A Contribution to the Embryology of the May Beetle
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PURDUE UNIVERSITY
THIS IS TO CERTIFY THAT THE TH E SIS PR EPA R ED U N D E R MY SU PER V ISIO N
Philip Luginbill, Jr.
BY
ENTITLED
A Contribution to the Embryology of the May Beetle
COMPLIES WITH THE UNIVERSITY REGULATIO NS O N GRADUATION T H E SE S
A ND IS APPROVED BY ME A S FU LFILLIN G THIS PART O F THE REQUIREM ENTS
FO R THE D EG R EE O F
Doctor of Philosophy
P rofessor
H
ead of
ijt
C h a r &e
Sceool
or
of
T h e s is
D epartm ent
19
TO THE LIBRARIAN THIS TH E SIS IS NOT TO B E REGARDED A S CONFIDENTIAL.
F S O F K S S O R U S O B lu tO B
GRAX>. SCSOOIi F O R K 9—3 -4 0 —131
A CONTRIBUTION TO THE EMBRYOLOGY OF THE MAY BEETLE
A Thesis Submitted to the Faculty of Purdue University by Philip Luginbill, Jr. In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
August, 1949
ProQuest Number: 27712220
All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.
uest ProQuest 27712220 Published by ProQuest LLO (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346
ACKNOWlEDGimNT
The author wishes to express his deepest appreciation to: Professor J. J* Davis, Head of the Department of Entomology, and Professor C. M. James of the Zoology Department for their inspiration and guidance; my father. Dr. Philip Luginbill, Sr., for his aid in technique and in collecting the May beetles; and my wife, Betty, for her encouragement and assistance throughout this study.
ABSTRACT The May beetles, Phvllophaga fervida (Fab. 178l) and Phyllophaga hirticula (Knoch I801), were used for this study. early in the spring, and P. hirticula
P. fervida appears
several weeks later.
By using
these two species. May beetles were available for egg production until the first part of July. In order to secure eggs for sectioning, May beetles were caged over a layer of sifted soil. o'clock.
This soil was resifted every day at five
The eggs collected were placed and incubated in two-ounce
ointment boxes containing soil. All eggs were fixed with hot Benin's fluid.
The chorion of each
egg was pricked with a minuten nadelin pin to increase permeability. Bouin's fluid was removed by washing the eggs with acid alcohol. tertiary butyl alcohol series was used to dehydrate all eggs.
The
Seven
grams of stock rubber solution, 5 grains of bayberry wax, and 4-5 grams of hard paraffin were mixed together and melted to form an embedding mass. A lard can containing a 50 watt bulb for a heating element served as an embedding oven.
To embed the eggs, they were placed in small
vials and covered with a layer of embedding mass. placed in the oven.
The vials were then
A vacuum was used to aid penetration in some of
the eggs of earlier stages.
All eggs were stored in the vials in which
they were embedded, still submerged in their embedding mass. Eggs less than two days old were embedded and sectioned individually. All other stages were embedded and sectioned four at a time.
No attempt
was made to orient eggs into the desired plane before sectioning.
Eggs
were ribboned into ten micron sections and were stained primarily with Delafield's haematoxylin with eosin as a counter stain. Before taking microphotographs, the light passing through the camera and microscope was centered on the ground glass by removing the eyepiece of the microscope. source.
A bright spotlight was used as a light
Sections to be photographed were circled with a red wax pencil.
Ortho-contrast film aided in securing detailed structure of the embryo sections. The egg of Phvllophaga is oval in shape and averages 2.4 mm. in length.
It becomes spheroidal in shape about the second day.
Spermatozoa
pass through the chorion by means of micropyles.A very prominent periplasmic membrane is formed in the May beetle egg just below the vitelline membrane.
The periplasmic membrane is continuous with a reticulum of
protoplasmic strands which ramify throughout the inner portion of the Qgg* Maturation of the female pronucleus occurs in or eiround the anterior periplasm.
The spermatozoa enters the egg at oviposition, and fertiliza
tion occurs almost in the center of the anterior portion of the egg. Meroblastic cleavage division begins immediately after the formation of the zygote.
Cleavage nuclei first reach the periphery of the egg in
the equatorial region.
The periplasm rearranges itself and forms pockets
to receive the cleavage nuclei. nuclei of the May beetle egg.
About 21 chromosomes are present in the After cell walls are laid down around the
cleavage nuclei, the egg enters the blastoderm stage. Some cleavage nuclei remain behind among the yolk granules.
These
nuclei are called primary vitellophags, and are later augmented by
secondary vitellophags which enter the yolk from the germ hand and blastoderm. Â polar cap is formed at the anterior end of the egg.
A crowded
condition soon develops in the entire blastoderm due to rapid cell division.
The cells divide more rapidly on the dorsal side and migrate
anteriorly pushing the polar cap posteriorly along the midventral line. Migration cannot provide all the space needed for the dividing cells, and as a result, groups of nuclei are pinched or crowded out into the yolk granules.
These
groups later return to the midventralline where
they aid in germ band formation. Indentations at the anterior and posterior ends of the germ band begin the formation of the embryonic membranes.
The anterior margin
of the anterior indentation and the posterior margin of the posterior indentation grow out to form double folds. each other, meet, and
fuse.
These folds extend toward
The inner layers of the two
amniotic layer, and the outer layers the serosal
folds form the
layer. As the embryo
grows older these two membranes fuse together forming one membrane. This amnio-serosal membrane later migrates into the yolk granules to form the dorsal organ. Pleuropodia attain their maximum development in the May beetle larva at the end of the eighth day.
They then decrease in size until the larva
is mature, at which time the pleuropodia appear to shrivel up and fall off, Entoderm formation begins with two indentations of the germ band as the embryo reaches its maximum length in blastokinesis.
One of these
indentations occurs at the anterior end just below the protocephalon, and the other at the posterior end forward of the amniotic germ band union.
The cells at the bottom of these indentations migrate to the inner side of the germ band, and the germ band closes behind them.
By active
proliferation of the cells, cell masses called anterior and posterior mesenteron rudiments are formed which send out ribbons of cells to produce the mid-gut. The lower layer of cells remaining after mesodermal formation is considered to be the ectoderm.
The central nervous system is one of the
most important ectodemal derivatives.
It is formed from the neural
ridges and middle cord, and has twenty pairs of ganglia in the young embryo.
The first pair of ganglia forms the protocerebrum, the second
pair the deutocerebrum.
These are the only two pairs of ganglia which
are anterior in position to the stomodaeal invagination.
The trito-
cerebrum is formed from the lateral connectives, and migrates forward to aid in the formation of the brain.
The ganglia of the mandibular,
maxillary, and labial segments unite to form the subesophageal ganglion. Three pairs of ganglia are found in the thorax. are distinct in the fully grown larva-
These do not fuse and
Eleven pairs of abdominal
ganglia are formed in the early embryo, but only ten remain distinct in the mature larva, as the last two abdominal ganglia fuse. The tracheal system is formed by invaginations of the ectoderm. There are nine pairs of spiracles in the mature larva. Germ cells first appear at the posterior pole about the time of germ band formation.
A few germ cells are visible above the mesoderm
at the posterior end of the germ band during blastokinesis. appear as gonads suspended from the splanchnic layer.
They later
TABLE OF CONTENTS Page INTRODUCTION..................................................
1
REVIEW OF LITERATURE...........................................
2
T E C H N I Q U E ....................................................
11
Egg P r o d u c t i o n .............................
11
Preparation of Eggs for S t u d y ...............................
12
Microtechnique.........................
14
Microphotography
.
..................................
RESULTS AND DISCUSSION ......................................... The E g g
.
Maturation and Fertilization
l6 l6 16
...............................
Cleavage and BlastodermFormation .
........................
l8 19
Germ Band Formation.....................
20
Embryonic Membranes andBlastokinesis
22
The Mesoderm
......................
. . . . . . . . . . . . . . . . . .
25
The E n t o d e r m ........... ...................................
27
The Ectoderm
28
......................................
Germ C e l l s .................
. . . . . . .
31
S U M M A R Y ......................................................
32
LITERATURE C I T E D ..............................................
35
APPENDIX......................................................
42
FIGURES Figure
Page
1*
Phvllophaga fervida Fah..............................
2.
Phvllophaga hirticula Knoch
•••••
...................
42 42
3a. Eggs of the May beetle, Phvllophaga fervida. The elongated egg was freshly laid; the others were two weeks old
....
43
3b. Eggs of the May beetle, Phvllophaga fervida. in clumps of earth in which they were l a i d ...........................
43
4.
Light t r a p ..............................................
44
5.
Gage for May beetles used for obtaining eggs . . . . . . . .
45
6.
Embedding oven and test tube of vials containing eggs in embedding m a s s ...............
7*
45
Test tube rack of vials containing eggs in embedding mass, and m i c r o t o m e .........
46
8.
Warming plate for slides
9#
Longitudinal section of egg just after oviposition showing
..........................
the cytoplasmic cap in the polar r e g i o n ............. 10.
........ . . . . . . . . .
48
Longitudinal section through polar region of egg showing tail of sperm lodged in micropyle
13.
47
Longitudinal section through the polar region showing rods of protoplasm just under a micropyle...............
12.
47
Longitudinal section through posterior half of egg showing saucer-shaped mass of cytoplasm
11.
46
...............
49
Sagittal section through polar region of egg showing head of sperm resting among the protoplasmic network * . • • . . . •
$0
Figure 14.
Page Sagittal section through posterior end of egg showing sperm laying directly under saucer-shaped micropyle and vitelline spheres
15*
. . . . . .
.....................................
51
Transverse section showing the two layers of the chorion, and the nucleus arriving at the periphery of the egg before ••••
52
16.
Enlargement of the same section as in Figure 1 5 .........
53
17*
Transverse section showing first polar body dividing and
undergoing maturation division . . .
