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INTRODUCTION Background and research needs • ALB introduced from China and is a serious pest of many hardwood tree species, ALB first detected in New York City (1996) and spread to Carteret and Woodbridge, New Jersey. Other infestations were detected in Chicago (1998), Toronto, Canada (2003), and most recently Worchester, Massachusetts (2008) • Survey and detection is still done by visual surveys, underscoring the need for semiochemical-based lures • Long-range attractants are the best hope for developing surveying and monitoring tools, and can be used in combination with fungal pathogens for potential biological control. ALB Pheromones – what is known • Contact sex pheromones in female ALB ellicit mating behaviors in males (Zhang et al. 2003) • Trail Pheromones: evidence for a trail following pheromone suggests a pheromone involved in the mate guarding behavior of the males, and oviposition deterrence of females. In lab bioassays, males followed trails of females, and females avoid areas with these pheromones (Nehme et al. 2006) • Volatile pheromones: male ALB produce two volatile pheromones (dialkyl ethers) that are produced in highest abundance by 10-day old males (Zhang et al. 2002), and in a 1:1 ratio are attractive to virgin females in bioassays using an olfactometer (Nehme et al. 2006), and there is some evidence of female attraction in the field (Nehme et al. 2007). Although there is some evidence of field attraction, we hypothesize these pheromones are short range attractants. New evidence for long-range pheromones: • Young virgin females have unique cuticular hydrocarbon profiles (Figure 1), and potentially encode information regarding age and mating status • Activation of the virgin female extracts produce volatile compounds that elicit male antennal responses and attraction by male ALB (vs. activated mated female extracts) in olfactometer bioassays, and was confirmed using synthetic compounds • Discriminant analysis among groups of beetles reveal six compounds (structurally related to the contact pheromones), and are hypothesized to have multiple roles in the mating sequence as contact pheromones, trail pheromones, and antedecents to long-range pheromones. • Six compounds are produced in maximum abundance by virgin females, which sharply reduce their production immediately after mating, and were the basis for the lures tested in China in 2008. HYPOTHESES • Synthetic virgin female pheromone lures are more attractive to ALB compared to mated female pheromone lures • Host plant volatiles and pheromone lures are more attractive than using the pheromone lures alone • Using the additional compounds from the discriminant analysis, we hypothesize activated extracts of virgin females that have maturation fed will enhance the attraction of the synthetic lures plus plant volatiles F i g u r e 1 . D i s c r i m i n a n t A n a l y s i s o f c u t i c u l a r h y d r o c a r b o n p r o f i l e s o f i n d i v i d u a l b e e t l e s = 7 . 3 7 0 6 , p FIELD RESPONSE OF THE ASIAN LONGHORNED BEETLE TO HOST AND FEMALE VOLATILES Jacob D. Wickham and Stephen A. Teale Department of Environmental and Forest Biology 1 Forestry Drive, Syracuse, New York 13210 METHODS Four field experiments were conducted in Ningxia, China to evaluate suspected pheromones and host attractants (Tables 1 and 2). Pheromone lures were placed on intercept panel traps in a randomized complete block design (RCBD). Release rates were determined gravimetrically and lures were changed weekly. July 2006 experiment 1: (4 treatments x 6 replicates) We compared pheromone blends of six compounds from mated and virgin females. July 2006 experiment 2: (7 treatments x 7 replicates) Pheromone blends were tested with five plant volatiles and linalool oxide. July 2007: (7 treatments x 7 replicates) Pheromone blend plus plant volatiles and linalool oxide were formulated to have more natural release rates. July 2008: (8 treatments x 6 replicates) The goal of the experiment was to augment the attraction of the lures used using new compounds that serve as antecedents to the volatile pheromones used in 2006 and 2007. In November 2007, we discovered that maturation-fed virgin females have unique cuticular hydrocarbon profiles (Figure 1), and six additional compounds significantly contributed to the discrimination of these females. Formulations of these compounds were prepared by extracting them from maturation fed virgin females and activating them. RESULTS July 2006 Experiments 1 and 2 – Virgin female blends were more attractive than lures baited with a blend of compounds derived from mated females (Figure 3), and the virgin female lured plus plant volatiles and linalool oxide attracted more beetles then controls (Figure 4). RESULTS CONT. July 2007 – Female pheromone lures with improved release rates captured more beetles compared to controls, and captured significantly more males (Figure 5) C hina 2007:M ale vs.Fem ale A LB captured 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 pv + LO LO partialfem ale blend fullfem ale blend partialfemale blend + pv + LO fullfem ale blend + pv+ LO control m ean beetle/trap/w eek m ales/trap fem ales/trap a*† a*† pv = plant volatiles LO = linalool oxide Treatments * 2 = 6, df = 1, p < 0.05; † treatment caught significantly more males than females 2 = 4, df = 1, p < 0.05 b Figure 5. Field test of female pheromone blends, the partial female pheromone blend is comprised of three synthetic antennally active compounds July 2008 – the naturally activated virgin female extracts plus the synthetic blends from 2007 captured significantly more beetles compared to controls, and captured significantly more males than females (Figure 6). 