The Pennsylvania State University

The Graduate School

Department of Entomology

DEVELOPING MONITORING TRAPS

FOR THE ASIAN LONGHORNED BEETLE

A Dissertation in

Entomology & Comparative and International Education

by

Maya E. Nehme

© 2009 Maya E. Nehme

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

August 2009

The dissertation of Maya E. Nehme was reviewed and approved* by the following:

Kelli Hoover Associate Professor of Entomology Dissertation Advisor Co-Chair of Committee

Edwin Rajotte Professor of Entomology, IPM Coordinator and CI ED joint faculty Co-Chair of Committee

Thomas Baker Professor of Entomology

Melody Keena US Forest Service Research Entomologist and Adjunct Faculty of Entomology Special member

David Baker Professor of Education, Professor of Sociology

Gary Felton Professor of Entomology Head of the Department of Entomology

*Signatures are on file in the Graduate School

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ABSTRACT

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiinae), commonly known as the Asian longhorned beetle, is a wood-boring invasive species introduced from Asia to North America and Europe through solid wood packing material. A. glabripennis is a serious pest both in China and the U.S. This research project was developed in response to the need for efficient monitoring traps to assess population density and dispersal in the field and to detect new introductions at ports of entry. The first stages of the project aimed at filling the gaps in our knowledge of the effect of semiochemicals on A. glabripennis adult behavior and exploring potential use of these chemicals for monitoring purposes. Semiochemicals studied were the male- produced putative volatile pheromone vbutan-1-ol and 4-(n-heptyloxy)butanal) and plant volatiles.

The first series of experiments were conducted using the male-produced blend, its two components and plant volatiles in choice bioassays against a hexane control. In Y-olfactometer and walking wind tunnel bioassays, virgin females were more attracted to the male-produced blend and its alcohol component than males. Virgin males were even repelled at higher doses.

These results suggest that the male-produced pheromone plays a role in mate-finding. When plant volatiles were offered in the Y-olfactometer, males were more attracted than females. Out of 12 plant volatiles tested, (-)-linalool, cis-3-hexen-1-ol and linalool oxide were moderately attractive to both genders, while 3-carene and trans-caryophyllene were only attractive to males.

Combining the male pheromone blend with (-)-linalool alone or with cis-3-hexen-1-ol attracted significantly more males than did the pheromone alone. Combinations of the pheromone and plant volatiles were also tested in the greenhouse, along with four trap designs, namely

InterceptTM Panel, hand-made screen sleeve, Plum Curculio and Lindgren funnel traps. The

former two trap designs caught significantly more beetles than the latter two.

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Subsequently, field trapping experiments were conducted in China in the summers of

2007, with Intercept™ panel traps hung on poplar trees and 2008, with Intercept™ panel traps hung on poplar trees, screen sleeve traps wrapped around poplar trunks, and Intercept™ panel traps hung on bamboo polls 20 m away from host trees. Traps were baited with the A. glabripennis male-produced pheromone alone or in different combinations with plant volatiles.

Traps baited with the male-produced pheromone alone caught significantly more females than control traps in both years. The addition of a mixture of (-)-linalool, cis-3-hexen-1-ol, linalool oxide, trans-caryophyllene and trans-pinocarveol to the pheromone significantly increased trap catches of virgin females. Screen sleeve traps baited with a combination of (-)-linalool and the pheromone caught the highest number of beetles overall in 2008, while traps placed on bamboo polls caught the lowest number. While the logistics for the most effective implementation of a trapping program using a mixture of the pheromone and plant volatiles require additional studies, these results indicate that this pheromone has considerable promise as a monitoring tool for A. glabripennis in the field.

In order for the results of this project to be effectively used in China and the U.S. for monitoring A. glabripennis populations, a participatory action research (PAR) program was designed to involve local communities, governmental agencies, academic institutions and the forest industry in monitoring efforts. The design includes a program steering committee, composed of members of all target groups that will carry out diagnosis, prescription, and implementation of the project. Process and Impact evaluations will be conducted continuously during the program timeline of one year, starting in the fall of 2009 and ending in the fall of

2010. Implementation, which involves setting up traps after determining best practices by the local groups, will be conducted during the beetle flight season in Summer 2010. Results of the trapping season will then be evaluated and documented for use in the following field seasons.

