Honey Bee Genetic Diversity and Breeding: Towards the Reintroduction of European Germplasm

Managed Pollinator Coordinated Agriculture Program (CAP) Updates

A National Research and Extension Initiative to Reverse Pollinator Decline

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by Walter S. Sheppard, Washington State University

CAP Updates: 24

As we reflect that the western honey bee Apis mellifera thrives in some regions of the world with minimal or no chemical treatments for mites and diseases, it seems reasonable to expect that we could make significant and sustainable improvements to our own managed pollinator population by more fully utilizing the resources of the honey bee genome.

One of the objectives of the Managed Pollinator CAP is to better understand the honey bee genetic resources available in the United States.  A number of cooperating University and USDA laboratories in this Coordinated Agricultural Project and partners in the bee breeding industry are involved in breeding efforts to increase tolerance to Varroa mites, resistance to diseases and to otherwise improve apicultural characteristics in our managed honey bee populations.  Fundamental to all breeding programs in agricultural animal and plant systems is the requirement that the population of interest contains adequate genetic diversity from which to make progress toward the “selection” of various desired traits.  Simply put, “genetic diversity” is the raw material for breeding.

The original distribution of the honey bee included Europe, Africa and parts of Asia, where as many as 28 distinct “subspecies” or geographic races are known to occur.  Records indicate that, with the assistance of sailing ships, North America beekeepers established a population of one of these subspecies (A. mellifera mellifera) in Virginia by 1622.  The subsequent history of beekeeper-assisted honey bee transport to North America is a fascinating topic in its own right, with most additional introductions taking place between 1859 and 1922.  By 1922, when further importations were restricted by the US Honey Bee Act, seven additional subspecies had been sampled and introduced to the US, including three that found eventual favor with US beekeepers:  A. m ligustica (Italians), A. m. carnica (Carniolans) and A. m. caucasica (Caucasians) (Sheppard 1989a, b).  

Figure1.  Nucleus colonies at WSU’s Smoot Hill Apiary on the 800-acre Hudson Ecological Reserve

As a by-product of these various introductions, a large population of feral honey bees became established in the US.  Genetic studies demonstrated that this feral population contained genetic markers indicative of various honey bee subspecies that constituted historical founding populations (Schiff and Sheppard 1995, 1996), thus providing evidence that the feral population could serve as a genetic reserve for bee breeders.  Unfortunately, the arrival and establishment of parasitic mites in the late 1980’s eliminated much of the feral honey bee population and consequently, this potential source of usable germplasm for breeding.  There is recent evidence that remnants of the US feral population may yet persist (Magnus and Szlansky 2005; Seeley 2007), which could restore the utility of this population to bee breeding.  A further challenge to the maintenance of a broad genetic foundation for breeding results from current large-scale queen production practices, whereby each queen “mother” is typically used to produce more than a thousand daughter queens.  Overall, it has been estimated that less than 500 queen mothers are used to produce around 900,000 daughter queens annually for commercial sale in the US. (Delaney et al. 2009). 

One approach to provide additional genetic variability to the breeding process is to import additional genetic variation or even intact honey bee stocks from Old World sources.  In recent years, one example of the latter has been the importation, screening, propagation and release of Russian Honey Bees by the USDA-ARS and industry partners.  The idea behind the importation of this novel honey bee stock from far-eastern Russia, was that these bees had a long history of contact with Varroa destructor mites in a cold climate in Asia that could have contributed to selection for a measure of tolerance to both Varroa destructor and tracheal mites.  Studies have supported this hypothesis, with Russian Honey Bees and their hybrids showing lower population growth of these pests, in side-by-side comparisons with some domestic strains (DeGuzman et al. 2007; Tarpy et al. 2007).   These honey bees are currently available from queen producer members of the Russian Bee Breeding Association.  Another approach to provide germplasm for US honey bee breeding is to make additional importations of genetic material from subspecies that previously found favor with US beekeepers and use this material to “supplement” current genetic stocks of honey bees.  Given the expense, regulatory and logistical difficulties that accompany the effort to import novel strains of adult honey bees, we undertook a limited approach to import only honey bee semen for instrumental insemination of US queens derived from existing managed stocks.  The story of these initial semen importations and the vision for future releases to assist US bee breeding efforts form the basis for the remainder of this article. 

