28 2.

8
1.0 :: 11111 . "'" 2.5
:; 11111 1111,2.5
W
loJ ~F2 I 22
.
I,l; ~I" 3.2 2.2
W
~ I~
&;.l
~. ,~
I!,;l

~ m~ I 2.0 ...~ I~
1.1 '"~~
- 1.1
u

101'-1.1.

--
I I .
14 16
11111'·25 11111 . 11111 . 111111.25 111111.4 111111.6

MICROCOPY RESOLUTION TEST CHART MICROCOPY RESOLUTION TEST CHART

NATIONAL BUREAU OF STANDARDS-J963-A NATIONAL BUREAU OF STANDARDS-J963-A
 UNITED STATES DEPARTMENT OF AGRICULTURE
WASHINGTON. D. C.

BREEDING THE HONFYBEE UNDER

CONTROLLED CONDITIONS
By W. 3". ~?l;AN. dpicIIUllri#, Dil-illian of Bee Culture, Bureau ot Entamolnuy

·4jONTENTS
Page Fage
lntroductron._......................,._ "" 1 New instrumenl~ and methods lor artificlnl
I:'tntus 01 work on breed)'ng und~, controlled insemination,-Continued.
conditions................................. -I l\[ucns plug•. _______._•• _________ •_____• 31.
With nnw",i insemiImtion. ............. 4
Marking Indiridl.'nU;___________________ ..__ 33
With artificial insemination ........... , 5 Drones. _...... __................__••__•••••. 3~'
Bret."tling work aC the b£1l culrure laboratory. n Obmining drones _____ ..._. _______....._ 3~

R\l,mlts of artitlcilli insemlnntlon .........:.. 12 Dcterm1lling !<Cnlnl maturity 0< drones.. 3S

On queens used in 19'.!7.19:?S. flnd 11129._. 12 Qneen renring lind testinn........... _....... 36
On queens IL"Cd in [Link].................. 13 Experiments in 1930......_._...___• ___,. 36
"\jJurtmen~ jll\-es_.._____ •• _., ••________ - 3i
(In queens [Link] 1931 and 1932,......... Ii
X~w in~ttuments nnd methods f~r Ilrtillcini Wintering·.1l: apartment hives...._•••__ • 40
Jnsemlnution. _.... _.. ~"'_ ... __.........._.._......-__.. _....."".. '21 QUeen renring in 1931. ___ ....... __----._ 41
Oln..<s holder • __......................._. 21 Hive records_____•• ______• ___• __• __•••__ 42
~rount for glllssllOlder._ ••••••••••__ ".. 2'! Y'L,'\bility· of spermatozo~. ____ ,. .. ~ __........ __.. _____ 42
[Link] und holdingubdomintll tips... 23 Success!\'e ins~minnti:lIls. _.. ______• ______.__ 43
Multiple set·UII ................__ • _____• 2i Mldng sperlll fn~m difTeront drones________ 4-1
False stage ••___.........__••••_•••_.____ 2S OptimulU age ot· !Iucen lor artificial ilL<:emina·
Pipette manipulator._. __.... __ ._. __ .___ 2S tion. ____...... ____•______ ......__ ...__.__ 44
:Pipette•••• _••••• _••• __._. __ ..... __ "'" 30 SucC".ssion of gellenltions_ .......... ____•••_.'. 4.;
Use 01 an anesthetic___ ... _. ___ '.' __"'" 30 Summnry___•___ ..________• _____..____.. ____ 46
Filling tbe (llpette._ ............. ______ • 31 :Lirerature cited ............. __ ......__....,. 4i

INTRODUCTION
l\.lthough the honeybee has been h:ept by lllall since long before
the Christian era) yet in the course Lr. the centuries little or no
change has been wrought in it by breeding if the development of
the so·called " :,;ol<1en " strain of Italians ill the Unitecl States and of
the ,; "eredelte " strains of the German brown race in central Europe
is excepted. The fact that in nature the honeybee mates only on
the wing is commonly given as the principal reason for this situation.
Even though bee breeders have relied on natural matings in their
work with the honeybee during the centuries s they would have made
progress more comparable with that accomplished in [Link] breeding
of man's other useful animals had it not been for two factors. In
the first place, even less than 100 years ago the bee breeder did not
have aclequate knowledge of reproduction. in the honeybee since he
did not know how drones develop. In the second place, until
recently he had devised nO means of distinguishit!g definitely between
l'Ilces of the honeybees and their various strains. "g"en Mendel (lItis,
if), p. 14£-157) \ the. father of modern genetics, lacked snch means
whelt he endeavored to work out problems of bee genetics in addition
to those on peas which brought him f " m e . ­
1 Italic llumiJer!! III pnrent1l1'ses r~fer to T,iterlltur(' CHI-d. p. -17.
12!l7S7-:12--1
 i i '\ , ,

II \[, ,d"i'- :.1;1:'. I 1"""I'!' 1,:1\0' [."\"1' 1""'11

". I"n;:; '110' L,':1
H;:: ;! -:' I:' \"'1' 1."1 ,·"rdill'·,1 I'· Ill,,· ]';1,'0' lit' tilt·
1.· I;:,,'I,!, II
."1: 1 \ ! I t l . ;1~'" it r~'~f1l!'l"d 1/:<11 tL:ll ht· \\(Jl'l\.,·d tlfl tliP Ilt'ntItt'r ttl'
I) ;1'; i,,·, ,I:,,' [!,dLlI. ;:", {':lI'lllllla:;, Ii,· I':!!,\ 1,:1:1' .. ;111'1 till' ('Yl'riall.
;\ i,liI "i. t .. til" l"t\:\" III'" "I " 'r,'~i"l. II,· 11,,·01 ,·"llIlli,·, ];.1'1'1
;1 'II r 1 ;t!l~!'lt :q'l;t'.'- ~·tl~!ll:tll'L'" :!' lli~ l·lt1i ... tl~l· ~'nt'lll'll at l~l'lll).
, F '::' 1. j

\[".I,'n. ;"\itili,' it fllrt,,:lt',,1. ,.j P'11J'",111"lillil ill dw hl)Ill'.dH"·

; ;1'1 It 11l'!,.!',I! ft 11 I!!' if; 11;i',~1 .. \\h"p ....... \\alll111f'1'.ta1tl I i'''~ I',J. J~ I'. l.::11
.;" 'I d,·" ,·t,·arl. t i." ,,!..:!.!:·bY!II!! 1111!"[ 1"11 III tIll' ,\11""11. TIll' '11t('~.
t.,,!! "I t·,.. t.1:·I" "I' llwt!ll!!,' [,"II,:lIIW'; Jltl:lli'\\"\'''oi Itlltil 11\"'1' n

1.. 1 ,\"'h'ld'''' ,'q,~" .. y in bi~ I'll , ... ft'r l-::Il"!IIl itt I~r!.lj. (o." •• ';,u ... ', \',th,la. ,1Iti ... )
I,"q :i . . h,d \\ilh ml ...... l .. 1~ (.r ,f':'> ..... :-;i,:iJ:;":' I', n. tIllll

l,1(ll'lt',.d ~ t':lr~ lat(·I', Whl'1l I IU].l'I· (1". jI • •,,; J (,~t;tldi~IJ{',l tl,,' fa!'l
til'll IL.. '!II""11 lit·,· 1l1;lt"~ "lIt~i,I,· \\111'11 ill Ili!!,·11t. 'fbi..; I'l'llltl .. lll had
":Illj,·./ ",,"'I! , I I k, "Ii ill! II!'~'·t'\,·\, :I~ 1:'~:lIIIlJll\,. \\llll~l' il'tl'",,,t ill it
" ./ ..:.,:,1,·01 llil1l t,. ;Ittl'm!'t til It:tv,' a '11l!'J'1l :tlld .11'011(' 1I1:11l'
:1. .1 :..:h-- ,il·:, lit. a lal,[". rrlll"'I' ai", tl'i.·,1 IIII' ~;1I1W l'\l't'rillll'llt
\"'('1/" l' ilo'kl.!,.!· Ie, 1;[Link] ,,,Intillli . .\]lotlli'1' illtJ 1I1 rtallt a,hall"" had
I ,i.:,,! :-1:1'" -1"'111\ 1,,·1',1\'" [Ild",!"- ,1i,,·()\I'\'v. \d 1I'1 I ~,.Jliradl. n,',
"p,.; 1.~. I" ('tll'i,1 " : . /'. :'1. ~!l,,\\,'d .],·Iit,ll'-(\' tllat :t 'lll'·,'lt ":111 jH'
t, Ir. 1 It,>!" :ll:." \\",1\,,), lana if it j, :":1\"11 t'1'1I1"'1' [''".\ :<lld ,',11',·
('I'i'. '!" .:.,:1, II. 11- hn:ll Ii f... \',0( 1t!lt il 1-.1:,. It,,\\,·\',,!,. \\a~ ll/i,·I'­
lj~ an!'ll~:I:! t!:-.\'q\.'r\· HZ:ld.· Jillith t .1 ..' • j'. j!'/~ I lll!lt l11i' drollP 1'"
l '

; .• ".1:,' I·d ; ,Irt!w! "",'I.,:ti,·alh', L;ltl,· i'l"'~I't·,- "o,tld 1..·, "1,,,,'1,·.1 Ill'
t l ,., ;"·"!'!',:",l"l· \\jll",nt at j..;I~1 l".II,\I.. .t!!·" .. t' tlll"l' f'Jlll' fllWl:llll"nt:d
( 1 1 ,! I. _,
I1 ," Ll:l'~ ""I ,'" l~Hill t;I"I(" a \\:lY L;t,~ 11l'PL lljll'llI,d III lLto 111'lt II{'Pfltll'r
f ,r d:-f;! ~14'-!tIJ!~' ;,'1 ~l':dt'Jy 11I·,t\\.·,1. \:It':''·:i.~ la .. ({lid -.ll':liIL""; (If
J • ...
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 3
the honeybee, as a result of studies undertaken to determine) eS'pe­
cially from the standpoint of biometrics, characteristics marlnng
them. The pioneer in this type of bee research was Koshevnikov,
of Russia. Among others who have nw,de contributions in this field
are Alpatov and Mikhailoff, also of nussia; von Buttel-Reepen,
Zander, and Gotze, of Germany; RytH·, of Czechoslovakia; Tokuda,
of Japan; and Phillips and Kellogg, of the United States.
'Within the past decade another tool has been placed in the hanels
of the bee breeder' with the establishment of the fact that queen bees
can be inseminated artificially. This makes possible full control
with regard to the drones used in breeding. It is true that artificial
insemination of the honeybee is, as yet, only an instrument of the
scientific investigator and that the commercial breeder must con­
tinue, for a time at least, to rely on natural inseminations for his
work. The way has been opell'3cl, nevertheiess, for the investigator
in any locality to keep pure stock of various races and strains as
well as to develop special strains of the honeybee. The commercial
breeder can thus, in turn, be provicl\ld with a continuous reserve
from which to replenish his stock if natural matings fail him.
Many problems await the bee breeder. He might develop strains
which would be O'entle: disease-resistant, capable of carrying larger
honey loads, captilile o:f flying longer distances, or which would have
tongues of the proper length to secure nectar from floral sources not
now availal:>le to the honeybee. For some regions it would seem
desirable to develop strains that fly at lower temperatures than do
bees now commonly found in the United States. This is of par­
ticular importance to those interested in bees as pollenizing agents.
In general, breeding of the honeybee in modern beekeeping prac­
tice ralls under two main headings: (1) Breeding under natural or
uncontrolled conditions, and (2) breeding under artificial or con­
trolled conditions. By naturul or uncontrolled conditions are meant
thos''', obtaining in nuture or ordinarily in the apiary, where virgin
quei:;:ls and drones fly freely and mating takes place only on the wing.
By artificial or controllerl conditions are meant both those which,
wllile permittinl? natural insemination to occur, obtain through some
restriction by the beekeeper as to the particular drone or drones
with which the queen may mate, the flight range of the queen, or the
flight range of both queen and drones, Ilnd those which obtain when
the beekeeper causes the transfer of spermatozoa from drone to queen
to be made otherwise than as in nature.
The following outline, in connection with the text which im­
mediately follows, may prove helpful in keeping cleRr the distinc­
tion between the various methods which are in use or have been tried
for breeding the honeybee: .
BIlElEDINQ OF THE HONh"YBEE

1. Uncontrolled condltlons (IIttie or no restrictions on queen or drones;

mllting on tlle wing).
2. Controlled conditions.
A. Naturnl insemination.
1. AVlIllable drones restricted (mating stations).
2. FIIght rnnge of queen restricted (tethering, etc.).
3. Flig-ht rang(s of both queen and drones restricted (tents,
greenhouses) .
4 TEOH~ICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

Controlled. conditions-Continued<.
B. Artificial insemination.
1. By placing drone organs in :position in queen.
u. [Link] separation from drone.
li. After separation frOm drone.
2. By removing sperm from dlrone organs first.
n. External npplicntior. (brush, squeezing out sperm, etc.).
li. Internal npplicatior. (injecting within genitrl! opening;).
It is to be noted that th~ ;:'Llcilile does not include snch methods
as daubing the eggs with sperm, since this is an attempt to accom­
plish fertilization of the egg itself anduot to simulate mating of the
queen bee.
STATUS OF WORK ON BREEDING UNDER CONTROLLED CONDITIONS
WITH NATURAL INSEMINATION

The mating stations of Europe furnish a practical example of

controlled conditions which restrict the drones with wtich the queen
may mate, but ·which still permit a natural insemination to take
place. These stations are in isolated localities in which the bpe­
keeper has endeavored to have only drones of his own choice present.
The virgin queen is taken there and is free to fly from her nucleus
and to mate on the wing with these drones. The breeding experi­
ments conducted a number of years ago in Texas by Newell (f39) ,
as well as those in progress on an island in Lake Ontario by Sladen,
of Ctllluda, at the time of his death in 1921, also come under this
category.
:Many good results are reported from the breeding work carried
on at these stations ill Europe, especially in Switzerland. The work
in Switz~r1and is linked ·with the name of Kramer (Goldi, 1.4),
who started the movement in his country to maintain in purity
and to improve the native Swiss bee by ha,ying selected stock mate
in isolated localities. (Fig. 2.) The results of this work from 1915
to [Link] accorc1ing to n survey of certain apiaries in Switzerland
which contllinecl colonies both of the improved (veredelte) and of
unimproved stock Udl, 7). 30) ,show that the honey crop of the im­
proved stock averaged higher per colony each year than that of the
unimproved stock, ranging from 20 per cent in 1923 to 100 per cent
more in 1924. These perc~ntages, howave1', were based on the rela­
tion of the average per-colony yield for improvec1 anc1 unimproved
stocks, respectively, to the sum total of the hvo averages, and the
number of improved colonies used in the survey was larger each
year than the number of unimproved colonies, being in 1928 about
two and one-half times and in 1924 more than seven times as large.
In the Unitecl States isolatecl locations free from wild swarms
or colonies of neighboring beekeepers are not easily accessible to
most queen breeders. Consequently the mating station l1as played
little or 110 part in beekeeping here, alth<;lugh certain commercial
queell breeders in this country have attempted to duplicate, in a
degree, conclitions found in an ideal mating station by endeavoring
to rid 'the territory in the immediate vicinity of their mating yards
of undesirable colonies of bees. Their methods have consisted of
buyin!!: up colonies found in the neighborhood, requeening near-by
colonies with desired stock free of charge, paying rewards for wild
swarms, and similar measures. 'l'he possibility that the queen nUly
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITiONS 5
mate with a stray drone even in a locality thought to be well iso­
lar,ed from local bees reduces somewhat the value of mating stations
:in a. scientific breeding program.
Att~mpts to mate queens tethered at the end of a pole are examples
of a partial re~triction of the flight range of the queen bee, while
attempts to hu.:te a, queen and chosen drones mat~ inside a tent or
other inclosure are 9xamples illustrating both a restriction of the
drones availabla for mating and a partial restriction of the flight
range of queen lmd d:t:ones. In all these cases, however, there is an
enclea,or to' have mating take place on the wing-in other words,
to effect a natural insemination. :Mendel was Qne of many who

[Link] !!.-;\lntlng station Itt Bruggen. bel St. Gnllen, S""ltzerlanll. (Krnmer) (Pub·
Iisher] by perlllisslvrt ()t V\,rE~in l)eutsch·ikhwelzer}8cher Bh.·III·ntreunrle)

haTe unsuccessfully attempted to obtain matings of queen bees withil:1

inclosures (19, p. 141'). This phase of controlled mating was also
im-estigatNl in the Bureau of Entomology in 1907 (17). Since no
satisfactorily verifie(l reports of success are available for matinf,fS
attempted under the conditions given in this pa,ragraph, no further
attention will be paid this category at the present time.
[Link] ARTIFICIAL INSEML"fATION

