Mark J. CarrollCarl Hayden Bee Research Center
USDA-ARS Tucson, AZ
*formerly of Center for Medical, Agricultural, and Veterinary Entomology (CMAVE)Chemistry Unit, USDA-ARS Gainesville, Florida
and University of Florida Department of Entomology & Nematology
Varroa mite attractants:potential solution for Varroa mite/
viral challenges to honey bees
I. Volatile (odor) collections in the hive environment
II. Comparisons of volatiles from bee brood
III. Responses of mites to host volatiles (semiochemicals)
IV. Using host volatiles for mite control in the hive environment
Varroa mite attractants:potential solution for Varroa mite/
viral challenges to honey bees
Varroa mite (Varroa destructor)
• parasite of capped bee brood and adult bees
• recent host switch from the Asian honey bee (Apis cerana) . to the western honey bee (Apis mellifera)
• western honey bees are highly susceptible
• leading known cause of colony mortality worldwide
• mite resistance to chemical treatments is growing
mite moves into the back of the host cell, movement stops (arrestant behavior)
mites emerge with the newly emerged host
mite feeds and produces young on developing bee brood
brood host emerges as adult bee host cell capped by worker bees
mites disperse to other adult bees
maternal mite encounters brood of various ages on phoretic host
detection, excitation, and invasion of brood host cell (attraction behavior)
photos of pupae courtesy of USDA
Identify the cues that the mite uses to acquire its brood hostCell invasion behaviors
1-3 daughters/round
Varroa cell invasion behavior
• attraction occurs over very short distances (less than 7 mm)
adult female mite - 2.0 mm wide
adult worker bee – 14 mm long
worker brood cell – 11 mm deep
• mites have strong brood caste preferences for larvae
drone > worker >> queen
• mites only invade during a narrow window of host development
worker brood – 15-20 hrs before capping through capping
drone brood – 40-50 hrs before capping through capping
Mites are attracted to volatiles from host brood
Near contact cue ~ 7 to 10 mm from target cell
Are mite attracted to brood odors alone?On-comb volatile infusion bioassay
odors collected off enclosed brood
volatiles slowly infused into single cells through capillary tubes (~2mL/min)
A C D B
B D
DC A
C A
B
volatiles from 3 brood ages (plus control) infused into single cells across empty comb
choice arena
• 40 free-roaming mites released
• position of mites in arena noted 30 minutes after release
Mites are attracted to odors from capping brood On-comb volatile infusion bioassay
0
10
20
30
40
50
60
70
80
90
100
air early 4th instar capping 4th instar pupae non-responders
mit
es in
infu
sed
or
adja
cen
t ce
lls (
%)
odor source
Mites find capping brood hosts in a chemically confusing environment
actively capping brood (aka “potential hosts”)
“… a sea of competing odors from different-age brood …”
• few brood of any particular age
• excess background odors from . hive materials
• handling? stress? artifacts?
Problems with identifying odor cues from brood comb (from a human perspective)
Isolated brood pulled from the comb = stressed larvae!
Avoid!
Aluminum observation frame (AOF)Collection and manipulation of colony odors
vacuum line
(pull)air line
(push)
glass plate
a push-pull airflow system
Aluminum observation frame Fits inside the perimeter of any deep frame
Multifunctional ports (air, vacuum, food, waste disposal,
odor sampling)
air flow ports
view into open port with bees
ports run along top edge of AOF
Push-pull airflow system Sampling or adding odors
airflow (adding odors)vacuum (sampling odors)
SuperQ filter capillary release
early 5th instar worker larvae
late instar worker larvae (pre-capping)
capped worker brood
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
capping worker larvae
Gas Chromatography of odors
pre-capping queen larvae
capping queen larvae
capping drone larvae
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
pre-capping drone larvae
Gas Chromatography of odors
0
5
10
15
20
25
30
35
40
45
CONTROL CA CB
% m
ites
dro
pp
ing
into
tra
p
jar volatile treatment
Brood volatiles CA and CB cause phoretic mites to move off adult beesMite bottom screen (jar) bioassay
b
b
a
p < 0.05, one-way ANOVA
Capping brood emit higher amounts of CA and CB than other developmental stages
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
worker larvae (2d before capping)
worker larvae (12h before capping)
capping worker brood capped worker brood
brood developmental stage(RELATIVE ATTRACTIVENESS TO MITES)
both compounds
compound A (CA)
compound B (CB)
(NOT ATTRACTIVE) (ATTRACTIVE)
(ATTRACTIVE) (LESS ATTRACTIVE)
vola
tile
em
issi
on
s (n
g/h
r/b
roo
d)
Capping drone larvae emit greater amounts of CA and CB volatiles than worker brood
0.0
1.0
2.0
3.0
4.0
5.0
6.0
worker larvae
(2d before capping)
capping worker
brood
drone larvae
(2d before capping)
capping drone brood
brood caste/developmental stage
(RELATIVE ATTRACTIVENESS TO MITES)
