ALBERT R. MANN
LIBRARY

Tia

TECHNICAL BULLETIN NO. 34. JULY, 1917

f MICHIGAN AGRICULTURAL COLLEGE

EXPERIMENT STATION

DIVISION OF ENTOMOLOGY

A STUDY OF THE FACTORS WHICH GOVERN MATING IN
aac THE HONEY BEE

BY
GEORGE D. SHAFER

EAST LANSING, MICHIGAN
1917

Cornell University

Library

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924003712084

TABLE OF CONTENTS.

The Project

Structural Characters of the Reproductive Organs. ...........-..000ee ee eueee

a. ‘Organsvof the Queen». 0.5... ahha kaw vas ae eS oe SA a ee Me ee
by (Qrparis: Of the. Drone cd weeded Gian soe idl ac had duadeeha does ag Rahat ay ee
Position Assumed by the Queen and Drone in Coition, on the Mating Flight....
Attempts to Control either Mating or “Fertilization” of the Queen Honey Bee. .
I. Classification of Certain Recorded Attempts to Control Mating............

a. Instances of Mating during Confinement in Onc Way or Another........
b. Instances of ‘Artificial Fertilization” by Hand................00.0000-
II. Attempts, at Controlling Mating, Carried Out Under This Project........

Hegends: forthe: Piguressssiv 03s eh-ohtietdass Ga seats aay eee bee A eater oA

A STUDY OF THE FACTORS WHICH GOVERN MATING IN THE
HONEY BEE.

BY GEORGE D, SHAFER.

THE PROJECT.

For the project as entitled above it was proposed to undertake a
study which should be directed, in the main, along the following lines
of work:

1. An examination of available literature relating to the problem.

2. A study of the primary and secondary reproductive organs of the
drone and the queen, with respect to relationships of the organs to each
other—in the case of the primary organs, including a histological as
well as an anatomical study where that seemed advisable:

3. An attempt to discover mating habits by observations under field
or apiarian conditions or by study after resorting to devices which
modify those conditions.

4. Certain experimental mating tests, designed in such conformity
with the instincts and other factors found to govern natural mating,
as might possibly lead to some practical method of control—i. e, a
method of mating a selected drone with a certain queen.

These lines of work have not been followed out fully to the author’s
satisfaction, but it seems best to report, at this time, some results ob-
tained during the past season—1916.

The facts are now well known that, normally, the virgin queen meets
the drone in the air on her “mating flight” and that on returning to
her hive from that flight, the successfully mated queen bears a part of
the torn drone organ in her vulva. Huber* has been credited with
having first related these facts, and his description shows that the dis-
covery was original with himself and his assistant. His little book
figured, particularly, one part of the drone organ as lost in commerce
with the queen (see his fig. 7, Plate II). His figure represents the
“bulb” shown herewith in Fig. "4, B. Von Siebold,j also, 1853, learned
by direct examination that “those definitely formed parts in the vagina
of the queen just matedt were nothing but the torn copulative organs
of a male bee (drone),” but neither he nor Huber carried their studies
in this connection further, and apparently no one has recorded any
attempt to do so, up to this time. Early in the work on this project, it
occurred to the writer that a careful examination of the torn male copu-
“sAppendix, “New Observations on’ the Natural History of Bees,” by Francis Huber, 1821
(Translation)—Langstroth in “Hive and Honey-Bee.’”’ (1862) says in _a footnote that Posel
and Jansha were acquainted with certain of these facts as early as 1784.

+“A True Parthenogenesis in Moths and Bees,” by Carl T. E. Von Siebold, 1857, (Trans. by

W. S. Dallas).
tItalics inserted to make the context clear.

6 EXPERIMENT STATION BULLETIN.

latory organ in the vulva of the newly mated queen might give Bee
evidence of the position assumed by the queen and drone relative to
each other in the moment of coition. The importance of gaining post-
tive knowledge in this connection is evident. Before attempting to
intercept queens returning from the mating flight in order to make the
examination just suggested, however, it was only natural to wunder-
take a careful first hand review of the structural characters of the re-
productive organs in the queen and drone—especially of the copulatory
organs in the drone. This report has to do, therefore, more particularly
with a study of the male and female reproductive organs and with the
relation of these organs in sexual union as demonstrating the relative
position assumed by the drone and queen in that act.

STRUCTURAL CHARACTERS OF THE REPRODUCTIVE ORGANS.

The structural features of the organs of both sexes of the honey-bee
have been studied, described, and illustrated by more than one student.
Swammerdam’s drawings of the reproductive organs of the queen and
drone, published in “The Book of Nature,” were the first illustrations
to exhibit the essential gross characters of those organs. Later,
Cheshire (1886) in the first volume of his “Bees and Bee-Keeping,”
published illustrations of both the male and female organs of the hive-
bee. In 1910, Snodgrass, Tech. Bulletin No. 18, U. 8S. Dept. of Agr.,
Bureau of Entomology, has also given original drawings of the repro-
ductive organs in the queen and in the drone honey-bee. Attention is
called to the drawings of these three workers in particular because their
illustrations of the organs mentioned have been accepted and copied
in manuals and books on bees in general use today.* A few observa-
tions made in the study carried on in this connection should, perhaps,
be recorded.

a, ORGANS OF THE QUEEN.

Cheshire has omitted one of the glandular vessels emptying into the
base of the sting (see Snodgrass—B. gl., Alkaline gland of the Sting)
in his Figure 42 entitled “Ovaries of the Queen, etc.,” p. 218, Vol. I of
“Bees and Bee-Keeping.” Also he has represented the poison sac as
emptying into the base of the sting on the ventral side of the vagina,’
or common oviduct, in the same figure which is designated an “under
side” view. In the queen bee, the base of the sting and the duct empty-
ing into it from the poison sac lie dorsad (instead of ventrad) to the
bursa copulatriv and the vagina. Figure 57 named “Reproductive
organs, sting, and poison glands of the queen, dorsal view,” as given
by Snodgrass in the reference cited, represents the organs in their true
relation. Swammerdam’s drawing of the ovaries of the queen, etc.
omits the alkaline gland of the sting mentioned above, but the other
structures and connections are faithfully represented as they appear in
a dorsal view where the muscles at the base of the sting have been sepa-
rated along the middle line and bent to each side. In a dorsal view
these muscles of the sting very often almost or entirely hide the pair
of little side pockets at the posterior end of the vagina—i. e., the pockets

amore
*See the footnote on page 10.