'When conditious are so controlled that both queen and drone are
deprived of flight, the insemination must be accomplished artificially.
This may be eifecte(l either by placing the drone organs in proper
position in the queen to accomplish insemination or by removing the
sperm from the drone organs and transferring it to the queen.
The methods proposed for bringing the drone~s organs into proper
position within the queen can be divided into two classes: (1) Caus­
ing these organs to evert in the proper position in relation to the
6 TEOHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

queen and then severing them from the drone, and (2) separating
them from the drone and then placing them in proper position in
the queen. The methods for first removing sperm from the drone.
and then transferring it to the queen can also be divided into two
classes: (1) Dropping or brushing the sperm on or near the ex­
terior genital opening of the queen, and (2) introducing the sperm
within the opening.
One of the early attempts at the artificial insemination of queen
bees, and one [Link] is often quoted, was that by Huber, who fol­
lowed the suggestion of Bonnet in 1789 that he endeavor to insemi­
nate a queen bee by introducing within her vagina, through the use
of a brush, .sperm from a drone. HUber (18, 'I). 1, p. 60) failed in
snch an attempt, as have doubtless most of his successors, regardless
of the method employed.
According to his statements before the Wanderversammlung der
Imker deutscher Zunge, held in Leitmeritz in 1927, Wankler, of
Germany (48): was one of the earliest modern queen breeders to
attempt to make use of instrum\}nts in the insemination of queen
bees. He affirmed that as early as 1885 he had resorted with some
success to mechanical means for inseminating queen bees. He also
referred to success attained by himself, even in the eighties, in fer­
tilizing drone eggs from drone cells by daubing them with sperm.
1Vankler (48) mentioned, among. his instruments, a pipette
(Spritze) to which could be attached a model (kunstliche Glied) of
the drone organs, a means for moving the pipette, and a block on
which to hold the queen. He stated that the semen is forced into
the pipette and then the model is attached and introduced into the
vagina of the queen, which is bound to the block. The sperm is next
forced out of the pipette into the model through pressure on a screw
knob. The pipette, having seryecl its function, is detached, but the
model remains attached to the queen at least long enough for the
semen to be taken up by the spermathecll. The queen is put back
on a frame of brood under a cage of wire screening (Pfeifendeckel)
fine enough to catch the model when it finaLly becomes detached from
the queen and drops off.
It is to be noted that the taL~ by vYankler was given after 'Vatson's
announcement of his discoyery lwd vYanlder referred to it. [Link]­
bruster,! however, in a preface to vYanlder's book (4'7, p. 5) written
in 1924, mentioned that Wanlder had presented him with one of the
pipettes made of silver and ,vith a model of It drone organ fashioned
in metal.
:McLain (~!2) claimed that he had achieved success in 1886 in ar­
tificially inseminating queen bees with an instrument consisting of a
fine nozzle fitted to a hypodermic syringe in place of the regular in­
jecting needle. After the nozzle had been filled with sperm from the
drone, a small tube was slipped over it. The queen was held in a
clamp made of the two halves of a block of wood. 2 inches square
and 4 inches long in which a hole had been hollowed out to hold her
body with the exception of the tip of the abdomen.. McLain also
reported success in having queen bees mate in a greenhouse, and even
in squeezing sperm from a. drone directly into the vestibule of It
queen bee.
 BREEDING THE HO:NEYBEE UNDER CONTROLLED CONDITIONS 7
No other nmeteenth-century attempts to mseminate queen bees
artificially need be discussed here, since in no instance were such at­
tempts repeated successfully by others to the conviction of the bee­
keeping world.
The first announcement of import:mce in this field in the twentieth
century was that of Jager and Howard (Je~) in 1914 regarding suc­
cess in artificially inseminatmg (De out of eight queen bees worked
with that summer~. These invest,gators reported that, of the first
3,000 eggs laid by this queen, all but 4 proved to be worker eggs. She,
was wintered successfully (16), but in the spring she laid drone eggs
as well as worker eggs and soon was laying drone eggs exclusively.
A;lter three weeks of dmne laying .she was dissected, but her sperm­ it
atheca was found packed with living spermatozoa. In this work \\
(16) the sperm from the drone was diluted with salt solution be·fore
beinO' taken up in a fine pipette. The queen was then held in the left
hand, while the pipette was introduced into the genital opening with
the right hand and the
sperm was forced out of
[Link] by hlowing
WIth the mouth.
During. the next two
years Howard and France
(16) p-ndeavoted to carry
this method further, but \ l
were successful with only \
3 out of 55 queens. · For FIGUUE 3.--Fol'ceps und block used by Shafer In
one 0 f t h e 3 success was llIuting tests (Shufer)
gauged by 'the finding of
sperm in the spermutheca upon dissecting the queen 26 days after the
attempted inser.'1ination. One of the others laid only 5 per cent of
worker eggs. In this work the sperm' was not diluted with salt
solution.
The success reported by Jager and Howard may have led Shafer
(45) to his investigations in the same field. Shafer's main work was
directed" through squeezing the drone-to evaginate the male Oi.'gan
into the vagina of the queen m the normal position." To keep the
abdominal tips of the queen ([Link]~and to hold the sting out or the
way in the process, a pair of forceps was moun;'.ld on one of the
inclmed surfaces of a triangular block in such a way as to allow
the forceps to open only a quarter of. an mch. (Fig. 3.) A small,
thin plate was attached to one point of the forceps to aid in holding
the sting back after the forceps had been introduced between the
abdominal tips. A binocular microscope was used during the attempt
at insemination.
Shafer later contrived a device which consisted of a No.5 insect
pin bent at right angles one-sixteenth inch from the head end and
inserted firml~ in a block. The pinhead could be inserted in the
"sting notch' and as the queen's abdominal tips separated she could
be moved so that the pin pressed against the sting and held it dorsal­
ward. Shafer did not report any successful insemination, however.
In 1923 Quinn (43) announced success in artificially mseminat­
ing queens by a method which involves causing the drone to evert
8 TECHNICAL BULLETIN 326, U. S. DEPT. OF AGRlCULTURE

jts genital organs into proper position in th~ queen for the transfer
of sperm to take place. No details of the method were published,
but the actual operation appears to have been performed by his
grandson, Harry Laidlaw, instead of by Quinn himself. In dem­
onstmtions witnessed by the writer in 1928 (1) anel in the spring of
1930 (8), Laidlaw supported the queen with his left hand while
making use of a pair of protractors to hold the abdominal tips
apart, somewhat as did Sluder (45). The drone was held in the
right hand while being caused to evert its organs in the proper
position in the queen. No microscope was used.
In the summers of 1930 and 1931 Lu,ieUaw was given temporary
employment as field assistant by the Division of Bee Culture at its
stll,tioll in Baton Rouge, La. His work durinO" 1930 led to the state­
ment in the anllual rep'ort of the Chief of the fiureau of Entomology
(134,1). 71) that" the reslilts obtained so far indicate thut the method
has considerable promise, for a partial degree of insemination can
be effected in all cases." In December, 1H31, Laidlaw reported be­
fore the American Association for the Advancement of Science that
he had improved upon his technic somewhat, having devised a small
spring to fit inside the queen's abdominal tips and hold them apart
nnd having adopted the use of a microscope.
Apparently [Link] has been the only sllccessful exponent of his
method thus far, although, according to a recent statement (B), .1.\.• Z.
Abushady, of Egypt, ,vas successful in 1930 in inseminating tht'ee
queen bees [Link] without instruments. Unfortunately no
details were girell as to Abushady's work, however.
Malyschev (133), OT Russia, in 1923 reported success obtained the
year before in the artificial insemination of one qneen bee. He
dissected ont the organs of a drone that had been caused to ejaCUlate
by being chloroformed and placed them in the propel' position in :t
queen that had been subjected to :mullonia. Malyschey reported
t,hat this queen laid eggs nOl'lnally in a nucleus and ·was then .trans­
ferred to fL queenless colony. He did not record her further hIstory.
In October, 1026, ·Watson satisfied n, special committee at, Cornell
University by a clemonstration that he had developed a method
which gave results far superior to any that had previously been
published (6). According to his method (49)the sperm is injected
with t"L microsyringe or pipette into the queen, which is bound to a
small wooden block. or miniature operatll1g table, hollowed out to
fit her body. (FiO". '4.) The queen is held in position on the block
by fL threael passecf several times around. her thorax and the anterior
part of her abdomen, the posterior tip of her abdomen being left
free. The injection is accomplished under a binocular microscope.
To iacilit"te insertion of the microsyl."inge, 'Yatson places a fine­
pointed forceps between the queen's abdominal tips in such a way
as to hold the sting back out of the way and to spread. the tips. The
microsyringe is held in a Barber pipette holder or micromanipulator
which allows movement in three directions. Sperm is drawn in and
is forced out of the tube OT the pipette, which is about 0.5 111m ill
diameter, by a fine wire plunger moved by a screw. According to
'Vatson's technic as first published (49), with the bulb of the drone
held on the inc1exfinger of the left hand, the end of the pipette is
 BREEDIXG '.rHE [Link] UNDER COXTROLLED GCNDITIONS 9
forced into the IllLlcns and u small qnantity is dnrwll in. The
pipette is then forcetL more deeply into the bulb and the full load of
sperm is hlken up. In It well-developed drone the white IllUCUS is
readily cliscel'llibLe in one end of the bulb and the cream-colored
sperm in the other end. The mucus is taken up .first so that it will
be forced ont of the tube lust and thus serve its purpose, as set forth
by Bishop (:J), of acting as a plug to the sperlU owing to its property
of congeiLling on contact with ail'. .
III slIb::;cquent work ":atson (:j,!). minimizes the importance of the
IllU('U3 pll1g~ and describes a small .• ::;aculus" which is formed in the
end of the bulb containing the spf!rm when pressure is applied at
tll't otlll'r !.'ui\' By cutting this sac antl inserting the pipette there,

l,'lGCUFJ 4.-L. R. WlltsOIl rlclIlQlIstrlltiIlg his lll!'lhol! of nrtifi('ial ins(,lIIillatioll or

fjU('('1l \)'!(>s. (Copyrig-ht by the AIll\'rii.'IlI1 BI'c .Journal)

imitead of pushing it through the mucus fit'::it it is possible to take

lip in the syl'in~e n lar~(> charge oj ;,p'2rlll which contains pmcti­
('uIly .110 mUl'US.
..:\:: to his oml Sllccess with his IlwtlH)(l. \'~nt!-ion (;iJ) states that,
of ol:! <]IIN'IlS operatCtL upon in HEW, he foun<I evidence, either by
till:' mif'l.·os('ope or by the l'p:tring of \Yorker bL'ood, of some c1egl'ep
o.t ill;,('lll i lIa tion in about half this numb(,t'. Of DO queens operated.
upon ill 1D2T. 6;) pel' (,pnt showed some tl!.'grt'c of insemination, and
(IT of !);~ (l1lt't'IlS opel'ate<1 upon in 1l):28 ::;ho\ye<I some insemination.
In it IH'j\ratp communication to till' writer. dated .Janllary 6, 1D3:2,
'Watson stated that, out of 42 qlleens tn'nted in 1929, 35 proved to
be inseminated in some degree and that tlte rC'll1aining 7 were lost.
In 1930 "\Ta tSOll, as repot'ted by Disbl'o\\'e (11), opel'ated 011 .4.0 queens.
Xine of these quel'lls W(,l'l' lost. uut tite n'maining :)1 showed SOllle de­
gl'l'(' of i ns('mi [Link]. About 12 !llJpea red to be normally insemi nated,
while about 12 Ol' 14: appeareel to be only about 2 per ('cnt inseminated.
In l!);n, [Link] to the e0ll1l1l11llieution mentioned aboye, 1Yatson
tl'p!ltp(l1:~ que(,lls, Of thC'!:'e. 8 pl'oYe<l to be inseminated in some de­
gree, 1 is l't'col'(\Nl H:; a faillin-, Hlld -± \\'ere lo;;t. \Vatson states that
1!!!HR7-:'12--2
10 TEOHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

"of the queens treated in 1931 only three, or possibly IOUI', were any­
thing like copiously inseminated."
To determine the .degree of insemination with the aid of the mi­
croscope l "Watson (50) compares the quantity of sperm in the arti­
ficially inseminated queen with that in the normally inseminated
queen. The spermatheca of the normally inseminated queen at the
beginning of egg laring is so filled with sperm that it is opaque,
while that of the VIrgin queen remains filled with a clear liquid.
It is, of course, difficult to judge accurately the degree to which the
spermatheca is really filled, although with practice results can be
obtained that will afford interesting comparisons. .
One feat performed by '[Link] through his method wa.s the trans­
fer of Ii ving spermatozoa from the spermatheca of one queen to that
of [Link] (50). On another occasion he was able even to rear
worker bees from eggs laid by a queen that had been artificially in­
seminated with spermatozoa from the spermatheca of a queen that
had been mated a ye..'tr before (53). To be able" to transfer or to
save in, this way the spermatozoa contained in the spermathecae of
queens mated with drones of certain desired strains or races should
prove valuable at times in breeding work in preserving and prop­
agating those strains 01' races.
The summer after 'Vatson's discovery, Prell (41) announced that
he, too, had successfully inseminated queen bees artificially by a
method which, from the description. given, seems to be like that
used by Malyschev. .Although Prell refers to :Malyschev's work, he
ascribes to him a method employing a pipette. Unfortunately,
Prell gives no details as to the queens or the number with which he
had success. He only reports that brood rearing began normally
within a few days after the queens had been released. In a report
of his work for 1928 Prell states (493) that in that year he attempted
unsuccessfully to inseminate drone-laying queens. He did not state
that he had been successful otherwise with his method during the
year.
[Link] the first person to verify Watson's method was the
writer (393). Within nine montns from the time Watson had given
publicity to his method, worker bees which were the offspring of a
queen bee that the writer had subjected to the 'Watson method were
emerging in. the apiary of the bee culture labomtory of the Bureau
of Entomology at Somerset, :Md. The results of this work were
not made public until the December meeting of the American [Link]­
ciation of Economic Entomologists in the following year (1928),
although the work had been mentioned previously by Alpatov (3)
and by Hambleton (15).
The only other published reports of success by the Watson method
are those by :Mikhailoff, of Russia, ana by Disbrowe, of Canada.
[Link] has informed the writer personally, however, that G. H.
Cale also has performed the operation successfully, if a microscopic
examination of the spermathecae of the queens may be used as a
criterion.
During his first season's work on the artificial insemination of
queen bees, conducted durin~ the summer of 1928, Mikhailoff (935, 937)
performed 113 operations WIth apparatus made by himself in follow­
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 11
ing Watson's book (49) as a guide. In addition he performed. 30
operations by Malyschev's method. These 143 operations were per­
formed on only 105 queens, which means that some queens. were
treated more than once. Success was had in 18 instances, as was
evidenced by the presence of sealed worker brood in 6 cases, and
by spermatozoa found in the spermathecae of 12 queens. All-worker
brood developed from the first eggs laid by 2 of the queens, but
success in the case of 1 of these is attributed to the ~falyschev
method, because it was used on the queen after an apparently un­
successrul use of the Watson method 4- days earlier.
Mikhailoff (126) continued his experiments in 1929 with the aid of
a helper, I. A. Licenko, and used a micro syringe made by Watson.
During the year 114. queens were operated upon, some of them more
than once, the total number of operations being 138. Twenty-four
of the queens, or 21 per cent, showed some degree of normal insemi­
nation. Mikhailoff states that eight of these queens g:lve all-worker
brood.
In 1930, according to a communication to the writer, Mikhailoff
treated about 100 queens. These treatments were 2'{ per cent suc­
cessful, as gauged from the actual rearing of worker brood. Of these
successfully treated queens 10 per cent gave evidence of complete
insemination.
Disbrowe's experiments date from 1929 (9). He states (10) that
approximately 25 queens were operated upon during that season. Of
this number more or less success was had with 5. In a later article
(11) he mentions having continu,'d the work in 1930, but giyes no
results.
BREEDING WORK AT THE BEE CULTURE LABORATORY

"\Vork on a bee-breeding program was begun at the Bureau of En­

tomology's bee culture laboratory at Somerset, Md., before the an­
nouncement of Watson's discovery. A study, then under way, of
queen rearing and of the biological and physical characteristics of
the leading recognized races (30,31,37,38,39) was one step in this
progl'llm. It was planned to make later a thorough trial of mating
stations on the European model, but when Watson's method was made.
public it "Was considered advisable to test out methods of [Link]
insenunation first and to endeavor to improve the technic of those.
fonnel acceptable.
In any bee-breeding program regardless of whether artificial or
natural insemination IS employed, there are numerous manipulations
which must be carried on. Even when artificial insemination is used,
it is not the greatest consumer of time in a large-scale breeding project.
The manipulations belonging to the technic for breeding the honey­
bee under controlled conditions, whichever type of insemination is
used, cover the maintenance, supply, and checking of the queens,
drones, and workers used in the work. In the technic employed by
the writer these manipulations include queen rearing to supply the
necessary virgins, maintenance of nuclei to test the queens treated,
marking queens, checking up on queens after the operation, mainte­
nance of supply colonies both to rear queens and to provide honey and
brood ior nuclei as required throughout the season, making provision
12 TEOHNICAL BULLETIJ:.r 326, ·U. S. DEPT. OF AGRICULTURE

to obtain sufficient drones of the righlj stock, marking drones, and

other necessary operations. As beekeepers know, the successful car­
rying out of all tIlls procedure is, in part at least, dependent on
weather and floral conditions.
As commonly performed by the beekeeper or queen rearer, many
of the manipulations just mentioned require too many bees, 01' too
much time and equipment, to be economically or efficiently used in an
intensive study of the genetics of the honeybee, Consequently the
'writer has endeavored to develop a technic for breeding the honey­
bee uncleI' controlled conditions which will permit the USe of'a maxi­
mum number of queens per queen breeder with a minimum require­
ment per qneen of time, bees, and equipment. This technic, together
with new technic for nse ,,-ith the "Watson method of artificial insem­
lllation, is described in the text that follows. A discussion of the
results obtained on queens used in the work 011 controlled mating is
given first, however.
In this bulletin, unless stated otherwise, or unless specific reference
is made to the spermatheca, the term" percentage of success" in con­
nection with the writer's work refers to the percentage of worker cells
in t11e first two weeks' total of sealeel brood. .A.s applied to the
spcrl11utheca, the term is used to denote the relative degree to which
the spermatheca appears filled with sperm, as compared with the
spermatheca of a normally inseminated queen. 'rhe actual emergence
of 'worker 01' of queen bees is, of course, the final criterion for the
purpose of the breeder or geneticist as to whether a queen is inSelni­
natec1 an(l as to the degree of insemination, but tIlls gauge can not
always be readily applied. Thus, when a queen lays few worker eggs
in relation to the number of drone eggs, the actual emergence of the
resulting workers is not always observed, and in such a case the story
is told by -the finding of 'worker [Link]. When a queen is killed
before she has laicl any eggs, an examination of her spermatheca is
the only way of determining whether or not she ha.s been inseminated.
"'When a, queen is laying mostly drone eggs and is using worker
cells for them, it is sometimes easy on casual observation to mistake
the cappings over the resulting pupae for worker cappings, but a
littleprnctice on the part of the observe"!: shoulc1 eliminate error from
this SOl1rce. On the other hand, spermatozoa may be found III the
spermatheca of an artificially inseminated queen that has been injured
sufficiently in the course of the insemination to prevent normal egg
hying. Such an lnseminutioll could not be called successful from
its utility in a genetical study, although it would be successful as
far as the introduction of living spermatozoa within the spel'matheca
is concerned.
RESULTS OF ARTIFICIAL INSEMINATION
ON QUEENS USED IN 1927, 1928, AND 1929

In 1927, 20 queens were subjected to the Watson method by the

writer. Positive results were obtained in 5 instances, inc1uding the
completely successful case mentioned previously. For 1 of the other
4 queens the degree of succeSs was 50 per cent, for 2 it was between
5 and 10 per cent, and for 1 it was below 5 per cent.
In 1928 experiments were conc1ucted on 16 queens. Six of these
queens were subjected to 'Yatson's methoel and the last 3 were
 BREEDIJ',G 'ttlE HONEYBEE UNDER CONTROLLED CONDITIONS 13
inseminated suct:'essfully. In 2 cases the degree of success was over
70 per cent and in 1 it was 100 per cent. The other 10 queens were
subjected to the Q,uinn-Laidlaw method, but without success. Since
then the writer' has done little with either this or the Malyschev
method. Details on the woi'k in 1927 and 1928, as well as the method
of rearing the queen bees and handling them in the nuclei, are given
elsewhere (32, 33, 34). . '
In 1929, 16 queens were operated on and placed in nuclei for obser­
vation. Of these, 1 was accidentally lost too soon to determine
whether or not it had been inseminated, 4 were lost by balling or
otherwise within 2 or 3 days after being put in the nuclei, 1 was lost
8 days later, and 2 were lost when their nuclei were robbed out within
3 days :tfterwards. This left only 8 queen bees on which to con­
tinue observations. Four of the 8 queens gave evidence of insemina­
tion: 2 of them_ beginning their [Link] careers as if 100 per cent
inseminated, while the other 2 appetLred to be about 50 per cent
inseminated.
ON QUEENS USED !N 1930

In 1930, 52 queens were operated upon by the 'Vatson method.

