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 Results and Discussion

(i) Selection of SASKATRAZ breeding lines for Honey Production, Tracheal Mite Resistance and Varroa Tolerance.


The Saskatraz apiary was established during the summer of 2004, and by October 15 all 35 colonies were normalized for both tracheal and varroa mite populations as described in Materials and Methods. No further chemical miticide treatments were used and measurements on mite infestation levels and honey production were initiated in May, 2005. The genetic diversity and gene flow in and out of Saskatraz is shown in Figure 1. The operation and logistics behind the breeding program are summarized showing the time frames involved. This model was developed over the past three years based on the progression of the program. Blocks A, B, C, D and E represent gene flow from different sources in to Saskatraz. All of these colonies were preselected (as described in materials and methods) prior to placing into the Saskatraz natural selection site for evaluation. Colonies were managed by Meadow Ridge using standard procedures for outdoor wintered colonies in Saskatchewan.  Fall feeding consisted of up to 30kg of 50% sucrose with Fumidil B and spring feeding up to 15kg of 50% corn syrup. No pollen or pollen substitute were used. Colonies were wrapped in insulated 4 packs on pallets prior to October 31, and opened in May for inspection and preliminary selections. Colonies were scored for wintering ability, cluster size and location, brood pattern, brood diseases (chalk brood), temperament, burr comb, queen characters (size, age, color, egg placement, etc.). In 2006 the first six breeding lines were released to commercial queen breeders and beekeepers, followed by ten additional lines between 2007 and 2009. Block F shows the gene flow out of Saskatraz, representing 14 breeding lines certified for distribution as of 2009. Queen breeders are encouraged to return lines subjected to out crossing and reselection (RC) to maintain genetic diversity and enrich for economic traits. These lines as well as those selected by Meadow Ridge outcrosses are reselected for evaluation at the Saskatraz natural selection yard site. Block G represents Saskatraz satellite yards set up by Meadow Ridge for close population mating and for maintenance of daughters (stock maintenance) of selected lines. The Saskatraz natural selection apiary is also used for close population mating procedures, to select for drone populations surviving high varroa infestations. Drones from colonies suppressing varroa population growth would be expected to enrich for varroa resistance in colonies headed by queens mated with the most fit drones from the Saskatraz drone population

Mite populations and honey production were monitored as described in Materials and Methods. Honey production data is summarized for 2005 and 2006 for non-selected (SAT-04, 24) and selected (SAT-14, 17, 23, 28, 30, 31, 34) colonies in Figure 2. The best selections for honey production were SAT-14, 30 and 31. SAT-31 superseded at Saskatraz in 2005, showed the highest honey production in 2006, but was lost prior to multiplication of daughters. The increased honey production and vigor of this colony after supersedure at Saskatraz is of interest. Controlled studies (Matilla and Seeley, 2007) on genetic diversity have shown “enhanced productivity and fitness” in colonies where the queens have mated with genetically diverse drones, such as the drone population at Saskatraz. We have reselected out crosses of SAT-30, and 14 daughters in 2008 and 2009.Both are showing increased honey production, and are currently undergoing revaluation at Saskatraz.

Figure 1:  Letters A to G represent isolated apiaries and the year of establishment at Meadow Ridge. Solid arrows indicate genetically diverse gene (GD) flow into Saskatraz, dashed arrows gene flow out of Saskatraz. (ii) denotes instrumental insemination. RC denotes recurrent selection. 1 Denotes no chemical miticides.

Figure 2.Saskatraz honey production measured during the summer of 2005 and 2006.

Figure 3.Varroa mite population growth for selected and non-selected lines were estimated by the natural drop method, and counted on a weekly basis between May 7 and Oct.15, 2005

Figure 3 shows mite population growth data measured by natural drop in 2005.Data from eight test colonies are shown, identifying high (SAT-06, 26), intermediate (SAT-17) and low (SAT-14, 23, 28, 30, 34) varroa population growth.  Figure 4 shows the same analyses done during the summer of 2006, after 21 to 24 months with no chemical miticide treatment. Although spring mite levels were low and comparable to fall 2005 levels an accelerated rate of varroa population growth occurred in July and August of 2006, in both selected and non-selected colonies. Non-selected colonies (SAT-24 and 04) and SAT-23 failed in early fall of 2006. Selected colonies (SAT-28, 31 and 34) showed the best suppression of varroa mite population growth in 2005, maintaining the same status in 2006 (figure 4).