DIVISION OF ENTOMOLOGY. 7

which, together with the posterior end of the vagina itself, form the
structure commonly named the bursa copulatrix, Fig. 5. The drawings
of Snodgrass and Swammerdam just mentioned, however, show clearly
the relation of the large pair of ovaries and the pair of oviducts leading
into the common median vessel or vagina—also the globular seminal
receptacle, or spermatheca, with its attendant white accessory glands.
The conmon duct of these little glands passes around underneath the
spermatheca, where, in conjunction with the latter, it connects with the
upper end of the short spermathecal duct which, in turn, opens through
the dorsal wall of the vagina very close to its anterior end. Swammer-
dam has represented the spermatheca pushed to one side in an attempt
to show its duct connecting with the dorsal part of the vagina. —

b. ORGANS OF THE DRONE.

Snodgrass has given the most instructive and accurate illustration
of the reproductive organs of the drone, natural position, dorsal view,
in his Fig. 56, A. Briefly, they consist of the penis, the ejaculatory
duct and a set of paired organs—the testes, the vasa deferentia, the
vesiculae seminales and, finally, the accessory mucous glands. The lat-
ter are connected by the slender ejaculatory duct with the large in-
vaginated penis which lies entirely within the abdomen along the
median line between the paired organs. Of this latter set of
organs, the accessory mucous glands are the larger. They have the
form of two long sacs, slightly constricted near the middle, having the
blunt closed ends turned anteriorly. The narrowed posterior ends of
the glands bend toward each other and meet on the median line close
above the base of the penis at the extreme caudal end of the body. The
walls of these glands, at the blunt rounded ends, are comparatively
thin (Fig. 6). Beginning at the region of the constriction, however, the
walls gradually become much thicker posteriorly, due principally to
the presence of a greater number of muscle fibres. In the region of
greatest muscularity, the fibres are arranged more or less regularly
into three layers. Figs. 7 and 8 are photomicrographs of a cross section
made about one-fourth the length of the gland from its posterior end.
They show on the outside a laver of longitudinal fibres; immediately
within that, circularly arranged fibres; and, finally, within that next
to the glandular cells lining the lumen, a layer of longitudinal fibres
marked “ec.” The lumen of the gland is usually packed to distention
with a white granular homogencous fluid. The active spermatozoa
originate in the pair of testes and pass through the coiled vasa defer-
entia into the long narrow, cylinder-like, curved vesiculae seminales or
seminal vesicles there to be stored in quantity. The vesicle walls are
muscular throughout their length, and of about uniform thickness.
Lying upon the basement membrane of the cells lining the lumen of
each vesicle (Figs. 13 and 14) is a layer of circularly arranged muscle
fibres which are cut lengthwise in cross sections of the organ itself.
Next outside the circular layer is a somewhat thicker layer of longi-
tudinal fibres, and outside of that is an envelope consisting mostly of
loose circularly arranged connective tissue fibres and muscle fibres in-
terwoven with many small tracheae. The right and left seminal vesicles
each connect by short bent ducts with the narrowed bases of the cor-

8 EXPERIMENT STATION BULLETIN.

responding accessory glands. These two glands empty ventrally, on
the median plane, into the ejaculatory duct, which passes at once
dorsally to the left and forward in a broad arch to where it joins the
anterior end of the resting penis. Snodgrass has given, also, at 104
in his Fig. 56 (reference) an excellent side view of the resting invagi-
nated copulatory organ showing its basal pouches (marked “LZ in his
figure) unfolded and lifted up. This organ, as well as the ejaculatory
duct, is essentially a chitinous tube surrounded by its layer of hypo-
dermal cells. Externally, the tube opens by a wide crumpled slit on the
median line far toward the posterior ventral side of the drone’s abdo-
men. In the resting condition the genital slit is entirely hidden by the
fused triangular plates bounding it on the anterior ventral side and
by the dorsal genito-anal cover plate. At the extreme posterior end
(just anterior to the external slit) the penis wall is very thin and from
its sides arise the large membranous pouches or premmophyses (42)
already mentioned. Attached about the wrinkled base of the penis
even up to the origin of the pneumophyses are many small tracheae the
special significance of which will appear later. These tracheae may
easily be seen under the binocular microscope in a properly prepared
dissection of the drone’s abdomen. Likewise, such a dissection, or refer-
ence to Snodgrass’ drawing or to Plate I of this bulletin, will show
how singularly the wall 6f the copulatory tube is differentiated in the
remainder of its length both as to shape and chitin structure. On
the ventral wall just anterior to the bases of the pneumophyses is an
oblong, somewhat quadrangular, thick chitinous plate (Fig. 3, xx).
Immediately anterior to this plate is a series composed of three narrow
cross-striped chitinous ridges and four more weakly chitinized ones
with corresponding creases—the whole set appearing, at first sight,
somewhat as a broken or interrupted spiral band (vy, Fig. 3). On the
dorsal wall opposite the anterior end of the ventral quadrangular plate
is a triangular plate of chitin (ww) having its broadest or basal portion
directed backward. Both of the plates and the stronger cross-striped
chitin ridges are clothed with spinules or hairs inside the resting tube.
The extreme forward end of the copulation tube is greatly enlarged
into a pear-shaped bulb (B, Fig. 3) which goes over in the ejaculatory
duct (2jD) at its anterior point. In the dorsal wall of the bulb is a
pair of symmetrical chitinous plates (tt) one lying on either side of
the median line. They are widest at their anterior ends and taper to
slender, parallel prongs (P) at the posterior ends. Also each plate
bears posteriorly by a thin arm a little chitin-wing (we) on the cor-
responding side of the bulb. The hypodermis of almost the whole dorsal
wall of the bulb presents a double longitudinal fold, which gives rise
not only to the symmetrical plates and little chitin wings already de-
scribed, but also toa large clastic thickening (named by Snodgrass) of
tough gelatinous-like consistency, This elastic thickening is shown in
raetip ee after eversion at ss" in Fig. 9 and in surface view Fig.
. the thinner portions of the penis wall are whitish in appear-
ance, while the heavier chitinized portions (i. e., plates) are yellowish
ne ee ae _ Finally, arising from the dorsal side of the

ately posterior to the bulb is a thin-walled doubly pinnate-

DIVISION OF ENTOMOLOGY. 9

lobed projection, (ww, Fig. 3) the lobes of which are turned toward
the bulb.

