Sensitization to Pheromone and Green Leaf Volatiles after Exposure to Green Leaf Volatiles in the ob

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Sensitization to Pheromone and Green Leaf
Volatiles after Exposure to Green Leaf Volatiles
in the obliquebanded leafroller, Choristoneura
rosaceana.

• NOAH E. RESSA
• Center for Integrated Plant Systems, Michigan
  State University

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Choristoneura rosaceana is a common pest on
apples worldwide.

• Major pheromone component is 98 (Z)11-14Ac and
  2 (E)11-14Ac.
• Does not respond well to pheromone mating
  disruption. Requires a high dose and results in
  85 inhibition.

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Leafrollers are pests of cultivated apples
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Female release of pheromone
Wind
Plume of pheromone
(E)11-14Ac 2 (Z)11-14Ac 98

ca. 70 nanograms/gland female
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Moth Antenna
-The most important olfactory organ -Sifts
odorant molecules from the air which are then
perceived by receptor cells
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Anemotaxis
Females emit pheromone into the wind from
specialized glands. Male moths follow this odor
plume until they come close to the source.
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Pheromone dispensing systems
Hand-applied dispensers
Sprayable formulations
Aerosol emitters
Attract and Kill
Paraffin emulsion
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Proposed mechanisms for mating disruption

• Sensory adaptation at the peripheral level
  affecting
• olfactory receptors
• Habituation affecting processing of and normal
  responsiveness
• to olfactory information reaching the central
  nervous system
• Camouflage of female-produced plumes
• False-trail-following of synthetic pheromone
  plumes by male
• moths

-Bartell 1982, Rothchild 1981, Carde 1990
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The main objectives of this study were

• Characterizing the EAG responses of male and
  female C. rosaceana and to a series of green leaf
  plant and fruit volatiles
• Determining the effect of sustained pre-exposure
  to various concentrations of plant volatiles on
  subsequent EAG responsiveness
• To determine whether sensitization induced by
  plant volatile exposure is octopamine-mediated

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Methods and Materials
1. Expose moths 1 h. to mixture of 9
volatiles or each volatile individually
2. Assay moths with the same volatiles and
pheromone after 1 min. exposure
3. Assay moths with the same volatiles and
pheromone 2nd time after 60 min. of exposure
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Plant volatiles chosen

• hexenal
• trans-2-hexenol
• 3. trans-2-hexenal
• 4. 1-hexenol
• 5. cis-3-hexen-1-ol
• 6. geraniol
• 7. benzaldehyde
• 8. linalool
• 9. limonene

Green leaf volatiles
-Dominate the EAG responsiveness of
oligophagous insects, polyphagous insects also
show selective reception
(Red Apple volatiles)

Non-apple fruit odors
-These volatiles are commonly tested in insects
(moths) for EAG activity -These volatiles are
widely believed to play an important role in
host-plant location in insects that exhibit
sensitivity by EAG
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Green leaf volatiles
Alcohols
cis-3-hexen-1-ol
1-hexanol
trans-2-hexenol
Aldehydes
trans-2-hexenal
hexenal
CHO
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Fruit volatiles
Limonene
Benzaldehyde
Geraniol
Linalool
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Pre-exposure to plant volatiles
Carbon filter
Air entry
Plant volatile enriched air.
Moths
Teflon container
Air exchange
Mesh Divider
Planchette with plant volatiles
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What does an electroantennogram (EAG) measure?
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What does an electroantennogram (EAG) measure?
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Stimulus delivery device used in EAG assays
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Stimulus cartridges are made by pipetting 20 µl
of various concentrations of plant volatiles or
pheromone in hexane onto strips of filter paper.
These are allowed to dry briefly then placed into
Pasteur pipettes and sealed until ready to use.
Air is passed through these cartridges to
stimulate moths olfactory receptor neurons (ORNs).
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2 ml puffs of air are blown through a stimulus
cartridge to a moth attached to an
electroantennogram (EAG). The EAG amplifies and
measures the depolarizations in the moths antenna.
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Choristoneura rosaceana males
6


