مؤتمر الجمعية العربية لتربية النحل األول 6-5 فبراير 2018 م مركز أبوظبي الوطني للمعارض - دوله اإلمارات العربية المتحدة

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1 The Project Objective: To develop productive, gentle honeybees with tolernce to mites nd brood diseses By: Albert J. Robertson The Ssktchewn Honeybee Breeding nd Selection Progrm 1 2

2 Current Honeybee Helth Issues Vrro Viruses Miticides Pesticides Nutrition 3 Outline Review of Ssktrz Breeding Ssktrz Hybrid Project Selection nd Moleculr Anlysis of Extreme nd Intermedite Phenotypes for Vrro Resistnce nd Susceptibility - Biomrker Development (Micro nd Kinome Arrys) nd Virus Screening Proteomic nd PCR nlyses for Pthogen Identifiction Mngement of Vrro Popultions by Selective Tretment Strtegies Using Vrro Tolernt nd Susceptible Phenotypes Screening of Hive Products nd Honeybees for Miticides (Toxicology Collbortion, U of S) Efficcy of Combined Tretments of Synthetic nd Orgnic Miticides Plese find Published Ppers nd More Informtion on the Ssktrz Project t 4

3 Current Objectives 1. To propgte, mintin nd improve (stbilize) productive nd vrro tolernt phenotypes (17 Ssktrz fmilies estblished), using re-current nturl selection nd newly discovered biomrker tools. 2. To continue identifiction nd chrcteriztion of key genes (genomic nlyses, microstellites, micro rrys), nd proteins (SDS-PAGE-LC-ESI-MS) nd enzymes (kinome rrys) involved in signl trnsduction mechnisms. 3. Investigtion of pesticide residues (OP-NI) in hive products nd bees. 4. Evlution of spring nd fll miticide tretments both lone nd in different combintions to improve efficcy. Determine rtes of vrro popultion regrowth fter miticide tretments in different Ssktrz breeding lines, including hybrids. 5 Ssktrz Breeding Progrm Primry Selection Criteri: 1. Honey Production 2. Wintering Ability 3. Mite Resistnce nd Suppression 4. Resistnce to Brood Diseses (chlk brood etc.) 5. Viruses nd Nosem Susceptibility Breeding methods used to select nd enrich for importnt trits (nturl selection, out crossing, bck crossing, recurrent selection, progeny nlyses nd closed popultion mting). 6

4 7 SK & MB 2004 Ssktrz Breeding Progrm Logistics Cndin Selections Bck Cross Selections MR 1992 Ssktrz Nturl Selection (No Miticides) Recurrent Selection Russin Selections B.C. Selected Drone Mothers (Diverse fmilies with desired trits) Outcross Survivors (50 Apiries) 8 See for review

5 Phoretic Mite Level (MHB) Honey Production (lbs) The Originl Ssktrz Apiry 9 Nturl Selection for Vrro Tolernce Binsville Phoretic Mite Levels nd Honey Production Dt TWR BV PRD BV BV TWR BV MRV BV NA HRP PRD MRT BV HRP HRP BHCH S96 S88 S28 G44 BHCH G50 SL25 S136 SY26 UP G8 SY26 SY26 S145 S April 4, 2017 Phoretic Mites September 15, 2017 Phoretic Mites Honey Production Colony Men Phoretic Mites Yrd Men Phoretic Mites Yrd Honey Production Men 0

