Author: Walter S. Shappard, Washington State University
Originally jontly published in American Bee Journal and Bee Culture February 2013
Under the broader goal of incorporating pathogen- and disease-resistance traits into US honey bee populations and increasing the genetic diversity available for bee breeding, the Managed Pollinator Coordinated Agricultural Project (CAP) includes an objective to characterize our current honey bee genetic resources. US populations are primarily derived from honey bees introduced during a very active period of importation that occurred between 1859, when the first Italian honey bees (Apis mellifera ligustica) were imported, and 1922, when the US Honey Bee Act restricted subsequent importations of honey bees. This federal legislation, originally put in place to prevent the introduction of adult honey bees infested with tracheal mites, was later expanded to restrict the importation of honey bee germplasm in the form of eggs and semen (after the arrival of Africanized honey bees in South America). To some extent the Honey Bee Act served its purpose, as tracheal mites did not reach the US until 1984. Following the introduction and widespread establishment of tracheal mites and then Varroa mites (1987), both managed and feral honey bee populations experienced major colony losses. Prior to these heavy losses, the feral population had been shown to be genetically diverse (relative to managed honey bees) and represented a possible breeding resource for US commercial bee breeders (Schiff et al., 1994).
As part of an ongoing effort to supplement US bee breeding populations with additional genetic diversity from Old World source populations, researchers at WSU, in collaboration with colleagues in the USDA-ARS and USDA-APHIS, are involved in the collection and introduction of germplasm from several European honey bee subspecies. The story of our initial importations can be found in a previous Honey Bee CAP article (February 2012). The continuation of this story, including updates on the use of semen cryopreservation in stock reconstruction form the basis of this current article.
Figure 1. Preparing to collect drones from an apiary in central Italy near Lake Scanno (L’ Aquila)
For a number of animals of agricultural importance, cryopreserved semen and instrumental (“artificial”) insemination are widely used in breeding and selection programs. In 1944, an average dairy cow produced less than 4,600 pounds of milk per year, while in 2007 the average dairy cow produced over 20,000 pounds per year (Capper, 2007). Although no single cause was responsible for this astounding “per cow” increase in milk productivity, a significant portion can be attributed to breeding improvements and progeny testing made possible by the widespread use of cryopreservation and artificial insemination. Overall, these reproductive technologies have been estimated to provide a four-fold increase in the rate of genetic improvement in dairy cattle, relative to natural mating (VanVleck, 1981). Cryopreservation and artificial insemination also play a role in breeding efforts in other agricultural animals, including turkeys, sheep, pigs, horses, and beef cattle. In honey bees, instrumental insemination has been widely used in genetic research and for the production of some breeder queens provided to commercial queen producers. These instrumentally-inseminated breeder queens are then used as grafting mothers by queen producers for specific commercial strains (such as New World Carniolan and Minnesota Hygienic) or as sources of traits of special interest to breeders for crossing (such as Varroa-sensitive hygiene (VSH)). Cryopreservation of honey bee semen has been an elusive target, although recent improvements in methodology now provide the means to incorporate this reproductive technology into both breeding and conservation efforts for honey bees (Hopkins et al., 2012)
Figure 2. Portion of a research apiary at the National Institute for Apiculture (Reggio-Emilia, Italy)
Our research group made collections of fresh semen from this honey bee subspecies in Italy in 2008-2010. The semen was then transported to the US and used within a week of arrival to inseminate virgin queens of “Italian” honey bee strains provided by US queen producers. Following release of the germplasm by USDA-APHIS, these queens were overwintered and provided to several cooperating queen producers for evaluation.
