SBIO 599

1. Development of Pesticide against Asian Longhorned Beetles (Anoplophora glabripennis) Using Entomopathogenic Fungi and Nematodes. SBIO 599

2. Outline • Introduction • History • New York’s Battle • Asian Longhorned Beetle • Development of Pest Control • Insecticides • Fungus: Beauveria brongniartii • Fungal product • Nematodes • Conclusion

3. Asian Longhorned Beetle (ALB) is native to China and Korea. ALB have caused serious damage on poplar (Populus) and many trees have been killed in Asia. History

4. New York’s Battle • In 1996, ALB was discovered in Brooklyn, NY. • Over 1000 trees have been removed, chipped, or burned in first year. • Probably kill 30% of all urban trees – economic impact of more than $40 billion in damages.

5. Asian Longhorned Beetle • Morphology - (L) 20-35 mm x (W) 6-12 mm • Black shiny body • Long antennae • White spots • Life Cycle Egg --> Larva --> Pupa --> Adult It requires 1-2 years to develop from egg to adult

6. ALB -cont. • Biology - fly ~300m in a single flight - live for up to 60 days - adult activity peaking in July * Adults mate on the trunks of host trees * Female lays eggs inside of trees * Eggs hatch with in 1-2 weeks * Larvae feed deep inside of trees and transform to pupae and then adults. * The new generation of adults exit through 6-18 mm holes by chewing host trees.

7. Why should we care? (USDA, 2001) 7

8. Outline • Introduction • History • New York’s Battle • Asian Longhorned Beetle • Development of Pest Control • Insecticides • Fungus: Beauveria brongniartii • Fungal product • Nematodes • Conclusion

9. Insecticides • For the pest control of ALB, several chemical insecticides were tested in US. • Imidacloprid is an insecticide manufactured by Bayer. It is sold under a variety of trade names including Admire, Advantage, and Gaucho. • However, none of these insecticides worked well for controlling ALB. molecular formula of Imidacloprid

10. Outline Introduction History New York’s Battle Asian Longhorned Beetle Development of Pest Control Insecticides Fungi Fungal product Nematodes Conclusion 10

11. Shimazu et al., 2002

12. Entomopathogenic Fungi Net mortality of the inoculated adults increased after around day 6. Many adults inoculated with both F-1101 and F-263 were infected and killed in moderate numbers.

13. LC50 were calculated from net mortalities to evaluate the infectivity of the fungi • The LC50 of B. brongniartii F-1101 to adults of ALB was around 104 conidia/ml.

14. Outline Introduction History New York’s Battle Asian Longhorned Beetle Development of Pest Control Insecticides Fungus: Beauveria brongniartii Fungal product Nematodes Conclusion 14

15. Development of fungal product as new commerciogenic insecticideHiguchi et al. (1997), Dubois et al. (2004), and Hajek et al. (2006).

16. Comparison chart 16

17. Field Tests of Sheet Cultures

18. Field Tests of Sheet Cultures (Higuchi et al., 1997) 18

19. Conclusion • The sheet cultures clearly provide a useful method of controlling ALB population by decreasing the proliferation, and it had high infectivity to ALB (Higuchi et al. 1997).

20. Comparison chart 20

21. Material and Method Two sheet cultures of B. brongniartii were used: • NBL 851(fungus was isolated from the cadaver of an Yellowspotted longicorn beetle, Psacothea hilaris) • WU 20 (fungus was isolated from the cadaver of an ALB) The number of oviposition per female estimated by Sum of all oviposition scars / Sum of all exit holes x 0.5 (Dubois et al., 2004) 21

22. Field Tests of Sheet Cultures -cont. • Two sheet cultures of B. brongniartii were used: NBL 851(fungus was isolated from the cadaver of an Psacothea hilaris) and WU 20 (fungus was isolated from the cadaver of an ALB)

23. Conclusion • The sheet cultures clearly provide a viable method of controlling reproductivity. • The sheet culture decreased population of ALB due to… - decreased reproduction by killing females before laying eggs - decreased female lifespan (Dubois et al., 2004) 23

24. Comparison chart 24

25. M&M • 2 different fungal bands were tested • B. brongniartii NBL-851(isolated from P. hilaris) and Metarhizium anisopliae VD-1 (isolated from ALB) • Chi-squared tests were used to compare observed reproduction in treatments with controls.

26. Field Tests of Sheet Cultures The number of Oviposition/female in both fungal treatments were approximately half those found in controls (chi-suared test: P < 0.05)

27. Conclusion • The sheet cultures clearly provide a viable method of controlling reproductivity. • The sheet culture decreased population of ALB due to… - decreased reproduction by killing females before laying eggs - decreased female lifespan (Hajek et al. 2006) 27

28. Summary chart 28

29. Entomopathogenic Fungi • The sheet culture containing entomopathogenic fungi controls adult ALB due to decrease their lifespan and reproduction. • However, it was NOT effective to ALB Larvae.