The values shown in Figure 5 are cumulative counts obtained by natural drop, at 7 to 10 day intervals, comparing the results obtained for 9 Saskatraz lines during the summers of 2005 and 2006. Figure 6 shows the average mite drop per day for selected and non-selected lines between May and October, 2006, showing the rapid increase in mite populations in sensitive colonies and suppression by tolerant lines. Mite populations tended to follow the increase in adult bee populations, a reflection of increased brood production required for varroa reproduction. The average mite drop per day per month dropped off with reduced brood production and adult bee populations. Sensitive colonies (SAT-04, 24) were dead by September, and selected colonies were showing reduced drop rates in October.

 

Figure 4. Cumulative varroa mite population growth measured as in figure 3, by natural drop in 2006

Figure 5.  Comparison of total varroa mite drop in 2005 and 2006 measured by natural drop.

Adult bee populations were also monitored for tracheal mite infestations and varroa mites by alcohol washes on a monthly basis. Tracheal mite infestations and varroa mite levels were determined by the provincial apiculture laboratory in Prince Albert under the direction of John Gruszka. Figure 7 shows Saskatraz hive locations and tracheal mite levels as determined in May (Blue) and October (Red) of 2005. Hive locations bordered by red are breeder selections (SAT-14, 17, 23, 28, 30, 34). All colonies were inoculated with 200 to 300 bees  carrying 60 % tracheal mite infestations in October, 2004.In the spring only 4 colonies tested positive for tracheal mites, the highest being 3% (SAT-33). In the fall one year after infestation 11 tested positive; however, 2 that tested positive in the spring were negative in the fall. The colony showing the highest infestation level in October (SAT-8=14%) came from a Saskatchewan breeder that has never tested positive for tracheal mites, and therefore no selection pressure for resistance.

Figure 8 a to e follows both tracheal and varroa mite infestations for adult bees between May and September 2006, for selected (SAT-14, 17, 23, 28, 30, 31 and 34) and non-selected (SAT-04, 24) colonies. Tracheal mite populations remained relatively low throughout the summer and fall, peaking in non-selected colonies SAT-04 and 24 and SAT-23 in July. No tracheal mite infestations were found in SAT-34, and other selected colonies maintained very low levels. SAT-17 had a Buckfast background and also showed good tracheal mite resistance. All of the selected Saskatraz breeders except SAT-23 showed good to excellent tracheal mite resistance. Danka and Villa, 2003, showed that honey bees resistant to tracheal mites were more responsive in autogrooming behavior when challenged with tracheal mites than sensitive bees. In this study, tracheal mite resistance and suppression of varroa mite population growth were correlated in SAT-28 and 34. Varroa mite populations on adult bees exploded in August, Figure 8d) resulting from large flushes of varroa emerging with hatching bees. Colonies reduced in brood rearing in August resulting in increased adult varroa mite infestation of the adult bee population. The alcohol wash results presented in Figures 8 support those shown for previous natural drop analyses. Colonies showing the lowest levels of adult varroa infestations, best suppressed the varroa population growth rate.

SAT-28 superseded in 2005, and in 2006. A number of SAT-28 daughters are being used as breeders by Saskatchewan bee breeders and we have reselected out crosses of this breeding line in 2007, 2008 and 2009, which show excellent over wintering ability and honey production. On May 23, 2006 John Pedersen and I, while evaluating Saskatraz colonies, noticed this colony showed aggressive behavior towards varroa mites. More than one worker bee was observed to simultaneously attack and bite an adult mite. SAT-34 and a number of her daughters have shown good to excellent suppression of varroa mite population growth; however, the temperament of the SAT-34 line was aggressive and required selection of less aggressive progeny.

Figure 6:  This bar graph shows the average mite drop per day from May until October 2006 for selected SAT-28, 30, 31, 34 and non-selected colonies.

Saskatraz Tracheal mite levels and hive locations

Mite Levels and Hive Locations.png

Figure 7:  Per cent tracheal mite infestations were determined on a monthly basis by sampling 100 bees per colony from May to October. May (blue), October (red) values for each colony at each location are indicated in the upper right hand corners of each hive location. Red squares and stars denote selected colonies.

Figure 8(a)

 

Figure 8(b)

Figure 8(c)

Figure 8(d)

 

 

Figure 8e

Figure 8(a) to (e) show the results of alcohol wash assays on 1 to 2 hundred bees sampled from selected and non-selected colonies between May and September 2006. Both percent tracheal (blue bars) and varroa (red bars) infestations for adult honey bees are shown.