In 2012, several members of the WSU bee research group (S.W. Cobey, B.K. Hopkins, W.S. Sheppard) again traveled to Italy and, with the assistance of Professor Raffaele Monaco (University of Bari) made collections of honey bee semen from the apiaries of a number of beekeepers in central Italy. (Figure 1) In addition, we visited the National Institute for Apiculture in Reggio-Emilia, Italy, where Drs. Marco Lodesani and Cecilia Costa are involved in a project to genetically characterize the endemic honey bees of Italy. They are collaborating with beekeepers and queen producers from around Italy to characterize local and regional populations of the Italian honey bee and to conserve these honey bees in the face of Italian importations of other European honey bee subspecies. Under European Union rules, honey bee subspecies and selected strains or hybrids (such as Buckfast) can now be sold throughout much of Europe, regardless of their genetic origins (http://www.buckfast.dk/en/global.html or http://www.buckfast.dk/en/bq-flash.html).
At the National Institute for Apiculture, we were able to make an extensive collection of honey bee semen from drones taken from their apiaries (Figure 2, 3). In addition to collecting fresh semen for immediate use upon our return to WSU, we also cryopreserved aliquots of the semen for use in the near or distant future (Figure 4, 5).
Figure 3. An excluder used to collect returning A. m. ligustica drones outside the hive (Reggio-Emilia, Italy)
Our collection of semen from these two subspecies prior to and including 2011 was previously described in a Honey Bee CAP article (February 2012). Given that only semen was allowed for importation, the initial expression of A. m. caucasica genetics from these imports was through insemination of daughter queens derived from a US Carniolan strain in 2010, yielding mated queens who produced colonies expressing 50% Caucasian genetic background. In 2011, virgin daughters of these crossbred queens were inseminated with fresh imported Caucasian semen, resulting in colonies that expressed a 75% Caucasian genetic background. In 2012, daughters produced from these queens were inseminated with previously cryopreserved A. m. caucasica semen, resulting in queens yielding colonies expressing an 87.5% Caucasian background. A number of virgin daughters of these queens were then produced and allowed to naturally mate in a semi-isolated area where an apiary incorporating Caucasian genetic material is maintained. These naturally mated queens are being overwintered in 2012 in several locations and will be evaluated in Spring 2013. A number of these overwintered queens will be selected as queen mothers and backcrossed to cryopreserved A. m. caucasica semen in 2013. From these queens, daughters will be produced in summer 2012 and naturally mated to the Caucasian pool to produce the queens and colonies that overwinter in 2013.
Queens that are instrumentally inseminated with fresh semen can be established in full-sized colonies and overwintered normally. However, although queens inseminated with cryopreserved semen can produce normal worker brood and serve as grafting mothers to produce a subsequent generation of queens, their useful life as a “head of state” for a colony (i.e. through a full winter in northern climates) is somewhat less certain, probably due to the limited amount of cryopreserved semen used for each insemination and possible cryo-damage that affects long-term sperm survival within a queen’s spermatheca.
Of the three subspecies for which we have been able to import semen for breeding purposes over the past 5 years, Apis mellifera carnica has been the most utilized, with progeny from instrumentally inseminated breeder queens being overwintered and tested by collaborating queen producers. At present, several queen producers in California are selling stocks that include some measure of genetic material from our 2008-2011 importations.
The reasons for the rapid adoption of imported A. m. carnica genetic material, relative to A. m. ligustica are multiple. First, the Italian honey bee has been the most popular strain with US beekeepers for over a century and US queen producers have developed their commercial strains over a long history serving beekeeper demand. They are prudently cautious to evaluate any outside source material, both for apicultural traits they want to be expressed in their stocks and for the combining ability of these imported stocks with their own. Secondly, given the number of queen producers that maintain Italian honey bee strains, they have been able to trade queens (i.e. genetic material) among themselves, when they felt the need to incorporate additional diversity in their breeding lines. In addition, hundreds of thousands of queens of Australian sourced “Italian” strains of honey bees were imported into the US between 2004 and 2010, with unknown effects on US breeding populations.
In the case of Caucasian honey bees, the requirement to reconstruct this strain in the US through an initial cross with Carniolan bees has already been noted. The initial importation of A. m. caucasica semen was made only in 2010 and, thus, we have had only limited time to evaluate the Caucasian honey bee within our breeding program. However, based on the interest expressed by a number of beekeepers and queen producers, we anticipate some release of this material to queen producers in 2013.
