University of Southern California Dissertations and Theses

Neurosecretion In The Life Cycle Of The Digenetic Trematode, Acanthoparyphium Spinulosum, Johnston, 1917

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Neurosecretion In The Life Cycle Of The Digenetic Trematode, Acanthoparyphium Spinulosum, Johnston, 1917

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Content NEUROSECRETION IN THE LIFE CYCLE OF THE DIGENETIC
TREMATODE ACANTHOPARYPHIUM SPINULOSUM
JOHNSTON, 1917
by
David Freed Steele
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)
August 1970
71- 16,437
STEELE, David Freed, 1940-
NEUROSECRETION IN THE LIFE CYCLE OF THE
DIGENETIC TREMATODE ACANTHOPARYPHIUM SPINULOSUM
JOHNSTON, 1917.
University of Southern California, Ph.D., 1970
Biology
University Microfilms, A X E R O X Company, Ann Arbor, Michigan
Copyright O by
DAVID FREED STEELE
1971
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED
UNIVERSITY O F SO UTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 9 0 0 0 7
This dissertation, written by
...................... Dayid.Free.d.StesJle.
under the direction of /as Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by The Gradu
ate School, in partial fulfillment of require
ments of the degree of
D O CTO R OF P H IL O S O P H Y
'.'In
Dtan
Date Augus.t...L9.7.Q..
DISSERTATION COMMITTEE
.....
I x J ) c«m4
ACKNOWLEDGMENTS
I should like to express my sincere appreciation to
the chairman of my dissertation committee, Dr. Walter E.
Martin, for his encouragement and help during this investi
gation .
I am also grateful to Dr. Russel Zimmer, Dr. Robert
Chew, Dr. Richard Tibby, and Dr. William Easton for serving
on my dissertation committee.
Finally, I am indebted to Dr. John Soule for the
photomicrographs.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS....................................... ii
LIST OF TABLES....................................... iv
LIST OF ILLUSTRATIONS................................... v
INTRODUCTION ......................................... 1
Chapter
I. HISTORICAL REVIEW ............................... 4
Neurosecretion in the Polychaetes
Neurosecretion in the Oligochaetes
Neurosecretion in the Hirudinea
Neurosecretion in the Nemerteans
Neurosecretion in the Nematodes
Neurosecretion in the Turbellaria
Neurosecretion in the Cestodes
Neurosecretion in the Trematodes
II. MATERIALS AND METHODS......................... 21
MateriaIs
Methods
III. RESULTS....................................... 27
IV. DISCUSSION................................... 48
SUMMARY................................................ 55
LITERATURE CITED ..................................... 74
iii
LIST OF TABLES
Table Page
1. Measurements of Ten Two-Day-Old Adults
of Acanthoparyphium spinulosum .............. 28
2. Measurements of Ten Four-Day-Old Adults
of Acanthoparyphium spinulosum.............. 29
3. Measurements of Ten Six-Day-Old Adults
of Acanthoparyphium spinulosum.............. 30
4. Measurements of Ten Eight-Day-Old Adults
of Acanthoparyphium spinulosum .............. 31
5. Measurements of Ten Ten-Day-Old Adults
of Acanthoparyphium spinulosum .............. 32
6. Measurements of Ten Twelve-Day-Old Adults
of Acanthoparyphium spinulosum.............. 33
iv
LIST OF ILLUSTRATIONS
Figure Page
1. Plot of Body Dimensions of Adult Acantho-
paryphium spinulosum Two, Four, Six,
Eight, Ten, and Twelve Days Old............. 36
2. Plot of Ovary Dimensions of Adult Acantho
paryphium spinulosum Two, Four, Six,
Eight, Ten, and Twelve Days Old............. 38
3. Plot of Anterior Testis Dimensions of Adult
Acanthoparyphium spinulosum Two, Four,
Six, Eight, Ten, and Twelve Days Old .... 40
4. Plot of Posterior Testis Dimensions of Adult
Acanthoparyphium spinulosum Two, Four,
Six, Eight, Ten, and Twelve Days Old .... 42
5. Genital Primordia in Two-Day-Old Acantho
paryphium spinulosum....................... 58
6. Genital Pore Primordium in Two-Day-Old
Acanthoparyphium spinulosum ................. 58
7. . Ovary and Primordium of Uterus and Ootype
in Four-Day-Old Acanthoparyphium
spinulosum................................. 60
8. Anterior Testis of Four-Day-Old Acantho
paryphium spinulosum....................... 60
9. Posterior Testis of Four-Day-Old Acantho
paryphium spinulosum....................... 62
Figure Page
10. Posterior Half of Four-Day-Old Acantho
paryphium spinulosum....................... 62
11. Cirrus Sac Primordium of Four-Day-Old
Acanthoparyphium spinulosum ................. 64
12. Cirrus Sac of Six-Day-Old Acanthoparyphium
spinulosum.................................. 64
13. Incompletely Formed Uterus and Ootype of
Six-Day-Old Acanthoparyphium spinulosum . . . 66
14. Sperm and Eggs in Eight-Day-Old Acantho
paryphium spinulosum....................... 66
15. Two of Four Additional Neurosecretory Cells
Which Appear in Nine-Day-Old Acantho
paryphium spinulosum; Cameron and Steele
Stain........................................ 68
16. Medially-Placed Neurosecretory Cells Seen
in Ten-Day-Old Acanthoparyphium spinu
losum; Cameron and Steele Stain ........... 68
17. Medially-Placed Neurosecretory Cell Seen
in Four-Day-Old Acanthoparyphium spinu
losum; Ewen S t a i n ......................... 70
18. Eight-Day-Old Acanthoparyphium spinulosum . . . 72
vi
INTRODUCTION
Neurosecretory cells have the regular functions of
ordinary nerve cells; however, they also possess some prop
erties of gland cells, in that they manufacture and release
a hormone. They can be affected by other hormones. The
phenomenon of neurosecretion is important, because the
neurosecretory cells thus make possible a relationship be
tween nervous and endocrine tissues. With these unique
properties, the neurosecretory cells hold an important posi
tion in the coordination of neuroendocrine interactions.
With the exception of the Coelenterata and Cteno-
phora, the phenomenon of neurosecretion has been observed
in all invertebrate and vertebrate groups. In general, the
neurosecretory system consists of a group of neurosecretory
cells and their axons which usually terminate near vascular
channels and form neurohemal organs. The neurohemal organ
acts as a storage place for the secretion until it is dis
charged into the circulatory system. The most frequently
1
2
cited examples of neurohemal organs are the corpus cardiacum
of insects, the sinus gland of crustaceans, and the neuro
hypophysis of vertebrates.
Neurosecretory material can be observed both in the
perikaryon and in the cellular processes. The selective
staining property of this material with such methods as the
alcian blue or aldehyde fuchsin techniques allows it to be
observed at the level of the light microscope. With the
electron microscope, the neurosecretory material appears to
consist of membrane-bounded, intracytoplasmic granules which
vary in size from 1000-3000&. Presumably these elementary
granules aggregate into larger inclusions of varying sizes
which become stainable and visible with the light micro
scope. It is thought that the hormonal component of the
neurosecretory material is a polypeptide which is subse
quently bound to a sulfur-rich protein carrier (neurophy-
sin) .
Evidence obtained by use of the electron microscope
suggests further that the neurosecretory material is synthe
sized much like other proteinaceous glandular products.
Electron micrographs show the material along the rough-
surfaced endoplasmic reticulum of the perikaryon and in
association with the Golgi apparatus where it presumably
3
receives a membrane. It is finally released into the cyto
plasm. The neurosecretory granules are then stored in the
cytoplasm until they are released. Although the typical
releasing site is the axon terminal, there is evidence to
suggest that release may occur along the axon or at the
periphery of the cell body (Bullock and Horridge, 1965;
Scharrer, 1967).
CHAPTER I
HISTORICAL REVIEW
Neurosecretion in the Polvchaetes
Neurosecretory cells have been observed in the adult
