variety of biological activities (Krell, 1996) and have an indispensable
role in pollination for agricultural and natural eco-systems (Morse and
A large colony population is necessary for a high yield of hive
products (Chen, 2001). Because of this, for many decades attempts in
The western (
) and the eastern (
) honey bee
are rare cases of insects that have been successfully domesticated.
Honey bees play an important economic role as a producer of honey,
royal jelly, beeswax and propolis, which have been proven to have a
ORIGINAL RESEARCH ARTICLE
Sustainable multiple queen colonies of honey bees,
Apis mellifera ligustica
Huo-Qing Zheng1, Shui-Hua Jin2, Fu-Liang Hu1* and Christian W. W. Pirk3
1College of Animal Sciences, Zhejiang University, Hangzhou 310029, China.
2Pinghu Breeding Apiary, Zhejiang 314200, China.
3Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa
Received 25 December 2008, accepted subject to revision 7 July 2009, accepted for publication 8 August 2009.
*Corresponding author: Email: firstname.lastname@example.org
Honey bee multiple queen colonies composed of several mated queens able to move around freely were produced by modulating biological
factors that evoke fighting and queen elimination within the colony, mainly by ablating mandibles of queens to avoid inter-queen rivalry.
Following this method, 128 colonies in eighteen apiaries were set up with multiple queens, all of which were mated and 6-12 months old. One
hundred of the colonies (78.1%) retained all introduced queens. In total, 658 out of 733 queens (89.8%) were accepted after their
introduction. The majority of these colonies experienced no queen loss for two months and most were still stable after six months. Of 80
colonies, 55 (68.8%) experienced no queen loss over the winter. These results show that our method is viable to produce sustainable
multiple queen honey bee colonies for commercial use. In addition this technique will help to increase our understanding of basic questions of
the evolution of sociality, such as division of reproduction and the evolution of polygyny.
Colonias sostenibles de abejas
Apis mellifera ligustica
con múltiples reinas
Colonias de abejas con reinas múltiples compuestas por varias reinas fecundadas capaces de moverse libremente fueron producidas
modulando factores biológicos que provocan la lucha y la eliminación de la reina dentro de la colonia, principalmente quitando las mandíbulas
por ablación de reinas para eludir rivalidad de la reina-reina. Siguiendo este método se establecieron 128 colonias con múltiples reinas en
dieciocho colmenares, que fueron apareadas con 6-12 meses de edad. Cien de ellas (78,1%) conservaron a todas las reinas introducidas. En
total, 658 de las 733 reinas (89,8%) fueron aceptadas después de su introducción. La mayoría de estas colonias no experimentó ninguna
pérdida de reinas durante dos meses y la mayoría seguía siendo estable después de seis meses. De 80 colonias, 55 (68,8%) no
experimentaron ninguna pérdida de la reina durante el invierno. Estos resultados muestran que nuestro método es viable para producir
colonias de abejas sostenibles con múltiples reinas para uso comercial. Además esta técnica ayudará a aumentar nuestra comprensión de las
cuestiones básicas de la evolución de la sociabilidad, como la división de reproducción y la evolución de poliginia.
Keywords: Multiple queen colonies, monogyny, polygyny, mandible, young worker
Journal of Apicultural Research and Bee World
48(4): 284-289 (2009) © IBRA 2009
many countries have been conducted in developing methods that
increase brood rearing beyond the natural capacity of a normal single
queen colony (Kovtun, 1949, 1950; Melnik, 1951; Spoja, 1953;
Wallrebenstein, 1958; Haydak and Dietz, 1967). One possibility to
achieve this is to create a colony where more than one queen
reproduces. Honey bee societies are, however, normally monogynous
(Ribbands, 1953). If several queens meet, which may happen
because virgin queens emerge simultaneously, or alien queens enter
the colony by colony merger (Neumann
., 2001), they typically
fight for reproductive supremacy until only one survives and
monopolizes reproduction in the colony (Winston, 1987). During the
fights, queens grapple together, using their legs and mandibles to
position themselves to sting each other. Stinging is the usual cause of
death, but is only successful when queens have a good purchase with
their mandibles on their opponents and can position themselves
suitably (Butz and Dietz, 1994; Gilley, 2001; Dietemann
Worker honey bees may also play a role during the elimination of
supernumerary queens by destroying queen cells (Tarpy and Fletcher,
1999), by affecting queen behaviour through
immobilization of queens (Gilley, 2001), by attacking queens with
balling behaviour (Robinson, 1984) and by interfering with the
outcome of inter queen competition through the “vibration
signal” (Allen, 1959; Painter-Kurt and Schneider, 1998) or withholding
trophallaxis (Tarpy and Fletcher, 1998).