Figure 4. Aliquots of honey bee semen ready for cryopreservation. Note the red and yellow internal rods used to mark samples during cryo-storage. The semen is the tan liquid near the bottom of each tube.
As we look to the future of agriculture, it is vital to recognize that current agricultural production methods are highly dependent on honey bee pollination and migratory beekeeping. Almond production alone requires the annual pollination services of well over half of the managed honey bee colonies in the US in late winter/early spring. Given the climatic requirements of almonds (over 80% of the world’s production occurs in California), it is unlikely that almond production will ever move to other locations throughout the US. Thus, in almonds, the need for honey bees, transportation and high density planting is likely to continue to be a driver of the commercial beekeeping industry. If and when agricultural production in other crops shifts toward less monoculture and small-scale producers, non-Apis bees and non-migratory honey bees will likely play a larger role in the food and forage crop pollination story, just as they already do in home gardens and small farmstead agriculture. However, at present, the critical issue of honey bee colony health remains of paramount importance to agricultural production, and beekeepers, researchers and industry continue to struggle to find sustainable, safe and effective means to keep honey bee colonies alive and thriving.
Among 28 subspecies of the honey bee, there exists extensive genetic variation across their Old World homelands, as a result of the diverse environmental and climatic conditions faced by this species over many thousands of years. Thus, honey bees adapted for northern Europe, the Mediterranean coastline, the Caucasus Mountains and sub-Saharan Africa have all accumulated specific genetic differences that underlie traits adaptive within their own particular history. In many cases, some of these traits are more or less “desirable” from the standpoint of beekeepers managing colonies in the US. Thus, while the tendency for high rates of reproductive swarming and “defensiveness” in Africanized honey bees (derived from sub-Saharan African A. m. scutellata) is clearly not a desirable trait for most US beekeepers, their ability to co-exist with parasitic Varroa mites without chemical treatment or intervention by beekeepers would be seen by most as a most welcome trait (Martin and Medina, 2004).
Although it remains an elusive target, one of the primary goals of bee breeding is to move toward the development of managed honey bee populations that are productive and useful from the standpoint of human needs, while also maintaining traits that allow them to thrive and sustain themselves without excessive intervention by beekeepers in the form of insecticides and drugs. The re-collection of original source populations that were the bases for early US honey bee introductions provides additional genetic material for beekeepers to evaluate for traits leading to that end. Likewise, cryopreservation provides a means to more fully utilize these genetic resources without the limitation of the very short “shelf-life” of fresh semen.
As we look to the future of bee breeding, the establishment of genetic “repositories” for honey bee germplasm will provide the means to cryopreserve the genetic diversity existing within particular honey bees subspecies or selected top tier “strains” of domestically selected honey bees. In common with genetic repositories that are maintained for other agricultural species, a repository for honey bees would provide the means to more easily conduct progeny testing, whereby the “outcome” of specific crosses can be evaluated and then the parental sources can be retroactively “scored” as to their value for future breeding. A honey bee germplasm repository would also allow queen producers to cryopreserve samples of their current honey bee stocks and bring them out many years later for the purposes of backcrossing or stock reconstruction. Repositories also permit easy transport and exchange of honey bee genetic material among queen producers across the country or around the world (following proper regulatory oversight). In a sense, cryopreservation and the establishment of genetic repositories provide the means to breed across “time and space”, allowing honey bee germplasm to be conserved for years or decades and then thawed for use as breeding needs dictate.
Figure 5. Cryopreservation in action. Semen samples are in the small black unit in the liquid nitrogen filled “cryobath” on the left. The computer is used to control the freezing rate.
Capper, J.L. 2009. More production per cow, not less, is the most environmentally friendly strategy. Western Dairy News - Hoards Dairyman. 9:w45-w46
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