One other queen was operated upon by the Malyschev method, but
she died from an undetermined cause too SOOIl to ascertain the
results. The data for each of the 52 qneens are given in Table 1-
Sixteen queens received some degree of insemination, as was evi­
denced by the presence of emerging workers, worker pupae, or sper­
matozoa in the spermathecae of the queens. Success in the case
of 10 of these queens was determined solely by observinO' under the
microscope Ii ving spermatozoa in their spermathec,te, in tYle case of :d
solely by observing worker pupae or emerging workers, in the case of
3 others by observing both sealed brood and sperm, and in the cnse
of 1 by observing both emerging brood and sperm. These data alone
show that insemination resulted in practically 31 per cent of the
work in 1930.
Flat cells, which were Tmdoubtedly those of worker brood, ,vere
observed in the case of 7 other queens. These cells did not ocCUr in
such abundance thnt workers were seen emerging, and such cells
as were opened for observation did not contain brood old enough for
the sex to be determined on superficial examination.. A microscopic
examination of 2 of these queens made, respectively, 33 and 124 days
after insemination revealed no spermatozoa. The other 5 queens
disappeared ,yithout such an examination having been made.. 1£ it is
admitted that these 7 queens were also inseminated, the percentage of
success for 1930 becomes 44.2 instead of being 31, as mentioned
previously.
Of the remainint! 2!1 queens, 5 (Nos. 4, 5, 9, 14, and 24) were found
clead and 3 (Nos. 17, 22, and 2'7) had disappeared within 2 days of
their attempted insemination. One queen (No.1) died within 2 days
of a second insemination attempted 14 days after the first one. Nine
more queens (Nos. 10, 23,25, 26, 28, 29, 3~, 41, and 51) were found ..
dead or had elisappearell within 3 to 9 days of the attempted insem­
ination. Queen No. 10 was found balleel to deatH. Three others of
the foregoing were given a second operation. Owing to other work,
queens Nos. 1,4,5,9; 10, and 14 were not examined under the miscro­
scope. One queen ~No. 47) was killed accidentalJ:' on the seventh
 TABLE I.-Record oj queens operated on in 1930 by the Wal80n method I ~
~

Period I Period 1
Period
Date of Date of Drones Drones Age of between betw~en Period lived Flatly Workers Sperm t;jj
Queen first second Ether 'J.'ypo of used for used for queen at first last lived after 'rotal sealed or seen In
No. Oross 2 IllSeml­ Insemi­ used first
plunger' Insemi­ second
Insemi­
first
Insemi­
and
second
inseml-
nation
after
egg last length
ofUfe brood virgins sper­ @
nation nation insemi­ seen reare? matheca
nation nation nation inseml- and egg laying
nations laying nation ~
1 - - - 1 - - 1 - - 1 - ·- - 1 - - '_ _1_ _1_ _1_ _ _ _,_ _,_ _._ _._ _._ _,_ _ ~
,t"'
Dal/s-
1._____
2______
I
IXL________ Apr. 20 May 10
IXL________ Apr. 30 Apr. 30
x G
Number
1
Number
?
DaU8
7
DaUB
14 ----------
DaUB
----------
Daus
2
Days
23 td

~
4 ,____ IXL________ :May 3 May 3
X
X
G
G
1 1 11
10
0o __________
3 156 1(159) 170 ----'X"---I == == == === =I: ==== =:= ==
5______ IXL________ May 5 _________ _ X G
1 1 ---------- 2 12
6______ IXL___________ do______________ _ 2 ? ---------- ---------- ---------- 1 ?
l'j
X G 1 ? 10 2 12 ? ----'X-- --1== == X
== ::==c====== =:
7______ IX!.________ May 0
I__ X G 1 ? 10 143 153 ? X ~
8______
9______ Ixe ________ May
IXL________ ~-------
May 227 _______• __ X
X
G
G
1 1? ----------
__________ ---------­ ---------- 19 ? !2j
10______ Ixe ________ May 28 _________ _ 1 5 __________ ---------- ---------- 1 2
X G 1 --------i- ---------- ---------­ 8 13 ,1:/0
11._____ exe _______ June 4 June 19
12______ exe__________ do______________ _ 'X
o
G
G
1
1
7 15
7 __________ 3 34 (37)
30
759
43
==== ==:= ==1= ======== =1----:it---­ I:':l
8 __________ ----- .. ---- ----------
"'"
13______ exe _______ Juno 5 __________
14______ exe __________ do____ • June 5 o G 1 --------j" o ----------
__________ ---------- 47 55
X W 1 9 ---------­ 2 11
15______ exe ___ ___ June 6 _________ _ ~
====~====[=====:=I----g----
~

16______ exe__________ do______________ _ X W 1 ? ---------- ---------- ---------- 745 ?

X W 1 10 7 117 (124) 7134
17:_____
18______
exe _______ June 10 _________ _
exe_______ June 13 _________ _
X G
G
1 ? ---------- ---------- -------.,,-­ 2 ? rn
19______ IXe ___________ do______________ _ X 1 15 ---------- ---------- ---------­ 38 53
20______
21._____
exe _______ June 18 _________ _
exe__________ do______________ _
X
o
W
G
1
1
21
21
---------- ---------- ----------
10 23
1
(33)
22
754 ====i====[:::=:::r--~-"-- ~
I'j
22______ Ixe ________ June 19
o G 1 22 ---------- ... _-- .----- ------ .. --- 33 55
1-3
X W 1 ---------- ------.--- 2 '1 __________ __________ 0
23______
24______
25______
IXL________ June 20
IXe ________ Juno 23 _________ _
exe _______ June 30 Juno 30
X
X
W
W
1
1 --------i-
?
4
7
----------
__________
__________ ---------- ----------
o ---- .. ----- ----------
__________
5
1
--. "-------1----------1---------­
9 __________ __________
8 _______________ .____
0
0 o
X G 11 ___ .______ __________ I'!J
26______ exe _______ July 1 July 1 X
1 8 o __________ ---------- 3 0
G 1 1 8 o __________ ---------­ 7 15

~
27______ exL _______ July 2 July 2 X ·1
G ] 1 9 o __________ ---------­ 10
=:===::::=I:===:==::: 1----~----
28______ eXL _______ July 3 July 3 X W I 1 10 ---------- 7 17
29______ eXI________ July 7 X W 1 13 o ----------
---------- __________ ---------­ 3 16

~
30______
3L____ exL_______ July 8 • July 8
eXL _______ July 9 _________ _ o W 1 14 -.--------­ 1 15
32______ eXL __________ do______________ _ o W 1 _-
15 ---------- ------_ .. ---------­ 2 17
33______ exL _______ July 10 _________ _ o G 1 13 ---------- ---------- ---------- 12 25
34______ exL __________ do____ - __________ o W 1 14 ---------- ---------- ---------­ 5 19 1-3
35______ exL __________ do______________ _ o
o
W 1 13 ---------- ---------- ---------- 11 24 ==:== ==== =C: =:==== =:1----~- --- C1
30______ exL _______ July II _________ _ o
W
W
1.
1
]5 ---------_ .. . --------- ---- .. ----­
HI ---------- ._- ... --_ .... - ------ .. ---
17
10
32
20 ~
37______ exw_______ July 14 __ "_____ ._ o W ] 18 24 62 (86) 7104 ----f---[:::::==I----~----
38______
39______
exw_______ July 15 _________ _
exw__________ do ______________ _ o
o
W
W
]
I
18
19
23
28
.68
23
91
((51 l 7 ]09
70 X

~ '.}
40______ 7-1XW________do_____ ----------1 25 ___ • ________ .------- _________ •
0 W 1 8 _.-.------ --_ ........ --- ...... ,..-- ......... 11
.L____ 7-1XW______ July 17 ----------1 0 w 1 22 ----""----- .......... .. .
6 ...__________
-----~ --- . - -~
4 26 ----X,---- .-
- ...... ,..- ......... ... ,..- .........
-,.. ..................
~

42______ WXW ______ July 18 ___ •______

7-IXW_________ do____________ ,.-_1 0 W 21 ____--- __ • 10 ? ? ----X---­ ~
43______
44______ 7-1XC______ July 23 July 251
0
0
w
w I 1
10 ---------- -- ...... --- .... - ---------.,
16 2 12 98
6
(110)
16
, 127 ----X--- ---·X---­ X gj
45______
46_____
7-1XL _________do_____________ ._
7-1XL______ July 24 __________ 0
0
W
W
I
2
15 14
17 ---------- --- .. - .... _-- - .. _..... -- .. - ..
109 (123)
4
138
, 21
X X --·-X-·-- t?;j
t='
47______ 7-1XL______ July 25 _____.---- {) .... - .... ---,..-
------30- --------0- 7 '16 ......... .,.. .... -- .. - ...... --- .. -- ---.. -_ ...... ­
W
~
0 1
48..____ 7-1XL_____ July 28 ----------1 0 w 2 20 (311) 159 X X
49______
50______
IXL________ July 31 __________ l
IXL___________ do_______________ , 0 W ~ 31 12 --_
.. - ............. 18 iii X _..------,.... ----X-·-­ ~
0 W ~ 31 ---------- ----- .... ---- ----- .. -- ... ­ {) 740

~
51._____ IX?_________ Aug. 1 ' 0 W 1 32 .. _-------- --------_ .. ---_ ....,-- .... II 41 - ......-----­
52______ IXL________ Aug. 4 __________ 0 W 5 22 ----------
22 ___ • _____________ .. ---.. - .. ----
----- .. _•.. -____________ 1 _- --
' 2!i ..,,'--- .... .. .... ., .. - ... -- X
53 10____ IX!.________ Aug. 5 _________ .1 0 W 3 1 '23 .. - ......... -- .... -----_ ..... _.. X
III
1X in'~icates positive, 0, negative results, except In column headed" Ether used," In which X Indicates that ether WII8 used Bud 0 that ether was not used. 0
"rhe first symbol used refers to stock of queen; second symbol to stock of drone. I=JtaUan, C~Caucaslan, W=whlte-eyed italian, 7-1~FI generation of queen No.7. .~
• Q=glllSS; W=wlre. t?;j

i
• Numbers in plU'enthescs not jnc1udcd In total length of life.
• Queen No.3 was operated on by Malyschev method. .
• No ether was used In first Insemination.
! Killed for dissection.
I Two additional [Link] on this queen were made on this date.
, Killed accidentally.
10 Actual total Is 52. ~
t='
t!j
~
0
0
~
~
0

~
t='
0
0
.~
t='
a
0
~
fJl

~
C.
-{) ,~.

". ~ ,.<.-. ,..,., ....... , • '., 0- .v. '''-'-. _~

16 TECHNICAL BULLETIN 326) U. S. DEPT. OF AGRICULTURE

day after the operation, but when fOlm(1 her spermntheca had dried
out too much for proper exnminntion. Queen No. 35 survived 15
days after the attempted inseminntion and was 1 of 7 of the fore­
going for which negative results were obtained under the microscope.
The remaining 9 queens lived from 10 to 47 days after their opera­
tions without showing any evidence of insemination. Four of these
9 queens (Nos. 12, 13, 21, and 40) disappeared; 1 (No. 36), when
fmmel, was too dry for examination, while the spermathecae of 3
(Nos. 8, 18, and 32) were not examined microscopically. No sperm
was seen in the spermatheca of queen No. 15.
In onty 1 (No. 45) of the 3 queens fr0111 which worke;;;:; or queens
were reared in 1930 did all-worker brood develop. QU'den No. 52,
however,which was killed accidentally on the dny after the bpern­
tion, proyed to contain such a large quantity of living spermatozoa.
in her spermatheca as to warrant the asslIDlption that aU or practi­
cally all r"er brood would have been workel·s. at least in the early
stages of her egg laying. '
In the case or most of dle other 13 queens in whose spermathecae
living spermatozoa were jound. the degree of insemination appeared
relatively small. Fi \'c of these queen:.,; were in hives 4 days or less
after being inseminatec1~ while one was in a hive only 9 days. .As
this period was scarcely long enough for these qneens to begin egg­
laying, there was no opportunity to determine the degree of in­
semination from the qunntity of worker brood reared. "T orker bees
h:lC1 been reared from one (So. ;14) of these queens, however. The
examination of the spermatheca of this qneen was. made more than
3 months after the insemination. Although flat-sealed cells were
found on brood reared from queens :Nos. 37 and 38, no worker pupae
or emerging workers were observed. On examination 80 days after
inseminatioll the spermatheca of No. 37 had a light, milky appear­
ance and liYing spermatozoa were fonnd, while 91 days after in­
semination the spennathec:a of Xo. 38 showed that the sperm had
collected into a l:5mall spherical mass floating in the clear spermathe­
cal liquid.
Another interesting case was that of a queen (No. 48) born on July
8 and inseminated on ,Jllly 28. On September (i only 5 flatly sealed
cells were found out of It total of 150 sealed. The queen was killed,
and on examination of her spermatheca a spherical mass of sperm
much larger than in the case of No. 38 was seen floating in tI~ clear
liquid which ordinarily characterizes the spermatheca of an"llRsem­
inatecI queen bee. Further examination showed living spermatozoa to
he present. Such cases are quite comparable to that of the queen bee
operated upon by Jager and Howard, which, as mentioned earlier,
contained spermatozoa in hel' spermatheca even after she had been
laying only drone eggs. . .
. [Link] queen (No. 11) was born on ~fay 28 and ll1semmated on
June 4. No e~gs being seen in the meantime, she was reinseminated
on June 19. E~gs were seen on June 22 but, although larvae were
seen later, none of these were found to develop into adults. The
queen was killed on July 26 and, although her spermatheca, showed
only a slight degree of cloudiness, living spermatozoa. were
discovered.
 'I

BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS .17

In queen No. 19 the sperm was found massed within' the sperma­
theca in a shape like a smoke ring which revolved rapidly~ This
queen and queens Nos. 52 and 53 were the only ones whose sperma­
thecae appeared under the microscope to be more than 10 .per cent
filled with spermatozoa. Of these three, the spermatheca of No. 52,
as mentioned earlier, appeared to be well filled, or between 75 and
100 per cent. That of No. 19 appeared to be at least 20 per cent
filled, while that of No. 53 appeared to be 10 to 20 per cent filled.
The spermatheca of the queen which produced all-worker brood (No.
45) was not examined micros~opically.
ON QUEENS USED IN 1931 AND 19"'~

In 1931 a total of 103 queen bees were treated, 97 by the Watson

method and 6 by the Malyschev method. (Table 2.) Of the latter,
5 disappeared within 1 to 29 days after the performimce of the
experiment, while the spermatlieca of the sixth (No. 11) showed
neO'atiTe results under the microscope.
Of the 97 queens on which the WatsoJ). method was used in 1931,
both queens and workers were ,~d4\from 8, or 8* per cent.
Workers but no queens were reared from 3 others, thus making 11
qneens, or 111;3 per c~nt of those treated, from which the F 1 genera­
tion was reared. Ad indicated by the proportion of sealed. drone to
sealeel worker cells which were found, some of these queens (Nos.
5,12,33,34,37, and 51) laid no drone eggs; others (Nos. 47, 79, and
84) laid only a very small proportion in relation to worker eggs-no
more, however, than certain normally inseminated queens in neigh­
boring colonies; while about half the eggs laid by 2 (Nos. 78 and 82)
proved to be drone eggs. Of the 11 queens, 5 (Nos. 51, 78, 79, 82,
and 84) were alive and laying at the end of the brood-rearing season
und entered tha winter of 1931-32.
In additiun to the 11 queens successfully inseminated as evidenced
by the production of F 1 queens or workers, the spermathecae of 25
other queens lost by balling, by accident, or otherwise were found to
contain livinO' spermatozoa. TIIis makes a total of 36 queens insemi­
nated, or slightly more than 37 per cent of indisputable insemination
for the season. If to this total are added 2 queens (Nos. 30 and
48) whose only indication of being inseminated was the presence of
some flatly sealed cells among their brood, the grand total for 1931
becomes 38, or slightly more than 39 per cent of successful insemina­
tion. These last 2 queens, however, laid relatively few eggs, al­
though sufficient to have rendered pos,~ible a continuation of their
particular stmin under favorable circumstances.
Of the 25 queens whose degree of insemination was determined
from their spermathecae, the spermathecae of one-fifth (Nos. 4, 29,
53, 86, and 89) were estimated to be 50 per cent or more filled with
sperm, those of about one-sixth (Nos. 45B, 52, 59, and 93) to be 25
per cent or more. filled, those of more than one-fourth (Nos. 3,21,40,
42, 11, 80, and 81) to be 10 per cmit or more filled, while those of
slightly more than the remaining third (Nos. 8, 10, 20, 22, 24, 44, 46,
88, and 99) were estimate? to be less than 10 per cent filled. Flatly
sealed brood was found In 2 of these cases (Nos. 10 and 20), al­
though no emerging adults were seen.
129i8i-32---3
 :j
'-::.:-

TABLE 2.-Record of queens used in 1931 1 I-"

_._-----. . ----; . . .~-:---

00

Queen
Date 0.(
first In­
Date or
second
,rypeoCIDroucs
Droucs Drones Age DC
t~~~e 1c:1- plung- Ur:duror
d
Period Period l'erlod
d queen tween
ufoer
be· be-
tween
at first first second last
tween I
be· Period l'urlod
lived lived Total Flatly I. Work­ ~~~Tn
after length ECaled ers or spar­
I
H
t<J
a
p;j
No. Cross' s9mlna­
atlon
Insem­
Ination tlon I
semlna- er or first
second third Insem. and
metho1d Insem-
and Insem­
Insem- Insem­ Inlltlon second third Inatlon
aCter
egg last
insem­
or life brood· virgins matbe­
seen reared 2!