Polygyny may occur naturally during supersedure and swarming,
but is only temporary, with monogyny eventually being reestablished
by a variety of means (Gilley and Tarpy, 2005) in a period varying
from hours to months (Hepburn and Radloff, 1998). The attempts to
artificially produce more productive multiple queen colonies therefore
required physical separation of the queens to prevent them fighting
(Wallrebenstein, 1958; Haydak and Dietz, 1967). Attempts also have
been made to obtain free running multiple queen colonies, but their
success was variable and none of the methods was widely accepted.
For example, Kovtun (1949, 1950) introduced several queens up to
1.5 years old, with wings clipped, into a hive consisting of combs of
emerging brood and honey with the empty cells filled with warm
water. It was claimed that there must be no workers in the hive
otherwise all the queens will be killed (Kovtun, 1949, 1950). This
method is not, however, practical since the comb is easily destroyed
by warm water due to the thermoplastic properties of wax (Pirk
2004). Secondly, the queens are prone to die due to the lack of care
from workers, even if they refrain or survive the fighting. Melnik
(1951) introduced three queens sequentially, one queen every two
days, into a queenright colony without any treatment. This colony
performed less well than single queen colonies for honey production
and all the queens were found dead outside the hive when a young
queen emerged (Melnik, 1951). Spoja (1953) successfully introduced
queens of varying ages with wings clipped by introducing them
among worker bees without using cages (Spoja, 1953), but this
method did not produce stable multiple queen colonies as many of
them did not survive the over wintering period (Spoja, 1953).
Here, we report an efficient method of obtaining stable multiple
queen colonies composed of several egg laying and freely moving
honey bee queens. Our multiple queen colonies were produced by
simultaneously modulating biological factors that normally prevent
polygyny in honey bees and providing a suitable social context within
the hive. This involved the reduction of the possible sources that
evoke fighting and queen elimination within the colony, i.e. removing
older intolerant workers (Robinson, 1984) and decreasing the great
fighting ability of queens (Dietemann
Materials and methods
The honey bee colonies used were of the Pinghu strain (from Pinghu
County, Zhejiang Province, China) derived from the Italian honey bee
Apis mellifera ligustica
). This strain has been selected by local
beekeepers in recent decades for high royal jelly production. Queens
were reared from grafted larvae, were allowed to mate freely and to
return to their individual colonies until their egg laying ability was
established at 6-12 months of age.
The colonies destined to host the multiple queens were prepared
as follows: combs of emerging brood were selected and shaken,
which triggers flight in the older bees, while young bees tend to
remain on the comb (Sigg
., 1997). The combs were then placed
in the hive box with the young bees still clinging to them. Young bees
were used to avoid workers balling and killing the queens, a
behaviour typical for older workers (Robinson, 1984). The host hives
were placed at a distance (5-10 m) from their original location to
ensure that all remaining foragers (older bees) did not re enter them.
One to three day old workers were however preferred to freshly
hatched individuals which may not be able to care for the queens
efficiently (Lindauer, 1953). The amount of combs and bees to be
used in the multiple queen colony depended on the number of queens
to be introduced. Four to six combs were used for three to six queen
colonies. Additional combs of honey and pollen were added beside the
brood combs to provide enough food because the colony was
deprived of foragers at the beginning.
