Chemical Ecology and Lure Development for Redbay Ambrosia Beetle

David Owens, Paul E. Kendra, Wayne Montgomery, Jerome Niogret, Elena Schnell, Tereza Narvaez, Daniel Carrillo, and Nancy Epsky

USDA-ARS, Subtropical Horticulture Research Station 13601 Old Cutler Road, Miami FL

E-mail: paul.kendra@ars.usda.gov David.owens@ars.usda.gov

Development of Attractive Field Lure 2009 - present

• Field tests (forest sites) • Lab and semi-field behavior

tests (Archbold Biol. Station, Lake Placid)

• Chemical sampling & analysis (Miami) – Lure emissions – Tree volatile emission

present

RAB Biology and Detection • One and done flight – uses

reliable cues to find hosts quickly – Harsh environment, limited energy

• Unique chemical ecology – No pheromone production – Are not attracted to fungal volatiles

long-range – Not attracted to ethanol (Hanula et al. 2008, Hanula & Sullivan 2008)

First Lures = Problematic Lures

• Volatile collection from redbay = 16 candidates (Hanula and Sullivan 2008)

• First lures contained several candidate terpenoids = Manuka (Myrtle bush) and Phoebe (laurel tree)

• Manuka lost attraction after 3 wk • Phoebe caught 6X more RAB, but no longer available

Phoebe Manuka Control

Bee

tles

/ tra

p / w

k

0

5

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25

a

b

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Xyleborus glabratus(mean + SE)

12-wk testHighlands Co., FL

(Kendra et al. 2012. J. Econ Entomol.)

Xyleborus glabratusWeekly Captures over Time

Week

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Beet

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/ wee

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PhoebeManukaControl

Freeze

Bolt Boring Bioassay • No-choice tests to assess RAB boring

behaviors – 1-gallon bucket – 1 bolt plus 10-15 female RAB – Recorded # RAB boring – Replicated 5x

Kendra et al. 2013 FL Entomol.

RAB Flight Window • Multiple species

attracted to wood volatiles

• RAB flies earlier (27 collection dates, Apr-Oct 2011)

• Useful method for obtaining RAB in host-seeking behavior, the perfect stage to evaluate attractants

1600 1630 1700 1730 1800 1830 1900 1930 2000

Num

ber

of F

emal

es C

olle

cted

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Time Interval

1600 1630 1700 1730 1800 1830 1900 1930 2000

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ber

of F

emal

es C

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X. ferrugineus

X. glabratus

1600 1630 1700 1730 1800 1830 1900 1930 2000

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X. affinis

Kendra et al. 2012 Environ. Entomol.

sunset

Development of Field Lures for RAB

• Avocado bolts are just as attractive as Phoebe oil

Phoeb Guat W.Ind Mex Manuk Contr

Beet

les

/ tra

p / w

eek

0.0

0.5

1.0

1.5

2.0

X. glabratus(mean ± SE)

8-wk testAlachua Co., FL

a

aab

b

c c

(Kendra et al. 2011. J. Chem. Ecol.)

Bioassays and Field Tests

Time (hr)

0 5 10 15 20 25

Perc

enta

ge o

f Fe

mal

es B

orin

g

0

20

40

60

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100 Silkbay (99%) – very attractive in field Swampbay/Redbay (91%) – so-so in field

Avocado (80%) – in lab, so-so in field

Camphor tree (50%) – very attractive in field Lancewood (44%) – not attractive in field

Live Oak (0%) – negative control

(Kendra et al. 2014 PLoS ONE)

Time (hr)

0 5 10 15 20 25

Perc

enta

ge o

f Fe

mal

es B

orin

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>70% boring on cut end; trees most susceptible to attack after pruning or injury!

• Differential field attractiveness

Silkbay

Camph

or.

Cal. Bay

Sassa

fras

Swampa

y

Redba

y

Avoca

do

Lanc

ewoo

d

Spiceb

ush

Live O

ak

Contro

l

Nor

mal

ized

Cap

ture

s

0.0

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1.0a a

bb b b

c c c

b

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Laurel Chemical Analysis

• Emissions of terpenoids from bolts correlated with RAB captures in field

• Major sesquiterpenes – α-cubebene (peak 2)

– α-copaene (peak 3)

Kendra et al. 2014 PLoS ONE

Evaluation of Other Essential Oils - 2012

• 7 essential oils – including cubeb, ginger, tea, angelica, orange

Cubeb oil = new attractant

(from berries of tailed pepper Piper cubeba)

