What is an EAB’s Favorite Meal and Why is it Important?

1. What is an EAB’s 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. Host Range Objectives 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

5. (1) Alternate Species of Concern 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 1st stage larval galleries.

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 1st stage larval galleries

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

10. No-choice lab bioassay Black walnut Black ash

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 m2 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. Landing Rates of EAB Adults Total adults Elm Hickory Green ash Pipe White ash Hackberry Black walnut

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 m2in Suspended logs 3 logs per tree on 5 trees in 2 sites; 30 total logs Green ash 195.5 ± 49.5 Elm 0.0 Walnut 0 ± 0

18. Host range field test: 1st & 2nd 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. • 1st 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 Green vs. White ash trees 4 neighborhoods over 3 years. 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 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. a 2 males 1.8 Host selection - landing b 1.6 b 1.4 No. of beetles on foliage (mean +SE) 1.2 c 1 c 0.8 c 0.6 0.4 0.2 0 a 2.2 females 2 ab ab 1.8 1.6 1.4 bc 1.2 c c 1 0.8 0.6 0.4 0.2 0 green black white blue European Manchurian species

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

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 16 a 20 a 14 18 12 16 14 10 b 12 total amount consumed (cm)2 proportion consumed (%) 8 10 b 8 6 6 4 4 2 2 0 0 green Manchurian green Manchurian

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

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. healthy 3 green 2.5 black white 2 blue 1.5 European Manchurian 1 0.5 0 -0.5 -1 -1.5 -3 -2 -1 0 1 2 3 4 stressed 2 1.5 1 0.5 0 -0.5 -1 -1.5 -2 -2.5 -3 -3 -2 -1 0 1 2 3 4

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. americana F. chinensis Chiam 1 Chiam 2

40. Hybrid study: Beetles released in cages

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

42. 30 25 20 15 10 5 0 16 14 12 10 8 6 4 2 0 F. americana F. chinensis Chiam 1 Chiam 2 Hybrid study: feeding a a a a amount consumed (cm2) (mean + SE) a a a a proportion consumed (%) (mean + SE) species

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

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