Two days after the colonies were prepared, queens were taken
out of their original colonies and introduced to different locations in
the host hives after a third to half of both mandibles were removed
with micro-scissors. The ablation of mandibles reduced their
propensity to fight and kill each other (Dietemann et al., 2008). In
addition, the large abdomens of the egg laying queens might further
reduce their ability to fight (Spiewok, 2006).
In order to test the viability of our method, this protocol was
implemented in the seventeen household apiaries in Pinghu, Zhejiang
and in our experimental apiary in Hangzhou between 2005 and 2008.
In each of these apiaries, one to three multiple queen colonies, each
Honey bee multiple queen colonies 285
consisting of four to seven queens were created each spring from
March to April to be exploited commercially for royal jelly production.
The number of multiple queen colonies varied in each apiary and year
due to how many were needed (20, 38, 39 and 31 in the respective
years). The acceptances of the queens introduced were recorded, and
the sustainability of the colonies successfully established was
monitored over six months (Table 1). The monitoring of 7, 10 and 14
multiple queen colonies created in the springs of 2005 to 2007 ended
in October each year because of the frequent queen loss caused by
robbing by other colonies when tea plantations were in bloom and
because of apiaries being displaced to other locations at this period.
The remaining 46, and another set of multiple queen colonies (n=5 in
2005, 11 in 2006, 18 in 2007), which were created in the autumn
when a strong pollen and nectar flow was present, were allowed to
overwinter with four to six frames of workers and ample food supply.
Their survival as well as the number of queens over wintering
successfully were recorded before colonies were used commercially
again in the next spring (Table 2).
The results gathered in the four years from the eighteen apiaries
showed the high success of the method. One hundred out of 128
colonies (78.1%) were successfully established without losing one
queen (Table 1). In 23 colonies (18.0%), one or two queens were
eliminated, and in only five colonies (3.9%) were all but one queen
eliminated (Table 1). Of the 123 colonies successfully created, 116
were kept without queen loss for two months and 97 retained their
queens for the whole six month period. The majority (55 out of 80)
overwintered successfully and no queen loss was recorded the next
spring. In fifteen cases, one or two queens per colony were lost
286 Zheng, Jin, Hu, Pirk
during overwintering. In seven cases three or four out of six to eight
queens were eliminated. In only three instances (3.8%) were all but
one queens eliminated (Table 2). One four queen colony survived for
two years without any queen loss. Discussion Multiple queen
colonies with two to eight queens cohabiting, free running and laying
eggs peacefully (Fig. 1.) have been produced since 1999 using the
methods described. We have now empirically demonstrated the
efficacy of this protocol, and a success rate of 78.1% was obtained
for the creation of these multiple queen colonies.
Multiple queen colonies with two to eight queens cohabiting, free
running and laying eggs peacefully (Fig. 1.) have been produced since
1999 using the methods described. We have now empirically
demonstrated the efficacy of this protocol, and a success rate of
78.1% was obtained for the creation of these multiple queen colonies.
Years Number of
colonies Results Number of
Kept without queen
loss in two months Kept without queen loss in six months
2005 20 All queens accepted 15 (75.0)
1-2 queens eliminated 4 (20.0)
All but one eliminated 1 (5.0)
2006 38 All queens accepted 30 (78.9)
1-2 queens eliminated 7 (18.4)
All but one eliminated 1 (2.6)
2007 39 All queens accepted 32 (82.1)
1-2 queens eliminated 6 (15.4)
All but one eliminated 1 (2.5)
2008 31 All queens accepted 23 (74.2)
1-2 queens eliminated 6 (19.4)
All but one eliminated 2 (6.5)
Total 128 Multiple queen colonies 123 (96.1) 116 97
Sustainability of multiple queen colonies created in springs of 2005 to 2008.
Seven queens (marked with coloured tags and encircled) coexisting
peacefully on one side of a comb.
