Chemical Ecology of glabratus and Implications for Monitoring and Management

Paul E. Kendra, Wayne S. Montgomery, Jerome Niogret, Elena Q. Schnell, and Nancy D. Epsky

USDA‐ARS, Subtropical Horticulture Research Station

13601 Old Cutler Road, Miami, FL email: [email protected]

------Conference on Disease and Natural Ecosystems: Impacts, Mitigation, and the Future

16-18 June 2015 Applied Approach • Target = Adult female RAB, host‐seeking flight • Dispersal flight is risky – Harsh environment (dessication, wind, rain) – Predation (e.g. dragonflies) – No food available • Strong selection – Reliable cues to find host trees quickly – Optimize timing of flight to minimize risks Chemical Ecology of RAB

– Initial research on RAB attractants (Hanula et al. 2008, Hanula & Sullivan 2008) • No evidence of pheromones (aggregation or sex) • No long‐range attraction to fungal symbionts • No attraction to ethanol (standard lure for ambrosia ) – Conclusions: • RAB can attack healthy, unstressed trees • Host tree volatiles = primary attractants • α‐copaene, calamenene are likely attractants from redbay; but cost/availability make them impractical for field lures • Manuka and phoebe oils identified as attractive baits for RAB ARS ‐ Miami,FL Research ‐ RAB attractants (2009‐pres.) Multidisciplinary approach

• Field tests (forest sites)

• Lab tests (Archbold Biol. Station, Lake Placid) – Behavioral bioassays (attraction, boring preferences) – Electrophysiology techniques to measure olfactory responses

• Chemical sampling &analysis (Miami) – Volatile collections – Gas chromatography‐ mass spectrometry (GC‐MS) 1. Method for Capture of Live RAB

General Method • Late afternoon (~4:00 pm) • Lay cotton sheet on ground • Bait = Freshly‐cut host wood • “Lure in” ambrosia beetles • Collect with soft brush

Kendra et al. 2012 FL Entomol. sunset Unique Observation: 14 12 X. glabratus Temporal Separation of 10 8 6 4

2 Number of Females Collected 0 • Multiple species attracted to 1600 1630 1700 1730 1800 1830 1900 1930 2000 wood volatiles 70 60 X. affinis

50 • X. glabratus flies earlier 40 (27 collection dates, Apr‐Oct 2011) 30 20

10 Number of Females Collected of Females Number • Useful method for obtaining 0 1600 1630 1700 1730 1800 1830 1900 1930 2000 RAB in host‐seeking behavior, 6 X. ferrugineus the perfect stage to evaluate 5 4 attractants 3 ‐ Lab bioassays 2 1 Number of Females Collected Females of Number ‐ Electrophysiology studies 0 1600 1630 1700 1730 1800 1830 1900 1930 2000 Time Interval Kendra et al. 2012 Environ. Entomol. 2. Electroantennography (EAG) Technique to measure olfactory response of antennal receptors (How well does an smell a particular chemical?) • Mount antenna between 2 electrodes • Place under purified air flow • Deliver chemical sample • Measure receptor potential

EAG lab at Archbold Biol. Station A B C

Sample EAG recordings from single RAB antenna EAG Technique Major challenge: small size of RAB antenna (0.3‐0.4 mm length) • Used a gold, 2‐pronged electrode • Electrode modified by attaching a flexible gold wire to one prong

Modified antennal electrode

Scanning electron micrograph of RAB antenna Validation of New EAG Methods

Comparative studies with 3 species: 2.5

a 2.0 Ethanol a X. glabratus, X. affinis, and X. ferrugineus a 1.5

• Ethanol = standard reference chemical 1.0 EAG Response (-mV) Response EAG • EAG responses to test chemicals could then be 0.5

0.0 expressed as a percentage relative to ethanol X. glab X. affin X. ferr

100 100 100 A.a Silkbay wood B.a Phoebe oil C. Manuka oil

80 80 80 a

60 b b 60 60 ab b b b 40 40 40 EAG Response (%) EAG Response 20 20 20

0 0 0 X. glab X. affin X. ferr X. glab X. affin X. ferr X. glab X. affin X. ferr

Kendra et al. 2012. Environ. Entomol. 3. Bolt Boring Bioassay • No‐choice tests to assess RAB boring behaviors – 1‐gallon bucket – 1 bolt plus 10‐15 female RAB – Recorded # RAB boring and location on bolt after 2, 4, 6, 8, 24 hr (Positive boring = half of the body inserted) – Replicated 5x

Kendra et al. 2013 FL Entomol. Composite Results – Bioassays

(Adapted from Kendra et al. 2014 PLoS ONE)