6 Phoretic Mite Level (MHB) Honey Production (lbs) Phoretic Mite Level (MHB) Honey Production (lbs) Nturl Selection for Vrro Tolernce 2017 Mrtin s Phoretic Mite Levels nd Honey Production Dt ?? MRT MRT BV MRK BV PRD PRD BV BV TWR BV BV PRD PRD S14 SY26 UP UP S113 S113 S96 G44 S136 S14 SK WAG G44 G44 G44 S28 G44 XX 17 XX April 3, 2017 Phoretic Mites September 7, 2017 Phoretic Mites Honey Production Colony Men Phoretic Mites Yrd Men Phoretic Mites Yrd Honey Production Men Colony Selection for Honey Production nd Wintering Closed Pop. Mting Priddy's Phoretic Mite Levels nd Honey Production Dt CHR GRT SBD CAL PRD PRD RCH BC RDG - - EBD TWR - BC BC G50 S136 S96 S136 HY S93 G20 Ssk-X S28 G8 S96 S28 G50 S93 UP S96 S April 3, 2017 Phoretic Mites September 14, 2017 Phoretic Mites Honey Production Colony Men Phoretic Mites Yrd Men Phoretic Mites Yrd Honey Production Men

7 Phoretic Mite Level (MHB) Honey Production (lbs) Ssktrz Hybrid Yrd for Closed Popultion Mting Drone Mothers Tower Phoretic Mite Levels nd Honey Production Dt S14-15 G44-15 S96-15 Y26x26 FC Y26x26 FC S14-15 S28-15 BC Y26x26 FC S96-15 BC S14-15 BC S28-15 BC G50-15 Pitt S14-15 BC S96-15 BC S96-15 BC April 16, 2016 Phoretic Mites August 19, 2016 Phoretic Mites Honey Production Colony Men Phoretic Mites Yrd Men Phoretic Mites Yrd Honey Production Men S65-15 BF Slide from USDA Bton Rouge. 14

8 Honey Production (lbs) Phoretic Mite Level (MHB) Slide from USDA Bton Rouge. 15 Effects of Adding VSH Trits to Nturlly Selected Lines N=15 N=15 N=15 N=15 N=15 N=15 N=6 N=15 N=11 N=2 N=15 N=10 G8 G11 G18 G20 G44 G50 St-X VSH Crosses Men Ssk-X S96 Ssktrz Men DP22 Apiry Men Honey Production July 2nd, 2013 Phoretic Mites Oct 1st, 2013 Phoretic Mites Men Phoretic Mites during 2013

9 Number of spores per bee Vrro Infesttion (%) Progeny Anlyses S Adult Bee % Vrro Infesttion for Eight S113 Dughters S113-1 S113-2 S113-3 S113-4 S113-5 S113-6 S113-7 S113-8 SM113 Colony ID 17 Nov.15/10 Apr.21/ Nosem Spore counts in live bees from S88 fmily S88-1 S88-2 S88-3 S88-4 S88-5 S88-6 S88-7 S88-8 Men Dec Jn Jn Feb Feb Colony ID

10 Percentge of Men - Where 0 is Men Percentge of Men - Where 0 is Men 150% Anlysis of Individul Ssktrz Fmilies for Phoretic Mite Levels nd Honey Production from 2015 to % 50% 0% -50% -100% SC11 G11 HY G34 G44 G50 G8 S136 S14 S28 S30 S65 S96 SASK-X SL25 SY26 UP HP N=6 N=5 N=24 N=11 N=9 N=13 N=11 N=18 N=22 N=11 N=11 N=35 N=12 N=27 N=27 N=33 Fll N=10 N=10 N=33 N=11 N=9 N=12 N=11 N=20 N=24 N=10 N=13 N=36 N=10 N=26 N=29 N=38 Spring N=11 N=10 N=33 N=11 N=6 N=12 N=9 N=20 N=24 N=9 N=13 N=34 N=10 N=27 N=26 N=34 Spring Smple Period (MHB) - First Smple Tken Ech Yer Yerly Honey Production Fll Smple Period (MHB) - Smpled Lte August, Erly September Ssktrz breeding lines were scored for spring nd fll phoretic mites nd honey production t 17 different piries between 2015 nd The men honey production nd phoretic mites were clculted for ech piry. 19 Individul fmilies were then scored ccording to percent of the yrd men for ech prmeter. Ssktrz SKHY UP Anlysis 60% Line Anlysis of Ssktrz Colonies from 2015 to % 40% 30% 20% 10% 0% -10% -20% -30% -40% Ssktrz Ssktrz Hybrid UP Ssktrz Honey N=262 N=40 N=33 Fll N=273 N=45 N=38 Spring N=264 N=43 N=34 Men Spring Smple Period (MHB) - First Smple Tken Ech Yer Men Fll Smple Period (MHB) - Smpled Lte August, Erly September Men Yerly Honey Production 20