polychaete supraesophageal ganglion (Bobin and Durchon,
1953; Clark, 1959; Gabe, 1954; Korn, 1958a, 1958b, 1963;
Schaefer, 1939; Scharrer, 1936, 1937; Takeuchi, 1965c), in
the infraesophageal ganglion (van Damme, 1962), and in the
larvae of several polychaete families (Korn, 1958b, 1960a,
1960b) . Furthermore, neurosecretory material has been
observed in nerve cell axons supplying the palps (Arvy,
1954) and in axo" applying the prostomial epidermis as
well (Defretin, ,.
There seems to be little agreement, however, on the
number and kinds of neurosecretory cells present in these
worms. The existing classifications tend to lead to con
fusion since most authors erect their own classification of
neurosecretory cells and base it either on cell morphology
or on cytochemical properties, or on both. Scharrer (1936,
4
5
1937), Schaefer (1939), and Bobin and Durchon (1953) de
scribed four types of neurosecretory cells. Three types
have been observed by Clark (1959), Gabe (1954), Hauenschild
(1959), and Hauenschild and Fischer (1962). Only two types
have been suggested by Defretin (1959) and Herlant-Meewis
and van Damme (1962a). This two-cell classification has
been supported with electron microscopic studies made by
Golding (1967a). Golding et al. (1968) and Golding (1970),
however, have described another fuchsinophilic cell which
may be neurosecretory. This latter cell type is located in
a gland covering the ventral posterior surface of the brain.
The structure of the gland suggests that it may be a hor
mone -release center.
Two of the original four-cell types described by
Scharrer (1937) have been shown to be different aspects of
a single cell cycle (Gabe, 1954) . Of these two cell types,
one is thought to have the components of a photoreceptor
system, although there is no experimental evidence for this
function (Dhainant-Courtois, 1965). A third cell type de
scribed by Scharrer (1937) is probably not nervous tissue
and has no endocrine function (Herlant-Meewis and van Damme,
1962a, 1962b; Hauenschild and Fischer, 1962). From these
findings, Clark (1965) suggested that only a single type of
6
neurosecretory cell exists. An excellent critical analysis
which compares the cell types of the various workers has
been prepared by Golding (1967a) .
In the Nereidae, Nephyidae, and Arenicolidae poly-
chaetes, the supraesophageal ganglia secrete a neuroendo
crine factor (hormone) which inhibits sexual maturation
(Durchon, 1952; Clark, 1956; Hauenschild, 1956). Although
no direct evidence exists, it is suspected that the hormone
is produced by neurosecretory cells in these ganglia (Clark,
1959; Durchon and Frezal, 1955; Hauenschild, 1959) . Ex
tirpation of the supraesophageal ganglia results in rapid
egg growth and abnormal yolk deposition in the female (Clark
and Ruston, 1963; Howie, 1962) and removal of the ganglia in
an immature male worm causes the precocious appearance of
spermatozoa (Durchon, 1951, 1952; Hauenschild, 1956) . In
the Syllidae, however, the pr©ventricular region of the
pharynx is the source of the sex hormone (gonadotropin) and
not the supraesophageal ganglia (Abeloos, 1950; Durchon,
1950, 1959; Junqua, 1957; Durchon and Wissocq, 1963) .
Despite these differences of hormone origin, it is generally
agreed that high hormonal levels have an inhibitory effect
upon gametic growth but that continuing lower levels are
necessary for gametic maturation (Clark and Ruston, 1963;
7
Clark, 1965) .
Regeneration in polychaetes is also thought to be
influenced by neurosecretory cells (Golding, 1967b, 1967c;
Clark and Bonney, 1960; Clark and Clark, 1959) as is meta
morphosis (Hauenschild and Fischer, 1962; Durchon, 1960).
It has been suggested that a single hormone controls both
metamorphosis and gametogenesis (Hauenschild and Fischer,
1962) . Kamemoto et aJL. (1966) suggest that osmoregulation
may also be controlled by neurosecretory cells, since
aldehyde-fuchsin-positive cells are seen to increase in
number when worms are placed in dilute concentrations of
sea water.
Neurosecretion in the Oligochaetes
The first description of neurosecretory cells in
oligochaete worms was made by Scharrer and Scharrer (1937) .
Since then many workers have confirmed the presence of these
cells in the brain (supraesophageal ganglion) (Aros and
Vigh, 1961a, 1961b; Aros et al.., 1965b; Dogra, 1967; Her
lant-Meewis, 1955, 1956a, 1956b; Hubl, 1953), the subeso-
phageal ganglion (Aros et al., 1965b; Herlant-Meewis, 1955,
1956a; Hubl, 1953, 1956; Takeuchi, 1965a, 1965b; Teichmann
et al.. 1966), the ventral ganglion chain (Aros et al..
8
1965b; Brandenburg, 1956; Teichmann et al., 1966), and the
circumesophageal connectives (Aros and Vigh, 1961a; Teich
mann et al.., 1966) .
There are several types of neurosecretory cells
present in the oligochaetes. The collective terminology
used in describing these cells is confused since various
authors have established their own criteria according to
size, location, staining affinities, and electron micro
scopic characteristics. Dogra (1967), Hubl (1956), and
Marapao (1959) describe three types of neurosecretory cells
(a, b, and c) in the earthworm. Other workers were able to
show two types (a, b) (Aros and Vigh, 1961a, 1962a, 1962b;
Herlant-Meewis, 1956a, 1956b), whereas some describe four
types of cells (Brandenburg, 1956; Otremba, 1961; Shanta-
kumari, 1963). Three types of neurosecretory cells were
described by Michon and Alaphilippe (1959), but were called
"a," "b," and "d." Using the electron microscope, Scharrer
and Brown (1961, 1962) reported only one type of cell pres
ent in the supraesophageal ganglion, while Oosaki (1966)
describes six types in the supraesophageal ganglion with
morphologically different inclusions, two of which he be
lieves to be "ordinary neurons."
Dogra's (1967, 1968) descriptions of neurosecretory
cells in Pheretima posthuma do not agree completely with all
such cell descriptions, but do summarize the basic charac
teristics of the a-, b-, and c-cell types found in a major
ity of the oligochaete worms.
a-cell: stains purple with paraldehyde fuchsin,
greenish-blue with performic acid-Victoria
blue (.cystine and cysteine present), has a
small nucleus, neurosecretory material in
distinct aggregates
b-cell: stains light purple or brick-red with par
aldehyde fuchsin, gives a negative response
to performic acid-Victoria blue (cystine and
cysteine absent), has a large nucleus, neuro
secretory material in fine granules
c-cell: negative to basic stain (paraldehyde fuch
sin), stains orange with Halmi's counter
stain, smallest of cell types, has an agran
ular cytoplasm
Most investigators agree that the neurosecretory
material of oligochaetes is of a peptide character consist
ing of protein-bound sulfhydryl and disulphide groups (Aros
et al., 1965a; Bianchi, 1963a, 1963b; Teichmann et al..
1966; Tork et al.., 1965). Myhrberg (1967) and Bianchi
(1967) suggest that another type of neurosecretory cell
exists which has monoaminergic neurosecretory material.
Teichmann and Goslar (1968) report the presence of mono
amines in the beta cells.
Alpha cells apparently regulate various stages in
10
reproduction since they demonstrate a cyclic secretion which
can be related with the spring and summer reproductive
periods (Herlant-Meewis, 1955, 1956a, 1956b; Hubl, 1953).
Removal of either supra- or subesophageal ganglia blocks
egg-laying and maturation of gametes (Durchon, 1962;
Herlant-Meewis, 1956a, 1959) . Michon and Alaphilippe (1959)
report the presence of neurosecretory material during dia
pause .
Neurosecretory cells of oligochaete worms are also
thought to effect regeneration of body parts (Harms, 1948;
Hubl, 1953, 1956), color change (Aros and Vigh, 1959,
1961b), and show accelerated production of neurosecretory
material in response to dehydration (Aros and Bodnar, 1960;
Aros and Vigh, 1962b). Schmid (1947) was also able to
demonstrate increased neurosecretion in novocaine- and
epinephrine-treated worms.
Neurosecretion in the Hirudinea