Figure 2.  Professor Raffaele Monaco in an apiary in south central Italy that served as a source for A. m. ligustica semen in 2009

The initial challenge prior to importation of aliquots of honey bee semen into the United States was to establish a means to safely contain imported material until final release could be granted by the USDA-APHIS (United States Department of Agriculture  – Animal, Plant Health Inspection Service).  To that end, isolated quarantine sites were established at Bald Butte and Smoot Hill in the wheat growing Palouse region of eastern Washington (Figure 1).  Both of these facilities encompass several hundred hectares of land, with lockable access, each surrounded by thousands of additional hectares of wheat, lentils, barley and other non-forage crops for honey bees.  As a result, the chances of interaction with other honey bee colonies were minimized.  As part of the protocol for importation, subsamples of all semen collected and (in some cases) daughter progeny from instrumentally inseminated queens were submitted to Dr. Judy Chen (USDA-ARS, Beltsville) for virus analysis, with results being transmitted to USDA-APHIS.  The inseminated clipped-wing queens were maintained in nucleus colonies under quarantine until final release, on a case by case basis, by USDA-APHIS.  By some measures, this approach may seem to be “overkill” to prevent the hitchhiking of pathogens, especially given that hundreds of thousands of Australian packages were imported into the US in recent years with no testing.  However, the current protocol and permitting system provides a verifiable mechanism suitable for future importations of honey bee genetic material for breeding. 

Figure 3.  Collecting A. m. carnica semen for importation. LR – Sue Cobey, Stane Plut and Ales Gregorc

The Old World honey bee subspecies named in the USDA-APHIS permit to WSU to support US queen breeding efforts are limited to A. m. ligustica, A. m. carnica and A. m. caucasica.  These three subspecies were all previously introduced into the US and their descendants were commercially relevant honey bee strains for over a century. 

A. m. ligustica

This honey bee subspecies was initially introduced into the US in 1859 and forms the genetic basis for the popular “Italian” honey bee strain used by most US commercial beekeepers.  The bees are generally gentle and respond to a honey flow or feeding by rapid expansion of the brood nest and adult bee population. In 2008 and 2009, we obtained semen from a number of locations in southern, eastern and central Italy with the assistance of Professor Rafaelle Monaco from the University of Bari (Figure 2).  The semen was transported back to the US and used to inseminate a number of virgin U.S. queens (2008) and F1 queens (50% US “Italian”: 50% A. m. ligustica) (2009).  In 2010, we obtained semen from A. m. ligustica drones near Bologna and upon transport back to the US, used it to inseminate F2 queens (75% A. m. ligustica: 25% US “Italian”).  Sue Cobey conducted the instrumental inseminations and a number of US queen producers provided the virgin queens (see Acknowledgements).  Germplasm from these importations was made available to cooperating queen producers.

A. m. carnica

The homeland of this honey bee subspecies is the European Alps and, worldwide, Carniolan honey bees are one of the most popular strains maintained by beekeepers.  The subspecies was initially introduced into the US in 1877, where it gained a reputation for gentleness and good overwintering characteristics.  In the US, one of the most successful efforts to promote the Carniolan strain has been the selected strain of  “New World Carniolan” honey bees maintained by Sue Cobey and a supporting group of commercial queen producers in California.  In 2008 and 2009, fresh honey bee semen from A. m carnica was obtained from the Institut fur Bienenkunde in Kirchchain, Germany.  The semen was transported to the US and used to inseminate virgin US “Carniolan” queens (2008) and F1 queens (50% US “Carniolan”: 50%  A. m. carnica) (2009).  In 2011, Sue Cobey and Brandon Hopkins travelled to the Slovenian Alps and collected additional A. m. carnica semen (Figure 3).  Some of this semen was frozen (cryopreserved) for later use and some aliquots were returned to the US in the fresh state and used to inseminate F2 queens (75% A. m. carnica: 25% US “Carniolan”).  This genetic material was incorporated into the New World Carniolan program and is currently available through collaborating queen producers. 