11 Ssktrz Hybrid Project Introduction To commercilize nd distribute the breeding stock to the end users. Every yer colonies re selected for honey production, overwintering bility, temperment, mite resistnce nd brood diseses. In 2017 we sent 130 queens to be reselected in Mrch The Cliforni Tech Trnsfer Tem, Bee informed Prtnership hs independently evluted our Ssktrz breeding stock in lte Februry erly Mrch. Colony Number 7 Colony ID S65 Robin 14 Brood Pttern Chlkbrood Presence (+/-) Temper -ment Pollen plcement Queen Presence (+/-) Queen Mrk Presence (+/-) Phoretic Mite Infesttion (MHB) %Mite Infesttion in Worker Brood %Mite Infesttion in Drone Brood Excellent - 1 Averge Tech Tem Hygienic Behviour Test 93%O / 80%R Observtion Green mrk on queen Y26 x 26 Mrtin 14 Good - 1 Averge %O / 99%R Y26 x 26 Mrtin 14 Excellent - 1 Averge %O / 100%R No drone brood; no visible mrk on queen - 37 G44 JHN 12-9 B.V. 14 Excellent - 1 Averge %O / 75%R 21 No Drone Cliforni Tech Trnsfer Tem Evlution included hygienic testing (uncpped, removed), colony strength (frmes of brood), brood pttern (1-poor to 5-best), queen sttus, temperment (1- best to 5- poor), color, vrro infesttion (Mites per Hundred Bees) nd nosem spore count. % Uncpped 22

12 23 24

13 S96 RCD-14 (Hygienic Behvior; 89%U+70%R) 25 Y26x26 Mrtins (Hygienic Behvior; 100%U+100%R) 26

14 S65 SW 09 (A), S65 ROB 14 (B), S28 PRD 14 (C), S28 MRT 14 (D), S28 MRT 14 (E), 27 S146 GP (F) Bee Hygienic Behviour Recpping Cells 28

15 #47-S96 CHR Benefits of the Ssktrz Hybrid Project Ssktrz hybrids re vluble source of drones. Useful for distributing lleles for selected trits (honey production, overwintering, vrro resistnce, brood disese) mongst breeding popultion. 30

16 Length of Survivl (Months) Biomrker Development Microstellites (SNP Discovery) Microrrys (trnscripts) Proteins Kinome Arrys (signl trnsduction) (DNA) (RNA) (Protein) (Signl Trnsduction) Phoretic Mite Level (MHB) 0 S88 S23A S14 Sy26 S85-09 S84C-4 S23A-3 JH-8-10 S S65 SAT-1 S88-4 G50-3 G4 Colony survivl length Mites per hundred bees t smpling time Mites per hundred bees during smpling yers 0 The colony survivl time nd vrro mite infesttion of selected honey bee colonies. 32 The survivl time (green br) is presented in months, nd the Vrro mite infesttion (blue brs) is presented in Mites per Hundred Bees (MHB). The light blue br shows the vrro infesttion rte t the smpling time. The drk blue br shows the vrro infesttion rte in the smpling yers (men±sem). A colony with single str is still live.