Neurosecretory cells were first seen in the Hiru
dinea by Scharrer (1937). Recent workers have also observed
these cells in the supraesophageal ganglia of the leech
(Hagadorn, 1958, 1962a, 1962b, 1966a, 1966b; Hagadorn and
Nishioka, 1961; Legendre, 1959; Nambudiri and Vijayakrishnan,
I 11
1958), in subesophageal ganglia (Czechowicz, 1963; Gersch
and Richter, 1961; Hagadorn, Bern, and Nishioka, 1963; Le
gendre, 1959; Perez, 1942), in the circumesophageal connec
tives (Czechowicz, 1963; Hagadorn, 1964), in the ventral
nerve cord (Mishra, 1967; von Tiimpling, 1965), and in the
segmental ganglia (Perez, 1942) . Recently, Duchesne (1969)
has reported neurosecretion in the caudal ganglia.
Two types of neurosecretory cells have been reported
in the species studied. Acidophilic beta cells are less
consistent in appearance than are alpha cells and are di
vided into two subclasses: beta^ (stains with Orange G, has
little or no tryptophan and cystine) and betaj (rich in
tryptophan, less numerous than beta^) (Hagadorn, 1966a).
Early experiments suggested that the paraldehyde fuchsin-
staining alpha cells could also be divided into two sub
classes (Hagadorn, 1962a, 1966a); however, Hagadorn (1966b)
and von Tiimpling (1965) believe that these subclasses repre
sent differing stages in a single cell cycle. This belief
is further supported by electron microscopic examination of
the central nervous system of the leech by Coggeshall and
Fawcett (1964) . Axonal transport is most obvious in the
alpha cells and their secretions are rich in cystine (Haga
dorn, 1966a) .
12
Cyclical neurosecretory activity has been observed
in the alpha cell system and was thought to be correlated
with the reproductive cycle (Czechowicz, 1963; Hagadorn,
1962b). Recent experimentation has shown restoration of
gametogenesis in brainless animals after brain macerate in
jections (Hagadorn, 1966a), and both alpha and beta cell
types have been seen within the ventral nerve cord in the
region of the ovisacs (Mishra, 1967).
Experiments in which leeches were exposed to hypo-
and hypertonic conditions suggested that neurosecretory
cells participate in the regulation of water metabolism
(Czechowicz, 1968). They are also thought to take part in
color changes (Gersch and Richter, 1961).
Neurosecretion in the Nemerteans
Neurosecretory cells have been demonstrated in the
nemertean cerebral ganglia and lateral nerve cords (Leche-
nault, 1962, 1963) . Neurosecretory material has been ob
served in the axons of these neurosecretory cells (Leche-
nault, 1962).
There is little agreement upon the number and kinds
of neurosecretory cells present in these animals. Bianchi
(1969) describes four sizes of neurosecretory cells; of
13
these, three possess neurosecretory material which cannot
be differentiated by histochemical techniques. Lechenault
(1963) describes two cell types based on size in large worms
and only one cell type in smaller ones. Both authors do
agree, however, that the neurosecretory material contains
both carbohydrate- and sulfur-containing proteins.
Experiments using nemerteans during non-reproductive
periods show hyperfunctioning of neurosecretory cells when
the animals are placed in dilute sea water. It was sug
gested that the neurosecretory cells have an osmoregulatory
function (Lechenault, 1965). Decapitation of sexually im
mature worms causes a precocious development of the gonads
and associated reproductive structures (Bierne, 1964, 1966).
It has further been demonstrated that a cycle of activity in
the cerebral organs corresponds with spawning in these ani
mals (Gontcharoff and Lechenault, 1958).
Neurosecretion in the Nematodes
Neurosecretory cells have been observed in the
dorsal and ventral ganglia of parasitic nematodes (Davey,
1964, 1966; Davey and Kan, 1967; Gersch, 1957; Gersch and
Scheffel, 1958; Ishikawa, 1961). The neurosecretory mate
rial of these cells is fuchsinophilic-positive and can be
14
observed in both nerve cell bodies and axons (Davey, 1964) .
Axonal transport of neurosecretory granules has been sup
ported with data from the electron microscope (Rogers,
1968). No fuchsinophilia can be demonstrated in the ganglia
of larval worms (Davey, 1966) . Apparently no histochemical
studies have been made.
During molting in these worms, neurosecretory cells
in the dorsal and ventral ganglia show a cycle of secretion.
At the same time, the excretory glands are known to produce
an enzyme (leucine aminopeptidase) which is necessary for
the molting process (Davey, 1966). In view of these simul
taneous phenomena, Davey and Kan (1967) suggested that ec-
dysis was under neurosecretory control since neurosecretory
cells failed to stain when animals were placed in a medium
unfavorable to molting. Later, Davey and Kan (1968) were
able to obtain leucine aminopeptidase by incubating nematode
head extract with excretory glands, thereby providing the
first direct evidence of a neurohormone and its target organ
in a nematode.
Recently, the removal of neurosecretory cells from
an insect host has been shown to affect the level of in
festation of a parasitic nematode. This is the first report
which suggests that there might be a dependence upon host
15
neurosecretory material by a parasitic worm (Gordon, 1968).
Neurosecretion in the Turbellaria
Neurosecretory cells have been observed in the
cerebral ganglion of the turbellaria (Grasso, 1965; Kaplon
ska, 1967; Lender and Klein, 1961; Liotti and Rosi, 1968a;
Liotti, Bruschelli, and Rosi, 1966), in the ventral nerve
cords (Grasso, 1965; Kaplonska, 1967; Vendrix, 1963), and
in the lateral nerves and plexuses (Grasso, 1965; Liotti,
Bruschelli, and Rosi, 1966) . Both unipolar and bipolar
neurosecretory cells have been found throughout the nervous
system (Grasso, 1965; Kaplonska, 1967; Liotti, Bruschelli,
and Rosi, 1966; Ude, 1964). Neurosecretory material has
been seen in axons (Battaglini, 1964; Lender, 1964; Vendrix,
1963) and in the parenchyma of these platyhelminths (Bat
taglini, 1964; Vendrix, 1963). Electron microscopic studies
have revealed electron-dense granules similar to vertebrate
and invertebrate neurosecretory material (Morita and Best,
1965; Oosaki and Ishii, 1965).
Two types of neurosecretory cells have been found in
the turbellaria; their classification was based upon mor
phology and their position within the central nervous sys
tem. Cell type "a" was found only at the periphery of the
16
cerebral ganglion, was monopolar, and had a nucleus dis
placed to one end of the cell. Type "b" was found only
within the ganglion mass. It was spindle-shaped with a
large, spheric, centrally-located nucleus. Both cell types
had neurosecretory granules of varying sizes within the
cytoplasm (Kaplonska, 1967; Vendrix, 1963). Cytochemical
studies showed the neurosecretory material of both cells to
have a glyco- or mucoprotein fraction (Vendrix, 1963) .
Neurosecretory cells are thought to influence plan
ar ian regeneration (Kaplonska, 1967; Lender, 1964; Liotti,
Bruschelli, and Rosi, 1966; Liotti and Rosi, 1968b, 1968c;
Ude, 1964). The cells undergo a cycle during the regenera
tion process in which they are seen to increase in number
and change in form (Lender and Klein, 1961) . Liotti and
\
Rosi (1968c) have been able to show a reduction in the
activity of neurosecretory cells in normal worms by lowering
the temperature. In regenerating worms, a similar reduction
of temperature slows the regeneration process and neuro
secretory activity. Increases in temperature enhanced both
regeneration and neurosecretory cell activity. The authors
suggested that these data demonstrated the direct relation
ship between regeneration and neurosecretory cell activity.
Neurosecretory cells are not present in young
17
animals, but when the genital apparatus is established they
make their first appearance and increase during the repro
ductive period (Kaplonska, 1967; Lender, 1964; Liotti,
Bruschelli, and Rosi, 1966; Ude, 1964) . Nerve fibers and
cells surrounding the ovaries and testes have been seen to