.............
pushing its second polar body out of the yolk into a cyto plasmic pocket during maturation.
The lighter area nearest
the center of the egg is the protoplasm surrounding the pronucleus which is dividing to give off its second polar body as it migrates toward the center of the egg
. . . . . . .
54
18.
Sagittal section of the second polar b o d y ...............
54
19.
Enlargement of same polar body as shown in Fig. I8 . • . . •
54
20.
Sagittal section showing the fusion of male and female nuclei
55
21.
Sagittal section showing the periplasmic layer beneath the vitelline membrane. At one end the periplasm is breaking up to form pockets to receive the cleavage nuclei...........
22.
Sagittal section showing periplasmic layer forming pockets to receive the cleavage nuclei . . . . . .
23*
55
...........
5b
The beginning of cleavage division following the formation of the z y g o t e ..........................................
5b
Figure 24.
Page 57
Stages of mitosis;............................... a.
Resting stage.
b.
Prophase.
c.
Telophase.
See figure l6.
See figure 27-
d . Anaphase. e. 25#
Metaphase.
Sagittal section to show cleavage nuclei approaching the periphery of the e g g .....................................
26,
Sagittal section to show cleavage nuclei after arrival at the periphery
27#
58
...........
Sagittal section through posterior end of the egg where the polar cap will later be formed. shown in the prophase stage.
On the left a nucleus is
The nucleus in the center of the
picture is in the telophase s t a g e ....................... 28.
58
59
Longitudinal section of posterior end of the egg during blastoderm formation showing large cells which might be germ 59
cells 29.
Sagittal section showing some stages of mitotic division in the periphery of the e g g ............... ..
30.
.
.
.
60
Sagittal section showing cleavage nuclei migrating to the periphery of the egg.
A later stage thanFigure 25
....
60
31 . Longitudinal section through the polar cap forming at the anterior end of egg
...........................
bl
32. Longitudinal section through the polar cap of the egg showing elongated masses of protoplasm among
thec e l l s ............
6l
Figure 33*
Page Sagittal section of the blastoderm showing the formation of "nuclei groups"
34.
.................................
Another sagittal section of the blastoderm showing the formation of a "nuclei group".
35*
62
.......................
Sagittal section through the polar cap.
.
62
Large cells are
believed to be yolk synitia approaching the cap
63
36.
The same section as figure 35
63
37#
Longitudinal section through the polar cap after "nuclei groups" have been formed.
. . . . . . . . . .
A typical "nuclei group" is shown
in the upper left hand corner of the section . . . . . . . .
64
38. Longitudinal section through the polar cap and dorsal side of the egg to show crowded condition of the nuclei.
A group of
nuclei can be seen approaching the polar cap, and the dorsal blastodermal cells appear to be crowding the top of the polar cap................................. ... 39.
. . . .
.
Longitudinal section of the blastoderm showing the anterior polar cap, and cells on the ventral side elongating.
Nuclei
of the blastoderm appear to be folding under the polar cap . 40.
65
Enlargement of cells on the ventral side of the blastoderm which are beginning to e l o n g a t e .......... ..............
41.
64
66
Transverse section at anterior end of the egg showing groups of nuclei approaching the periphery of the egg. blastoderm are sending out cytoplasmicstreamers. appears on the ventral side of the egg
...
Nuclei of Polar cap 86
Figure 42.
Page
Transverse section near anterior end of the egg showing "nuclei groups" breaking up and passing into the periphery of the egg
67
.........................
43. Sagittal section showing germ cells at the posterior pole during germ band formation . . . . . . . . . . . . 44.
67
Longitudinal section showing the completed germ band with the polar cap now on the side in the region where the head will form.
The indentation above the polar cap is the beginning
of the formation of the amnion
.........
68
45. Transverse section at anterior end of the germ band showing the horseshoe-shaped mass of cells along the midventral line
68
46. Sagittal section through germ band showing amniotic fold starting its migration toward the posterior e n d ..........
69
47. Transverse section near the middle of the embryo showing the neural ridges and the amniotic fold beginning to form 40.
...
69
Longitudinal section through the germ band showing the anterior amniotic fold approaching the posterior fold which has just formed.
The cells on the inner side of the germ
band are believed to be the cells which will form the mesoderm 70 49. Transverse section near the middle of the egg showing the lower layer and the inner layer. has divided into two lamellae.
The inner layer or mesoderm Coelomic sacs are beginning
to form at the lateral edges of the mesoderm
............
71
Figure
50 .
Page Transverse section near the middle of the egg showing coelomic sacs connected by a mesodermal bridge.
The neural
groove lies between the two neural ridges which are composed of neuroblastic cells . . . . . . 51.
.................. •
Developing embryo laying on top of the yolk cells showing sunken medial nerve cord at the bottom of the neural groove
52.
72
Transverse section of the anterior end of the embryo showing neuroblasts separating from the ectodermal layer.
The medial
cord and coelomic sacs are also visible . . . . . . . . . . 53*
72
73
Transverse section at the posterior end showing coelomic sacs below and layer formation in the mesoderm a b o v e .........
73
54. Sagittal section of the embryo showing coelomic sacs, and the indentations at the anterior and posterior ends which furnish cells for the formation of the anterior and posterior mesenteron rudiments 55*
. . . . . .
...................
74
Sagittal section of the posterior end of the embryo at the same stage as figure 54 to show cells with large nuclei and clear cytoplasm resting on the mesodermal layer. believed to be germ cells
56.
These are
.........
75
Sagittal section of the embryo showing formation of the mesenteron ribbons from the anterior and posterior mesenteron rudiments............
57•
76
Sagittal section of the posterior end showing the posterior mesenteron rudiment attached to the proctodaeal invagination and sending out a mesenteron r i b b o n...................
.
77
Figure 58.
Pag® Sagittal section of the anterior end of the embryo showing
the anterior mesenteron rudiment attached to the stomodaeal invagination and sending out a ribbon to form the walls of .....................
the mid-intestine 59*
77
Transverse section through the left side of the embryo. The splanchnopleure is located next to the yolk. cells are shown under the splanchnopleure. visible.
Blood
Cardioblasts are
A large cell which is believed to be an oenocyte
is shown in the developing body cavity
78
. . . . . . .
60.
Same section as 58 , but showing the ventral nerve cord, also
61.
Longitudinal section of the posterior end showing the proctodaeum and the amniotic-serosal membranes starting to fuse .
62.
78
79
Transverse section of dorsal side showing amniotic-sarosal membrane migrating to the dorsal side where it will sink 79
into the yolk and be absorbed » . ....................... 63.
Sagittal section showing the protocerebrum, deutocerebrum, subesophageal ganglion, and first abdominal ganglion
64.
...
80
Sagittal section through anterior end showing the brain and the subesophageal ganglion.
Connectives from which trito-
cerebrum is believed to develop is shown at the base of developing brain 65.
. . . . . . . . .
.............
.
80
Invagination showing commissure running under the stomodaeum connecting the ganglia of the tritocerebrum.............
8l
Figure 66.
Page Horizontal section showing paired tritocerebrum and also a pair of nerve cords leading forward to the location of
the frontal ganglia 67*
...................
8l
Horizontal section through stomodaeal invagination showing deutocerebrum on each side.
Nerve cords originating at
tritocerebrum are also shown on the outside of the deuto cerebrum 68.
82
...............
Horizontal section showing protocerebrum on each side of the stomodaeum
82
.................
.
69.
Section showing neuropile of the connectives and commissures
70.
Sagittal section of the pleuropodia on the first segment of the abdomen . . . . . . .
71.
........
...................
84
Transverse section of the dorsal side of developing embryo showing beginning of the heart f o r m a t i o n ...............
74.
84
Transverse section showing absorption of the amnioticserosal m e m b r a n e .....................
73*
83
Transverse section showing gonad attached to the splanchnic layer in the segment
72.
. . . . . . . . .
83
85
Transverse section through the anterior end showing paired indentations of the lateral sides. the beginning of tracheal formation.
These indentations are The dark mass in the
yolk on the dorsal side is the disintegrating amnioticserosal membrane.
The ventral nerve cord appears on the
ventral side, and part of the stomodaeal invagination is located above the nerve cord
...................
85
Figure
75.
Page Transverse section showing the heart forming on the dorsal s i d e ...........
76.
..
....
86
Sagittal section of the head showing connectives leaving subesophageal ganglion. also shown
78.
86
Longitudinal section of the heart at the anterior end of the embryo
77*
.
The brain, mouth, and esophagus are
.................
.....
87
.....
Transverse section of sixteen day old larva to show gonad located on the left side of the section just outside the mid-intestine
79.
. . . . . . . . .
.
87
Sagittal section of a portion of the thorax and abdomen in a fully developed embryo showing nuscles and spiracle on prothorax
80.
...........
88
Sagittal section of mature larva showing heart, mid-gut, ventral nerve cord, part of brain and frontal ganglion, mouth, esophagus, hindintestine, and a n u s ..........
89
A CONTRIBUTION TO THE EMBRYOLOGY OF THE MAY BEETLE
INTRODUCTION This study of the embryology of the May beetle was undertaken for purely scientific reasons.
Until the present time no papers have been
published on embryonic development of the May beetle, Phvllophaga sp., nor any of the other Scarabaeidae.
No doubt, the great difficulty
encountered in securing eggs in sufficient quantity and in making sections did much to discourage such a study.
It was hoped that by
working with this group of insects, some new facts concerning the formation of the invertebrate embryo would be discovered. The May beetles Phvllophaga fervida (Fab., 1781) and hirticula (Enoch, 1801) were chosen for this study because they are two of the most common May beetles in Indiana.
Phvllophaga fervida is an oblong,
shiny, smooth, dark brown beetle about 22 millimeters long (Figure l), and it appears in early spring.
Phvllophaga hirticula (Figure 2) is an
oblong, reddish-brown to dark chestnut brown, shiny beetle, I6 to 19 millimeters in length, possessing several longitudinal lines of erect hairs on each elytron.