17 Ctegory Gene S88- /G4- Signl Trnsduction (Pup) S88+/ G4+ Honey Bee Protein GB17702-RA 2.40 Cdherin-87A-like DB Neurobechin-like GB14355-RA Anosmin-1-like GB11723-RA 6.88 Apolipoprotein D-like isoform 2 Lipids (Pup) Cytochrome P450 (Pup) GB18070-RA 2.23 Acyl-CoA Delt(11) desturse-like GB13246-RA 0.47 Phospholipse A1 member A-like isoform 1 GB Esterse E4-like GB11754-RA 0.31 Cytochrome P isoform 1 GB12136-RA 4.08 Cytochrome P450 6A1 Immune (Pup) GB13473-RA 2.07 Apidecins type Reltive gene expression of Esterse E4 trnscripts in different honeybee tissues Hed Thorx Abdomen G4 w G4 wo Susceptible phenotype G Hed Thorx Abdomen S88 w S88 wo Tolernt phenotype S88 34

18 Dr Xio Qiu nd Snjie Jing Food nd Bio products, University of Ssktchewn 35 Differentil expression nlyses of Esterse E4 in wide rnge of colony phenotypes Reltive expression levels P<0.05 with mites without mites b b G4 S88-4 S65 SAT-1 JH-8-10 b b S G50-3 S88 S23A b b b S84C-4 S14 S85-09 S23A-3 b Sy26 G8 b S86C-1 S86E 36

19 Kinome Anlysis of Colony Phenotypes nd Honey Bee Immune Responses There is growing pprecition tht cellulr kinse ctivity (the kinome) is one of the most informtive levels to chrcterize host. Provides informtion on signl trnsduction. Informtion on how different honey bee phenotypes respond to vrro, viruses nd other pthogens Printing nd Vlidtion of the Bee Specific Peptide Arry. A) The rrys were printed by commercil prtner (JPT Technologies). For ech rry ech spot is printed in triplicte within ech block. Ech block is then printed in triplicte for nine technicl repets of ech peptide. This imge, tken s qulity control step in rry production, illustrtes the consistency nd reproducibility to peptide spotting. B) An imge of dt scn of representtive rry tht hd been used for nlysis of 38 whole bee smple. All of the rrys of this work were of comprble qulity with respect to the clrity nd consistency of peptide phosphoryltion. A cler nd consistent pttern of extents of peptide phosphoryltion is pprent cross the three printed blocks.

20 Clustering of Kinome Dt. Kinome dtsets were subjected to hierrchicl clustering nd PCA nlysis. Pupe from two colonies (G4 nd S88) were selected for either the presence (+) or bsence (-) of Vrro mites. A) Het Mp Clustering Averge Linkge + (1 - Person Correltion) ws used for clustering both the bee-tretments (columns) nd the peptides (rows). Ech column represents the kinome ctivity of individul pupe (n=3/tretment). Cluster nlysis segregted kinome profiles first by colony phenotype (S88: Tolernt; G4: susceptible) nd then segregted G4 lrve by response 39 to Vrro infection. B) Principle Component Anlysis: Seprtion of the smples on the bsis of phenotype is clerly observed with further distinction with the susceptible, but not tolernt, smples on the bsis of infection sttus. Tble 1: Peptides with Differentil Phosphoryltion between Vrro Mite Susceptible nd Resistnt Honeybees. Accession # P Accession # p Accession # p Accession # p P P Q P Q Q P P P Q9Y2H P P P Q Q P O Q P O Q P Q9UPZ P O P Q8WUM Q P Q P Q Q9P0L P Q920L O P P Q P P P Q P Q6P9R O P P P P Q9NQU P P Q Using n expnded honeybee specific peptide rry developed for this project kinome nlysis ws performed on whole orgnism smples representing bees (n=3) representing rnge of susceptibilities to vrro mite infesttion. From most susceptible to most resistnt these phenotypes were G4, S88-1, S651B6 (susceptible), S9612 nd S65S1 (Intermedite susceptibility), nd GS14, S23A, S88 (resistnt). T-tests were performed to identify peptides with differentil ptterns of phosphoryltion between the phenotypes. A totl of 54 peptides were identified which demonstrted consistent differentil (p<0.05) phosphoryltion between the bees of the three susceptible 40 phenotypes vs bees of the three resistnt phenotypes [Tble 1].