be filled with neurosecretory granules during this time
(Grasso, 1965). Neurosecretory material has also been shown
to increase when the worms are in hypotonic surroundings
(Ude, 1964) .
Neurosecretion in the Cestodes
Neurosecretory cells have been observed in the sco-
lex of cestode platyhelminths (Davey and Breckinridge,
1967). The cells were found in a cluster in the rostellum
and their axons were part of a nerve tract which led to the
lateral ganglia of the central nervous system. These cells
were seen to have a cyclical secretion associated with the
development of the adult worm. The neurosecretory material
found in the cell bodies could be demonstrated in adult
worms three days after infection. After fuchsinophilia
developed, the neurosecretory cells were seen to enlarge and
neurosecretory material could be demonstrated in the axons
sixteen to eighteen days after infection; however,
18
fuchsinophilia decreased in older adult worms.
Davey and Breckenridge (1967) do not suggest a spe
cific function, but do associate strobilization with the
first appearance of neurosecretory material and the shedding
of the first proglottid with the first release of neuro
secretory material. Because the cells were bipolar and
anteriorly prolonged into filaments, a characteristic of
sensory cells in cestodes, the authors suggested they have
a sensory function. They further described identical cells
in the cysticercoid which were, however, devoid of neuro
secretory material.
One other study has demonstrated the presence of
electron-dense vesicles which resemble neurosecretory vesi
cles in the nerve processes and presumed neuromuscular junc
tions of another cestode (Morseth, 1967) . Apparently no
histochemistry or description of cell characteristics has
been done in this group.
Neurosecretion in the Trematodes
Two neurosecretory cells have been demonstrated in
the posterior ventral margins of the cerebral ganglion of
the adult trematode Dicrocoelium lanceatum (D. lanceolatum
or D. dendriticum) (Ude, 1962). The cells, one on each side
19
of the ganglion, stained with paraldehyde fuchsin. The
neurosecretory material, in the form of fine granules, was
evenly distributed throughout the cell bodies and could be
seen in the axons . Axonal swellings similar to Herring
bodies were observed in the abdominal nerve cord. Homo
geneously staining droplets of neurosecretory material were
also seen around the periphery of slit-like cavities in the
abdominal nerve cord and the author suggests that these
cavities may be storage places of the secretion.
Electron microscopic investigations of the anterior
portions of the adult Fasciola hepatica have demonstrated
neurones (beta) in the cerebral ganglion and surrounding
parenchyma which may have a neurosecretory function (Gresson
and Threadgold, 1964) . Differing types of these cells were
seen and the authors believed that these represented various
stages of activity of the same cell. Similar neurones were
also observed in close proximity to the ovary. These find
ings were not supported with specific staining techniques
for neurosecretory material. Electron-dense vesicles have
also been reported in tissue resembling nerve processes in
the cercaria of Fasciola hepatica (Dixon and Mercer, 1965).
The authors suggested that these vesicles resembled neuro
secretory vesicles described in other invertebrate groups.
20
The tissue containing the vesicles was not in the cerebral
ganglion area and could not be identified positively as
nervous tissue.
Fuchsinophilie neurosecretory material has also been
reported in the cell bodies and axons of neurosecretory
cells of Haematoloechus sp. (Bonner, 1968).
Despite all the research on the phenomenon of neuro
secretion and its probable relationship to reproduction,
apparently no one has investigated the appearance of neuro-
secretion in the life cycle of trematode parasites. Fur
thermore, it would seem logical that representatives of the
platyhelminth phylum should be the subject of more research,
in view of the fact that neurosecretion probably begins in
this group.
In the author's estimation, Acanthoparyphium spinu-
losum Johnston, 1917, is an ideal experimental animal since
all stages of the life cycle (Martin and Adams, 1960, 1961)
are easily obtained in large quantity. Furthermore, the
snail Cerithidea hegewischi californica acts as both first
and second intermediate host for this parasite and hatchery-
raised chicks will serve as host to adult worms.
CHAPTER II
MATERIALS AND METHODS
Materials
Naturally-infected snails (Cerithidea hegewischi
californica) were collected from brackish-water ponds near
the Bolsa Chica Gun Club a few miles north of Huntington
Beach, California. The snails were returned to the labora
tory, isolated in finger bowls, and kept in sea water which
was changed once a day.
Rediae were obtained by crushing the snails and
opening their digestive glands. Cercariae could be recov
ered from the water in the finger bowls after they were shed
from the snails. Metacercariae were found encysted in the
snail's radular muscle and surrounding connective tissue.
Adult worms were grown by feeding infected snail radular
muscle to chicks which were newly hatched and had not yet
been fed. All chicks were obtained from a local hatchery.
21
22
Methods
Adult worms two, four, six, eight, ten, twelve, and
fourteen days old were recovered from the duodenum and upper
jejunum of the chicks and placed in physiological saline
(0.9 grams NaCl in 100 ml. 1^0). All specimens were fixed
in either Heidenhain's (Susa) or Bouin's fixatives. Adult
worms used for serial sectioning were carefully removed to
a clean microslide and gently straightened with fine paint
brushes. Worms were held and fixed in such a manner that
longitudinal sections passing through their anterior ends
would reveal the oral sucker, pharynx, and cerebral gang
lion. Adult worms used for whole mounts were placed on a
microslide with a drop of water, flattened dorso-ventrally
with a coverslip, and fixed so that internal organs could be
viewed to best advantage. Rediae and cercariae were dropped
into fixative without any attempt to position them. Meta-
cercariae were left intact within the radular muscle and
fixed in situ.
All specimens fixed in Susa fixative had to be
washed in 50 per cent alcohol for one hour and then post
treated in 70 per cent iodine-alcohol for several hours so
that the mercury of the fixative could wash out of the tis
sue. All tissues to be sectioned were then washed and
23
stored in 70 per cent alcohol until they were embedded.
Wholemounts were left in 70 per cent iodine-alcohol
for only two hours since they tended to bend if left for
longer periods of time. They were washed in 70 per cent
alcohol until the remaining yellow tint left by the fixative
was removed and stained in Mayer's paracarmine for five
minutes. The wholemounts were then destained in 70 per cent
acid-alcohol, washed in 70 per cent alcohol, dehydrated,
cleared in xylene, and mounted in Permount.
Wholemount aspects were measured with an ocular
micrometer and the following data were collected: diameters
of the oral suckers and ovaries, lengths and widths of the
body, pharynx, acetabulum, anterior testis, and posterior
testis. An average measurement and range for each were
determined and put into tabular form.
All tissues used for sectioning were embedded in
Tissuemat and sliced at 7 microns. Clean microslides were
given a thin coat of egg albumin and then preheated to 50°C.
Ribbons were floated on warm water and the finished slides
were dried at 48°C. All tissues were stained within twenty-
four hours after sectioning.
24
Methods for determining the
presence of neurosecretion
Cameron and Steele's paraldehyde fuchsin.— The par
aldehyde fuchsin method of Cameron and Steele (1959) has
been used primarily for the staining of invertebrate neuro
secretions. It combines Gabe's (1953) modification of the
