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4-1-1956

A STUDY OF EGGS OF ASCARIS

LUMBRICOIDES VAR. SUUM WITH THE
ELECTRON MICROSCOPE
Rodney A. Rogers
Zoology Department, State University of Iowa, Iowa City, Iowa

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Rogers, Rodney A., "A STUDY OF EGGS OF ASCARIS LUMBRICOIDES VAR. SUUM WITH THE ELECTRON MICROSCOPE"
(1956). Journal of Parasitology Archives. Paper 618.
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1he Journal ot Yarasitology

Volume' 42

APRIL, 1956

Number 2

A STUDY O F EGGS O F ASCARIS LUMBRICOIDES VAR. SUUM

W I T H T H E ELECTRON MICROSCOPE*

Zoology Department, State University of Iowa, Iowa City, Iowa

The egg of Ascaris has been widely used as a subject for cytological investigations. Nelson (1851) described the development of the egg of Ascaris nzystax.
Leuckart (1886) and Wharton (1915) reported the Ascaris luinbricoides egg to
be surrounded by a shell and an outer, albuminous layer. The appearance of unfertilized eggs of this species was described by Otto (1932) and Keller (1933).
The number of layers surrounding the egg of ascarids has been reported as three
(Nelson, 1851, Ascaris 'mystax; Leuckart, 1886, Ascaris luwibricoides; Ackert,
1931, Ascaridia lineata; Wottge, 1937, Parascaris megalocephala; Christenson,
1940, Ascaris lumbricoides), four (Kreuzer, 1953, Ascaris lu~.izbricoides;Frenzen,
1954, Ascaridia galli, and five (Zavadovsky, 1928, Ascwis). A variety of techniques has been used by different authors in studying the egg layers, including
microscopic observations of the penetration of substances into the egg, polarizing
and fluorescent microscopy, centrifugation, and chemical tests on the membranes.
The chemical composition of the layers has been investigated by several workers
(Faurk-Fremiet 1913, 1913a; Chitwood 1938; Christenson, et al. 1942; Jacobs
1940; Timm 1950; MonnC and Honig 1954; Fairbairn 1955) and, in general, have
led rather consistently to the conclusion that there are three membranes. Wottge
(1937) described the endogenous formation of the egg layers of Ascaris wegaloccpha2a, whereas Christenson (1940) believes the outer layer of the Ascaris egg to
be of exogenous forn~ation.
Morita (1953) examined paraffin sections of the egg shell of Ascaris megalocephala with the electron microscope and concluded that the layer "consists of completely individualized microfibrils." In the present investigation, thin sections were
examined with the electron microscope to determine structural details of ova and
the order of formation of the egg coverings of Ascaris luwzbricoides var. szlu7tz.
MATERIALS A N D METHODS

Living adult Ascaris were 'removed from 'the intestines of freshly killed pigs
and thin slices from various parts of the egg-containing uterus were placed in fixative. The eggs were fixed in either 1% osmium tetroxide according to the method
of Palade (1952) for 2 hours, or Carnoy's fixative (acetic acid 2070, absolute
Received for publication, August 29, 1955.
* A revised portion of a thesis submitted in partial fulfillment of the requirements for the
Ph.D. degree at the State University of Iowa, 1955.
t Present address: Biology Department, Drake University, Des Moines, Iowa.
97

98

T H E J O U R N A L OF PARASITOLOGY

alcohol 80%) for 18-24 hours. Following fixation, the ova were washed, dehydrated, and infiltrated with a mixture of 72% n-butyl- and 28% methyl-inethacrylate. The eggs were sectioned at 0.025 microns with an International Minot rotary
microtome, the thin sections then placed on celloidin-coated grids and examined
with the aid of a model EMU-2B RCA electron microscope.
OBSERVATIONS

Organization of the Ascaris egg.