100 Silkbay (99%) Swampbay/Redbay (91%) 80 (80%)

60 Camphor tree (50%)

40 Lancewood (44%)

Percentage of Females Boring Females of Percentage 20

Live Oak (0%) 0

0 5 10 15 20 25 Time (hr)

>70% boring on cut end; trees most susceptible to attack after pruning or injury. 4. Comparative Field Tests • Replicated field tests with freshly‐cut wood bolts • Silkbay most attractive = positive control; captures normalized • Determined relative attraction of 9 species within

a 1.0 a

0.8

b b b 0.6 b b

0.4

bc 0.2 Normalized Captures Normalized c c c 0.0 . s y o h k or a a ay s rol fr b t ph l. Bay mp cad Silkbaym a ssa Red vo ive Oa Con C a wa A icebu L Ca S S p LancewoodS Kendra et al. 2014 PLoS ONE 5. Chemical Analysis • Emissions of terpenoids from bolts correlated with RAB captures in field • 4 sesquiterpenes – Major • α‐cubebene (peak 2) • α‐copaene (peak 3) – Minor • α‐humulene (peak 6) • calamenene (peak 8) Kendra et al. 2014 PLoS ONE Current Hypothesis ‐ Host Location, Acceptance

Long‐range: RAB in flight attracted to the odor plume from host trees. Terpenoids: α‐copaene, α‐cubebene, α‐humulene, calamenene (Hanula & Sullivan 2008, Kendra et al. 2011, 2014); eucalyptol (Kuhns et al. 2014); other monoterpenes (Martini et al. 2015)

Mid‐range: RAB uses visual cues to find hosts of appropriate diameter (Mayfield and Brownie 2013)

Short‐range: RAB detects (1) terpenoid gradients to find best site for attack (avocado: α‐copaene and α‐cubebene) (Niogret et al. 2013), and (2) fungal odors = food attractants (Hulcr et al. 2011, Kuhns et al. 2014)

On contact: RAB ‘taste’ and ‘feel’ the wood to confirm suitability (eucalyptol, Kuhns et al. 2014)

Summary: Host must smell, look, taste, and feel ‘right’ before boring initiated. Development of Field Lures for RAB

• First lures = phoebe oil, manuka oil (Hanula & Sullivan 2008) • However, tests in FL (2009‐2010) indicated that manuka lures not very effective

2.0 X. glabratus a (mean ± SE) a 1.5 8-wk test ab Alachua Co., FL

1.0 b

Beetles / trap / week / trap / Beetles 0.5

c c

0.0 Phoeb Guat W.Ind Mex Manuk Contr

(Kendra et al. 2011. J. Chem. Ecol.) Follow‐up Tests Confirmed Results

25 350 (mean + SE) Xyleborus glabratus a 300 Weekly Captures over Time 20 Freeze 12-wk test 250 Highlands Co., FL Phoebe Manuka 15 200 Control

150 10 Beetles / week / Beetles 100 Beetles / trap / wk

5 b 50

c 0 0 0 2 4 6 8 10 12 14 Phoebe Manuka Control Week (Kendra et al. 2012. J. Econ Entomol.) • Phoebe caught 6X more RAB • Manuka lost attraction after 3 wk • Unfortunately, phoebe lures no longer available, manuka lures only option for RAB monitoring Evaluation of Other Essential Oils

Field tests in 2012 compared 7 essential oils for RAB attraction: (Homemade lures –2 ml oil)

• Manuka • Phoebe • Cubeb • Ginger root • Angelica seed • Tea tree • Valencia orange

(Agricultural Research 2012) 12 a X. glabratus (mean + SE) a 10 4-wk test (25 Apr - 23 May 2012) a

8

6 b b

4 Beetles/ trap / week bc c c 2

0 Man Cub Phb Gin Ang Tea Ora Blk Essential oil (Kendra et al. 2013. Am. J. Plant Science) Cubeb oil identified as a new RAB attractant (from berries of tailed pepper Piper cubeba) Field Tests 2013 Collaboration with Synergy Semiochemicals Corp. (BC, Canada)

New lure = distilled cubeb oil in bubble lure (enriched in sesquiterpenes) Field tests to compare efficacy and longevity of commercial lures: • Cubeb bubble lure • Manuka lure • Phoebe lure • Unbaited control Test 1 (Highlands County, FL)

7

a 6 Xyleborus glabratus (mean + SE)

8-wk Field Test (5 reps) 5 Archbold Biol. Station (Lake Placid, FL) (28 Feb - 25 Apr 2013) Xyleborus glabratus - Weekly captures 4 60 Cubeb Manuka 3 50 Control b RAB / trap / RAB / wk 2 40