Figure 9(a)

Figure 9(b)

Figure 9(a) and (b) are microscopic ventral images of varroa mites collected by natural drop on Saskatraz test colonies.  Figure 9(a) is an example of an intact mite showing mouth parts and eight legs, in (b) the varroa mite has two severed legs on the right side and is missing mouth parts.  The damage results from honey bees biting varroa mites during autogrooming activities or as aggressive behavior towards the mites.

When assessing varroa mite population growth by natural drop in 2006, 100 mites from each sticky board were sampled and microscopically analyzed every 6 to 7 days for 65 days (late July on) for mites damaged(Figure 9) due to grooming behavior (Figure 10), and for the number of light immature mites present (Figure 11). Figure 10 shows that SAT-28, 30 and 34 damaged more mites than SAT-24 and 06, but the number of mites damaged by SAT-14 and 17 were similar to non-selected colonies. Figures 11a and b show that colonies with high numbers of immature mite drop (SAT-06, 24) showed greater increases in varroa population growth and fewer damaged mites relative to the number of immature mites and total mites in the hive. SAT-34 and 28 (Figure 11c and d), respectively, showed higher numbers of damaged mites and lower numbers of immature mite drop throughout the summer, with damaged mites equaling or exceeding immature mite drop in some cases. SAT-28 and 34 also showed more variability in the number of mites damaged compared to immature mite drop, which may reflect more grooming activity or hygienic behavior depending on environmental conditions throughout the summer. SAT-31 (Figure 11f) showed the next best grooming activity, but similar levels of mite population growth to SAT-30 (Figure 11e). SAT-14 and 17 showed phenotypes for grooming and immature mite drop intermediate to selected and non-selected colonies (Figure 11g and h),respectively.

Hygienic testing of selected Saskatraz colonies (SAT-28, 30, 31, 34) and non-selected lines SAT-04 and 24 were performed in 2006. These results were described in detail previously (Robertson, A. 2007). Briefly, SAT-34 was most hygienic followed by SAT-28. These colonies showed intermediate honey production, but excellent suppression of varroa mite population growth, where as SAT-30 showed intermediate hygienic behavior and excellent honey production.  The most hygienic hives (SAT-28, 34) showed the highest percentage of chewed mites due to grooming behavior and the lowest overall percentage of light mites ( cf Figure 11 c and d).

 

Figure 10:  Samples of varroa mites from Saskatraz test colonies were collected every six to seven days and scored for damaged legs and mouth parts.  Damage to the dorsal shield was not scored as due to grooming activity.  The values plotted are mean plus or minus SEM( Standard Error of the Mean), n=10.

Figure 11(a). SAT-06

 

Figure 11(b) SAT 24

Figure 11(c) SAT 34

 

Figure 11(d) SAT 28

Figure 11(e) SAT 30

 

Figure 11(f) SAT 31

Figure 11(g) SAT 14

 

Figure 11(h) SAT 17

Figure 11 a and b show the number of varroa mites in the hive for non-selected colonies (Figure 11a and b, SAT-06 and 24) and a colony selected for mite tolerance (Figure 11c, SAT-34). Varroa mites were collected by natural drop on sticky boards on a weekly basis for data presented in figures  11a to h, inclusive)

 

An experiment was performed on Saskatraz test colonies to determine if some of the varroa tolerant lines showed phenotypes similar to Apis cerana (Roth, 1999). Apis cerana limits varroa population growth by removing varroa infested worker brood, but allows drone brood to become more infected. Grooming, and hygienic traits (detection, uncapping and removal of varroa infested brood) may help cerana to survive varroa infestations and co-exist with the parasite. Apis cerana also “entombs” varroa infected drone brood.

Preliminary experiments were performed by random sampling (n=4) and counting the number of varroa in worker and drone brood (100 brood cells per sample) over the summer. Saskatraz 34 (Figure 12) shows a high drone brood infestation in August, but suppresses the number of varroa present in worker brood. Some studies with the varroa sensitive hygiene trait (VSH),which refers to bees that detect and remove reproductive varroa from sealed worker brood with high efficiency (Boecking, 2000) are consistent with this observation. In studies of the effects of drone and worker varroa infested brood on the VSH phenotype, Harris (2007) showed that VSH bees removed more worker pupae infected with varroa than drone infected brood.