vola
tile
em
issi
on
s (n
g/h
r/b
roo
d)
both compounds
compound A (CA)
compound B (CB)
(NOT ATTRACTIVE) (ATTRACTIVE) (ATTRACTIVE)
(ATTRACTIVE)
Detecting the sense of smell Electrophysiological responses to odors
Adrian Duehl - USDA CMAVE
olfactory sensillae in the pit organ
location of pit organ on forelegSEM by Adrian Duehl
SEM from Dillier et al, 2003, Swiss Bee Research Centre
Mite foreleg electrophysiological responses to CA and CB
EAG (EFG) - Responses to synthetic chemical puffs
location of sense organs on forelegs
Control
CA
CB
Mites detect CA and CB with their forelegs
EAG (EFG) responses
green line and black bar
indicates odor puff
0
2
4
6
8
10
12
1 to 5 1 to 100 1 to 1000 1 to 10,000 Blank
odor concentration (in solution)
EAG
am
plit
ud
e (
-mV
)But mites only detect high concentrations of CA
p<0.05, one-way ANOVA
a
b bb
b
Behavioral responses to synthetic CA and CB
Mites become excited by CA and CB at a close distance Casting – twitching of forelegs
Final approach
Casting path straightens out and velocity increases at the last second
Problems in evaluating a near contact chemical cueToo fast, too small to easily observe
Open odor sources – contact fatal Point odor sources - too localized
Need to spread out mite response in time and space
EthoVision – video analysis of mite movements
mite tracks in user-defined arena & odor zones
Diffusion bioassays
• odors diffuse up through mesh floor
• relatively even odor presentation
• mite never contacts odor source
• mite starts on central glass pin, moves off
mite moving off central glass pin
odor sourcesmite starts on pin
Nitex mesh
arena floor
Mites are attracted to CA volatiles Dual choice diffusion bioassay
0
10
20
30
40
50
60
70
80
90
100
control CA
% o
f ti
me
in
od
or
sou
rce
zo
ne
odor source
CA
CA
CON
CON
odor source
configuration
CA
CA
CON
CON
odor source
configuration
Most, but not all mites, display arrestant behaviors (stopping) when
moving over CA volatiles
No choice diffusion bioassayMite responses to a single odor source
Without attractant or arrestant cues, mites tend to move out of the arena
odor zone
outer wall
pin & disc starting point (neutral zone)
(neutral zone)
Mites remain in near contact with CA No choice diffusion bioassay (EthoVision)
0
10
20
30
40
50
60
70
80
90
100
control CA
% o
f tim
e in
od
or z
on
e
arena odor source
p < 0.001, t-test
Mites move slower during exposure to CA volatilesNo choice diffusion bioassay (EthoVision)
0.0
0.5
1.0
1.5
2.0
2.5
control CA
arena odor source
velo
city
(m
m/s
)
p = 0.022, t-test
Mites are immobile more often during exposure to CA No choice diffusion bioassay (EthoVision)
0
10
20
30
40
50
60
70
80
90
100
control CA
imm
ob
ile (%
of t
ime
)
arena odor source
p = 0.005, t-test
Mite responses to CA – m2p video clip
Using CA to control mites in the hive environment
I. FloodingII. In-hive trap
flooded airspacecontrol airspace
Flooding – disruption of behaviors by saturating the sense of smell with synthetic chemicals
Flooding bioassay
• enclose mite-infested adult bees with capping brood in observation frames
• treat each frame’s airspace for 36 hrs
Control - air only
Flooded - synthetic CA
• count the mites that invaded brood cells during the experiment
release of CA into airflow of flooded AOF frame
detection of cell capping by comparing photos
Flooding the brood comb airspace with synthetic CA reduces mite cell invasion
0
5
10
15
20
25
30
35
40
45
control flooded% o
f re
cen
tly-
cap
pe
d b
roo
d in
fest
ed
frame treatment
p=0.028, t-test
… yet colony functions (larval rearing and capping)
continue as usual
• One of two treatments added in solvent to the bottom of single cells (12 cells/tmt)
Control – solvent only
CA - CA in solvent
• 100 free-roaming mites released directly onto frame
• mite positions noted 2 hours after release
Are mites attracted to cells treated with CA?
.
0
10
20
30
40
50
60
70
80
90
100
inside control cell inside CA cell outside cells
% o
f m
ites
mite position
Free-roaming mites are attracted to CA-treated cells
p=0.031, t-test
Mites in CA-treated cells remain at the bottom (81%) … and often turn upside-down
treated cell outlined in blue
Challenges for in-hive trap development
Building a better bait (formulation)
• Improve activity with minor volatile synergists
• Provide odor release over a full mite reproductive cycle (3 weeks)
• Achieve consistent release under a variety of environmental conditions – stable colony temperatures help
mid-summer 105 Fearly spring 40 F
Bees.The other half of the equation.
• Proximity - must attract the bees near the bait (within ~5 mm) without direct contact
• Bait must not be repellent to bees
• Bait must not disrupt colony function
Is “bribery” the answer?
Varroa volatiles – an ongoing collaborative project
Carl Hayden Bee Research Unit
Tommy Deeby
... and more to follow!
CMAVE Chemistry Unit
Peter Teal
Adrian Duehl
Alex King
Tredina Davis
Shelley Olson
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