21 Clustering of the kinome profiles of bees of different phenotypes t different developmentl stges. 41 Principl component nlysis. The first three principl components re shown. The points re s follows: resistnt phenotypes (red) nd susceptible phenotypes (green). 42

22 Adult Bee Vrro (%) Effects of Miticides on Vrro Popultion Growth Men Percent Vrro Infesttion (%) on Adult Bees t 8 Different Apiries nd SAT 88 (2010). Vrro% My2010 Vrro% Jun.2010 Vrro% Oct.2010 Not Treted 41. Treted with Apivr Apiry 44

23 Reltive Expression of Trnscripts in Response to Miticide Tretments SY26 S14 JH-8-10 Reltive expression level of trnscripts from two selected biomrker genes, AmApoD nd AmCYP6A1 in pupe bdomens from two vrro tolernt (SY26 nd S14) nd one susceptible (JH-8-10) honey bee colony in response to Apistn, Apivr, nd Thymovr miticide tretments. Reltive gene expression (men±sem, N=3) ws normlized by the expression of internl reference genes (ctin nd RpS5), nd error brs indicte the expression vribility of ech gene. A. SY26; B. S14; C. JH Significntly different (p<0.05) vlues re depicted by different letters bove columns. The tretment comprisons used the LSD (lest significnt 45 difference) method for difference nlysis of ech gene. DWV in Hed Tissue w/ nd w/o Vrro Mites DWV in Hed Tissue in Miticide Experiment DWV in Abdomen Tissue in Miticide Experiment Quntittive mesurements of DWV in two vrro tolernt (SY26 nd S14) nd one susceptible (JH-8-10) colony in response to vrro mite infesttion nd miticide tretments. y xis: Ct vlues for DWV detection (men± SEM, N=3); x xis: three colonies (SY26, S14 nd JH-8-10). A. DWV in the hed with nd without vrro mite; B. DWV in the hed with nd without miticide tretments; C. DWV in the bdomen with nd without miticide tretments. Notes: Dt points followed by different letters re significntly different (p<0.05). The multi-tretment comprisons of Ct vlues used the LSD (lest significnt difference) method46for difference nlysis.

24 Ct vlue of DWV detection Quntittive mesurement of DWV infection in hed tissues in three different colonies in response to the vrro mite infesttion Honeybee Colony ID Ct Vlue of DWV in noninfested bees Ct Vlue of DWV in miteinfested bees Fold chnge of DWV (infested/non-infested) Y ± ±0.22 b S ± ±0.35 b 2, JH ± ±0.70 b 3,430, Quntittive mesurements of DWV infection in hed tissues in three different colonies treted with Apivr, Apistn nd thymol miticides Honeybee Colony ID Non-treted bees Apivrtreted bees Apistntreted bees Thymoltreted bees Fold chnge (Apivrtreted/nontreted) Fold chnge (Apistntreted/nontreted) Fold chnge (Thymoltreted/nontreted) Y ± ± ± ± S ± ±0.22 b 19.11±0.25 c 22.66±0.14 d JH ± ±0.49 b 9.19±2.60 c 21.50±0.29 d 1,176, , DWV Anlysis of Ssktrz Phenotypes with nd without Mites DWV Infection in Honeybee Hed Tissue * without mites with mites * * * * * * * * 5 0 S88 S23A S14 Sy26 S85-09 S84C-4 S23A-3 JH-8-10 S S65 SAT-1 S88-4 G50-3 G4 48

25 Proteomic nd PCR nlyses for Pthogen Identifiction 49 Virus nd Nosem species identified by Proteomic nd PCR Anlyses of Honey Bees from Western Austrli nd Ssktchewn Colonies with nd without Vrro. Vrro destructor, n ecto prsitic of the domestic honey bee, hs invded most countries round the world with the exception of Austrli, lthough some isolted res in some other countries still do not hve the prsite. Recent studies hve concluded tht the Vrro mite spreds viruses throughout infested colonies nd increses the virulence of deformed wing virus[3], cusing honey bee helth problems nd incresed overwintering losses. In our kinome rry studies the dt implies tht Vrro mites my cuse immune suppression in susceptible phenotypes, mking these colonies more susceptible to pthogens[2]. In this study our objective ws to investigte wht honey bee viruses were detectible in honey bees smpled from pckge bees imported from Western Austrli in 2014, colonies from Ssktchewn with no detectble Vrro infesttion, nd colonies with high nd low Vrro mite infesttions. Our pproch ws to use RT-PCR to detect pthogenic RNA viruses with known sequences nd to further investigte the presence of possible unknown (virus flor) or recombinnt forms of previously chrcterized honey bee viruses by proteomic methods. We lso tested Vrro collected from the infested colonies to determine wht viruses were crried by Vrro in highly infested colonies nd those with low infesttions. In ddition,the presence or bsence of nosem species ws determined in honey bee smples s well s Vrro. 50

