Table Of ContentTABLE OF CONTENTS
Introduction............ 1
Acknowledgements.......... 2
Materials and Methods................ 3
Brief Resume: Incretory Structures in Insects • 12
Gross Anatomy of the Retrocerebral Complex . . 14
The Generalized Concept ................. 14
The Complex in the A d u l t ............. 15
The Corpora Allata of the Honey Bee • • • • • • 20
Historical.............................. 20
Adult A n atomy.......... .23
Adult Histology and Cytology ..........24
Embryogeny.............................. 26
Developmental Anatomy, Cytology,
and Histology......................27
The Corpora Cardiaca of the Honey Bee • • • • • 30
Historical.............................. 30
Adult A na tomy..........................32
Adult Histology and Cytology........... 34
Embryogeny; Developmental Anatomy,
Cytology, and Histology........... 34
The Paracardial Commissures ............ 36
Morphological Aspects of the Complex • 36
Summary...................................... 41
Bibliography ............................ 42
P l a t e s .................. , . . . 4 7
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AN ANATOMICAL, HISTOLOGICAL, AND EMBRYOLOGICAL STUDY
OF THE RETROCEREBRAL COMPLEX IN THE
HONEY BEE (APIS MELLIFSRA L.)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
by
BLAKE BJ. HAN AN, B.A., M.Sc.
The Ohio State University
1952
Approved by*
INTRODUCTION
Personal experiences in various phases of applied
beekeeping came at a time when fundamental research
was being conducted on insect parahormones and great
medical advances were being made through the appli
cation of vertebrate hormones to humans. The applied
aspects of endocrinology to the field of apiculture
appeared to be a fertile field for research. Cer
tain questions were particularly intriguing. To what
extent were incretory structures present in the honey
bee? Could such secretions as they produce be used
in the artificial regulation of processes and behavior
such as ovulation, secretion of food and venom, and
swarming? An analysis of the proper approach to the
field of research involved fundamental studies on
incretory structures of the honey bee, their physi
ological role, and the applied aspects of endocrinol
ogy in the field of apiculture. Arduous search
through the literature revealed little reliable infor
mation on the anatomy of such structures in the honey
bee as are known to produce parahormones in other
insects. Thus a study of the structures would have
to be undertaken before an understanding could be
achieved as to the possible role of parahormones in
the honey bee.
The so-called retrocerebral complex of the
honey bee was chosen initially for investigation
and the entire research program confined to its
structures and relationships.
It is hoped that the results of this investiga
tion will lead into research on the physiological
role of these structures and thence, possibly,
into the applied aspects of endocrinological api
culture.
ACiQTO'WXEDGEMENTS
Sincere gratitude is extended to all who have
had a part in the completion of this work. Prom
among such individuals who have provided insight,
assistance, cooperation, inspiration, and materials,
the writer wishes to express his special apprecia
tion to the following: Professors Winston E. Dunham,
Charles A. Reese, Willard C. Iflyser; and John A.
Knierim, a graduate assistant; as well as to Ernest
and Ronald Powler.
2
MATERIALS AND METHODS
COLLECTING
Honey beeB in which adult structures were studied
were secured from several sources* Old workers were
collected from flowers, and young workers were obtained
from brood nests of bee colonies* Drone bees, regard
less of age, were collected from the Ohio State Uni
versity apiary and the writer’s apiary. Virgin queens
were reared by using a modified Doolittle culture
method and also by procuring queen cells from swarming
colonies and incubating them at 92° * 1°P. Spent
queens were obtained from beekeepers as a result of
their requeening programs* Larvae and pupae of known
ages were collected in colonies in several apiarieB,
FIXATION
Adult material collected early in the study was
fixed in a 10^ formalin cold-blooded Ringer* s fixative.
ThiB was not too satisfactory because staining tech
niques used later were found to be more effective after
Zenker’s fixation. Even with double fixation, mater
ial fixed originally in formalin was not nearly of the
cytological quality as that fixed originally in Zen
ker’s. The advantage of formalin fixation lay in
its rather non-critical fixation time limit. Con-
sequently, the formalin technique was employed when
material was collected by others and fixation time
could not "be controlled.
After fixation in either solution, all tissues
were stored in 70% alcohol. Tissues fixed in
Zenker’s fluid were washed in iodized 70% alcohol
before storage.
Larvae and pupae obtained by the author were
fixed for 12 to 18 hours in Zenker* s solution, then
washed in iodized 70% ethyl alcohol, and finally
stored in 10% alcohol.
