The early embryology of Aulocara elliotti (Thomas) (Orthoptera: Acrididae) with studies on the effects
of maternal age and environment of the developmental rate of the egg
by Margaretha Harders Wessel
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Entomology
Montana State University
© Copyright by Margaretha Harders Wessel (1973)
Abstract:
The early embryology of Aulocara elliotti was investigated using histological methods. A staging
criteria was formulated for this period. The female pronucleus was observed about 1/5 the egg length
from the posterior part of the egg, while the first cleavage division of the zygote nucleus was observed
later in the same vicinity.
Chromatin was eliminated during the second cleavage division. Cleavage nuclei were first noticed in
the posterior periplasm and only later were present in the anterior periplasm although they were never
as numerous there. Cell membranes were not observed in the presumptive serosa before differentiation
of the embryonic rudiment. One nucleolus was observed in presumptive serosal cells.
. Comparisons of the developmental rates of eggs from females of different ages and reared at different
densities were made and it was found that eggs from females reared at one pair per cage developed
fastest when laid during the middle of the fecund period. Eggs from females reared at a density of six
pairs per cage developed fastest when laid during the early part of the fecund period and thereafter the
rate of development declined steadily.
The incorporation of tritiated uridine and thymidine during early development was determined with
autoradiographic methods. Tritiated uridine was first incorporated into RNA during blastema
formation, Tritiated thymidine was incorporated into DNA during the entire time period (6 days). 'A
posterior-anterior gradient of the incorporated 3H-thymidine was observed, A large number of eggs
developed abnormally after being exposed to the isotope in Ringer’s solution, No conclusions
therefore, could be drawn concerning maternal effects (age, density) on RNA and DNA synthetic
patterns. THE EARLY- EMBRYOLOGY OF AULOCARA ELLIQ TTI (THOMAS) (ORTHOPTERA:
''/ACRIDIDAE) WITH STUDIES ON THE EFFECTS OF MATERNAL- AGE '
AND ENVIRONMENT. ON THE DEVELOPMENTAL RATE OF THE EGG '
by
MARGARETHA HARDERS. WESSEL
A thesis submitted- to the Graduate Faculty in partial
fulfillment of the requirements for the degree
.of
DOCTOR OF PHILOSOPHY
in
Entomology
Head, Major Department
- 'M l. J? S i
J f /.
Chairman, Examining Committee
Graduat^Dean
MONTANA STATE -UNIVERSITY
.Bozeman,- Montana
June, 1973
ill
ACKNOWLEDGMENT.
My deepest gratitude and affection are offered to my major pro­
fessor, Dr. Saralee Visscher, for her enthusiasm, confidence and un­
failing support during the course of this study.
Her questions and
knowledge concerning insect embryology kindled my curiosity and ex­
panded my horizons.
I am indebted to D r s . S. Visscher, P. D. Skaar and G. Roemhild
for the critical reading of the manuscript and to Dr. W. Dorgan for
his assistance.with the autoradiography.
Additional members of my
committee, Drs. H. Watling and L. Jackson, made helpful suggestions.
I wish to thank D r s . S. Chapman and J. Schaeffer for help in
interpreting some' of the data and micrographs, Mr. D 0 Eritts for
doing most of the photographic work, and Mrs.. Della White and Mrs.
Nina Bradley for typing the manuscript.
I am. appreciative of. my fellow students,. J, Bromenshenk and
J. Mussgnug, for providing some biological materials, and of Dr. J. H.
Pepper for stimulating discussions.
The financial support of the U. S= Department of Health, Education
and Welfare, through a Title IV NDEA Fellowship, and of- the College of
Graduate Studies and Agricultural Experiment Station, Montana State
University, are gratefully acknowledged.
Finally, I wish to recognize the support and encouragement of my
family and friends while this work was in progress.
iv
TABLE OF CONTENTS
Page
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ACKNOWLEDGMENT............. ..
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LIST OF TABLES ................. ..
LIST OF FIGURE S
ABSTRACT
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INTRODUCTION ,
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MATERIALS AND METHODS
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DESCRIPTIVE
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Introduction . . . ... . . .
Results 6 0 o’ O O O O . O 0' O
Maternal Influence . . .
Delayed Oviposition . ,
Autoradiography
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Tritiated uridine .
Tritiated thymidine
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Events of Oogenesis and Egg Deposition
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Temporal Pattern of Development
Discussion . . o o o . o o o .
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EARLY EMBRYOLOGY:
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Biological Material . . . . . .
Descriptive and Maternal Effects
Autoradiography . . . . . . . . .
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EARLY EMBRYOLOGY:
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Discussion
Abnormalities . . . .•
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SUMMARY
.
APPENDIX
A .
APPENDIX
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APPENDIX
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ft ft ft ft 104
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ft
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LITERATURE CITED . . .-v . f
t ft ft ft ft ft ft ft ft .ft" ft ft ft:
ft- ft.
A
6
' 105
A 0 a a
106
V i
LIST OF TABLES
Table
Page
I.
Reproductive
data for 1970
81
II.
Reproductive
data for 1 9 7 1 . . . . . . . . . . . . . . . . .
82
vii
LIST OF FIGURES'
Figure
Page
1. . Egg pod and newly-laid egg of A'= e V b io ttt.
2.
3.
4.
5.
14.
Diagram of stages A to I of the early embryology of
A #* & IslsQI/ (Sis o e s e « e ■ o • 1 6 o' o ' o o o - o o e . o o e
0
e
o
o
2.0
A longitudinal section of the chorion of a newly-laid
egg of A. s IsTsTsOisisis ... . . .- . ., . . . . .. .. . . .
. .' .
23
A longitudinal section of the, chorionic cap of a newlylaid egg of A. S tZ io tt-L -'
25
A longitudinal section of the periphery of a newlylaid egg of A '-e Z Z -C o tti
26
6.
Basophilic droplets and second cleavage division in a
longitudinal secion of a- six-hour egg of A. e Z Z io t t i
. . . .27
7.
The female pronucleus in a longitudinal section of a
newly-laid egg. of A.-. e t Z i o t t i '.' . ... v
29
8.
Polar body in a newly-laid egg of A.. e t Z - io t t i
30
9.
Chromatin elimination during second cleavage division in
a longitudinal section of an egg of A. eZ Z iott-L . . . . . .
32
. Interphase
10
cleavage nucleus in the anterior part of the
egg of A . eZZrottTs
. ... . . . . . . . ...
. .. .. .- . . 33
11. Interphase nucleus in the anterior part of the egg
of. A . ■SZZ-IsOtt^ls .
O
O
6
.
o'
.
O
Cr
o'
6
o'
O
O - O 1 O
O
34
O- 1 O'
12. Peripheral nuclei at the•posterior end of the egg
of A.- eZZ-Lottis
.
e
o
e
o
a
o
e
d'
o
e
o-o
e
o • o'-
o
36
o
13.
Periplasm of the anterior end of the1 egg,of A. ■e Z Z io t t i . .
37
14.
Blastema nucleus at the periphery of a three-day-old
egg of Ao- SZZ^lsOtt^ls. 0 O . . . O' O O' O' O' O' 6 - 0 O 0" 0 O O
38
15.
Beaded blastema nucleus at periphery of a four-dayold egg of A . SZZlsOttls - O O .' 0 . 0 0 0 0 0 0
O' . 0
0
O
0
O
- 0' OO
39
viii
LIST OF FIGURES
(Continued)
Figure
16.
17.
18,
19.
20.
21.
22.
23o
24o
25o
Page
Doughnut^shaped nuclei in posterior part of a threeday-old egg of A. e V t i o t i z i ............... ............. ..
. . 40
Mitosis at a 90° angle to the periphery of a threeday-old egg of A. e Z X to tt i which produces1 a daughter
blastema nucleus and a secondary vitellophag . . . . . .
o . 42
Mitosis tangentially to the periphery of a one-dayold egg of A, e V l i e t t i which produces two blastema
nuclei . . . . o . . . , . . , . . , . . . , , . . .
. o 43
Internal nuclear aggregate in a five-day-old egg
of A o &Is'Is'lsOIylSl* 0 0 0 0 . Q' 0 , 0 , a . . . . . .
. . 45
Peripheral nucleus with distinct nucleolus in a
four-day-old egg of A.- e l t i o t t i .
.
Cuboidal cells of the embryonic rudiment in posterior
end of a six-day-old egg of A. e l l - t o t t i
. . . . . . . .
. . 47
Nuclei in mosaic germ disc in the posterior end of an
eight-day-old egg of A. e t H o t t i . . , . ...............
, . 50
Germ band and serosal cells in the posterior part of a
ten-day-old egg of A. e l t - i o t t ’i . . . . . . . . . . . . . .
. . 52
Embryo, amnion and serosal cells in a twelve-day-old
egg of A o■a %."IsisOis1*%
* . . . o . . . . . , * . . . . . . . . .
. . 53
Displaced periplasm in egg of A, e tV L o tv i- used for
autoradiography
. .• o . . . . . . . . ... . c . . . o■ .
Internal aggregation of. nuclei in a five-day-old egg
of A. B V L -Io tti
27o
28.
. . 46
Developmental stage reached by known-age eggs of
A. e 'V b io tti during early development
. . 66
.- . 70
. o . 76
Developmental stage reached, by known-age eggs of
A. e lt- io tt- L during early development . . . . . . . . . . . . .
. .
76
ix
LIST OE FIGURES
(Continued)
Figure
29.
30.
31.
32.
33.
Page
Developmental stage reached by- known-age eggs of
A. e V lto t-b i during early development. . . . . . . . . . .
Developmental stage reached, by.known-age eggs* of
A. e 'i l'l o t t - i during early development
. .
. . .- 77
Developmental stage reached by known-age eggs of
A. e ’l V io 't'b i’ during early- development. . . . ., .
Developmental■stage reached by known-age eggs of
A. BVl1L o tiy l during early development . . .. . .•
o
o'
Composite graph of. Figs.. 27-32 . . . . ..... ., .
Longitudinal section- of a five-hour-old egg of
A o B“l llsO l IyU o . . . . cf - o o o o o . - a o d O e
.- .
78
.. .
79
6 o' . O
Longitudinal section of posterior part of a six-dayold egg Of A o- ’ BI VlyOfct1L - . . o o o
.-.
o o- - o . o - o
e
O O shown in Fig.
.- 78
.. . 84
35^, Longitudinal section of a three-day-old egg of
A. BVliofcti--
36.
77 ■
o
o
86
. . 87
36 . . . . . 88
37=
Anterior part of the same egg as
38.
Displaced periplasm in posterior part of a four-day—
old egg of A . BI I 1Lo I Vl >o . . . . . . . .
= .-.
= . . .- .
89
39.
Developmental stage reached by known-age eggs of.
A. BVl1LotfcL exposed to a solution of 3H-Uridine ininsect Ringer, s . . . .. o . .- .
. .-. .
. . .-. = . = . .. 91
40.
Developmental stage reached by known-age eggs of
Ac - O l l 1L o t t 1L exposed to a solution of 3H-thymidine
in insect Ringer’s . .
. . . . . .
O O
91
X
ABSTRACT
The early embryology of A u loca va e V l i o t t i was investigated using
histological methods. A staging criteria was formulated for this
period.
The female pronucleus was observed about 1/5 the egg length
from the posterior part of the egg, while the first cleavage division
of the zygote nucleus was observed later in the same vicinity.
Chromatin was eliminated during the second cleavage division. Cleavage
nuclei were first noticed in the posterior periplasm and only later
were present in the anterior periplasm although they were never as
numerous there, Cell membranes were not observed in the presumptive
serosa before differentiation of the embryonic rudiment.
One nucleolus
was observed in presumptive serosal cells.
. Comparisons of the developmental rates of eggs from females of
different ages and reared at different densities were made and it was
found that eggs from females reared at one pair per cage developed
fastest when .laid during the middle of the fecund period.
Eggs from
females reared at a density of six pairs per cage developed fastest
when laid during the early part of the fecund period and thereafter
the rate of development declined steadily.
The incorporation of tritiated uridine and thymidine during early
development was determined with autoradiographic methods. Tritiated
uridine was first incorporated into RNA during blastema formation,
Tritiated thymidine was incorporated into DNA during the entire time
period (6 days). 'A posterior-anterior gradient of the incorporated
3H-thymidine was observed, A large number of eggs developed abnormally
after being exposed to the isotope in Ringer’s solution, No conclusions
therefores could be drawn concerning maternal effects (age, density) on
RNA and DNA synthetic patterns.
INTRODUCTION
The importance of the, Acrididae has been recognized throughout
the history of mankind in accounts of grasshopper plagues devastating
crops and rangelands.. A voluminous literature has been published con­
cerning aspects of the ecology, morphology, physiology and behavior of
both nymphal and adult acridids.
Some of' the. more readily observable
characteristics of the development-of. the egg such as water.and tem­
perature requirements, incidence and duration of diapause, and external
morphogenesis of the embryo also.-have been investigated in. a number of
species.
The research on the Acrididae has been reviewed by Uvarov
(1966), Chapman (1969) and Hemming; and Taylor (1972).
In 1961, Roonwal-
compiled the Bibliographla Acrididiorum with.supplements in 1961 and
1968, citing the literature to the Acrididae-.
Few descriptive studies have been done on the histology of early
embryogenesis of- the Acrididae»
Roonwal (1936) published an extensive
monograph on the early development- of the African migratory locust,
L o o u sta Y n igvatovia (R & E-), and. in 1937 he published his' studies on the ■
organogensis of that same species .
These works were reviewed, by
Johannsen and Butt (1941) in "Embryology of Insects and Myriapods."
Slifer and King (19.34), in a short paper,,, discussed the early embry­
ology of the.differential grasshopper M elanoplus d i f f e v e n t ia li s (Thomas) , a species widely distributed in the United-States,;. and in
1963, Van Horn (Visschef) completed detailed investigations of the-
-2-
histology of organogenesis' in A ulooava e l l - t o t t i ' (Thomas), a species
indigenous to the western United. States and Canada..
At Montana State University in the 1950’s, A. e l l i o t t t ^■one. of the
ten most important economic pests of rangelands (Anderson, 1961), was
selected for intensive studies in- order to try to gain an understanding
of the factors underlying the wide fluctuations in' population numbers
observed in this species.
In 1952 Anderson and Wright included data
on. A ulocapa Q ill- Io ttiV- in their investigations of behavior and damage of
Montana grasshoppers and Anderson. (1961,- 1962, 1964, 1972) reported on
relationships between grasshoppers.and vegetation.
Studies on the viability of newly-hatched nymphs of A .' e l l t o t t - i
under conditions of stress were published by Hastings and Pepper (1964),
while the structure and performance of a specific adult population in
the field was investigated by Mussgnug (19.72).
Bromenshenk (in pro­
gress) is investigating the communication and behavioral characteristics
between individuals of a population of A, ■e l l t o t t - i while Hastings (1971)
studied the fecundity of ffemales mated to males of a different popu­
lation.
The least understood aspect of the biology of A.- e l l- io t t i -
appeared to be that of the embryology and, therefore, a series of in­
vestigations were conducted dealing with different aspects of embryonic
development..
The size and weight of the egg, the numbers of eggs produced,
the morphological and physiological features of the diapause egg, as
-3-
well as developmental respiratory patterns, were, described by Roemhild
(1961, 1965a, b, 1967, 1968)„
He hypothesized that hormone depletion may­
be a contributing factor in diapause initiation and concluded that the
diapause itself was maintained by the differences in pH values present
in compartments of the eggs formed by; embryonic membranes„
Biochemical and physiological aspects of. development were investi­
gated to determine metabolic patterns before, during and following
diapause.
Svoboda (1964) and Svoboda, Pepper and Baker (1966) reported
on the lipids in the egg during d e v e l o p m e n t w h i l e •Bunde (1965) and
Bunde and Pepper (1968) described the biosynthesis and occurrence of
free amino acids- during embryogenesisv
The effects of temperature on
oxygen consumption in.the egg.were studied by Laine (1966).
Horvath
(196,7) examined the development of muscles in the embryo of A, eZ-Z-tettf,
Leopold (1967) used histochemical methods to study postembryonic ovarian
development and oogenesis- in A. ■e V L ie tti,*
Quickenden (1969, 1970) and
Quickenden and Roemhild (1969) investigated the occurrence of carbo­
hydrates- in eggs and their relationship to maternal age and density,
while Robinson (1970) recorded the distribution,.rate of synthesis and
characterization of proteins in eggs of A.. e tU o iy b i.
Urban (1970)
v
followed- the ontogeny of six hydrolytic enzyme's during- embryogenesis,
using histochemical and electrophoretic techniques. ■
Van Horn (Visscher), (1963, 1966a) reported on the histology and
morphogenesis of embryonic development of AutoaaTa e t l i o t t i . from the
—4—
time of germ disc formation until hatching and established the staging
criteria for the embryogenesis of this species.
Using these criteria,
comparisons of the growth and variability of embryos from a single wild
population in two different years were' made.
The effects' of maternal
aging on the pattern of embryonic development was studied (Van Horn,
1966b).
Investigations were begun to describe the fecundity, viability
and developmental rate of embryos obtained from single pairs of adults
from different wild populations exposed to varying environmental con­
ditions.
It was found that photoperiod, temperature,.aging and crowding
of the parental generation brought about marked changes in the rate of
development of the embryonic offspring.
Young females from some popu­
lations produced eggs with high incidence of sterility in their first
egg pods (Visscher, 1971).
Alteration of embryonic growth in the progeny by diverse environ­
mental factors, as well as aging, suggested that the stimuli were
probably acting.upon the maternal neuroendocrine system and, in turp,,
altering the kinds or amounts of materials incorporated into the egg
system.
Gland volume changes were observed during the post-diapause
development of A. . e V l i o t t i
(Van Horn, 1968) and the influence of endo­
genous hormones upon embryonic development was suggested.
The possi­
bility that exogenous hormonal or other growth factors from the mother
—5—
could play an important role in the regulation of the rate of embryonic
development was also hypothesized=
Experiments using' applications of
analogues of juvenile hormone revealed that embryonic, morphogenesis of
A= eZ.Z--LOtti was profoundly altered (Visscher, 1972) and histological
analysis demonstrated that endocrine gland changes accompanied these
morphological effects.
These results supported the hypothesis that-
maternally-contributed growth factors may determine the rate and
pattern of early-embryonic development of A= e ZZiotti and, thereby, be
of great importance to the population success: ©f this species.
Before experimental studies could be undertaken to demonstrate•
such a mechanism, a basic understanding of the events of early embry­
onic development: in this species had to be obtained.
The descriptive
studies reported in this thesis, therefore, were undertaken to gain­
understanding of- the developmental processes occurring during early
embryogenesis of A =. eZZioiii and to. establish-, a basis for experimental
analysis of early development, in this species =
The scope of this thesis encompasses the following:
1.
Examination and description of the early- embryogenesis
of A= e ZZ-Zoft-Z using histological methods =
2.
Creation of a staging criteria for early embryonic
development.
3.
Determination of the developmental rates of eggs from
crowded and uncrowded parents, during the early stages
-6-
of development.
4o
Comparison of developmental rates from young, middleaged and old females «•
5.
Determination of patterns of ENA synthesis and DNA
synthesis in early eggs to learn whether these are
affected by maternal factors =
6o
Establishment of the beginning of new RNA synthesis in
early eggs«
MATERIALS AND. METHODS-.
Biological Material
Fourth and fifth instar nympha of A. e V tio ttV were collected from
a field near Billings, Montana in early June of 1970 and 1971„
These
nymphs were reared in a greenhouse insectary at Montana State Univer­
sity in clear Incite cylindrical cages (ll" high, 8 V
diameter) and
placed on a removable pan filled with soil from the collection site
according to methods of Visscher (1971).
A vial with water and fresh
western wheatgrass (Agropyrum s m it h i- i ,. Rydberg) was provided every other
day.
In 1971 the temperature regime fluctuated diurnally from 75°F to
850Eo-
Due to mechanical failures, a larger range of temperature was
experienced during 1970, (60-lO4°F).
Neither photoperiod nor humidity
was regulated and therefore correspond approximately to the local
conditions in the insectary.
In 1970 twelve pairs of adults were reared with one pair per cage
(designated hereafter as "single"), and 24 pairs of adults, were main­
tained under crowded conditions with six pairs per cage ("crowded").
In 1971, 20 pairs of adults: were reared-with one pair per- cage and
only two cages of crowded pairs, six pairs per cage, were maintained.Only adult males were replaced when they died.
Egg pods were collected each morning at nine o ’clock and at other
appropriate times when needed, by.sifting, the soil from the cage pans.
— 8—
Egg pods were stored upright, in plaster of Paris blocks.according
to methods of Visscher (1971)s kept in an incubator at 25°C constant
temperature and watered every other day,
At scheduled intervals the. egg pods were removed.from the-incubator
to obtain eggs of a known period of development after, which the ootheca
was removed with watchmaker forceps,
Descriptive■and Maternal Effects
Eggs were fixed for 12-20 hours in a solution of 85 volumes
dioxane saturated with picric acid, 10 volumes of 40% formalin■and five
volumes of concentrated formic acid.
The methods of Anderson (1964),
a modification of Griffiths and Carter (1958), were used with minor
changes throughout this study in the preparation of serial sections.
After one hour of fixation the chorion was pricked with a glass needle
or removed completely with watchmaker forceps,
Following 12 hours of
fixation the eggs were washed in three changes of dioxan, dehydrated
in Cellosolve (Sargent) for four changes of at least two hours each and
placed in a 2% solution of celloidin in Cellosolve at 30°C overnight i
Three changes of benzene, for a total of 15. minutes, were used for
clearing.
To prepare the- eggs for embedding, they were placed in a
solution of equal volumes of benzene and a paraffinrceresin mixture,
Because excessive' heat during the embedding procedures altered the
structural components of the egg, causing the yolk to be powdery and
reftactile, a low melting point mixture of ceresin wax and paraplast
-9-
was used.
The blocks were stored a t .4°C until they were ready to be
sectioned and the face of the block was cut to expose the egg.
The,
block was then placed in a 5% Tergitol 7 - ethane-diol (J. T. Baker
Chemical Co.) solution for 12 hours,and soaked in distilled water for
an additional 24 hours or more to facilitate sectioning.
Serial sections 5-8 y thick were cut with an AG. Spencer rotary
microtome.
The ribbons were attached to the slides with albumin,
except those for autoradiography, dried overnight and then stained
with Harris' and Delafield's hematoxylin and eosin Y.
Slides were
permanently mounted with Adams Histoclad and viewed with a Zeiss
binocular microscope (ocular 12.5, objectives- neofIuar 10, 16, 40 and
Apo 100).
Photographs were taken with a Zeiss 35 mm camera and Pan-X
film.
A large number of other fixatives, dehydrating, infiltrating,
embedding and wetting agents were tried, including the cupric-phenol
methods of Slifer and King (1933). and Roonwal (1935) but none proved
x
to be satisfactory.
Egg structures in each individual section were recorded on
specially prepared figures (Appendix A) and important structures were
photographed.
'
Autoradiography
Eggs to be used for qualitative autoradiographic experiments were
I
incubated at 25°C until the desired age was reached after which they
-10-
were removed- from the ootheca,
Originally it was planned to inject
the eggs with Ringer's solution and trltiated uridine and thymidine
through a micro-injection apparatus but the high turgor pressure of
the egg and the fragility, of the shell made, this impossible, even after
a period of desiccation. . The eggs were, therefore, exposed to a radio­
active solution after a short period of desiccation according to methods
used by Bunde (1965). with A.- e llio . t t - i- eggs.
The absorption solution
consisted of either trltiated Thymidine (methyl - HS) or trltiated
Uridine (-5-H3): in Ringer's solution■to- indicate DNA or RNA synthesis,
r e s p e c t i v e l y B o t h radioisotipes were obtained from Amersham/SearIe
and the vials contained 250 yCi in a 10% aqueous solution with a
specific activity of 27 Ci/mmol (3H-T) a n d '24'Ci/mmol (3H-U) .. A 1.5
ml solution containing a concentration of 100 yCi of trltiated materials
/10 ml of Ringer's solution was pipetted into five depressions of
tissue culture dishes (L'inbro multi-dish-disposo trays, Limbro Chemita-I
,Co.). containing six depressions each.
One depression in each dish was
used as a control and contained only Ringer's solution.
Ten eggs were, selected for each of the six compartments in the
series on the basis of. maternal factors involved (age, density).
The
eggs were incubated for periods of four hoursj 12 hours, 24 hours, 48
hours, 72 hours and 96 hours.-
On completion.of the exposure period
the eggs were prepared for sectioning as described previously.
The
sections were:placed on precleaned slides on boiled distilled water
—11—
instead of albumin and picric acid was removed from the tissues by­
passing them through two changes of ethyl alcohol, 70% and 50%, for
five minutes each.
Liquid nuclear track emulsion (Kodak NTB 2) according to methods'
of Prescott (1964) was used to coat the slides after which they were
air dried and stored in black slide boxes for- the required exposure
time (thymidine 10 days; uridine 2Q days).
Silvergrain reduction above
background concentration was taken as evidence of isotope presence.
Slides were developed with D-Il Kodak developer, rinsed in dis.
tilled water and fixed in Kodak fixer.
They were rinsed in running tap
water for 20 minutes, rinsed in distilled water, and stained with
Harris’ hematoxylin and eosin Y.
All of the autoradiographic procedures
were carried out in complete darkness to minimize background.
.EARLY EMBRYOLOGY;
'
DESCRIPTIVE
Events of Oogenesis and Egg .Deposition
Eggs of A„' e V l- io tti. are produced by two ovaries,. each having an
average of five oyarioles (Leopold, 1967)=
The ovary is of the.
panoistic type, characteristic of the more,.primitive .orders, such asthe Orthoptera.
The panoistic type ovary,'.unlike the meroistic type,
does not have specialized "nurse oells" and, t h e r e f o r e t h e folli•
■
•
•
'
’ ■
...'
cular epithelial cells of the vitellarium are thought to constitute
the only trophic tissue for the developing oocytes.
‘
■.
According to Leopold, the overioIe is divided into a gefmarium
;
and a vitellarium-as-.early as the second nymphal instar» -At this
stage, the nuclei of the oocytes within the vitellarium are in the postpachytene stage of the first, maturation division, and will, remain in
that state until after they leave the vitellarium^
Prefollicular tissue surrounds the primary oocytes in the
vitellarium beginning at the second instar„■ in the fifth instar, cells
can be recognized in this tissue and it differentiates further into
the definitive follicular epithelium soon after the adult molt.
time the
At this
primary oocyte have increased greatly in size, primarily due
to an increase in cytoplasm.
Yolk deposition commences two to three
days after the adult mbit in the ultimate and penultimate oocytes and,
accompanying vitellogenesis,- the oocytes enlarge rapidly in size.
At
the time of laying they contained large amounts- of -yolk characteris tic
'
— 13—
of eggs of the Acrididae9 rendering them most difficult to section.
Leopold observed that the follicular epithelium laid down the chorion
upon completion of yolk deposition,
At the posterior end of each follicle, established according to
the orientation of the female, there are cells which appear to be
different from the remainder of the follicle cells, being somewhat
larger and mostly of a columnar shape.
These are thought to produce
the specialized chorionic cap which later overlies the hydropyle
connection with the developing embryo.
In most grasshopper species the
chorion has a sculptured appearance characteristic, of the species
(Tuck and Smith9 1939) reflecting the arrangement of follicular cells
of the ovary.
In A. e llit O t t i, the chorion appears to be smooth with the
exception of the chorionic cap.when magnified 6X-50X,
During oviposition, the eggs are enveloped by a moist, frothy
material called spumalihe, said to be secreted by the accessory glands
(Johannsen and Butt9 1941).
Soil particles adhere to the outside layer
of the spumaline and this mixture hardens into a protective egg pod
(Fig. I).
Each ovariole seems to synchronize the release of the most mature
egg, possibly by muscle contraction; therefore, the eggs are laid in
groups, the egg pods containing from 0-12 eggs, with an average of
eight eggs per pod (Van Horn, 1966a).
Occasionally pods are laid
without any eggs, perhaps because the female was disturbed during laying.
-14-
Figure I,
Egg pod and newIy-Iaid egg of A„ e l l i o t t i ,
-15-
Definition of Terms
Blastema
- a layer of peripheral nuclei not separated by
cell walls (Rempel and Church, 1969b)
- designates an association of embryonic, cells with
a definable functional state (Krause and Sanderi
1962)o
- stage at which the nucleate periplasm is still
without cell membranes=
The egg at this stage
becomes a syncytium (Weissman in. Ando, 1962)„
Blastoderm
- peripheral cell layer surrounding the yolk, which
differentiates.into the embryonic and extraembryonic regions (usually follows blastema stage)„
It appears that in A„
differentiation in
the embryonic area occurs before the rest of the
blastema forms cell membranes.
Uniform Blastoderm
- the cells are evenly distributed over the entire
surface (Anderson, 1972)=
Differentiating
Blastoderm
- a differential concentration of cells in a local­
ized area at the surface, with a more attenuated
distribution elsewhere (Anderson, 1972). ■
Cleavage Center
- cleavage of the zygote occurs in this region=
- the first, identifiable center of control
(Counce, 1961)=
—16—
Alternative Term:
Cleavage Energld
Furchungszentrum (Krause. 1938a)=
- cleavage nucleus and its associated island of
cytoplasm (Krause, 1953).
Embryonic
Germ Anlage
- part of blastoderm, destined to become embryo after
differentiation of blastoderm into embryonic and
extraembryonic areas.
Alternative Term:
Endoplasmic
Reticulum
Embryonic Primordium
- a network of internal cytoplasm in which deuto­
plasmic components are suspended (Krause and
Sander, 1962).
. Alternative Term:
Protoplasmic or Cytoplasmic
Reticulum (Johannsen and Butt., 1941) =
Periplasm
- a yolk free cytoplasmic layer at the.periphery of
the egg.
Alternative Terms-:
Keimhautblastema (Patten) ;
Cortical Layer, Cortex (Counce, 1961)=
Percentage Egg
Length (n% E= L=)
- the length of each egg is translated into 100
units, 0 representing the posterior pole and 100
the anterior.
Specific locations are given as
n% E=' L. (Counce, 1973).
Vitellophags
- nuclei with encompassing islands of cytoplasm
which lie within the yolk mass; and act as agents
of yolk digestion (Anderson, 1972).
-17- .
Alternative Term;■■ Yolk Nucleus (Counce, 1961) „
Primary (I0)
Vitellophags
- vltellophags whicb arise directly by the further.
division of cleavage energids which remain within,
the yolk mass as their, fellow energids migrate to
the surface (Anderson, 1972)
Secondary (2°)
Vitellophags
- vitellophags originating from mitosis of the peri- ■
pheral nuclei and which migrate back into the yolk.
Yolk Mass
- yolk with scattered cleavage energids (Anderson,
1972).
Alternative Terms: Yolk Plasmodium; Yolk-Endoplasm
System.
Results
External Appearance of the Egg
Fixed newly-laid eggs of A, e l l i c b t t i 'included in this study ranged
from 4,9 mm to 5.8 mm long, and from 1,5 mm to 1,7 mm wide (average, based
on 12 eggs, was 5.3 mm x 1.6 mm) (Fig. I).
They have a moist,, pale
yellow appearance when first removed from the, pod, but the surface be­
comes lighter in color on exposure ,to the air. . The egg appears curved
longitudinally and is rounded at the anterior end while slightly pointed
at the posterior end,
The outer, covering, or chorion, df the-egg., is a
relatively tough, somewhat transparent membrane secreted by the maternal
follicular cells.
When viewed with the dissecting, microscope (SX^SOX),
-18-
the chorion of the egg of A a e t l i o t t i . presents a smooth appearance
except for the posterior area where a distinct sculptured appearance
is evident=
This posterior area of the egg is called the chorionic
cap and it is this region which is directed downward in the egg pod.
when the egg is laid=
The apex of the chorionic cap is brownish in
color and darker than the remainder of that structure=
Measurements
f
of the length of the chorionic cap indicate that it covers an average
•
of 4% or =24 mm of the length of the egg (average, based on 12 eggs)=
Around the base of the chorionic cap 50 to 60 micropylar openings are
present in the chorion which allow sperm penetration through the egg
coverings (range based on 12 eggs).=
In eggs 30 days old, these open­
ings have a darker appearance than the base of the cap and, consequently
are more obvious =
It is not known whether eggs of A= eV IZot-bi are
synchronized during their maturation period, i=e=, whether they arrive
at one certain stage before they are laid=
It has been observed, how­
ever, that there are developmental.differences between eggs within a
pod within the first day after oviposition (see Experimental Results)=
A 0 e Z Z io t t i females usually lay one pod every other day under labora­
tory conditions =■ This may vary, however, depending on the population
source=
Staging Criteria
Manipulation of the events occurring during early development of.
insect eggs frequently has been undertaken by experimental embryologists
-19-
Comparisons of experimental results of Aerididae to those from other
species, however, are difficult because standard criteria of the early
development in the Acrididae are lacking„
Chapman and Whitham (1968)
developed a standard criteria for staging acridid embryos and incor­
porated the results from 18 studies and 10 species reported on in the
literature=
The events of the early embryology, however, were grouped
as stage 0«
The early development of eggs of A 0. e V L 'to tti. can be categorized as
stages A to I, based on discrete morphological and developmental events
observed in the present studies 0
The number of individual embryos from
which these stages are derived is noted in each category (Fig0
2)« It
has been customary to divide the early developmental, periods of insect
eggs according to time periods, i»e0, hours, or days if the development
was of long duration, thereby assigning a particular specific time
period to each embryological event =
With the recent findings concerning
the regulation of developmental rates of. A 0 e U - i o t t i
(Visscher, 1971)
it has become clear that separate developmental stages in this species'
cannot be described on the basis of time alone 0
The following stages were determined in early eggs of A 0 e l l - i o t t i i
Stage A
Maturation:
While maturation- divisions were not-observed the
female pronucleus
in the newly-laid egg was found located in
the posterior part of the egg, about midway between the median
line and the periphery=
Structures which stained similar to
N
Stages
Figure 2,
Diagram of stages A to I of the early embryology of A 0 e l l i o t t i ,
B N=
blastema nucleus, C = chorion, Ch C = chorionic cap, C N = cleavage nucleus,
E R = embryonic rudiment, F P = female pronucleus, G B = germ band, P B = polar
body, S C = serosal cell, S V = secondary vitellophag, V = vitellophag, Y =
yolk, Z = zygote. Stage criteria are based on the observations of five eggs each.
-21-
polar bodies were present at the periphery of the egg.
Stage B
Fertilization;
The zygote nucleus was observed in the same
vicinity as the female pronucleus .was previously located and
it is thought, therefore, that the fertilization process takes
place in that same area.
Stage C
First cleavage divisions
The zygote nucleus is dividing into
two cleavage nuclei.
Stage D
Second cleavage division;
present in the yolk.
Two to four cleavage nuclei are
Eggs of A. e H i o t t i . seem to lose
synchrony of division early and consequently the number of
cleavage nuclei are varied after this time.
Stage E
Late cleavage divisions;
Cleavage nuclei are present through­
out the posterior, part of the egg.
Differences between 1°
vitellophags and future blastema nuclei are not apparent.
Stage F
Initiation of blastema formation:
The first entry of cleav­
age nuclei into the peripheral periplasm takes place.
Cleav­
age nuclei in the remainder of the egg are still scattered
throughout the yolk.
The nuclei in the anterior portion of
the yolk are often large and present a diffuse appearance
during interphase.
Stage G
Completion of blastema:
Nuclei are present in the entire
peripheral periplasm. . Mitosis in the blastema continues and
as a result, both additional blastema nuclei and 2° secondary
-22-
,vitellophags 'are formed.
Stage H
Formation of embryonic rudiment:
Differentiation of an
embryonic area from the extreme posterior blastema takes
place. ■ The nuclei are packed close together in a single
■ layer with little cytoplasm between them, while the cells
take on a characteristic cuboidal ..appearance.
Stage I
Multilayered germ band:
through repeated mitoses.
The germ band becomes multilayered
The lateral edges of the germ band
roll into the yolk.
The Newly-Laid Egg
Membranes.
The chorion of the egg 0-% hour after laying, when
examined in longitudinal serial sections with the light microscope,
consistes of three layers (Fig. 3).
Based on measurements on six eggs,
the outer layer or exochorion is 1%-p thick and has a dense appearance.
The middle layer or endochorion is 4% p thick, appears textured, and
takes an intense eosin stain while the.third or inner layer is very
thin (.5 y) with an opaque appearance resembling the outer layer.
These chorionic layers are usually of uniform thickness around the egg
except at the posterior pole.
There, in the area where the future
embryo and the hydropyle will appear, the mean thickness of the endo­
chorion of five embryos was 95 y at the apex.
The exochorion has
about the same thickness throughout the egg except at the posterior
end where it is folded and penetrated by pores seeming to terminate in
-23-
Figure 3.
A longitudinal section of the chorion of a newly-laid egg of
A„ e l l i o t t i o
Ex C = exochorion, En C = endochorion, I L =
inner layer of chorion.
400X.
-24-
the endochorion (Flg= 4)=
The mlcropyles occur In a ring around this
posterior porous region of the exochorion=
In serial sections,.however,
they are difficult to locate, perhaps because they may close very soon
after oviposition.
The inner layer of the chorion appears to be the
same thickness around the egg=
A very thin vitelline membrane (Fig= 5) (I p or less, based on 4
eggs) is sometimes visible in regions where the periplasm is sparse and
the yolk globules, particularly lipids, are often closely packed
against the periphery=
During histological preparation of the egg the lipids are dissolved
leaving vacuoles adjacent to it, outlining the vitelline membrane=
The periplasm is a layer of cytoplasm located between the vitelline
membrane and the yolk=
pale pink with eosin='
Its matrix appears homogeneous and stains a
Otcasionally small, droplets which stain blue are
visible along the margin of this layer which raise questions concerning
their identity (Fig= 6)=
In a few instances, periplasmie extensions
can be seen extending centripetally between the yolk globules,=
Nucleus=
T h e ■maturation divisions were not.observed in sections
of the newly-laid egg.
Leopold (1967) reported that the oocyte nucleus
seems to be in the post-pachytene stage of the first maturation divi­
sion before the egg leaves the vitellarium, and therefore, the
maturation divisions may have taken place as the egg descended in the
oviduct or during the first minutes after oviposition= ■The female
-25-
ExC .
Figure 4»
A longitudinal section of the chorionic cap of a newly-laid
egg of A. e l l i o t t i *
Ex C = exochorion, En C *= endochorion,
Po = pore.
260X,
— 26-
Figure 5.
A longitudinal section of the periphery of a newly-laid egg
of A„ e l l i o t t i .
V M = vitelline membrane, C = chorion, Y G
yolk globules.
260X.
-27-
r
Figure 6.
Basophilic droplets and second cleavage division in a
longitudinal section of a six-hour egg of A. e l l i o t t i ,
N = nucleus, Cy = cytoplasm.
650X.
— 28-
pronucleus was observed about one-fifth of the lerigth of the egg from
its posterior end and about 115 y from the periphery.
Twelve distinct
chromosomes were present with little cytoplasm surrounding them (Fig. 7)
Small amounts of chromatin, thought to be polar bodies, also were ob­
served at the periphery of the egg, about the same distance from the
pole as the female pronucleus (Fig. 8).
Yolk.
When eggs are immersed in xylol the chorion becomes trans­
parent, permitting observation of the dense, core of yolk, wider at the
posterior pole and tapering towards the anterior pole.
The yolk, or
deutoplasm, did not appear homogenous throughout the egg, but displayed
a definite polarity in distribution of,yolk particles.
The polarity.of
yolk particles also was apparent in sections from posterior portions
of the egg; they sectioned much more readily than those in the anterior
portion.
.Generally the yolk.in the. anterior,part of the egg was com­
posed of large, closely packed yolk globules which easily shattered
during preparation of serial sections, while the yolk in the posterior
part had a smoother appearance, with smaller globules.
In transverse sections of the egg, stratification of yolk was ob­
served in a few instances.
The larger globules were found in a band
below the periplasm while the. smaller globules were found centripetally.
At the 50% egg length (E. L.), the inner core contained larger yolk
particles.
-29-
Figure 7
The female pronucleus in a longitudinal section of a newlylaid egg of A. e l l i o t t i o
N = nucleus, P B = polar body.
650X.
— 30—
PB
Figure 8,
Polar body in a newly-laid egg of A 0 e l l i o t t i .
body,
650X.
P B =
polar
-31-
Temporal Pattern of Development
It seems likely that fertilization of the female prdnucleus
occurred sometime during the first six hours of development, probably
soon after oviposition.
While neither sperm nor the actual process of
fertilization was positively identified, cleavage of the zygote nucleuswas observed during this period and at least two cleavage divisions were
completed.
Synchrony■of division appeared to-be lost early, usually
before the egg was 2.4 hours old, as evidenced by the presence of di- .
viding and interphase nuclei (thus stages A-D took place in the first
six hours),
.
The abundance of yolk and the apparent absence of distinct cyto­
plasmic connections between cleavage nuclei may have contributed to the
early loss of synchrony,
During the interphase the cleavage nuclei
took on a diffuse appearance, with the surrounding cytoplasm appearing
stellate-shaped, and no distinct outer cell membrane could be seen
(Figs, 9, 10),
Small basophilic bodies, thought to be eliminated
chromatin, were observed during the second cleavage division (Fig, 11);
these were never observed at a later stage.
The outward migration of the cleavage nuclei and their first arrival
in the periplasm at the posterior end of the egg generally took place
before the egg was 24 hours of age (stages E-F)«
During cleavage and
migration the nuclei seemed to be distributed randomly in the yolk and
neither spheroid nor elipsoid configurations of nuclei were noted at this .
-32-
«
«
I
Figure 9.
Chromatin elimination during second cleavage division in a
longitudinal section of an egg of A„ e l l i o t t i .
(egg is less
than one hour old) 650X„
-33-
Figure 10.
Interphase cleavage nucleus in the anterior part of the egg
of A. e l l i o t t i .
Longitudinal section. N = nucleus, P periplasm.
100X.
-34-
Figure 11»
Interphase nucleus in the anterior part of the egg of
A= e l l i o t t i '
Enlargement of Fig, 10, Cy = cytoplasm,
400X.
-35-
time.
Peripheral nuclei were observed in the posterior half of the egg
near the site of the first cleavage divisions.
While rapid mitotic di­
visions took place in the posterior peripheral nuclei, forming a thin
syncytial membrane around the yolk, hereafter referred to as blastema
(Fig. 12), the anterior cleavage nuclei divided more slowly, invading
the periplasm of the anterior pole of the egg only after the egg was two
days old.
The anterior periplasm (18 .p thick average, based on three
eggs) often had a cloudy appearance prior to penetration by the cleavage
nuclei, but this condition was transitory (Fig. 13).
Initially the cleavage nuclei and their cytoplasmic islands
(approximately 30 p diameter, based on five eggs) were isolated from
one another at the periphery o f .the egg and the cytoplasm appeared to
penetrate deeply into the yolk with the nucleus (7 y) centrally located
in the cytoplasm (Fig. 14) after repeated mitoses.
However, this cyto­
plasm no longer appeared to extend deeply into the yolk, but seemed to
be incorporated into the periplasm, and that layer, therefore, appeared
to become thicker than before.
After invasion of the periplasm the
nuclei of the blastema became quite varied in appearance; they moved
closer to the outside margin of the egg, stained more darkly and the
chromatin often displayed ring, beaded or knobbed configurations (Figs.
15, 16),
It is possible that sequential changes take place during the
development of the peripheral nuclei, because different types of nuclei
seemed to predominate in eggs of different ages.
These cytological
-36-
Figure 12.
Peripheral nuclei at the posterior end of the egg of A.
e H io tti,
Longitudinal section. Eighteen hours old. Ch C =
chorionic cap, N = nucleus, M N = mitotic nucleus.
125X.
-37-
Figure 13.
Periplasm of the anterior end of the egg of A. e l l i o t t i *
Longitudinal section of the same egg as Fig. 12. 125X.
— 38-
Figure 14,
Blastema nucleus at the periphery of a three-day-old egg
of A, e l l i o t t i .
Longitudinal section. 500X/
I
X
Figure 15.
Beaded blastema nucleus at periphery of a four-day-old egg
of A. e l l i o t t i ,
Longitudinal section. N = nucleus. 430X
?
Figure 16=
Doughnut-shaped nuclei in posterior part of a three-day-old
egg of A. e l l i o t t i *
Longitudinal section= N = nucleus,
260X,
-41-
stages may be indicative of differentiative changes which occur as
these cells take on new functions as blastoderm and then as serosal
and embryonic cells.
Not all cleavage nuclei appeared to take part in the formation of
the blastema.
Some remained in the yolk becoming primary vitellophags
or primary yolk nuclei.
Another source of vitellophags in the yolk of
A, e V L ie t t i. was identified during'mitosis of the peripheral nuclei;
depending on the direction of the spindle, peripheral nuclei were ob­
served dividing either at a 90° angle to the periphery to produce a
secondary vitellophag and a daughter blastema nucleus (Fig, 17), or
dividing tangentially with an outer surface to produce two peripheral
daughter nuclei (Fig, 18),.which then could divide again.
Secondary
vitellophags remained capable of division and mitoses were noted in
these cells throughout the formation of the germ disc
although not in
large numbers.
Nuclei of the blastema-seemed to be unevenly-distributed at first..
They occurred singly or in associations of two, three, or four nuclei,
probably related daughter nuclei.
They always seemed to be more
numerous in the posterior end of the egg and became less numerous to­
ward the anterior end.
In eggs of stage F (about three days old) these
nuclei generally seemed to be spherical in shape as evidenced in both
transverse and longitudinal sections.
However, at later stages (h and
I.) elongated nuclei, tangential to the periphery, were observed.
—42-
Figure 17.
Mitosis at a 90° angle to the periphery of a three-day-old
egg of A. e l l i o t t i which produces a daughter blastema nucleus
and a secondary vitellophag.
Longitudinal section. P =
periplasm.
500X.
—43—
*
Figure 18.
Mitosis tangentially to the periphery of a one-day-old egg
of A, e l l i o t t i which produces two blastema nuclei. Longi­
tudinal section.
500X.
— 44-
In three eggs of stage G (about four days old), aggregations of
about 12 yolk nuclei were noted In the posterior part of the egg..
In.
two instances the aggregates were located close to the extreme posterior
periphery of the egg and in another (in an egg used in autoradiographic
experiments), the aggregate was situated deeper in the yolk at about
the 35% level (Fig. 19).
Nucleoli in blastoderm nuclei were first noted in stage G eggs,
and only one distinct nucleolus per nucleus was ever seen (Fig. 20).
In eggs of stage Q- (four day old average), the periplasm in the
posterior, part of the egg appeared to thicken, becoming a layer clearly
distinct from the- surrounding deutoplasm.
Differentiation of the
blastema into definite embryonic and extraembryonic areas was observed
to begin at that time,;
apparent.
Several aspects of this differentiation were
Nuclei at the extreme posterior periphery formed a layer
with little cytoplasm between them and it could be surmised from the
cell shapes that cell membranes had formed.
At this time these cells
presented a characteristic cubbidal appearance except in the transition
zone between embryonic and extraembryonic areas; there they became
squamous in appearance and the shape of their nuclei was intermediate
between the tangentially-elongated nuclei of the presumptive- serosa and
the rounded and smaller appearance of the future, germ disc nuclei (Fig.
21).
—45-
Figure 19,
Internal nuclear aggregate in a five-day-old egg of A,
This egg was used for autoradiographic experi­
ments and shows incorporation of tritiated thymidine into
the nuclei. Longitudinal section.
IOOX.
e llio tti*
—46-
Figure 20.
Peripheral nucleus with distinct nucleolus in a four-day-old
egg of A. e l l i o t t i .
Longitudinal section.
640X,
-47-
Figure 21.
Cuboidal cells of the embryonic rudiment in posterior end
of a six-day-old egg of A. e l l i o t t i .
Longitudinal section.
260X.
-48Cells of. the developing germ disc numbered about 19 in three
embryos immediately after differentiation of the embryonic area with two
or three cells on either side in the transition zone=
Large
round yolk nuclei about 20 p in diameter (based on five eggs)
were consistently present immediately below the cuboidal cell layer or
below the transition zone=
It was difficult to establish with certainty
whether nucleoli were present in these yolk cell nuclei.
The single layer of germ disc cells characteristically had mitotic
divisions at this time =
Mitoses also were observed in the extra embry­
onic area in eggs which were younger than seven days of age.
In three eggs obtained from crowded parents, all stage H' and five
days old, a second layer of nuclei (16x7 p) was observed separated from
the presumptive germ disc cells by the yolk.
Because the embryonic
cells are not yet cuboidal in shape this could possibly be a yolk-cellmembrane (Johannsen and Butt, 1941), an ephemeral structure existing
only during differentiation of the blastoderm,
Vitellophags were not numerous at this time (usually between 10
and 15 were observed, based on five eggs).
Establishing a developmental sequence from the formation of the
germ disc until the embryo was 12 days old presented more difficulty
than it did in younger eggs.
With increasing age, the variability in
the rate of development of embryos became mere pronounced, develop­
mental changes seemed to occur at.a rapid rate during this.time period,
-49-
and because fewer older eggs were sectioned, a pattern of development
was less obvious.,
Continued mitoses in the germ disc resulted in the formation of
an uneven layer of cells=
In three embryos of stage H', the- early germ
disc was 9 p in width and 250 y in length, while it was not yet a
distinct two-layered tissue=
Pycnotic yolk nuclei were observed at this time and continued to be
present even in the oldest eggs examined (12 days)=.
They frequently
occurred at the posterior pole of the egg in the vicinity of the germ
disc, but also were noted less frequently near the anterior pole and at'
other locations in the egg=
Because dividing vitellophags were present
at this time, it seems conceivable that older vitellophags were replaced
with younger ones=
In addition, the possibility exists that these
pycnotic nuclei represent nuclei which were fixed while in an unstable
state=
Observations from three embryos of late stage H. showed the germ
disc to be composed of irregularly arranged cells forming an uneven
multilayered tissue" about two cell layers thick, which tapered towards
the lateral margins =
The cells in cross sections did not seem to be in
a definite euboidal. shape but fitted together as in a mosaic (Fig= ■22).=
The serosal nuclei appeared to increase in size with the age of the egg
and stained much darker than the germ disc nuclei or the yolk nuclei'of
the same egg=
Figure 22.
Nuclei in mosaic germ disc in the posterior end of an
eight-day-old egg of A. e l l i o t t i .
G D = germ disc. 260X.
-51-
In stage I large serosal nuclei (30x15 y, based on five samples)
were seen around the entire periphery of the egg.
nucleoli which stained intensely.
They had dense
The germ disc at this stage of
development seems to correspond to the external morphological stage I
of Van Horn (1966a).(Fig. 23).
After elongation of the germ disc took
place (stage 2, Van Horn) forming the protocephalon and the protocorm,
the margins of the germ disc appeared in cross section to roll inward
into the yolk, while the center region of the disc remained close to
the serosal cells of the posterior end of the egg.
The small, much elongated nuclei of the amnion (5x10 y) were ob­
served forming a membrane seeming to connect the serosa at a central
point of the extreme posterior pole with the longitudinal edges of the
germinal tissue.
The germ band consisted of two or three irregularly
arranged layers of cells.
The amount of cytoplasm was notably scarse
and, therefore, the germ band seemed to consist mainly of nuclei.
The germ band appeared to be U-shaped with the longitudinal edges
rolled more deeply into the yolk than in the previous stages.
The cells
at the margins turned back on the germ band at the points where the tran­
sition into the amnion was made, while the central portion remained
near the surface of the posterior pole below the serosal, cells (Fig.
24) ,
Mitotic figures were observed in the embryonic cells of several■
embryos.
I
In the center part of the germ band, a cluster of cells was
-52-
Figure 23.
Germ band and serosal cells in the posterior part of a tenday-old egg of Ao e l l i o t t i o
Longitudinal section. Y C =
yolk cell, S C = serosal cell, E = embryo, 260X.
-53-
SC
Figure 24o
Embryo, amnion and serosal cells in a twelve-day-old egg of
A. e l l i o t t i ,
Longitudinal section. A = amnion, S C =
serosal cell, E = embryo.
260X.
Il Il /-V
_54observed projecting towards the yolk, probably the first inner layer
cells.
The nuclei of these cells were the same size as those in the
germ band but they were grouped in: a loose irregular pattern.
Large, round, yolk nuclei and some pycnotic yolk nuclei were seen,
most often in the vicinity of the germ band.
The descriptive portion of this study of the early embryology of
A., e l l i o t i y t was concluded with the examination of the germ band in 12day-old eggs.
The study of the external and internal morphology, with
discussion of the organogenesis of embryos of A. e t t i o t t i from stage I
to hatching was completed by Van Horn (1963, 1966a).
Discussion
The chorion of different insect eggs has been reported to consist
of as few as one layer in the beetle L y t t a v i r i d a n a
(Sweeny e t a t , 3
1968) and as many as seven identified layers in the bug Bhodnius
TpvoZiazus (Beament, 1948a) , indicating how structural diversity exists
within the Class Insecta.Most studies- on the oogenesis of insect eggs have concluded that
the chorion is- secreted by the maternal follicle cells (King and Koch,
1963; King, 1970).
Chemically the chorion seems to be non-chitihous
and is made up predominantly of structural proteins, carbohydrates and
lipids according to King and Koch (1963).
Some confusion of terms exists in the literature concerning .the
naming of the different layers'of the chorion, but with the:help of.
-55-
drawings and photographs, homologous layers in different species can
be resolved.
The chorion of the eggs of the Acrididae has been the subject of
a number of studies:
(Jahn, 1935; Slifer, 1949, 1958; Matthde, 1951;
Salt, 1952; Roonwal, 1954a, b; Hartley, 1961, 1964; Hinton, 1962, 1969;
Slifer and Sekhon, 1963).
layers:
It has been.found to consist of at least two
the exochorion, a rather thin, dense layer; and a thicker inner
layer, the endochdrion.
The endochorion at the ultrastruetural level
appears to be composed of fine struts which provide the egg with a
plastron respiratory system (Hartley, 1961; Slifer and Sekhon, 1963;
Hinton, 1969).
A.
eVliotti.
Bunde (1965) and Robinson (1970) found that in eggs of
tritiated materials in solution were trapped in the shells
during an absorption period accounting for a large increase in dry
weight of the shell.
In eggs of D. m ig v d t o v ia (Hartley, 1961; Roonwal, 1954a) and of
M elanoplus d l f f e n e n t i a l i s
(Slifer, 1949) an additional outer layer,
the extrachorion was present when the eggs were examined after oviposition.
It was granular in appearance and shrank during embryogenesis.
Because this layer could not be found in ovarian eggs immediately before
oviposition, it was suggested that this layer.was secreted in the ovi­
ducts (Hartley, 1961).
Eggs of A. e ' l l i o t t i examined in the present
study do not seem to possess this extrachorion, possibly beicause-the
ootheca is an extremely protective structure compared to that of many
-56-
other members of the Aerididae.
Examination of fixed microscopic sections of the chorion of A 0
e H i o t t i revealed a narrow layer of dense material inside the endochorion as shown in Figure 3»
During fixation procedures the chorion
would often be separated from the rest of the egg but this inner layer
would always remain an integral part of the chorion.
It seems possible
that this layer could be equivalent to the primary wax layer (Slifer9
1948; Beament, 1946a; Davies, 1948; Matthed9 1951) which is produced
during oogenesis.
However, in autoradiographic studies to be dis­
cussed, in newly-laid eggs of A., -eV U iottn, which were immersed for four
hours in Ringer’s solution with tritiated thymidine and subsequently
sectioned, it was found that the radioactive material was present through
out the egg and large amounts were located in the eggshell itself.
There exists the possibility that the radioactive materials entered the
egg through the micropyles and other areas of. the chorionic cap but a
gradient was not noted in the sections, • Robinson (1970) found that pre­
diapause eggs of A, e V L t o t t i less than 20 days old have fragile shells
and lose weight rapidly upon desiccation and he attributed this to the
absence of serosal cuticles.
If a primary wax layer is present in eggs
of A, e V U io tti, this should be an important feature in preventing desic­
cation of the eggs.
Robinson (1970) and Bunde (1965) did note that pre­
diapause eggs seemed highly variable with respect to the absorption of
the isotopes they were working with,
Slifer (1949) observed that iodine
-57-
did not seem t© enter the egg of M. d - iffe v e n t'ia t- is before the fifth day
of development and attributed this to the protection of the primary wax
layer, but she also found that the eggs stopped developing after ex­
posure to the reagent„
She did not hypothesize on the cause for the
arrest of development»
The pores observed in the exochorion of the chorionic cap in eggs
of A„- e H t o t t i - appear to be similar to those Slifer found in
d iff e v e n t ia lis
(1949b)„
She observed that "the whole exochorion in
this region resembled a sieve."
She also cites work by Jannone on
B o c io s ta u v u s in which he found similar pores and believed that they
served for the passage of air into the endochorion.
Smith, Telfer and
Neville (1971) discussing the aeropyles in H yalophora c e c ro p ia observed
that "points of junction of groups of three follicle cells are marked
by the presence in the developing and mature chorion of lengthy follicle
cell villi, and it is clear that the channels in which these lie repre­
sent the aeropyles described by Hinton (1970)".
As shown in Figure 4,
the exochorion of A. O llio tt- C is extremely folded in this region,
corresponding to the sculptured appearance of the chorionic cap when
viewed externally and which is caused by the particular configuration
of the maternal follicle cells.
The pores in the exochorion extend
far into the endochorion and were present in eggs of all ages examined
histologically (newly-laid till 12 days old).
When viewed externally,
micropyles in the chorion of newly-laid eggs of A. e l l i o t t i were
-58-
difficult' to observe, but with increasing age they became darker and
were easily seen.
Andb (1962) noted that the number of:micropyles in
Qdonata may be variable, even in eggs from a single batch.
This was.
also observed in A. e l l i o t t t where the numbers ranged from 50 to 60 with
a mean of 56, based on observations on 12 embryos.
The origin of the vitelline membrane of A.-- e lV L o t t i remains unknown.
It was first observed in the newly-laid egg, with the exception of the
region around the posterior poles, as a dense, thin, (I y) membrane and
remained visible throughout the time period studied in this thesis CO12 days).
The thickness did not appear to vary but this is difficult
to establish with certainty with the light microscope.
Recent electron microscope studies of the vitelline membrane in
oocytes of other insect species discuss its origin.
Okada and
Waddington (1959) studying Dvos&pkiZa thought it to be a product of
the oocyte itself as did Rempel and Church (1965) with L y t t a v tv id a n a
and Machida (1940) in Bombyx,- while Hopkins and King (1966) working with
Bombus considered that both the oocyte and the follicles contributed to
the formation of the vitelline membrane.■ Most investigators, however,
seem to agree that the maternal follicle cells elaborate the vitelline
membrane by secreting pre-vitelline secretion droplets (Raven, 1961;
King and Koch, 1963; Favard-Sereno, 1966; Beams and Kbssel, 1969).
The secretion droplets are thought to leave the follicular cells by
reverse pinocytosis and eventually coalesce, forming the vitelline
-59-
membrane between the oocyte and the follicle cells„
Slifer and Sekhon
(1963) observed the vitelline membrane, which Slifer had earlier called
the resistant endochorion, in newly-laid eggs of. Af. d / L f f e v e n t ia t is ^
It
V
seemed to be a very thin (.05 y ) , dense layer and later appeared to be
incorporated into the cuticle.
Gerrity e t a t*
(1967) found that the vitelline, membrane
changes, in appearance within 15-30 minutes after oviposition.
They ob­
served that it appeared to be porous in freshly-laid eggs but that during
the next half hour "the pores become progressively smaller until the
membrane becomes solid and continuous".
In eggs of A.' e l t ' l o t t i the
membrane appeared to be dense, however, the egg was usually 15 minutes
old when fixed and the membrane could have condensed during this time
period.
The egg of A. S tt-L o tti, possesses a thin periplasm- characteristic
of the more primitive orders of winged insects.
the cortical layer or "Keimhautblastema".
It is sometimes called
In some insect's it is- di­
vided into two layers, such as Ande (1962) found in the Odbnata9.and
consists of an outer eosinophilic and an inner basophilic layer.
Mahowald
(1963)
observed an abundance of endoplasmic reticulum in the
periplasm of 5. m etanogastev with the electron microscope and noted
that "most of the basophilia of the cytoplasm is due to numerous unat­
tached ribosomes which are usually in- clusters rather than scattered
evenly in the cytoplasm".
Bier (1953) referred to small intensely
-60-
basophilic balls situated beneath the periplasm proper.
Basophilic
globules were found in the preblastoderm periplasm of A.- e V l i o t t i during
the course of this study and Leopold' (1967) reported unidentified baso­
philic droplets in the posterior part of the oocyte.
It seems likely
both could be analogous to. the ribosomes reported by Mahowald.
Another
possibility exists that the basophilic droplets in eggs of A 4 e l t t o t t i
are droplets of cytoplasmic DNA or DNA precursors.
The fast mitotic
rate after the initiation of cleavage, particularly during the formation
of the blastema, would be facilitated by distribution of cytoplasmic
DNA.
L fHelias (1970) reviews evidence for the presence of. low molecular
weight DNA in the cytoplasm of cells of mosquito larvae.
This DNA
appears to be bound to histones and is physiologically released follow­
ing periods of. active growth of the larvae.
Krause and Sander (1962)
in their review of ooplasmic reaction
•
'
...
'
systems in insects wrote that "yolk-rich" eggs have only a plasmalemma
and.do not possess a layer of peripheral periplasm referred to as
ectoplasm.
It is of interest, therefore, that sections of eggs of A.
e l l i o V t t at six hours show a thin peripheral layer of material which
does not resemble yolk.
It is thought that this layer is the periplasm.
Endoplasm, or internal cytoplasm, in eggs of A.- e l l i - o t t i was ob­
served only immediately around the nuclei in the yolk and not as a
cytoplasmic reticulum connecting the nuclei.
Although early loss of.
synchrony during the cleavage process could indicate a paucity of
-61-'.
cytoplasmic materials connecting the individual nuclei it seems likely,
that some connumication exists between them.
A n :increase in the thickness of the primary periplasm as observed.
in eggs of A.' e i t ' t o t t ' i seemed to be the result of cleavage nuclei with
their, complement of endoplasm reaching the periphery.
Such an increase
in periplasm;during formation of the blastema also was observed by Ando
(1960) and reviewed again by Krause and Sander (1962).
Krause and
Sander considered the increase in primary periplasm one of three most significant particle movements in the early insect egg, the other two
being migration of the cleavage nuclei and the appearance of secondary
periplasm.
In the more advanced orders of insects (e.g. Mecoptera, Lepidoptera,.
Diptera,' Hymenoptera) an "inner" periplasm is formed between the blasto­
derm and the yolk (Ando, 1962) which may be synonymous to the secondary
periplasm which Krause and Sander describe as being formed below the
germ anlage.
Counce (1961, 1973) discussed the periplasm, which she called
cortex, in relation to its influence on the destiny of the nuclei enter­
ing into it and its changing role in controlling the determination of
nuclei between maturation divisions and blastema formation.
Nuclear
materials (maturation products, supernumary sperm, etc.) which enter
early periplasm in B^ m e la n e g a ste r are destined to deteriorate, while
the OUt^migrating cleavage nuclei, entering the same periplasm but at:a
—62—
later date, are not subjected to this process but undergo mitosis and
eventually give rise to the blastoderm and secondary vitellophags»
It is thought that some stimulus provides for the initiation of
maturation in insect oocytes after a period of inactivity during
vitellogenesiso
This stimulus is reported to be of a diverse nature„
According to Wigglesworth (1965), the sperm seems to provide the
stimulus to maturation in the insect egg, while Kuwana and Takami
(1968) state that eggs of Bombyx m o ri only proceed "to the later stages
of meiosis after sperm entry".
By contrast, King and Slifer (1934) ob­
served that eggs of virgin females of Af„ d iffe T e n t- ia t- is underwent
meiosis in a normal manner without the presence of sperm and concluded
that the laying is the apparent stimulus to the completion of meiosis,
Visscher (1971) found that embryos in some eggs from virgin females of
A, B t l- I o t t i, could develop to stage 11 or 12 in a 30 day period at 25°C,
Counce (1961) reviewed two other instances in which sperm entry does
not seem to be required from the initiation of maturation (Doane, ZV
m e la rw g a s te r % Strasburger and Korner, Z?„ -fu n e b ris ) and she noted that
in many orthopteran eggs development may extend beyond the blastoderm
stage without the presence of sperm.
It is not known if sterility
found in some eggs of A, e l l i o t t i was caused by a lack of sperm pene­
tration or if some other, developmental process was involved,
As shown in Figure 7, 12 chromosomes were observed in the female
pronucleus of. the egg of A, e l l i o t t i *
This number, corresponds with
-63-
results obtained from squash preparations of testes,
Orthoptera
appear to have the X-O pattern of sex determination.(Makino, 1951),
The exact location at which, meiosis occurred in eggs of A-, e t H o t t i
w a s ■not determined,
Krause and Sander (1962) referred to the cyto­
plasmic island in which meiosis occurs as "maturation plasm" and placed
it near the site where the oocyte nucleus "dissolved" prior to meiosis.
In. A, B lZ -L o ttt the oocyte nucleus migrated to the posterior part of the
oocyte during the first days of adult life (Leopold, 1967) and seemed to
be located about midway between the median plane and the periphery just
prior to oviposition.
The female pronucleus was later observed at about
the same location and, therefore, it was difficult to establish with the
material available if meiosis occurred internally, or if it proceeded in
association with the periplasm and the female pronucleus migrated back
into the deutoplasm where syngamy then could take place,
Counce (1961,
1973) places the maturation plasm in association with the cortex and
states that the four nuclei resulting from meiosis are "arranged in
linear order at roughly right angles to the long axis of the egg,. Be­
cause the innermost nucleus is in a different milieu it becomes the
female pronucleus while the cortex determines that the other three
nuclei will become polar bodies,"
The presence of polar bodies in the periplasm such as were noted
in A, B lZ -L o ttl are not a clear indication, of the location of meiosis,
Hagan (1951), described maturation and fertilization in oviparous
(
-64-
insects and noted that before fertilization the nucleus, is located near
the center of the egg, but that the position of the nucleus shifts
during the maturation process.
The first cleavage division in eggs of A. Q lZ i-O tt1I is observed
within six hours of oviposition and is located in the same area where
previously the oocyte nucleus was observed.
The presence of a cleavage
center in some species of Orthoptera has been established by Krause
(Tachycines, 1938; Notonecta, 1957) but Moloo (1971) was unable to
locate such a center in the acridid S d kisto o e vo a gvogcccia*
Examination of sections of eggs of A. e l l i o t t i during the second
cleavage division revealed a small basophilic body in close proximity
to the chromosomes (Fig. 11).
This was thought to represent chromatin
eliminated during cleavage, similar to that found by Rempel and Church
(1969a) in L y t t a v ir id a n a * -
They observed that in their species "chroma­
tin usually seems to be eliminated by. only a few daughter chromosomes".
Synchrony of cleavage is lost by this time and the peripheral
nuclei are found in interphase and different stages of mitosis.
The
distribution of blastema nuclei remains uneven and eventually provides
for the presence of a differentiating blastoderm.
The varied nuclear
configurations observed during cleavage and blastoderm formation of
the cellular blastoderm may be related to the formation of the nuclearmembrane similar to that discussed by Schwalm (1969),
This author,
studied cleavage in L i m ig r a t o r ia with the electron microscope and
-65-
observed the formation of karyomeres, individual chromosomes with
double membrane envelopes, during mitosis.
In the early stages nuclei
are formed by the uncoiling and subsequent coalescence of the karyomeres
and he postulates that the kidney-shaped nuclei of Sauer (1966) could
have been formed in this manner.
In eggs of A.
eVl-lotti
it appears that
the doughnut-shaped and other irregularly shaped nuclei could have been
produced from the fusion of karyomeres.
In later stages when differ­
entiation of the blastoderm has begun, Schwalm observed that nuclei
could be formed by the clustering of chromosomes without the uncoiling
of the karyomeres.
An additional observation of Schwalm seems to have
bearing on the appearance of nuclei in eggs of A.
he reports
the presence of pre^nucleoli in nuclei long before differentiation into
embryonic and extra^embryonic areas occurs and emphasizes the corre­
lation between nucleoli and RNA synthesis.
The presence of large numbers
of nucleoli can be accounted for by the hypothesis that several pre­
nucleoli fuse in the formation of one functioning nucleolus.
appearance of nuclei in eggs of A..
The,beaded
before differentiation into
embryonic and extra-embryonic areas occurs could be explained by
Schwalm's observations.
The means by which migration of cleavage nuclei to the periphery
is accomplished is still a matter of conjecture.
In A.
eVl-iotti.
the
migrating cleavage nuclei appear to be drawn to the periplasm even if
that is experimentally displaced (Fig. 25).
Hypotheses brought forward
-66-
Figure 25.
Displaced periplasm in egg of A. e l l i o t t i used for auto­
radiography. Note nuclei of the inner blastema and
incorporation of initiated uridine.
Sevensday-old egg.
Absorption period is four days. Emulsion exposure period
is 20 days.
I B = inner blastema, Cl A = clear area, P =
periplasm,
125X.
— 67—
by insect embryologists to explain the migration of cleavage nuclei
include:
centrifugal influence propelling the nuclei toward the
periphery (Patten 1884; Eastham, 1927; Roonwal9 1936)9 the attraction
of the periplasm upon the nuclei (Sehl9 1931),. the passive drifting
of the nuclei due to local changes in and near the nuclei (Miller,
1939; Wellhouse9 1953), independent outward movement of the cleavage
cell (Rempel9 1951; Wolf, 1969) and Agrell (1964) holds that the
principal mechanism involves the shifting of the nuclei from an area
of decreasing cytoplasmic content (caused, by mitosis) into a shell
of a cytoplasm-rich region around the egg.periphery■>■ He adds that
this does not exclude auxiliary mechanism,,
Wolf (1969) postulates four mechanisms which could account for
the movement of energids:
1= the movement of the daughter nuclei
with the help of the spindle apparatus during anaphase,
movement through cytoplasmic streaming.,
2„ passive
3« active ameboid movement-
by the pigmented cytoplasm (the pigment is caused by the presence of
small protein-yolk globules which continue to be present around the
nucleus in W a o h tie tta p e w s ic a ,
4« a mechanism associated with the
many-layered complex nuclear membrane system.
He favors the last
mechanism and emphasizes that during the period when cleavage nuclei
change their function from mitotic activity to considerable migration,
the nuclear membranes take on a different appearance.
The membrane
loses the common two-layered structure with pores and is transformed
— 68—
into a many-layered membrane, containing tubular material which extends
into the ooplasm=
Both .primary and secondary■vitellophags were observed in early eggs
of A. e llt - o itt - c
formation;
Anderson (1972) reviews three modes of vitellophag
1= only primary vitellophags. appear to be formed from the
cleavage energids by mitosis9
2= all cleavage energids migrate to
the periphery and only secondary vitellophags repopulate the yolk=
3= both primary and secondary vitellophags are present, a condition
which appears to be the most common and includes eggs from A 0
eZtiottio
Krause and Sander (1962) stated that "probably those energids
which could not collect sufficient ectoplasm or did not gain, contact
with the plasmalemma turn into secondary vitellophags="
ettiotti
In A=
mitosis of the blastema nuclei at 90° angles to the periphery
appears to be responsible for the formation of secondary vitellophags=
The primary functions of the vitellophags are said to involve the
digestion of yolk materials to furnish cytoplasm, precursors and
energy for the metabolic processes, and the liquifaction of the yolk
in the area of germ band formation (Counee„ 1961)=
In a number of species, including A= e t t i o t t i ^ it is difficult
to distinguish with certainty vitellophags from other nuclei in the
egg but in other species' they present different morphological appear­
ances (Urban, 1970; Anderson, 1972; Krause and Sander, 1962; Ando,
-69-
1960; Wellhouse, 1953; Butts 1949)=
Agrell (1964) discussing his
observations on the polypoidy of yolk nuclei states that in the cricket
the yolk nuclei are at least octoploid,,
Polyploidy of yolk nuclei of
A 0 e Z Z -to tti. has not been determined.
Yolk nuclei aggregates or spheroids,, such as were observed in
eggs of A 0 Q lX -Io tt-I, are known to exist in other insect eggs (review
by Ando, I960),
possibilities.
Speculation concerning its function points to several
It could be an expression of an abnormally developing
egg or it could contribute nuclear material to assist in the formation
of the embryonic rudiment,. It should be noted at this time that in
the autoradiographic experiments on eggs of A, e X X to tt-l nuclei of the
spheroid were actively incorporating tritiated thymidine (Fig, 26),
An alternative explanation of the phenomenon involves the possibility
that the speroid is composed of yolk cells (Ando, 1960, 1962) digesting
yolk in preparation for the spatial requirements of the embryonic
rudiment or the increased mitotic rate of embryo formation,
Ando (1962) writes that in Ep-io-phXebia the cells of the spheroid
are ephemeral before blastoderm formation and only reappear at the
time of differentiation of the ventral plate,.
reviewing a paper by Seidler, mention a
Krause and Sander (1962)
spheroid but this appears
to be composed of cleavage energids .
Eggs of A, e X X io t t i belong to the "differentiating blastoderm"
type as opposed to the "uniform cellular blastoderm" described by
— 70-
Figure 26o
Internal aggregation of nuclei in a five-day-old egg of
Enlargement of Fig. 19. Concentration of
silvergrains became off center due to failure to remove
paraffin before dipping in liquid emulsion. Initiated
thymidine. Absorption period is four days. Emulsion
exposure period is 10 days.
250X.
A. e l l i o t t i *
-71-
Anderson (1972), in which a differential concentration of peripheral
nuclei exists„
Mitotic figures are noticeable in all parts of the
periphery but occur in- greater numbers at the extreme posterior end
where the embryonic area will differentiate»
It seems likely that a
posterior-anterior gradient of factors which may regulate mitosis
(DNA,. hormones, pH differences, etc,) is present in the egg and in­
fluences the differential distribution of the blastema nuclei„
The formation of cell membranes between nuclei of the extraembryonic region (Ando, 1960; Mahowald, 1963a; Rempel and Church, 1969)
was not observed in A., 'eZZfotff«
Examination with electron microscopic
techniques are necessary to determine when these membranes form=
In the
embryonic region, however, the cells had a definite cubeidal shape and
delineation between yolk and cytoplasm appeared to be present=
The increased size of the peripheral nuclei after the formation of
the serosa is. very noticeable in eggs of A= e l l i o t t i *
In other insects
this is thought to occur by endomitosis or amitosis of those nuclei
(Rempel, 1951; Roonwal, 1954; Krause and Sander, 1962; Agrell,- 1964) =
Using histochemical methods, Urban (1970) found the serosa to be one
of the most hydrolytically active tissues in eggs of A =- e Z-Hotti=
stated:
He
"The serosal membranes, as contrasted to the amnio tic and
provisional dorsal closure, appeared to contain more- hydrolytic enzyme
activity than any other tissue during pre-diapause development."
— 7 2—
Polivanova (1965) found in the three-day-old egg. of the bug,
E u ryga steT i-ntegrie eips., that the serosal membrane showed intense,
aromatic esterase activity.
The serosal membrane of A 0 e V L io tt-i seems
to be multifunctional and may begin secretory functions at the time of
blastema formation coincident, with the initiation of new RNA synthesis
and nucleoli formation.• Agrell (1964) determined that serosal cells
in the cricket were at least octoploid and polyploidy seems to be
characteristic of secretory cells in general.
The large basophilic
nuclei of the serosal cells of A. e t t t o t t i - may be indicative of their
polyploidy.
EARLY. EMBRYOLOGY:
EXPERIMENTAL
Introduction
Among the Acrididae- it is well documented that variability- in
developmental rates exists in. embryos in laboratory as well as in
field populations (Slifer, 1932; Roonwal, 1936; Van Horn, 1963, 1966a,
1966b; Visscher, 1971; Tyrer, 1970)„
This variability has often been
attributed to genetic differences between eggs, to exposure of the eggs
to different environmental conditions or to unknown factors.
In recent
years, however, it has been shown that embryonic variability in devel­
opmental rate may also be caused by environmental conditions experi­
enced by the mother (Hunter-Jones, 1958; Eyles, 1963; Edney, 1969;
Van Horn, 1966b; Visscher, 1971).
Visscher (1971) has reviewed 24 papers of studies with inverte­
brates indicating that parental factors may determine or affect
development in the offspring and observed that "maternal determination
of the rate and/or pattern of filial development may be a general
phenomena among insects."
Temperature, humidity, nutrition, photo-
period, crowding, and aging in the parental generation- are among the
factors known to influence- the development of the p r o g e n y I n A.
e V L ito tti. it has been shown that the embryonic growth rate may be largely
determined by- the mother (Visscher, 1971) and that it may be affected
by such factors as maternal density, photoperiod and aging.
-74-
Observations by Van Horn (1966b) and Visscher (1971) on the mater­
nal influence, on the developmental rate of embryos of A. e U i o t t i , were
mainly based on,eggs 30 days of age, reared under constant temperature
(25°C).
The mean stage of development,- based on the criteria developed
by Van Horn (1966a) for this period varies, from population to. popu­
lation and from year to year but appears to fall mainly between stage
9 to stage 13.
In .the Coleopterans L e y tin o tO J tSa d e e im lin e a ta and
Bermestes .macsuljxta, (Lpckshin, 1966) and in the cricket G ryV lus
dom estieus (!Hansen-Delkeskamp, 19-67) it was shown, that new synthesis
of ENA is
being initiated during blastoderm formation, indicating
that the developmental.rate thereafter -may be partly dependent on ,the
genome of the egg nucleud.
It seemed important,- therefore, that exper­
iments be performed to assess the influence of the maternal physio­
logical condition and environment on,the early developmental rate of
the offspring before gastrulation and to investigate thp temporal
pattern of ENA synthesis.
The incorporation of 3H-thymidine was also
followed to determine, the ,pattern of DNA synthesis.
Results
Maternal-Influence'
The 60 eggs from the 1971 population which were used for the .
descriptive part of this thesis were also studied relative to the effect
of maternal influence.
A few sections each of a number of.additional
: -75eggs (145 eggs) from the same soiirce were examined to:indicate the
stage, to which they had progressed.
Rate of development in eggs from females raised in "single" cages
(I pr/cage) and from "crowded" cages (6 pr/cage) appeared, to he
affected by both density and maternal age.
In eggs from .females, reared
with one male the rate of d e v e l o p m e n t w h e n classified by the standard
staging criteria, appeared to be slower during, the early part of the fecund period (Fig. 27) than during the middle part of the fecund
period (Fig. 28); it again seemed to slow down when approaching ces­
sation of egg deposition (Fig., ,29).-which usually ,occurred, shortly before
the death of the female.
In ..eggs./from, crowded females the rate of- development, appeared to
be most rapid during the early part of the fecund period (Fig. 30) but
seemed to slow down during.the,middle part of the maternal' reproductive
life .(Fig. 31). ■ Development in the last: part of the fecund period
appeared to be slowed still further (Fig. 32).
A composite of all data included in the above results is shown in ■
Fig. 33.
Solid symbols represent all data on the influence of crowding
on rate of development.
Open- symbols portray all data of embryos from
adults reared in single pairs.
Statistical analysis pertaining to all
data are presented, in Appendix C.
One five-day-old egg obtained early
in the fecund period from crowded adults was. retarded about.five stages
in its development and four eggs from the crowded group did not appear
— 76—
O
S in g le
Pairs
2
HG-
O
O
3
Egg
Stage
O
FO
^
O
2
2
O
O
0
1
O
O
2
2
O
O
O
O
7
8
O
2
D-
O
C
O
O
B
A
O
o
I
I
I
I
I
I
6
12
18
I
2
3
Mrs.
I
r
5
6
Days
Tim e
Figure 27.