The egg cytoplasm of Ascaris luw$bricoides var. suum is surrounded by three
layers: An outer protein coat, a middle chitinous shell, and an inner lipoid lining.
These layers, as observed in the living state by means of light and phase microscopes, are shown in Figure 1. The protein coat and the chitinous shell when
observed in eggs removed from the uterus of the adult worm, are usually colorless
and transparent. Striations are observed in the lipoid layer. Between the cytoplasm and the lipoid layer in the mature, fertilized egg is a fluid-filled cavity, the
perivitelline space. Some of the egg layers and the perivitelline space are absent
in unfertilized eggs (Fig. 2).
Structure of the egg by electron ,tlzicroscopy.
Protein Coat

The external margin of the protein layer is usually mamrnillated (Fig. 3). The
~nammillationsvary greatly, appearing obtuse, undulate, or very irregular. The
surface is granular, and when interrupted, the ends of fibers arising from the protein coat can be observed. In fully-formed eggs, a distinct separation between the
protein and the shell layer is observed, and the protein coat terminates abruptly
at the outer margin of the shell.
The protein coat appears as a very dense, reticulated material (Figs. 4 and 5)
containing granules without apparent structure. The fibrils of the reticulum have
a diameter of approximately 150 Angstrom units ( A ) and branch irregularly from
the granules. The coat appears uniform in density except where it is very broad,
in which case the branches are more widely separated at the outer margin.
Chitinous Shell

The chitinous shell attains a thickness of approximately three to four microns

in the mature egg. The inner surface of the shell is limited by a membrane (Fig.
4) containing small granules and fibrils. The outer surface of the shell is bordered
by a dense zone or membrane (Figs. 4 and 5), approximately 0.4 micron in thick-.
ness, containing granules and fibrils. At the margin of this membrane, the junction
between the protein layer and the shell is marked by a narrow line (Fig. 5) approximately 200 A in thickness. The membrane is joined at intervals by the
chitinous fibers of the shell. These chitinous microfibers (Fig. 5) vary in diameter
from approximately 75 A to 400 A. The majority of fibers branch when reaching
a length of 250 A or less to form a loose, irregular reticulum. A lamellation of
the chitinous fibers was not observed.
Electron micrographs which are believed to represent successive stages in the
formation of the chitinous shell are shown in Figures 6 through 11. Figure 6 shows
small granular projections attached to the plasma membrane along its entire

ROGERS-ASCARIS

L U M B R I C O I D E S EGGS

99

margin. These surface projections may extend outward from the plasma mei11brane, or they may be elongated, attached as an inverted-U (Fig. 7 ) , or scattered
irregularly along the membrane. The projections may have been formed from the
dark granules within the cytoplasm of the egg. A material which forms around
the surface projections (Fig. 8 ) , is possibly a precursor of the chitin fibers and
may have been carried to the surface by cytoplasn~icvacuoles (Wottge, 1937), or
secreted by the projections. The chitin structure as well as the disappearance of
the surface projections, is observed in Figure 9. Later stages in the process of
shell formation are shown ill Figures 10 and 11, where the chitinous shell has become much thicker. The plasma membrane is visible on the inner surface and the
protein material is beginning to be attached on the outer surface of the shell. The
embedding medium has not been removed from the latter two figures; hence the
fibrous arrangement of the chitinous shell is not as immediately apparent as in Figure 9. A section through sperm is shown external to the egg in the upper part of
Figure 7. All photographs in Plate I11 are taken at the same magnification, and
the appearance of size differences is due to the part of the egg through which the
section was cut.
Lipoid Layer

The lipoid layer is soluble in all common fat solvents, and therefore dissolved
in the process of preparing the eggs for sectioning. The layer is not fixed by one
percent osmium tetroxide or Carnoy's fixative, and the method of freeze-drying
was found not to be satisfactory for its study. It was, therefore, not possible with
any of these methods to obtain electron micrographs of the lipoid layer.
Perivitellilze Space

The perivitelline space, which is filled with fluid as a result of "deutoplasmolysis" (Lams, 1952), appears optically empty; no structures of any kind were
observed with the methods of fixation used.
Cytoplasm

Large numbers of granules and vacuoles are located within a very coarse
cytoplasmic reticulum (Figs. 12 and 13). The vacuoles, varying in size from 0.3
to 1.7 microns in diameter, are optically empty; a fine, fringed border is sometimes
seen surrounding the inside of the wall. The vacuoles may be concentrated in the
central portion, or scattered at random in the cytoplasm.
The granules of the cytoplasm, called "brown granules" by Boveri (1910), vary
in diameter from 0.1 to 0.8 microns. No structural details were observed and
whether or not some of the granules are mitochondria is uncertain.
In the immature egg, large, homogeneous, ovoid structures called "hyaline
spheres" by van Beneden (1883) were observed (Fig. 13). They vary in diameter
from 1 to 4 microns and are enclosed by a granular membrane. At the outer margin
of some immature eggs, material which appears similar to that contained within
the spheres was observed. The hyaline spheres are dissolved in acetic solutions
(FaurP-Fremiet et al., 1953, 1954) and were observed only with osnlium tetroxide
fixation.
The nucleus, or pronucleus (Fig. 14), approximately four microns in diameter,
is surrounded by a granular membrane. It contains dark bodies, presumably
chromatin material, as well as a loose protoplasmic reticulum.