When taking the number of queens accepted
introduced into account, the success ratio reaches 89.8 % (Table 3),
which is close to the anticipated success rate of 95-100 % when
introducing queens into single queen colonies (Morse, 1979). Provided
they are given sufficient food and close attention, these colonies are
sustainable enough for long term use (Table 1.) and all queens can
survive overwintering (Table 2.). Queens accepted by the colonies laid
eggs normally in their polygynous colonies. This method has been
accepted by Chinese beekeepers and has been regularly used for
several years in hundreds of apiaries in the Zhejiang and Jiangshu
Given that many attempts to create multiple queen colonies have
already been made by using both the free-running queen approach
(Kovtun, 1949, 1950; Melnik 1951; Spoja, 1953) and the physical
separation of queens (Haydak and Dietz, 1967; Wallrebenstein, 1958;
Honey bee multiple queen colonies 287
Farrar, 1953), it is important to consider why our method is
successful. Spoja (1953) suggested that queens need not be of the
same age. We would, however, strongly suggest using queens of the
same age; more than six months old. Young queens behave more
aggressively to their rivals (Spiewok, 2006) and the equality of their
status enhances acceptance by workers.
Another key factor is the age of the workers. The sensitivity of
young worker honey bees to respond to pheromonal stimuli is very
low compared to older workers, both for the perception of queen
pheromones and for recruitment for defensive and aggressive
behaviour (Robertson, 1984; Free, 1987). Host colonies with young
workers for the multiple queens could therefore be described as
essentially “neutralized” with respect to queen-worker interactions
compared to colonies having a normal age distribution among the
Various methods have been adopted to treat queens to make
them cohabit, including using cages (Kovtun, 1949), clipping one or
both of the wings (Kovtun, 1949), amputating stings (Lensky and
Darchen, 1963) or simply introducing them among bees (Spoja,
1953). Our data showed that mandible ablation is an efficient way to
prevent queens from fighting to the death. Queens with ablated
mandibles refrain from engaging in lethal contests that typically
characterize their reproductive dominance behaviour and coexist
peacefully within a colony, while intact queens fight until only one
Results of overwintering of multiple queen colonies.
Years Number of colonies
over-wintered Results Number of
colonies (%) Possible cause
2005 14 No queen loss 7 (50.0) /
1-2 queens eliminated 5 (35.7) Unknown
3-4 queens eliminated 2 (14.3) Other workers drifted in
All but one eliminated 0 (0) /
2006 31 No queen loss 21 (67.7) /
1-2 queens eliminated 6 (19.4) unknown
3-4 queens eliminated 3 (9.7) unknown
All but one eliminated 1 (3.2) inappropriate thermal insulation
2007 35 No queen loss 27 (77.1) /
1-2 queens eliminated 4 (11.4) unknown
3-4 queens eliminated 2 (5.7) Robbing and inappropriate mite
All but one eliminated 2 (5.7) unknown
Total 80 No queen loss 55 (68.8) /
1-2 queens eliminated 15 (18.8) /
3-4 queens eliminated 7 (8.8) /
All but one eliminated 3 (3.8) /
Year Queens introduced Queens accepted (%)
2005 118 106 (89.8)
2006 226 209 (92.5)
2007 218 192 (88.1)
2008 171 151 (88.3)
Total 733 658 (89.8)
Acceptances of queens introduced into hosting colonies.
There are some other factors that enhance the success, such as
the season; a strong pollen and / or nectar flow facilitates damping
differences in colony odour (Bethe, 1898). In the area of southern
China below the Changjiang River multiple queen colonies are mainly
created between March and May when rape, the major floral source,
is blossoming, and between September and October when the main
flow is tea. Spraying some honey water onto the queens and workers
before queen introduction triggers grooming behaviour and gives time
for the odour of the queen to become acceptable to the workers
(Morse, 1979), thereby enhancing the acceptance.
Improving reproductive speed and maintaining strong colonies are
preconditions for maximizing colony productivity. The increase in egg
laying maintains strong colonies, the development of a large field
force possibly improves the productivity as well as enhancing disease
resistance in the colony. An increase in the genetic diversity in the
colony enhances work efficiency (e.g. Fuchs and Schade, 1994; Jones
., 2004) as well as resistance against diseases and parasites (Baer
and Schmid-Hempel, 1999; Palmer and Oldroyd, 2003; Hughes and
Boomsma, 2004, 2006). Mandible ablation has no significant effect on
the egg laying ability of queens, and the rate of egg production of
three queen colonies and five queen colonies averaged 199 % and
328 % of that of a single queen colony, respectively (Hu and Zheng
unpublished). Despite the fact that egg laying rate is less than directly
proportional to the number of queens per colony, keeping several of
them in a colony is an effective way to improve the egg laying output
of a colony (Hu and Zheng unpublished).
