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Methods and Materials

(i)  Saskatraz apiary establishment and gene pool development

An apiary location operated by Meadow Ridge was chosen to establish the Saskatraz natural selection program. This apiary is located in an isolated area with native parklands and saline lakes to the south, east and west. The area is located in one of the major Saskatchewan migratory bird fly ways, with surrounding lakes and marshes. Willows, shrubs, poplars, native roses, both white and yellow blossom clover and native grass species are abundant. To the north of the apiary, typical parkland farm land predominates, producing mainly cereal grains and canola, with some alfalfa hay and pasture land. The apiary itself is well sheltered on all sides and surrounded by mature spruce, caragana and poplar, at one time being a family home stead. The site is not occupied and is seldom used except during harvest, because of grain bins located on the west side of the site. Isolation is necessary to protect the gene pool (closed population mating procedures) and to prevent infestation of other bee keepers with varroa mites, tracheal mites and associated pathogens.

Saskatraz was established in 2004 with 35 pre-selected colonies from fourteen different Saskatchewan and Manitoba queen breeders. Colonies were numbered in order of addition to the apiary. For example, Saskatraz -01 (SAT-01) stands for the first colony selection entered into Saskatraz. This gene pool included colonies naturally selected for over wintering ability in Saskatchewan, as well as selections for honey production, temperament, chalkbrood resistance and over all hive health. Since they were selected by different operations throughout Saskatchewan they would be expected to be genetically diverse. Thirteen producers donated 24 nuclues (nucs) bee hives and Meadow Ridge donated 11, as follows:  Dwight Sollosy(1); Yves Garez(2); Steve Clifford (2); Trevor Rehaluk(2); Ron Bacon(1); Corey Bacon(1); Carl Meyers(1); Tim Wendell(4); Tony Lalonde(1); John Pedersen(6); Len Proctor(1); Wink Howland(1); Lester Martens(1); Meadow Ridge(11). Reselected Russian stock (2000 to 2004) and breeding lines from the Manitoba Queen Breeders Association (MBQA) were also included in the breeding program. The MBQA is working with Dr. Rob Currie at the University of Manitoba. In 2005, 14 more selections were placed at Saskatraz with crosses made between Russian and German (Carnica) lines (semen) imported by Yves Garez, from Dr. Ralph Büchler, Kirchain, Germany The selections from the Russian (Rinderer et al., 1997) and German lines were introduced to add varroa and tracheal mite tolerance to the gene pool.

Since 2005, additional selections from new Canadian lines and reselections of Saskatraz stock have been added (Meadow Ridge, John Pedersen, John Polson, Robert Colbert, Jim Wood, Brent Mckee, Len Proctor, Tim Wendell, Yves Garez, and Robert Hamilton, Dr. Rob Currie, University of Manitoba (U of M.). Meadow Ridge maintains Saskatraz breeding stock by providing 50 yard sites for stock maintenance, outcrossing, backcrossing and closed population mating procedures.

All Saskatraz colonies showed trace to low levels of Varroa mite infestations, when measured by natural drop analyses, between August 7th and September 15, 2004. All colonies were treated with Apistan (2 strips per colony) for 32 days (September 15 to October 15) to normalize varroa populations. Two independent samples of 100 bees per colony failed to detect tracheal mite infestations. On October 15, 2004 all colonies were infected by adding 200 worker bees from a colony provided by John Gruszka showing 58 to 60% tracheal mite infestation

Meadow Ridge maintained four apiaries with “near pure” Russian lines established by back crossing selections released from Baton Rouge, USDA between 2000 and 2005. Initial stock importation was funded and implemented as a joint effort by the Saskatchewan and Ontario Beekeepers Associations. These Meadow Ridge apiaries were made available for re-selection of Russian stock for evaluation at Saskatraz, and used for both out crossing and back crossing procedures between 2004 and 2007.

In 2005, more honey bee semen was imported from Dr. Büchler’s program in Kirchhain, Germany. This program involves selection for varroa tolerance, honey production, grooming and hygienic behavior, and has been in progress for about 15 years. Susan Cobey assisted us in making 35 new crosses with this semen, (G-08 and G-72) by instrumental insemination of virgin queens from the following selected lines (yellow-green-05, yellow-blue-05, UM (University of Manitoba)-163, 234, 147, SAT 28, 30 and BTP-30). Yellow-green-05 and yellow-blue-05 virgin queens were derived from Russian breeder queens obtained from Charlie Harper (Queen breeder for USDA, Baton Rouge, Louisiana) in 2006.UM lines were obtained from Dr. Rob Currie at the University of Manitoba, SAT-28 and 30 virgin queens were obtained from SAT-28 and SAT-30 selections made at Saskatraz. BTP-30 is a breeder queen obtained by crossing a Buckfast queen with Russian P-30 drones. Some daughters of this cross were inseminated with G-08 and G-72 semen.

(ii)  Measuring honey production and mite infestations

Honey production was measured from July until September each year (2005-present) by weighing individual honey supers from all Saskatraz colonies every time surplus honey was removed. The error associated with the variation in empty super weights was calculated to be 2.3 (mean), 1.7 (standard error), n=20.