~
used lnatlon
Inatlon Inatlon laying Ination ea
_ _ _ _ _ _ 1 _ _-- Ill!mm- Inscm- Bnd
1___1-_1 __I_____1
1 ___1_ _ 1 __ Inatlons lnatJons egg
laying
- - -,---, ---,---,. ---, ----, ---
1_________ eXL ____ ,,_____ May
Numbtr
2 Number
________ NU1llbtr DaVB DaVB I DaYB DaVB DaV8 DaVB DaV8 ~
2________ 3
-jiiiiii-T ----------1 w ........... _-- .. 13 ----_ ... _- ---- ...... 8_... --------
________ ... _------ 5 III o ~
___________ Ma~' 5 June 12 W 22 1 2 52 _____ .._____ .___
CXI_-----------
3_________ cxe
3A _______ exe ___________ May
4_________ exe ___________ May
6
15
W
W
3
________
________ --------
--------
4
16
146
30
-----""-- -------- -------- --------
________ -------- -------- --------
--------
-------- 10
9
1
------.-- --------1-------­
25 ________ ________
15 ________ ________
X
0
X ~2!
18 - .. -- .. --_ ... - ---------- 2 _____ " __
5_________ exe___________ May W 2 ________ ... _------ 6 ________ ------- .. -------- ----- ... -- 2 8 X X _______ _
6_________ exe ___________ May 18 ---------- - .. -----_ .. - W 1 _______ • -------- 6 ________ -----,..-­ 14 5 I (19) 25
7.________ cxe ___________ May 19
-june--4- M 1 .... _-----
________ ------- .. ---- ... ---
7 ________..
------- 3 9 ---0--- ---0--' ---0--- CI>
May
8_________ cxe ___________ May
9_________ exe ___________
10_________ exe___________ May
19
19 May 20
20
W
W
W
1
11 ________
1 ________
..------~
6 ]01 --------
fi7 ________
________
--------
8 ________ -- .. _---- --------
-------,.
... ,._-----
11
0
7
40 ________ ________
13
14 ---X--- -------- ---X---
X
. I.\:l
CI>

ll________._ exe ___________ May 20 W 11 ______ .- -------- 13 163 -------­ 184

12_________ exe ___________ May M
________ 8 ________ --------
_... _----- ~
---x---.,---x--- ~ -­
20
13_________ exe ___________ May 21 -junii--5- W 21
1 ________
07 ________
-------- --------
15 -------- 3 98
0
(101)
17
122
14_________ exc ___________ May 22
22 -i,-fUy"23-
---------- W
________
- .. ------•
1 _______ ________ ---_ ... _--
1 -------- -------- 10 17 -------- -------- ----- ... rn
15_________ exe_____, _____ May 1\1 21 7 -------- 1 9 ..... _--- .... ---_ .. --- - .. _----­
________
. .. -------

~
16_________ CXo___________ May 25
-june--5- w .. -------
2 ________
10 ------- -------- --------
8 ________ -------- 8 18 -------- -------- _.... _--,.. ..
May 28 M 1 11 10 4 (14) 33 - .. ------ -------- ---- .. _-­
17_________ exe ___________ May 283 ---------- M 1 1________
18_________ OXL___________ __________ ---------- 2 _____ • __ -------- ________ ------- .. --------
127 -------- -------- 7 19
10_________ cxe ___________ Juue 3 __________ ---------- W 1 _______ • -------- -------- -------- 23 30 t-3
20_________ exo___________ June ---------- M 21 -------- - .. ----- ... -------- -------- 29 50 ---x--- -------- ---X--­
21. ________ cxe___________ June
22_________ exe ___________ June
23 _________ exe ___________ June
24________ exe ___________ June
5 June 29 ----------
5 __________
8 __________ ----------
-_
8 __________ .. --- .. ---
----------
W
W
W
W
. 22
1
1
1
________
________
_______ •
----------
--------
_.. _-----
24 ------- ..
11 ________
3 ________ -------- --------
3 ,._______ -------- --------
18 110
--------
--------
5 ________ -------- -------- --------
(128)
1
4
5
153 _______________ .
6 ________ ________
4
7
X
X
~
g;
25_________ exe ___________ June 10 ---------- ------ .. --- W
1
1
________
________ ... _..... _--- 6 ________ -_ ... _---- ------_ .. ------- .. 3 ==::=:== ::::==== ---X--­
8 ________ ________ 0 ~
26_________ exe ___________ June 11 ---------- ---------- W 2 ________ -------- 6 ________ -------- -------- -------­ 2 8
a
27_________ exe ___________ June 11 ---------- ---------- W o ________ -------- -------- - .. ------ 7 13
28_________ exo ___________
June
29_________ exe ___________ June
11 ---------- ----_ .. ----
12 ---- .. ----- ----------
W
W
2
2.
________
________
--------
--------
--------
7 ________ -------- -------- --------
7 ________ -------- -------- -------­
5
14
11
21
g
1 ________
==:==:=: :::=:::: ---X--­
~
30_________ exe___________ June 12 --------""- ---------- W 1 --------
________ 7 . ________ --------
o -------- -------- 1 8
31.________ axe ___________ June 12 June 12 ... _-------- W 31 ________ 20 90 (125) X ••_______
132 _______ 0 0
:12 _________ OXL___________ June 15 ---------- --- .. ------ W ._----- .. 10 ------- ... -------- ---- .. -- .. ---- .... -- 28 38 _______________ - 0
2 ________
33 _________ ex!. ___________ June
June 18 ____________________
19 W 2 ________ .... ------ 14 ---- .. --- ..------- '"'------- -------­ 11 25
34 _________ oxI___________ 1 June W 2 ________ ---_ .. -- .. 14 ..-----_ .. --- .. ---- 0 34 (40) 54 X X ?
221__________ ---------- W 6'/ --_ .... --- -------­ 12 (21) X . ________
X X
35_________ axe___________ June 23 June 23 __________ W 1 1 --------
______ ._
7
o •______ •_______• 9
..,------- 10
? _______
17
0

,,);
30••••••••• OXO •••__...... JUII~ 231 Juno 23]•••••••- •• 71 18 ........ ........
xu
:17••••_.... OXO._••••_.... '1 lJulle 23 . __ ••••••
CXO__ ......... JUliO 24 •• _•••• _...._•••••••
~lS ....__ •• _
'"ww I ,........,
1 ........ •?
0 ,........,........ ,.... ,•••
., ........ ~ .._..
7 _....... .........
]5 12
.17 II
6
('J7)
(2.l)
65
~
x 000
X
30......... CXl...... _•••__ JUliO 24 __ •• _••_••• , __ •• __ •• W 1 3 ........ ........ 0
~ I....··ot::::::t:::::::t::::::·
_ _ _ .... l

40._______ •
41.•••_._ ..
ex!. ...... _.._.
cxe ...........
OXI ••••• _••••• ,
JUliO
JUliO
24 Juuo 24 ......... .
26 ..........._•• , ••• ,.
'\'
W
W ..-... t:::::::
~ ~.l 1..······1····....1·..•··..1
..·....·
0 ........................
2
7 • ................
J!) _...............
........ ........
x
0
x E
42•••••••••
43._••__._.
44_._._....
45._•••••__
ax C ___•••••• _.
JUliO
J'UIIO
OX!. ___•__•• ___ JUliO
CXC ____ ••••__ • JUliO
25 JIIIIO 25 .".,,_._,
26 ••_.__•••• _. ______ __
20 •••••••• _. __ .~",._.
20 •___ •••••••••_••__ ••
W
W
4
4 -- - ....... -- 1-" -_ .... - ... 1........ --_ ...... 1-"::-- .... _­
2
7
4
"
11 _....... " __''''
8 ........ ........
0
x ~
W 2 1.... ___ .... _1 .. ____ .. _. 7 12 10 ................ 0 !j)
axe...__.._... •
:::~::: :::~::: ~
45A ....._. 1 W ? G? 1 U
._...... ........ ........ ........
1-- .. --- .... 1--.. -- ......

~
45B_._. __• exe........... I? W ? 0'/ ./ U
46••••••• __ 12-lxe._._.__ •• July
47. __•••_._ axe.__........ July a
1 j"jiliy··"!i"'....•.. •..
W
W
1
1
1 [Link] U 1 ........ ........ ........
20 ........ ........
7
(IISI ~7
14
exc........... Mny 4 ·jii!v..:i·,·.... ·.... W
g 00
4S•••••••.• I I 14 roll 12 SO (42 110 x .................
40......... axe........... ' ? IJuly fl W ./ 1 ,./ ? ............__ ....".... 0 ~ ~
exe ......... __ I': iJuly 6
" :::::::: ~~~~~~~~ ...~... ~
? ........ _..._.., _.~..... 14
50.........
51.........
52.........
53.........
axo .......... _'July 0 (il
CXC........... •? lJuly 13 ..........
axc ........... JUlie 18 July 0 1 July 13 W
W
WO
W
1
?
./
I
1
1
~ I·..··"j'
?
•?
13
?

21
? .I;l ................
./ ........ ........ ........
4 ........ ........
7
11
~ "W ·"X"· '''x·'' :::~:::
54....._••• rxo ...._....... July 17 •••••_............'. w 1 3 7 J!)
55.•••...•. lXCh.....•.•__• July 15 July 21 •• , ••••••.
56......... IXO .•.••••••••• July 10
57••••••••• CllXO •••••••••• July 30
July 16
Aug. 5
a
July 21
AUI!. 0
o
w
1
2
2
2
3
7
·..·....1
6 ..···..·..
6 I
1..···..·,·......·
........................
o 5 ... _____ ..... _.. _... _.... ..
1 .................
3
10
7
11
g
~
E
58••••••••• 12-IXL••••••••• Aug. 4
51l.•••••••• 47-1XO ••••••• _ Aug. 6
..............."••••
••••••••••••••••••••
w
w
1
I
6
6
8
21
o
14
~ ::::::::\::::::::I·"X"· ~
w
~
00••••••••• 47-1XO ••••••••• Aug. 6 ••••••• _•••••••••••• 1 6 ~ ........ ........ 0
61.•••••_•. 47-IXO ••••••••. Aug. 6 .................... w 1 U o ~ ............................... - ------_ ..
62••••••••• IXO •••••••••••• Aug. 7 ••_••••••••••• , ••••• w 1 o 4 U ........ ..._.... 0
03••••••••• OXL ••••••••••• Aug. 7 """"" •••••••••• w 1 o .. ___ .. __ ... 1,;,-.:'''''_'' "'''1 .... _ .. ____ 1_ ... _____ _ 8 17 (:)
64••••••••• 51-1XO ......... Aug. 10 ...............'••••• w 1 5 5 J!)
···:::::1::::::::1····8'.. o
65••••••••• 51-IX!. ••••••_._ Aug. 10 •••••••••••••••••••• w 1 3 8
66••••••••• OXL••••••••••• Aug. 10
67••••••••• 51-IXe•...••••. Aug. 13
•••••••_••••••••••••
••••••••••••••••••••
w
w
1
2
"
5
8
:l.1
Ii
28
~
... 0 !2l
~
68••••••••• 51-IXO••••••••• Aug. 13 ••••••••_••••••••••• w 1 7 14 ~
::::::::I::::::::1"'0'"
~
69......... 5J-IXI.......... Aug. 14 Aug. 14 •••••••••• w 1 1 1_______ _
8 ..''''0'[Link] 1 9
a
• ::::::::I::::::::r l
70......... 51-IXL......... Aug. 14 ••••••••....•••••••. 1 8 20 28
71......... a XL........... .\ug. 14 .••••••••• """"" w I 16 W
72......... OX!.••••••••••• Aug. 14 ............, ••••••• w I 8 13 21
73•••••••__ 51-IXL._•••.••• Aug. 18
74•••___ ••_ 4i-IXC......... Aug. 19
75••••••.•. 51-IXL......__ . Aug. 24
....................
... _._ ••• _ ••, •••••••
Aug. 24 ..__ ......
w
w
w
I
I
I I,........
8
8
13
........I........,
o ........ .........
.......,.........
......., 1~
11
~
M
~
(:)
o
!2l
76... __ •••• 47-IXC ......... Aug. 24 Aug~ 24 .......... w 1 o ........ ........ ........ 11

i
1 ........ 14
w
.............·..
x ···1-·.. ··...
I
77......... 47-IXC ......... Aug. 24 Sept. 21 .......... I 2 ........ 13 28 ..................._............
78......... 47-IXI.......... Aug. 24 .............._..... w 1 13 X .......

:::::~: l~:::~~I~:~~~:i-I~~l~l: ~~:~:~:

79......... IXL............ Aug. 26 ....._.............. w 1 21 X X ..._....
................ : x
SO......... IXC ............ Aug. 27 ' ' ' ' ' ' ' ' ' ' .......... w 1 19 ~
81._....... IXL........... Aug. 28 w ..····ii"!:::::::: ............... ~ X
82......... IXL............ Aug. 28
83......... 47-1XC ......... Aug. 28
Sept. 23 ...... _...
....._..............
Aug. 28 ..........
w
w
1
1
1 'iT""'"
.... - ..........
19
21
13
"
M~ X X
See footllotes ut ella oC tuble. ......
CO
 ~ .-. ",~

TABLE 2.-Record of queens u8ed in 19S11-Continued t-:)