Expected outcomes range from technical development of an effective protocol for monitoring

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to empowerment of local communities and farmer cooperatives that will benefit a large variety of Forestry-related activities.

In Summary, this thesis led to the discovery of promising lures and trap designs for the monitoring of A. glabripennis populations both in the U.S. and China. It also contributed to our

knowledge about the role of male-produced pheromones in mate-finding in this species. The PAR

program designed, if applied, should benefit all groups affected by this serious pest and fill the

existing gap for effective monitoring strategies.

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TABLE OF CONTENTS

LIST OF FIGURES ...... viii

LIST OF TABLES...... x

Acknowledgments ...... xi

Chapter 1 Literature review ...... 1

Introduction...... 1 Economic Impact...... 2 Need for monitoring techniques...... 4 Anoplophora glabripennis ...... 5 Taxonomy ...... 5 Host range ...... 6 Life cycle...... 7 Management of invasive species...... 10 Management of Anoplophora glabripennis ...... 10 Use of pheromones in management ...... 16 ALB Adult behavior and semiochemical-based communication...... 18 Synergism between insect pheromones and plant volatiles ...... 20 Public outreach and management of ALB ...... 22

Chapter 2 Attractiveness of Anoplophora glabripennis to male-produced pheromone and plant volatiles ...... 26

Abstract ...... 27 Introduction...... 28 Materials and Methods...... 31 Insects...... 31 Response of A. glabripennis adults to the two putative male-produced pheromone components...... 32 Response of A. glabripennis adults to plant volatiles...... 35 Response of A. glabripennis adults to a combination of the male-produced compounds and plant volatiles in the Y-olfactometer...... 36 Comparison of trap designs and lures in the greenhouse...... 36 Statistical analyses...... 39 Results...... 40 Response of A. glabripennis adults to the two putative male-produced pheromone components...... 40 Response of A. glabripennis adults to plant volatiles in the Y-olfactometer...... 44 Response of A. glabripennis adults to a combination of the male-produced compounds and plant volatiles in the Y-olfactometer...... 46 Comparison of trap designs and lures in the greenhouse...... 48 Discussion ...... 51 Acknowledgments...... 56

Chapter 3 Field testing of Anoplophora glabripennis male-produced pheromone and plant volatiles ...... 57

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Abstract ...... 58 Introduction...... 59 Materials and Methods...... 61 Comparison of lure treatments for Intercept panel traps in 2007...... 61 Comparison of lure treatments, trap design and placement in 2008 ...... 62 Field observations of mate-finding, flight and dispersal behaviors of A. glabripennis...... 65 Statistical analyses...... 66 Results...... 67 Comparison of lure treatments for Intercept panel traps in 2007...... 67 Comparison of lure treatments for Intercept panel traps in 2008...... 68 Comparison of trap designs and placement in 2008...... 71 Influence of tree infestation level on trap catches...... 75 Field observations of mate-finding, flight and dispersal behaviors of A. glabripennis...... 77 Discussion ...... 78 Acknowledgments...... 83

Chapter 4 Find the beetles/ fā jué jiă chóng!: A Plan for Participatory Action Research to detect and manage Asian Longhorned Beetle in China and the U.S...... 84

Introduction...... 84 History and description of the Asian longhorned beetles problem ...... 84 Specific aims of the campaign ...... 87 Background ...... 89 Significance of the project...... 96 Preliminary studies in China and a comparison with the U.S. and other countries ...... 98 Project Design and Methods ...... 104 Overview...... 104 Goals and Objectives...... 107 Evaluation ...... 109 Data Collection...... 110 Data analysis ...... 111 Data sharing ...... 112 Limitations and Challenges...... 112 Project timeline ...... 114 Suggested basic protocol for ALB monitoring...... 114