Close supervision is however necessary to maintain multiple queen
colonies since they are sensitive to internal and external factors. Lack
of attention by the beekeeper can result in the loss of one or some
queens at any time of the year, but especially over winter. From the
point of view of beekeeping practice, we do not recommend keeping a
large number of multiple queen colonies in an apiary because of the
extra amount of labour they require. It is nevertheless useful to keep
a limited number of them as supporting colonies to provide extra
brood (the future workforce) or replacement queens to other colonies
when needed. Multiple queen colonies can also be used as a source of
young workers to produce package bees, of which the demand
exceeds the supply in spring in some countries such as the USA due
to the increasing demands of pollination and the loss of bees resulting
infestation and associated diseases (Harrison,
2005; Lumpkin, 2005). In addition to their commercial applications,
multiple queen colonies are of great interest in theoretical research on
reproductive skew, enabling us to deepen our understanding of how
reproductive conflicts are resolved in insect societies.
We are grateful to Quan-Qing Jiang, Lin-Jie Fu, Kong-Nian Wang, Zai-
Fa Qian, Zhao-Sheng Cheng, Jin-Long Zhong, Fang-Jin Yao, Shou-Gen
Xu, Fu-Jin Shi, Xiao-Di Wang, Yin-Chu Han, Zai-Zhong Weng, Fu-Qi
Lu, Ming-Hua Wang, Xin-Xi Xu, En-Jie Xu, Shui-Guan Wang, Yu-Gen
Jiang in Pinghu, Zhejiang for access to their bee colonies. Many
thanks to Dr. Vincent Dietemann for his helpful comments on the
manuscript. This work was funded by the National Natural Science
Foundation of China (FLH) and by a Claude Leon Fellowship (CWWP).
ALLEN, M D (1959) The occurrence and possible significance of the
‘shaking’ of honey bee queens by workers.
BAER, B; SCHMID-HEMPEL, P (1999) Experimental variation in
polyandry affects parasite loads and fitness in a bumble bee.
397(6715): 151–154. DOI:10.1038/16451
BETHE, A (1898) Dürfen wir Ameisen und Bienen psychische
Pflügers Archiv European Journal of
70(1-2):15–100. DOI: 10.1007/BF01709580
BUTZ, V M; DIETZ, A (1994) The mechanism of queen elimination in
two queen honey bee (
CHEN, S L (2001)
The apicultural science in China
. China Agriculture
Press; Beijing, China. 326 pp. [in Chinese].
DIETEMANN, V; ZHENG, H Q; HEPBURN, C; HEPBURN, H R; JIN, S H;
CREWE, R M; RADLOFF, S E; HU, F L; PIRK, C W W (2008) Self
assessment in insects: honey bee queens know their own
FARRAR, C L (1953) Two queen colony management.
FREE, J (1987)
Pheromones of social bees
. Chapman and Hall;
FUCHS, S; SCHADE, V (1994) Lower performance in honey bee
colonies of uniform paternity.
25(2), 155-168. DOI:
GILLEY, D C (2001) The behaviour of honey bees (
) during queen duels.
107(7), 601–622. DOI:
288 Zheng, Jin, Hu, Pirk
GILLEY, D C; TARPY, D R (2005) Three mechanisms of queen
elimination in swarming honey bee colonies.
461–474. DOI: 10.1051/apido:2005033
HARRISON, B (2005) U.S. beekeepers make history with first
Australian bee imports.
American Bee Journal
HATCH, S; TARPY, D R; FLETCHER, D J C (1999) Worker regulation of
emergency queen rearing in honey bee colonies and the resultant
variation in queen quality.