Tracheal mite infestations were monitored on a monthly basis between May and October of each year. All colonies were sampled by collecting a minimum of 100 adult bees in glass jars containing 70% methanol. Tracheal mite analyses were carried out at the provincial apiculture laboratory in Prince Albert, Saskatchewan, under the direction of John Gruszka. Tracheal mites were detected following procedures similar to those described by Peng and Nasr 1985. Varroa mites were also counted after alcohol washes by straining out the bees and counting the remaining varroa.

Varroa mite populations were also assayed on a weekly basis, by natural drop, following procedures described by Martin, 1998. Commercially available Apinovar boards were placed under all Saskatraz colonies to monitor mite populations. The screened bottom boards allow dead or groomed mites to fall on a coroplast sheet which can be removed like a drawer from the bottom board for mite counting and analyses. Mites were counted and cumulative drop was recorded for each colony. Mature and immature (white mites) were recorded as well as total mites. In some cases, the percent mite infestations in worker and drone brood were also assessed. To identify varroa sensitive hygienic (VSH) phenotypes the number of mites per brood cell was recorded. Mite damage was also microscopically assessed using a phase contrast microscope, and mites were selectively sampled for molecular analyses.

 (iii)  Hygienic and grooming behavior assays

Assays for hygienic behavior were carried out as described by Spivak and Boecking, 2001, and for Varroa Sensitive Hygiene following procedures outlined by Ibrahim and Spivak, 2006. In 2005 hygienic tests using liquid nitrogen to kill selected brood areas to test Saskatraz colonies were carried out and sub sequentially modified in 2006. Brood was killed (whole frame) by repeated freezing and thawing at-20C and +4C, respectively over a 2 week time frame. Colonies were tested for uncapping and removal of dead pupae by placing test frames in third story supers with excluders placed over the second super. This method is demonstrated in Robertson( 2007). Dr. A. Ibrahim performed hygienic assays on Saskatraz breeding lines and out crosses in 2007, defining VSH phenotypes as colonies that uncapped and removed 95% or better of freeze killed pupae in 24 hours. VSH phenotypes were also identified by opening one hundred or more sealed brood cells and scoring for the number of varroa mites per cell as described by Harbo and Harris,2005, on selected and non-selected Saskatraz breeding lines.

Grooming assays were performed on 4 daughters from each of 6 Saskatraz breeding lines in the fall of 2008. Although a number of grooming assay procedures exist for laboratory work (Aumeier, 2001; Mondragon etal 2005); we are developing grooming assays that work at the colony level with a minimum amount of invasiveness or manipulation. All selected colonies were normalized for varroa mite populations by Apistan treatment in the fall (late-September to early November). Varroa infested (varroa nursery) colonies were used as a source of inoculums for these studies. Each colony was inoculated with a sufficient number of adult bees carrying varroa mites to introduce 300 adult mites to the test colonies, which were indoor wintered. This simulates in part, natural methods of Varroa mite infection. Mite drop was assessed every 24 hours for 21 days using apinovar boards. All mites dropping on boards were microscopically assessed for damage using a stereo microscope. In the spring (April) all surviving colonies were assessed for varroa mite infestations to determine varroa population growth rates.

(iv)  Molecular Marker Analyses

The technical aspects of DNA isolation and analyses, Polymerase Chain Reaction (PCR)and Reverse Transcriptase, (RT-PCR), marker analyses of this project were carried out on a contract basis by GenServe Laboratories., Saskatchewan Research Council, Saskatoon, Saskatchewan, by Bruce Mann in association, with Dr. Yves Plante. General methods are as described In DNA MARKERS: Protocols, Applications and Overviews. 1997. A brief description of materials and methods used will be described here. A Qiagen DNeasy tissue kit was used to extract DNA from 50mg of drone pupae or larvae. The DNA markers used (108 microsatellites) in the initial screening were selected from Solignac et al 2003 and Genbank. DNA was quantified by flourimetry and assessed for quality by agarose gel electrophoresis. Initial PCR reactions were performed using a 55C annealing temperature and 1.5m M MgCl2, DNA products were separated on polyacrylimide sequencing gels using the LICOR system and fragments scored using GeneImagIR (Licor). DNA was extracted from drone pupae collected from Canadian lines at Meadow Ridge and from Russian lines sent to us from the USDA, Baton Rouge, La. USA.  Russian lines reconstructed by backcrossing procedures at Meadow Ridge using embryo’s obtained from Baton Rouge, were also sampled. Other samples included German lines (semen), Russian x Canadian hybrids, alcohol preserved tissue samples of Scutellata and Scutellata hybrids from the Yucatan area of Mexico (Felipe Brizuela), and Saskatraz breeding lines. Microsatellite analyses were performed using methods similar to those of De La Rue et al 2001, Franck etal 2001 and Estoup etal 1995. Dendograms were constructed using a TREECON software package (Van de Peer and Wachter, 1990).

RT-PCR procedures were carried out at GenServe Labs, SRC by Bruce Mann and at the Veterinary Infectious Disease Organization (VIDO), University of Saskatchewan, (U of S) Saskatoon, Saskatchewan by Wayne Connor according to published procedures (Chen etal. 2005).RT-PCR kits were purchased from Invitrogen. Sequencing of amplified bands was performed to confirm identity.

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