original aldehyde fuchsin stain of Gomori (1950) with the
counterstain of Halmi (1952). Basic fuchsin, concentrated
hydrochloric acid, water, and paraldehyde are combined in
the Gabe method. The Halmi counterstain was originally de
signed to stain acidophilic granules found in certain cells
of the rat and mouse hypophysis. The counterstain consists
of a mixture of light green SF yellowish and orange G dis
solved in a phosphotungs tic -acetic acid mixture. With this
technique, neurosecretory material stains dark purple and
most non-nervous tissues orange.
Ewen's paraldehyde fuchsin.— The paraldehyde fuchsin
stain of Ewen (1962) was made primarily for staining neuro
secretory products in insects. It has been used for such
staining in platyhelminths (Bonner, 1968). The stain is
prepared in a manner similar to that of Cameron and Steele;
however, in the Ewen preparation, glacial acetic acid is
25
added to the staining mixture for more selective staining of
the neurosecretory material. Other innovations of Ewen call
for slides to be retained ten minutes in a mordant consist
ing of phosphotungstic acid and phosphomolybdic acid, and
for an acid-alcohol bath for the suppression of background
staining. The Halmi counterstain is used for one hour as
opposed to Cameron and Steele's thirty seconds. This method
allows for more contrast between neurosecretory products and
the neuropile mass. Other tissues are well differentiated
and the slides may be used for microanatomical studies.
Again, the neurosecretory material stains dark purple to
blue.
For best results with both the Cameron and Steele
and Ewen's stain, fresh Gomori's solution and sodium bi
sulfite were prepared daily immediately before staining.
All slides had to be stained within forty-eight hours after
sectioning or results were poor. Apparently there is an
auto-oxidation of the tissues if they are left in the air
for longer periods (Ewen, 1962) .
Humberstone's Victoria blue technique.— The per-
formic acid-Victoria blue staining technique of Humberstone
(Dogra and Tandan, 1964) is a modification of the performic
26
acid-alcian blue method of Adams and Sloper (1955) which was
used specifically to demonstrate the presence of cystine or
cysteine in paraffin sections of the hypothalamus of man,
rat, and dog. This improved technique as well as that of
Adams and Sloper have been used to demonstrate the protein
nature of the neurosecretory product of invertebrate neuro
secretory cells. The technique employs an iron-resorcin
lake of Victoria blue applied to oxidized sections to reveal
the presence of cysteic acid.
Before staining any trematode tissue, the Cameron
and Steele method was first used on known neurosecretory
cell centers in the rat brain. The results of these test
tissues were positive. The stain was then used on 7-micron
Tissuemat sections of rediae, cercariae, metacercariae, and
adult Acanthoparyphium spinulosum. It was obvious after
examining the first few slides that the stain also colored
body structures and other secretory cells to which neuro
secretion could not be attributed. Therefore, the author
has confined his attention to neurosecretory cells which
show obvious relationships within the central nervous sys
tem.
CHAPTER III
RESULTS
With the exception of the reproductive organs and
their associated ducts, all other organs of the adult worms
can be found in both the cercaria and metacercaria. In the
adult worm, the growth of most body features involves an
enlarging of these already present structures, whereas the
entire reproductive mechanism is seen to differentiate from
genital primordia and grow in size.
Tables 1 through 6 illustrate the results obtained
from measurements of two-, four-, six-, eight-, ten-, and
twelve-day-old adult worms. When looking at average meas
urements, body length and ventral sucker length and width
almost double in size between the second and fourth day.
During the same time other organ measurements, with the
exception of the gonads, show a less rapid growth. In
creases of one-half millimeter in body length occur from the
fourth to the sixth, sixth to eighth, and eighth to the
27
28
TABLE 1
MEASUREMENTS OF TEN TWO-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(mm) (mm)
Body length 0.630-0.890 0.760
Body width 0.168-0.252 0.221
Oral sucker diameter 0.047-0.059 0.054
Pharynx length 0.034-0.050 0.043
Pharynx width 0.031-0.044 0.035
Ventral sucker length 0.090-0.131 0.111
Ventral sucker width 0.078-0.124 0.109
Ovary diameter 0.018*
Anterior testis length 0.021*
Anterior testis width 0.021*
Posterior testis width 0.021*
Posterior testis length 0.024*
*Based on five adults.
TABLE 2
MEASUREMENTS OF TEN FOUR-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(mm) (mm)
Body length 1.190-1.650 1.420
Body width 0.350-0.448 0.388
Oral sucker diameter 0.062-0.078 0.069
Pharynx length 0.050-0.068 0.058
Pharynx width 0.034-0.050 0.043
Ventral sucker length 0.162-0.218 0.197
Ventral sucker width 0.187-0.233 0.206
Ovary diameter 0.034-0.050 0.045
Anterior testis length 0.053-0.109 0.074
Anterior testis width 0.047-0.093 0.070
Posterior testis length 0.062-0.134 0.090
Posterior testis width 0.044-0.081 0.063
30
TABLE 3
MEASUREMENTS OF TEN SIX-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(mm) (mm)
Body length 1.890-2.240 2.100
Body width 0.480-0.560 0.520
Oral sucker diameter 0.068-0.100 0.088
Pharynx length 0.065-0.081 0.073
Pharynx width 0.037-0.053 0.045
Ventral sucker length 0.250-0.290 0.270
Ventral sucker width 0.250-0.290 0.280
Ovary diameter 0.050-0.084 0.067
Anterior testis length 0.140-0.220 0.200
Anterior testis width 0.140-0.240 0.200
Posterior testis length 0.150-0.280 0.250
Posterior testis width 0.110-0.210 0.160
31
TABLE 4
MEASUREMENTS OF TEN EIGHT-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(nun) (nun)
Body length 2.380-3.110 2.710
Body width 0.520-0.670 0.630
Oral sucker diameter 0.078-0.130 0.105
Pharynx length 0.068-0.096 0.085
Pharynx width 0.037-0.062 0.056
Ventral sucker length 0.280-0.320 0.030
Ventral sucker width 0.280-0.350 0.320
Ovary diameter 0.081-0.128 0.107
Anterior testis length 0.240-0.350 0.290
Anterior testis width 0.220-0.310 0.280
Posterior testis length 0.310-0.430 0.370
Posterior testis width 0.180-0.270 0.230
32
TABLE 5
MEASUREMENTS OF TEN TEN-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(nun) (nun)
Body length 2.870-3.280 3.060
Body width 0.590-0.700 0.650
Oral sucker diameter 0.087-0.112 0.099
Pharynx length 0.075-0.090 0.084
Pharynx width 0.053-0.072 0.062
Ventral sucker length 0.280-0.320 0.030
Ventral sucker width 0.340-0.360 0.350
Ovary diameter 0.103-0.115 0.110
Anterior testis length 0.280-0.380 0.340
Anterior testis width 0.250-0.340 0.290
Posterior testis length 0.360-0.480 0.430
Posterior testis width 0.210-0.270 0.230
TABLE 6
MEASUREMENTS OF TEN TWELVE-DAY-OLD ADULTS
OF ACANTHOPARYPHIUM SPINULOSUM
Range Average
(mm) (mm)
Body length 2.770-3.250 2.950
Body width 0.590-0.770 0.670
Oral sucker diameter 0.096-0.134 0.113
Pharynx length 0.090-0.103 0.097
Pharynx width 0.047-0.078 0.066
Ventral sucker length 0.290-0.350 0.330
Ventral sucker width 0.320-0.390 0.360
Ovary diameter 0.100-0.128 0.117
Anterior testis length 0.280-0.380 0.320
Anterior testis width 0.270-0.320 0.290
Posterior testis length 0.350-0.430 0.410
Posterior testis width 0.200-0.250 0.220
34
tenth day (Figure 1). After this time body length becomes
more or less stable. Most organs reach their adult size by
the eighth or tenth day (Figure 18).
The greatest amount of growth occurs in the repro
ductive system. The lengths and widths of the anterior and
posterior testes and the ovary diameter increase in size by
nearly three and one-half times during the second through
fourth days (Figures 2 through 4). Between the fourth and
sixth days there is almost another trebling in testes sizes,
while the ovary diameter increases by only 50 per cent.
Another large increase in ovary size occurs between the
sixth and eighth days, when it nearly doubles in size (Fig