26 Phoretic Vrro Level on Colonies used for Proteomic Study 51 Virus nd Nosem Anlyses Pthogenic Virus Pthogenic Fungi Smple Type Colony Nme Deformed Wing Virus Isreli Acute Prlysis Virus Blck Queen Cell Virus Acute Bee Prlysis Virus Vrro destructor Mcul-like virus Nosem Apis Nosem Cerne WA RC Honeybee UF6A G20-3 Vrro UF6A G20-3 Tble 1. Pthogenic virus nd fungi detected in honeybees from Western Austrlin pckge bees in 2014 (WA), Ssktchewn bees free of Vrro (RC), nd with high (UF6A) nd low (G20-3) Vrro infesttions nd in Vrro collected from infected colonies. Viruses were detected by proteomic nlyses nd RT-PCR 52 nlyses.

27 Vrro Proteins Found in Highly Infected UF6A Honeybees ccession # protein description protein mss peptide mss peptide score expect vlue # of peptides gi cytoplsmic ctin, prtil E-15 8 gi vitellogenin gi hemelipoglycoprotein precursor, prtil ; lrge lipid trnsfer protein gi cytochrome oxidse subunit I, prtil (mitochondrion) gi cetylcholinesterse, prtil Honeybee Proteins Found in Vrro from UF6A # Accession # Protein Description Species Protein Score Protein Mss Protein Protein Mtches Sequences mjor royl jelly protein 1 precursor Apis mellifer hexmerin 110 Apis mellifer vitellogenin precursor Apis mellifer PREDICTED: LOW QUALITY PROTEIN: polipophorins Apis mellifer hexmerin 70b precursor Apis mellifer trnsferrin 1 precursor Apis mellifer mjor royl jelly protein 3 Apis mellifer PREDICTED: myosin hevy chin, muscle isoform (X1 to X13) Apis mellifer odornt binding protein 14 precursor Apis mellifer mjor royl jelly protein 2 precursor Apis mellifer silk fibroin 3 precursor Apis mellifer PREDICTED: polipophorins-like Apis dorst mjor royl jelly protein 5 precursor Apis mellifer PREDICTED: LOW QUALITY PROTEIN: trnsferrin Apis flore PREDICTED: protein NPC2 homolog Apis mellifer PREDICTED: unchrcterized protein LOC Apis mellifer PREDICTED: unchrcterized protein LOC Apis dorst phospholipse A2 precursor Apis mellifer mjor royl jelly protein 7 precursor Apis mellifer PREDICTED: glucose dehydrogense [FAD, quinone] isoform 3 Apis mellifer PREDICTED: chymotrypsin inhibitor Apis mellifer hexmerin Apis mellifer PREDICTED: puttive ATP-dependent RNA helicse DHX30-like Apis mellifer short-chin dehydrogense/reductse Apis mellifer het shock protein cognte 5 Apis mellifer PREDICTED: fructose-bisphosphte ldolse-like isoform X2 Apis mellifer PREDICTED: chitinse-like protein Idgf4-like, prtil Apis mellifer PREDICTED: nucleoplsmin-like protein-like isoform X1 Apis mellifer odornt binding protein 13 precursor Apis mellifer