0 1 2 3 4 5 6 7 8 M ated Fem ale Blend V irgin Fem ale Blend V irgin Fem ale single cm pd Control m ean beetles/trap/w eek + 1 S.E. b a* b bc * χ 2 = 9.44 df = 3 p < .024; ANOVA assumptions not met (too many zeros) Figure 3. Experiment 1 - Field test of virgin and mated female pheromone blends in China, 2006. C hina 2006:fem ale blend trials w /hostvolatiles 0 1 2 3 4 5 partial fem ale blend + pv partial fem ale blend fem ale blend + pv fem ale blend fem ale blend + pv+ LO fem ale blend + LO control m ean b eetles/trap /w ee a** b pv = plant volatiles LO = linalool oxide Treatment s Figure 4. Experiment 2 - Field test of virgin female compounds and plant volatiles and linalool oxide ** 2 = 12, df = 1, p < 0.01 b a* a* † pvlo = plant volatiles plus linalool oxide * 2 = 6, df = 1, p < 0.05; † treatment caught significantly more males than females 2 = 4, df = 1, p < 0.05 Figure 6. Field test of virgin female pheromone blends in China, 2008 (n=6). New compounds, in the form of female extracts, were added to the lures. Note: “Lab Act. Virgin” means that virgin female extracts were activated using a laboratory-controlled reaction, whereas “Nat. Activ. Virgin” means virgin female extracts were allowed to undego a natural activation to generate the volatiles (one female equivalent per lure). CHINA 2008 CONCLUSIONS • Virgin female blends were more attractive than mated female blends. • A multicomponent blend attracted more beetles than a single compound. • Synthetic female pheromones plus plant volatiles and linalool oxide were more attractive than controls. • Lures with natural release rates of synthetic female pheromones plus plant volatiles and linalool oxide captured more beetles compared to controls, AND captured significantly more males. • The new formulations containing the naturally activated female extracts including the compounds found in young maturation-fed virgin females plus synthetic female pheromones, plus plant volatiles and linalool oxide captured more beetles compared to controls, AND again captured significantly more males. ACKNOWLEDGEMENTS • Alphawood Foundation • National Science Foundation East Asia & Pacific Summer Institute (NSF-EAPSI) • Cornell University: Ann Hajek and Sana Gardescu • SUNY-ESF – Technical assistance: Paul Bryant, Max Collignon, Dominick Skabeikis, and Cameron Blank • Beijing Forestry University – Host professor Xu Zhichun, and students Sample chromatograms demonstrating differences between newly eclosed, pre-feeding male (TOP) vs. older male after following maturation feeding (BOTTOM) newly eclosed male (pre- feeding) maturation fed male com pound purity natural source source release rate fem ale com pound 1 98% fem ale Aldrich 10 fem ale equivalents/day fem ale com pound 2 98% fem ale Aldrich 10 fem ale equivalents/day fem ale com pound 3 89.93% fem ale synthesized 10 fem ale equivalents/day fem ale com pound 4 92.40% fem ale synthesized 10 fem ale equivalents/day fem ale com pound 5 94.25% fem ale synthesized 10 fem ale equivalents/day fem ale com pound 6 50.22% fem ale synthesized 10 fem ale equivalents/day cis-3-hexen-1-ol 98% host Aldrich 500 m g/day transcaryophyllene 80% host Aldrich 500 m g/day cam phene 95% host Aldrich 500 m g/day linalool 97% host Aldrich 500 m g/day linalool oxide 95% host/fem ale TC I(Japan) 15 ug/day delta-3-carene 99% host Aldrich 500 m g/day 2008 virgin fem ale cm pds 100% fem ale fem ale extract 1 fem ale equivalent/day Table 1. List of compounds used in field experiments in China Experiment1 Experim ent2 single com pound (com pound 4) X partial fem ale blend (com pounds 1,2,4) X X X X full fem ale blend (com pounds 1-6) X X X plantvolatiles plus linolool oxide (pvlo) X X X 2008 virgin fem ale com pounds X July 2006 Sum m ary ofcom pounds used in experim ents July 2007 July 2008 Table 2. Lure formulations used in field experiments in China LITERATURE CITED Nehme, M.E., K. Hoover, T. Baker, A. Zhang, and M. Keena. 2006. Laboratory bioassays on the maleproduced pheromone of Anoplophora glabripennis 2006 [cited. Available from http://esa.confex.com/esa/2006/techprogram/paper_25809.htm. Nehme, M.E., K. Hoover, A. Zhang, and M. Keena. Field bioassays on ALB male-produced pheromone 2007 [cited. Available from http://esa.confex.com/esa/2007/techprogram/paper_31615.htm. Zhang, A., J. E. Oliver, J. R. Aldrich, B. Wang, and V. C. Mastro. 2002. Stimulatory beetle volatiles for the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). Z Naturforsch 57 (5-6):553-8. Zhang, A., J. E. Oliver, K. Chauhan, B. Zhao, L. Xia, and Z. Xu. 2003. Evidence for contact sex recognition pheromone of the Asian longhorned beetle, Anoplophora glabripennis(Coleoptera: Cerambycidae). Naturwissenschaften 90 (9):410-3.