0

I
all
Period Period Period
t\~:;;n I I 1-3
t::J

I
Type Drones Drones Drones \ge of be· be· I' ·1 I Period' , S1
Date of Date of Date pf plung. used useo used ~ueen tween tween last If~ed lived '1'otal Flatly 'Vork· sJe~n 0
Qneen Cross , Jlrst In· second third In· er or for for for I1t Jlrst first second insem. after after length sealed ers !>r spero IIi
No. semina.! Insern.! semina.! method Jlrst second third Insern. and and inatlon egg last of life [Link] vlrgms lOathe. ~
Btlon Ination tlon Ilsed 3 Insern· Insern· Insern· Inatlon second tblrd and laying lnsem· . seen ..reared 11
Inatlon Inatlon Ination .ins~rn • .Insern. egg. flmti01! j C S
:>
Inatlons nations laying
t"'
- - - - -------1---,---,---,---,---,· - - - , - - - , - - - , - - - - ' - - _ '_ _ _1..; --.-.-.,.....1---1---1 ....
b:I
Number Number
~~~~~~~) V Davs DaV8DaVB I DaVB DdVB DaV8 I ! 0
84••••••••• lXO •••••.•••.•• Ang. 20 ------"'--- --------- . 'W
w
1 Da 180 18 ..•••••.••••..•• (II) X X .I ••••••••
~
85••••.;•••. 51-IXL•••••.••• Ang. 31
'Aug:'iii' 'SorC'i'
I ••••••••
I """j" •.••••••
22
20 """1' ..
.. --------
I
------- -------
23 '••••••••••••.••; 0
----_ . -
------ ....----
t::J
86••••••••• OXO •••••••••.•
87•.••••••• OXO •••••.•.__•
•?
Sept. I
W
I
? 1 27
----'----
16 36
--------
. . ..
. ....... X
...........
-------- "'. .......... 1-3 ?'

······i·' i~
W
······1· ······8· ------=;.. .
SIl•••••••• 37-IXL•••••••••
89•••••••.• . 47-1XL. .•••••••
Ang.17 'Atig:'25' ·S;;)~i"."i·
Sept. 4
W
w
?
) ...... j. .••••... \l
.... 7 -- ----
3(1 ------
..................... --- ..
~---- ... X
3
- .... -----.-
14 ........... --- -----.-­ X
H
Z
Sept. 8 'sjijjC's' ·Sept.··g· ······0· .... -...... . -. 20 ,. ....... --_ .. ---- ...-.. ....-..... -.
-~

9(1.. ••••••• 51-IX!...••••••• W ) 1 14 J 11 c.>

. --_ ...... ... _----- --_ ... ---- lfl .--- .. -- ... -_ .. _-- ....
~.- ,..-~-

91 •••••.••. 51-1XO••••••••• Sept. 9

Sept. 10 'SarCiii'
w 1 ........ ...... -"" .. -~
15 '-.~-."
o. ....•...
" 1
-_ .. -_ ... -- .. _------
0 toO
9'~ ••••••••• 61-1XO ••.••••••
93••.•••••• 47-IXO •.•••••• ~ Sept. .10 Sept. 10
W
w
) J •••••••.
1
J6
17 o .....••• .... -. - ... -- -- ....... _-,..
-~--"''''''.
tl
(10)
23
---- .. --- ...------- ···X'" ,.C>
94••••••••• 47-lXO ••••·••••• Sept. 16 Sept. 10 'Soiif:iii' 0 I "-"']' 8 0 o •......• -----_ .... 36 44 .......... --- -----_ ..- --_ .. -...... q
95••.•.•••• 82-lXL••••••••• Sept. 20 W .... _---. -.... ,..- ..... 6 •••••••• ............ _. . --- . -..... .. _----_ . 6 11 ... . -...... -. --- . --- .. 0 .,'
·w - ... •••.•••. .. ------ .. . ------- ..-- . -- .... 43 -- . -,..--" _. _---_ . ......... _--­
" "

--............ -,. ......._.. .........

00•.•••...• 82-1X!....•••••• Sept. 30 2 ...... _.. __ ~ 7 31l
97••••..••. 82-IXO••••.•••• Oct. 1 W "--_ ...... 8 ••.••••• . ---- . -. -------- .... ---_ .... 20 28 .. .. .. --- ... ------ ....
'- 0 !Il
----- . ------_ 17 ---_ .. . -.. -_ ........
..-----.. _-.. -.. ....-.. ..... -------
98•••••••.• 82-lXO ••••.••.• Oct. U •••••••••• -- ..-------- W ....... ~

- ...... 13 .. -------- 4
_---
A ...... _ ..... _
"'
911••••••••• 47-1XO ••••••••• Oct. ]5 --_
.... . -------
.. . W ...... ----. ..-- ......... 17 .. -----_ . -------- 8 26 ----- . -,. --- .
~

X t:I
.... _----- .---- . --- . ------- . -- .. . t::J
---~
100 II...... 7!HXI.......... Oct. 15 W ... --- .... 15 21 rA} i.. --....... -.. -.. -......... ~ .-,. .. -.. ---
-------~ ~ -~-
'd
I X Indicates positive 0 negatlve:results. t-3
I First symbol relers to stock 01 queen; second symbol to stock of drone. C=Caucasian; Ch=Chinese; Cp=Cyprlnn; Q=Oermnn brown; I=Ita][an; 4i-l, ctc.=FI generation 0
01 queen No. 47, or of queen represented by llrst number. I::j
I O=glass (Watson); W=wire (Watson); M=Malyschev method.
• Numbers in parentheses not included In total lengthlol lfle. :>
• Put In a nursery cage alter operation and no record taken. G:>
I Virgin used came from other than queen·rearing colony. ~
H
, 38 days old at second insomination. 0
I 31 dnys old at secoOli Insemination.
• 34 days old at second insemination.
0
ID Still alive on Nov. 1, 1931, but missing on lo'eb. 10, 1032.
t"'
H'
1\ Still alive on Feb. 10, 1932. •
12 Actual totlll Is 103.
d
~
:=:l

~-.~?
 BREEDING THE HO~""EY1::EE mmER COXTROLLED CONDITIONS 21
Of the 59 queen bees operated upon by the ""\Vatson method in 1931
and for which no positive results are recorded, the spermathecae of 26
proved to be clear on examination. Two of the remaining 33 queens
were inseminated late in the year, and in the slackening of brood
rearing, which occurred soon after all were successfully introduced,
the results of the attempted inseminations were not determined.
These queens, however, were still in their nuclei at ·the beginning of
",inter. The spermathecae of the other 31 queens were not examined
microscopically.
AlthGUgh the sep ·jIl is not yet over, it seems in order to state that
353 queens were subjected to the Watson method in 19;:12 before
September. 'With two exceptions only one operation was given each.
In the two ex~ptions two operations were given bat neither proyed
successful. Of the 353 queens, 115, or nearly one-third, [Link] success­
fully inseminated. From 43 of the 115, adult workers or queens,
or both, were reared. The majority of the 43 laid only worker eggE-"
The egg-laying performance of some of them could not be differen­
tiated from that of naturally inseminated queens. Certain of these
queens were given 10 frames. As about 50 queens yet remain to be
checked, the total number of successes for the season should prove
somewhat higher tllan that just given.
:XEW INSTRUMENTS AND j\IETHODS FOR ARTIFICIAL
INSEMINATION
In 1927 the technic originally described by Watson was carried
out in the main, with some minor [Link]. For instance, no
attempt was made to feed the queen while she was bound to the
operating block, as the writer has repeatedly observed that the queen
bee can go without food with no apparent injury during the. time
required for the operation. illumination was obtained by using
a standard microscope lamp and at times even an ordinary desk
lamp without any special reflector or filter. A. microscope lamp is
still bein~ used and is brought as near the queen as necessary for
sufficient lllumination during the operation. N'o ill effects from heat
have been detected. In private discussion with the writer Watson
stated in 1931 that h~ now uses a lamp that is attached to his
microscope.
GLASS HOLDER

In 1928 the first major modifications in the technic were made.

One of thesl' as described elsewhere (313, 33), does away with the
necessity of binding the queen down with thread to the operatin&"
table. ~\. small glass tube, devised by J as. I. Hambleton, is usect
instead. The queen is backed into this tube until her abdomen
projects just far enou~h from the narrowed opening at the other end
for the operation to De performed. .:.\. stopper formed of another
piece of glass tUbing prevents her escape once she is in position.
As originally described, the glass stopper was held in the outer
tube by a small wooden wedge. The writer later adopted the plan
of dipping the end of the stopper in melted paraffin so that a coating
is formed which will sene to retain the stopper in the outer tube
when it is shoved in the desired distance. In hot weather it may
be necessary to recont the stopper frequently if paraffin with too low
a melting point is used, and, at times, the wooden wedge can be used
 TECHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

in aUdition. It has been found most convenient of all, however, to

fasten the stopper in place by a small piece of DeKhotinsky cement.
The stopper is thus held firmly in place during the operation and is
easily loosened by turning it enough to crack the cement.
The writer has found that
the queen is easily made to
back into the tube by letting
her first run into another simi­
lar tube. When her head
touches the constricted end she
usually begins to back of her
own accord, and if the two
tubes have been brought to­
gether in the meantime, she
will continue backing until she
is in proper position in the
holder.
MOUNT FOR GLASS BOLDER

At first the glass holder

was mounted on a triangular
FIGURE 5.-Glass hO~~~~:upported on wooden wooden block (fig. 5), which
permitted an elevation at three
different angles. The tube could be turned to any desired angle in the
plane of the face of the block on which it rested, because it was bound
to the block only by rubber bands. Such a block serves very well.

FlOURS 6.-1I10unt for glass holder with queen shown In holder

In 1931 the wooden block was discarded in favor of a standard

dissecting lens stand. (Fig. 6.) This apparatus, entirely of metal,
consists of a long arm fitted to an upright rod by a rack and pinion,
the upri~ht itself being supported by a heavy base. At the end of
the arm IS a revolvable clamp which holds the glass tube and permits
it to be turned about the arm as an axis. The tube can also be readily
 BREEDING THE HONEYBEE. UNDER CONTROLLED CONDITIONS 23
set at any desired angle in relation to the arm, since it is held only by
pressure from the two sides of the clamp. ..
In 1932 a different type of mount was devised which is now used
exclusively. Included in its construction (fig. 11) is a narrow
strip of,. wood with a hole in each end through which passes an up­
right metal rod. The strip can be readily pushed up and down on
these rods, but yet fits tightly enough on them to stay at any desired
height. Through the center of this strip is a narrow slot about 2
inches long for the insertion of a thumbscrew which also passes
through a smaller wooden block. The thumbscrew is provideg with
a wing nut for fastening the two blocks firmly to each other:·, anv
vertical angle. 1'he slot permits a certain horizontal adjustt..eht of
the smaller block. in the lengthwise direction of th.:: long block. The
glass holder fits into a hole through the smaller block, and can be
rotated in it. To hold the tube in the desired position in this hole,
a thumbscrew is provided. Since this mount allows the holder to be

A' B
FIGURE 7.-CUps: A, Old type; B, new type. Both enlarged

adjusted in position for height, to be rotated on its own longitudinal

axis, to be revolved in a vertical plane, and to be moved for a limited
distance horizontally in the lengthwise direction of the longer block,
ample provision is made for bringing the glass holder into proper
position in relation to the pipette.
SPREADING AND HOLDING ABDOMINAL TIPS
CLIP

Another innovation dating from 1928 was the use of aU-shaped

clip of fine wire with each end bent at a right angle to form a small
prong. These prongs were so inserted as to hold theabdOIninal tips
of the queen bee apart while the pipette was manipulated into posi­
tion. 1.'he sides of the U were bowed outward to give some tension
and, when the prongs were in position between the abdominal tins,
projected at right angles from the queen. Instead of making" a
simple U-bend in the clip, the writer later found it advisable to
secure more tension by making a small loop at what corresponds to
the bottom of the U. (Fig. 7, A.)
Such a clip was first suggested by L. C. Spencer, of Louisiana, for
use with the Quinn-Laidlaw method, but, as described earlier (31a,
33), its use with the Watson method has proved more convenient than
using a pair of forceps for the same purpose. This type of clip,
however, possibly owing to the wire used, proved a trifle too heavy,
24 TECHNICAL BULLETIN 326 J U. S. DEPT. OF AGRICULTURE

since movements of the queen often caused it to swing downward

with the points of its prongs serving as pivots and then to drop out at
inconvenient moments.
In 1931 the writer devised an entirely new type of wire clip which
stays when put in position. It is U-shaped with the two sides of the
U straight and bent back on themselves nearly as far as the closed
end. At this point the rebent sides are bent upward at :right angles
and are left just long enough to be grasped conveniently with the
points of a fine dissecting forceps. The width of this clip approxi­
mates the lateral width of the vestibule of the queen, while the length
is approximately the height of ~he vestibule when the queen's abdomi­
nal tips are spread apart. (FIg. 7, B.)
Tht, clip is easily introduced between the ql,[Link]'s abdominal tips.
During this procedure it is held in a pair of dissecting forceps.
With the point of a transferring needle the abdominal tips are first
spread apart just enough to permit the insertion of the closed part
of tbe U between the Stlllg and the dorsal abdominal tip. Then,by
pressing against this tip with the closed end of the clip and by
pressing at the same time a~ainst the ventral abdominal tip with
the transferring needle, the tIpS are spread far enough apart so that
the sides of the clip can be dropped down within the sides of the
vestibule. (Fig. 8.)
When in position the rebent ends of the sides of the clip rest against
the queen's ventral abdominal tip, while the closed end of the clip
rests against her dorsal abdominal tip. The ti!Se of the clip rests on
the membranous covering of the posterior end of the body cavity.
The pressure of the tips plus that of the sides of the vestibule holds
the clip in this position regardless of respiratoi'y movements by the
queen. The swinging movement, which was an objectionable feature
of the older clip, is absent. The sting and sting palpi are free, but,
possibly owing to stretching of sides or ends of the vestibule, remain
out of way and at the snme time are kept from injury to themselves.
HOLDING ROOK

In connection with the clip, the writer in 1930 introduced the use
of a teasing needle with its point bent at a right angle to form a
hook. By means of this needle, or holding hook, the queen's ventral
abdominal tip is pulled to the left the desired distance when the
pipette is brought within the vestibule. The clip may be removed
at this time since the pipette itself then serves to hold the queen's
dorsal abdominal tip to the right. The sting remains out of way
between this tip and the pipette. In 1931, as a means for keeping it
in the proper I)osition without holding it by hand, the hook was
fastened to an adjustable clamp mOlmted on a supporting rod. When
thus held it is easily brought into place by moving thl'l base t.o which
the rod is attached.
In 1932 a method was devised for spreading and holding the
queen's abdominal tips apart 'by the use of two holding hooks alone.
In connection with the mount devised that year for the glass holder,
a small wooden block to support a holding hook (fig. 11) was
nttached to each of the two metal rods. These blocks can be shoved'
HI) and down or be turned on the rods, but fit tightly enough to stay
in the desired position. 'fhe holding hooks resemble teasing needles,
 BREEDING THE HONEYBEE UNDER CONTROLTJED CONDITIONS 25
having wooden handles. and points 0:£ fine spring wire bent in proper
shape to hold the abdominal tips of the queen without injuring
her. The handle passes thrqugh a hole in the block large enough
for freedom of movement in inserting the hook between the queen's ah.­
dominal tips, while a thumbscrew is provided to fasten it securely in
position. The ventral abdominal tip is held by one hook and the dor-

FIGURE S.-Insertlng the clip

sal abdominal tip by the other. Thus, merely by means of these two
holding hooks the tips are easily spread and held apart the proper
distance and the clip is eliminated altogether. When the pipette is
in position, the hook holding the dorsal tip can be withdrawn.
In the procedure just described the combined use of two hooks,
or of a hook and a clip, does away with the need of a forceps dur­
ing the insertion of the pipette, and the qneen's genital opening is
129787-32-4
26 '£EOilNICAL BULLETIN 326, U. S. DBPT. OF AGRIOULTURE

more readily located than when her abdominal tips are kept apart
by a pair of forceps held by hand. [Link], this procedure
speeds up the bt'inging of the pipette into proper position in relation
to the genital opening, and permits the operator to leave the micro­
scope during the operation at any time after the insertion of the

FIOUllfJ D.-View of apparatus during inseminatIon of queen with technic as modified

in 1931

pipette and, on returning, to find relatively the same view still

under the microscope, provided the queen has not had too great
freedom of movement. The view presented in the field of the
microscope is the following: At the right, the pipette; in the center,
the end of the queen's abdomen protruding from the glass holder
with her dorsal abdominal tip at the right and her ventral abdomi­
nal tip at the left i and, at the extreme left, the hook. (Fig. 9.)
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 27
MULTIPLE SET-UP

The time required for the part of the insemination accomplished

under the micl.'oscope varies considerably, depending upon the quan­
~ity of sperm in the pipette, !tow properly t?e pipette has been
mserted ill the queen, how rapIdly the sperm IS forcedQut of the
pipette, and similar factors. Sometimes the insemination has been
completen in half an hOUl', but oftenc,it has taken longer. This
does not include time spent in getting the queen ready for the oper­
ation, filling the pipEtte, and other preliminary manipulations.

1,'IQURE10.-Unit of multiple set-up at first devised in 1932_ There is shown the

microscopel false stage~ new pipette manipulator, rod support for manipulator,
micropipette, glass holoer for queen but without queen, mount tor glass holder,
holding hook with its support, and. ne~-stYle lamp

From experience thus far, the writer prefers that the operation
should not be hurried.. During the actual operation the screw of
the pipette is turned slowly for the purpose of forcing the sperm
out just fast enough for it to enter the queen without leaking ;back
out. If it does begin to leak, the operation is stopped to allow the
sperm and mucus exposed to the air to harden, as described later
under the heading" Mucus Plug." For this and other reasons, the
operator must often wait during the operation before proceeding
further.
In order to utilize any extra time on hand during an operation,
the use of a multiple set-up for the work in 1932 was adoptea so that
several queens might be operated upon more or less simultaneously.
This multiple set-up contains several individual units (fig. 10),
each of which consists of the apparatus used in the artificial in­
semination of a queen. To reduce the expense of each unit, however,
28 . TECHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

and to provide greater convenience in the work, the writer devised a

pipette manipUlator and also a false stage which is not attached to
the microscope but yet serves as the base for the support of the
manipUlator. The stage and manipulator are described under sep­
arate headings. The method of holding each individual queen is
that already described which permits the operator to [Link] the micro­
scope once the pipette is properly inserted in the queen. The false
stage is so constructed that, even if th~ Barber micromanipulator is
used, only one miscroscope and one lamp are needed for the entire
set-up regardless of the number of units. The writer's equipment,
however, includes a lamp for each unit because it is much more con­
yenient and saves time.
By the use of a multiple set-up any tendency to hurry the opera­
tion should be reduced because the operator has more than one
queen with which to occupy his time. The number of queens to be
provided for in one set-up will depend upon the skill and convenience
of the operator. The set-up used by the writer in 1932 consisted of
six units.
FALSE STAGE

In the writer's equipment for work with the Watson method in

1932, instead of the false stage pictured in Figures 9 and 12i which
slips tightly over the base of the microscope stand and ho ds the
Barber micromanipulator, there is used a stage (figs. 10 and 11)
made of a sheet of metal supported at each end by wooden blocks of
a height sufficient for the base of the microscope stand to be slipped
in and out :readily beneath the stage without touching it. Instead
of using wooden blocks for supports, the sheet of metal may be bent