It will thus be seen that the dorsal and ventral walls of the penis
tube bear distinguishing characters. This is equally true after the
organ becomes evaginated, as it does in the act of copulation. In that
act it is generally believed that the whole tube of the penis turns out
through the genital slit into the vagina of the queen. That is, the
pnheumophyses evaginate into the pouches of the bursa copulatrix—the
pinnate lobe and the bulb everting in turn and pressing deeper within
the vagina. It is well known that if a flying, excited drone is caught
and held between the thumb and finger or in the hollow of the hand,
the copulatory organ will sometimes be suddenly everted with force.
The abdomen contracts rigidly to less than half its former size as the
organ is expelled, and the insect quickly dies. AJso, as a drone is held
between the thumb and finger, pressure on the abdomen (from before
backward) may often be used successfully to fully expel the organ—
especially when the tracheal system is well filled with air. Cheshire,
at “E” Fig. 41 (citation) and Michaélis,* Plate XXVI, have both given
illustrations of the drone organ in the fully extruded condition. In
order to help show the relation of the structural parts in the extruded
penis, an illustration of the same is also given herewith, Fig. 4. Pres-
sure, brought to bear upon the contents of the drone’s abdomen through
the contraction of the abdominal wall muscles, normally starts the
process of evagination. As the abdomen contracts, the confined air
in the vesicles of the tracheal system becomes compressed}; the blood
in the body cavity, however, inoves freely to the area of least resist-
ance—to the base of the penis. In that moment the crumpled tubular
penis wall, next the genital slit, is pushed through the slit and swells
out behind—expanded with blood. Eversion has begun, and with it the
small tracheae at the base of the penis are stretched lengthwise to the
limit. They yield and break as the large, ventral, quadrangular, spine-
covered plate rolls out and, thus, they release the compressed air of
the tracheal Vesicles within the evaginating fold of the partly protruded
penis. Instantly, the pneumophyses dart forth and curve downward in
the form of two hollow white horns, filled with aii and blood. The
cross striped ventral ridges and the dorsal triangular plate appear.
Each everting portion of the penis draws after it the next anterior
portion down through the part of the tube already evaginated until,
finally, the doubly pinnate-lobed appendaget begins to show. Then
the bulb snaps out and the process of evagination stops at the point
where the former anterior end of the bulb—now the distal end of the
extruded organ—connects with the narrow ejaculatory duct. The
opening of that duct may be seen in the upturned tip of the fully ex-
truded penis (0, Fig 4), and from there through the lumen of that
organ the slender ejaculatory duct itself may be traced to where it
still attaches to the accessory glands at the posterior end of the ab-
domen—sometimes the ends of the paired organs are found partially
forced into the distended base of the penis. The position of the ejacu-

*Michaélis, Georg. Bau und Entwicklung des Mannlicben Begattungs—apparates der Honig-
biene. Zeitschr. Wiss. Zool. LX VII, 1900, pp. 489-60.

7Djathchenko, Sophie, 1906 in Ann. de L’Institut Agron. de Moscou, figures the closing ap-
paratus of the Stigmatal tracheae of the bee.

tThis appendage evaginates normally just as the bulb snaps,

10 EXPERIMENT STATION BULLETIN.

latory duct in the fully evaginated male organ is indicated by dotted
lines in Fig. 4. Figs. 12 and 9 show the ejaculatory duct in longitudinal
and in cross sections of the evaginated organ, respectively. Michaélis
(page 456, citation) understood this position of the duct in the extended
organ, but he did not figure it. The elastic thickening, which gave a
bulbous or convex form to the dorsal side of the penis bulb in its rest-
ing condition, now presents itself as a concavity or cup in that same
side of the everted bulb (c. Fig. 4). Above the outer or distal edge of
the cup is the opening from the ejaculatory duct (0, Fig. 4), and before
the opposite edge stands the fully expanded doubly-pinnate lobe facing
toward the month of the duct, while from the edges of the cup on either
side of the pinnate lobe arise the pair of little chitin wings (we). The
plates which bear these little wings lie partly covered by the elastic
thickening, now, with their slender free prongs (p, Fig. 4) directed
proximally.

The above description of the extruded drone organ and of the act of
extrusion does not agree in certain essentials with the description
given by Cheshire. Microscopic sections of the ejaculatory duct show
that its walls are not muscular (Fig. 10). Both longitudinal and cir-
cular muscles occur in the walls of the seminal vesicles and in the
walls of the posterior portions of the accessory glands, however; but
these muscles serve another purpose* and are not in a position to assist
in the evagination of the penis. On page 202, Vol. II, Cheshire states
that “The bean, and the remaining parts, from o to mm, are surrounded
by a membranous sheath, which remains intact after the expulsion
mentioned.” In accordance with that statement, his drawing, A, Fig.
41, p. 199, shows two walls from “o to a”—i.e., the bulb and the remain-
der of the resting copulation tube is represented as lying within a
sheath which, itself, connects with the wall of the ejaculatory duct.
Now, dissections and microtome sections of the resting male organ
show the wall to be single (see Fig.“11), and, indeed, the wall of the
ejaculatory duct goes over into the wall of the bulb. Unfortunately,
the drawing A, Fig. 41 of Cheshire, mentioned above, has been copied
as late as 1908 in Berlese “(li Insetti,” Vol. I, p. 864.