30. Results from Shimazu et al. (2002)

31. Outline Introduction History New York’s Battle Asian Longhorned Beetle Development of Pest Control Insecticides Fungus: Beauveria brongniartii Fungal product Nematodes Conclusion 31

32. Entomopathogenic NematodesComparison Chart

33. M&M (filter paper bioassay) (Solter et al. 2001) 33

34. 8 /10 8 /10 2 /10 1 /10 (Solter et al. 2001) 34

35. Conclusion • S.carpocapsae and H. marelatus had highest infectivity and mortality against ALB larvae. • The larvae exposed to S.carpocapsae and H. marelatus died quickly. • LD50 (median lethal dose) was ~19 nematodes for ALB larvae. • S.carpocapsae and H. marelatus had an ability to invade, kill, and reproduce in ALB larvae. (Solter et al. 2001) 35

36. Entomopathogenic NematodesComparison Chart 36

37. M&M (filter paper and diet cup bioassay) (Fallon et al. 2004 & 2006) 37

38. Fig. 3. Mortality (bar) and nematode infectivity (line) of A. glabripennis larvae exposed to S. feltiae, S. riobrave, S. carpocapsae, S. glaseri, H. marelata, and water on filter paper (A), and in diet cups bioassay (B). Single larvae were each exposed to 100 IJs (N = 7 larvae/ treatment). Line points headed with the same letter are not different among treatments [P < 0.0001; (A), P < 0:0001; (B) ]. Bars headed by an ‘‘*’’ are significantly different from the control (P > 0.05). (Fallon et al. 2004) 38

39. (Fallon et al. 2006) 39

40. Results • S.carpocapsae and S. feltiae had highest infectivity and mortality against ALB and cottonwood borer larvae. • The larvae exposed to S.carpocapsae and S. feltiae died quickly against ALB larvae. • LD50 (median lethal dose) was 9-17 nematodes for ALB larvae. (Solter et al. 2001) 40

41. Conclusion • S.carpocapsae and S. feltiae had an ability to invade, kill, and reproduce in ALB larvae and cottonwood borer larvae. • Moreover, the percent mortality against cottonwood borer showed ~50%. Solter et al. (2006) suggested that S.carpocapsae and S. feltiae may have an ability to invade to more specific insect. (Solter et al. 2001) 41

42. Entomopathogenic NematodesComparison Chart 42

43. Outline Introduction History New York’s Battle Asian Longhorned Beetle Development of Pest Control Insecticides Fungus Fungal product Nematodes Conclusion 43

44. Summary (Fungi) • B. brongniartii against ALB were highly effective as a insecticide (Shimazu et al., 2002). • The sheet culture of B. brongniartii in field trials demonstrated that adults ALB were infected and died more quickly and fewer reproduction (Dubois et al. 2004; Higuchi et al. 1997; Hajek et al. 2006). • These results suggest that this unique application method holds promise for controlling the Asian Longhorned Beetle.

45. Summary (Nematodes) Nematodes (H. marelatus,S. carpocapsae, and S. feltiae ) were highly effective against ALB larvae (Solter et al. 2001; Fallon et al. 2004). The LD50 examination showed a potential to be commercialized as a pesticides (Solter et al. 2001; Fallon et al. 2004). These results suggest that the liquid culture of entomopathogenic nematodes could be a great product for controlling the Asian Longhorned Beetle. 45

46. Conclusion • Using biopesticides containing entomopathogenic fungi or nematodes has a great potential to control ALB and other pest instead of removing, chipping, or burning of infested host trees. • These studies provide a means of making possible a more effective pesticide in regions where chemical methods were previously inapplicable.

47. THANK YOU!! Questions?

48. References • Dubois T., Zengzhi Li, Hu Jiafu, and Ann E. Hajek. 2004. Efficacy fiber bands impregnated with Beauveria brongniartii cultures against the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cermbycidae). Biological Control 31: 320-328. • Fallon J. Declan, Leellen F. Solter, Leah S. Bauer, Deborah L. Miller, James R. Cate, and Michael McManus. 2006. Effect of entomopathogenic nematodes on Plectrodera scalator (Fabricius) (Coleoptera: Cermbycidae). Journal of Invertebrate Pathology 92: 55-57. • Fallon J. Declan, Leellen F. Solter, Melody Keena, Michael McManus, James R. Cate, and Lawrence M. Hanks. 2004. Susceptibility of Asian longhorned beetle, Anoplophora glabripennis (Motchulsky) (Coleoptera: Cermbycidae) to entomopathogenic nematodes. Biological Control 30: 430-438. • Haack, Robert A., Kenneth R. Law, Victor C. Mastro, Sharon H. Ossenburgen, Bernard Raimo, J. 1997. New York's battle with the Asian long-horned beetle. Journal of Forestry 95 (12): 11-15. • Hajek, A. E., B. Huang, T. Dubois, M. T. Smith, and Z. Li. 2006. Field studies of control of Anoplophora glabripennis (Coleoptera: Cermbycidae) using fiber bands containing the entomopathogenic fungi Metarhizium anisopliae and Beauveria brongniartii. Biocontrol Science and Technology 16 (4): 329-343. • Higuchi Toshio, Takeshi Saika, Shuji Senda, Tomohiro Mizobata, Yasushi Kawata, and Jun Nagai. 1997. Development of biorational pest control formulation against longicorn beetles using a fungus, Beauveria brongniartii (Sacc.) Petch. Journal of Fermentation and Bioengineering 84: 236-243. • Rynk, Robert. 2003. Meet the beetles. BioCycle 44 (4): 46-51. • Shimizu Mitsuaki, Zhang Bo, and Liu Yi-ning. 2002. Fungal Pathogens of Anoplophora glabripennis (Coleoptera: Cerambycidae) and Their Virulences. Shinrin SougouKenkyuujyo Kenkyuhoukoku1 (382): 123-130. • Solter, L. F., Melody K., James R. C., Michael L. M., and Lawrence M. H. 2001.Infectivity of Four Species of Nematodes (Rhabditoidea: Steinernematidae, Heterorhabditidae) to the Asian Longhorn Beetle, Anoplophora glabripennis (Motchulsky) (Coleoptera: Cerambycidae). Biocontrol Science and Technology 11: 547-552. • United States Department of Agriculture. 2001. Wanted: The Asian Longhorned Beetle. USDA-APHIS Program Aid No. 1655: 1-16.