This beetle becomes active several weeks after
P. fervida. By using these two species, beetles were available for egg production until the first part of July.
The embryological development
of both species is essentially the same, as corresponding stages of the two species were compared, and they appeared to be identical. May beetle adults emerge from the soil at dusk during early spring and summer.
They feed on the foliage of trees and shrubs until dawn.
The beetles then promptly return to the soil where the females lay their
eggs (Figure 3a).
These hatch in from two to three weeks.
The young
grubs feed on humus in the soil during the first year, burrowing deep into the ground to pass the winter.
The following spring the larvae
migrate upward and feed on the roots of plants until fall, when they once more burrow down into the soil to pass the winter.
The third year,
the larva feeds during the spring, pupates in mid-summer, and changes to adult in late summer and early fall.
The adult beetles remain in the
soil until the following spring at which time they emerge, and repeat the life cycle. This study was begun in 1946 and continued until the present time* Much time was spent on equipment and problems in technique before the actual study of the embryology of the May beetle was undertaken. REVIEW OF LITERATURE Von Baer, who elaborated Pander's idea of the germ layer theory is generally referred to as the "Father of Embryology", but according to Needham (1934), insect embryology begins as far back as Aristotle. Insects were classified as animals which "produce eggs which have no hard shells and which increase in size after being laid", according to Aristotle, One of the first men to work with insect embryology was Herold (1815) who worked on the order Lepidoptera.
Hummel (1835) worked on
the roach, and Kdlliker (I843) published a comparative study of insect and vertebrate development.
Weismann (1863) made a very comprehensive
study of dipterous embryology.
Kowalevsky (l8?l), whose research dealt
with worms and arthropods, was the first investigator to study fixed and sectioned material.
From I87I to the present time, important contributions
have been made by many investigatore,
In the review that follows, some
of these contributions which are of interest in the study of the embryology of the May beetle are mentioned. As the zygote nucleus reaches the center of the egg, it undergoes mitotic division.
The daughter or cleavage nuclei begin their migration
toward the periphery of the egg.
Butt (193b) on Brachvrhinus and
Huettner (1923) on Drosophila reported synchronous cleavage divisions. Blochmann (I887) first observed that all cleavage nuclei are connected to the protoplasmic reticulum of the egg.
Wheeler (1889b), in his
outstanding work on Blatta. and Nelson (1915)» on Apis, reported that the migrating cleavage nuclei first reached the periphery of the egg forward of the equator of the egg.
Work on Hvdrophllus by Heider (I889)
and on Calendra by Wray (1937) showed that the cleavage nuclei arrived first at the egg surface around the equatorial region.
Heider (I889)
reported that the nuclei arriving at the periphery are closely crowded together, and that there is a formation of two protoplasmic layers around the periphery of the egg. Most authors seem to readily agree that the origin of the primary vitellophags is from the cleavage nuclei which remain in the yolk after the completion of the blastoderm.
Wheeler (1889b) on Lentin otarea.
Nelson on Apis, and Kessel (1939) on the flea are but a few that report this method of vitellophag origin.
Many workers have reported the
formation of secondary vitellophags by the immigration of nuclei into the yolk from the ventral plate.
Butt (l93b). Nelson (1915)»
Lassmann (I93b), who worked on Melophagus. report this secondary vitellophag formation.
Metchnikoff (1866) while working on Miastor was the first investi gator to recognize germ cells and trace their development until they formed gonads.
Butt (1934) describee a granular plate of cytoplasm at
the posterior end of the egg in the Sciara which he believes to play a major role in the production of the germ cells from the cleavage nuclei. Nelson did not find such a cytoplasmic mass in the Apis.
Many insect
embryologists, including Heider, found that the nuclei located at the posterior end of the egg could not be distinguished from the other cleavage nuclei.
Wray (1937), in a study on the Calendra. reports a
mass of cells which are distinctly different in appearance from the surrounding blastodermic cells at the posterior end of the egg.
He
describes them as "larger in size and retaining a cuboidal shape, whereas the surrounding blastodermic cells soon change to a columnar or prismatic shape".
He also describes the mass as projecting out from
the blastoderm margin in the direction of the vitelline membrane. These, he believes to be germ cells which later move into the yolk mass and become situated on the inside of the blastoderm.
His observations
seem to be in agreement with Butt (193b) on Brachvrhinus. Johannsen and Butt (1941) list two types of germ cell differentiation. Miastor
In the
(Hegner I9I2 ), one germ cell gives rise to all the germ cells
produced by the insect.
In the other type, several cleavage cells form
the germ cells by migration into the posterior polar plasm, each forming a germ cell as in the Brachvrhinus (Butt 193b). Krause (194?) found that within the order Coleoptera, examples of all types of germ cell origin could be found.
Using Nelsen's (1934)
outline of the nmin general stages at which sex cells can first be
identified in insects, he lists the species of Coleoptera belonging to each stage as follows: The first main stage at which germ cells arise is the period of blastoderm formation, the germ cells being segregated as caudal pole cells. To this group belong the following species with the author who described the origin; 1. 2. 3. 4. 5*
Calligraoha mult1ounctata Leptinotarsa decemlineata Calandra granaria Calendra callosa Brachvrhinus ligustlei
Hegner (I908) Hegner (I908) Inkmann (1933) Wray (193b) Butt (l93b)
The second stage is thatimmediately after blastoderm formation, the germ cells segregating from primitive blastoderm cells in the caudal region. To this group belongs; 1.
Donacia crassipes Donacia crassipes
Friederichs (1906) Hirschler (I909)
The third period occurs shortly after the germ-band, as such, is demarcated from the blastoderm, but before segregation of the inner layer is initiated. The germ cells are segregated from the primary ectoderm in the caudal portion of the germ-band. An example of this group is: 1.
Tribolium confusum
Hodson (1934)
The fourth stage of germ cell origin is that during the period of the inner layer and mesenteron segregation, but before the forma tion of the coelomic sacs: 1.
Tenebrio molitor Saling (1907) (Nelsen puts the species in this group, but Saling admits that the gonad anlage cannot be identified with certainty as early as this stage.) The fifth period occurs after the coelomic sacs have formed. The germ cells are derived, or associated with - when first detected - the coelomic sac mesoderm. Examples are: 1.
Hvdrophllus piceus Heider (I889) Hvdrophllus piceus Grabor (I891) 2. Lina sp. Graber (I89I) 3# Tenebrio molitor Saling (I907) (Probably this is an example of group four.)
The sixth period occurs during post-embryonic development. Coleoptera are represented in this group.
No
Kessel (1939), on the flea, describes the germ-band as forming by the crowding of the nuclei toward the ventral midline and the polar surfaces of the egg.
The cells of the median dorsal region flatten out
and remain quiescent while those on the ventral side where the embryo will form, elongate and become columnar.
More rapid cell division on
this ventral side produces a thickened area which is called the germband.
Nelson (1915) reports a similar germ-band formation in the Apis. Johannsen and Butt (1941) classify orders of insects on the basis
of their embryonic membranes.
They list eleven separate groups and
place the Coleoptera in the third group composed of orders having "serosa and amnion fully developed, fusing either ventrally or at head's end just before revolution; secondary dorsal organ formed; germ-band superficial".
Hirschler (I909) reports a primary dorsal organ forming
in Donacia: however, primary dorsal organs seem to be lacking in most Coleoptera embryology.
Secondary dorsal organs, formed by absorption
of the serosa in the yolk, are reported by Hirschler (Heider and many other authors.
I889),
By contrast, Wray found that in the Calendra
the serosa remained intact until hatching and did not form secondary dorsal organs. There are four methods of gastrulation in the insect according to Kessel (1939):
"1.
The lower layer is formed by active proliferation
or emigration of cells from the blastoderm along the median line of the ventral plate." "2.
Wheeler (1889b) reported this in Blatta.
The lower layer arises by an emigration of cells from the
blastoderm along the median line of the ventral plate.
In this case a
distinct groove is formed, but its lips are not approximated to form a
tub©,**
Auten (1934) reports that this type was observed in Phormia*
”3*
There occurs an actual invagination of the ventral midline
blastoderm to form a groove which changes into a closed tube by the approximation and fusion of its lips."
Holder (I889) reports this for
Hvdrophilus as does Wray (1937) for Calendra. "4.
The middle portion of the ventral plate becomes separated
from the lateral blastoderm.
This middle plate then sinks inward while
the lateral plates grow together and fuse over its outer surface." Nelson (1915) reports this for Apis also. Coelomic sacs in Hvdrophilus as described by Holder are formed secondarily by clefts that appear in the solid mesoderm.
Carrière (1890)
found in Chalicodoma that the coelomic sacs occur exactly in the position occupied by the lumen of the tube formed during mesodermal formation. Butt (1934) reported that there are no coelomic sacs formed in Sciara. Wray (1937) reports that in Calendra the coelomic sacs form by the folding over of the lateral margin of each mesodermic segmental rudiment, thus enclosing a cavity.
According to Johannsen and Butt (1941),
coelomic sacs generally occur in all gnathal and thoracic as well as in all abdominal segments except the last one or two.
Kessel in the flea
reports a coelomic cavity in the deutocerebral or antennal segment* Wray reports a coelomic sac in the first antennal and the second antennal or intercalary segment. The origin of the me sent eron rudiment in the insect embryo is a much disputed and an important question, for it is from this mesenteron or entoderm rudiment that the entodermal layer is believed to be formed. Brauer (I925) lists three important theories for the development of the
mesenteron prior to 1884* 1* That of Dohrn, according to which the mesenteron was derived from the yolk cells or cleavage cells remaining in the yolk and taking no part in the formation of the blastoderm (1866)* 2. That of Kowalevski, according to which the mesenteron was derived from the splanchnic layer of the mesoderm (187I). 3* That of Ganin, (l8?4) who described the mesenteron as arising from the inner ends of the stomodaeal and proctodaeal invagination. Brauer also reports some of the more important views on mesenteron formation of other authors since 1884.