Chapter 5 Conclusions ...... 117

References cited...... 121

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LIST OF FIGURES

Figure 1-1: Life cycle of Anoplophora glabripennis...... 8

Figure 2-1: The modified walking wind tunnel, composed of a 1.5 m long glass tube connected to a plastic beaker closed by a 12 V fan at the end. Chemicals were applied on the cotton wick and beetles introduced at the free end of the tube and allowed to walk inside for 20 minutes. Tubes were virtually divided into three compartments: I (initial), M (middle) and NS (near source)...... 33

Figure 2-2: Trap designs used in the greenhouse trapping experiment. From left to right: the Lindgren funnel trap, the hand-made screen sleeve trap, the InterceptTM Panel trap, and the circle trunk curculio trap ...... 38

Figure 2-3: Mean number of visits of A. glabripennis adults to the source in the walking wind tunnel at 4 different chemical amounts (0.05, 0.5, 1, and 2 g) during 20 minutes. Treatments include the male pheromone (A. glabripennis male-produced pheromone blend), alcohol (4-(n-heptyloxy) butan-1-ol), and aldehyde (4-(n- heptyloxy) butanal) offered against a hexane control. Each pheromone treatment and chemical amount combination was tested separately for virgin males and virgin females (n=10 for each)...... 42

Figure 2-4: Percentage of virgin males and virgin females that chose plant volatiles when offered against a hexane control in a Y-olfactometer. Error bars represent standard errors of the mean. Letters over bars represent significantly different responses between males and females at the 95% confidence level. Percentages that are significantly higher or lower than 50% are labeled with an asterix. Numbers under plant volatiles names represent the number of beetles of each gender tested...... 45

Figure 2-5: Response of A. glabripennis virgin males and virgin females to combinations of the male-produced pheromone blend with plant volatiles offered against the pheromone alone in a Y-olfactometer. Error bars represent standard errors of the mean. Letters over bars represent significantly different means between treatments at the 95% confidence level ...... 47

Figure 2-6: Mean trap catches per week for the four trap designs tested in the greenhouse: the screen sleeve trap, Intercept Panel trap (or Intercept PT), Circle trunk trap (or Curculio trap) and Lindgren funnel trap. Error bars represent standard errors of the mean. Letters over bars represent significantly different means at the 95% confidence level...... 49

Figure 2-7: Mean trap catches per week per lure for the Intercept panel (top) and screen sleeve (bottom) traps. Error bars represent standard errors of the mean. Letters over bars represent significantly different means within lure treatments at the 95% confidence level. (MP= A. glabripennis male pheromone blend; L= linalool; LO = linalool oxide; Hex= (Z)-3-hexen-1-ol; HA= (Z)-3-hexenyl acetate; MPL = male

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pheromone blend + linalool; MPHex = male pheromone blend + (Z)-3-hexen-1-ol; MPHA= male pheromone blend + (Z)-3-hexenyl acetate; ref. Table 1 for amount ...... 50

Figure 3-1: Intercept TM PT traps (left) and screen sleeve traps (right)...... 63

Figure 3-2: Mean number of A. glabripennis adults caught in Intercept TM PT traps in China (July 2007), based on total caught per lure change. Controls consisted of unbaited traps. Live female and live male traps contained 5 live females or males, respectively, in small cages attached to the trap. The pheromone treatment consisted of 10 µg of the male-produced pheromone blend; the alcohol treatment of 10 µg of 4-(n-heptyloxy) butan-1-ol; the aldehyde treatment of 10 µg of 4-(n-heptyloxy) butanal; and MPPL of 10 µg of the pheromone and 100 µg of each of linalool and (-) -trans-pinocarveol. All chemicals were applied on rubber septa. Error bars represent standard errors of the mean. Letters over bars represent significantly different means at the 95% confidence level among lure treatments for each gender (small letters for males and capital letters for females)...... 68

Figure 3-3: Mean number of male and female Anoplophora glabripennis caught in Intercept TM PT traps hung on host trees (Graph A) and screen sleeve traps (Graph B) at Site A in July and August 2008, based on total caught per lure change. L=linalool; MP=male-produced pheromone; MPL= male-produced pheromone + linalool; Mix= linalool + (Z)-3-hexen-1-ol + (-)-trans-pinocarveol + linalool oxide + trans-caryophyllene; MM= male-produced pheromone + linalool+ (Z)-3-hexen-1-ol + (-)-trans-pinocarveol + linalool oxide + trans-caryophyllene. Error bars represent standard errors of the mean. Letters over bars represent significantly different means at the 95% confidence level across lure treatments for each gender. An asterisk over the bar represents significantly different means between males and females. Male trap catches were not significantly different among lures (no letters over bars)...... 70