1 30 c RAB / wk RAB 0 20 cubeb manuka blank

10

(Kendra et al. 2014. J. Pest Science) 0 012345678910 Week

• Cubeb bubble lure captured 3 times more RAB than manuka Test 2 (Glades County, FL)

2.5

Xyleborus glabratus (mean + SE) a 2.0 8-wk Field Test Fisheating Creek State Park (8 Mar - 3 May 2013) 1.5

1.0

Beetles / trap / week / / trap Beetles b 0.5 b

0.0 Cubeb bubble Manuka patch Control

(Kendra et al. 2014. J. Pest Science)

• At very low population levels, cubeb bubble lure captured 6 times more RAB than manuka Test 3 (Highlands County, FL)

X. glabratus weekly captures

160 a 18 Xyleborus glabratus (mean + SE) Cubeb 140 Phoebe Manuka 16 12-wk Field Test Blank 120 14 Highlands Hammock State Park 100 12 b 10 80

8 Beetles / week / Beetles 60 c

Beetles / trap / wk 6 40 4 c 20 2 0 0 123456789101112 Cubeb Phoebe Manuka Control Week (Kendra et al. 2015. J. Econ. Entomol.) • Cubeb > phoebe > manuka • Cubeb field life of 3 months Lure 1.6 30 α-copaene Cubeb Emissions B 1.4 Manuka 1.2

25 g) • Manuka:  1.0 0.8 20 g) 0.6  High initially, ( Quantity 0.4 expon. decay 15 0.2 0.0 (large surface area: ( Quantity 10 volume ratio) 30 40 50 60 70 80 Time (d) 5 • Cubeb: Low initially, 0 0 1020304050607080 slow, steady Time (d) release (Kendra et al. 2015. J. Econ. Entomol.) (smaller surface area: volume ratio) Conclusions Cubeb bubble lure • More attractive than the manuka lure • Has a field life of at least 3 months, due to extended low release of sesquiterpenes • Best product currently available for detection of RAB (and less expensive than the manuka lure)

However, cubeb lure contains a complex mixture of terpenoids; specific attractive chemicals had not been confirmed. Further Improvement of RAB Lure (Collaboration with Synergy)

Goal = Elucidate the primary attractants in cubeb oil. • Used fractional distillation to separate whole oil into 17 fractions (based on chemical boiling point) • Fractions formulated as bubble lures – EAG analyses – 2‐choice bioassays • Only 2 fractions were significantly attractive to RAB; both were high in α‐copaene and α‐cubebene • Decided to focus on α‐copaene first New Lure Evaluations 16 A a 14 Two prototype lures prepared 12 10 / trap / wk / • 8 b Copaiba oil b (9% α‐copaene, no cubebene) 6

X. glabratus X. 4

2 • Proprietary oil product c 0 (50% α‐copaene) 50% Cop Cubeb Copaiba Control

180 B 160 50% Cop Cubeb 12‐wk field test 140 Copaiba Control 120

• Compare new lures to cubeb lure / wk 100

(10% α‐copaene, 10% α‐cubebene) 80

60 X. glabratus X. Conclusions 40 20 • (‐) α‐copaene = primary attractant 0 123456789101112 • Increased copaene = improved lure Week Kendra et al. J. Pest Sci. (in review) THANK YOU! Sources of Funding • USDA National Plant Disease Recovery System • Florida Avocado Administrative Committee Recent Publications • Kendra, P. E., W. S. Montgomery, J. Niogret, E. Q. Schnell, M. A. Deyrup, and N. D. Epsky. 2014. Evaluation of seven essential oils identifies cubeb oil as most effective attractant for detection of Xyleborus glabratus (Coleoptera: : Scolytinae). J. Pest Sci. 87: 681‐689.

• Kendra, P. E., W. S. Montgomery, J. Niogret, G. E. Pruett, A. E. Mayfield III, M. MacKenzie, M. A. Deyrup, G. R. Bauchan, R. C. Ploetz, and N. D. Epsky. 2014. North American Lauraceae: Terpenoid emissions, relative attraction, and boring preferences of redbay , Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae). PLoS ONE 9 (7): e102086.

• Kendra, P. E., J. Niogret, W. S. Montgomery, M. A. Deyrup, and N. D. Epsky. 2015. Cubeb oil lures: Terpenoid emissions, trapping efficacy, and longevity for attraction of redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae). J. Econ. Entomol. 108: 350‐361.

• Kendra, P. E., W. S. Montgomery, M. A. Deyrup, and D. Wakarchuk. Improved lure for redbay ambrosia beetle developed by enrichment of α‐copaene content. J. Pest Sci. (in review).