He states that Kowalevski in I886
described the origin of the mesenteron rudiments of the Musca arising from the anterior and posterior parts of the middle mesodermal plates; Heymons (I895) in work on the Orthoptera found that the mesenteron was derived from the blind inner ends of the stomodaeal and proctodaeal invaginations.
The mesodermal rudiments are reported to be arising
from the proliferating areas of undifferentiated blastoderm at the two ends of the ventral plate in the mason bee, Chalicodoma (Carrière and Bürger 1897)5 it was believed by Nelson (Embryology of the Honey Bee) that the anterior rudiment is derived from the cells of ventral blasto derm by immigration, eind that the source of the posterior mesenteron is the posterior end of the ventral plate.
The walls of the mesenteron
are formed in all insects with but a few exceptions by pairs of ventro lateral ribbons arising from the two mesenteron rudiments.
Nelson found
that in the Anis the two mesenteron rudiments each form a median dorsal ribbon instead of a pair of ventrolateral ones.
Wray also reports this
method in the Calendra. Wheeler (1889b) reported that several body regions became evident in the Lentinotarea embryo even before the differentiation of the germ layers
Nelson found the first true segmentation occurring just before the lower layer was formed in the Apis.
In the Brachyrhinus (Butt
1936),
segmentation begins in the thoracic region and proceeds both backward and forward.
In the Calendra (Wray
1937), the segmentation appears in
the head region and proceeds posteriorly until the thoracic and abdominal regions are demarcated.
Johanns en and Butt state that twelve
abdominal segments have been reported in Lepesma. Grvllotolpa. and Chalicodoma. The twelfth segment is the terminal anus bearing segment without appendages, but most Pterygota have eleven segments.
The number
of segments which enter into the composition of the head has caused much controversy.
The segments which make up the gnathal region are distinct;
therefore, the question centers on the number of segments which make up the procephalic region*
Johannesn and Butt summarize the various
opinions on the subject as follows i Until recently workers have also been in general agreement as to an antennal and an intercalary segment lying immediately in front of the mandibles. The part lying in front of the antennae, however, has been variously interpreted: as a primary head segment by Weismann (1864), Heymons (1895) and Heider (1889)5as an ocellar segment by Viallanes (1891 and Packard (I898); as an ocular segment by Holmgren (I908); as the acron plus the preantennal segment by Heymons (I9OI); as the brain emd labral segments by Patten (1884), Wheeler (1889b), and Carrière (1898); as an acron, labral, and preantennal segment by Wiesmann (1926); and labral, clypeopharyngeal, and frontoocellar segments by Verhoeff (I905)* Furthermore, Folsom called the part in front of the antennae the "oral segment" but recognized an interpolated superlingual segment between the mandible and msucillae. Finally, Janet accounted for four in front of the antennal segment, or nine in all. Blastokinesis, which Wheeler (I893) called all the movements or flections of the germ band during development (Johannsen and Butt 1941), usually involves the invagination of the germ band as in Odonata, the lengthening and rotation of the germ band accompanied by the growing over of the amniotic fold, as in the Lepidoptera, or only a slight
10
movement caused by the growth in length of the embryo which usually remains on the ventral side of the egg with the head at or near the cephalic pole. The ventral nerve cord development is essentially the same for all insects.
Kessel gives a very good description of ventral nerve
cord formation in the flea.
The fate of the median cord seams to be a
question that is not easily decided.
In the Apis (Nelson 1915), it
appears that the median cord takes part in the formation of the commissures in the intraganglionic sections.
In interganglionic
regions, it appears that the median cord is united with the epidermis. Wheeler (1893) reported that the intersegmental regions of the median cord are taken up into the central portion of the ganglia to form functional ganglion cells.
In the head region (Heider, I889), it has
been observed that the frontal ganglion is formed by invaginations of the dorsal wall of the stomodaeum.
Comstock and Kochi (I902) in their
discussion of the skeleton of the head give
credit for the naming of
the three principal divisions of the head to Viallanes who designated them as the protocerebrum, deutocerebrum, and the tritocerebrum. Wheeler (1889b) showed that the protocerebrum innervated the compound eye and ocelli, the deutocerebrum the antennae, and the tritocerebrum the labrum.
Comstock and Kochi (I902) reported the subesophageal
ganglion innervated the mandibles, maxillae, and the labium.
Folsom
(1900), on the mouth parts of Anurlda. found the superlinguae arise as a pair of appendages between the mandibles and the maxillae, and also found a pair of primary ganglia between the maxillary and mandibulary segments.
Snodgrass (1935) states that the primitive arthropod brain
11 included only the ganglia contained in the protocerebrum and deuto cerebrum*
He believes the ganglia of the second antennal segment, the
tritocerebrum, to be the first segment of the ventral nerve cord as they are united by a commissure that lies beneath the stomodaeum.
Kessel
{1939) found that in the flea the tritocerebrum developed from the connectives. TECHNIQUE Egg Production The beetles used for egg production were collected from a govern ment light trap at Creencastle, Ind., and also from light traps set up near the insectary at Purdue University (Figure 4).
A few beetles were
collected by hand from the leaves of trees at night with the aid of a flashlight. The cages used for egg production were glass globes, I8 inches tall, and open at both ends. cloth.
The top opening was covered with cheese
The cage rested on an earthenware flower pot saucer, 11 inches
across and 2 inches deep, into which a two-inch layer of soil had been sifted (Figure 5)* A fresh branch of persimmon leaves was added every other day to each cage in order to insure a fresh supply of food for the beetles. The soil was kept compact and moist at all times, because the beetles would not lay in loose, dry soil. A series of egg laying observations was made by sifting the soil of different cages at different hours during the day.
From this it was
discovered that the May beetle did not begin to lay eggs in any great numbers until approximately two o'clock in the afternoon.
All eggs were
12
collected for this study starting at five o'clock in the afternoon each day.
Only one collection of eggs could be made each day, as the beetles
would not lay again until the following day, once they had been disturbed. Eggs were collected by sifting the soil with an 18-mesh screen.
The
soil passed through the screen, but the eggs, which were embedded in small balls of soil, remained on top of the screen.
By gently crushing these
lumps, the eggs on the inside were exposed and collected with the aid of a wet camel's-hair brush.
They were then placed in two-ounce tin oint
ment boxes containing moist soil.
Thirty eggs were placed in each tin,
and these tins, with the time and date written on the top, were placed All eggs were kept at a temperature of 78 degrees
in an incubator.
Centigrade and at a humidity of 80 percent.
They were sprayed each day
with a solution of merphenyl nitrate and water to prevent attack by the Rhijopcrs NfQ/-fC6hS fungus Rhvaopue niger-% Preparation of Eggs for Study As the eggs reached the stage of development desired, they were removed from the incubator, placed in a watch glass, and killed with hot Bouin's fluid. While still in the hot Bouin's fluid, each egg was punctured ten times on the side opposite the embryo with a minuten nadelin pin under a binocular microscope.
As development progressed, the embryo could
be detected through the chorion.
After six hours, the Bouin's fluid
was removed, and the eggs were transferred to two-inch open-mouth glass vials containing &cid alcohol.
Acid alcohol was made by adding 1 drop
of concentrated hydrochloric acid to 100 cubic centimeters of 50 percent ethyl alcohol.
The eggs were washed by changing the acid alcohol every
13 eight hours until the yellow Bouin's fluid ceased to color the solution. This was a vary important step.
The first year's collection of eggs was
ruined by failing to completely remove the Bouin's fluid.
These eggs
were not punctured as described above, and therefore, the washing alcohol did not penetrate*
As a result, the Bouin's fluid remained in the egg
and hardened the yolk, making it impossible to get adequate infiltration with an embedding paraffin.
Many chemicals, oils, and stains were used
in an attempt to soften the yolk, but only one stain, Borax-Carmine, had any softening effect. After washing with $0 percent acid alcohol, the eggs were dehydrated using the tertiary butyl alcohol series as recommended by Johansen (1935)< All eggs remained in each solution of this series for twenty-four hours. Many embedding masses were tried before one was found which would hold the yolk granules in place while sectioning. finally selected was produced as follows:
The mass which was
A stock rubber solution was
made by heating a mixture of one part crude rubber and three parts hard (52 to 54 degrees Centigrade) paraffin in a 56 degree Centigrade oven for one week. blocks.
The liquid layer was decanted, cooled and stored in
Seven grams of this stock rubber solution, 5 grams of bayberry
wax, and 45 grams of hard paraffin were mixed together and melted to form the regular embedding mass. The 100 percent butyl alcohol, vdiich was the last solution in the tertiary butyl alcohol series, was removed from the two-inch open-mouth vials and replaced by the regular embedding mass described above. vial was labeled and placed in an embedding oven at 53 degrees Centigrade.
Each
14 The embedding oven was made from a small size lard can (Figure 6). The inside of the can was lined with sheet asbestos.
A shade from a
goose-neck lamp placed inside face down on the bottom over a 50-watt bulb served as the heating element.
The heat from this bulb held the
temperature of the can within one degree of 53 degrees Centigrade.
A
wire mesh shelf was placed about six inches above the heating element. A large glass petri dish, the bottom of which was lined with asbestos, was placed on top of this shelf, and the embedding vials were placed in the petri dish.
In this manner, no direct heat from the heating element
reached the eggs in the embedding vials.
A three-inch flap was cut in
the top of the lard can to facilitate handling of the vials.
The top
was not lined with sheet asbestos. A vacuum was used to aid penetration in some eggs, especially those of the early stages.
When this was done, the embedding vials were placed
in an Srlenmeyer flask instead of on the petri dish inside of the oven. The vacuum was produced inside the flask by using a rubber stopper, rubber hose, and a water vacuum pump. The vials were removed from the embedding oven after six hours. When a vacuum was needed, it was never used more than two of these six hours.