at each end for this purpose. If the Barber micromanipulator is
used, provision is made for attaching a removable support for it,
while. for the pipette manipulator described in the next section pro­
vision is made for attaching to the stage the rod which supports the
manipulator. This stage is made long [Link] to attach to it the rod
for the holding hook at the left of the operator, or to serve as a base
for the. support of the holding hook in case a movable support is used.
It is also long enough not to tip up if the Barber micromanipulator is
being employed with it, but is not wide enough to hold the lamp or
the base of that type of mount for the glass holder shown in Figure
6. If desired, it can be bolted or clamped to the table or other sup­
port on which it rests. When the stage is equipped with a rod at the
right for the new pipette manipulator (fig. 11), and one afthe left
for the holding hook, these two rods also serve to hold the new
mount devised for the glass holder in 1932. In the multiple set-up
these stages are placed ina row, or otherwise, for conveniently trans­
ferring- the microscope from one to the other, as ·well as to provide
for shifting the miscroscope lamp if only one is used.
PIPETTE MANIPULATOR

The new pipette manipUlator devised by the writer in 1932 con­

sists of two small oblon~ wooden blocks joined together by a thumb.
screw and wing nut whICh permits fastening the two pieces. at vari­
ous angles in relation to each other. (Fig. 11.) .one of the blocks
is made into a clamp which is fitted to an upright rod attached to
 :BREEDING THE HOlo'"EYBEE In,J)ER CONTROLLED CONDITIONS 29
the false"stage. By means of another bolt and wing nut this clamp
can be fastened firmly at any height on the rod and at any horizontal
. angle. Through the free end of the other block is a hole just large
enough. to permit the pipette to be shoved through freely, a small
screw being provided to fasten the pipette at the proper position.
An additional clamp just beneath that holding the manipUlator. on .
the rod serves to hold the manipulator at the proper height' when
rotating it in a horizontal plane.
The ~d supporting these clamps is threaded at one end. This end
is paSsed through a hole in the. false stage. Q,nd a nut fitted to it per­
mits fastening the rod rigidly in place. If this. rod and another
for a holding hook are fastened to separate bases like those used for

FIGURE 11.-Equlpment devised nnd used by writer In 1932, showing false stage,
mount for glass holder, two holding hooks' and their mount.~, and pipette manipu­
lator with extra clamp just beneath It on its supporting rod. The pipette is
slightly modified from the standard Watson type as regards the style of aetlvating
[Link] and the. fact that it passes through a wooden Instead of a metal cap flUe!!
[Link] the outer glass sheath

the holding llOOk in 1931 (fiO'. 9), these two rods can be used as a
support for the new type of mount for the glass holder (fig. 11)
devised in 1932 and the stage may be dispensed with. However,
the writer has found it more convenient to use the false stage, since
it is more rigid and lessens the strain on the long strip of the mount.
It ispossiblc to operate the new manipUlator when its supporting rod
is mounted on a separate base alone in conjunction with the mount
for the glass holder used in 1931 (fig. 6) and a separate base for a.
holdin~'hook, but the lack of rigidity plus the risk of hitting either
base WIth the microscope or body makes this arrangement much less
desirable than the use of. the false stage. .
This type of pipette manipulator evidently does not give so fine
an adjustment as does the Barber micromanipulator, but its cost is
negligible, and, if properly made, it will serve the purpose if used by
a person with a steady hand. It has the further advantage of rota­
tion in a vertical plane. It was the only manipulator used by the
writer in 1932.
30 TEOHNICAL BULLETIN 326, U. S. DEPT. OF AGHlCULTUUE

PIPETTE

.A:i, one stage of the work it seemed desiruble to usc in the miero­
syt:~lge u "luss tube of smaller diumeter than the one furnished by
'Ylltson. A smaller pipette meunt using a correspondingty slllaller
plunger. After some experimentiIw in 1929, glnss tubes of less thun
hult the diameter of those furnished by \Vlltson were mude and fitted
with ~lass pllln~ers (34.). All the writer's work in 1929 wus carried
out wIth such pIpettes. In 1930, 20 queens (Table 1) were opel'llted
on with them, Ilnd 5 in 1931.. ('ruble 2.) Some success was had.
1Vutson hud experimented with glnss plungers but glwe them up
because he considered them too ll'llgile (4<9). He attempted to wind
his fTIIlSS plun n'ers with thread to secure better suction, wherells the
writ~r hus hal'success in using them unwmpped.
]j"'OI: use with the small tubes glass plungers present certuin desir­
able features, umong these being their rigidity when in lhe tube and
the ellse with which they can be dl'Uwn out from a piece of glass rod
to fit any such tubes. On the other hund, the tubes can be aruwn to
fit the plungers. One end of the plunger is carefully smoothed off
in 11 flame ulld the other end is fastened by DeKhotinsky cement
directly onto a glass stem drawn out to the proper shape to permit
the spt'ing to uct Oil it, Otherwise, construction of these small
pipettes is the same us thllt in the standard 'Wutson pipette except
thut nn outer piece of glllss tubing is fitted over the plunger tube to
give it greater strength.
Although snch pipettes uppeur to o ITer certain udvuntages, it is evi­
dent thut if the tubes are kept short enou~h to permit convenient
munipnlution und to reduce danger of brealmge they will not hold
so much sperm us does the originul 'Vatson pipette. The smaller the
dinmeter of tho pipette, the mOl'O this criticism Ilpplies. Considel'll­
tion of the qUlUltity of sperm IlvlLiluble in tho pipette at the time of
the operation in rei uti on to sllccess of the insemination WIlS one renson
for tho exclusive use of tho regular 'VutSOIl pipette throughout the
latter part of 1930 and for most of the work ill 1931.
However 1 since the results in 1929, although on a small number of
queens, indICate thnt, within the limits of the tiperm nVllilable, satis­
factory results cun bo obtained by tho Use of the Slllaller pipettes if
employed under pI'Opel' conditions, the quostion of size of l?ipette
merits further study, }"'or the work in 1932, consequently, plvettes
woro prepared which are smllller than the stnndul'd 'Vatson pIpette
but largor than tho pipettes used in 1929. l>.lungel's for these new
pipettes were made of nichrome wire.
USE OF AN ANESTIIETIC

AltIwugh at times in preyious work it had seemed that greater

success nllght be Ilttained through Ilnesthctizillg the queen, this was
iirst attempted by the writer in 1930. Ether wus the only agent used.
A swub of cottOll bllttin~ that hud been dipped in ether wus shoved
into tho glass tube holdmg the queen and WIlS left there until her
movements, hnd practically censed except fOl' tho mpid protrusion
Ilnd retrnction of her sting.
'rwenty-foUl' queens W01:0 opel'llted lIpon with the aid of ether, 15
with the small pipette and 9 with the regula~' 'Vlltson pipette. (Table
1.) Of these queens 6 proved to be inseminllted, 4: of these successes
 BREEDING THE HONE1:'"BEE U2oi-nER [Link] CONDITIONS 31
being with the small pipette and 2 with the standard Watson pipette.
Twelve of the other 18, or 50 per cent of the total, succumbed throu~h
balling or otherwise within 5 days after the final operation. Eight
of the 12 were operated upon only once, 3 were operated upon twice
on the same day, while 1 had two successive operations 14 days apart.
Of 28 queens operated upon without ether in 1930, only 4 (Nos.
30~ 31, 33 and 41), about 18 per cent, were lost, through natural
t
causes within 5 days after the operation. Two [Link] (Nos. 46 and
47) were accidentally killed within 7 days of the inseminatioI:1, while
2 others (Nos. 52 and 53) were killed for dissecti!on purposes on the
day following the insemination.
Owing to the high mortality so soon after the operation, possibly
due in part to the after effects of the ether, the work for the re­
mainder of 1930 was conducted without the use of ether. It may be
that the anesthetic was not properly applied. The use of some other
anesthetizing agent, such as carbon dioxide as used by Hambleton
#

for several years in various types of work on honeybees, might prove

advantageous, but in the satisfactory results reported by Watson,
'Mikhailoff, and Disbrowe no [Link] is made of the use of an
anesthetic and the work of the writer in 1931 was all accomplished
without it.
FILLING TRE PIPETl'E

Watson mentions holding the bulb of the drone on his index

finger while filling the pipette. The writer finds it more convenient
to retain the bulb by the forceps after pulling it loose from the drone
and to fill the pipette while the bulb is being thus held. (Fig. 12.)
An incision is first made in the sperm sac With fine scissors. If the
incision is made at the end of the bulb containing mucus, caJ."e is
taken to cut through the mucus at a point where the sac is at least
as large in diameter as the pipette so that the latter. will slip in
easily. Sometimes there is too much mucus in a drone in relation
to the. sperm, or it congeals so rapidly that the pipette can not readily
be forced through it to the sperm. In such cases another drone has
to be obtained.
On several occasions in 1930, and more frequently in 1931, the
writer cut an opening for the pipette through the " saculus " of the
sperm sac (lescribed by Watson (5!2). Although in many instances
it was found possible, on inserting the pipette into the "smculus,"
to take up large loads of sperm apparently free from mucus, this
was not true in every case. The writer is therefore not yet in a
position to say whether or not this method gives more success than
does the method by which the pipette is inserted into the sperm from
the other end of the sac without any special attempt to take up
some mucus first. Success has been achieved with both methods. .
MUCUS PLUG
In a previous publication (34) the writer mentioned that he did
not consider the use of mucus essential to success in artificial insemi­
nation by the pipette method. This viewpoint, as stated earlier in
this bulletin, is that most recently expressed by Watson (593). This
does not mean that the mucus plug is held unimportant. In the
writer's opinion it does play, in nature, the important rOle ascribed
to it by Bishop (5). In the Watson method of artificial insemination
it i:-; Lliflieult to secure this plu!!. ho\\'e"er, and a certain degree of
"llC('ess hus been attained [Link] it. If its llSP were easier, perhaps
!:illCCC'SSf'lll insemination by the pilwtte method mi!!ht be more fre­
quC'nt amI tlw dl'!!!'ee of inseminatiOI\ unifol'mly highpr, III this re­

., .. -: l>'~'

'~""--.t"": ~'1

;o;pP('ttlte Qllinn-LlIi(liaw and the ::\falyseilC'v Illl'thocls nppear to 11t1\'e

an a(\YHnta!.!;l' O\'PL' I-Itp \rnl::-;on method,
III till' \\,['il('I"s ,,'ol'k with the \\'(1t"on Illrthod, l)Hl'ticlllaL"ly in 1930
and IP;H wl1(>11 tl In I.'ge Illlllllwr of QIlC'(,I1S wel'p treated, no special
pll'ort wn:-; Illad!' to !!t't n, IllllCll:-; pltl!'!;, It: dllrin!! the oJll'[Link] sperm
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 33
bebrins to leak back around the pipette, the exposure to the air soon
causes it to harden on the surface sufficiently to keep the remainder
from running out of the genital opening. Possibly this hardening is
due to the presence of some mucus that has mingled with the sperm
in the process of filling the pipette. In any event it seems best to
provide a superabundance of sperm to take care of any wasted by
leakage.
It is now the writer7s usual practice to delay the withdrawal of
the pipette for a few minutes after the completion of the operation
and also whenever sperm begins to ooze out around the pipette. This
allows the sperm to harden around the end of the pipette and thus
form a sort of collar through [Link] the pipette can be pulled out
gradually without bringing the entire mass of sperm along with it.
The pipette is removed by raising it slightly and loosening it from
surrounding mucus with a teasing needle with bent point if this
can not be done readily with the sharp end of a transferriug needle.
In disregarding the mucus plug it seems essential to apply the
end of the pipette squarely to the genital opening and then so to
manipulate the plunger that the sperm enters without being forced
back out around the end of the pipette in too large a quantity.
With the improved apparatus described in this paper, this procedure
is rendered much easier to achieve than under the older technic,
especially since the genital opening is more readily found.
l\IARKING INDffiDUALS
In any breeding experiment it is important to know: ,'as exactly as
possible the genetIC history of the individuals being bred from. rrws
implies being able to distinguish between various individuals. There
are other reasons why one must be able to pick out with certainty
this or that bee for use in breeding work. In the first place, accord­
ing to Bishop (5) and others: the drones must be' of a certain age
before they are sexually mature. Even queens are held to have
optimum age limits for mating. In the second place, because of
drifting:, in an apia'ry of bees of the same race one can not be sure
that a drone found in a given hi\'e belongs to that hive. The writer
hiS found marked drones from one hive in as many as three other
hives 12 hours after marking, and further search might have shown
more cases of such drifting. Other drones were found to stay in one
hive o\,er a period of weeks. Workers often make themselves at home
in hiYes other than their own. Even when queens are confined to
their hives by queen excluders, they sometimes drop to the ground
nnd may be lost when their hives are being manipulated.
Such considerations as the iore;?oing render it imperative to have
some means of distinguishing delmitely given individuals within a
hive or individunls of {JOe hive from those of another. In the case
of a queen bee this can be done easily) since her wings can be clipped
to prevent flight awny from the hive and a distinctive roark can be
painted on her. Drones and workers present more of a; problem in
this respect, since theI:e are so many more of them than queens.
One way to make sure of a drone's origin is to rear and 'keep it
behind a queen excluder. This was proved practical in 1929 in at
least one instance, and in two instances in 1930. However, the need
of flight on the part of the drone is an objection to this method.
34 TEOHNICAL BULLETIN 326) U. S. DEPT. OF AGRICULTURE

A better method, and one which allows the drone free flight
throughout its life, is·to !;iive it a distinguishing mark when it emerges.
Mikhailoff mentions havmg done this in 1929 (135). The writer did
not begin any extensive use of such a method until 1930. As used
in 1931 it consisted in mark-ing the thorax of the drone with the
number of its mother and placing on the abdomen a number to mark
the week of emergence. Although this method gives the age of the
drones only by weeks, and somewhat roughly at that, it can be of
service in determining the relative range of age in weeks during
which drones are best for use. Those desiring to know the exact
age of any drone in days can give it a separate number on emergence.
Queens and workers were also marked bv numbering.
The method used for numbering drones, queens, and desired work­
ers is an adaptation of that gi,'en by Von Frisch (13, p. 42). Ten
pigments are chosen, one to represent each of the 10 digits. By
combining these in dots on the thorax, any desired number can be
represented. Un its
nre representee1 by a
dot of the proper color
on the left rear of the
thorax, tens by a dot
on the right rear, hun­
dreds by a dot on the
left front, and thou­
sands by a dot on the
right front of the
thorax. For instance,
if the numeral " one"
FIG(;UE 13,-;\farkcd drones is represented by a
blue pigment, bee No.
1 would carry a blue dot on the left rear of the thorax and bee No.
11 wOli1d carry a blue clot on the right rear and also one on the left
rear of its thorax, bee No. 111 would be marked as described for No.
11 plus Ii. blue dot on the left front of its thorax, and No. 1111 would
carry three blue dots as for No. 111 plus one blue dot on the right
front of its thorax. A similar system can be used for marking the
abdomen. (Fig. 13.)
DRONES
OBTAINING DRONES

It is not always easy to secure drones in sufficient quantity dur­

ing the active season, especially at its beginning and end. In an
endeavor to get an early supply in 1930, drones were purchased
from the South about three or four weeks before their usual appear­
ance in the apiary of the bee culture laboratory at Somerset. They
were successfully united to strong colonies and consequently the sea­
son for artificial insemination opened earlier than would have been
the case under natural conditions. Since, in purchasing drones com­
mercially, it is not always possible to get the desired stock, this pro­
cedure is practical only for certain purposes.
J... ate in 1931 limited trials were made of stimulative feeding to in­
duce drone rearing during a normal slackening of brood rearing.
 BREEDING THE HONEYBEE UNDER OONTROLLED OONDITIONS 35
A certain measure of success seemed to attend these efforts, since
drones were present in these colonies until late in the fall. The
exceptionally warm weather may have been a contributing factor.
When the first queens reared in early spring in 1932 were ready
for treatment, drones were on hand from an overwintered queen
which had been successfully treated the previous fall but which
proved a drone layer in spring. 'I'his points to one way of securing
an early supply of drones.
Prior to 1931 most of the drones used from the Somerset apiary
were caught by hand at the hive entrance as they were returning from
a flight, either on the wing or just as they alighted. Such drones
proved more satisfactory than those taken from combs. This
method was slow at times, especially when drones were scarce or
when their flight was not active; so in 1931 queen-and-drone traps
were put on the hives at the entrances just before the hours of great­
• est flight, which have been found to OCcur at Somerset shortly after
noon. Usually more than sufficient drones were easily collected in
this way, ancl by keeping the excess caged overnight in proper con­
tainers it was possible to have available the next morning a supply
of drones suitable for the work on artificial insemination. Candy
was supplied as food for the drones kept overnight and, when it
seemed necessary, some workers were caged with them as attendants.
Usually sufficient workers for this purpose were collected in the
traps. An electric incubator provided proper temperature for the
drones on cool nights. On several rainy days in 1931 the work
was continued by the use of such drones when otherwise it would
have been necessary to suspencl operations pending better weather.
DETERMINING SEXUAL MATURITY OF INDIVIDUAL DRONES

Since in picking drones at random from a hive it is not always

possible to get only those that are sexually mature even if their cal­
endar age is: marked, it is desirable to have some simple criterion
as to their sexua~ maturity-in other words, some indication as to
whether or not It drone, regardless of a~e in days, will furnish a
supply of active spermatozoa. In experIments conducted thus far
there has been found less correlation between calendar age and
physiological age in drones, as .far as sexual maturity is concerned,
than might be anticipated. For example, some drones 2 or 3 weeks
old were found without sperm or with less than was possessed by
younger drones. .
Statements have appeared that drones which ejaculat.e on decapita­
tion are sexually mature. In 1930, of five queens (Nos. 48, 49, 50,
52, and 53) inseminated with sperm from such drones, all proved
to be inseminttted to It greater or losser degree. In the operation
on the other queen (No. 47) for which a record was kept, sperm
from a drone not. in this class was used, but the queen proved to be