Higher levels of brood infestation were found in both worker and drone brood in a non-selected colony (SAT-04) in June and July (figure 12). In August there was no drone brood detected in SAT-04, and the worker brood showed over 60% infestation levels. Worker brood infestation levels were less than 9 per cent +/- 4.1 SEM in SAT-34. Table 1 shows the data for 14 Saskatraz test colonies determined throughout the summer. SAT-28 shows a phenotype similar to SAT-34, maintaining low levels of varroa infestation in the worker brood. Both of these colonies showed good suppression of varroa population growth, grooming behavior and hygienic behavior. SAT-14, 17 and 30 maintained low levels of worker brood infestation until late July and August. Soon after drone brood infestation levels increased, drone brood production in the colonies decreased and worker brood varroa levels increased to higher levels (Table 1). Apis ceranae entombs infested drone brood, Apis mellifera does not, and drones that successfully emerge carry high numbers of phoretic mites serving as a source of infection to neighboring colonies, and worker brood within the colony. Table 1 shows that by mid August varroa mite susceptible colonies (SAT-04, 14, 17, 23, 24, 25, 30) were critically infected.

 

Figure 12:  Drone and worker brood was randomly sampled between June and August 2006 to determine if differences exist between colonies in percent varroa mite infestations of worker and drone brood. SAT-34 showed low levels of infestation in worker brood, but high levels in drone brood by August.  A non-selected colony, SAT-04, showed a general increase in worker brood infestation by August.  No drone brood was present in SAT-04 in August. LSD p-05 (least significant difference)

 

The total number of colonies at the Saskatraz yard site from which honey was harvested in 2006 was 49. Colony numbers are constantly changing because of queen failures, failed supersedures and wintering losses. All colonies entered in 2004 have numbers 35 and lower, by August 30, 2006 SAT-72 was the highest recorded entry from which honey was harvested. Colonies requeened or nucs moved into the yardsite were given consecutively higher numbers.

 Table 1: Comparison of % Brood Varroa Mite Infestation in Saskatraz Test Colonies May-to August 2006.

Saskatraz Colony ID

May 29

June 14

July 19

Aug. 16

Brood type

worker

Drone

worker

drone

worker

drone

worker

drone

01

17+/-3

61+/-4.7

04

24+/-4.9

66+/-4.2

17+/-3

74+/-9

55+/-3.7

ND

06

12+/-2.3

33+/-3.4

14

3 +/-1.0

13+/-1.4

0

24+/-2.8

3+/-1.9

37+/-5

29+/-13

36

16

22+/-5.8

32+/-4.9

13+/-1

39+/-5

17

3+/-1.0

6+/-2.0

2+/-0.71

37+/-3.0

9+/-1

21+/-1.9

48+/-2.8

80+/-8.0

23

0

ND

5+/-1.9

56+/-8.1

37+/-2.8

77+/-9.8

24

16+/-2.8

53+/-9.4

34+/-3.8

ND

25

14+/-1.2

54+/-4.8

82+/-2.6

96

26

23+/-9

79+/-9.5

28

0

23+/-1.9

5+/-2.5

30+/-2.6

9+/-5.3

ND

30

2.7+/-1.2

17+/-2.4

6+/-2.6

41+/-4

7+/-3

65+/-2.5

84+/-7.1

ND

31

51+/-20

ND

34

1+/-1.0

12+/-2.8

1+/-1

6+/-3.5

9+/-4.1

95

Sample size = 100
Values reported are mean =+/- SEM; n=4
ND = no drone brood present
∆ = No Data

Close population mating of virgin queens from 2005 and 2006 selections (SAT-14, 17, 23, 28, 30, 34) were made by backcrossing at Saskatraz after June 19, 2006. The objective of these crosses was to mate the best queen selections with drones from colonies best tolerating high varroa infestations, thereby enriching for varroa tolerant phenotypes in the original selections. Varroa preferentially infest drone brood putting a high selection pressure on drones from varroa sensitive colonies. Table 1 shows the most varroa tolerant colonies had the lowest drone infestation levels, and would be expected to produce the largest number of drones fit for mating with virgin queens from selected colonies. This should be an effective method for combining both drones and queens with the best varroa tolerance. However, this proved to be difficult, with poor mating success and frequent supersedures. Out of 60 mating nucs only 24 queens successfully mated and established. Extensive analyses of progeny from these close population mated queens is still in progress. This natural selection method produced not only colonies with improved mite tolerance, but with superior honey production.SAT-61,63,65,87 and 98 show excellent honey production and good varroa mite suppression (Figure 13 and 14). SAT-65, 84 and 93 showed excellent varroa suppression, with SAT-84 having a VSH (Varroa Sensitive Hygiene) phenotype, but less than average honey production. SAT-65 produced excellent honey yields and good varroa tolerance.