In case of almost all the dissections made of drones, taken before
the hive entrance, the bulb of the resting sexual organ was found to
not contain spermatozoa in quantity, and no “spermatophore” was
found. Cheshire’s spermatophore} as described on page 203, Vol. II
appears, in reality, to possibly be the elastic thickening of the wall
of the resting bulb. In the few instances in which spermatozoa were
present in the dissected resting bulb, it is believed that they were
forced there by incomplete muscular contraction of the “paired organs”
during the process of opening the drones’ abdomens. Several times,
while studying the organs of a drone that had been opened under water,
the paired organs contracted somewhat so that—through the partially
transparent wall of the ejaculatory duct—the granular sperm fluid
oe be seen as it was forced from the accessory glands. All of this
eads to the belief that the pair of seminal vesicles and the posterior

*See line 4, p. 15; also, last part of ie alas 11.
0,

: quotes Leuckart as describing a sperma-
ge, in case of the honey-bee. I bave not seen. Leuckart’s
drone’s reproductive organs

DIVISION OF ENTOMOLOGY. Il

ends of the accessory glands (not the “bean” or bulb)* are the regular,
normal, and final reservoir for spermatozoa in drones able to fly.

_ Since the bulb was regularly found to be empty, rather than fillel
with spermatozoa or any definitely arranged compact spermatophore,
the question may well arise as to the actual reason for existence of the
bulb-cavity and the swollen ventral wall of the same. It is possible that
during the final marriage flight the bulb-cavity may receive some
spermatozoa; that may not be determined—however, a necessary and
actual use of the cavity may be observed by holding a drone and care-
fully watching the large dorsal elastic thickening and the accompany-
ing dorsal plates as the bulb is made to pass slowly through the process
of eversion. It will be seen that the thickening and plates together,
although elastic, can not make a short bend during evagination as does
the thinner portions of the wall, but that in effect, they must somer-
sault or swing, in a body, around the “prongs” of the plates as a pivot
within the cavity afforded by the expanded ventral wall of the bulb. In
other words, the large elastic thickening of the bulb with the pair of
accompanying plates could not evert if the thinner opposite wall of the
bulb were not correspondingly enlarged. The use of the elastic thick-
ening and the pair of bulb-plates, themselves, will appear later (see
page 15).

When the characters of the reproductive organs of the drone and
queen had thus been reviewed carefully and fixed in mind, measures
were taken to observe queens leaving the hive on the mating flight and
to intercept them on their return in order that the exact orientation
of the torn male organ in the vagina of the queen might be studied.

POSITION ASSUMED BY THE QUEEN AND DRONE IN COITION, ON THE MATING
FLIGHT.

The queens used in making observations on the “mating flight” were
obtained directly from nursery cages kept in a hive body above the
brood chamber of a strong colony, and separated from it only by a
queen excluder. These queens were introduced into hive nuclei, con-
taining three to five regular Langstroth frames, either by the intro-
ducing cage, or by- the smoke method. Over the entrance to each nucleus
was kept an Alley’s Improved Queen and Drone Trap—the trap en-
trance at the back stopped to a small opening. The traps were used
in order that the virgins might not take flight except when the nucleus
was under observation. Other work made it impossible to watch the
nuclei all the time during that period of the day when the virgins were
most apt to attempt to fly. The plan, therefore, made it possible to
conserve virgins for those periods when observations could be made and
in that way much time was saved. Any immediate or recent movement
of the queen from the nucleus into the trap was quickly made known
by a certain excitement of the workers in the trap-entrance and at

*Two other considerations would make it seem unlikely that the cavity of the bulb should
be regarded as the final reservoir for spermatozoa before their ejection into the vagina. First,
the large amount of sperm fluid found in the vagina of the “newly mated’ queens examined
(as explained later, p. 12) seemed much more than could be contained by the space of the bulb-
eavity. Second, no such muscles as are found in the walls of the seminal vesicles and acces-
sory glands (Figs. 138, and 7 and 8) would be needed to pass the spermatozoa along slowly to
the “bulb,” there to be stored. The heavy musculature in the walls of these paired organs in-

dicates’a function of squirting the sperm fluid through the ejeculatory duct with considerable
force.

12 EXPERIMENT STATION BULLETIN.

such moments she was usually eager to fly when quietly se ae sles
the trap. If, on the other hand, the queen had been in the ls aoe
time, having come out when the nucleus was not under aa ie a if
presence was almost always marked by a small cluster of worke ee
flight did not often occur in the latter case, and as a rule Ee c) oe
better to simply allow her to run back into the nucleus—no difficulty baer
experienced with any queen when the trap was momentarily remov

and she was placed in the nucleus entrance. : Ff

Of course, when a virgin was once released and took wing, wate
had to be kept for her return home, but the trap would then prevent
her entry until she could be captured for study, or allowed to “run in
according to the wish of the observer. . ; :

Nine virgins were observed in eighteen flights. Five of the nine
were lost, but the other four were captured “newly mated,” with the
torn drone organ present in the vulva, before their respective nuclei.
The vaginae of two* of the four queens were dissected open carefully
in order to examine the part of the drone organ present and in both
cases the bulb only, of that organ, was found. A study and comparison
of the two showed that both bulbs had been everted. Studied with
reference to orientation in the vagina, the gelatinous-like thickening
forming a concavity or cup, in each case, faced dorsalward and anterior-
ly. The convex side of the bulb was turned ventrally and_ slightly
toward the mouth of the vagina. The “prongs” of the two symmetrical
bulb-plates were directed posteriorly and dorsally—their curved points
against the dorsal wall of the vagina. Almost all the membranecous
parts had been stripped from the gelatinous-like thickening and chitin
plates of one of these bulbs. In case of the other, however, the penis
had been torn off between the pinnate-lobed appendage and the bulb,
while the ejaculatory duct also had been torn away at its point of at-
tachment with the latter. The part of each bulb which faced up and
anteriorly, toward the inside of the vagina (i. e., the cupped part of the
bulb), was covered with the thickish, white, coagulated sperm-fiuid.
This fluid, in fact, packed all the part of the vagina anterior to the
bulb, and in one queen the oviducts also were quite distended with it.
Figs. 17 and 18 show cross sections of one oviduct of this queen
through the portion most widely distended with the sperm-fluid. Both
spermatozoa and the granular secretion from the accessory mucous
glands are shown to be present mixed together. The seminal receptacle
of this same queen was sectioned and studicd. But very few of the
spermatozoa had gained access to itj—showing that the receptacle was
not filled by the first discharge of the spermatic fluid into the vagina
in this case, at least.