46(4), 372–377. DOI:
HAYDAK, M H; DIETZ, A (1967) Two queen colonies, requeening and
American Bee Journal
HEPBURN, H R; RADLOFF, S E (1998)
Honey bees of Africa
Verlag; Berlin, Germany. 147 pp.
HUGHES, W O H; BOOMSMA, J J (2004) Genetic diversity and disease
resistance in leaf cutting ant societies.
HUGUES, W O H; BOOMSMA, J J (2006) Does genetic diversity hinder
parasite evolution in social insect colonies?
Journal of Evolutionary
19(1): 132-143. DOI: 10.1111/j.1420-9101.2005.00979.x
JONES, J C; MYERSCOUGH, M R; GRAHAM, S; OLDROYD, B P (2004)
Honey bee nest thermoregulation: diversity promotes stability.
305(5682): 402-404. DOI: 10.1126/science.1096340
KOVTUN, F N (1949) How to make and use multiple queen colonies.
26(9): 29-30. [in Russian].
KOVTUN, F N (1950) Letter to the editorial office (multiple queen
27(2): 112 [in Russian].
KRELL, R (1996)
Value added products from beekeeping
. Food and
Agriculture Organization of the United Nations.
LENSKY, Y; DARCHEN, R (1963) Étude préliminaire des facteurs
favorisant la creation de sociétés polygynes d'
Annales de l'Abeille
6(1): 69-73. [in French].
LINDAUER, M (1953) Division of labour in the honey bee colony.
34(4): 63-73; 85-90.
LUMPKIN, D (2005) Overcoming obstacles during 2005 almond
American Bee Journal
MELNIK, M I (1951) Managing multiple queen colonies.
28(9): 36-37. [in Russian].
MORSE, R A (1979) Rearing queen honey bees. WICWAS Press;
Ithaca, NY, USA.
Honey bee multiple queen colonies 289
MORSE, R A; CALDERONE N W (2000) The value of honey bees as
pollinators of US crops in 2000.
NEUMANN, P; PIRK, C W W; HEPBURN, H R; RADLOFF, S E (2001) A
scientific note on the natural merger of two honey bee colonies
Apis mellifera capensis
PAINTER-KURT, S; SCHNEIDER, S S (1998) Age and behaviour of
(Hymenoptera: Apidae), that perform
vibration signals on queens and queen cells.
PIRK, C W W; HEPBURN, H R; RADLOFF, S E; TAUTZ, J (2004) Honey
bee combs: construction through a liquid equilibrium process?
91(7): 350-353. DOI: 10.1007/s00114-004-0539-3
PALMER, K A; OLDROYD, B P(2003) Evidence for intra colonial genetic
variance in resistance to American foulbrood of honey bees (
): further support for the parasite / pathogen hypothesis
for the evolution of polyandry.
268. DOI: 10.1007/s00114-003-0418-3
RIBBANDS, C R (1953)
The behaviour and social life of honey bees
Bee Research Association Limited; London, UK.
ROBINSON, G E (1984) Worker and queen honey bee behaviour
during foreign queen introduction.
263. DOI: 10.1007/BF02223610
SIGG, D; THOMPSON, C M; MERCER, A R (1997) Activity dependent
changes to the brain and behaviour of the honey bee,
The Journal of Neuroscience
SPIEWOK, S (2006) When killers grow old: decrease of fighting ability
in honey bee queen ontogeny.
The IUSSI Congress
, Washington, DC.
SPOJA, J (1953) Observations on the operation of multi queen
TARANOV, G F (1951) Biological and economic characteristics of the
high mountain grey Gruzinian bee.
28(2):28-36 . [in Russian].
TARPY, D R; FLETCHER, D J C (1998) Effects of relatedness on queen
competition within honey bee colonies.
537–543. DOI: 0003–3472/98/030537+07
WALLREBENSTEIN, W (1958) My contribution to the multiple-queen
XVII International Beekeeping Congress
WINSTON, M L (1987)
The biology of the honey bee
University Press; Cambridge, UK.