uninseminatec1. In 1931, of the 38 instances of successful [Link]­
tion by the 'Vatson method, drones which had ejaCUlated on decapi­
tation were used in 25. The reaction of the drones was not recorded
in 3 of these instances. For 16 of the 26 queens whose spermathecae
were found clear in a post-mortem examination, such ill·ones had
also been used. The reaction .of the drones used was unrecorded in
only one of these instances. Since the proportion of the drones in
36 TECHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTU~E

question to the total number used in the unsuccessful cases cited for
1931 is about the same as their proportion to the total number used
in the successful cases cited for the same year, these data, indicate
that, for 1931 at least, it was immaterial for the accomplishment of
artificial insemination which type of drone was used.
QUEEN REARING AND TESTING
EXPERIMENTS IN 1930

To economize on the quantity of bees necessary in the work, the

writer in 1930 adopted the use of what he terms a "nursery colony"
for rearing and keeping virgin queens, as well as for testing in­
seminated queen bees. This colony had no laying queen of its own.
It WIIS made up in the spring in two standard Langstroth hive bod­
ies with 10 frames full of stores in the lower story and 10 frames
full of sealed brood with adhering bees in the upper one. Until
summer, when the quantity of sealed brood available decreased,
frames of sealed brood were added as necessary to insure a sufficient
number of youn.g bees to carryon the work given them. This colony
was fed at no tlme. .
"When queen-rearing operations were be~un, one of the frames in
the second .hive body was replaced by a ·trame filled with bars for
cell cups wlth grafted larvae. Grafts were made weekly or oftener
as desired. About the third. day after being sealed, the accepted cells
were put in ~~lley nursery cages in a· nursery frame and left in the
second hive body. Three such frames were in this hive body most of
the season, room being provided by removing the original frames as
necessary.
As the season advanced a third hive body was added to the nursery
colony for the purpose of taking care of the inseminated queens.
When first put on, this body contained brood and honey. As queens
were insemmated they were placed in California introducing cages
directly on a comb of sealed brood about ready to emerge but wlth
no other bees on it. Room was made in the hive body for these cages
by removing the original frames as necessary. The California cage
holds one Langstroth frame. It is made of galvanized metal except
ior the two sides, which are of wire screening. The comb is inserted
at the top of the cage, which is provided with a sliding cover made
of the galvanized metal. A 10-frame standard hive body holds 6
such cages plus 1 Langstroth frame.
Up to the close of the main honey flow, whenever an appreciable
quantit.v of the broo(l in a cage had emerged, the comb, with queen
and adhering bees, was removed and set in another cage similar
except that one of the screene(l sides was replaced by excluder zinc.
This new cage was then put back in the space formerly occupied
by the old. cage, and thus worker bees from the nursery colony and
those which had emerged in the cage could intermingle and pass in
and out freely. The queens were left in these cages until they
began to lay or until it was apparent that tlie attempted insemina­
tion was not successful. 'Then a queen began to lay, she was put
in a nucleus, together with her comb from the nursery cage and a,
frame or two of bees from the nursery colony.
 BREEDING THE HO~"'"EYBEE UNDER CO:S-TROLLED CONDITIONS 37
As a matter of practice, the sides macle of excluder zinc were faced
in the same direction in any hive body which contained more than
one cage with such a side. On one occasion the nursery colony con­
tained fh'e queens in these modified cages, and three of the queens
were layin~. In addition the colony contained other queens in the
regular California cages in another hive body.
, Satisfactory results were obtained with this system until after the
\;>; )se of the main honey flow. when it was found that the bees were
iricline(fto ball some of the qt1eens UPO'n gaining admittance to them
throuc,h the excluder cages. This may have been due in part to the
pl-cse;ce of a comparatively large quantity of olel worker bees in the
hive. :1 condition aggravated by the fnct that sealed brood became
less available from the supply colonies at this period of the season.
Since the nursery colony was not fed at any time. the effect O'ffeed­
inc' in inducing accq)tance of the queens ,vhen tlie main honey flO'w
ha~I passed was not determined. After the main honey flow, there­
fQre. the use Qf the cage with an excluder-zinc side was discontinued
and each queen was transferred directly to her own nucleus box as
soon as sufficient yOlmg bees had emerged from the comb in her
screened cage to keep up the activities of an independent nucleus.
APARTMENT HIVES

In 1031, as an outgrO'wth of the use of the equipment just described,

the writer de"ised what may be called" apartment)) 11i"es, which
are made up of Qne or more compartment hive bodies. A compart­
ment hive bQdy is a hive body separated lengthwise into two or more
divisiQns or compartments by wire screening. (Fig. 14.) Ordi­
nary copper windO'w screening has served this purpose nicely. Strips
of bent tin are slipped over the edges of the screening anel sQldered in
place. The bottQms of the hive boc1ies and, consequently, Qf each
compartment are also cO"'et'ed with wire screening. Each compnrt­
ment hO']<1s one O'r more full·depth frames l nnd thus the tQtalnllmber
of such cQmpartments PO'ssible in a 10·frame hive body varies frQm 2
to 10. The bees are prQyided with an entrrulCe to' each complll'tment
by a· smH 11 hole bored through the hive bO'dy. A small blQck at the
entrance sel',Tes as an alighting board, and over the entrnnce is fitted
a piece. of queen excluder. A separate coYer of thin WOQd is fitted to
each compartment and a standal'd outer CQver fits o,'er the whQle.
On the basis Qf the size of cQmpartments fQnnd in each, the com­
partment hiTe bodies used thus far by the writer have consisted Qf
those with five 2-frame cQmpnrtmentsl thO'se with two 5-frame com­
partments, and thQse with twO' 3-frame compartments plus Qne
4-fl'ame COmpJlrtment.
The compartment hive bO'dies are set directly o,'er a queen-right
colO'ny frQm the hive of which the inner cover has been removed.
Any heat rising from the colony belQw will pass through the screen
intO' the compartment hive body, and heat will also pass frO'm CQm­
partment to CQmpartment. These hive bQdies may be tiered up.
(Fig. 15.) In such a case the individual covers to each cQmpartment
except those Qn the tQP hi"e body can be removed, SO' that the screen­
ing on the bottom Qf the hive bQdy abo'-e fits snugly against the top
of the screenin!! fQrming the wans of the divisions in the hive body
38 TEl'l I XJt'.U. Il{"U,r,:nx :l~lj, to, S. 111-:[''1'. OF AGRICGr;rurU~

-
 BREEDIXG THE HO}'''EYBEE UNDER COYTROLLED CONDITIONS 39
below, preventing the passage of bees :from compartment to com~
partment. In 1932 the largest number of hive bodies thus tiered up
on one colony and manipulated by the writer was four, each con~
taining five 2-frame nuclei. Counting the queen in the colony on
the bottom, at one time there were 21 queens on this one stand, each
with its 0"""11 separate organization of bees. Hence the name
"apartment" hive. .

,V"Grmr lJ.-A i)'COlJlpartment hh-e body tiered on a full-strength colony. The hive
body Is inside of the winter prot~cth'e collar. The glass tube which extends
through the collar as Ii paSsage for the bees shows at the entrance at which there.
Is no e.-cclnder zinc. .aluminum tags US('il to keep records IIi the hive are visible
on the frames

The apartment hives have proved to be as good as, or even better

than, the ordinary type of nucleus dllring the robbing season, since
the bees for the most part have defended each compartment well. A
little care must be exercised, of course, in opening the compartments
at this seaSOn. Little trouble was e:~;perienced in 1931 by bees from
one compartment running into~ a neighboring compartment when
adjoining compartments were exposed for purposes of manipulation.
In 1932, in the case of tiered-up compartment hive bodies, all com~
partments that were not being manipulated were kept covered.
It may be that all bees in the apartment hive have more or less of
a common hive odor. In any event the bees in each compartment
40 TECHNICAL BULLETIN 326, 11. ~. DEPT. OF AGIUCULTURE

~eem to remain there without any more than normal drifting else­
'\\"here~ even though the entrances were often not very far apart.
The queens arc introduced into the compartments by means of a
)Iillerintrodueing cage. The workers are usually denied access to
the ('Hildy for three days by fastening a, piece o.f tin across the candy
hole. Frames of emerging brood are added to the compartment;; as
11ecessary to insllre the presence of young bees when a queen is
ueing introduced or at other times.
The 1.18e of apartment hives is recommended for only those bee­
keepers who will give them the proper cal'e and attention.
WINTERING IN APARTMENT HIVES

An expel'il1lrl1t to winter queen bees in apartment hive;; Ivas llnder­

takrll j'or the wintrl' of 1981 and 1932. Two compartment hive

F'lOl~nt;; lH.-\\"int"PI'illg in Hp:lt'hnellt hin's: ,A, ~\partlllellt hive COll8isting of n. U-COIll­

IHlI"tnlPllt hint bo(ly OYl'r H (lol"llwl ('oion,Y:' B. telf,\ScopC l'Ovcr and tray remo,"cd to
~h()\I" Inail'lrina I I nll~l' ('on'I'~ Of eomlJal'tnll,'n t~

bodie'S. earh '\'ith fiye 2-framr nuclei, two with two 5-frame nuclei,
lIlid. 011(' with h\'o 8-frnl1lC' nuclei plus one 4-frnme 1I1leleus were
dlOSCll. ElIc·h of thC' fin:~ compartment hive bodies was over a 10­
-frame colollY in rt double-wnll hive. and a packed double-,yu1l col­
InI' wn!'j liUr(L around it, A tray of sawdust covered the top of each
hi.\'(~. and o\'er this a, tcleseope covel' was slipped, A hole drilled
through the collar. into which glass tubing was fitted, connectecL the
entrance of eaeh compal:tment with til(' outside. A piece of queen
exdu<1eL' ,yas placrcl OYeL' the outer opening of the tube ill some cases.
(Fig. 10.)
Thr wintrl' was mildcr than is common in the region of Somerset,
)[(1.. but since therc was conseqnently more flight activity than
uSlial fot' this pCI'j(Hl of the year the Iveathel' can not be considered
in it:::eIl' as haying l)(lell conducive to sllceessful wintrring, Never­
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 41
theless, the condition on March 1, 1932, of all those nuclei which had
plenty of bees on November 1, 1931, was highly satisfactory, with
the exception of one 5-fraine mj,cleus. This [Link], which contained
queen No. 51, appeared to ha';e sufficient bees on entering winter,
but on February 10 it was queenless and onlv a handful of living
bees remained. Of the 17 experimental nucleI, this. 5-frame nucleus
and a 2-frame nUcll'llS were the only ones not strong enough in the
spring to cal"ry OIl brood rearing successfully. Perhaps the bees on
hand in each nucleus in the fall were somewhat olc1pr than was
thought. Each of the 10-frame colonies below the compartment
hive bodies wintered [Link]. The especially strong condition
of some of the 2-frame nuclei ,vas a. pleasant surprise and showed
clearly the valne of this method for wint('ring with a minimum of
bees the queens being tested, provided there has been no neglect in
insuring .the presence of sufficient young bees in the nuclei by the
beginning of winter.
QUEEN REARING IN 1931

In line with the other efforts to reduce the number of worker

bees and the labor required in the work, all queen cells upon
being _sealed are put in Alley nursery cages and then placed in an
electric incubator and left there at brood-rearing' temperature until
they hatch. This incubator has glass sides. top, and door, and
has sufficient room inside to hold two hive bodies. For queen-rearing
purposes, however, this space is divided by a number of shelves to
hold the nursery cages. The incubator is located in a building
nc1jacent to the apiary. .
An attempt was made in 1931, but somewhat unsuccessfully, to keep
virgins in the incubator after hatching. Fresh candy was given
for food, and worker bees 'were provided as attendants. On one
occasion all of about 50 virgins so left. died within three or four
days after emergence. Thereaftel' the newly emerged virgins were
given fresh candy and their cages placed in nursery-cage frames.
These in turn were placed in a super directly above the brood nest
of a queen-right colony, but separated from it by a· queen excluder.
Frames of sealed brood, takeu from the brood nest below, were placed
next to the nursery-cage frame. The chief disadvantage of this
method is the tendency of the beps to desert the caged virgins when
the 'weather becomes cool.
In 1932 virgins were successfully kept in early Sp'rillg in 3-frame
queenless nuclei in apurtment hives. A special frame was used which
consisted of an old brood frame with enough comb cut out of the
lower portion to allow the insertion of two nursery cage bars from
a standard Alley nursery frame.
In 1931 queen-rearing operations were often conducted with larvae
from several queens at once. It was desired to have larvae from any
one queen reare(1 by only one colony. However, to have maintained
a separate colony for rearing the larvae transferred from each of
these queens, as described in the work for 1930, would have taken
too nutch time and too many bees i hence use was made of 2-frame
nuclei in compartment hive bodies. Each nucleus was kept strong
in young bees. '.rhe lower portion of one comb in each compartment
,vas cut away and a removable cell bar was inserted next to a thin
 .....

42 TECHNICAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

strip of wood, which in turn was securely fastened next to the cut edge
of the comb. Good results were obtained from these queen-rearing
nuclei. In 1932; 3-frame compartments were used instead of the
2-frame compartments, since it is easier to maintain sufficient bees
in the larger nuclei.
Frequently it is desirable to carryon transferring operations on
cold, rainy, or dark days, or during the robbing season. In order to
do this the writer devised a wooden support to hold a brood frame
at a convenient angole on a stand during the process. The stand is
located in the same room as the electric incubator. With a small
movable electric lamp in one hand and the transferring needle in the
other, transferring is accomplished under convenient conditions.
HIVE RECORDS

For keeping records within the hive concerning cell bars, que~n
cells, and queens, tags cut out of aluminum tape and bearing num­
bers or letters, or both, have proved useful. Numbers and letters can
quickly be marked on such tags by a metal stamping outfit, or by
scratching with a nail or other sharp instrument. The tags are
easily fastened to top bar, cell cage, or queen cage by a. thumb tack.
The number assigned to any queen and her race or origin, as well
as dates of her birth, insemination, first egg laying, and the like,
may be thus recorded. In the case of cell bars, the number or ori­
gin of the queen from which the larvae are derived, the date of
grafting, and the date of first sealing of cells on the bar can be
placed on the tags. (Fig. 15.)
When there are many nuclei to be looked after, the use of thumb
tacks on the outside of the hive will be found helpful as a means
of indicating quickly those colonies needing special attention. Thus
an absence of thumb tacks on a compartment may indicate that all
was normal within at the last examination. One tack may mean that
the queen has been successfully introduced, two tacks may have some
other meaning assigned it, and so on. The position of the tacks may
be given a certain meaning. The advantage of the tacks is that
[Link] are easily withdrawn but stay where placed unless forcibly
removed.
VIABILITY OF SPERMATOZOA
Certain experiments performed in Europe (35, 36) indicate that
spermatozoa from drones may remain alive at least a number or
hours in various electrolytes. Bishop (5) has reported that sperma­
tozoa in a salt solution mounted on a slide were still alive after two
hours' contact with ice. In order to determine how long honeybee
spermatozoa would remain alive under conditions somewhat similar
to those in the glass pipette, the writer mounted some on a slide with
a cover glass. Under these conditions the spermatozoa at times were
found motile more than three hours later at room temperature.
Of particular interest in this connection are the cases of queens
Nos. 29 and 86, used in the work for 1931. One of these queens (No,
29), after an operation which seemed normal and satisfactory, was
found dead in her Miller introducing cage the next morning at 8.30.
A -eost-mortem examination under the microscope was not made
untIl 11 a. m., at which time her spermatheca was estimated to be 50

,\
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITIONS 43
per cent filled with sperm and an abundance of live s'permatozoa
were observed in highly active motion. Even more striking was the
case of queen No. 86, which was found dead at. 3 p. m. on September
1; She was in a:!l Alley nursery cage in a colony at that time and
was left there until the next day at 11 a. m., when a microscopic ex­
amination of her spermatheca indicated that it was 15 per cent filled
with sperm. A great abundance of living spermatozoa was seen.
It is to be noted that these spermatozoa had survived 20 hours in the
spermathecaof a dead queen in a ca ge between the frames of a hive.
Certain results given by Bishop ~5, p. ~47) are in line with the
findings presented for these two queens. On one occasion he sub­
jected a "fertile" queen to a temperature 3.6 0 F. (20 C.) below
freezing, for 15 minutes. He states that none of her eggs laid subse­
quently were found to be infertile.
SUCOESSIVE INSEMINATIONS
The writer has given elsewhere (39) a review of the numerous
instances in beekeeping literature on the multiple matings of queen
bees. That insemination results when the sperm from different
drones is introduced into queen bees on successive occasions not on
the same day has been indicated in work both by Watson (513) and by
the writer. In 1930 the writer attempted this on three occasions and
had good results in two of them. (Table 1.) For one of the suc­
cessfu! instances the interval between operations was 2 days, while
for the other one it was 15 days; for the unsuccessful case it was 14
days. In addition seven queens were given two operations in one
day, but in only one case was the treatment successful. Three of the
queens were found to have clear spermathecae, and three disappeared.
In 1931, 33 queens were operated upon twice by the Watson
method, and 8 of these in turn were operated upon three times.
(Table 2.) Si.~teen of the 33, or about 48.5 per cent, proved to be
inseminated. This is to be compared with 34.4 per cent of ascer­
tained success for the 64 queens operated on only once, 22 of these
having been found inseminated. In the case of 14 of the 33 queens
the second operation was performed on the same day as the first,
while a third operation was performed later on 3 of these, but all 3
disappeared. Of the remaining 11 queens operated on twice in the
same day, 4 proved to be inseminated, 4 were lost, and 3 had clear
spermathecae.
Of the other 5 queens which were given a third operation,3 proved
to be inseminatpd and the other 2 dIsappeared. In one of these suc­
cessful cases (No. 86) sperm was found so massed in the queen's
( spermatheca that the degree of insemination ·was estimated to be
at least 75 per cent. Queen No. 53 was found to be somewhat less
inseminated, while the other case (No. 88) was classed as less than
10 per cent successful.
The results lor 1930 are not comparable with those for 1931
because of the use of ether in some cases. The results for 1931,
although not conclusive in themselves, seem to be in line with Wat­
son's statement (5~) that repeated inseminations increase the degree