The other two of the four queens referred to in this connection were
not dissected. It Was deterinined for both, however, that the everted
bulb was the part of the male organ that showed as a “white pellicle”
in the vagina. One of these queens was caught upon her return trip
and held, while, with a pair of forceps, the bulb was gently worked
loose by pushing downward until it could be slipped out of the vulva.
As in the former two queens, the concave part of the bulb was turned

fie cee ens ee hydrate (water) solution-—1 ta 20
e queen ha cen out on her mating flight 22 mi a8 " 3 g
nucleus, mated ;—she was then killed within ave mice WPSh SNe Atee GAUSHE Hefore ‘het

DIVISION OF ENTOMOLOGY. 13

-dorsalward and forward, facing inside the vagina—i. e., toward the at-
tachment of the seminal receptacle (spermatheca).

The fourth queen had been gone only 15 minutes when she returned
mated. In her case it was not found necessary to remove the bulb
from the vagina in order to determine that its orientation was the saine
as in the three instances already described, and that it also was everted.
As soon as the bulb had been examined by turning it a little from side
to side with the forceps, this queen was placed at the entrance to her
nucleus and allowed to run in. Three minutes later the nucleus was
opened ;—it was the purpose to determine, if possible, what disposition
would be made of the bulb which had been left in the vulva. Workers
were found to be following the queen about very attentively, some of
them nipping at the torn male organ which seemed now about half
out of the vaginal opening. Very soon the queen passed around on
another frame, and when caught sight of again, the white penis-bulb
was lying on the hive bottom just beneath her. Whether a worker ac-
tually pulled the bulb from the queen was not observed, but she was
free from it within scarcely more than five minutes after she entered
the nucleus. Undoubtedly this bulb had been loosened a little in the
vagina by the forceps during the examination mentioned; but it was
noticed also that the elastic gelatinous-like thickening. was slightly
shriveled when the bulb was picked up from the hive bottom. In order
to determine whether this mating would really prove successful, the
queen’s wings were clipped and she was then introduced into a strong
nucleus, the hive entrance of which was covered with a queen and drone
trap. She was found out in the trap only once, three days after the
introduction. On the twenty-first day she had three frames of sealed
worker-brood besides other larvae and eggs. It would seem almost cer-
tain, therefore, that spermatozoa had been stored in the seminal re-
ceptacle.

If we consider the four cases just described together, now, the
practically similar statements with respect to orientation of the bulbs
in the vaginae will appear most significant only when considered with
the determination (stated in each instance) that the bulbs were fully
everted. This determination itself rests upon evidence afforded by
two observed conditions which should perhaps be clearly repeated in
this connection: ,

First, The pair of symmetrical bulb-plates, within the vagina, were
placed so that their “slender prongs” pointed dorsally and toward the
opening of the vagina in each case—i. e., toward the position that the
base of the copulatory organ must have occupied before it was torn
away, and that relative arrangement of the plates can occur only in the
fully evaginated organ (see Fig. 4)—not in the partially evaginated
organ.

Second, The concave surface of the gelatinous-like thickening was not
enclosed by the membranous portion of the bulb-wall, but the convex
side of the thickening was covered by that membrane (except in the in-
stance mentioned when the latter was almost entirely torn away)—a
condition, again, which can be true only of the fully evaginated organ.

One other newly mated queen, with male organ attached, was caught

14 EXPERIMENT STATION BULLETIN.

r of
rly preserved* for me by Mr. M. H. Hunt, a queen breeder 0
pedtecd Medipau. In this case the evaginated penis seemed 0 ae
been torn off in the region between the cross-striped chitinous io a
and the quadrangular plate. The somewhat shriveled, tom en 6) :
organ projected backward from the mouth of the queen's cae nee
was pierced from above by the sting. Fig. 19 is a alee Sia . f the
queen’s abdomen, showing the projecting male organ and the tip - ;
sting sticking through at Stn. Near the tip of the sting, on the side o
the organ corresponding with the ventral side of the queen, there ap-
pears what certainly seems to be the cross-striped chitinous ridges. It
will be remembered that these ridges are on the same side of the evagi-
nated penis as the convex portion of the bulb (see. Fig. 4). Microscopic
sections, made longitudinally in a dorso-ventral direction through the
entire abdomen of this queen, were attempted in order to show the drone
organ in place within the vagina. Unfortunately, the most valuable
part of these sections was ruined so that they do not give direct evidence
in regard to the orientation of the bulb. The cross-striped chitinous
ridges, however, indicate that the copulatory organ, in this case also,
was placed in the vagina exactly as was determined in the four instances
where the entire evidence had to be gained from the penis bulb alone,
since in those instances the convex side of the evaginated bulb was
turned ventrally (and outwardly) relative to the queen's vagina.

It hag already been given as the general belief that the penis tube
turns out or everts through the genital slit directly into the vagina of
the queen. The pouches of the bursa copulatrix of the queen, the man-
ner of extrusion of the drone organ, its size and its irregularity in form,
as well as the helplessness of the drone almost immediately after ex-
trusion, make it practically certain that this view is the correct one.
In accordance with this view it would be necessary for the genital slit
of the drone to be placed close to the outer vaginal opening before ex-
trusion of the male organ. That being the case, evidence has already
been presented by which it is possible to decide definitely the position
assumed by the queen and drone relative to each other during coition.

The evidence from the five instances described shows that each drone
presented himself in such a manner as enabled him to place the male
organ within the vagina with the dorsal side of the former against the
dorsal side of the latter—with the cup of the evaginated penis-bulb and
the mouth of the ejaculatory duct directed upward and inward toward
the duct-opening of the seminal receptacle. In order to ever accomplish
such a result there seems to be but one possible position which the drone
and queen may assume, relative to each other, at the moment of coition
—they must meet and clasp face to face. In that position, the abdomens
press together, naturally. The grooved tip of the fused plates bordering
the drone’s genital slit passes easily through the “sting notch” (sn, Fig.
15) to push the sting dorsalward and force the queen’s genito-anal cover
shields apart. Forthwith, we may imagine, the drone’s abdomen might con-
oa tne plant Crampton of the penis tube. This, then, would cause
ea - Hes rangular plate to press ventrally until it opened the

ne bursa copulatrix and allowed the pneumophyses to dart

“Killed in botling hot picric alcohol, ma

in 95% alcohol with distilled water, dau saat mixing a saiurated solution of DPicric acid