After removal from the oven, the lower ends of the vials were
placed in ice water until the embedding mass solidified.
The vials were
then turned upside down in a test tube rack until they were needed for sectioning (Figures 6 and 7).
When stored in this manner, the possibil
ity of the eggs being hardened by liquids was eliminated. Microtechnique Vials containing eggs to be sectioned were returned to the embedding
15 oven and reheated for two hours at a temperature of 53 degrees Centi grade.
Several of these eggs were then removed and embedded in paper
boats, as described by Kennedy (1932).
Eggs less than two days old
were more difficult to section and were, therefore, embedded and sectioned individually. four at a time.
All other stages were embedded and sectioned
No attempt was made to orient the eggs in the desired
plane before sectioning.
Eggs were sectioned and stained, and only
those properly oriented were saved for study.
All eggs were ribboned
into ten micron sections using a Spencer rotary microtome (Figure ?). These ribbons were placed on slides which were first smeared with Mayer's albumen (Kennedy, 1932) and then covered with water.
The slides were
then transferred to a modified warming plate. This modified warming plate was made as follows:
a plate of
l/8-inch glass, 20 by 15 inches was placed on the top of a cardboard box. In the bottom of the box was placed a wooden board on which four light sockets had been fastened 3 inches apart.
Light bulbs of 15* 25* 40,
and 60 watts were screwed into these sockets.
When lighted, these bulbs
gave a graduated temperature range from 53^ C. to 56® C.
on the surface
of the glass plate above (Figure 8). The staining procedure as outlined by Kennedy (1932) was followed from this point.
Delafield's haematoxylin with eosin as a counter stain
was used for the staining of most sections; however, Mayer's haemalum with a counter stain of Fast Green FCF were cuLso used to some extent. Heidenhain's iron haemat oxylin was tried, but found unsatisfactory as it colored the yolk granules' so dark that they dominated the entire picture and obscured all nuclei.
16 Microphotography Photographs were taken with a bellows type camera mounted so that it could be raised or lowered over the eyepiece of a microscope.
A
very bright spot light was placed approximately 45 inches away from, and shining directly into, the flat mirror of a microscope.
The light
beam was centered in the camera by removing the eyepiece of the micro scope so that the rays were concentrated on the ground glass.
A clear
light bulb was used, as a frosted bulb did not produce a clear image. After replacing the eyepiece, the condenser below the stage of the microscope was lowered to diffuse the light rays passing throu^ the camera. All sections to be photographed were circled with a red wax pencil so that they could be readily located when the slide was placed under the microscope. Five by seven structure.
ortho-contrast, cut film was used to secure detailed
Six seconds was the usual period of exposure.
course, varied somewhat with the density of the slide.
This, of
Prints were made
on glossy velox paper. Numbers 2 and 4. RESULTS AND DISCUSSION The Egg The egg of Fhvllophaga fervida is oval in shape and averages 2.40 millimeters in length, and 1.55 millimeters in width (Figure 3&). It becomes spheroidal in shape the second day due to absorption of water from the soil, and increases in size as it grows older until it becomes as large as 4.0 millimeters in diameter (Figure 3b)* When laid, it is glistening white and is covered with a sticky substance to which the soil particles adhere.
17 The outer surface of the egg shows the usual slightly raised ridges which enclose polygonal spaces formed by the ovarian follicle cells.
In
a section, the chorion appears as a single membrane made up of two laminal membranes (Figure 15) which are united by slender rods.
The
outer layer stains pink with eosin, but the inner layer does not take a stain.
Beneath the chorion lies a second protective covering*
This is
the vitelline membrane (Figure 9) which is very thin and noncellular. Spermatozoa pass through the chorion and vitelline membranes by means of micropyle openings.
The micropyles, which are on the outer
surface of the chorion, may be volcano-like in structure or sunken saucer-like craters.
The chorion contains an opening at this point
through which the spermatozoa pass. Just inside the vitelline membrane lies what might be termed a "plasma membrane", irtiich is composed entirely of protoplasm.
This
membrane is 6 to 9 microns thick and exists in the egg from the time of oviposition until the formation of the blastoderm.
It can be seen only
by overstaining with Delafield's haematoxylin or the use of FCF Fast Green (Figure 21).
This superficial protoplasm is known as periplasm
or cortical ooplasm. anterior polar plasm.
The periplasm at the anterior pole is called the In some Fhvllophaga eggs, a small dense proto
plasmic cap is formed in the polar plasm under the micropyles (Figure 9)*
In one sectioned egg, an irregular saucer-shaped mass can
be seen lying partly in the periplasm and partly in the yolk (Figure 10). Whether these two masses are the same could not be determined.
They both
disappeared shortly before the arrival of the cleavage nuclei. The periplasm is continuous with a reticulum of protoplasmic
18 strands which ramify throughout the inner portion of the egg (Figure I3 )* Contained within the reticulum are vitelline spheres or granules which serve as stored food for the developing embryo. in size (Figure 14).
The spheres vary greatly
Short rods of protoplasm are sometimes present at
the polar region where the sperm enters the egg (Figure 11). Maturation and Fertilization The location of the pronucleus at the time of oviposition has not been determined with any degree of certainty.
In some eggs, it was
observed in the periphery of the egg (Figure 15) near the polar region. In others, it was not observed at all, probably due to improper staining for nuclei.
Shortly sifter oviposition, the pronucleus surrounded by
cytoplasm migrates out to the periphery of the egg (Figure 15) and gives off the first polar body.
It then migrates back toward the center of
the egg giving off a second polar body during this migration.
The first
polar body which remains at the periphery, divides, also forming a second polar body.
This second polar body is pushed out of the cytoplasmic
periphery to a position under the vitelline membrane where it is separated from the remainder of the egg (Figure 1?). cell until it degenerates.
It remains in a cytoplasmic
The remaining polar bodies migrate around
the yolk granules, some growing very large in size (Figures I8 and 19). All polar bodies appear to have degenerated by the end of the second day* As the egg is oviposited, the spermatozoa enter the micropyles. Figure 12 shows the filament of the spermatozoa lodged in a volcano-type micropyle.
Figure 13 shows the head of a spermatozoon lying a short
distance from the lodged filaments among a cytoplasmic network at the polar end of the egg.
A spermatozoon is shown in figure 14 breaking
19 through a saucer-type micropyle; this is partially engulfed by a mass of cytoplasm which may have come from the cytoplasmic cap previously mentioned.
As this was not the only sperm found in the egg, polyspermy
is indicated.
The actual process of fertilization or the fusion of the
female and male gamete nuclei is shown in figure 20. in the center of the anterior portion of the egg.
It occurs almost
Fusion causes the
eurea surrounding the gametes to glow. Cleavage and Blastoderm Formation Cleavage is the term given to the mitotic division of the nuclei after the formation of the zygote.
Various stages of mitotic division
are shown in figures l6 , 24 and 27.
Immediately following the fusion of
the gametes and formation of the zygote, meroblastic cleavage beings (Figure 23)*
All cleavage nuclei are connected by protoplasmic strands
to the reticulum, and all are covered with protoplasmic strands which trail behind them as they migrate to the periphery. moves together toward the periphery (Figure 25)*
The first group
The first place to be
reached on the surface of the egg is between the poles in the equatorial region (Figure 26).
The periplasmic ring, which is present around the
egg from the time of oviposition, rearranges itself to form pockets of protoplasm.
This occurs before the nuclei reach the periphery (Figures
21 and 22).
When the nuclei enter the periplasm, they are equally
spaced and are not crowded together as in the Hvdrophilus (Heider, I889), Synchronous nucleus division was not observed during the migration. However, this probably occurred as indicated by the uniform migration of the outer ring of individual nuclei.
A chromosome count was made in the
cleavage nuclei approaching the polar region.
About twenty-one
20 chromosomes are present in the nuclei of the May beetle egg. Butt (1936) and other embryologists have reported germ cells forming as the cleavage nuclei reach the posterior polar region.
Cells resembling
germ cells were observed on one slide only during blastula formation at the posterior polar region (Figure 28).
These will be discussed later.
Division of the nuclei continues (Figure 29) after the nuclei enter the periphery of the egg until they are correctly spaced for the formation of cells.
The cell walls are then laid down.
This process starts from
the outside and proceeds inwardly, enclosing each nucleus with its adjacent cytoplasm.
After the completion of the cell walls, the egg
enters the blastoderm stage. As in most other insect eggs, all cleavage nuclei do not reach the periphery of the egg and enter into the formation of the blastoderm. Some remain behind among the yolk granules.
These nuclei are called
primary vitellophags, and are later augmented by secondary vitellophags which enter the yolk from the germ band and blastoderm.
Butt (1936)
also observed secondary vitellophags in Brachyrhinus. When these nuclei enter the yolk, they leave most of their cell cytoplasm behind them. There is no difference in appearance between the primary and secondary vitellophags, as they both appear very dense and stain heavily with Delafield's haematoxylin. Germ Band Formation The blastoderm now begins to change in appearance.
All nuclei
divide so rapidly, a crowded condition soon develops in the entire blastoderm, especially on the dorsal side (Figure 38).
On the ventral
side, due to this crowding (Figures 39 end 40), the cells elongate and
21 change from low type columnar cells to high type columnar cells. Cell division proceeds much more rapidly at the anterior end of the egg, and soon a cap of cells is produced (Figure 3i) which is similar in appearance to the mesenteron rudiment described by Kessel (1939) in his work on the flea, and to the head lobe which Johannsen and Butt (1941) described under their general discussion of embryology.
As the cap
grows, the cells of this polar cap lose their form, and their nuclei appear crushed and irregular in shape.
During the development of the
polar cap, two circular masses of granular material which resemble in appearance yolk synitia as described by Nelson (1915)» form along the inner edge of the cap (Figures 35 and 36). their significance is unexplainable.