100

T H E J O U R N A L OF P A R A S I T O L O G Y
DISCUSSION

The nature of the egg envelope and therefore a knowledge of its ultrastructure
are of interest. The controversy which exists concerning the number of membranes in nematode eggs was summarized by Jacobs (1940) who indicated, "the
difference in the number of membranes described by various authors can . . . be
attributed to the variety of techniques used in studying the eggs".
The electron micrographs presented herein support the belief that there are
three layers surrounding the egg cytoplasm of Ascaris lumbricoides var. suum, viz.
the outer protein coat, the middle chitinous shell, and the inner lipoid layer. The
report of Frenzen (1954) that the egg envelope of Ascaris lumbricoides includes
four layers is not supported by the present work. The portion of the egg indicated as a separate layer by him is probably the zone of separation between the
protein coat and the chitinous shell. This zone, although more dense, is not believed to represent a distinct egg layer, since close observation at high magnification
reveals a typical chitin structure. Furthermore, the report of Zavadovsky (1928)
that the egg cytoplasm of Ascaris is enclosed by five layers is not supported by the
present investigation, since a lamellation of the chitinous shell was never observed.
Jaskoski (1952) suggested that the outer protein coat represented an important
auxiliary barrier against the passage of materials in or out of the ascarid egg.
Germans (1954) concluded that the "slime layer" confers protectio~~
against desiccation. Wottge (1937) reported that the protein coat contained a lipoid substance,
and this material might be located in the interstitial vacuoles of the protein reticulum. The network arrangement of the protein coat with interposed lipoid material,
as well as the limiting membranes separating the egg layers, may lend support to
these conclusions.
The endogenous formation of the protein coat of the egg of Ascaris ?+aegalocephala, advanced by Wottge (1937) was based on the presence of a protein covering around unfertilized eggs. Chitwood ( 1938), however found eggs without
this layer and suggested that the coat was a uterine secretion added after the two
inner layers had formed. In support of this latter point of view, Lowry et al.,
(1941), observed granules within the cytoplasm of the uterine cells of Ascaris
equorum, and suggested that the granules represented an albun~inous substance
which is secreted into the uterus, and surrounds the eggs as they pass along it.
The electron micrographs of the pig Ascaris egg fail to show the protein coat surrounding either the oocyte as described by Wottge (1937), or the outer margin of
those eggs in which the chitinous shell was in the process of formation. I n view
of these observations, the order of formation of the egg envelope is believed to
occur as follows: the chitinous shell is formed first, followed by the appearance of
the lipoid lining, both of these layers of endogenous formation; the protein layer
of the egg is added last as a product of uterine secretion.
Kreuzer (1954) suggested that the chitinous shell provides mechanical protection to the egg and preserves stability of form. Wottge (1937) referred to this
layer as the "hotnogeneous membrane" because of the clearness and transparency
of the layer, and Schmidt (1936) indicated that the shell substance was homogeneous and composed of non-fibrillar material. Observations of the chitinous shell
with the electron microscope indicate that the shell is distinctly fibrillar. The suggestion of MonnC and Honig (1954) that the shell of Ascaris consists of chitin

ROGERS-ASCARIS

LUMBRICOIDES EGGS

101

fibers and protein lamellae which alternate with each other seems not to be warranted on the basis of these observations. The photograph published by Morita
(1953) as a cross-section through the shell, actually appears to be a photograph
of the outer protein layer. The size of the chitin fibers, as reported by this author,
probably represents the measurements of the protein strands of the outer coat, and
consequently the lower limits are much too large. In addition, it is believed that
the cross-section of the shell, in any case, would not have the appearance pictured.
Monnt! and Honig (1954) indicated that the lipoid coat is the portion of the
egg envelope which protects the egg against penetration by harmful chen~icals,and
Kreuzer (1954) suggested that this layer produces a completely effective diffusion
barrier to ions. The ultrastructure of this region will be of interest when fixation
techniques for the study of this material are devised.
Faurk-Fremiet, et al. (1953, 1954) reported that the hyaline spheres were distributed at random in the cytoplasin of the oocyte, and passed to the periphery
after fertilization, where they coalesced and disappeared into the perivitelline space.
The role and significance of the spheres remain undetermined.
SUMMARY

