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