They soon disappear, and
During polar cap development,
elongated masses of protoplasm form among the irregular nuclei in the polar cap (Figure 32), and they also disappear as the number of nuclei in the polar cap increases. As rapid cell division continues throughout the blastoderm, the cells on the dorsal side of the egg migrate along the periphery toward the polar cap.
During this migration, the polar cap is pushed poster
iorly along the nddventral line. needed for the dividing cells. occurs.
Migration cannot provide all the space As a result, an unusual phenomenon
The cells in the periphery arch inwardly at several points and
give an undulated appearance to the blastoderm (Figures 33 and 34). Then groups of nuclei are pinched off or crowded out into the yolk granules (Figure 37).
These groups, which begin migrating around the
yolk as soon as they are formed, are each composed of from four to six teen nuclei.
Butt (1936) observed single nuclei which he termed
22 secondary vitellophags moving in from a crowded germ band, and degener ating within the yolk.
These particular ball-type groups differ from
the secondary vitellophags as observed by Butt in that they do not degenerate and are grouped together.
After several hours the nuclei in
these groups swell and become less dense.
Protoplasm clusters around
them, giving them a moss-like appearance.
The groups then migrate back
to the periphery of the egg (Figure 41).
The cells on the ventral side,
together with most of those covering the anterior polar region, pass out cytoplasmic streamers giving the blastoderm a ragged appearance.
The
groups of cells migrating around in the yolk become attached to these streamers, break up, and pass into the cytoplasmic periphery once more (Figure 42). completedt
Some groups pass into the polar cap.
Thus a cycle is
the blastodermal cells divide in the periplasm, pass out
into the yolk granules, pick up additional protoplasm, and return again to the periphery where the germ band is being laid down.
By this migra
tion, blast ode m a l cells are concentrated on the midventral line forming the germ band. During the formation of the germ band, at the posterior and of the egg, large cells appear which differ from the remainder of the blastoderm in that they are irregular in shape and have a less dense appearance. These are believed to be the germ cells (Figure 43). Embryonic Membranes and Blastokinesis The embryo of the May beetle becomes covered by two embryonic membranes at an early stage in the development of the germ band.
The
outer of these membranes is known as the serosa, and the inner one is termed the amnion.
Both are cellular in nature and are composed of
23 squamous type cells.
They are both produced from that portion of the
blastoderm which is not involved in the formation of the ventral plate. The amnion is continuous with the germ band.
The serosa, although it
is continuous with the amnion during its formation, later loses all connection with the embryo and forms an entirely independent covering just inside the vitelline membrane. The embryonic membrane formation begins with a shallow indentation (Fig. 44) at the anterior end of the germ band above the polar cap.
The
anterior margin of this indentation soon grows out to form a double fold (Figures 46 and 47), the anterior amniotic fold.
The inner layer of this
fold, which is continuous with the ventral plate, will become the anterior part of the amnion, and the outer layer will become the serosa. The anterior amniotic fold now extends posteriorly and laterally along the side of the egg.
Another indentation occurs on the posterior end of
the germ band when the anterior amniotic fold has covered two-thirds of the germ band (Figure 48).
The posterior margin of this indentation
forms the posterior amniotic fold which advances anteriorly to meet the anterior amniotic fold.
There is no partial involution of the embryo
as in the Odonata and Hemiptera. region.
The two folds join in the posterior
As they join, the intervening walls rupture.
The inner or
amniotic layers and the outer or serosal layers of the two folds fuse to make two complete membranes which protect the embryo.
The serosa is
formed from the outer layer of the amniotic folds and partly from the blastodermal covering of the egg.
These membranes are completed during
the third day of the embryonic period. In contrast to the serosa, the amnion covers only the surface of the germ band, and is continuous with it.
The area enclosed by this membrane
24 is called the amniotic cavity# These embryonic membranes exhibit important differences as to their fates#
Both the serosa emd the amnion are still complete at the end of
the sixth day#
On the seventh day, the amnion and the serosa fuse
together directly over the amniotic cavity and form one protective membrane (Figure 6l).
By the end of the tenth day this fusion is com
pleted at which time the amnio-serosal membrane ruptures and is pulled toward the dorsal surface of the egg (Figure 62).
As it migrates
upward, this membrane becomes very thick and appears to have changed to low type columnar cells.
As the dorsal wall of the embryo is completed
by the growth of the ectoderm and middle layers, the membrane assumes an umbrella-like appearance and is then pulled into the yolk where it is dissolved (Figure 72).
This degeneration membrane is called a dorsEil
organ. Two other folds appear at this time which have no connection with either the amnion or the serosa.
Each is formed directly from the
ectoderm of the embryo, and is composed of a single layer of low type columnar cells.
They appear to be similar to the paired evanescent,
embryonic évaginations of the first abdominal segment reported by Graber (1889) and Wheeler (1889a). These évaginations are called pleuropodia and are believed by Johannsen and Butt (1941) to serve as organs of excretion or secretion during embryonic life. they furnish a hatching enzyme.
Blunck (1914) believes
In the May beetle larva the pleuro
podia attain a maximum development at the end of the eighth day (Figure 70).
They then decrease in size until the larva is mature, at
which time they appear to shrivel up and fall off.
25 At the same time that the embryonic membranes are forming, the posterior and anterior ends of the germ band lengthen.
The anterior
end moves half way around the periphery of the anterior pole.
The
posterior end passes down the ventral side toward the germ cells, around the posterior pole, and continues anteriorly on the dorsal side until it has covered one-third the distance between the poles (Figure 54).
By the end of the sixth day, the posterior end of the
germ band has withdrawn to a position directly behind the anterior end of the embryo which is now located in the mid-polar region along the midventral line (Figure 56).
This rotation of the embryo is called
blastokinesis. The Mesoderm As the germ band is being covered by the amnion, the mesodermal layer is formed in the following manners
the nuclei on the inner side
of the germ layer which have a denser appearance than the other nuclei, line up along the midventral line (Figure 48).
A cytoplasmic layer
appears along the inner edge of the germ band, and the nuclei migrate inwardly into it forming the mesodermal layer.
This manner of meso
dermal layer formation seems to be identical to that reported by Wheeler (1889)in Blatta. As the mesodermal layer is forming, the cells along the midventral line at the anterior end of the germ band migrate inward and form a horseshoe-shaped group of cells on the inner side of the germ band (Figure 45).
These cells were observed to see whether or not
they formed a mesodermal tube as reported by Heider (I869) in Hvdrophilus. No such tube was formed. divides into two lamellae.
Soon after the formation, the mesodermal layer This division starts at the lateral edges
26 and proceeds inward (Figures 49 and 53)*
Eighteen pairs of coelomic
sacs, connected intrasegmentally and intersegmentally by very narrow passageways, are formed on the lateral edges of the mesoderm between the two layers.
One pair of coelomic sacs are located forward of the
stomodaeal invaginations in the antennal segment, and the remaining seventeen pairs are located posterior of the stomodaeal invagination. The intrasegmental passages exist only a short time, however, as reorganization and proliferation of the mesoderm soon transform the two mesodermal layers and passageways into a single bridge of dense mesodermal tissue (Figure 50)*
The coelomic sacs increase in size
(Figure 50)> and the cells of the dense mesodermal bridge become loosely connected.
The inner half of the coelomic sacs, or that half
nearest the yolk cells, becomes detached and folds back dorsally along the entodermal layer covering the yolk to form the splanchnic mesoderm. Muscles of the alimentary canal are formed from this layer.
The cells
of the mesodermal bridge lying intrasegmentally between the two coelomic sacs and above the neural area divide along the midventral line and pass into the cavities formed by the opening of the coelomic sacs.
Only a single layer of mesodermal cells is left covering the
neural area.
Between this single layer and the neural tissue, a cavity
is formed by this redistribution of mesodermal cells which is called the epineural sinus.
The mesodermal cells which pass into the ruptured
coelomic cavities and come to rest on the ectoderm, form the somatic mesodermal tissue.
Muscles (Figure 79) of the body wall and appendages
arise from this mesodermal layer. By the end of the seventh day the lateral sides of the embryo have
27 grown one-fourth the distance around the yolk.
Blood cells are now
conspicuous (Figure 59)» and what might be interpreted as cardioblasts can be seen in the upper end of the lateral sides of the embryo between the somatopleura and splanchnopleura (Figures 59 and 6o).
A group of
mesodermal cells (Figure 59)» appear directly under the cardioblasts. These cells will later aid in the formation of the heart. By the twelfth day the layers of the lateral sides have thinned out to single cell layers and have almost encircled the yolk*
The
amnio-serosal membrane has folded into the yolk to form the secondary dorsal organ (Figure 72).
The cardioblasts have become elongated,
curved, and grouped, preparatory to uniting and forming the heart (Figure 73)#
Development continues, and the lateral sides meet ©ud
fuse to form the heart (Figures 75» 76 and 80) by the end of the six teenth day. The Entoderm Entoderm formation begins with two indentations of the germ band as the embryo reaches its maximum length in blast okinesis (Figure 54). One of these indentations occurs at the anterior end just below the protocephalon, and the other at the posterior end, slightly forward of the amniotic germ band union.
The cells at the bottom of the indenta
tions migrate to the inner side of the germ band, and the germ band closes behind them.
By active proliferation, cell masses called the
anterior and posterior mesenteron rudiments are formed which send out ribbons of cells running lateroventrally of the yolk (Figures 57 and 58)* The ends of these ribbons approaching each other from opposite directions meet and fuse, connecting the posterior and anterior mesenteron rudiments
28 By lateral proliferations, these ribbons replace the layer of mesodermal cells over the epineural sinus, and by continued lateral proliferation form the mesenteron or mid-gut (Figure 80).
The replaced mesodermal
cells form blood cells and pass into the body cavity. The Ectoderm The lower layer of cells remaining after mesodermal formation is considered to be the ectodermal layer.