A. m. caucasica

This honey bee subspecies is endemic to the Caucasus Mountains and was initially introduced into the US in 1882.  It has general characteristics similar to the Carniolan honey bee, including gentleness and excellent overwintering characteristics.  Historically, it was also noted for a propensity to collect copious amounts of propolis.  While once available as a commercial “strain” in the US, the Caucasian honey bee is no longer readily widely available, so our breeding efforts included retrieval of this genetic material and propagation via a Carniolan genetic background.  The initial collection of A. m. caucasica semen took place in 2010 from locations in the Republic of Georgia, with the assistance of Mr. Eric Olson (a commercial beekeeper from Washington State) who had local family ties in Tblisi, Georgia, Dr. Ivane Nicoladze and Shalva Ioseliani, a Georgian queen producer (Figure 4).   In 2011, the collecting trip was repeated, with the addition of a cryopreservation component for portions of the semen collection (Figure 5).  In 2010, the collected semen was used to inseminate US “Carniolan” virgins and in 2011, the collected semen was used to inseminate F1 daughters of these initial crosses (50% US “Carniolan” and 50% A. m. caucasica).  This genetic material is currently being evaluated in the WSU honey bee breeding program and will be made available through collaborating queen producers.  

Figure 4.   A. m. caucasica apiary high in the Caucasus Mountains near Mestia, Georgia.  Note the medieval “Svanetian Towers” used as defensive structures in the 9th through the 12th centuries.   Caucasian queen producer Shalva Ioseliani is in foreground.


Traditionally, honey bee semen used for instrumental insemination in breeding and experimental protocols has been used “fresh”.  That is, as collected semen loses about 50% viability per week under normal “room temperature” storage conditions, the allowable window for making use of collected semen is generally around 2 weeks.  In most of our semen importation efforts, one week was allocated for the actual collecting trip and the semen was used to inseminate waiting virgin queens back in the US during the second week.  This presented a limitation on the importation process that was recently removed with the development of a viable means to cryopreserve honey bee semen and subsequently create multiple generations of queens from frozen semen in a short time  (Hopkins and Herr 2010; Hopkins et al – In Press).   Starting in 2011, subsamples of all semen collections were cryopreserved and placed in storage in liquid nitrogen for future use as part of a honey bee germplasm repository.

Figure 5.  WSU PhD student Brandon Hopkins transfers recently cryopreserved honey bee semen from a programmable temperature module into liquid nitrogen for long term storage in a Tblisi, Georgia hotel room.

Honey Bee Breeding

There are many challenges to maintaining honey bee colony health within the modern agricultural system of the United States.  Many of these challenges derive from the demand for the efficient and timely pollination of large monoculture crops, including the need for extensive highway transportation of beehives, the requirement that colonies (in almonds) be stimulated to be in a physiological state conducive to pollination activity several months earlier than their “normal” annual cycle might dictate, overall limited nutritional opportunities due to placement on single crops and an ever present stress due to pesticide exposure both in the hive and field environment.  Researchers participating in the Managed Pollinator CAP continue to evaluate such factors affecting colony health in many parts of the country.  As a result, there is a growing body of evidence from these and other laboratories that demonstrate the potential and realized negative consequences of specific parasites, pathogens, nutritional stresses and pesticides, both singly and in combination.  All of these potentially interacting factors impinge on a US honey bee population that has a genetic background reflecting both historical and beekeeper-selected processes.  Research has shown that the maintenance of genetic diversity at the colony level can be an important contributor to colony health (Tarpy, 2003; Tarpy and Seeley, 2006).  The addition of genetic diversity at the population level can increase the prospect that genetic material will be available for the subsequent selection of traits useful to apiculture.  However, it will take a concerted effort by bee breeders, queen producers and researchers involved in selection programs to improve the genetic stocks of honey bees available in the US.  As we reflect that the western honey bee Apis mellifera thrives in some regions of the world with minimal or no chemical treatments for mites and diseases, it seems reasonable to expect that we could make significant and sustainable improvements to our own managed pollinator population by more fully utilizing the resources of the honey bee genome.


