Prevention of deformed wing virus infection in larval and ... Desai - Deform… · Prevention of deformed wing virus infection in larval and adult honeybees (Apis mellifera L.) by

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

mRNAof 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

TTTTT

GFP= Green Fluorescent Protein

Rearing worker honey bees in lab

1 day old larvae 3 day old larvae5 day old larvae

Pre-pupae Pupae

Adult bee emerged

Effect of dsRNA on wing deformity

11.1

42.1

31.6

9.87.0

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

efo

rme

d b

ees

a

a

b bb

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

Su

rviv

al

dis

trib

uti

on

Fu

nc

tio

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

Re

lative D

WV

con

cen

tra

tio

n (

log

)

aa

bb

bb

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

TTTT

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

Su

rviv

al d

istr

ibu

tio

n F

un

cti

on

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

Re

lative v

iru

s c

on

cen

tra

tio

n (

log

)

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 FundingNSERC-CANPOLINCanadian 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