28 % Survivl Mites per Hundred Bees (MHB) Mngement of vrro popultion growth by genetics nd selective tretment strtegies Objective: - To control vrro with resistnt stock nd nturlly occurring miticides (oxlic nd thymol) Miticide Tretment Effects in Vrro Susceptible nd Tolernt Colonies DU - Ssktrz TL - Austrlin GM - Austrlin Before Tretment-2011 After Tretment-2011 Spring Surviving Colonies Spring 2012 DU - Ssktrz TL - Austrlin GM - Austrlin 56

29 Vrro mites per 100 bees Effects of Apivr nd Thymovr vs Control 9.00% 8.00% 7.00% 6.00% Before Tretment After Tretment-October 2013 After Tretment-April % 4.00% 3.00% 2.00% 1.00% 0.00% Control Apivr Thymovr 2013 Phoretic Vrro Infesttion of Adult Bees in Merv's Apiry Error brs re mens ± stndrd error, n= Miticide Residues Concentrtion (ng/g, wm) Compound Before tretment After tretment Control Apivr Thymovr LOD (ng/g) Amitrz ND ND 15.4 ND 4 2,4 DMA ND ND ND ND 50 2,4 -DMPF ND ND ND ND 4 Thymol Tble 1. Concentrtions of miticides (ng/g, wm) in bees before nd fter tretment with Apivr nd Thymovr October LOD = Limit of detection

30 Wintering Survivl Rte(%) % Survivl 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Control Apivr Thymovr Wintering Survivl Rte of Fll Miticides Tretment(2013) - No Tretment(Control), Apivr nd Thymovr. The Control(no tretment) survivl rte ws 80%, Apivr tretment ws 93%, nd Thymovr tretment ws 67%. 59 Efficcy of Combined Fll Miticide Tretments nd Effects on Colony Winter Survivl Tretments Tested in 2015 Apivr (2 strips) Apistn (2 strips) Apivr (2 strips) + Oxlic Acid (3.2% w/v) Apivr (2 strips) + Thymovr (1 wfer) Apistn (2 strips) + Thymovr (1 wfer) Apistn (1 strip) + Thymovr (1 wfer) Apistn (2 strips) + Oxlic Acid Thymovr (2 wfers) + Oxlic Acid Apistn (1 strip) + Thymovr (1 wfer) + Oxlic Oxlic Acid + Apistn (2 strips) fter two weeks This initil experiment ws tested on totl of 50 colonies, 5 per tretment to gin 60 n ide of the most effective combintions.

31 # of Surviving Colonies in Spring Phoretic Mite Level (MHB) Combined Tretment Experiment Winter Survivl nd Men Mites per Hundred Bees For Combined Tretments Before, After, nd in Spring Apivr (2) + Apivr (2) + Thymovr (1) Oxlic Apivr Apistn (2) + Apistn (2) + Oxlic then Thymovr (1) Thymovr (1) Apistn (2) + Oxlic Apistn (2) Thymovr (2) + Oxlic Apistn (2) + Apistn (1) + Oxlic Thymovr (1) 0.0 Men Mites per Hundred Bees Before Tretment Fll 2015 Men Mites Per Hundred Bees After Tretment Fll Men Mites per Hundred Bees In Spring 2016 (April 7th) Number of Surving Colonies out of 5 Efficcy of Combined Spring Miticide Tretments nd Effects on Colony Winter Survivl Tretments Tested in 2016 Apivr (2 strips) Apivr (2 strips) + Oxlic Acid (3.2% w/v) Apivr (2 strips) + Thymovr (1 wfer) Apivr (2 strips) + Thymovr (1 wfer) + Oxlic Three different combintions were determined to be effective enough to tril further. We then selected totl of 60 colonies, 15 per tretment plus n dditionl 15 for control group tht received just Apivr. This experiment hs continued for two yers with n identicl tretment pplied in the spring of the second yer. 62

