Chr) appear as dense granules. Food vacuoles
(Fv) and many small vesicles (Ve) with dense material are
present. 20,000X.
189
190
m m M
This dissertation has been
microfilmed exactly as received 6 7 -1 3 754
MATSU DO, Hitoshi, 1933-
ON THE ULTRASTRUCTURE AND MORPHOGENESIS OF A
MARINE CHONOTRICHOUS CILIATE PROTOZOAN,
Lobochona prorates.
University of Southern California, Ph.D., 1967
Zoology
University Microfilms, Inc., Ann Arbor, Michigan

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Asset Metadata

Creator Matsudo, Hitoshi, 1933- (author)

Core Title On the ultrastructure and morphogenesis of a marine chonotrichous ciliateprotozoan, Lobochona prorates

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1967-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, zoology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mohr, John Luther (committee chair), Bils, Robert F. (committee member), Garth, John S. (committee member), Gorsline, Donn S. (committee member), Pray, Thomas R. (committee member), Zimmer, Russel L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-147974

Unique identifier UC11360175

Identifier 6713754.pdf (filename),usctheses-c18-147974 (legacy record id)

Legacy Identifier 6713754.pdf

Dmrecord 147974

Document Type Dissertation

Rights Matsudo, Hitoshi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA