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 ELLIQTTI (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? Si 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 Drs. 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 Drs. 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
V IT^L O O O- O O 6 O 6 O O O O' Q O O 9 0 O . 9 0 0 ° 0 ° 0 0 ii
ACKNOWLEDGMENT...... » . O 0 0 • « » • <* ° e 0 iii
LIST OF TABLES ...... • ° O <> O «■ 0 0 O .° ° ° vi
LIST OF FIGURE S o o o o o o o o o o • 6 © • < » 0 0 ° 0 6 6 0 0 vii
ABSTRACT O O O O O O O' O ^ O O O O O v 0 O' . (> O-' 0 0 6 0 0 0 ° °' X
INTRODUCTION , . 6 » . ■« . . „ » O . 6 6 0 0 6 0 ° 0 I
MATERIALS AND METHODS » . • 6 ■O <) o' O . 6 6 6 0 0 6 ° 0 7
Biological Material ...... > O • 0. 7
Descriptive and Maternal Effects O O t» * '© O O O .0 O O 0’ O 8
...... 0. Autoradiography • ° • 0 ° .° 0 ° 9
. ... EARLY EMBRYOLOGY: DESCRIPTIVE • 6 O <1 6 O ' 0 0 ° O „ 0 ° O- 12
Events of Oogenesis and Egg Deposition 6 0. 0. 12 Definition of Terms ...... o 0 © < e . . 0 O- ' 0 O O 6 6 15
ReSUltS 0 . e' o . o . o o o'. o 0 O O C> O O O . 0 O O 0 O O 6 17
External Appearance of the Egg .O- <) 6 e 6 O O e O O O 0 . 17 Staging Criteria ...... O O © (I o- O O O O O O - O O O 18
The Newly-Laid Egg , . . O O 1 O O O O O O O O O O 22
Membranes . . . o' 0* O O O O o - O O O ■ O 22 Nucleus . o . . = 'o . O O- e. 6 0 -0 O- O O O 0 24
Y O Ih O O O O O .' 6 O © < 0' "o' O 0 O 0 O 0- O 28
Temporal Pattern of Development I ■ 0' O O O O 0 0' O O O 31 Discussion . . o o o . o o o . 0' « 0 ° > 0 6 0 54
EARLY EMBRYOLOGY: EXPERIMENTAL . . ° • 0 0. o- »■ 0 o- 0 ° 73
Introduction ...... o . 0 . . 73 Results 6 0 o’ O O O O . O 0' O 6' O 0 0 « O e O ... O- O O O 74
Maternal Influence . . . . 0 •O O , O- 0 0- 0 . O O ‘ 0 O 74
Delayed Oviposition . , . O O O I O O ' O - O o- O O O O 83
Autoradiography ...... O O O o- O O1 0 O O O O 83
Tritiated uridine . . *. O ,» . O 0 6‘ 0 0’ O- O O- 0 83
Tritiated thymidine . O O 5 . O " O ■ O O O Oj O ■ O 0* O 85 V
TABLE OF CONTENTS (Continued)
Page
Abnormalities . . . .• i . . .• » ...... 85
Discussion e e 6 e o e ' o ' o ■ ,e- o- o e . a - o e e o' o C o o o ■ o o 90
SUMMARY . o . o '.' . •„0 OA 1 A A O''O a O 4 O 6 6 ft O o . o . o 99
APPENDIX A . o -o o o ft A ft ft' O 6 A" .ft ft. ft A ' ft ' 6" 6 ■ 6 o o 103
APPENDIX Bo-. o o . . o ft ft ft ft 104
APPENDIX C- o . ... o o .ft ft ft 6 ft A A" ft A ft ft ft ft ft - ft. A 6 ' 105
LITERATURE CITED . . .-v . ft ft ft ft ft ft ft ft ft .ft" ft ft ft: A 0 a a 106 Vi
LIST OF TABLES
Table Page
I. Reproductive data for 1970 81
II. Reproductive data for 1971...... 82 vii
LIST OF FIGURES'
Figure Page
1. . Egg pod and newly-laid egg of A'= e V b io ttt. 14.
2. Diagram of stages A to I of the early embryology of A # * & IslsQ I/ (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
3. A longitudinal section of the chorion of a newly-laid egg of A. s Is TsTsOisisis ...... - . ., ...... ' . 23
4. A longitudinal section of the, chorionic cap of a newly- laid egg of A. StZiott-L -' 25
5. A longitudinal section of the periphery of a newly- laid egg of A'-eZZ-Cotti 26
6. Basophilic droplets and second cleavage division in a longitudinal secion of a- six-hour egg of A. e Z Z io tti . . . .27
7. The female pronucleus in a longitudinal section of a newly-laid egg. of A.-. e t Z io t t i '.' .... v 29
8. Polar body in a newly-laid egg of A.. e tZ - io tti 30
9. Chromatin elimination during second cleavage division in a longitudinal section of an egg of A. eZZiott-L ...... 32
10. Interphase 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-O1 O O O- 1 O' 34
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 o 36
13. Periplasm of the anterior end of the1 egg,of A. ■ e Z Z io tti . . 37
14. Blastema nucleus at the periphery of a three-day-old
egg of Ao- S Z Z^lsO tt^ls. 0 O . . . O' O O' O' O' O' 6 - 0 O 0" 0 O O O 0 O 38
15. Beaded blastema nucleus at periphery of a four-day-
old egg of A . SZZlsOttls -O O .' 0.000000 O' .0 0- 0' O O 39 viii
LIST OF FIGURES (Continued)
Figure Page
16. Doughnut^shaped nuclei in posterior part of a three- day-old egg of A. e V t i o t i z i...... 40
17. Mitosis at a 90° angle to the periphery of a three- day-old egg of A. e Z X to tti which produces1 a daughter blastema nucleus and a secondary vitellophag ...... o . 42
18, Mitosis tangentially to the periphery of a one-day- old egg of A, e V lie t t i which produces two blastema nuclei . ...o...,..,..,...,,... . o 43
19. Internal nuclear aggregate in a five-day-old egg
of A o & Is 'Is'lsO Iy lSl* 0 0 0 0 . Q' 0 , 0 , a...... 45
20. Peripheral nucleus with distinct nucleolus in a four-day-old egg of A.- e l t i o t t i - . . . . 46
21. 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
22. 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
23o Germ band and serosal cells in the posterior part of a ten-day-old egg of A. e lt - io t t ’i ...... 52
24o Embryo, amnion and serosal cells in a twelve-day-old egg of A o ■ a %. "IsisO is 1*%* ...o.....,*...... 53
25o Displaced periplasm in egg of A, e tV L o tv i- used for autoradiography . .• o ...... c . . . o■ . . . 66
Internal aggregation of. nuclei in a five-day-old egg of A. B V L-Iotti .- . 70
27o Developmental stage reached by known-age eggs of A. e 'V b io tti during early development . o . 76
28. Developmental stage reached, by known-age eggs of A. e lt-io tt-L during early development ...... 76 ix
LIST OE FIGURES (Continued)
Figure Page
29. Developmental stage reached by- known-age eggs of A. e V lto t-b i during early development...... 77 ■
30. Developmental stage reached, by.known-age eggs* of A. e'il'lott-i during early development . . .- 77
31. Developmental stage reached by known-age eggs of A. e’lVio't'bi’ during early- development- . . . . ., . .- 78
32. Developmental■ stage reached by known-age eggs of A. BVl1Lotiyl during early development ...... • o o' .- . 78
33. Composite graph of. Figs.. 27-32 ...... , . .. . 79
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 .. . 84
35^, Longitudinal section of a three-day-old egg of A. BVliofcti-- 6 o' . O 86
36. Longitudinal section of posterior part of a six-day- old egg Of A o- ’ BI VlyOfct1L - . . o o o .-. o o- - o.o-o e O O - o o . . 87
37= Anterior part of the same egg as shown in Fig. 36 . . . . . 88
38. Displaced periplasm in posterior part of a four-day— old egg of A . BI I 1Lo IV l > o...... = .-. = . . .- . 89
39. Developmental stage reached by known-age eggs of. A. BVl1LotfcL exposed to a solution of 3H-Uridine in- insect Ringer, s . . . . . o . .- . . .-. . . . .-. = . = . .. 91
40. Developmental stage reached by known-age eggs of Ac - O ll1Lo tt1L exposed to a solution of 3H-thymidine in insect Ringer’s ...... O O 91 X
ABSTRACT
The early embryology of Aulocava eV lio tti 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,
Loousta Ynigvatovia (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 Melanoplus diffeventialis-
(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 Aulooava ell-totti' (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. Aulocapa Q ill-IottiV- 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.' elltott-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 llto tt-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 ll- io tti- 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 developmentwhile • 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 tlio tti. 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 lio 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= eZZiotti 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= eZZ-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.
4 o Comparison of developmental rates from young, middle-
aged 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 sm ithi-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 overnighti
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.- ellio.tt-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,
respectivelyBoth 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 as- the Orthoptera. The panoistic type ovary,' . unlike the meroistic type, does not have specialized "nurse oells" and, thereforethe 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 post- pachytene 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. At this time the 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 llitO tti, 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 extra-
embryonic 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 - a differential concentration of cells in a local Blastoderm 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: Furchungszentrum (Krause. 1938a)=
Cleavage Energld - cleavage nucleus and its associated island of
cytoplasm (Krause, 1953).
Embryonic - part of blastoderm, destined to become embryo after Germ Anlage differentiation of blastoderm into embryonic and
extraembryonic areas.
Alternative Term: Embryonic Primordium
Endoplasmic - a network of internal cytoplasm in which deuto Reticulum 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 - the length of each egg is translated into 100 Length (n% E= L=) 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) - vltellophags whicb arise directly by the further. Vitellophags division of cleavage energids which remain within,
the yolk mass as their, fellow energids migrate to
the surface (Anderson, 1972)
Secondary (2°) - vitellophags originating from mitosis of the peri- ■ Vitellophags 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 Aa e tlio tti. 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= eVIZot-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 tti 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. eV L'totti. 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. A0 e U - io tt 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; Two to four cleavage nuclei are
present in the yolk. Eggs of A. e H io 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: The germ band becomes multilayered
through repeated mitoses. 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= Its matrix appears homogeneous and stains a
pale pink with eosin=' 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= The■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„ elliotti. 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 newly- laid egg of A. e llio t t io 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 . P B = polar body, 650X. -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 nucleus- was 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 t t i , 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, eVLietti. 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,; Several aspects of this differentiation were apparent. 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, e l l i o t t i * This egg was used for autoradiographic experi ments and shows incorporation of tritiated thymidine into the nuclei. Longitudinal section. IOOX. —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. -48-
Cells 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-cell- membrane (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. They had dense
nucleoli which stained intensely. 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. In the center part of the germ band, a cluster of cells was
I -52-
Figure 23. Germ band and serosal cells in the posterior part of a ten- day-old egg of Ao elliottio 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
_54-
observed 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 llio tiy t was concluded with the examination of the germ band in 12- day-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 Lytta viridana (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). It has been.found to consist of at least two layers: 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). Bunde (1965) and Robinson (1970) found that in eggs of
A. eVliotti. 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 igvdtovia (Hartley, 1961; Roonwal, 1954a) and of
Melanoplus dlffenentialis (Slifer, 1949) an additional outer layer, the extrachorion was present when the eggs were examined after ovi- position. 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 endo- chorion 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., - eVUiottn, 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 to tti 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-iffevent'iat-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 to t t i- appear to be similar to those Slifer found in diffeventialis (1949b)„ She observed that "the whole exochorion in this region resembled a sieve." She also cites work by Jannone on
Bociostauvus 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 Hyalophora cecropia 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 tti 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 CO-
12 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 Lytta vtvidana 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/Lffeventiatis^ 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. It is sometimes called the cortical layer or "Keimhautblastema". 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. metanogastev 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 lio 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 li 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 it '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^ melanegaster 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„ diffeTent-iat-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 tl- Io tti, 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 melarwgaster % 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
was■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 lZi-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 Sdkistooevoa 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 Lytta viridana*- 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 nuclear- membrane similar to that discussed by Schwalm (1969), This author, studied cleavage in Li migratoria 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. The,beaded appearance of nuclei in eggs of A.. 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 llio tti 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, 2„ passive movement through cytoplasmic streaming., 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 Waohtietta pewsica, 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. ellt-oitt-c Anderson (1972) reviews three modes of vitellophag formation; 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 A0 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=" In A= ettiotti 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 eZZ-totti. has not been determined.
Yolk nuclei aggregates or spheroids,, such as were observed in eggs of A 0 QlX-Iott-I, are known to exist in other insect eggs (review by Ando, I960), Speculation concerning its function points to several possibilities. 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, eX X tott-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,. Krause and Sander (1962) reviewing a paper by Seidler, mention a spheroid but this appears to be composed of cleavage energids .
Eggs of A, e X X io tti 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 A. e llio tti* 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. -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 extra- embryonic 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 = - eZ-Hotti= He stated: "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,
EurygasteT i-ntegrieeips., 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 progenyIn A. eV Litotti. 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 io 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 LeytinotOJtSa deeimlineata and
Bermestes .macsuljxta, (Lpckshin, 1966) and in the cricket GryVlus domestieus (!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 : -75- eggs (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 developmentwhen 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 Single Pairs O O 2 H- 0 O 1 G - O O O 3 2 2 F - O O O O O Egg 2 2 Stage ^ O O O 2 D- O O C O B
A O I I I I I I I I r o 6 12 18 I 2 3 4 5 6 7 8 Mrs. Days
Time of Development
Figure 27. 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 Single Pai
age
Hrs. Days
Time ol Development
Figure 28. Developmental stage reached by known-age eggs of A. e l l i o t t i during early development. Eggs were obtained from middle- aged females reared one pair per cage (single, middle). Sample size is noted above each symbol. Total sample size - 41 eggs, -77-
□ Single Pairs I - □ □ I H- □ □ □ I 1 G- □ □ □ 3 3 2 I F - □ □ □ □ □ □ Egg 3 2 I E - □ □ □ □ Stage 1 D- □ □ 2 C - □ □ B - A □ T I T T T T O 6 12 18 I 2 3 4 5 6 7 8 Hrs. Days Time at Development Figure 29. 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 of D evelopm ent Figure 30. 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 Pairs I - A (e p r /c a g e ) 2 H - A A
G - I F - A A Egg E - S ta g e ▲ D - ▲ A i C - ▲ A
B - 3 A - A I T I I I I I I T I T o 6 12 18 1 2 3 4 5 6 7 8 H r$. Days Time at Development Figure 31. Developmental stage reached by known-age eggs of A. e llio tti during early development. Eggs were obtained from middle- aged females reared six pairs per cage (crowded, middle). Sample size is noted above each symbol. Total sample size = 30 eggs.
Crowded Pairs I (6 PR/CAGE) H G I I F Egg E S ta g e
D
C
B I A ■ I I I I I I I I I I I I-- 0 6 12 18 I 2 3 4 5 6 7 8 Mrs. Days
Tim e at D evelopm ent Figure 32. Developmental stage reached by known-age eggs of A. e llio tti 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
Maternal Fecund Period Early Middle Late Density: Crowded @ Single Q
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. Crowded females laid an average of
9.7 pods. Conclusions regarding the maternal influence on fecundity cannot be drawn from these data because: I. three single females died early (after producing 0-1 pods), 2. the eggs contained within the pods were not counted and 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-
Table I. Reproductive data for 1970. These eggs were used in developing histological techniques.
Maturation Date Cage Density 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
i -82-
Table ii. Reproductive data for 1971. These eggs were used for the descriptive studies, maternal effect studies, and the autoradiographic studies.
Maturation Date Cage Density of Female 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, during- examination 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 llio tti, 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 3H- thymidine 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 lio 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 llio tti 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-
Maternal Fecund Per od • O B 96 Density: Early Middle Late OOO A • • 72 Crowded # ^ B
E Single O ^ O O OO x ▼ • P B O 48 O u O O r e • 0 B T 24 O OO m Hrs e • O B 12 O OO
OB • 4 OO D
A BCDEFGH Egg Stage 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.
Maternal Fecund Peri od • O B 96 Density: Early Middle Late ood
Crowded # ^ I • 0 D 72 > E Single O ^ Q OOD x P • O B ° 48 u OOP r e e o e T 24 O OO
m Hrs • ■ e <
12 O OO
■ • 0 4 O OO
A BCDEFCHl Egq Stage
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
Onoopeltus fasoiatus that eggs which were laid late in the fecund period took longer, to develop, while Gassier (1966d) observed in L. migratoria 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 elan ogaster,■ 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. elliotti, 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. BrVl1Io tti. 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 BTaohystola magna which are also present in the native habitat of A., ell-iott-l, may have had a disturbing effect on the oviposition. behavior of females of
A. O lt-Iott1Ia 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■ and.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 Drosophila) and eggs produced by species with -95-
panoistic ovarioles lacking nurse cells, such as A= e V tto tti- , 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= migratovta) 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 Leptinotarsa deeimtineata. and Tenebrio m olitor new RNA synthesis seems to be initiated during blastoderm formation. Previously stored RNA was utilized before that event.' 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 fa 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 llio tti 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, etli-otti exposed to radioactive isotopes in solution are similar to those obtained by Ktithe (1966). with eggs of the beetle, Bevmestes fvishehi, 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
In - 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, melanogaster in.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 middle-
aged '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 Autooara ett-Lotti, 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. The initiation of new RNA was established. The major findings and conclusions are listed below:
1. Under low magnifications the chorion of the egg of'Ar^elVlotti
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,.
4o- Nine separate stages were delineated (stages A - D covering
the period of oviposition through stage I (Van Horn, 1966a).
5 o 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).;. -IQl-
17o 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 one-
day-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 tti*
Number- Grasshoppers Species of Cages per Cage
Aevo^edeVlus ctavatus 2 5 pairs
A geneotettZ x deovum 6 ■ 5 pairs
Amphitovnus aolovadus. I 5 pairs
Boopedon sp. I 3 males .
B vaehystola magna ■ I 23 males & females
Dvepanoptevna femovatum 3 26 males & females
Hadvotettix tvifaseiatus 2 28 males & females
Melanoplus bivittatus 10 50 pairs
Melanoplus paehavdii 2 Unknown
Metatov pavdalinus 2 17 males & females
Xanthippus covallipes I 2 pairs APPENDIX C
Regression Coefficient Fecund Period Density B(x, y)
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 F Signifi Fecund Period Density Versus Fecund Period Density Value cance
middle single - . early . single 1.6 middle single early crowded 1.3 middle single - late single .6 middle 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 LITERATURE CITED
Agrell, Io 1964o Physiological and biochemical changes during insect developmento In Physiology of Insecta, Vol.-I-, pp„ 91-148. M„ Rockstein.* ed. Academic- Press, New York.
Albrecht, F-. 0., M. Verdier and R. E. Blackith.. 1959, Maternal control of ovariole number in the progeny of the migratory locust. Nature, 184:103-104.-
Anderson, D. S. 1964. Serial sectioning of refractory locust eggs. Quart. J„ Micr.. Sci., 105(3):379-380.
Anderson, D. T. 1972. The development of hemimetabolous insects, In Developmental Systems:Insects, Vol„ I, pp. 95-163. S. J, Counce and C. H. Waddington, eds. Academic Press, New York,
Anderson, N. L. 1961. Seasonal losses in rangeland vegetation due to grasshoppers. J.. Econ. Ent., 54:369-378.
______. 1962. Grasshopper-vegetation relationships on Montana grasslands. Ph.D. Thesis, Montana State College.
______. 1964. Some relationships between grasshoppers and vegetation. Ann. Ent. Soc. Amer.., 57:736-742.
______. 1972. Acridids as pests:, the evaluation of range losses. In Proe. of the Int. Study-Conf. on the Current and Future Problems of Aeridology, London,. July, 1970. C. F. Hemming and T-. H. C. Taylor, eds. Centre for Overseas Pest Research, London.
______and J. C. Wright. 1952. Grasshopper- investigations on Montana rangelands. Mont. Agr. Exp., Sta, Bull. No.: 486.
Ando, H. I960. Studies on the early embryonic development of a scorpion fly, Panorpa pryeri Maclachlan (Mecoptera, Panoipidae) Sei. Rep. Tokyo Kyoiku Daig., 9:227-242.
______. 1962. The comparative embryology of Odonata with, special reference to a relic dragonfly EpiophZebia superstes Selys. Japan Soc. Prom. Sci., Tokyo. -107-
Beament j J. W. L, 1946.. The formation and structure, of the chorion of the egg in an Hemipteran Rhodnius prolixus. Quart. J. Micr. Sci., 87:393-439.
______. 1948. Penetration of insect egg shells and penetration of. the chorion, of Rhodnius pvoliscus Stahl. Bull. Ent. Res.-, 39: 359-383.
Beams, H. W. and R. G. Kessel. 1969. Synthesis and. deposition of oocyte envelopes (vitelline membrane, chorion) and the uptake of yolk in the dragonfly (Odonata: Aeschnidae) J.» Cell Science, 4:241-264.
Bier, K. 1963. Beziehungen zwischen NahrzellkerngrSsse und ausbildung ribonukleinsaurehaltiger Strukturen in den Oozyten von Form ica rufa rufor-pratensis■ minor Gossw.- Verb. Dtsch. Zool. Ges»■ in Freiburg,. 1952, pp. 369-374.
Bunde, D. E. 1965. Free amino acid content and biosynthesis in eggs of the grasshopper Aulocara e llio tti Thomas during development. Ph.D. Thesis, Montana State University. 65 pp.
.______.,and J. H. Pepper. 1968. Biosynthesis and occurrence of free amino acids in eggs of the grasshopper Aulocara e llio tti. J, Insect Physiol., 14:1635-1649.
Butt, F. H. 1949, Embryology of the milkweed bug,.Oneopeltus ■ faseiatus. Cornell Univ. Agr.. Exp. Sta.-, Memoir 283. ■
Gassier, P. 1966. Variability des effets de groupe (effats Immddiats et trasmis). sur Loeusta m igratoria m igratorioides. (R. et F.) Bull. Biol. Fr. Belg., 100:519-552.
Cave, M,- D. and E. R». Allen. 1971. Synthesis of ribonucleic acid in oocytes of the house cricket (Aeheta domestio u s ), Z. Zellforsch, 120:309-320.-
Chapman, R. F. 1969„ The Insects: Structure and Function.. American Elsevier Publ.- Corp, Inc., New York. 819 pp. * pp.. 335-355.
______. and F. Whitham.- 1968. The external morphogenesis of grasshopper embryos. Proc6- R. Ent.-- Soc. Lond. (A) 43: (10-12): 161-169. —108—
Clark, A.- and M. Rockstein, 1964. Aging, in insects. In The Physiology of Insecta Vol. I, pp. 227-281. Academic Press, New York.
Clarke, J. M., J„ M. Smith and P. Travers. I960. Maternal effects on the rate of egg development in BvosoTphita obsouva. Genet, Res. Camb., 1:375-380.
Gounce, S. J. 1961, The analysis- of insect e m b r y o g e n e s i s Ann. Rev. Entomol., 6:295-312.
______. 1973. The causal analysis of insect embryogenesis. In Developmental Systems: Insects, Vol. II, pp. 1—156. Counce and C, H. Waddington, eds. Academic Press, New York.
Davies, L. 1948. Laboratory studies on the egg of the blow fly* Luoilia sevicata* J. Exp. Biol., 25:71-85.
Eastham, L., E. S. 1927. ■ A contribution to the embryology of P ie v is vapae* Quart. J. Micr. Sci.,- 71:353-394.
Edney, E. B. 1969.. The effect of parental temperature upon filial egg period in Bvosophita melanogastev.. Physiol. Zool,, 42(3): 257-265.
Eyles, A, C. 1963. Incubation period and nymphal development in Nysius huttoni White (Heterdptera:Lygaeidae:Orsillinae) .. New Zealand J. Sci.-, 6 (3) :446-461.
Favard-Serdno D. 1966. Rdle de I'appareil de Golgi dans la secrdtion du chorion de I’oeuf chez Ie Grillon (Insects, Ort,hopt&re). In Electron Microscopy, Vol. II, pp. 553-554. R. Uyeda, ed. Maruzen, Tokyo,
Gerrity, R. G., J. G. Rempel, P. R. Sweeny and N. S. Church. 1967. The embryology of. Lytta vividana-Le Conte (Coleoptera:Melo-idae). IT. The structure of. the vitelline membrane. Can. J. Zool., 45:497-50-3.
Griffiths, I. and M. E. Carter. 1958, Sectioning refractory animal tissues. Stain Techn., 33:209-214.
Hagan, H-.- R. 1951. Embryology, of the Viviparous Insects. The Ronald Press Co., New York. 472 pp. Hansen-Delkeskamp,. E.' 1968» Synthese rlbospmaler RNS in normalen und embry.olosen Keimen von Aoheta domest-Lca -L„.. Wilhelm Roux Arch. EntwMech-o Org., 161:23-29.
1969 . Synthese'. von KNS und Protein wahrend der Oogenese und frtihen Embryogenese- von Aaheta domest-vea*. Wilhelm Roux Arch.■ EntwMech. Org..,. 162:114-120.
1 •' ,. H. W. Sauer a.nd F. Duspiva, 1967, Ribonuclein- shuren in der Embrypgenese von Aoheta' domestioa L. Z. Naturforschg 22b:540-545.. . ' /,.
. Harris, S. E.. and H. S. Forrest.-' 1967. RNA and DNA■ synthesis in developing ,eggs of the milkweed bug, Onoo^erLtus fa s o ia tu s (Dallas) Science, 156:1613-1615..'. - '
Hartley, J. C. 19.6.1,■ The shell of acridid eggs. Quart. J. Micr. Sci., 102(2):249-255« -
Hastings,- E. 1971. Laboratory studies of compatibility of males' and females of , two populations of. AuZooava eZlAotti (Orthoptera: Acrididae).- Ann.- Entomol.- Soc. Am.-, 64(1).: 155-157,
. ", ; -. and J, H. Pepper.: 1964. Population studies on the big- ' headed ■ grasshopper, AuLooava etLiottt*', • Ann. Entbmolv Soc. Am., 57(2)r216-220.
Heinoneni, L. and 0. Halkka. 1964. Early stages of oogenesis and metabolid DNA in the oocytes, of the house cricket,- Aoheta dom estious (LO . Ann,- Med. exp. Fenn. , 45:101-109.
Hemming, C .' F. and T , H C .• Taylor, eds. • 19.72. Proceedings of the International Study Conference on the Current and Future Problems of Acridology. Centre for Overseas Pest Research, London.
Hinton, HoE.. 1962. Respiratory-systems of insect egg shells. Science Progress, 50(197):96-12.3.
.______1. . .\ 1969. Respiratory systems.of insect shells. , Ann. Rev. Ent,-, 14:343-368. '
' '■ - .. : - - . . ' . ' ■' - ■ •.. 1970. Insect egg shells.. Scientific American, 223:84-91, . ... : - 110-
Hopkinsj C. R 0 and Po E. King. 1966. An electron-microscopical and histochemical study of the oocyte periphery in Bombus te r r e s tv is during vitellogenesis. J, Cell. Sci., 1:201-216.
Horvath, B. Z. 1967. Studies- on the development of muscle in embryos of Autooava e ltio tti Thomas (OrthopterarAcrididae) using the fluorescent antibody technique.- Ph.D. Thesis, Montana State University. 69 pp,
Hunter-Jonesj P.- 1958. Laboratory studies on the inheritance of phase characters in locusts. Anti-Locust Bull. No. 29. 32 ppv
Jahn, T. L. 1935. Nature and permeability of. grasshopper egg membranes. II. Chemical composition of membranes. Proc. Soc. exp. Biol., N. Y. 33:159-163.
Johannsen,-. 0. AV and F. H. Butt. 1941., Embryology of Insects and Myriapods. McGraw-Hill, New York'.- 462 pp.
King, R. C. 1970. Ovarian Development in Bvosovhila metanoaastev. Academic Press,. New York,
_.______= and E. A. Koch. 1963, Studies on the ovarian follicle cells o f .BvosophyLla. Quart, J^ Micr. Sci., 104:297-320.
King, R. L. and E. Slifer, 1934'. Insect development: VIII, Maturation and early development of unfertilized grasshopper eggs. J. Morph., 56 (3) :,603-619,
Krause,- G. 1938. Einzelbeobachtungen und typische Gesamtbilder der. ntwicklung von Blastoderm und Keimanlage im Ei der GewMchshaus- heuschrecke Taohyoines asy'namovus Adelung. Z. Morph, Oek.- Tiere, 34:1-77.
______. 1953. Die Aktionsfolge zur Gestaltung des Keimstreifs von Taohyoines (Saltatoria), insbesondere das morphogenetische Konstruktionsbild bei Duplicitas parallels. Wilhelm Roux Arch. EntwMech. Org.-, 146:275-370.
______. 1957. Neue Beitrage zur Entwicklungsphysiologie des Insektenkeimes. Verhandlungen der. Deutschen Zoologischen Gesellschaft in Graz. Dunrihaupt, Kothen. -Ill—
Krause, G. 1961. Preformed ooplasmic reaction systems in insect eggs. Symposium on Germ Cells and Development:302-337o Institute Intern, d ’Embryologie and Fondazione A. Baselli (1960).
______. and K. Sander. 1962. Ooplasmic reaction systems in insect embryogenesis. In Advances, in Morphogenesis, Vol. II. M. Abercrombie and J. Bracket, eds., pp. 259-303. Academic Press, New York.
Kuthe, H= W. 1966. Das Differenzierungszentrum als selbstregulierendes Faktorensystem fur den aufbau der Keimanlage im Ei von Bevmestes f v is o h i (Coleoptera). Wilhelm Roux Arch, EntwHecti.- Org., 157: 212-302.
Kuwana, J. and T. Takami. 1968. Insecfa-. In Invertebrate Embryology, M. Kumd and K. Dan, eds,, pp.: 405-484. Clearinghouse for. Federal and Technical Information, Springfield,. V a .
Laine, I. I. 1966. Temperature effects on the oxygen consumption during, development of Auloaara e llio tti (Thomas). Ph.D. Thesis, Montana State University, 74 pp.
Leopold, R. A. 1967. A study of the ppsfembryonic ovarian develop ment and vitellogenesis - of Aulooava e llio tti- (Thomas) (Orthoptera: Acrididae). Ph.D.. Thesis, Montana State University, 96 pp.
L'Helias, C. 1970= Chemical aspects of growth and development in insects = - In Chemical Zoology, Vol. V., pp. 343-400. M.- Florkin and B. T. Scheer, eds. Academic Press, New York.
Lockshin, R. A= 1966. Insect embryogenesis: Macromolecular synthesis during early development. Science 154:775-776=
Machida, J. 1941. On. the formation of yolk and the egg membrane in the domesticated s ilk w o rm -Bombyx m o ri-L * J , Coll. Agric.. Imp, Univ.- Tokyo. 15:83-131.
Mahowald, A. P. 1963. Ultrastructural differentiations during formation of the blastoderm in the Brosephila melanogastev embryo, Dev. Biol.,- 8:186-204.
■ 1973. Oogenesis. In Developmental Systems:- Insects, Vol. I, pp, 1-47= S.. J. Counce and C, H. Waddington, eds. Academic Press, New York. -112-
Makino, Sajiro, 1951= An Atlas of Chromosome Numbers in Animals0 The Iowa State College'Press, Ames, Iowa0 p„ 290«
Matthee, J 0 J 0 1951, The structure- and physiology of the. egg of Looustana ipandattna (Walk). Science Bull, ,316, Agricultural Research Institute, Pretoria,. South Africa,
Miller,■ A, 1939, The egg and. early development of the. stonefly 'Ptevonaroys pro tens,-- J 0 Morph0 , 64:555-609,
Moloo, S, K 0 1971, The degree of determination of, the early embryo of Sohistooevoa gvegavia'(FoCskaL) (OrthopterasAcrididae), J 0 Embryoli exp. Morph,, 25(3):277-299,
Mussgnug, G, L 0 1972, The structure and performance of -an adult population, of Autooava e ttio tti (Thomas) (Orthoptera, Acrididae) near Billings, Montana, M.S, Thesis, Montana-,State University, 97 pp,
Okada, E. and C, H 0- Waddington,. 1959, The submicroscopic structure of the D vosophila egg, J, Embryol0 exp. Morph,, 7:583-597,
Patten, W 0 1884, The 1 development of phryganids- with a preliminary note of Blattagevmaniod^ Quart, J. Micr0 Sci0., 24:549-602,
Polivanova, E 0 N,- 1965, The functional significance of the embryonic, membranes of insects, Doklady Akademii Nauk SSSR, 160(I):243- 245 o- (translated),
Prescott , D, M 0 1964, Autoradiography with liquid emulsion, ■ In Methods in Cell Physiology, VoI, I, pp, 365-370, Dv M, Prescott, ed, Academic Press, New York,
Quickenden9 K, ■ L.„ 1969, Studies of carbohydrates in eggs of 'Aulooava e l l i o t t i (Thomas) (Orthoptera, Acrididae)- in relation to develop ment, temperature, maternal age and crowding, Ph0D, Thesis, Montana State University82 p p ,
______, 1970, Carbohydrates in the eggs of the grasshopper Aulooava. e llio tti, during development, Jv Insect Physiol, 16:171-183,
. and G, R,. Roemhild, 1969, Maternal age and density effects on carbohydrates partitioned to eggs of the grasshopper, Aulooava e llio tti. J, Insect Physiol,, 15:1215^1223, -113-
Raven , C. P., 1961. Oogenesis: The storage of developmental information. Pergammon' Press Ltd., London. 274. pp.
Rempel, J. G v 1951. A study of the embryology of MamestTa e o n fig u v a ta (Walker) (Lepidoptera, Phalaenidae). Can. Entomol., 83:1-19.
______. and N. S. Church. 1965. The embryology of L y ita v iv id a n a Le Conte (Coleoptera:. Meloidae). I, Maturation fertilization and cleavage. Can. J. Zool., 43:915-925.
______. 1969a. The embryology of L y tta v iv id a n a Le Conte (Coleoptera: Meloidae).. IV. Chromatin elimi nation. Can. J. Zool., 47:351-353.
______1969b. ' The embryology of L y tta w ivid a n a Le ..Conte (Coleoptera : Meloidae) . V-.. The blastoderm germ layers, and body segments. Can. J. Zool., 47:1157-1171.
Robinson, R. W. 1970. The distribution, rate'of synthesis and characterization of protein from the eggs-of Ayloaara: eVLiotti (Thomas) (Orthoptera,- Acrididae), during-development. Ph.D. Thesis, Montana State University. 86 pp.
Roemhild, G.- R. 1961. Studies on the eggs of the grasshopper species Autoeccra- e l t i o t t i Thos.. Ph.D.. Thesis * Montana.-State-University, 94 pp.
______. 1965a. Cyclical temperature effects on diapause • termination of eggs of AurIoeava e t t i o t t i (Thomas). J. Insect Physiol. * 11:1633-1639
______. ■ 1965b» Respiration of the eggs and parts of eggs of Autoeceva e t t i o t t i (Thomas). Physiplv Zool., 40 :182-188.
____ .______. 1967. Compartmentation in the eggs of- Autoeava- e t t i o t t i (Thomas). Physiol. Zool., 40:182^188.
■ -______. 1968. Cationic effects and levels in developing eggs of the grasshopper Autoeccva e ld io tti, ' J. Insect Physiol.-, 14: 1035-1043..
Roonwal, M. L. 1935. Fate of the embryonic- membranes in insects. Current- Sci., 4:317-318. -114-
Roonwalj-Mo L-.. 1936. Studies on the embryology of the African migratory- locust, Loousta migratovia migratorioides (R&F) (Orthbptera, Acrididae). I. The early development, with a new theory of multi-phased gastrulation among insects. Phil. Trans, Roy. Soc;.- (London), B,. 127:175-244. .
■ ______■ 1937. Studies on the embryology of the African migratory locust, Loousta Wigvatoria migvatorioides (R&F) (Orthoptefa, Acrididae). IIk Organogeny. Phil.. Trans. Roy. Soc. (London), B, 127:175-244.
______.- 1954a. The egg-wall of the migratory Iocnsti Loousta migvatovia migvatovioides (R&F) (Orthoptera, Acrididae),. Proc. Nat. Inst. Sci. India, 20(4):361-370.
______. 1954b. Size, sculpturing, weight and moisture content of the developing eggs of the. desert locust 'Sohistooevoa gvegavia (Forskal) (Orthoptera, Acrididae). Proc.- Nat. Inst. Sci. India, 20:388-398.
______. 1961. Bibliographia Acrididiorum.- From: Rec. Indian Mus., -56 (1-4) 1958:1-611. •
______. and R. K. Bhanotar. 1968. Third supplement to Roohwal8s Bibliographia Acrididiorum. Rec. Zool. Survey India, 61(1&2):191- 2851
Salt, R. W. 19.52. Some aspects of moisture absorption and loss in eggs of MeZanoplus bivittatus (Say). Can. J»-Zool., 30:55-82..
Sauer, H, W.- 1966. Zeitraffer-Mikro-Film-Analyse embryonaler differ- enzierungsphasen von GvyZZus domestious* Z. Morph. Oekol. Tiere, 56:143-251.
Schwalm, F. E-. 1969.» Changes in the formation- of -nuclear membranes and ultrastructure of chromosomes during the . early development of . locust eggs (Loousta migvatovia)* Wilhelm Roux Arch. EntwMech. Org., 162:41-55.
.Sehl, A. 1931. Furchung und Bildung der Keimanlage bei der Mehlmotte Ephestia huhnieZZas nebst eine-r allgemeinen ubersicht uber den - Verlauf der Embryonalenentwicklungi Z. Morph. Oekol. Tiere, 20:535-598. -115-
Sllfer, E 0- H„’ 1932. Insect development. IV.. External- morphology of grasshopper embryos of known age.and with a known temperature history, j. Morph., 53:1-21.
, _____ . 1948. .- Isolation 'of-: a wax-like, material from the shell of . the grasshopper egg. Discuss. Faraday. Soc., 3:182-187.
______. 1949a. Changes in certain of the grasshopper egg coverings during development as indicated by fast green and other dyes. -J.. Exp. Zool., 110:183-203.
. 1949b. Variations,.. during development, in the resistance of the grasshopper egg to a toxic substance.; Ann. Entom. Soc. Amer.-, 42:134-140.
■ 1958. Diapause in the eggs of Metanoplus differentiaHs (Orthoptera,-: Acrididae). J. Exp. Zool., 138:259-282.
• _____ and R. L. King. 1933. Grasshopper eggs and the paraffin method.. Science, 78:366.
______- ■ _____ 1934. Insect development, VII, Early stages in development of grasshopper eggs of known age and with a known temperature history. " J. Morph., 56:593-601.
______. and:S.,S. Sekhori.. 1963. The fine structure of the membranes which cover the egg of the grasshopper Metanoiptus diffeventiatis, with special reference, to the hydropyle. Quart. J. Micr. Sci., 104(3)^321-334.
Smith, D. S., W=. H. Telfer and A-. C. Neveille.. 1971. Fine, structure of the chorion, of. a moth, Eyatophora oeorop-iac Tissue & Cell, 3(3):477~498.
Smith, R. ■ L. 1971. Synthesis of 5S and transfer RNA during embryo- genesis-in the milkweed bug, Qncopetinis faso-iatus; DeV. Biol., 25:502-513.
Svoboda, J. A. 1964; Studies on some of- the' lipids.of the egg of the grasshopper species Autooana eVt-iotti (Thomas). Ph.D. Thesis, Montana State College, Bozeman.. 71 pp.
______., J. H. Pepper and G. L. Baker. 1966, On the lipids of the egg of the- grasshopper,. Autooara etUotti* : J. Insect Physiol.-, 12:1540-1545. —116—
Sweeney, P, R., N= S= Church, J= G=. Rempel and R= G= Gerrity= 1968 = The embryology of Lytta V1Lvtdana. Le Conte (Coleoptera: Meloidae) = III= The structure of the chorion and the micropyles= Can= J=. Zool., 46:213-217.
Tsien, H= C= and J= M=. Wattiaux= 1971= Effect of maternal age on DNA and RNA content of. DvosopkLZa eggs= Nature (New Biology), 230: 147-148=
Tuck, J= B= and R= C= Smith. 1939= Identification of the eggs of mid-western grasshoppers. Kans= Agr. Exp= Sta= Tech. Bull= 48=
Tyrer, N= M= 1970= Quantitative estimation of the stage of embryonic development in the locust,. SokLstooevoa gvegavia, J= Embryol = exp. Morph=,-23(3):705-718.
Urban, R= E= 1970. Histochemical and electrophoretic analysis of hydrolytic enzymes during the embryogenesis of AuZooava eZZLotti (Thomas) (Orthoptera, Acrididae)= Ph=D= Thesis, Montana State University= 131 pp=
Uvarov, B= PV 1966= Grasshoppers and Locusts. Vol = . I= University Press, Cambridge. 465 pp. .
Van Horn, S=-N= 1963= The embryogenesis and embryonic variability of AuZooava eZZLotti Thomas (Acrididae,. Orthoptera). Ph=D = Thesis, Montana State College, Bozeman=. 172 pp.
______= 1966a. Studies on the embryogenesis of AuZooava eZZLotti (Thomas) (Orthoptera,. Acrididae), I. External morpho genesis. J= Morphol=, 120:83-114'.
. 1966b. Studies on the embryogenesis of AuZoeava eZZLotti (Thomas) (Orthoptera,-Acrididae) =: II= Developmental variability and the effects of maternal age and environment =: J. Morphol=.., 120:115-134 =
______o ■ 1968= Changes in gland volume during the post diapause development in embryos of AuZooava eZZLotti (Thomas) (Orthoptera, Acrididae)= Proc=- Montana Acad, Sci=, 28:72-75=
Visscher (Van Horn), S= N= 1971= Studies on the embryogenesis of. AuZocava eZZLotti (Orthoptera:- Acrididae), III= Influence of maternal environment and aging on development of the progeny. Ann= Ent= Soc= Am., 64(5.) : 1057-10.74 = -117-
Vlsscher, S. N= 1972» Analysis- of embryonic diapause in Auloeava (Thomas) (Qrthopteras Acrididae). s Histological, quantitative, and experimental= 14th International Congress of Entomology, Canberra= p» 138i
Wellhouse, W= T= 1953. The embryology of Thevmobia domestiea Packard, Ph=D= Thesis, Iowa State University. 110 pp.
JJigglesworth, V= B= 1965= The Principles of Insect Physiology= Methuen & Co= Ltd= Distributed in U= S= by Barnes & Noble. 741 pp.
Wolf, R= 19.69= Kinematik und Feinstruktur plasmatischer Faktorenber- eiche des Eies von W aehtliella 1Qevsieaviae L= (Diptera) I= Das Verhalten ooplasmatischer Teilsysteme im normalen Ei= Wilhelm Roux Arch = EntwMech, Qrg.,, 162:121rl60»
Wright, T=. R= F= 1970= The genetics of embryogenesis in Dvosophila^ Adv= Genet=, 2:261-395=
Zinsmeister, P= P= and R= Davenport = 1971. RNA and protein synthesis in the cockroach ovary. J. Insect Physiol., .17:29.-34 = D378 W516 Wessel, Margaretha H cop. 2 The early embryolog of Aulocara elliotti
AND ADPWKW
:: ; Lu
j ’~ * I' -^xj- / / /-- V ci'yyjru^/r