DIVISION OF ENTOMOLOGY. 15

into the “side pouches.” Thus, might the whole process continue until
the elastic cup of the bulb itself snapped over in its proper position and
directed the mouth of the ejaculatory duct toward the inner cavity of the
vagina. It is at that moment, most probably, that the muscular walled
seminal vesicles and accessory glands contract and send their contents
through the ejaculatory duct in such quantity as to fill the whole inner
vaginn and distend its walls. Also, it must be then that the winged,
concave, elastic bulb-thickening (assisted dorsally by the bilobed append-
age*) performs its principal function as an effective stopper to hold the
sperm-fluid within the vagina. When the drone falls back helpless, and
the copulatory tube becomes torn, the thinner portions of the tube walls
collapse; but the thick elastic part of the bulb remains firm—the “slen-
der prongs” of the bulb-plates holding securely against the dorsal wall
of the vagina—until after the gelatinous-like portion begins to shrivel,
when the whole bulb becomes dislodged.

In looking up references upon the mating of queen honey bees, two
quite definite observations were found recorded—both agreeing with the
determination given here as to the position necessarily assumed by the
queen and drone in sexual union. The essential facts of these two
records follow:

Mr. 8. A. Shuck (Am. Bee J., 1882, p. 789) records that he fastened
a virgin five days old on a thread ten feet long. He then secured the
thread at the end of an eighteen-foot pole which he raised in his bee
yard at about 2:30 o’clock on an afternoon when numerous drones were
flying. The drones chased the queen at intervals, and finally one of. them
caught her apparently face to face for a few seconds only; the drone
quickly swung back, head downward, snapped and fell to the ground.
The queen then bore the “evidence of her mating.”

Mr. E. A. Pratt (Gleanings in Bee Culture, and A B C and X Y Z of
Bee Culture by Root) says: “On June 21, 1888, I saw this mating take
place. The queen issued from the hive, took two circles and came within
five feet of my face, and was there met by a drone. They seemed to
face each other, clinging by their fore legs, their bodies being perpen-
dicular, and in this shape flew from my sight. * * * * *”

ATTEMPTS TO CONTROL EITHER MATING OR “FERTILIZATION” OF THE QUEEN
HONEY BEE.

The usual and normal method of mating of the queen honey bee is
on the wing and in the open. Many bee-keepers have asserted that it
can occur only in that way. The bee journals, however, record many in-
stances of successful attempts to control either the mating of queens, or
their fertilization, through confinement, or by some hand method. None
of these attempts have yet proven practical. Discredit or doubt ‘has
been placed upon some of the claims; and rather generally, they have
been ignored. Nevertheless, many of the recorded attempts seem to de-
serve consideration; and it may, perhaps, be profitable (without making
a complete review) to briefly classify the means used in the case of

*The flans of the bilobed appendage appear to have the purpose of pushing out to fill all
irregularities around the “prongs” of the bulb-plates between the “little wings” in order to
prevent a leak of the sperm-fluid there when the latter is sent in quantity, with much force,

into the anterior vaginal cavity. As has been pointed out, page 10, the bilobed appendage and
its little flaps are directed toward the bulb as they evert.

16 EXPERIMENT STATION BULLETIN.

twenty-four instances taken, mostly, from Gleanings in Bee Culture and

the American Bee Journal.
I. Classification of Certain Recorded Attempts to Control Mating: :
a. Instances of mating during confinement in one way or another.

: orkers allowed to fly in the open, but queens and drones con-
Ree - “Queen and Drone Toxcluders” so that they could fly only as
large enclosure of screen, cloth-netting, or glass. McLain, Am. Bee 2
Vol. 24, 1888, p. 487—6 queens successful; Davitt, Adv. Bee Culture
by Hutchison, 1905—100 queens in one summer; Church, Gleanings 1
Bee Culture, Vol. 34, No. 24—1 queen. ; :

2. Workers, queens and drones all confined in greenhouse, or in large
tent. McLain, Am. Bee J., Vol. 23, 1887—1 queen out of 6—later, 3
queens out of 6; Hohenshell, Am. Bee J., Vol. 23, 1887—2 queens.

3. Queen and drones confined in a small nucleus above a colony by
screen or by a Queen and Drone Excluding honey-board, but allowed to
fly in a small glass or cloth covered mating box placed over the nucleus.
Cramer, Am. Bee J., Vol. 17, p. 19—~ queens; Filbert, Am. Bee J., Vol.
16—1 queen; Hasbrouck, Am. Bee J., Vol. 14, 1873—many queens fertil-
ized, also had many failures; Arms, Am. Bee J., 1881, p. 50—has had
good satisfaction.

4. Virgin queen and drone, without workers, confined together at
vight time in a small mating chamber—glass or cloth-covered top. Has-
brouck, Am. Bee J., Vol. 15, p. 5193 queens; Fincastle, Am. Bee J.,
XVII, 1881—5 queens; Brown, Gleanings in Bee Culture, 1884, p. 674—
1 queen.

5. Virgins not able to fly from hive because of imperfect wings or be-
cause wings were clipped. Cook, Gleanings in Bee Culture, Vol. X, p.
544—1 queen; also Am. Bee J., Vol. 21, p. 154—rarely in confinement in
hive; Brown, Gleanings in Bee Culture, VIII, p. 33—1 queen; Davis,
Am. Bee J., Vol. 15—1 queen; Bagby, .\m. Bee J., Vol. 14, p. 553—1
queen.

6. Queen tied to a thread so that she could fly only above the home
yard. Shuck, Am. Bee J., 1882, p. 789—-I queen.

b. Instances of “Artificial Fertilization” by hand:

1. By squeezing drops of sperm-fluid from the drone organ into the
vulva of the queen. Balch, Am. Bee J., Vol. 23, 1887—had practiced this
on queens with defective wings for 15 or 20 years; McLain, Am. Bee J.,
Vol. 28, and Baldwin reported by McLain—-certain of 25 queens.

2. By squeezing contents of seminal vesicles (dissected out) directly
into the vulva. McLain, Am. Bee J., Vol. 23—3 queens.