I
4
of
D evelopm ent
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from young fe­
males reared one pair per cage (single, early)„ Sample size
is noted above each symbol. Total sample size = 38 eggs.
A
S ingle
Pai
age
Hrs.
Days
Tim e
Figure 28.
ol
D evelopm ent
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from middleaged females reared one pair per cage (single, middle).
Sample
size is noted above each symbol. Total sample size - 41 eggs,
-77-
□
S ingle
Pairs
I-
Egg
Stage
H-
I
G-
□
□
□
1
□2
□
12
18
ED-
□
C-
□
□
□
3
3
2
I
□I
□
□
□
3
□2
□
□
□
I
T
T
T
T
2
3
4
5
6
7
8
□
F-
□1
□
□
I
□
BA
□
T
O
6
I
Days
Hrs.
Tim e
Figure 29.
at
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from old fe­
males reared one pair per cage (single, old). Sample size
is noted above each symbol. Total sample size = 38 eggs.
Hrs.
Days
Tim e
Figure 30.
D evelopm ent
of
D evelopm ent
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from young fe­
males reared six pairs per cage (crowded, early). Sample
size is noted above each symbol. Total sample size = 32 eggs,
-78-
Crowded
I -
Pairs
A
(e p r /c a g e )
2
A
H-
A
G-
I
A
FEgg
S tage
▲
E -
▲
D-
A -
A
i
A
▲
C B -
A
3
A
o
I
T
12
6
I
I
I
I
I
I
T
I
T
18
1
2
3
4
5
6
7
8
H r$.
Days
Tim e
Figure 31.
at
D evelopm ent
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from middleaged females reared six pairs per cage (crowded, middle).
Sample size is noted above each symbol. Total sample size
= 30 eggs.
Crowded
I
Pairs
(6 PR/CAGE)
H
G
I
I
F
Egg
S tage
E
D
C
B
I
■
A
I
0
I
6
I
I
I
I
I
12
18
I
2
3
Mrs.
I
5
I
I
6
7
I-8
Days
Tim e
Figure 32.
I
4
at
D evelopm ent
Developmental stage reached by known-age eggs of A. e l l i o t t i
during early development.
Eggs were obtained from old fe­
males reared six pairs per cage (crowded, old).
Sample size
is noted above each symbol. Total sample size = 34 eggs.
Egg
Stage
M a te rn a l Fecund Period
Density:
Early
C ro w d e d
@
Sing le
Q
Mid dle
Late
Time of Development
Figure 33»
Composite graph of Figs. 27—32. Age data can be separated from density
data by combining the appropriate symbols.
— 80—
to contain any recognizable nuclei after a period of development
ranging from three to eight days.
One of these eggs was from the
early fecund period, two from the middle and one from the late fecund
period.
Three of the eggs were from cage "V" in which the female
grasshoppers had all died by August 21 and one from cage "U" in which
the last female survived until September 5.
Females in cage "V" laid a total of 61 pods, while those in cage
"U" laid a total of 55 pods.
Females reared as single, pairs laid from
0-13 pods with an average of 7.7.
9.7 pods.
Crowded females laid an average of
Conclusions regarding the maternal influence on fecundity
cannot be drawn from these data because:
early (after producing 0-1 pods),
pods were not counted and
I. three single females died
2. the eggs contained within the
3. too few females were included in the
sample.
Delayed Oviposition
Reproductive data for parental adults reared in 1970 and 1971
are presented in Tables I and II.
Females from the population in 1970
matured on an average 12 days later than those reared in 1971 (July 15,
1970 as compared to July 3, 1971).
In 1970 the first eggs were laid
on July 31, while in 1971 first eggs were deposited on. July 6.
As
described earlier in the Materials and Methods, temperature fluctuations
in the insectary were greater in 1970 than in 1971 and in 1970, 12 other
-81-
i
Table I.
Reproductive data for 1970.
These eggs were used in
developing histological techniques.
Cage
Density
Maturation Date
of. Female
Number of Pods Laid
A
I pair per cage
7-9
5 misshapen, 5 normal
B
I pair per cage
7-9
4 misshapen, I normal
C
I pair per cage
7-16
4 misshapen. I normal
D
I pair per cage
7-16 ■
I misshapen, 0 normal
E
I pair per cage
7-16
0 misshapen, 0 normal
F
I pair per cage
7.-23.
3 misshapen, I normal
G
I pair per cage
7-11
4 misshapen, 5 normal
H
I pair per cage
7-11.
2 misshapen, 0 normal
I
I pair per cage
7-16.
I misshapen, 7 normal
J
I pair per cage
7-17
3 misshapenj 3 normal
K
I pair per cage
7-18
9 misshapen, I normal
L
I pair per cage
7-19
2 misshapen, 4 normal
■M
6 pairs per cage
7.-9 to 7-19
4 misshapen, 2 normal
N
6 pairs per cage
7-9 to 7-15
9 misshapen, 3 normal
0
6 pairs, per cage
7-15 to 7-20
5 misshapen, ■5 normal
P
6 pairs per cage
7-11 to 7-15
0 misshapen, 0 normal
-82-
Table ii.
Cage
Reproductive data for 1971. These eggs were used for
the descriptive studies, maternal effect studies, and
the autoradiographic studies.
Maturation Date
of Female
Density
Number of Pods Laid
A
I pair per cage
6.-24
8
B
I pair per cage
6-24
12
C
I pair per cage
6.-29
2
D
I pair per cage
6-30
. 13
E
I pair per cage
7-1
9
F
I pair per cage
7-1 '
9
G
I pair per cage
7-1
10
H
I pair per cage
7-2
13
I
I pair per cage
7-2
11
J
I pair per cage
7-4
5
K
I pair per cage
7-5
10
L
I pair per cage
7-5
11
M
I pair per cage
7-5
8
N
I pair per cage
7-7
I
0
I pair per cage
7—10
0
P
I pair per cage
7-9
12
Q
I pair per cage
7.-9
11
R
I pair per cage
7-9
6
S
I pair per cage
7-8
I
T
I pair per cage
7-7
7
U
6 pairs per cage
6-24 to 7—6
55
.V
6 pairs per cage
6— 25 to 7^-8
61
■
•
— 83-
species of grasshoppers were being reared in the insectary concurrently
with the adult A, ■ e V H o tti.
(Appendix B) „■ In addition to the obvious
differences in reproductive performance of these two populations, duringexamination of eggs obtained from six different adult females and fixed
at 24 hours of age, .embryos were found at' stages 10- or 11 of development
(according to staging, criteria of Van. Horn, 1966a)=
It was expected
because of their ages that they would be in early stages, of development,
certainly as young as stage I.
These eggs were either among those first
laid or were laid following deposition of a misshapen egg pod.
Misshapen
egg pods often contained crumpled eggs and these were not fixed for
study.
It is not known, therefore, whether those eggs contained embryos
or not.
Autoradiography
Tritiated uridine.
The concentrations of reduced silvergrains
more numerous than those in the background of the histological sections
of eggs exposed previously to tritiated uridine were accepted as evi­
dence of RNA synthesis.
While this technique does not permit identi­
fication of different species of RNA, it does indicate the site of
RNA production at the moment of fixation.
Grain counts from randomly
distributed sample areas of five eggs gave no evidence of RNA
incorporation at stages A-E (Fig.. 34).
.Incorporation was observed
first during stage F when cleavage nuclei had entered the
—84-
Figure 34.
Longitudinal section of a five-hour-old egg of A. e l l i o t t i ,
The egg was allowed to absorb 3H-Urldine in an insect
Ringer's solution for four hours. No appreciable amount of
the isotope was incorporated into RNA. Absorption time is
four hours. Emulsion exposure time is 20 days. 260X„
—85-
periplasm and blastema formation was occurring (Fig. 35).
Subsequent
stages showed considerable incorporation in both the peripheral
nuclei and the periplasm.
The presence of reduced silvergrains, above
the background amount,was observed in the yolky portion of the egg in
stages F - I .
Nuclei which were undergoing mitosis did not show any
RNA ■incorporation.
,
Tritiated thymidine.
Concentrations of reduced silvergrains above
background count were accepted as evidence of the incorporation of
tritiated thymidine into DNA.
All stages, observed (A-I) showed
incorporation of the isotope; there seemed, however, to be a longitu­
dinal gradient present in the.egg,, decreasing from posterior to anterior
pole, with respect to the quantity of. tritiated thymidine incorporated
into the nuclei (Figs. 36, 37).
Abnormalities.
A high incidence (about 50%) of abnormally devel­
oping eggs was observed during the autoradiographic experiments.
Abnormalities were sometimes evidenced by the presence of a clear area
without yolk at the posterior part of the egg.
When these eggs were
sectioned, aberrant developmental patterns were clearly visible.
In a
large percentage of the eggs the nuclei never reached the-posterior
periphery but seemed to make an inner posterior blastema in. the yolk
(Figs. 25, 38).
When nuclei did pass into the smooth area behind the
interior blastema, they appeared to become pycnotic.
In a few
— 86—
,> ■ & /
Figure 35.
Longitudinal section of a three-day-old egg of A. e l l i o t t i .
Incorporation of 3H-Uridine is visible in nuclei and peri­
plasm. Absorption time is three days. Emulsion exposure
time is 20 days. N = nucleus, P = periplasm. 260X.
— 87—
t
Figure 36.
Longitudinal section of posterior part of a six-day-old egg
of A. eZZiotti.
Note heavy incorporation of 3H-thymidine
into nuclei.
Emulsion exposure time is 10 days. Compare
with Fig. 37. N = nucleus.
IlOX.
-88-
Figure 37o
Anterior part of the same egg as shown in Fig. 360 Note
the absence of heavy incorporationo A gradient of 3Hthymidine is present from posterior to anterior.
Emulsion
exposure time is 10 days. N = nucleus. IlOX.
-89-
Figure 38 o
Displaced periplasm in posterior part of a four-day-old
egg of A, e V l i o t t i ,
An inner blastema is being formed.
Absorption period of 3H-thymidine is four days. Emulsion
exposure time is 10 days. P = periplasm, N = nucleus,
110X.
-90-
instances nuclei did not enter the anterior periplasm and an interior
blastema was also formed in that area.
It appeared that in eggs where,
nuclei did not reach the periphery in the posterior or anterior part
of the egg, the periplasmic oval made visible by the incorporation of
isotopes was smaller than normal and incorporation was not present
beyond the boundaries of the periplasm and germ rudiments did not form,
No conclusions concerning effects of maternal influence (age,
density) on the initiation of new RNA synthesis in developing early
eggs of A. e l l i o t t i could be drawn from the data collected (Figs, 39,
40).
The large number of abnormally developing.eggs prevented the
collection of an adequate sample of each developmental age group in
all of the experimental categories (maternal age at time of laying
and maternal crowding).
It is also conceivable that those eggs which
appeared to be normal were, in fact, affected in their developmental
rate by the dessication-absorption technique.
Discussion
The relationship between variability in developmental rate during
early embryogenesis (0-12 days old) and the influence of maternal
environment in eggs of A,, e l l i o t t i ., reported on in' this study, confirms
earlier observations made from embryos of this species at 30 days of
age (Van Horn, 1966a; Visscher, 1971).
Evidence from other species also indicates that both maternal age
and maternal crowding in insects may affect the developmental rate of
-91-
M a t e r n a l F e c u n d Per od
Densi t y:
O
#
^
Single
O ^ O
•
B
48
•
24
m Hrs
•
e
12
4
OO
•
D
OO
A
B
O B
O
O B
0
OOO
OO
O
OO
u
r
e
T
A
B
Crowded
▼
•
E
x
P
OOO
O
72
• O B
Early Middle Late
•
96
B
C
D
E
F
G
H
Egg S tag e
Figure 39=
Developmental stage reached by known-age eggs of A. e l l i o t t i ,
exposed to a solution of 3H-Uridine in Ringer's solution=
Each symbol represents one sample.
96
72
•
#
^
•0 D
OOD
I
>
O ^ Q
• O B
48
O O P
u
r
e
T
O B
ood
Early Middle Late
Crowded
Single
E
x
P
°
M a t e r n a l F e c u n d Peri od
Densi t y :
e o e
24
O OO
•
<
12
O
OO
■
m Hr s
e
■ • 0
4
OO
O
A
B
C
D
E
F
C
H
l
Egq St age
Figure 40=
Developmental stage reached by known-age eggs of A= e l l i o t t i
exposed to a solution of 3H-thymidine in Ringer's solution.
Each symbol represents one sample=
-92-
the offspring.
A review by Clark and Rockstein (1964) includes work
undertaken by Richard and Kolderie who found in mass, cultures of
O noopeltus f a s o ia tu s that eggs which were laid late in the fecund period
took longer, to develop, while Gassier (1966d) observed in L. m ig r a t o r ia
that aging produces a.reduction in incubation time.
Edney (1969),
reporting on the effect of maternal aging (defined as a function of time
and temperature) in D. m e la n o g a s te r,■ reviewed some additional evidence
to support the conclusion that maternal aging affects the rate of
development of. the egg, but also mentions three reports in which such
influence could not be determined.
In A. e l l i o t t i it was shown that aging of. the female affects the
developmental rate of the embryonic, offspring at 30 days of age (Van Horn,
1963, 1966b; Visscher, 1971) and influences, the amount of.trehalose and
glycogen incorporated into the egg (Quickenden,.1969; Quickenden and
Roemhild, 1969).
Crowding also has been identified as a maternal factor affecting
developmental rate in the offspring in insects.
Visscher (1971) found
that in A. e l l t o t t i the rate of development was faster in eggs from
mothers kept at a density of. two pr/cage under short-day conditions and
slower when the density was one pr/cage, but under long-day photoperiod.