ures 2 through 4). From the results obtained, it appears
that the reproductive system reaches its asymptotic growth
by the eighth day.
In the two-day-old worm three areas of genital
primordia can be seen (Figure 5). These primordial cells
stain more darkly than surrounding tissue and have no regu
lar form. There is another region of darkly staining cells
immediately posterior to the bifurcation of the gut near the
mid ventral surface of the body (Figure 6). This area be
comes the future genital pore.
In the four-day-old adult, the most anteriorly
Fig. 1.— Plot of body dimensions of adult Acantho-
parvphium spinulosum two, four, six, eight, ten, and twelve
days old.
35
M M
BODY DIMENSIONS
length --------
width --------
3
2
1
DAYS
FIGURE 1
Pig. 2.— Plot of ovary dimensions of adult Acantho-
paryphium spinulosum two, four, six, eight, ten, and twelve
days old.
37
MM
38
OVARY DIAMETER
. 10
J 05
. 01
8 12 6 IO 2 4
DAYS
FIGURE 2
Fig. 3.— Plot of anterior testis dimensions of adult
ftcanthoparyphium spinulosum two, four, six, eight, ten, and
twelve days old.
39
ww
40
ANTERIOR TESTIS
length -------
w id t h -------
1 0
DAYS
FIGURE 3
Fig. 4.— Plot of posterior testis dimensions of
adult Acanthoparvphium spinulosum two, four, six, eight,
ten, and twelve days old.
41
M M
42
POSTERIOR TESTIS
length -------
w id th --------
. 4 2
. 2 4
.03
lO
DAYS
FIGURE 4
43
placed primordial cells have differentiated into two re
gions: a recognizable ovary with a circular outline, and
a group of cells immediately posterior to the ovary which
have started to become the future uterus, ootype, and ovi
duct (Figure 7). Posterior to the ovary, the anterior and
posterior testes have differentiated from the remaining two
genital primordia, and have assumed a more or less spherical
and recognizable shape (Figures 8, 9, and 10). Lateral and
posterior to the ventral sucker a group of darkly stained
cells can be seen. Although roughly organized, it is ap
parent that these cells will differentiate into the future
cirrus sac (Figure 11). In some specimens the seminal
vesicle is partially differentiated.
In the six-day-old adult, the seminal vesicle,
cirrus sac and cirrus, and prostatic gland cells are visible
(Figure 12). In some worms the vas efferens is completely
formed. Single ducts from each testis extend anteriorly
from the lateral side of the testes and unite with each
other anterior to the anterior testis. This newly formed
vas deferens extends anteriorly and joins with the cirrus
sac. No sperm can be seen in the seminal vesicle. The
remaining primordial cell area posterior to the ovary has
not completely differentiated into oviduct, uterus, and
44
ootype (Figure 13). The vitellaria are also developed and
extend from the level of the ovary to almost the ends of the
ceca on both sides. Yolk globules are visible within the
vitellaria of some of the six-day-old worms. The genital
pore has also differentiated.
By the eighth day differentiation of the reproduc
tive system is complete. Sperm can be seen in the seminal
vesicle and eggs are present in some worms (Figure 14).
Using the Cameron and Steele stain, two neurosecre
tory cells were seen in six- and eight-day-old Acanthoparv-
phium spinulosum. The cells appeared on the posterior med
ial margins of each cerebral ganglion which is in close
proximity to the anterior tip of the pharynx. In nine-day-
old worms, four additional neurosecretory cells appear (two
in each ganglion) in more anterior portions of the brain,
making a total of six such cells within the central nervous
system (Figure 15). These same six cells are present in
ten- (Figure 16) and twelve-day-old specimens. In thirteen-
day -old worms, the more medially placed cells disappear,
leaving what appear to be the same four neurosecretory
cells, first seen in nine-day-old specimens, within the an
terior portions of the cerebral ganglia.
In longitudinal sections passing through the oral
45
sucker, pharynx, and cerebral ganglia, the neurosecretory
cells, when stained with the Cameron and Steele method, are
fusiform and possess a light-blue nucleus. The neurosecre
tory material stains dark blue and the ganglionic mass light
blue. In cross section, the neurosecretory cells at the
medial margins of the ganglia appear bipolar. Neurosecre
tory material could be seen in their axons.
Neurosecretory cells were not observed in rediae,
cercariae, and metacercariae with the Cameron and Steele
method. A cercaria of Parorchis acanthus was also stained,
but it also was devoid of any observable neurosecretory
cells.
Other structures such as gland cells of the oral
sucker, subcutaneous gland cells, and the integument are
stained blue with the Cameron and Steele method. Muscular
organs and the parenchyma stain a light orange and integu
mentary spines red. Immature stages stained in a similar
manner to those of the adult worms.
Worms from the definitive host four, five, six,
eight, twelve, and thirteen days old were stained with the
Ewen method. Similar results were obtained in six-, eight-,
twelve-, and thirteen-day-old sections as were previously
mentioned for the Cameron and Steele stain. No
46
neurosecretory cells were seen in four-day-old worms.
Neurosecretory cells were first observed in five-day-old
worms and were seen to be the same pair of medially placed
cells as previously described in six- and eight-day-old
specimens (Figure 17). All neurosecretory activity disap
pears in fourteen-day-old adults. In order to ascertain
whether neurosecretory activity resumed in older worms,
adults sixteen, eighteen, and twenty-two days old were sec
tioned and stained. The results were negative.
The Ewen method is a somewhat more selective stain
than that of Cameron and Steele. Less blue color is taken
up by body structures, although secreting cells other than
neurosecretory cells do take the stain. The cerebral gang
lia stain a blue-green and neurosecretory cells have a red
nucleus with neurosecretory material staining dark blue.
Again, muscular organs and the parenchyma have an orange
color and the integument stains blue.
Humberstone's Victoria blue stain was used to stain
neurosecretions in a ten-day-old adult Acanthoparvphium
spinulosum. The neurosecretory material of both the medi
ally and anteriorly placed cells stained blue-green. Neuro
secretory granules found in the axons of the lateral nerves
also took the stain. Because of the specificity of
47
Humberstone's method in which cystine and cysteine are
stained, the protein nature of the neurosecretory material
is established. The only other structures which took the
stain were the subcutaneous gland cells.
CHAPTER IV
DISCUSSION
Neurosecretory activity is first seen in the cere
bral ganglia of five-day-old pre-adult Acanthoparvphium
spinulosum. At this age the posterior and anterior testes
and the ovary have assumed recognizable shapes after having
differentiated from three genital primordia. Accessory
reproductive structures have not differentiated, however.
The appearance of neurosecretory cells at the time the
gonads are differentiating could be expected in view of the
fact that neurosecretory activity has been correlated with
gametogenesis in most of the invertebrate phyla including
some Platyhelminthes.
Liotti, Bruschelli, and Rosi (1966) have demon
strated that planarians never have neurosecretory activity
as larvae; however, its appearance coincides with the estab
lishment of the genital apparatus and increases during the
reproductive period. Decapitation of planarians causes
48
49
regression of gonadal differentiation; however, with the
subsequent regeneration of the head, the gonads will develop
normally (Grasso, 1966) . In cestodes, the appearance of
neurosecretion can be correlated with the first formation of
the proglottids which eventually house both male and female
reproductive structures (Davey and Breckenridge, 1967). In
leeches, neurosecretory activity appears just before gameto-