1. Studies of ova of Ascaris lunzbricoides var. suuvvz with the electron microscope support the belief that the egg envelope is coiiiposed of three layers which in
order of forniation include a middle chitinous shell, an inner lipoid layer, and an
outer protein layer. The protein coat and the chitinous shell are bordered by limiting membranes.
2. The protein coat of the egg, whose margin ii~aybe sinooth or maminillated,
appears as a dense, reticulated material, with fibrils having a diameter of about
150 A.
3. The chitinous shell is coinposed of highly branched microfibers which vary
in diameter from approximately 75 A to 400 A. A lamellation or orientation of
the chitin fibers in any one plane of the egg was not observed. Electron micrographs, which may represent successive stages in the formation of the chitinous
shell, are presented.
4. The lipoid layer, in the living state, has a striated appearance. A suitable
method for the fixation of the layer, however, was not found.
5 . Electron micrographs fail to reveal structures of any kind in the perivitelline
space of the egg.
6. Large numbers of granules and vacuoles are located within a very coarse
cytoplasmic reticulum inside the egg itself. The hyaline spheres, observed in immature eggs, are described.
7. The highly branched arrangement of the protein coat and the chitinous shell,
as well as the limiting membranes separating the egg layers, may help explain the
extreme resistance of Ascaris ova to many environmental changes.
ACKERT,
J. E. 1931 The morphology and life history of the fowl nematode, Ascaridia lineata
(Schneider) . Parasitology 23: 360-379.
BENDEN,
E. VAN 1883 Recherches sur la maturation de l'oeuf et la fkcondation (Ascaris megalocephala). Arch. Biol. Gand. 4: 265440.
BOVERI,T . 1910 Uber die Teilung centrifugierter Eier von Ascaris megalocephala. Arch.
Entwicklngsmechn. Organ. 30, 2. Teil : 101-125.

