1. What is an EABs Favorite Meal and Why is it Important? Therese M. Poland Deborah G. McCullough Andrea C. Anulewicz Deepa S. Pureswaran

2. EAB Host Range China - Fraxinus spp. Japan & Korea – Juglans - Pterocarya - Ulmus North America - ??

3. Why Study Host Range? Know what species are at risk Understand susceptibility of urban & forest trees Improve survey and detection Enhance management options Propogate and enhance resistance

4. Assess EAB feeding preference & larval development (1) Alternate species of concern (2) Compare North American Species (3) Stressed & vigorous trees (4) North American & Asian ash Host Range Objectives

5. No-choice lab bioassay Caged adult female & male with wood section & ash leaf. American elm, black walnut, shagbark hickory, hackberry & privet tested. Reared until females died. After 4 weeks, eggs counted & bark dissected to assess 1 st stage larval galleries. (1) Alternate Species of Concern

6. EAB eggs on or just under bark

7. Mean no. eggs per cm2 no-choice test; n = 5 No. eggs per cm2

8. Evaluated 1 st stage larval galleries

9. Density of galleries per 100 cm2 no-choice test; n = 5

10. No-choice lab bioassay Black ash Black walnut

11. Caged-Stem Bioassay Screen cages confined 3 pairs of beetles on each tree Dead beetles replaced June-Sept. Stems dissected: 4 blocks in Nov. 6 blocks in March Repeated in 2004 & 2005

12. EAB Gallery Density per m 2 on Caged Stem Bioassay

13. Host Range Field Tests Ash, Elm, Walnut, Hickory, Hackberry logs & drain pipe set on t-posts at 4 sites. Adult landing rates monitored using Tanglefoot. Logs dissected.

14. Green ash White ash Elm Black walnut Hickory Hackberry Pipe Landing Rates of EAB Adults Total adults

15. Gallery Density in Logs Strapped to T-Posts

16. Host Range Field Tests Green ash, elm & walnut logs suspended in heavily infested ash tree canopies; Will EAB females make a mistake? Logs dissected.

17. Mean density of EAB galleries per m 2 in Suspended logs Green ash195.5 ± 49.5 Elm 0.0 Walnut 0 ± 0 3 logs per tree on 5 trees in 2 sites; 30 total logs

18. Host range field test: 1 st & 2 nd stage larvae inserted into green ash, elm & black walnut

19. Larval insertion: green ash, elm & black walnut trees & logs

20. Alternate Species Results Adults will oviposit on alternate species under no-choice conditions. Oviposition mistakes occur but rare. 1 st stage larvae fed readily on ash species; a few larvae attempted to feed on other species but development impaired.

21. (2) Preference Among North American Ash Species 2003: canopy dieback was significantly higher in green ash than white ash 2004: canopy dieback increased in both species, still higher in green ash 2005: canopy dieback reached 100% for most green ash and rose sharply in white ash Green vs. White ash trees 4 neighborhoods over 3 years. Similar pattern for exit holes and woodpecker attacks.

22. Preference Among North American Ash Species White vs. Blue ash trees 2 woodlots over 2 years. 2004: exit holes and woodpecker attacks were significantly higher in white ash than blue ash 2005: exit holes and woodpecker attacks increased in white ash but not blue ash and the difference was more significant.

23. Host selection and feeding preference on ash spp. Green – F. pennsylvanica Black – F. nigra White – F. americana Blue – F. quadrangulata European – F. excelsior Manchurian – F. mandshurica

24. Experimental set up beetles released

25. Host selection - landing –number of beetles on each ash species counted every 2h during the day for 48h Feeding preference – amount consumed –Leaves were scanned before and after the experiment –Amount fed determined

26. species 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 greenblackwhiteblueEuropeanManchurian ab bc a cc females 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 a b b c c c males No. of beetles on foliage (mean +SE) Host selection - landing