A density of two pr/cage may be closer to the normal field conditions of
A. e l l t o t t i than one pr/cage because this species is thought generally to
aggregate in subpopulations in the field (Mussgnug, 1972).
-93-
Quickenden (1969) and Quickenden and Roemhild (1969) , in addition
to investigating the influence of aging in A. e l l i o t t i ,
studied the
effects of. crowding on the amounts' of trehalose and glycogen incorporated
into young eggs (1-7 days old).
Crowding.seemed not to affect glycogen
content, but trehalose levels were higher in the first 2/3 of the fecund
period in eggs from females kept at six pr/cage than those of lower
densities.
During the last part of the fecund period, however, this
level declined rapidly and fell below the value obtained from the lower
densities.
Quickenden suggested that this effect might result from
differential Stimulation of. the maternal brain-corpora cardiaca axis
imposed by crowding.
He proposed further:
"It- is suggested that
factor(s) responsible for prediapause growth rates and perhaps differ­
entially incorporated in yolk may be under the same control as factor(s)
responsible for trehalose mobilization and incorporation in yolk;"
Crowding of A. BrV l1Io t t i . females appeared not to affect the amounts of
lipids (Svoboda, 1964; Svoboda e t a l* , 1966) nor the amount of free
amino acids in the egg (Bunde,, 1965; Bunde and Pepper, 1968).
The unusual results observed in eggs of the 1970. population in
which stage 10.and stage 11 embryos- were found in one-day-old eggs,
may possibly be accounted for by the environmental stresses under which
this population was reared.. Temperature fluctuations in the insectary
were large, and perhaps more important, the room was crowded with clear
lucite cages with screen tops containing high densities of 12 different
-94-
species of grasshoppers..
The constant visual, auditory or chemical
perception of some of the more predatory species, such as B T a ohystola
magna which are also present in the native habitat of A., e l l - i o t t - l , may
have had a disturbing effect on the oviposition. behavior of females of
A. O lt - I o t t 1I a
Excessive soil probing and the indiscriminate laying of
single eggs and misshapen clusters on the grass and the sides of the
cages were indicative of anomalous behavior.
Kuwana■a n d .Takami (1968)
reviewed studies on species with rapid embryonic development, in which
development may take place while the egg is held in the- vagina (this
should not be confused with normal viviparity).
Anderson (personal
communication) observed that females of S'. gTegav-la exhibited anomalous
ovipositing behavior in the field and appeared to lay eggs which
contained embryos in advanced stages.
A search of the literature did
not reveal any published accounts of advanced embryonic development
in- newly-laid acridid eggs.
If the rate of development- is determined in part by the mother’s
contribution■to the egg, then the yolk and the endoplasm must-be of
extreme importance.
Clarke e t a
t (1960) have shown that "the rate of
development of D ro s o p h ila eggs from laying to hatching, is in the main
determined by the female laying the eggs, and only to a small extent
by the genotype of the embryo."
There.are,: of course, basic differences
between the eggs produced, by species with meroistic ovafioles having
nurse cells (such as D ro s o p h ila ) and eggs produced by species with
-95-
panoistic ovarioles lacking nurse cells, such as A= e V t t o t t i- , but
maternal contributions from the hemolymph in addition to those from
the follicle cells may turn out to be important factors in determining
the rate of development of the egg =
The cytoplasm or yolk may exert its influence on the developmental
rate of the egg through one or more of its constituents (nucleic acids,
proteins, carbohydrates, lipids, enzymes, hormones, "growth factors",
organelles, etc =)=
Krause and Sander (1962), reviewing ooplasmic reaction
systems in insects, relate that "there is obviously some extra-nuclear
storage of material containing or constituting growth information,"
while Albrecht e t d l . t
(1959) state that "the. transmission of phase
status to the progeny (in L= m ig r a to v ta ) is held to occur through the
accumulation of extra-chromosomal material in the egg="
Wright (1970),
reviewing the genetics of embryogenesis in VvosaphiZa which possesses
a highly determinate development, writes that "a considerable number
of changes that occur during early embryogenesis are inherent in the
organization of the egg and will take place whether the egg is fertil­
ized or not and whether the cytoplasm is populated by cleavage nuclei
or not.
The synthesis of RNA in the oocytes of insects possessing the
panoistic type ovary has been reviewed by Mahowald (1973).= .Additional
studies were published by Heinonen and. Halkka (1964)., Cave and Allen
(1971), and'Zinsmeister and Davenport (1971).
It appears, from these
-96-
studies that the oocyte, nucleus actively synthesises RNA (mainly
ribosomal) during oogenesis', in contrast to eggs from meroistic ovaries
where most of the RNA content seems to be provided by the nurse cells
(Mahowald, 1973)=
Few studies are published on the analysis of RNA
synthesis during early embryogenesis in early eggs from panoistic
ovaries (Hansen-Delkeskamp, 1968, 19.69; Hansen-Delkeskamp e t a t . ,
1967).
Generally, however, they seem to agree with findings obtained
from eggs possessing meroistic type ovaries (Lockshin,. 1966; Harris and
Forrest, 1967; Lane Smith and Forrest, 1971;, Counce, 1973).
Lockshin reports that in the coleopterans L e p tin o ta r s a d e e im tin e a ta .
and T e n e b rio m o lit o r new RNA synthesis seems to be initiated during
blastoderm formation.
event.'
Previously stored RNA was utilized before that
Hansen-Delkeskamp came, to similar conclusions.. The autoradio­
graphic techniques used to visualize isotope incorporation in the
present study were similar to those used by Lockshin..
Studies by Harris and Forrest (1967) on the milkweed bug QneoperLtus
f a s e ia tu s indicated that during gastruiation a burst of. new RNA
synthesis occurred.
With their biochemical techniques it was possible
to identify this RNA as ribosomal RNA.
They noted that the possibility
exists that during blastoderm formation some type of, low molecular RNA
(not r-RNA) may be synthesized.
It seems conceivable that this is
the type of RNA which appears in eggs of A. e l l i o t t i during blastoderm
formation and which Lockshin observed in his studies.
-97-
It is interesting that some aspects of the abnormal development
obtained in eggs of. A, e t l i - o t t i exposed to radioactive isotopes in
solution are similar to those obtained by Ktithe (1966). with eggs of
the beetle, Bevmestes f v is h e h i, irradiated with ultraviolet light„
Kuthe observed that the cleavage energids did not form a normal
blastoderm at the. periphery, but instead, formed an "inner blastoderm"
away from the periphery of. the egg.
The inner blastoderm differ­
entiated into the embryonic and extra-embryonic areas, but subsequent
embryonic- stages showed that the primitive embryonic rudiment developed,
abnormally.
He concluded that the periplasm was not necessary, for
blastoderm formation, but its destruction apparently adversely affected
development of- the embryo at a later time.
Isotope incorporation into
blastema nuclei in the egg of. A. e l l i o t t i *shows that the synthesis of
RNA and DNA occurred following exposure of. the egg to the isotopes in
solution.
The abnormal position of the blastema, medial to the peri­
phery in both the controls and experimentally treated eggs.,, in addition
to the subsequent failure of the eggs to develop germ rudiments, would
seem to emphasize rather than negate the importance of factors in the
peripheral cytoplasm to the regulation of early embryonic development
I n - A* ■e t t i - o t t i .
It was attempted in the present- study to find positive or negative
indications of maternal effects upon the initiation of RNA synthesis
in the egg.
Tsien and Watteaux (1971) investigated RNA and DNA contents
—9 8-
in unfertilized eggs of L, m e la n o g a ste r i n .relation to maternal age at
the time of oviposition and reported that eggs from young and old fe­
males have a higher DNA and lower RNA content than eggs from middleaged 'females (the periods corrected for differences between virgin and
mated females)=
Because the techniques used with eggs of A, e l t t o t t t
initiated teratological development-, different methods must be used
before sufficient data concerning the role of maternal influence on
RNA synthesis can be. obtained..
The demonstration that embryonic RNA synthesis in A. e t t i o t t i . is
initiated during blastema formation, coincident with the first
appearance of the nucleolus, appears to be the first such report on
a member of the Acrididae.
SUMMARY
A descriptive histological study was undertaken of the early
embryology of the grasshopper A u to o a ra e tt- L o tti, from a population
obtained near Billings, Montana in 1970 and 1971.
A staging criteria
for early embryonic development was established for this species.
Determinations of the effects of maternal age and density on the
developmental rate of the young embryo were made using this staging
criteria.
Additionally, autoradiographic studies were conducted to
determine the patterns of. RNA and DNA synthesis.
new RNA was established.
The initiation of
The major findings and conclusions are listed
below:
1.
Under low magnifications the chorion of the egg o f ' A r ^ e l V l o t t i
appeared to be smooth with the exception of the sculptured
chorionic cap.
2.
The chorion of the egg is composed of three layers:
the
exochorion, the endochorion and a thin,, dense unidentified
layer.
3.
About 55 micropylar openings are present around the base of
the chorionic cap,.
4 o- Nine separate stages were delineated (stages A - D
covering
the period of oviposition through stage I (Van Horn, 1966a).
5o
A thin, vitelline membrane was observed around, the periphery
of the egg internal to the chorion.
—
100
—
6.
A thin periplasm was present in the majority of eggs examined»■
7.
Blue-staining basophilic droplets were observed in the peri­
plasm of the young egg.
8.
The female pronucleus was observed about 20% of the egg length,
from the posterior part of the egg. and midway, between the
median line and the periphery.
Twelve distinct chromosomes
were present.
9.
Polar bodies were observed at the periphery of the egg about
20% egg length.
10.
Chromatin seemed to be eliminated during the second cleavage
division.
11.
The cleavage nuclei entered the posterior periplasm first
and only later were observed entering the anterior periplasm.
12»
The density of the blastema nuclei showed a posterior-anterior
gradient,
13.
A variety of nuclear configurations were noted during the
blastema period.
14.
Both primary and secondary vitellophags were observed.
15.
Yolk aggregations (spheroids) were noted in three eggs of
stage G.
16»
One nucleolus was present in nuclei of: presumptive^ serosal
cells.
Nucleoli were not identified in.other cells during the
time period studied (1-12 days of. development).;.
-IQl17o
Cell membranes seemed to be formed in the embryonic area during
differentiation of. the blastema but did not seem to be present
in the presumptive serosa prior to differentiation of the embryonic
rudiment,
18.
Maternal age and rearing density appeared to affect the
developmental rate of the egg during the early embryo-logical .
period studied.
19.
Eggs from middle-aged females reared at a density of one pair
per cage developed slightly faster than eggs from the early
or late maternal fecund period..
20.
Eggs from, young females reared at a density of six pairs, per
cage developed faster than eggs from middle-aged females and
much faster than eggs from old females.
21.
In 1970 stages 10 and 11 embryos-were observed in 16 oneday-old eggs obtained from females reared under stressful
environmental conditions.
This appears to be the first evi­
dence of advanced embryonic development in newly-laid eggs of .
the Acrididae.
Speculations are discussed concerning the
possible cause of delayed oviposition..
22.
Tritiated uridine was first incorporated into RNA during blastema
formation.
-102-
23.
Tritiated thymidine was incorporated into the. DNA of"the
nuclei during the entire time period studied (6 days)„
A
posterior-anterior gradient of incorporated tritiated
thymidine was observed.
24.
A large number of eggs developed abnormally after being
exposed to the radioactive isotopes in Ringer's solution.
25... No conclusions are made, of the effect of maternal age and
rearing density on the pattern of RNA and DNA synthesis.
APPENDIX A
Title:
Sample of sheet used to record histological sections.
M IVs/esssl
e n u rv>JL>e.r
APPENDIX B
Title:
Species and numbers- of grasshoppers reared in 1970 in the
same insectary room with A. e V L io t t i*
Species
Numberof Cages
Grasshoppers
per Cage
Aevo^edeVlus c ta v a tu s
2
A g e n e o te ttZ x deovum
6
Am phitovnus aolovadus.
I
5 pairs
Boopedon sp.
I
3 males .
B v a e h y s to la magna ■
I
23 males & females
Dvepanoptevna femovatum
3
26 males & females
H a d v o te ttix t v if a s e ia t u s
2
28 males & females
M e la n o p lu s b i v i t t a t u s
5 pairs
■
5 pairs
10
50 pairs
M e lan oplus p a e h a v d ii
2
Unknown
M e ta to v p a v d a lin u s
2
X a n th ip p u s c o v a llip e s
I
17 males & females
2 pairs
APPENDIX C
Fecund Period
Regression Coefficient
B(x, y)
Density
early
single (Figo 27)
.2950
middle
single .(Fig. 28)
.3116
late
single (Fig. 29)
.2878
early
crowded (Fig. 30)
.2742
middle
crowded (Fig. 31)
.2756
late
crowded (Fig. 32)
.2812
All regression slopes were compared and found not to be significant.
Analysis of Variance
Fecund Period
Density
Versus
Fecund Period
Signifi­
cance
1.6
early
crowded
1.3
late
single
.6
single
middle
crowded
.8
middle
single
late
crowded
2.0
early
single
early
crowded
1.7
early
single
late
single
.3
early
■single
middle
crowded
.9
early
single
late
crowded
1.5
early
crowded
-
late
single
6.63
*
early
crowded
-
middle
crowded
3.9
*
early
crowded
—
late
crowded
5.67
late
single
late
crowded
.05
middle
crowded
late
crowded
.2
single
middle
single
middle
single
middle
-
-
-
.
.
F
Value
single
middle
early
Density
**
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Wessel, Margaretha H
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