On May 18, 2007 inspection of the Saskatraz yard site revealed that all of the colonies but seven died between November 2006 and May 2007. The surviving colonies consisted of a selection SAT-65, provided as a queen by a collaborating queen breeder and established at Saskatraz on July 10, 2006. SAT-65 swarmed during the summer, and a daughter mated at Saskatraz re-established the colony. The remaining survivors were nucleus’s made up at Saskatraz in 2006. Extensive post mortem analyses are still in progress. We recently partnered with VIDO to perform virus and microsporidia analyses on colonies dying or collapsing because of varroa infestations. The results of these studies will be reported when complete. We visually assessed the dead colonies when the yard was first opened and found hives full of pollen and honey with no evidence of dead bees. Last inspection of the yard on November 10, 2006 indicated most colonies; particularly the selected breeders had good wintering populations. We found no evidence of dysentery, starvation, nosema or queen failures. Only a few colonies had significant numbers of dead bees, these colonies likely resulted from failed queens or failed supersedures.

On June 7, 2007 all remaining colonies were treated with Apistan to normalize varroa mite populations. Saskatraz stock outcrossed to Meadow Ridge apiaries in 2006, to maintain the Saskatraz gene pool, were reselected in 2007 for further testing at the Saskatraz natural selection yard site. Back crosses to Russian stock and closed population mated Saskatraz daughters, as well as new selections were added in 2007. The Saskatraz apiary produced an average of 200lbs per colony in 2007.

Figure 13:  Total honey production per colony in 2008. SAT-63, 65, 94, 96 and 98 all produced over 400 lbs.

During the fall of 2008 varroa mite populations were closely monitored at the Saskatraz yard site by analyzing adult honey bee populations (alcohol washes), natural drop (Figure 14) and brood infestation levels. SAT-65, 84, 88, 96, and 98 produced economical honey yields (Figure 13) and showed the best suppression of varroa mite population growth (Figure 14). Some colonies showed no detectable varroa, but no significant honey production (SAT-82). The percent varroa infestation in Saskatraz worker brood was assayed in detail for SAT-63, 65, 84, 86, 87, 88, 90, 91, 94 and 96 (Figure 15).

Figure 14:  Total varroa drop per week for Saskatraz test colonies in 2008. SAT -65, 82, 84 and 93 showed the lowest drop per week from May 28 to Sept 30.

Figure 15:  One hundred brood cells were randomly sampled for varroa infestation from Saskatraz test colonies on September 16, 2008. Error bars are SEM, n=5. Red bars show colonies showing visual signs of virus; Deformed Wing Virus, (DFW) or testing positive for Israeli Acute Paralytic Virus (IAPV) by RT-PCR (Figure 18). No virus was detected in SAT-65, 84 or 93.

SAT-65, 84 and 93 showed the lowest percent varroa infestation in worker brood. Varroa reproduction per brood cell was also analyzed in order to select for colonies which suppress varroa mite reproduction (SMR), now called VSH or Varroa Sensitive Hygiene (Figure 16). Eleven colonies (100 cells per colony) were randomly sampled (SAT- 63, 65, 84, 86, 87, 88, 90, 91, 94, 96) and assayed for the number of varroa per cell. SAT-65 and 84 showed the lowest levels of varroa per cell. SAT-84 showed the lowest level of infestation with a mean number of 2 varroa per cell. This phenotype fits the definition of a VSH phenotype. This phenotype removes the most reproductive varroa mites from worker brood cells, leaving only brood cells with low numbers of varroa mites. This results in the overall suppression of varroa mite population growth. It is thought that the mites with low reproductive rates may undergo delayed oviposition, but it is not known whether this is due to the pupae parasitized or the varroa mite (Jeff Harris, 2010. Orlando meetings).

Figure 16:  Brood comb from all Saskatraz colonies was randomly sampled for varroa infestation and the number of varroa per cell determined by stereo microscope analyses. One hundred cells were analyzed per colony. Mean values are plotted, error bars are SEM.

Pre-emergent pupae infected and not infected with varroa were sampled for both morphological (phase contrast microscopy) and molecular analyses (RT-PCR for viruses). Pre-emergent pupae that were heavily infected with varroa (8 mites/cell) showed morphological (Figure 17) anomalies (deformed wings, extruded proboscis, deformed and atrophied abdomens). Ten Saskatraz selections with levels of varroa infestation between 15 and 90% were screened for the expression of IAPV (Israeli Acute Paralytic Virus) and Deformed Wing Virus (DFW) by RT-PCR at GenServe Labs, SRC (Figure 18). Some critical observations were made. Colonies showing visual symptoms (DFW) of virus infections (SAT-63, 90, 91, 94, 96) had high levels of varroa infestation (65 to 95%) and tested positive by RT-PCR for IAPV and DFW virus infections. However, 2 colonies (SAT-65, and 84), showed no virus symptoms or expression of viruses by RT-PCR. Figure 18.