Funding for this work was provided by the USDA Managed Pollinator CAP, grants from the State Beekeeping Associations of Washington, Oregon, California and Oregon, from the California Almond Board, the National Honey Board, the Foundation for the Preservation of Honey Bees and from Project ApisM.  Overseas field work was made possible by contributions of time and bees from numerous beekeepers and other individuals, including:  R. Buchler, S. Cobey, C. Costa, E. Frost, A. Gregorc, B. Hopkins, S. Ioseliani, R. Kalashyan, M. Meixner, R. Monaco, E. Olson, I. Nicoladze.  S. Cobey conducted instrumental insemination. Virgin queens for instrumental insemination were provided by S. Cobey and US queen producers: Tom and Suki Glenn, Pat Heikem, Koehnen and Sons, Inc., Jackie Park, Valerie Severson, Pat and Bonnie Stayer and Glenda and Shannon Wooten.  US field work, apiary and stock maintenance relied on oversight from Beth Kahkonen and Elizabeth Frost.  Additional field assistance was provided by N. Boyle, M. Taylor, J. Vierya, and B. Hopkins.


Literature Cited


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Delaney, D. A., Meixner, N.M. Schiff., and W. S. Sheppard.  2009.  Genetic characterization of commercial honey bee (Hymenoptera: Apidae) populations in the United States by using mitochondrial and microsatellite markers.  Ann. Entomol. Soc. Amer.  102: 666-673. 

Hopkins B.K., C. Herr (2010) Factors affecting the successful cryopreservation of honey bee (Apis mellifera) spermatozoa. Apidologie. 41, 548-556.

Hopkins B.K., C. Herr, W.S. Sheppard.  Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen. Reprod. Fertil. Dev.  In Press.

Magnus, R. and A. L. Szlanski.  2010.  Genetic evidence for honey bees (Apis mellifera L.)of Middle Eastern lineage in the United States.  Sociobiology. 55: 285-296.

Schiff, N.M. and W.S. Sheppard. 1995. Genetic analysis of commercial honey bees (Hymenoptera: Apidae) from the southern United States. J. Econ. Entomol. 88: 1216-1220.

Schiff, N.M. and W.S. Sheppard. 1996. Genetic differentiation in the queen breeding population of the western United States. Apidologie. 27:77-86.

Seeley, T.D.  2007.  Honey bees of the Arnot Forest: a population of feral colonies persisting with Varroa destructor in the northeastern United States.  Apidologie.  38: 19-29.

 Sheppard, W. S. (1989a, 1989b) A history of the introduction of honey bee races into the United States.  American Bee Journal.  129: Part 1;617-619 and Part 2; 664-666. 

Tarpy D.R. (2003) Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth. Proceedings of the Royal Society of London Series B-Biological Sciences 270(1510), 99-103.

Tarpy D.R., T.D. Seeley (2006) Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens. Naturwissenschaften 93(4), 195-199.

Tarpy , D.R., J. Summers, and J.J. Keller (2007)  Comparison of parasitic mites in Russian-hybrid and Italian honey bee (Hymenoptera:apidae) colonies across three different locations in North Carolina.  J. Econ. Entomol.  100: 258-266.