One of the most important
ectodermal derivatives is the nervous system.
About the time of the
appearance of the proctodaeal and stomodaeal invaginations, the neural groove forms along the midventral longitudinal line, and extends from the stomodaeum to the proctodaeum.
This is the first step in the form
ation of the nervous system from the ectoderm (Figures 50 and 51)#
This
groove is formed by two longitudinal thickenings
(Figures 47 and 50)» of
the ectoderm along the midline of the germ band.
The medial unthickened
portion forms the groove.
The longitudinal thickenings are called
neural ridges and are formed by the active proliferation of specialized ectodermal cells called neuroblasts below the surface layer (Figures 50 and 5 2 ).
These neuroblasts also form the middle cord by active prolif
eration from the floor of the neural groove. At segmentation, all the layers of the germ band,except the ento derm, are affected.
During this process the neural ridges are meta-
merically constricted into segmental divisions.
The layer of ectoderm
over the neuroblasts forms the epidermis and is therefore called the dermoblast.
The neuroblasts form the definitive nerve cells.
In the
intrasegmental region the neuroblasts are very active and soon give rise to numerous nerve cells.
Two ganglia are produced in each segment.
29 and the ganglia of all segpiente are connected successively by less thickened interganglionic portions called connectives (Figures 69 and 77)#
Each pair of ganglia are connected transversely by coiniaissural
neuromeres. The neural ridges increase very rapidly in width, and with the formation of the neuropile, fuse closely to the midline to form a definite ganglion (Figures 60, 74 and 80).
Their connectives retain
their individuality* The central nervous system consists of twenty pairs of ganglia at the ninth day, but by the end of the seventeenth day, the last two abdominal ganglia have fused together leaving a total of nineteen ganglia in the mature larva.
The first pair of ganglia make up the greater part
of the supra-esophageal gemglion. This pair soon unites to form the bilobed protocerebrum (Figure 68) of the ocular segment.
A single
commissural mass connects these two halves of this neuromere. pair of ganglia (Figures 63 and 67) form the deutocerebrum. mere innervates the antennae.
The second This neuro
It is believed that these two pairs of
ganglia are the only ones of the nerve chain which have their origin anterior in position to the stomodaeum.
The tritocerebrum, another pair
of ganglia, appears to be formed from the lateral connectives posterior to the stomodaeal invagination (Figure 66) in agreement with Kessel (1939) in his work on the flea.
They are considered to be ventral
ganglia because the transverse tritocerebral commissure passes below the esophagus (Figure 65)*
A nerve cord originates from each ganglion of
the tritocerebrum and extends forward to a location above the stomodaeal invagination.
Bach of these cords has a clavate ganglion at the distal
30 end (Figure 6?).
These ganglia are believed to be frontal ganglia*
Heider (I889) describes these ganglia as originating by invagination of the dorsal wall of the stomodaeum* The fourth, fifth, and sixth pairs of ganglia of the mandibular, maxillary, and labial segments form the gnatho-cephalon.
These ganglia,
although originating separately, soon unite to form the single subesoph ageal ganglion (Figures 63 , 64, 77, and 8o). The seventh, eighth, and ninth pairs of ganglia develop into the three thoracic ganglia.
They do not fuse, but preserve their identity
and are distinct in the fully grown larva. Eleven abdominal ganglia belong to the abdominal region; ten are distinct in the mature larva, as the last two ganglia fuse together. The tracheal system is another derivative of the ectoderm, and it arises as paired invaginations of the ectodermal layer.
These invagina
tions are located along the lateral margins of the germ band.
Their
mouths form the spiracles (Figure 74), and the invaginations themselves sink below the surface, branching again and again to form the complex respiratory system of the May beetle larva.
Nine pairs of spiracles
were counted on the fully matured larva, eight pair on the abdomen and one pair on the prothorax. The stomodaeum and the proctodaeim (Figure 80) are formed by the deepening of the indentations in the germ band mentioned under entoderm formation.
Both are considered to be of ectodermal origin.
The ectoderm gives rise to a number of other structures which have not been considered in detail during this study.
Some of these are the
hypodermis, the tentorium, the corpora allota, labial glands, and
31 perhaps the Malpighian tubules. Germ Cells The germ cells in the May beetle larvae cure difficult to trace during the development of the embryo.
In most insects, for example the
flea (Kessel, 1939)» they appear during the formation of the blastoderm at the posterior pole and are easily detected by their Icurge size, eparce distribution, prominent nuclei, and their unusually clear cyto plasm.
No such cells are visible during the formation of the blasto
derm in the larvae of the May beetle.
In a few sections some cells
which appear larger than the remaining blastula cells can be seen at the posterior pole, but they do not possess any of the other above named characteristics (Figure 28).
Odsome is not noticeably present
in the egg as the cleavage nuclei move into the periphery at the posterior pole, and the cleavage polar nuclei do not change in appear ance at either pole.
The rate of division at the anterior pole increases,
however, to begin the formation of the anterior cap already mentioned (Figure 31). Cells somewhat larger than the other blastodermal cells, which possess very prominent nuclei, appear at the posterior pole about the time the germ band is being formed (Figure 43).
During blastokinesis,
as the posterior end of the germ band passes the posterior pole, some large cells similar to the above mentioned, force their way through the amniotic layer into the amniotic cavity.
These are believed to be the
germ cells beginning their migration into the germ band, but they could not be traced after their entrance into the amniotic cavity. As the posterior end reaches its limit of extension in blastokinesis,
32 a few germ cells appear at the posterior end of the embryo on the meso dermal side (Figure 55)*
When the splanchnic mesodermal layer begins
its migration dorsally for the formation of the body cavity, the germ cells appear in the splanchnic mesoderm at about the fifth body segment and form clusters on each side of the developing body cavity.
These
clusters, or gonads, are later enclosed by an envelope of mesodermal tissue and are suspended from the splanchnic layer (Figure 71) by a narrow band of mesodermal cells. StMÆÂRT Phvllophaga fervida (Fab. I781) and Phylloohaga hii*ticula (Knoch 1801) are the species studied.
No embryonic difference was noted between
these two species. Chorions of all eggs were punctured to increase their permeability. Rubber paraffin was used as the embedding mass. was used for the dehydration series.
Tertiary butyl alcohol
Centering the light beam passing
through the microscope and camera was the most important step in microphotography. The Phvllophaga egg is of the centrolecithal type, and spermatozoa pass through its chorion by means of micropyles.
The egg possesses a
very prominent periplasmic membrane. Maturation division occurs at the periphery of the egg and ferti lization takes place almost in the center of the anterior portion of the egg. Meroblastic cleavage begins immediately after fertilization. Cleavage nuclei first reach the periphery between the poles in the equatorial region.
The periplasmic membrane forms pockets to receive
33 the cleavage nuclei.
About twenty-one chromosomes are present in the
nuclei of the May beetle egg. Primary vitellophags are formed from cleavage nuclei which remain behind among the yolk granules.
Secondary vitellophags are formed by
cells which enter the yolk from the germ band or blastoderm. A polar cap is formed at the anterior end of the egg.
Rapid cell
division in the blastoderm causes groups of nuclei to be crowded out into the yolk granules.
These groups return to the periphery along
the midventral line to aid in germ band formation. The embryo is covered by two membranes, the serosa and the amnion. These membranes later fuse and are absorbed by the yolk forming the dorsal organ. During blastokinesis the germ band lengthens along the periphery of the egg until its posterior end has covered the posterior pole, and the anterior end has reached the mid-polar region at the anterior pole. It then retracts until it covers only the ventral side of the egg. The mesoderm is formed by nuclei migrating inward from the germ band.
Eighteen pairs of coelomic sacs are developed in the mesoderm.
One pair is located forward of the stomodaeal invagination, the remainder posteriorly. The entoderm is formed by active proliferation of cell masses called mesenteron rudiments which are produced by cells migrating in ward from the germ band. The ectoderm is the lower layer of cells remaining after mesodermal formation.
One of its main derivatives is the central nervous system
which is formed from the neural ridges and the middle cord.
Two pairs
34 of ganglia form anterior to the stomodaeum. brum and the deutocerebrum.
These are the protocere-
A third pair, the tritocarebrum, which
originates posterior to the stomodaeal invagination, moves forward and fuses with the protocerebrum and deutocerebrum to form the brain.
The
ganglia in the gnathal segments fuse to form the subesophageal ganglion. The three thoracic ganglia remain distinct.
Eleven abdominal ganglia
are found in the abdominal region in the early embryo, but only ten exist in the mature larva, as the tenth and the eleventh ganglia fuse. The tracheal system arises from paired invaginations of the ecto dermal layer.
Nine pairs of spiracles are found in the fully mature
larva. Germ cells are first distinguishable at the time of germ band formation.
They next appear above the mesodermal layer at the posterior
end of the germ band during blast okinesis. together to form gonads.
Later the germ cells group
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li108-111. Blunck, Hans 1914. Die Bntwicklung des Dytiscus marginalis L. vom Ei bis zur Imago. I. Th. Das Embryonalleben. Zeitschr. f. wiss. Zool.
lilt76-151.
Brauer, Alfred 1925*
Studies on the embryology of Bruchus quadrimaculatus Fabr. Ann. Entomol. Soc. Amer. I8 (No. 3)*283-312.
Butt, F. H. 1934. Embryology of Sciara (Sciaridaet Diptera).
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1936. The early embryologieeuL development of the parthenogenetic alfalfa snout beetle, Brachvrhlnus ligustici L.
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Die Entwicklung der Mauerbiene (Chalicodoma muraria Fabr.) im Ei. Arch. f. Mikrosk. Anat.
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and Bürger, 0. 1897• Die Entwicklungsgeschichte der Mauerbiene (Chalicodoma muraria Fabr.) im Ei.
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*Dohrn, Anton 1866. Zur Embryologie der Arthropoden.