Figure 17:  Picture of a pre-emergent pupae (SAT-94) removed from a brood cell infested with 8 varroa mites.

IAPV and DWV Bee Viral Screening Image Cropped.jpg

Figure 18:  Screening of pre-emergent pupae from varroa tolerant (SAT-65, 84) and sensitive (SAT -90, 91) breeding lines for IAPV using RT-PCR (Bruce Mann, SRC).

 

Figure 18 also shows IAPV screening of adult bees preserved in alcohol, from samples of Africanized bees from the Yucatan area of Mexico, Apis ceranae (unknown source) and attendants from queens shipped from southern Chile, Hawaii (Kona), Australia and California (Strachan). A trace of IAPV was found in an attendant from Chile, while all others showed no detectable viruses. SAT-86 pupae parasitized with varroa tested positive, where as pupae not infected showed no detectable virus. Both pupae parasitized with varroa and not parasitized showed IAPV infection in SAT -90 and 91. Y. varroa is a sample of varroa mites from a producer who experienced some high colony varroa levels in 2007. Varroa feces collected from SAT-90 brood comb also showed a strong signal for IAPV infection. This finding indicates that the potential exists for brood comb to be carrying viruses for infecting subsequent brood production. Experiments are in progress to determine the infectivity of virus particles in varroa feces. Varroa samples collected from Ontario (Alison Skinner) in 2001 and 2004 showed the presence of IAPV virus in 2004, but not 2001.

In the fall of 2008 the varroa levels increased to critical levels (both in brood and on adult bees) in most colonies, and an attempt to rescue these colonies was made by treating with organic acids (Figure 19). Formic acid treatment did not increase the varroa drop rate except in SAT-96 between September 30 and October 17. A slight increase was noted in SAT-65, 61 and 86 between October 17 and 25. After oxalic vapor treatment on October 25 varroa drop rates increased most dramatically in SAT-61. There was an overall increase in the mean drop rate for all colonies showing the effectiveness of oxalic treatment.

In the spring of 2009 only three colonies were found to survive the winter SAT-85, 88 and 96. These colonies were established at Saskatraz in 2007, and showed lower levels of mite drop in the fall of 2008, but significant virus infections. Scanning electron microscopy showed considerable damage to surface hair on the bees from colonies treated with oxalic vapor compared to untreated colonies (data not shown). Formic and oxalic treatments may have imposed considerable stress on the bees in the fall contributing to their death. SAT- 65, and 84 both died even though varroa mite (Figure 19) and virus levels (Figure 18) were low. The fall treatment protocol was not successful and may have resulted in high mortality rates. The Saskatraz yard site was restocked during July of 2009, with reselected colonies managed under standard commercial procedures. All colonies received three formic acid treatments (mite wipe pads) and Apistan during the spring of 2009. SAT- 85, 88 and 96 did not receive any treatments except with organic acids as described in Figure 19, in the fall of 2008.

SAT-110, 113, 121, 125, 126 all produced over 370 lbs in 2009 (Figure 20). SAT-125 was a daughter of SAT 30, out crossed in 2006 and reselected at Meadow Ridge in the spring of 2009. This colony was selected for high honey production in 2006.

Figure 19:  Cumulative varroa drop at Saskatraz apiary between May 28 and November 23, 2008. Vertical black bars show times (September 30 and October 17) of treatment with formic acid (mite wipe pads-60% formic acid), followed by oxalic vapor (O) on October 25, 2009. Oxalic vapor treatment was performed by Calvin Parsons.

Figure 20:  Total honey production per colony in 2009. SAT-125 and 126 produced the most honey per colony in 2009. The survivors SAT- 85, 88 and 96 produced 211, 142 and 112 lbs respectively

Figure 21:  Survivor colonies SAT- 85, 88 and 96 showed total varroa mite counts of 1252, 540 and 544 respectively. Two colonies provided by a collaborating queen breeder for testing SAT-109 and 126 showed varroa mite levels increase to 4970 and 3482 respectively.