32 Honey Production (lbs) Mites per Hundred Bees (MHB) Phoretic Mite Levels (MHB) Survivl Percent Summry of Phoretic Vrro Levels (MHB) nd Colony Survivl After Four Different Miticide Tretments from April 12th, 2016 to September 5th, % 80% 90.0% 78.8% % % % 56.3% % % Men n=12 Men n=11 Men n=12 Men n=12 A - Apivr B - Apivr + Oxlic C - Apivr + Thymovr D - Apivr + Thymovr + Oxlic Men MHB April 12th, 2016 Men MHB August 20th, Men MHB April 1st, 2017 Men MHB July 24th, 2017 Men MHB Sept 5th, 2017 Two Yer Survivl Percentge (April 2016 to October 2017) 22.5% 11.3% 0.0% 450 Summry of Phoretic Mite Levels (MHB) nd Honey Production After Four Different Miticide Tretments in Men n=12 Men n=11 Men n=12 Men n=12 A - Apivr B - Apivr nd Oxlic Liquid C - Apivr nd Thymovr D - Apivr, Thymovr, nd Oxlic Liquid Estimted Honey Production During 2017 Seson (±5%) Men Mites per Hundred Bees April 1st, 2017 Men Mites per Hundred Bees July 24th, 2017 Men Mites per Hundred Bees Sept 5th, 2017 Men Mites per Hundred Bees over the 2017 Seson (n=3)

33 Conclusions: Possible to identify productive nd mite tolernt colony phenotypes, nd improve by estblished breeding procedures (bck crossing, out crossing, re-current selection nd progeny nlyses). Colony phenotypes re difficult to stbilize becuse of considerble vribility in progeny, from selected breeders. This my be due, in prt, to multiple mting (subfmilies), queen events (supersedure), high recombintion rtes, episttis, nd genotype environment interctions (epigenetic effects). Evolutionry chrcteristic? New selection tools (biomrkers) for identifying phenotypes expressing genes involved in vrro tolernce, pthogen resistnce immunity, nd productivity should help to stbilize phenotypes nd ssist with breeding procedures. Improvement in techniques for the dignosis of honey bee pthogens will help to improve the qulity of breeding stock, nd bee helth in generl. Use of non synthetic miticides (orgnic cids, thymol, etc.) in timely fshion, with tolernt stock will improve honey bee helth nd colony longevity. Combined miticide trils re showing better efficcy, good colony survivl, nd no 65 observble effects on bee mortlity. This should dely development of resistnce by vrro. Acknowledgements Ssktchewn Agriculture (ADF), Agriculture Council of Ssktchewn (MB, AB, BC nd Yukon), Medow Ridge Enterprises Ltd., SBDC, Bee Mid Honey nd Ssktchewn beekeepers. GenServe Lbs (Bruce Mnn, Dr. Yves Plnte, nd Dr. Steven Creighton, SRC). VIDO (Dr. Philip Griebel, Dr. Scott Npper nd Wyne Connor). University of Ssktchewn Food nd Bio product Sciences (Dr. Xio Qiu, Snjie Jing nd Jin Wng). Toxicology Center, University of Ssktchewn (Dr. John Giesy, Dr. Grry Codling nd Yhy Ngr). Mohmmd Mostjern (R. A ) nd Dr. Syed Qsim Shh ( ). Yng Tn, Colton Rutherford, Héloise Grez Ssktchewn Bee Keeper Colony Contributors & Coopertors. Dr. Abdullh Ibrhim (Reserch Associte, Summer 2007). John Pedersen breeder stock multipliction nd selection (2006). Eric Pedersen, Lyse Boisvert, Mtthew Polinsky nd Vik Cummins, Elizbeth Bygrski, Lim Blim, Meghn McCullough, nd Dvid Hilger, summer students ( ). Fmily members & Medow Ridge stff; Tom, Jenny, nd Cecili Robertson, Neil Morrison, Rob Pece. Collbortors: John Gruszk (Prince Albert, Ssk) Dr. Solignc (Pris, Frnce), Dr. 66 Rlph Buchler (Germny), Dr. Rob Currie (U of M), Dr. R. Dnk (Bton Rouge, LA), S. Cobey (Dvis, CA), Geoff Wilson (Prince Albert, Ssk).