genesis in the testes and ovaries, with amounts of neuro
secretory material being highest during the breeding period.
A neuroendocrine factor is thought to be responsible, since
removal of the brain from adult leeches before the breeding
season stops all gametogenesis (Hagadorn, 1962a) .
The two neurosecretory cells seen in five-day-old
Acanthoparvphium spinulosum are also present in six- and
eight-day-old worms. Between five and eight days the acces
sory organs associated with the reproductive system differ
entiate and in eight-day-old specimens, both sperm and eggs
are present. The number of sperm and eggs is, however, not
great. No more than six eggs can be seen in worms of this
age. It seems likely, then, that early gametogenesis and
differentiation of accessory reproductive organs are under
the influence of two neurosecretory cells. The possibility
exists that neurosecretory cells control the early
50
differentiation of the three genital primordia. Even though
no neurosecretory activity was observed in worms younger
than five days, elaboration of neurosecretory material in
small quantities could begin and not be detected by the
staining methods used in this research.
The effect of neurosecretion upon the differentia
tion of the gonads and accessory structures is well docu
mented in other invertebrates. In decerebrate polychaetes,
both immature sperm and eggs fail to ripen and in worms with
mature gametes, spawning is inhibited (Howie, 1962).
Spawning is restored after brain homogenates are injected
into the coelom. After brain extirpation there is abnormal
growth of partially matured oocytes as well as abnormal yolk
deposition in the eggs of these worms (Clark and Ruston,
1963; Hauenschild, 1956). Decapitation of sexually mature
oligochaetes has several effects upon the reproductive sys
tem: mature oocytes are histolyzed, eggs in the oviducts
degenerate and will not be laid, and maturation of spermato-
gonial cells and spermatocytes ceases. Spawning stops im
mediately in these decerebrate worms and somatic sexual
organs disappear, leaving the worm looking as it did at
puberty (Herlant-Meewis, 1959). Rude and Lender (1964) have
shown that implantation of brain from a mature animal into
51
the coelom of a brainless one brings about the formation of
spermatids where only spermatogonial cells had existed. In
insects, neurosecretory cells control gametogenesis, yolk
deposition (Lea, 1963, 1967; Highnam, Lusis, and Hill,
1963), oviposition (Highnam, 1962), and activity of the
accessory sex glands (Engelmann, 1959).
In nine-, ten-, and twelve-day-old Acanthoparvphium
Bpinulosum. four additional neurosecretory cells are seen,
indicating an increase in neurosecretory activity. These
additional cells appear just before large numbers of eggs
and sperm are produced. The number of eggs gradually in
creases after the ninth day. It is possible that large in
creases in the number of gametes are dependent upon further
neurosecretory activity. Increases in the amount of neuro
secretory material during the reproductive cycle are also
seen in other invertebrates. In planarians, increased
amounts of neurosecretory material have been seen during the
reproductive period (Kaplonska, 1967) . Nerve cells and
their fibers surrounding the ovaries and testes are also
filled with neurosecretory granules at this time (Grasso,
1965) . In cockroaches, increased amounts of neurosecretory
material are present within the cerebral ganglia and have
been related to oocyte growth. There is a noticeable
52
decrease in amounts of neurosecretory material in storage
when the ovaries contain growing eggs (Adiyodi and Adiyodi,
1968) . Large amounts of neurosecretory material appear in
cells of the pleuro-parietovisceral ganglion mass in pulmo-
nate gastropods. This secretion rises in amount until the
late spermatozoan stage, when copulation occurs, then rap
idly decreases until only small amounts are found. A rela
tionship between the maturation of female gametogenesis and
the increases in neurosecretory activity has also been sug
gested (Smith, 1967). In leeches, both the number of stain-
able neurosecretory cells and the amount of neurosecretory
material increases during the reproductive period. The
cells start secreting before gonadal development and the
amount of secretion reaches a peak during the breeding sea
son (Hagadorn, 1962). In entomostraca crustaceans, in
creases in the amount of neurosecretory material have been
observed at the beginning of egg maturation with smallest
amounts when eggs are discharged into the brood pouch
(Streba, 1957). With these and other reports of increases
of neurosecretory material during the reproductive cycle of
invertebrates, it would seem that the correlation between
neurosecretion and the reproductive phase of adult develop
ment cannot be denied.
53
All neurosecretory activity stops in fourteen-day-
old Acanthoparvphium spinulosum. An increase in the number
of eggs found in the oviduct, however, continues for several
days. This evidence suggests that neurosecretory material,
even though not being elaborated in the neurosecretory cells
of the cerebral ganglion, could still be present in the
axons of the lateral nerves or in nerves surrounding the
ovary and testes, as was observed in planarians by Grasso
(1965). In trematodes, Ude (1962) has demonstrated slit
like cavities near the abdominal nerve cord which are filled
with neurosecretory granules. These apparently act as
storage areas for neurosecretory material. Such structures
were not seen in Acanthoparvphium spinulosum. although
separately spaced aggregates of neurosecretory granules were
seen in the lateral nerves.
The positive results obtained from the Humberstone
Victoria blue stain establish for the first time the protein
nature of neurosecretory material in trematodes. A similar
stain (alcian blue; Adams and Sloper, 1955) has been used
successfully in planarians (Vendrix, 1963), but no cyto-
chemical studies of neurosecretory material have been made
in cestode platyhelminthes. The Humberstone stain has also
been used to demonstrate the protein nature of insect
54
(Dogra, 1968; Dogra and Tandan, 1964; Dogra and Ewen, 1970)
and oligochaete neurosecretions (Dogra, 1967).
Even though neurosecretion has been demonstrated in
parasitic trematodes, no one has attempted to correlate the
appearance of neurosecretion with any particular feature of
the life cycle. This research demonstrates for the first
time the absence of neurosecretion in cercariae, rediae, and
metacercariae. It further shows neurosecretion appearing in
pre-adult worms at the time the reproductive system is dif
ferentiating, and also demonstrates an increase of neuro
secretory material at a time just prior to the production
of large numbers of eggs and sperm. The protein nature of
the neurosecretory material is also shown for the first
time.
SUMMARY
1. No neurosecretory material is observed in
rediae, cercariae, and metacercariae of Acanthoparvphium
spinulosum.
2. Neurosecretory cells are first observed in five-
day-old Acanthoparvphium spinulosum. Five-, six-, and
eight-day-old Acanthoparvphium spinulosum have two neuro
secretory cells. Nine-, ten-, and twelve-day-old worms have
a total of six neurosecretory cells in the cerebral ganglia.
Thirteen-day-old worms have four such cells.
3. Neurosecretory activity disappears in fourteen-
day -old worms.
4. The appearance of neurosecretory activity can be
correlated with establishment of the gonads. Increased
neurosecretory activity occurs just before large numbers of
eggs and sperm are produced.
5. The protein nature of the neurosecretory mate
rial has been established for the first time in this group.
55
56
Fig. 6.--Genital pore primordium in two-day-old
Acanthoparvphium spinulosum.
Fig. 5.— Genital primordia in two-day-old Acantho
parvphium spinulosum.
Abbreviations Used
PP Posterior testis primordium
AP Anterior testis primordium
OP Ovary, uterus, ootype primordium
GP Genital pore primordium
57

