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Prevention of deformed wing virus infection in larval and ... Desai - Deform · PDF file Prevention of deformed wing virus infection in larval and adult honeybees (Apis mellifera

Jul 16, 2020

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  • Prevention of deformed wing virus infection in larval and adult honeybees (Apis mellifera L.) by dsRNA ingestion

    Suresh D. Desai*, Young-Jae Eu ***, Steven Whyard** and Robert W. Currie*

    * Department of Entomology, University of Manitoba,

    ** Department of Biological Sciences, Winnipeg, Canada R3T 2N2

    *** Center of Applied Microfluidic Chemistry, Chungnam National University, Daejeon, South Korea

  • Introduction • Colony losses -2006-2010

    – USA-34% - 46%

    – Canada-34 -36%

    – Manitoba-35% -36%

    • Varroa, Viruses, Nosema, presence of pesticides, poor nutrition and other stressors

    Currie, Pernal, & Guzman-Novoa, 2010, VanEngelsdorp, 2009

    http://www.wildaboutbritain.co.uk

  • DWV damage on bees • DWV- crumpled wings, reduced body size

    – Early death of deformed bees

    – Short life span of normal winged bees

    • Death of colony

    • Honey production

    • Death in overwintering colonies

    Genersch et al, 2006, Ball and Bailey, 1997; Kovac and Crailsheim, 1988, Highfield, et al.2009

  • RNA interference

    siRNAs are incorporated into RNA-induced silencing complex (RISC)

    dsRNA

    mRNA of virus

    siRNAs 21-23bp

    Adapted from Science video

  • Experimental design

    • Larval experiment

    • Replicated twice

    • Adult bee experiment

    • Replicated once

  • Larval experiment

    • 5 Treatments 1. dsRNA DWV+DWV

    2. dsRNA GFP+DWV

    3. DWV only

    4. Control-no treatment

    5. dsRNA only

    T T T T T

    GFP= Green Fluorescent Protein

  • Rearing worker honey bees in lab

    1 day old larvae 3 day old larvae 5 day old larvae

  • Pre-pupae Pupae

    Adult bee emerged

  • Effect of dsRNA on wing deformity

    11.1

    42.1

    31.6

    9.8 7.0

    0.0

    5.0

    10.0

    15.0

    20.0

    25.0

    30.0

    35.0

    40.0

    45.0

    dsRNA DWV + DWV

    dsRNA GFP+ DWV

    DWV only control-no treatment

    dsRNA-Toxicity

    % D

    ef o

    rm e

    d b

    ee s

    a

    a

    b b b

    only

    A B C

  • Effect of dsRNA on survival in DWV infected larvae

    0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0.8

    0.9

    1.0

    dsRNA only

    Control

    DWV+dsRNA

    DWV

    S u

    rv iv

    a l

    d is

    tr ib

    u ti

    o n

    F u

    n c

    ti o

    n

    Days

    DWV+GFP

    dsRNA

    DWV

  • Relative DWV concentration (Larvae)

    0.0

    0.2

    0.4

    0.6

    0.8

    1.0

    1.2

    1.4

    1.6 Normal winged bees

    Deformed bees

    R e

    la ti v e D

    W V

    c o n

    c e n

    tr a

    ti o

    n (

    lo g

    )

    a a

    b b

    b b

    dsRNA DWV+DWV dsRNA GFP+DWV DWV only

    RNAi Treated RNAi Control DWV Control

  • Adult bee experiment

    • 50 bees/treatment

    • 1µg dsRNA/bee

    • Sugar syrup and water

    • Dead bees collected daily

    • 4 Treatments 1. dsRNA DWV+DWV

    2. dsRNA GFP+DWV

    3. DWV only

    4. Control-no treatment

    T T T T

  • Effect of dsRNA on survival in DWV infected adult bees

    2 3 4 5 6 7 8

    0.0

    0.2

    0.4

    0.6

    0.8

    1.0

    Control

    DWV+dsRNA

    DWV+dsRNA GFP

    S u

    rv iv

    a l d

    is tr

    ib u

    ti o

    n F

    u n

    c ti

    o n

    Days

    DWV only

    dsRNA

    DWV

  • Relative DWV concentration (Adult bees)

    0.0

    0.2

    0.4

    0.6

    0.8

    1.0

    1.2

    1.4

    1.6

    1.8

    2.0

    2.2

    2.4

    DWV onlydsRNA GFP+DWV

    R e

    la ti v e v

    ir u

    s c

    o n

    c e n

    tr a

    ti o

    n (

    lo g

    )

    a

    b b

    dsRNA DWV+DWV

    RNAi Treated RNAi Control DWV Control

  • Conclusion

    dsRNA against DWV:

    • Suppresses viral replication

    • Marginal increases in larval survival

    • Reduces wing deformity

    • Increases longevity of adults

    • Is not toxic

  • Acknowledgments Funding NSERC-CANPOLIN Canadian Bee Research Fund Manitoba Beekeepers Association, Saskatchewan Beekeepers Association, NSERC,ARDI, UMGF, ACAFF,

    •Rob Currie •Rob Hill •Steve Whyard •Judy Chen •Joachim de Miranda •Young-Jae Eu •Nitzan Paldi •Rheal Lafreniere •David Ostermann •Santosh Kumar •Doug Durnin •Amanda Van Haga •Manitoba beekeepers

  • Thank you