Pupae furnished by Dr. W. C. Myser of Ohio State
University had been labelled as to age and had been
fixed for 18 to 24 hours in Peterson* s fluid
(K.A.A.D.) which has the following formula:
Kerosene, commercial grade 1 part
Glacial acetic acid 2 parts
Ethyl alcohol, 95% 10 parts
Dioxane 1 part
Following fixation in this fluid, the material had
been rinsed quickly in water and stored in 10%
alcohol.
It was discovered that Peterson* s fluid was not
a suitable fixative for critical histological or
cytological study of honey bee tissues. It has many
good qualities as a larval fixative. Cells thus
fixed take nuclear stainB well, but do not produce
as clear-cut cellular characteristics under magni
fications greater than approximately 100X as do
either Bouin*s or Zenker’s solutions.
TECHNIQUE; ADULT TISSUES
Attempts were made to section the entire head
capsule and contents. This procedure was unsuccess
ful because of the heavy aclerotization; therefore
the following method was used to remove the retro
cerebral complex from the head. While the anesthe
tized insect was held in the fingers, the occipital
region of the head was clipped off. The mouth parts,
ventral head region, and then the lateral aspects of
the head were similarly removed. This left in place
adjacent to the neck, the greater portion of the
brain and esophagus, the retrocerebral complex, and
a more or less square portion of the frons and
antennal region that still hung to these parts anter
iorly. The insect was pinned in a small dissecting
dish in 70% alcohol and, with the aid of a dissecting
microscope, the frontal wall of the head capsule was
then detached and the remaining soft tissues includ
ing the complex were clipped free of the thorax at
the neck.
5
After the complex was removed from the head
capsule, it was dehydrated through an alcohol series.
The tissues were kept for 10 to 15 minutes first in
8 5 then 95^, and finally in 100/& ethyl alcohol.
After dehydration, the tissues were cleared in
xylene for 5 to 10 minutes. Cedarwood oil was used
initially hut in the case of these particular adult
tissues, xylene was found to he more rapid and at
the same time cause no great amount of overhardening
or shrinkage.
Tissues thus dehydrated and cleared were trans
ferred directly to the melted imbedding medium,
52° - 55°F. Fisher’s Tissuemat. Infiltration time
on adult tissue was never less than two hours and
more frequently overnight.
The problem of orienting small pieces of tissues
was difficult. This was eventually solved by putting
the tissue, together with the Tissuemat, into a warmed
3 mm slide well. At the moment the Tissuemat had
cooled sufficiently to hold the material in the
desired position,a drop of cold water was pulled acroBB
the surface of the Tissuemat with a pipette to harden
the surface rapidly. The whole slide was then im
mersed in cold water to reduce crystal size. This
procedure may not be new but it has several things in
its favor when imbedding small material. It reduces
the chances of poor orientation and thus cuts down
the number of replicas needed to obtain one satis
factory series. The imbedded material is also very
easily labelled and stored in the slide well.
After imbedding, the block was cut from the well
and trimmed for sectioning. This was facilitated by
the lower side of the block being already flat on
removal from the slide well. Sections of adult tis
sues were finally standardized at 6 microns in
thickness. Sections under 6 microns did not sec
tion or flatten well, whereas those above 8 microns
were too thick for critical cytological studies.
A number of nuclear stains were tested on these
tissues. The stains that yielded the best results
were iron hematoxylin, methylene blue, and phospho-
tungstic acid hemotoxylin. The latter stain was
finally used exclusively. Methylene blue produced
beautifully stained neural tissue, but staining
times were so critical and the washing effects of
alcohol were so acute, that only a small percentage
of the finished slides were of high quality. Most
of the hematoxylins (Ehrlich*s, Delafield's, etc.)
produced too diffuse an effect for critical cyto
logical use. Both iron and phosphotungstic acid
hematoxylin (Jones, 1950) yielded very clear, clean-
line staining. The latter stain was chosen primarily
"because it produced a greater degree of differential
staining, especially in muscular, glandular, and
neural tissues. Cytoplasmic counter stains of eosin,
and orange G. were tried but discontinued since they
added little, if any, value to the differential
staining effect mentioned above. All sections were
mounted serially in Piccolyte,
TECHNIQUE« POST-EMBRYOLOGICAL TISSUES
Basically, the slide preparation technique was
patterned after Dr. Myser* s methods in his study on
the post-embryogeny of the honey bee. The following
brief outline will describe the handling of larval
and pupal material.
From storage, after employing either K.A.A.D.
or Zenker* s fixation, pupal heads and necks were
clipped from the thorax and dehydrated upward through
an alcohol series. From absolute alcohol the speci
mens were passed through gradually increasing con
centrations of cedarwood oil.
Later xylene was substituted for the slower
cedarwood oil technique. This was satisfactory,
however, only when the tissues had been dehydrated
completely (4 hours or more in two washes of abso-