Pig. 7.— Ovary and primordium of uterus and ootype
in four-day-old Acanthoparvphium spinulosum.
Fig. 8.— Anterior testis of four-day-old Acantho
parvphium spinulosum.
Abbreviations Used
0 Ovary
UP Uterus and ootype primordium
AT Anterior testis
59
Figure 8
Fig. 9.— Posterior testis of four-day-old Acantho
parvphium spinulosum.
Fig. 10.— Posterior half of four-day-old Acantho
parvphium spinulosum.
Abbreviations Used
PT Posterior testis
AT Anterior testis
O Ovary
UP Uterus and ootype primordium
61

Fig. 11.— Cirrus sac primordium of four-day-old
Acanthoparvphium spinulosum.
Fig. 12.— Cirrus sac of six-day-old Acanthoparvphium
spinulosum.
Abbreviations Used
CP Cirrus sac primordium
CS Cirrus sac
63

Fig. 13.— Incompletely formed uterus and ootype of
six-day-old Acanthoparvphium spinulosum.
Fig. 14.— Sperm and eggs in eight-day-old Acantho
parvphium spinulosum.
Abbreviations Used
UP Uterus and ootype
S Sperm
E Egg
65

Fig. 15.— Two of four additional neurosecretory
cells which appear in nine-day-old Acanthoparvphium spinu
losum: Cameron and Steele stain.
Fig. 16.— Medially-placed neurosecretory cell seen
in ten-day-old Acanthoparvphium spinulosum: Cameron and
Steele stain.
Abbreviation Used
NS Neurosecretory cell
67

Fig. 17.— Medially-placed neurosecretory cell seen
in five-day-old Acanthoparyphium spinulosum: Ewen stain.
69

Pig. 18.— Eight-day-old Acanthoparyphium spinulosum.
Abbreviations Used
OS Oral sucker
P Pharynx
GP Genital pore
VS Ventral sucker
U Uterus
CS Cirrus sac
O Ovary
E Egg
00 Ootype
AT Anterior testis
PT Posterior testis
V Vitellaria
71
Figure 18
73
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Creator Steele, David Freed (author)

Core Title Neurosecretion In The Life Cycle Of The Digenetic Trematode, Acanthoparyphium Spinulosum, Johnston, 1917

Degree Doctor of Philosophy

Degree Program Biology

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

Tag biology, general,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Martin, Walter E. (committee chair), Chew, Robert M. (committee member), Easton, William H. (committee member), Tibby, Richard B. (committee member), Zimmer, Russel L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-469657

Unique identifier UC11362486

Identifier 7116437.pdf (filename),usctheses-c18-469657 (legacy record id)

Legacy Identifier 7116437

Dmrecord 469657

Document Type Dissertation

Rights Steele, David Freed

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

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