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54*849-851»
Fabricius, J. C. 1781. Species Insectorurn, Melolontha fervida Fabr. (Phvllophaga fervida Fabr.), Tomus I, p. 36. Folsom, J. W. 1900.
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***Ganin, M. 1874. Ueber den Mitteldarm der Insekten.
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Graber, V, 1889. Ueber den Bau und die phylogenetische Bedeutung der embryonalen Bauchanhange der Insekten. Jour. Roy. Micr. Soc.
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37
1891.
Beitrage zur vergleichenden Embryologie der Insekten. d.
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Hegner, R. W. 1908. Effects of removing the germ-cell determinants from the eggs of some chrysomelid beetles.
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1912. The history of the germ cells in the paedogenetic larva of Miast or. Science.
36 »-^24-126*
Beider, K. 1889.
Die Entwicklung von Hydrophilus piceus L. Berlin, pp. 1-98.
Klg. Acad. Wiss*
Jena.
■**Herold, Dr. 1815. Entwickelungsgeschichte der Schmetterlinge,
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Gassel u. Marburg. i^Heymons, R. 1895. Die Embryonalentwickelung von Dermapteren und Orthopteren unter besonderer Beruckslchtigung der KeimblatterbiIdung. Monograph!sch bearbeitet.
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* 1901. Die Entwicklungsgeschichte der Scolopendre.
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22 (No* 4 )*627-744.
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38 Hodson, A. C. 1934. The origin and differentiation of the sex organs of Tribolium eonfasum. Ann. Entomol. Soc. Amer.
27*278-288.
Huettner, A. F. 1923. The origin of the germ cells in Drosophila melanogaster. Jour. Morphology.
37*385-423.
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Johannsen, 0. A. and Butt, F. H. 1941. Embryology of insects and myriapods. Hill Book Co., Inc.
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Kennedy, C. H. 1932. Methods for the study of the internal anatomy of insects. Mimeographed by H. L. Hedrick, 254 E. Oakland Ave., Columbus, Ohio Kessel, E. L. 1939. The embryology of fleas. 28(No. 3 )*1-78.
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39 Knoch, A. W. 1801«
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APPENDIX
42
Fig. 1.
Phyllophaga fervida Fab. (1) X 2
\ d
XI# K 4'.
Fig. 2.
Phvllophaga hirticula Knoch X 2
(1)
(l) These pictures are furnished through the courtesy of the Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture,
43
Fig" 3&" Eggs of the May beetle, Phvllophaga fervida. The elongated*egg was freshly laid; the others were two weeks old. ' '
Fig. 3b* Eggs of the May beetle, Phvllophaga fervida. in clumps of earth in which they were laid, v2 / (2 ) These pictures were contributed by Professor J. J. Davis, Head of Entomology Dept., Purdue University. They were taken in I9II when he was with the U. S, D. A. Bureau of Entomology.
44
Fig. 4.
Light trap*
45
m
Fig* 5» Cage for May beetles used for obtaining eggs.
Fig* 6* Embedding oven and test tube of vials containing eggs in embedding mass.
46
Fig* 7* Test tube rack of vials containing eggs in embedding mass, and microtome.
Fig* 8 * Warming plate for slides,
47
. ‘ï .TACk*
Fig* 9* Longitudinal section of egg just after oviposition showing the cytoplasmic cap in the polar region, x 110.
Fig. 10. Longitudinal section through posterior half of egg showing saucer shaped mass of cytoplasm, x 110.
48
4 t m
%
Fig. 11. Longitudinal section through the polar region showing rods of protoplasm just under a micropyle, x 625
V£
Fig. 45. Transverse section at anterior end of the germ band showing the horseshoe-shaped mass of cells along the midventral line, x 150.
69
i a
I
m
s i
m
Fig. 46. Sagittal section through germ band showing amniotie fold starting its migration toward the posterior end. x 100.
Fig. 47. Transverse section near the middle of the embryo showing the neural ridges and the amniotie fold beginning to form. X 150.
70
Fig. 48. Longitudinal section through the germ band showing the anterior amniotie fold approaching the posterior fold which has just formed. The cells on the inner side of the germ band are believed to be the cells which will form the mesoderm, x 100.
71
M
Fig. 49 . Transverse section near the middle of the egg showing the lower layer and the inner layer. The inner layer or mesoderm has divided into two lamellae. Coelomic sacs are beginning to form at the lateral edges of the mesoderm, x I60.
72
Fig. 50* Transverse section near the middle of the egg showing coelomic sacs connected by a mesodermal, bridge. The neural groove lies between the two neural ridges which are composed of neuroblastie cells. X 135-
Fig. 51* Developing embryo laying on top of the yolk cells showing sunken medial nerve cord at the bottom of the neural groove, x 135*
73
\ V
^ r.
» *
V
v^>^«eC
. ♦
X
i,
Fig* 52. Transverse section of the anterior end of the embryo shoving neuroblasts separating from the ectodermal layer. The medial cord and coelomic sacs are also visible, x 135*
i
Fig. 53# Transverse section at the posterior end showing coelomic sacs below and layer formation in the mesoderm above, x 55#
74
#
Fig. 54. Sagittal section of the embryo showing coelomic sacs* and the indentations at the anterior and posterior ends which furnish cells for the foirmation of the anterior and posterior mes enteron rudiments, x 55*
75
* ’1?,:
iJ
Fig. 55* Sagittal section of the posterior end of the embryo at the same stage as fig ure 54 to show cells with large nuclei and clear cytoplasm resting on the mesodermal layer. These are believed to be germ cells. X 600 •
76
I
Fig. 56. Sagittal section of the embryo showing formation of the mesenteron ribbons from the anterior and posterior mesenteron rudiments, x 55#
77
n
Fig. 57. Sagittal section of the posterior end showing the posterior mesenteron rudiment attached to the procto daeal invagination and sending out a mesenteron ribbon. X 150.
Fig. 58# Sagittal section of the anterior end of the embryo showing the anterior mesenteron rudiment attached to the stomodaeal invagination and sending out a ribbon to form the walls of the mid-intestine, x 150.
e
h
Fig. 59# Transverse section through the left side of the embryo. The splanchnopleure is located next to the yolk. Blood cells are shown under the splanchnopleure. Gardioblasts are visible. A large cell which is believed to be an oenocyte is shown in the developing body cavity, x 500-
CJ
6
%
Fig. 60. Same section as 58» but showing the ventral nerve cord, also, x 130.
79
#
Fig. 6l. Longitudinal section of the posterior end showing the proctodaeum and the amniotieserosal membranes starting to fuse, x 130.
%: lii Fig. 62. Transverse section of dorsal side showing amniotic-serosal membrane migrating to the dorsal side where it will sink into the yolk and be absorbed, x l60
80
V
#
Fig. 63. Sagittal section showing the protocerebrum, deutocerebrum, subesophageal ganglion, and first thoracic -abdominal- ganglion, x I30.
■
m
-
k
Fig. 64. Sagittal section through anterior end showing the brain and the subesophageal ganglion. Connectives from which tritocerebrum is believed to develop is shown at the base of developing brain, x I30 .
81
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Fig. 65. Invagination showing commissure running under the stomodaeum connecting the ganglia of the trito* cerebrum, x 110.
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Fig. 66- Horizontal section showing paired tritocerebrum and also a pair of nerve cords leading forward to the location of the frontal ganglia, x 110.
82
Fig. 67• Horizontal section through stomodaeal in vagination showing deutocerebrum on each side. Nerve cords originating at tritocerebrum are also shown on the outside of the deutocerebrum. x 110.
-
-
Fig. 68. Horizontal section showing protocerebrum on each side of the stomodaeum. x 110.
83
Fig* 69* Section showing neuropils of the connectives and commissures* x 110.
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Fig. 70. Sagittal section of the pi euro podia on the first segment of the abdomen, x 110*
84
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Fig. 71. Transverse section showing gonad attached to the splanchnic layer in the segment.
X 110.
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.
Fig. 72. Transverse section showing absorption of the amniotic-seresal membrane, x 110.
85
Fig. 73# Transverse section of the dorsal side of developing embryo showing beginning of the hea.rt formation, x 110.
I
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Fig. 74. Transverse section through the anterior end show ing paired indentations of the lateral sides. These indent ations are the beginning of tracheal formation. The dark mass in the yolk on the dorsal side is the disintegrating amniotic-serosal membrane. The ventral nerve cord appears on the ventral side, and part of the stomodaeal invagination is located above the nerve cord, x 110.
86
4#
i
Fig. 75* Transverse section showing the heart forming on the dorsal side* x 115#
Fig. 76. Longitudinal section of the heart at the emterior end of the embryo.
X 1000.
87
1
Fig. 77* Sagittal section of the head showing connectives leaving subesophageal ganglion. The brain, mouth, and esophagus are also shown, x 130.
Fig. 78# Transverse section of sixteen day old leirva to show gonad located on the left side of the section just outside the mid-intestine, x 115*
88
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Fig. 79# Sagittal section of a portion of thé thorax and abdomen in a fully developed embryo showing muscles and spiracle on prothorax. X 130.
89
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y 5^5
Tig. 80. Sagittal section of mature larva showing heart, mid-gut, ventral nerve cord, part of brain and frontal ganglion, mouth, esophagus, hind intestine, and anus, x 50.
VITA Name - Philip Luginbill, Jr. Born - August 17, 1917, Columbia, South Carolina. Education - Grade School - Columbia, South Carolina and Monroe, Michigan. High School - West Lafayette, Indiana. B. S. in Agriculture - Purdue University, 1939# M. S. in Agriculture - Purdue University, 1941. Professional Organization
- Sigma Xi Thomas Say Entomological Society Theta Alpha Phi
Experience - Field Assistant in Entomology with Purdue University two summers. Grasshopper Control with U. S. D. A. - two summers. Officer in Navy - four years. Defense Plant Work.