102

THE J O U R N A L O F P A R A S I T O L O G Y

CHITWOOD,
B. G. 1938 Further studies on nemic skeletoids and their significance in the chemical
control of nemic pests. Proc. Helminth. Soc. Washington 5: 68-75.
CHRISTENSON,
R. 0. 1940 Nemic Ova. Chapter 12 in Chitwood and Chitwood, An Introduction to Nematology, Section 1. Baltimore, Md. Monumental Printing Co. pp. 175-190.
CHRISTENSON,
R. O., EARLE,H . H. JR., BUTLER,R. L. JR., A N D CRELL,H. H. 1942 Studies on
the eggs of Ascaridia galli and Heterakis gallitzae. T r . Am. Micr. Soc. 61: 191-205.
FAIRBAIRN,
0 . 1955 Embryonic and postembryonic changes in the lipids of Ascaris lulnbricoides
eggs. Canadian J. Biochem. and Physiol. 33: 122-129.
FAUR~-FREMIET,
E. 1913 Le cycle germinatif chez I'Ascaris megalocephala. Arch. Anat. Micr.
15: 435-758.
1913a Le formation de la membrane interne de l'oeuf d'Ascaris megalocephala. Compt.
Rend. Soc. Biol., Paris 74: 1183-1184.
, EEEL,J., A N D COLAS,J. 1953 Les inclusions protCiques de l'oeuf de Parascaris
equorum. Compt. Rend. Acad. Sci., Paris 237: 629-631.
-, A N D ---1954 Les inclusions protCiques de l'oocyte de Parascaris equorum.
Exper. Cell Research 7: 153-168.
FRENZEN,
K. 1954 Untersuchungen zur Morphologie und mikroskopische~l Anatonlie von
Ascaridia galli Shrank, 1788. Zool. Jahrb. 73: 395-424.
GERMANS,
W . 1954 Laboratoriumsuntersuchungen uber die Resistenz der Eier des menschlichen
Spulwurmes Ascaris luntbricoides L. Ztschr. Parasitenk. 16: 93-110.
JACOBS,
L. 1940 The chemistry of the egg membranes. Chapter 12 in Chitwood and Chitwood,
An Introduction to Nematology, Section 1, Baltimore, Md., Monumental Printing Co.
pp. 186-187.
JASKOSKI,
B. J. 1952 The protein coat in development of Ascaris lumbricoides eggs. Exper.
Parasitol. 1: 291-302.
KELLER,
A. E. 1933 A study of the occurrence of unfertilized Ascaris eggs. J. Lab. and Clin.
Med. 18: 371-374.
KREUZER,
L. 1953 Zur Kenntnis des chemischen Aufbaus der Eihulle von Ascaris luvibricoides.
Ztschr. Vergleich. Physiol. 35: 13-26.
1954 Zur Ketlntnis der physikalisch-chemischen Eigenschaften der Eihulle von Ascaris
lumbricoides. Ztschr. Vergleich. Physiol. 36: 21-26.
LAMS,H . 1952 Etude morphologie du cytoplasme de l'oeuf d'Ascaris megaloccphala au cours
de la ficondation. Acta Anat. 14: 141-167.
LEUCKART,
R. 1886 The parasites of man and the diseases which proceed from them, etc.
Edinburgh, Young J. Pentland (translation by Wm. E. Hoyle). 771 pp.
LOWRY,
O., BEAMS,H. W. A N D KING,R. L. 1941 The fibrillae of the uterine cells of Ascaris
equorum (variety u n w a l c ~ ~ Js ). Morph. 68: 585-591.
M O N N ~L., A N D HONIG,G. 1954 On the properties of the egg envelopes of various parasitic
nematodes. Ark. Zool., Stockholm 7: 261-272.
MORITA,S. 1953 The electron microscopy of cell membrane (1) The homogetleous membrane
of the egg cell from Ascaris megalocephala. Med. J . Osaka Univ. 3: 669-673.
NELSON,H . 1851 On the reproduction of the Ascaris nzystax. Proc. Roy. Soc. Lolldo116: 86-87.
OTTO,G. F. 1932 The appearance and significance of the unfertilized eggs of Ascaris lumbricoides (Linn.). J. Parasit. 18: 269-273.
PALADE,
G. E. 1952 A study of fixation for electron microscopy. J. Exper. Med. 95: 285-298.
SCHMIDT,W . I. 1936 Doppelbrechung und Feinbau der Eischale von Ascaris ~vregalocephala.
Ein Vergleich der Feinbaues faserigetl und filmartigen Chitins. Ztschr. Zellforsch. u,
Mikr. Anat. 25: 181-203.
TIMM,R. W. 1950 Chemical composition of the vitelline membrane Ascaris lu~rtbrrcoidesvar.
suis. Science. 112: 167-168.
WHARTON,
L. D. 1915 The development of the eggs of Ascaris lutnbricoides. Philippine J. Sc.,
Sect. B : Trop. Med. 10: 19-23.
WOTTGE,
K. 1937 Die stofflichen Veranderungen in der Eizelle von Ascaris nzegaloccpltala nach
der Befruchtung. Protoplasma 29: 31-59.
ZAVADOVSKY,
M. 1928 The nature of the egg-shell of various species of Ascaris eggs ( T o s a s c a r i s
limbata Raillet & Henry, Belascaris tnystax Zeder, Belascaris nzarginata Rud., Ascaris
suilla Duj.). Trudy. Lab. Eksper. Biol. Zoopark Moskov. Zooparka. 4: 201-205. (Not
seen.)

ROGERS-ASCARIS

LUMBRICOIDES EGGS

PLATE
I
FIG1. Living fertilized egg. P protein coat, S chitinous shell, L lipoid layer. x 1500
FIG. 2. Living unfertilized egg. P protein coat. x 700
FIG. 3. Fertilized egg. P protein coat, S chitinous shell, C cytoplasm. Carnoy's fixative.
Embedding medium not removed. x 12,000

PLATE
I1
FIG. 4. Mature egg. P protein coat, S chitinous shell. Carnoy's fixative. x25,600
FIG.5. The fibrous structure of the protein coat P and the chitinous shell S are shown.
Osmium tetroxide fixation. x 64,000
PLATE
I11
FIGS. 611. Successive stages in the formation of the chitinous shell. Figures 6-7, Osmium
tetroxide fixation. Figures 8-11 Carnoy's fixative. Embedding medium not removed in figures
10-11. All figures x 12,000.
PLATE
IV
FIG. 12. Mature egg. P protein coat, S shell, N nucleus. Carnoy's fixative. ~ 7 2 0 0

FIG.13. Cytoplasm of an immature egg in which the egg envelope has not yet formed. H
hyaline sphere, V vacuole, G brown granule. Osmium tetroxide fixation. x 12,000
FIG.14. Nucleus of egg enclosed by granular membrane. N nucleus. Carnoy's fixative.
x 18,400