The surviving colonies SAT 85, 88 and 96 produced 211,142 and 112 lbs of honey respectively, all below the yard average (254lbs). When first inspected in the May 2009 these colonies were very weak averaging about 3 partial frames of bees and small patches of brood. Varroa mites were visible on the adult bees and deformed wings were noted. These colonies all showed some virus infections (DWV, deformed wing virus) in the fall of 2008 and significant levels of varroa mites in the brood (Figure 15).They were not treated for varroa mites, worked down to a single box, fed  and expected to die. On July 3, 2009 the colonies had recovered, being full of brood and bees with bees hanging out the entrances. On inspection no visible mites were observed. The data from the spring of 2009 is difficult to interpret because of the organic acid treatments made in the fall of 2008, which may have imposed other complicating stresses on the Saskatraz colonies. A detailed post mortem analyses was performed on the dead colonies in the spring of 2009, (unpublished data) indicating severe loss of the hairs on worker bee external surfaces. Visual damage to the bees antennae was also observed in scanning electron microscope studies. These damages could result in loss of thermal protection, water balance and sensory mechanisms The Saskatraz survivors showed excellent tolerance to all these stresses and were able to make a strong recovery.

Although low levels of varroa mites were present in all colonies reselected for testing at  Saskatraz, and varroa populations  were normalized by spring treatments with Apistan and formic acid, two colonies  SAT-109  and 126 showed high varroa population growth rates. The queens heading these colonies were provided by a collaborating queen breeder in 2007 and reselected for Saskatraz in 2009. The colonies showed excellent honey production, but were sensitive to varroa. This line was traced back to a Russian release (yellow) made in 2004, which should have had some tolerance to varroa. The colonies from which the test lines were selected were managed with chemical miticide treatments up until June 2009. Most of the other colonies were daughters of Saskatraz breeders out crossed between 2006 and 2008 at Meadow Ridge and subjected to recurrent selection prior to re-introduction into Saskatraz. These colonies were also managed under commercial conditions, but with different miticides treatments. This data suggests that certain chemical miticide treatments and removing varroa infestations (selection pressure) results in decreased varroa tolerance over time. This requires further investigation and return of varroa tolerant selections released since 2006 for retesting at the Saskatraz natural selection site.

COMPARISON OF SASKATRAZ FAMILY HONEY YIELDS TO PROVINCIAL AND MEADOW RIDGE YIELDS

Figure 22 compares the mean honey production in Saskatchewan over the last 20 years to that of Saskatraz over the last 5 years. Although location effects are not accounted for the Saskatraz apiary out yielded the provincial averages every year, since 2005 without chemical miticide treatments. During years where varroa mite levels reached critical levels in the fall of the year (2006 and 2008), honey production continued to exceeded provincial averages.

Figure 23 shows the results of comparing Saskatraz honey production as a percent of provincial and Meadow Ridge values. Comparing to Meadow Ridge values helps assess management effects, and climatic effects within a 100 mile radius. The trend has been for Saskatraz production to move closer to Meadow Ridge values, even though Saskatraz colonies are not treated with chemical miticides. This effect can be explained by considering the effects of recurrent selection for honey production. In 2005 honey production was only 65 percent of Meadow Ridge; however, outcrossing colonies selected for honey production and reselecting for wintering ability and honey production should serve to further enrich for genes involved in these multi-genetic traits. This process not only maintains the majority of the gene pool originally selected from Saskatraz, but maintains genetic diversity, which has significant effects on hive performance (Matilla and Seeley, 2007). The mean honey production is now similar for Saskatraz and Meadow Ridge, and is showing mean Saskatraz honey production as 30% more than the provincial average.

Figure 22:  Honey production in Saskatchewan between 1989 and 2009 (source Saskatchewan Agriculture) reported as average production per year from approximately 63,000 colonies. The 20 year mean plus or minus SEM is shown. The average honey production per year at Saskatraz represents values calculated from colony numbers which varied between 25 and 35. The mean represents honey production between 2005 and 2009 plus or minus SEM.

Figure 23:  Shows the average honey production at Saskatraz per year as a percent of that for provincial and Meadow Ridge averages.

Figure 24 shows the results of honey production data collected from selected Saskatraz colonies and reselected daughters from out crosses of these original breeders. The honey production of selected Saskatraz families is compared as a percentage of provincial and Meadow Ridge averages. The Saskatraz colonies and their daughters selected for honey production (SAT-14, 17 and 30, 94, 86 and 125) consistently produced 40 to 60% more than the provincial average and 20 to 40% more than Meadow Ridge. Saskatraz colonies  originally selected for both  varroa tolerance and honey production and reselected daughters from them (SAT-28, 34, 85, 87, 93, 96, 112) consistently produced from 30 to 40% more honey than provincial  averages, but did not show any increased production when compared to Meadow Ridge values. This implies that increased varroa tolerance comes at a cost of reduced honey production. A 30% increase in honey production with a current average value of approximately $30 million per year in Saskatchewan, would translate into a considerable economic impact (9 million dollars per year).