27. 0 2 4 6 8 10 12 ab a cd bc d males 0 2 4 6 8 10 12 14 greenblackwhiteblueEuropeanManchurian a a a a b b females Amount of foliage consumed (%) species Feeding preference

28. (3) Stressed and Vigorous Trees Trap trees with different stress treatments: Girdled, herbicide, methyl jasmonate, wounded Plantation study: Girdled, fertilized, and control trees of different ash species

29. (4) Beetle performance on American vs. Asian Species Raised beetles (n=40) on green and Manchurian ash until they died Leaves changed and retrieved each week Measured amount consumed, weight gained / lost over two weeks and longevity

30. Foliage consumption 0 2 4 6 8 10 12 14 16 18 20 proportion consumed (%) 0 2 4 6 8 10 12 14 16 total amount consumed (cm) 2 a b a b greenManchurian greenManchurian

31. Beetle performance 0 1 2 3 4 5 6 greenManchurian weight loss at age 2 weeks (mg) 0 2 4 6 8 10 12 14 16 18 20 greenManchurian longevity (days) a a a a

32. Results Total amount and proportion of foliage consumed was greater in beetles fed on green ash compared to Manchurian No difference in weight or longevity

33. What may be going on? Higher nutritive value of Manchurian ash? Presence of anti-feedants in Manchurian ash? Higher consumption of green ash by larvae mortality of trees in North America?

34. Compounds Identified and Quantified in Different Ash Species hexanol E-2-hexenal Z-3-hexenol E-2-hexenol butoxyethanol Z-3-hexenyl acetate hexyl acetate E-β-ocimene nonanal / linalool nonatriene Z-E-α-farnasene Green White Black Blue European Manchurian

35. Z-3-Hexenol

36. Z-3-hexenyl-acetate

37. -1.5 -0.5 0 0.5 1 1.5 2 2.5 3 -3-201234 green black white blue European Manchurian -3 -2.5 -2 -1.5 -0.5 0 0.5 1 1.5 2 -3-201234 stressed healthy

38. Hybrid study North American ashes are preferred relative to Chinese ashes What would happen with hybrids? Crossed F. americana with F. chinensis to obtain two putative hybrids chiam1 and chiam2 Tested beetle landing and feeding on the four genotypes Compared volatile profiles by aeration

39. Host selection by landing and feeding preference F. americanaF. chinensisChiam 1Chiam 2

40. Hybrid study: Beetles released in cages

41. Hybrid study: landing Observations over time Mean number of beetles

42. Hybrid study: feeding amount consumed (cm 2 ) (mean + SE) 0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 16 F. americanaF. chinensisChiam 1Chiam 2 proportion consumed (%) (mean + SE) species a a a a a a a a

43. Hybrid study: Aerations Super-Q

44. Compounds quantified hexanal e-2-hexenal z-3-hexenol e-2-hexenol z-3-hexenyl acetate hexyl acetate e-β-ocimene nonanal / linalool farnesene Gas chromatography

45. Hybrid study: Comparison of volatiles F. americana 0 5 10 15 20 25 30 35 40 F. chinensis 0 5 10 15 20 25 30 35 40 chiam1 0 5 10 15 20 25 30 35 40 chiam2 0 5 10 15 20 25 30 35 40 hexanal z-3-hexenol e-2-hexenal e-2-hexenol z-3-hexenyl_acetate hexyl_acetate e-ocimene linalool nonanal farnesene Amount (ng / g dry wt)

46. Hybrid study - Results Landing and feeding: no significant differences Hybrids have taken on some characteristics from F. americana parent Volatile profiles of hybrids different from either parent – may not be simply inherited

47. Conclusions EAB feed and develop in all ash species No evidence of larval survival or development on any non-ash species EAB prefer Green>Black>White>>Blue ash EAB prefer N. American ash to Asian ash Hybrids may be intermediate and volatile characteristics are not simply inherited Surveys should focus on preferred trees (green ash) More research needed to develop resistance and attractive lures