X. glabratus (mean + SE)4-wk test (25 Apr - 23 May 2012)

Essential oil

Man Cub Phb Gin Ang Tea Ora Blk

Beet

les

/ tra

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eek

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aa

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(Homemade lures – 2 ml oil)

Field Tests 2013 • Collaboration with Chemtica and

Synergy Semiochemicals – Field test comparisons with distilled

cubeb oil in bubble lure

Xyleborus glabratus (mean + SE)

8-wk Field TestFisheating Creek State Park

(8 Mar - 3 May 2013)

Cubeb bubble Manuka patch Control

Beet

les

/ tra

p / w

eek

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0.5

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12 wk Test, Highlands Co.

• Cubeb > phoebe > manuka • Cubeb field life of 3 months• Less expensive • Standard lure for RAB since 2015

(Kendra et al. 2015. J. Econ. Entomol.)

Cubeb Phoebe Manuka Control

Bee

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18 a

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Week

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120

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160Cubeb Phoebe Manuka Blank

Further Improvement of RAB Lure (Collaboration with Synergy)

Goal = Identify primary attractant in cubeb lure

• Cubeb is a complex mix of terpenoids, at most 10% α -copaene and α -cubebene

• Separated cubeb oil into separate parts with different chemicals

• Choice tests and Olfactory response tests

• The two attractive parts were high in 1. α -copaene and 2. cubebene

New Lure Evaluations Two prototype lures prepared • Copaiba oil

(9% α-copaene, 0% cubebene)

• Proprietary oil product (50% α-copaene)

12-wk field test • Compare new lures to cubeb lure

(10% α-copaene, 10% α-cubebene)

50% Cop Cubeb Copaiba Control

X. g

labr

atus

/ tra

p / w

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16 a

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Week

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gla

brat

us /

wk

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50% CopCubebCopaibaControl

A

B

Kendra et al. J. Pest Sci. 2016

α -Copaene Gradient in Trunk

• Beetles use vision and chemistry to concentrate attacks on tree trunks

• α -cubebene and α -copaene concentration greatest in the trunk, lowest on twigs/leaves

RAB in Avocado

• What about avocado? – RAB is relatively rare in avocado – Wilt-infected trees do not

necessarily have RAB infestation (Carrillo et al. 2012).

– Can we detect RAB in avocado?

We don’t see this in avocado

Detection of RAB in avocado - 2015 • Deployed traps in a grove at the beginning of an

outbreak – α -copaene, α -copaene + ETOH, cubeb + ETOH, silk

bay

• Only α -copaene lures captured RAB! (but not many)

• α -copaene also captured fewer non-targets

One year later…

Wood seeking missile • Host choice: look, smell, and taste right • Beetle is detectable at new wilt-sites • α -copaene lure could possibly be used to alert

a problem • RAB does not breed well in avocado • Initial infestation from RAB, infestation spread

from because of other factors

But Wait…There’s Even More

• Euwallacea fornicatus = shot hole borer

• picked up with α -copaene • First discovered in FL in 2006

– Avocado 2010

Lure Evaluation - 2014

• Daniel Carrillo tested commercial ambrosia beetle lures – Ethanol UHR – Ethanol + Chalcogram – Ethanol + Conophorin – Ethanol + Cubeb

– Ethanol + Quercivorol

• Quercivorol used by the folks in CA, traps

capture hundreds of beetles/week

But Wait…There’s Even MORE!

Field Testing Lures 2 wk test, Spring 2016, site A

• α -copaene, quercivorol, combination, and blank

Quer + Cop Quercivorol Copaene Control

Bee

tles

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60a

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Field Testing Lures 4 wk test, Summer 2016, site A

• Population slightly lower

• Combo lure more attractive

Quer + Cop Quercivorol Copaene Control

Bee

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p / w

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bc

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Field Testing Lures 8 wk test Summer 2016, site B

• Low population • Wilt affected

grove • Combo more

attractive than either querc. or α -copaene

• Combo is additive or synergistic

Quer + Cop Quercivorol Copaene Control

Bee

tles

/ tra

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eek

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Host Attraction Hypotheses and Current Research

• Quercivorol – shorter range attractant – Produced by Euwallacea -infested wood

• α -Copaene – longer range attractant – Alert to a possible host

• Others? – Bioassays for attractive alternative hosts – Identify additional terpenoids – Guide lure evaluation

Questions?