The initial concept of establishing a large diverse gene pool at Saskatraz, selecting the best colonies for honey production and varroa tolerance, using natural selection, out crossing and reselecting progeny to maintain and improve the selected gene pool and to maintain genetic diversity has proven successful. Improved honey production, wintering ability and varroa tolerance improves economic benefits for bee keepers. Healthy colonies produce economic honey crops and provide improved pollination efficiency. However, this approach to bee breeding requires a large colony base and millions of dollars of infrastructure and high labor costs. The project was designed initially using selections from 14 queen breeders who had been overwintering honey bees in Saskatchewan for many years. These colonies were selected for overwintering ability, honey production and resistance to brood diseases (chalkbrood). None of these colonies showed significant tolerance or resistance to varroa mites, but some showed good tracheal mite resistance. The best varroa mite tolerance was introduced through the introduction of Russian stock in a joint venture with the Ontario Beekeepers Association, and by obtaining German stock (drone semen) from Dr. Ralph Buchler, Kirchhain, Germany. Semen importation was made possible by the efforts of Yves Garez, a Saskatchewan queen breeder. The selection process could be improved significantly by identifying biomarkers to assist selection of important traits.

Figure 24:  Saskatraz families selected for honey production (SAT-14, 17 and 30) and varroa tolerance (SAT-28 and 34) compared as a percent of provincial and Meadow Ridge production.


IDENTIFICATION OF MOLECULAR MARKERS FOR MONITORING GENETIC DIVERSITY AND CHARACTERIZING IMPORTANT PHENOTYPES.

 

Microsatellite marker analyses has been carried out in collaboration with Dr. Yves Plante and Bruce Mann, GenServe Laboratories, Saskatchewan Research council on a contract basis since 2003. Only data relevant to the current report is described here. Detailed methods and results  showing identification of 20 informative microsatellite markers for genotyping different honey bee populations will be published elsewhere

Figure 25:  A three dimensional plot showing the grouping of 5 different honey bee populations using 20 informative markers.

Figure 25 shows the results of screening 5 different sets of drones collected from sources of stock selected for introduction into the Saskatraz natural selection yard site. The statistical programs used for generating these plots were provided by Dr. Yves Plante, some of which can be accessed at http://bioinformatics:psb.ugent.be/psb/Userman/treecon. The plot clearly shows the genetic diversity between the different populations. The unknown samples represent hybrids generated by Russian Canadian crosses, and the New Zealand bee samples clustered between the Russian and Canadian samples.

The 20 informative markers were also used to genotype Saskatraz breeding lines Figure 26. Drones collected from each of 14 Saskatraz breeding lines. SAT-28, 34, 65, 84 and 96 all showed some degree of varroa tolerance, but no grouping similarities. Saskatraz selections sensitive to varroa population growth (SAT-04, 24, and 90) grouped closer together, but close to some lines showing varroa tolerance, such as SAT 84. This study indicates more markers are required to increase the resolution required to discriminate between different phenotypes, or higher resolution methods are required. However, the 20 informative markers can be useful to identify individual Saskatraz breeding lines. Figure 27 shows the map and linkage group location of each of the 20 informative markers. The map (Figure 27) was constructed using information at the www.ncbi.nim.nih.gov website and microsatellite sequences.

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Figure 26:  Genetic relatedness (genotyping) between Saskatraz breeding lines.

Figure 27:  The 20 informative microsatellite markers used to identify different honey bee populations were mapped using sequencing information from the honey bee genome project.

 

Current research activities involve progeny analyses of out crossed and re- selected Saskatraz breeding lines for grooming behavior, morphological characteristics, hygienic behavior, VSH phenotypes, molecular marker analyses (microsatellites, microarrays, kinome arrays) and testing Saskatraz breeding lines for susceptibility to virus infections. Mohammad Mostajeran, a research associate on the project is working on morphometrics, grooming behavior and VSH phenotypes and we are collaborating with VIDO (Dr.Philip Griebel and Wayne Connor) and the University of Saskatchewan, Food and Bioproducts (Dr. Xiao Qui and students) on virus immunity and microarrays, respectively. Extensive data has been collected in 2008 and 2009 on progeny analyses and will be described in future newsletters.


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