3. By diluting sperm-fiuid and using a pipette or syringe to intro-
duce the fluid into the vagina. McLain, Am. Bee J., Vol. 23—6 queens

out of 27; Jager and Howard, Science, N. 8., Vol. XL, No. 1037, p. 720
—l1 queen.

*The sizes of the confining cages described tn the references cited i i
_ aa i in eas any 80 ft. high to a 2 quart elise a He en ee noe
. I. Root Co., is now conducting a matin nt i Arge gre y
fh tence Gate one ees arn g § experiment in a large greenhouse nearly 600

Phillips (Beekeeping by Phillips, p. 69) ha rted : ae
covered enclosure, Pithoek naceeces Pp ) has reported attempts at mating, in a large glass

DIVISION OF ENTOMOLOGY. 17

While none of the methods outlined have been of practical use in
queen breeding as yet, the instances of success in certain cases (as
claimed) should be sufficient to encourage further trials and study. In-
deed, great benefit might be expected from any method that would
enable a queen breeder to practice definite selection of the drone as well
as the queen in mating merely his “breeding stock.”

II. Attempts, at Controlling Mating, Carried Out Under This Pro-
ject.

Before the close of the season (1916) fourteen virgins were used in
numerous attempts to bring about mating with a particular, selected
drone in each attempt. The conditions under which these trials were
made follow:

1. All the young queens were confined to nuclei of young worker
bees, before and after the tests, by Queen and Drone Excluders. Tests
were carried on before the window of a small canvass house on the
apiary grounds. Also, the virgins’ wings were clipped when the test
would permit that.

2. Alert drones were selected.from those caught (a few at a time)
at the hive entrance as they were taking flight, or just returning to the
hive. Then, they were kept on wing before the confining window until
needed.

3. Virgin queens which were old enough for the mating flight—
usually those, 3 to 10 days old, that were attempting a flight were used.

4. The trials were made on hot, sunny days during that time of day
when most drones were flying and when it was found that the virgins
usually issued for the mating flight—11 a. m., to 3:30 p. m.

5. The attempt was made in every case to bring the queen and drone
together face to face.

Two methods of bringing the queen and drone together in the correct
position were attempted. By one method each bee was fastened at the
end of a fine, elastic wire holder (Fig. 1, p. 19) and then set to humming
the wings. The holders permitted such freedom as would induce the in-
sects to keep flying—or trying to fly; yet, the operator might so direct the
movements of both queen and drone as to cause them to bump together,
on the wing, face to face. Only a comparatively few trials of this kind
were carried out. No case of mating was obtained, although the drones
often clutched the queens. One queen killed two drones in such at-
tempts.

By the other method the operator endeavored—through squeezing the
drone—to evaginate the male organ into the vagina of the queen in the
normal] position. A queen was held ventral side up between the thumb
and forefinger of the left hand; a drone was held dorsal side up, but
otherwise in the same manner by the right hand. As the insects were
brought face to face, the queen’s dorsal and ventral genito-anal cover
shields were separated; the tip of the drone’s abdomen was then in-
serted in the opening and his organ evaginated by skillful pressure of
the thumb and finger. The task proved to be a difficult one, since a
queen is very active with the tip of her abdomen and with her sting.
It was necessary, of course, to keep the sting pressed dorsalward dur-

18 EXPERIMENT STATION BULLETIN.

ing the operation, and the drone organ had to be everted at just ae
right moment—when the sting was slightly protruded—otherwise, :
bulb would not press deeply enough into the vagina to hold; and the
organ might be prevented from entering the vaginal opening at all. Two
different devices, or instruments, for separating the queens’ cover-shields
and pressing the sting dorsalward were tried. -

Fig. 2 shows a block holding a pair of sharp-pointed forceps which
have a very thin plate soldered to the lower edge of the right-hand prong.
near the tip. With the prongs pressed together, the tip of the forceps
could be inserted into the “sting notch” ventral to the sting (the queen
being held ventral side up) to force the cover plates apart. A peg in
the block allowed the forcep-prongs to open about one-fourth inch. The
male organ might then be made to enter betircen the tip of the prongs
while the thin plate (a, Fig. 2) held the sting dorsalward. .\ pair of
Bausch and Lomb “Binocular magnifiers” were found most helpful in
this connection.

The other device consisted simply of a No. 5 insect pin, with the
head bluntly pointed, bent at right angles one-sixteenth of an inch from
the head end, and inserted firmly in a block as shown at i, Fig. 2.
The bluntly pointed pin-head could be inserted in the “sting notch,”
and as the queen’s cover shields separated, she could be moved so that the
pin passed over the sting and held it dorsalward. This device was the
simpler of the two, and served the purpose well; however, no matings
that proved successful were obtained with either device. In the case
of two queens in particular, the male organ was torn off and the in-
verted bulb was held in the vagina exactly as has been described in nor-
mal matings. Later examinations of the queens’ seminal receptacles
proved that no spermatozoa had been stored in them. One of these
queens was kept (before dissection) in her nucleus until she had pro-
duced about one-third of two frames of drone brood in worker comb.
The brood came out normal, except that the individual drones were
small. Living spermatozoa were found in their seminal vesicles. Two
other queens with which controlled mating had been attempted, were
kept in their respective nuclei until a small amount of drone brood had
been started in worker cells. The abdomens of all these “drone laying”
queens were somewhat enlarged, and dissection showed a few fully de-
veloped eges in some ovarian tubules, although the ovaries were not as
fully enlarged as are those of fertile laying queens. Most of the virgins
used in the tests, however, continued to come out into the queen traps,
at intervals, in attempts to make mating flights evidently, until they
wore themselves out. ;

Why was it that even the matings which were apparently normal in
every way proved unsuccessful in storing the seminal receptacles with
spermatozoa ?

McLain and Baldwin (Am. Bee J., Vol. 23, p. 567) state that in press--
ing out drones they found only a comparatively few that vielded a fluid
seminal secretion resembling albumen. Others gave no secretion, or
only a thick curdy supply. It was with the fluid resembling albumen
when pressed from the drone organ, that these men claim to have had
success. McLain belicved that drones Yielding practically no fluid, or
only a curdy supply, through evagination of the male organ by hand-

DIVISION OF ENTOMOLOGY. 19

pressure, were not able to produce the abundant, more fluid sécretion
because they had not been properly fed by the worker bees.

