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

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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

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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

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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

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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

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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)

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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)

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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

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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?

.

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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