of success. In describing his work for 1927 (51), Watson men­
tions that he operated on some queens from 1 to 10 times.
44 TEClHNIOAL BULLETIN 326, U. S. DEPT. OF AGRICULTURE

It is possible that many of the queens that disappeared in 1931

before any check could be made as to their insemination, as well
as many of those that were found dead in the cage, may have been
lost in attempts to get out through the excluder to take a mating
flight. That many of the queens found dead in the nuclei had tried
to get out or had suffered rough treatment from their bees seems evi­
denced by the fact that the painted markings had been rubbed off the
thorax in nearly all cases. It seems of significance in this connection
that practically all those queens which laid eggs were readily accepted
by the. nuclei in which they were introduced.
}lIXING SPERM FROM DIFFERENT DRONES
'Vithollt following the genetic composition of succeedint? genera­
tions, it is hard to say in the case of insemination from tne sperm
of different drones 011 successive days whether or not the sperm.
from each drone was active. In the case of 8 of the queens operated
upon in 1930 the sperm from more than one drone was loaded into
the pipette at one filling. Seven of these operations were successful.
Sperm from 2 drones was used in 5 cases, 4 of which proved suc­
eessful; ~perm from 3 drones was used for 2 queens, and each of these
qneens was found to be inseminated. In one of these instances all the
first brood sealed proyed to be worker. For the eighth queen sperm
from 5 drones was used, and this operation was also successful.
In 1931 the sperm from more than one drone was loaded into the
pipette in the operations on 29 queens. Eleven of these queens
proyed to be. inseminated. On two occasions the sperm from 3
drones was used. and in each case a successful inseminlltion resulted.
In no instance ,,-as the sperm from more than 3 drones employed.
The foregoing examples indicate that a· mixture of sperm from
different drones does not destroy the vitality of all the spermatozoa
at least. In each case the pipette '1ms loaded by ta1.~ng up the sperm
from the drones in snccession. It happened tliat all the drones used
for IlllY one operation came from the same hive.
In llis 24 5u('cessful inseminations in 1029, ~Iikhailoff (95) used
sperm from 2 01' more drones for 22 of the queens. For 1 queen
he reports the use of only 1 ell·one. while for 1 he does not give
the number. In a later article (J28) he states that he used the sperm
from 2 to 4: drones for e,'el',Y queen.
OPTBIU}[ AGE OF QUEEN FOR ARTIFICIAL INSEMINATION
It is frequently stated in beekeeping books that the queen usually
mates earlv in life and that if she delays more than three weeks she
is apt to become a drone layer. Thus, Phillips (.W, p. 49) gives the
normal age of the queen for mating as 5 to 8 days. In a compilation
made by Armbruster (4) of data from mating stations covering six
[Link], 9 eluys ,yas the age of the queen at which the greatest numbet·
of matings was found to occur. If these conditions hold in natural
mating, it might be expected that more success would be attained in
artificial insemination with queens only a few days old than with
older queens.
:Menc1el (19, p. 1-4-'); 54), however, held that a queen remains CI1­
vable of being ins('[Link] lip to the twenty-eighth to the thirtieth
 BREEDL.~G THE HONEYBEE UNDER CONTROLLED CONDITIONS 45
day after emerging, and that for some queens this time limit is even
longer. LikeWIse, although in the data by Armbruster (4) Oil
nlitural matings of the queen bee the. earliest age at which any of thl!
queens mated was 3 days, the oldest age. is given as 32 days. The
queens successfully operated upon in 1929 by Mikhailoff (f!6) ranged
from 7 to 29 days in age, while Watson (4fJ) reports that he has been
successful in the artificial insemiIllltion of queens even 37 days old.
In 1930 a record of the data of emergence and of attempted arti­
ficial insemination was kept by the writer for 43 queens whiell were
opel'llted Oil only once or only once plus a second operation within
two days of the first, and in 1931 for 82 queens which were operated "
on only once or, in some instances, once again within one day of the
first operation, making a total of 125 queens. ~~s a matter of fact,
only 1 of the 43 queens listed for 1930 (No. 44) received a second
operation, whUe 5 of the 82 listed for 1931 (Nos. 8, 14, 46. 86, and.
90) received a second operation. Queen No. 86 received' a third
operation. In Table 3, which gives the results obtained from
queens of various ages, these six queens have been considered as re­
cei ving their successful operation on one occasion, since the timl!
interval between the opemtions is small. Of these 125 queens, 47
proved to be inseminated. .
TAnL~: 3.-Sl/.CCC8.~ (lUllinc(/. ·//;i/.h quecJl8 O][Link] on in 19;]0 anrl1981 oJ/ly OJ/Co
01' within t//;o t1aljs at (I [ll'ccolling opcration in IIflC [lcrioa s[lcai{iaa'

Total
queens inw
[Link]
. 1 opern-
Age of queens
seminated tlons

Per ant
23.7
34. i
i5.0
80.0

,·~I

From these results it is apparent that the greatest degree of suc­

cess was attained by llslng queens at lenst 17 days olel. The total
number of queens (100) under 17 days of age which were used, how­
ever, was four times as large as the number (25) of those which
were 17 days old or over, but the percentage of success with the
younger ~ueens was only 28 us compared with 76 for the older queens.
It is interesting to note that No. 47, operated on in 1931, even though
mated when 29 days old, proved to b& one of the most prolific layers
of worker eggs of the queens mated that year. In 1930 two queens
were successfully mated at the age of 31 days. On the other hand,
in 1931 a queen 1 day old was successfully inseminated. The data
presented here, although bused on a small nnmber of cases over 17
day;.; of age, indicate that the age limit at which queens can be in­
selllinatec1 artificially is higher than that commonly accepted as the
limit for natural insemination.
SUCCESSION OF GENERATIONS
Regardless of any method for inseminating queen bees at will or
of !lny methocl for carrying" qlleens through either the active or the
inactive season with a minimum of labor, time, and equipment, suc­
46 TECHNICA.L BULLETIN 326, U. S. DEPT. OF [Link]

cess in breeding the honeybee and other forms is dependent upon a

third factor-upon having a succession of generations. The writer
has been successful in. the first factorlp.';;lltioned since the beginning
of his work in this field, while, as for the second factor, an artificially
inseminated queen was wintered over in the first year of the work.
However, F ~ queens from artificially inseminated mothers were not
obtained until 1930. These were all reared Yrom one mother. Six
of them were successfully operated upon that year and from one of
these an F 2 queen was reared. 'rhis queen was born too late for
mating that year. however.
In 1931, as already stated, F~ queens were reared from eight arti­
ficially inseminated queen bees. Unfortunately none of these was
reared before JuLy. and since August was marked by an unusual
scarcity of drones. 'the inseminations during the lattel.' part of the
season were some,,~hat restricted both as to number of attempts and
as to degree of success.
Of the 97 queens used in the work with Watson's method in 1931,
32, or practically one-third, were F ~ queens. ,Vorkers of the F z gen­
eration were obtained from one of these (No. 78) in October. It
was then so late in the season that no attempt was made to rear F z
queens from this queen, but she was still alive at the beginning of
winter. Insemination in the case of 6 others of the 32 was deter­
mined by a microscopic examination of their spermathecae, making
a total of 7 of the F~ queens, or 21.9 per cent, which were found to be
inseminated. Eight of the remaining 25 gave no evidence of insem­
ination on a post-mortem examination; 15 disappeared; while the
other 2 (Nos. 1'7 and 92), inseminated late in the fall, enterecl the
winter season without having commenced egg laying.
In the work as conducted in 1932 there was no difficulty in obtain­
ing a. succession of generations. Of four successive O'enerations of
queens reared in that year from a. certain strain, the first three had I
been successfully inseminated by the Watson method before Sep­
tember, as evidenced by the rearing of virgins and workers. Cer­
tain other strains were carried three generations, and a number were
carried two. Under ordinary conditions five or six generations
should be secureclreadily in a season such as characterizes Somerset,
Md. Under optimum conditions during the active season one gen­
eration a month 'is not out of the questIOn, although one every five
or six weeks appears satisfactory at present.
sml~IARY

In breeding the honeybee under controlled conditions either

natural or artificial insemination can be used. The only feasible
method thus far developed which makes use of natural insemination
is that of using mating stations~ An important objection to this
method is the difficulty of being absolutely sure that no wild swarms
or colonies kept by beekeepers exist within & radius which would
make possible crossings other than those desired.
For absolute certainty as to drone and queen, artificial insemi­
nation can be employed. Two main methods have been developed
for accomplishing artificial insemination: (1) Removing the organ
containing the sperm from the drone and placing it in proper position
 BREEDING THE HONEYBEE UNDER CONTROLLED CONDITION'S 47
in the queen to accomplish insemination, as done by Malyschev and
by Quinn-Laidlaw j and (2) l:l:;,moving the sperm completely from
the drone organ and thel}[Link] it within, the queen, as done
by Watson. Thus, far onlll the Watson method has been l"epeatedly
used with success in the Hands of others.
The technic originally described by Watson has been modified by
the use of new instruments and methods. :Methods have also been
developed to use a small number of bees and to reduce the quantity
of eguipment required in rearing and testing queens used in breeding
work.
Spermatozoa will remain alive under the cover glass or in the
spermatheca of a dead queen for several hours at ordinary summer
temperature.
Successive artificial inseminations of a queen, at least before she
befcins egg laying, can be made with good results.
Sperm from differenii drones can, be mixed toget(her without
apparent effect on some of the spermatozoa at least.
'Vorker brood was reared from a queen inseminated when 29 days
old. In 1930 and 1931 greater success was had with queens 17 days
of age or older than with younger queens .
.A. succession of generations of the honeybee is possible through
the use of artificial insemination. , Four generations is the largest
numbl:..'t." yet obtained at Somerset, Md., in one season, although under
optimum conditions it should be possible to obtain a generation a
month during the active season.
LITERATURE CITED
(1) .ANONYMOUS.
1928. LOUISL\NA. MEm'ING A SUCCESS. Amer. Bee Jour. 68: 4~5.
(2) CAnUSHADY, .A. Z.]
1931. (EDITORIAL NCYl'E.) Bee Kingdom 2: 35.
(3) ALPATOV; W. W.
1928. [CONTROLLED lI:.\TING OF HONEnlElil QUEENS.J Pchelovodnoye Delo
8 :.344-349. [In Russian.]
(4) AnYllRUSTEB.. L.
1927. DIE BIENElNZUOliTmiG Ill: JAHRE 1926. Jahrb. Wiss. u. Praktische
Tier~cllt. 19 (TeH 2, Kleintierzucht) : 182-188.
(5) BISHOP, G. H. '
1920. FERTILIZATION L"< TIlE lIO~"EY-BEE. I. THE lI:ALE SEXUAL ORGANS:
THEID HISTOLOGICAL STRUCIrURE .\ND PHYSIOLOGlOAL FUNCTIONING.
H. DISPOSAL OF THE SEXUAL FLUIDS IN THE ODGANS OF THE FElLU.E.
Jour. Expt. Zool. 31: 225-[266J, 267-286, iIlus.
(6) CALE, G. H.
1926. THE FIRST SUCCESSFUL ATTE1[PT TO cmmROL TIlE HATING OF QUEEN
BEES. Amer. Bee dour. 66: 533--534.
(7) ClIRIST, J. L.
1798. ANWEISUNG ZUR :1>-UTZLIOlIEN UND ANGElNElIlI:STEN BIE~ZUClIT•
• • • Aufl. 3, 372 p .• illus. Leipzig.
(8) DALTON, d.
1930. SOUTHERN STATES CO~'"FEREINCE AT BATON ROUGE. .Amer. Bee Jour.
70 : 169, 191, 198.
(9) DISBIIOWE, H. B.
1929. ARTIFIOL\!. INSElI:INATlON OF Q~lIEEB. Beekeeper 37: 128.
(10)
1929. ARTIFICIAL [Link] OF QUEE~lIEES AT ONTARIO AGRICULTURAL
COLLEGE. Amer. Bee Jour. 49: 599.
(11)
1931. CON'IROLLED lI:ATING OF QUEEN BEES. Beekeeper 39: 53-54.
(12) DZlERZON, J.
1849. NEUE 'It"ERBEBSERTE BIENEN-ZUCHT. • . . Auft. 3, 304 p., ilIus. Neisse.
48 TEOHNICAL BULLETIN 326, n. S. DEPT. OF AGRIOULTUJlE

(13) FRISCH, K. VON

1931• .AUS DEY LEIlEN DEB .BI&.~EN. Au11. 2, 159 p., illus. Berlin.
(14} GOLD!, R.
1914. DIE WIRKSAMKEIT VOX HEB&X ULRICH KRAMER. Schweiz. Bienen­
Ztg. 37: 396-401.
(15) HAMBLETON, .T. I.
1924,). .BEE BIlEEDING BY ARTIFICIAL INSEYIXATION OOXE EXI'ERIME:'i'TALLY.
U. S. Dept. Agr. Yearbook 1928: 13&-141.
(16) HOWARD, C. 'V., . 1 .xO FRANCE, L. 'V.
1!)]7. FERTILIZATION ai' QUEEN BEES. Amer. Bee Jour. 57: 376-37S.
(17) How.\RD, L. O.
100S. REl'ORT OF THE ENTOMOLOGIST FOR 11l0S. U. S. Dept. Agr. Ann.
Rpt., 47 p.
(IS) Hwnm, F .•
1814. XOUYELLES OBSEB\'ATIONS SUR LES ABEILLES. Ed. 2, 2 Y. Paris,
Geneva.
(19) ILTIS. H.
1H2·!' GREGOR .10HANX ~IEXDEL. LEBE:'i', WEBK UND 'VIIIKUNG. 426 p.,
illus. Bel:1in.
(20) ,TAGEII, F., AND HOW..\IID, C. 'V.
1!)].1- TIlE AIITIFICIAI. FEUTILIZATION OF QUEEX BEES. Science (n. s.)
40: 720.
(21) KRAMEB, U.
1930. DIE UASSE:'i'ZCCHT DEB SCllWEIZEB HIKER. Auff. 7, re\'ised by)!.
,Tiistrich. 20S p., iIlus. Auruu. •
(22) )!CfJ,\lN, N. ,,'.
1887. IIE1'OltT ON EXl'ERUIENTS IX AI'ICULTUilE. U. S. Dept. Agr. Rpt. Ent.
1886: 5S3-5n2, illus.; also in Amer. Bee Jour. 23: 567-569, 1887.
(2.3) )L\LYSClIE\', S. I.
1!124. [ARTIFICI..\ L IXSEltn',\TIOX OF THE rrONE·...BEE.] C'helo\'ek i Priroda
1924: 579-586. [In RllsshlD, original not seen.]
(2·1) )[Link], C. L.
]1)::10. IIt:l'OItT Ot' THE CHIEF OF THE I1URE.\lJ OF ENTmroLOGY. 1:. S. Dept.
Agr. Anu. Rpt., 76 p.
)[Link], A. S.
1029. [THE A1tTIFICIAL IXSElIT:'i'ATION OF QUEEX BEES.] Opitnaia Paseka
1fi29: 10-22, ill us. [In Russian.]
(26)
1,1)30. [Sl-CCE.'iSES .\XI> FAILGRES IX 'rnE .ARTIFICIAI. INSEllIXATlOX OF
QFEE.~S IX TIlE lIO~EYBEE.] Opitnaill Paseka 1930: 15-19, illus.
[In Russian.]
(27)
1031. INSTRUlIE:'i'TAL INSEllD,ATIOX OF QUEEX BEES AT THE' TL"'L.\' STATIOX.
Amer. Bee Jour. 71: 72.
(28)
1931. i'BER DIE YEIIEltIlUNG DEit WErSS:\.UGIGKErTBEr DEB HOSIGBIE:'i'E (A1'1S
~IELLlFERA). Ztschr. Imlukth'e Abstam. 11. Yererbungslehre
59 : 100-202.
(29) Xt:WELL. "r.
1915. INHERITA..'CE IN THE HO:'i'EY BEE. Science (n. s.)' 41: 21&-219.
(30) XOLAN, "'. J.
1925. DE\·[Link]:'i'T OF QuEEN IlEARING IN THE =ITED STATES. YlIth In­
ternatl. Congo Beekeepers Sept. 1st to Sept. 4th 1924
[Proc.] : 29~07. In French in same volume p. 83-95; also in
L'~\piculteur70: 143-148; 177-1S1; 242-246. 1926.
(31)
1928. SEASOXAL BROOf)-REAIII:'i'G ACTIYITY OF THE CYPIU.\N HONEYBEE. ,Tour.
Econ. Ent. 21: 392-403, illus.
(32)
1929. SUCCt:SS IN THE ARTIt'lCIAL JNSElIINATION OF Ql:EEX BEES _\'T THE
BE."E CULTURE LABORATORY. Jour. Econ. Ent. 22: 544-551.
(33)
1929...\RTIFICIAL ISSElUNATION: WATSOX :lIETHOD IS DESCRIBED. Amer.
Hone)' .Prollucer 3 (4, 5, 6) : 3-4.
(34)
1929. [Link] Y [Link] Ol'[Link] \'('ELlcn llATEK. Ycela ~rora\'skii72
(30) :.398-101, ill mi. Trllllsl. into Czech by S. Sondek.
 BREEDING THE HONEYBEE UNDER. CONTROLLED CONDITIONS 49
"
~OLAX, ,V. J.
1929. THE BERLIN CONFER&"'CE OF THE AI'IS CL"CB. Amer. Bee Jour. 69: 541­
543, 568-560.
(36)
H)2\). BEEKEEPEIts OF MANY NATIONS MEET. Gleanings Bee Cult. 57: 640-
M'>
(37) - ­
1030. BEE ItACEB YARY IN .PIIYSICAL CHARACTERS .'Xl} IN BEHAVIOR. U. S.
Dept. .Agr. Yearbook 1030: 128-130, illus.
(38l-­
1930. RAOES O~' TInJ HONEYBEE. Penn. Beekeeper 5 (No.1) : 1-4.
(39)-­
1932. :lIULTII'LE ;.\1A'ITNGS OF THE QUEEN BEE. Rpt. :Md. State Beekeepers'
.Assoc. 2'&rd Anllual Meeting, Baltimore, January 7, 1932: 20-34.
(-10) PHILLIPS,E. 1l'.
1931. m:EKEEl'fNG; A DISCUSSrollO OF THE LIFE OF THE HONEYBEE AND
O~' TUE I'ltOD(,OTION OF HONEY. 490 p., illus. New York.
(41) PUELf., H.
1927. IlIE XUNTSLICHE llEFRUCHTIING· DER. illENENKONIGIS. Leipziger
Bienl'n·Ztg. 42: 2'25-230; 111130 in nliirkii;cbe Bienen-Ztg. 17: 236­
241;. :lnd Cong. Internutl. Zool. (1927) 10 (pt. 1): (455)-462.
1920.
(4:!)
1920. J'AliRESBElUOU'l' J Il~S trnEU DIE BIENE~AUSDLICHE TATIGKEIT D[
ZOOL(jGISOUEN INSTITt:T DEit FORSTLIOUEN HOCHSCHULEl THARANIYr.
Leillziger Biencn·Ztg. 44: 229-231.
(4a) QnNN, C. W.
102:~. }L\ND·FEltTILISATIOX O~' QUEENS. Bee "'orlel 5: 7(;.
(44) RtA Ulll;It, R. A. 1!'.. DE
1740. :\IE:\IOIKtJS .1'OlJR SERVIR A L'HISTOlRE DES INSECTE.'3. (l Y., Paris.
(45) SHAr";U, G. D.
1!U7. A ST1JIIY O~' TIlE F,\CTORS WIllell GO\,ERX :\rATING IN T'rlE HONEY BEE.
nIich. Agr. Expt. Sta. Tech. Bul. 34, 19 p., ill us.
(46) SWAlUlEltII.\.\l, J.
1j58. THE HOOK OF NATURE: OR. THE HISTOltY nt' INSECTS: UEDUCED TO
flISTTXGT cru\SSES, CONFIUMED BY 1'AltTICULAIC INST.-\NCEB, DIS,
PLAYED IN THE ANATOllICAL ANALYSIS OF ll.-\NY SPECIES • • •
English transl. from the Dutch and Latin original editions by
Thomas Flloyd. He\·. and impro\'ell . . . hy .Tohn Hill. 2 pts.,
illus. I..on<!oll.
(47) "~ANKLER, "'.
1024. IIlE KONIGlN, Si;[Link] FeR .rUNGER DEit NEUZEITLICHEN
WEISELZUOIIT. AntI. 4, stark venn. nnd umgc:lrb., 122 11., illus.
}'reiburg.
(48)
1\)27. rm~ Kl'[Link] lI"-YRUCHTUNG DER IHI'..'NESKOl'nOIN.
B ienen·Va tel'
50: 2(>1-2(>5; also in report of 65 Wanderversummlung der Bien­
enwirte deutscbm' Zunge, 6 p. Leitllleritz.
(40) 'L\TSOS, I•. R.
1027. (.'()NTUOLLED MATING OF Qt:EENBEES. 50 p., illus. Hamilton, Ill.
(50)
102T. OONTUOI.I.E1) MATING IN TUE HONEYIlEE. Iowa State Apiarist Rpt.
1927 : 36-41.
(iJl)
10:!8. CONTUOLLED MATING IN HONEYBEES. Quart. Re\". BioI. 3: 377-300,
illus.
(52)
1020. NEW CONTRIllljTIONS TO THE TEOlDo'IQl:E OF INSTUUMENTAL INSElII·
NATIOS OF QUEENBEES. Jour. ECOll. Ent. 22: 9'14-0"<14, illus.
(53)
lO:!!l. INSTRl:)lENTAL INSEMIXATION OF QUEESBEES. Internatl. Congo Ent.
(1928) Trans. 4: [07G]-077.
(54) ZIWAXSKY. F.
1872. mmtlQIlT UBER JIIE MON,\TYERS,Unrr;U~G A:\[ -1 Al'ltIr., 1 Si2. Die
Honigbiene yon BrUnn 6: {}fr-67.
ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE
WHEN TmS PUBUCATION WAS LAST PRINTED

Secretary of Agriculturc____________________ .ARTHUR M. HYDE.

Assistlmt Bccl·ctU1'Y ________________________ R. W. DUNLAP.
Director Of Saicntifio Work ____________ ~ __ A. F. WOODS.
Director of RcuuZtlto'f'1J Work__________'- ____ 'VALTER G. CAMPBELL.
Direotor of Ewtfmsiol1 Work________________ C. 'V. 'YARIIURTON.
Director Of PcrSOImeZ anit BIISilll!SS [Link]- ,V. ,V. STOOKBEIlGER.
traHon.
Dfrectol' Of Inlormation____________________ M. S. EISENHOWER.
SoUcitOl·__________________________________ E. L. MARSllALL.
Bureal' of AuriouUltral ECOIlOllLic8___________ NILS A. OLSEN, Ohief.
Bureau Of AuricuUtl1'aL EnUhweri1lu _________ S. H. ZlIcCROlty, Ol/;icf.
!Jureau of A1~imaZ 11:411stry_________________ JOHN R. MOllLEB, Oltief•.
Bureau of BiologicaL SIU'VCy___ ~ ___________ PAUL G. REDINGTON, Ohief.
Bureau Of Ohen~iJJtry anit Boi/s______________ H. G. KNIOHT, Oltier.
OfT!ce of 0(01)[Link] E(l)tC1l,~iOI~ WOI·k _______ C. B. SMITH, Ohicf.
Burea", of Dairy !lIdustry__________________ O. E. REED, Ohief.
Burell" Of Entolllology__,.. ___________________ C. L. MARLATT, 0111ief.
OfT!cc of EwjJcrimcllt Btati07ls______________ JAMES T. JARDINE, Oltief·
Fooe amt Drrtu AIZrninistrat-ion_____________ 'YALTER G. CAMPBEIL, Director o(
Regulatory! Work, in OMrge.
Forest Bc/·vU:c_____________________________ R. Y. STU<\IIT, Ohi~(.

Burcalt of Grain Futllrc.~ Adminisll·(liioll____ J. 'V. T. DOVEL, dhie(.

[Link] of Home Eoollomic8_________________ LOUISE STANLEY, Oltief·
Lib.,-(1Iry_______ .:. ___________________________ CLARIBEL n.
BABNEl'T, Librarian.
Bureatt of Plant Indtlstry__________________ WILLIAM A. TAYLOR, OMef.
Bureat~ of Plallt QlIaral'tillc________________ LEE A. STRONG, Oltie(.
Burean of PulJlio Roail8_~------------------ THOMAS H. MAODoNALD, Ohie(.
lVeatluw Burcau _________________________ CHARLES F. l\IARVlN, OMef.

This bulletin is a contribution from

Bureau of Entonaolof/Y _____________________ C. L. l\IABLA'l'T, Oltie(.
Divi.!lion of Bee Oulture_______________ J. I. HAMBLETON, Benior Jipicul-
Writt, in Oharue.
50

u. s. GOVf:lUUUNT rR'NlIHG onlCEt Btll

],'or snlq by; the Superlntcndcnt oC Documcnts, Wnshlngton, D. C. - - • - • rrlcc r; centB

t

..
..
t
t "
! •