Observation of the actions of drones during a honey flow and just
after a honey flow can not fail to show that drones must be well fed
in order to be rapid, alert and business-like on the wing. Still, with the
most promising alert drones, hand pressure usually failed to cause an
abundant discharge of seminal fluid exactly like that obtained (for ex-
ample) when a drone held in the warm hand sometimes performed the
act himself. In seeking a reason for this the writer examined, the semi-
nal vesicles and accessory glands of a number of select, active drones
after they had suffered evagination of the copulatory organ through
hand-pressure. Sometimes one or more of the glands and vesicles were
found to have been partly crushed—or, pressed far into the everted penis
by the side of the ejaculatory duct—usually, without having emptied
themselves through the contraction of their own muscular walls. Of
course, a small amount: of the crushed gland secretion was often forced
through the ejaculatory duct, but the pressure stimulus as applied to
the drone’s abdomen did not appear to be a sure or specific stimulus to
“set off’ the powerful muscles in the walls of the seminal vesicles and
accessory glands. It must be evident that without the proper contrac-
tion of those muscles, and the consequent emptying of the stored sperm-
atozoa and granular mucous together through the ejaculatory duct, no
mechanical evagination of the penis, however complete, could be of any
avail. Moreover, if successful mating is to be carried out (even in
limited practice) while the queen and a properly selected drone are
held in the correct position, there must be found some stimulus other
than hand-pressure—a stimulus which will cause evagination of the
male organ, and discharge of sperm-fluid by the contraction respectively
of the abdominal wall muscles, and the muscular walls of the seminal
vesicles and accessory mucous glands.

Piged

Fic. 1. Holder and fine elastic wire used in directing the movements of the drone and queen.
The loop of the silk thread ‘‘a’’ passed around the body between the thorax and abdomen. The
tiny metal disc “b’’ rested against the posterior dorsal portion of the thorax just beneath the
wings. The V-shaped notch ‘“‘c’’ served for fastening the thread.

Fic, 2. Forceps and block used in mating tests. t=thin metal blade on the lower edge
of the right forceps prong—described in text. i=Pin used in certain mating tests.

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I aALVId

PLATE II

Fia. 6. Cross section of Accessory mucus gland near the anterior or blind end, showing
the comparatively thin wall (w) of the gland in this region. m=granular mucus in the gland
cavity shrunken away from the walls of the gland during process of fixing. .

Fic. 7 and 8. Cross sections of accessory_mucus gland made near the posterior end of the
gland where the wall is quite muscular. a=Outer longitudinal muscle layer; b =Layer of cir-
cular muscles; c =Inner longitudinal muscle layer; d =Mucus gland cell layer; Fig. 8 shows the
fringe of the mucus cell layer; g c = Mucus cavity or lumen of the gland. ,

Fic. 9. Cross section through the bulb of an extruded penis; Ac gl=The posterior end of
an accessory mucus gland which was forced down into the penis, in this case, when that organ was
extruded by hand pressure; ss =gelatinous-like thickening of the bulb; we = Wings of chitin Plates
and tt =chitin plates of inverted bulb; Ej D =Cross section of ejaculatory duct (see Fig. 4).
w =thin wall of bulb.

Fie. 10. Longitudinal section of a small part of the ejaculatory duct. hh =Hypodermal
cell layer; ch =chitin; cl =canal.

Fie, 11. Small portion of the penis tube wall from “‘s’’ near the region of the cross striped
atea‘'vv” Fig. 4, or Fig. 12; h =hypodermal layer; ch =chitin; v =one of the chitinized bars of vv.

Fie, 12. Longitudinal dorso-ventral section through the extruded penis entire. Labeling
as in Figs. 3 and 4. The Ej D is shown through almost its entire length. By examining suc-
cessive sections, it could be followed right up to the base of the penis. Very little of the elastic
gelatinous-like thickening of the bulb is shown since the Ej D slips over against one side of that
thickening in the extruded penis as may be seen in cross section, Fig 9. The lobes of the doubly
pinnate lobed appendage (uu) are not everted here but show inside the appendage.

Fie. 13. Cross section of seminal vesicle. a =epithelial cell layer lining the vesicle cavity
(ve); bm =Basement membrane of epithelial layer; b =Inner layer of circular muscles, ¢ =longi
tudinal muscle layer; d =Outer layer of circular muscle and connective fibres interwoven with
tracheae (t).

Fic. 14. Part of cross section of seminal vesicle (1-8 objective) to show regular radiate
arrangement of spermatozoa (SP) often found, Labeling same as in Fig. 18. The outer circular
layer (d) was mostly torn away in this section.

Figs. 6 to 14 inclusive are photomicrographs.

PLATE III

Fie. 15. Photograph of posterior end of abdomen of queen. Dep and Vcp =Dorsal and
ventral genito-anal cover Pee s=sting; Sn =sting notch. : ‘ :

Fie. 16. Drawing of posterior end of drone’s abdomen showing the relative position which
the copulatory organ must assume when extruded into the vagina instead of into the open air.
In the latter case the drone organ extends backward (curving slightly dorsalward) from the tip
of the abdomen in the position represented in Fig. 4. Dep =Dorsal genito-anal cover plate.

Fig. 17. Cross section of oviduct (from queen just returned from mating) filled and greatly
distended with coagulated granular sperm fluid.

Fig. 18. Small portion of section same as Fig. 17, but with higher power of microscope
objective (1-6). Vw=Distended vaginal wall.

Fia. Abdomen of newly mated queen received from Mr. Hunt. In this case the drone
organ (Pen) had been torn off just proximad of the ventral cross-striped ridges (vv), and was
pierced by the sting at ‘Stn.’ In the other newly mated queens the “bulb”? was the only part
of the drone organ left behind in the vagina.

. ee 20. An enlargement of the tip of the abdomen shown in Tig. 19. Labeling same as
in Fig. 19.
Figs. 17 and 18, photomicrographs.

Plate Il

Photomount
Pamphlet
Binder
Gaylord Bros., Inc.
Makers
Syracuse, N. ¥.
PAT. JAN 21, 1908