5




4












EAG Amplitude (mV)
3





2
1
1 mg

O
100 ug



O

O
O
10 ug
O


O
O



O
1 ug
O
O
0



O
O
O


O
60 min after exposure


O

O
O
O

Control
O

O
Nine-mix pre-exposure dosage
10 mg
Hexanal
100 mg (neat)
Hexenol
Pheromone
Trans-H-ol
Cis-H-ol
Linalool
Trans-H-al
Geraniol
Benzalde
Blank
Limonene
EAG Cartridge Treatment
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Choristoneura rosaceana females
3.5






3


2.5



2
EAG Amplitude (mV)
1.5
1
0.5
1 mg



O
O

O

O
O
100 ug



O
O
O


O
O
10 ug



O
O

O
1 ug

O
O
0


O

O
60 min after exposure

O
O

O
Control
10 mg
Nine-mix pre- exposure dosage
Hexanal
100 mg (neat)
Hexenol
Cis-H-ol
Trans-H-ol
Linalool
Trans-H-al
Geraniol
Benzalde
Blank
Limonene
Pheromone
EAG Cartridge Treatment
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Discovery of Octopamine Literature

• Injection of the biogenic amine octopamine (OA)
  into the head cavity
• of moths increased action potential frequencies
  of stimulated
• pheromone-sensitive moth ORNs (Pophof, 2000
  Grosmaitre et al.,
• 2001 Pophof, 2002) and/or increased receptor
  potentials (Pophof,
• 2002).
• OA is also known to improve the detection and
  behavioral reactivity to
• pheromone blends in male moths (Grapholita
  molesta and
• Trichoplusia ni). (Linn and Roelofs, 1984
  1986).
• Octopamine (OA) is a major biogenic amine of
  insects and known to
• function as a neurotransmitter, neuromodulator,
  and neurohormone
• (Evans, 1985).

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7
a
a
Choristoneura rosaceana males
a
1
a
6
a
a
a
a
a
5
b
a
a
b
a
a
b
4
b
a
EAG Amplitude (mV)
b
b
a
b
b
b c
a
3
c
c
c
c
c
a
2
c
b
b
c
c
b
c
c
a
c
Treatment
b
b
b
c
1
c
c
c
b
b
c
5 min after OA
c
c
c
b
c
c
b
1 min after PV pre-exp
b
0
c
b
b
5 min after HR
b
b
b
Control
5 min after CP no PV pre-exp
b
Hexanal
1 min after PV pre-exp with CP injection
Hexenol
Pheromone
Cis-H-ol
Trans-H-ol
Linalool
Trans-H-al
Geraniol
Blank
Benzalde
Limonene
EAG Cartridge Treatment
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Summary of Octopamine Data

• Plant volatile and pheromone stimulation of
  whole-antennae of live
• male C. rosaceana five min after injection of
  OA increased
• depolarization of olfactory receptors similarly
  to that induced by pre-
• exposing moths to a mixture of nine plant
  volatiles.
• Injection of the OA antagonist, chlorpromazine
  (CP), blocked the
• sensitizing effect of plant-volatile exposure
  as evidenced by normal
• EAG amplitudes immediately following sustained
  pre-exposure to
• sensitizing concentrations of plant volatiles.

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Future questions

• Use of plant volatiles to modulate (sensitize)
  pheromone
• perception
• -Create more potent pheromone disruption systems
• -Create more sensitive traps
• Is the full blend of volatiles needed to induce
  sensitization or are only a few compounds
  responsible.

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Acknowledgements
Lukasz Stelinski, Dr. Jim Miller and Dr. Larry
Gut Marcia Ewers Frank Ewers and Patrick
Webber Everyone in CIPS Everyone in 499
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The End