Figure 3-4: Mean numbers of male and female Anoplophora glabripennis caught in Intercept TM PT traps hung on host trees (trap type 1) at Site B, China in July and August 2008, based on total caught per lure change. L=linalool; MP=male-produced pheromone; MPL= male-produced pheromone + linalool; Mix= linalool+ (Z)-3- hexen-1-ol + (-)-trans-pinocarveol + linalool oxide + trans-caryophyllene; MM= male-produced pheromone + linalool+ (Z)-3-hexen-1-ol + (-)-trans-pinocarveol + linalool oxide + trans-caryophyllene (see Table 3-2). No significant differences were found among treatments or between genders...... 71

Figure 3-5: Total trap catches per day per trap design/placement at Site A (Qing Tong Xia, Ningxia, China) from July 11 – Aug 7, 2008 over all lures. Treatments included differences in trap design and placement as follows: Intercept panel traps hung on host trees (n = 36); Intercept panel traps hung on bamboo poles 20 m far from host trees (n =18); and screen sleeve traps wrapped around host tree trunks (n = 18) ...... 74

Figure 4-1: PAR model...... 107

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LIST OF TABLES

Table 2-1: Lure treatments used in the greenhouse trapping experiments...... 38

Table 2-2: General linear model results: Effects tests for time spent near source and number of visits to source over the three highest chemical amounts (0.5, 1, and 2 μg)...... 41

Table 2-3: Percentage of A. glabripennis virgin males and virgin females that chose the male-produced blend, the alcohol alone, or the aldehyde alone when offered 1 g of each against a hexane control in a Y-olfactometer (Mean percentage ± SEM)...... 43

Table 3-1: Lure treatments used in field experiments in China in 2008...... 64

Table 3-2: Categories of host trees assigned according to infestation level...... 65

Table 3-3: Mean total trap catches of A. glabripennis for each trap design/placement at Sites A and B (Qing Tong Xia, Ningxia, China) for all lures for the entire trapping season of July 11 – Aug 7, 2008...... 72

Table 3-4: Generalized linear model results from analysis of trapping data to test main effects of lure treatments (6 total) and trap designs (hung on host trees) on total, female, and male trap catches at Site A...... 73

Table 3-5: Site details and correlation with trap catches at both sites in 2008 ...... 76

Table 4-1: List of groups to be involved in the PAR both in the U.S. and China...... 106

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Acknowledgments

I would like to extend my gratitude to all those who contributed to the completion of this work. I want to thank the Entomology department and the Comparative and International

Education Program direction, faculty, staff and fellow students for making this journey fruitful and enjoyable.

I would like first to thank Dr. Gary Felton, Entomology department head, with whom I had the first communications before applying to Penn State and who was always supportive and encouraging throughout the three years of my PhD program. On this note, I would like also to recognize the great contribution of my advisor at the American University of Beirut, Dr. Nasri

Kawar, who encouraged me to apply to Penn State.

None of the work I’ve done would have been possible without the continuous support, expert advice and encouragement of my very special and distinguished advisor, Dr. Kelli Hoover.

Thank you for believing in me, for sharing with me your knowledge and expertise and for giving

me all the great opportunities (travel, learning experiences and life experiences).

Dr. Melody Keena, U.S. Forest Service Northern Research Station, also contributed

immensely by offering space and insects in her lab for my work, and continuous support and

advice - I wouldn’t have been able to finish my degree in three years if it wasn’t for her help! I

would like also to acknowledge the hard work of the staff at the US Forest Service Northern

Research Station Quarantine Laboratory in Ansonia, CT in maintaining the insect colonies.

I owe my enrollment in CI-ED to Dr. Edwin Rajotte, who introduced me to the program and followed my progress throughout. I would also like to thank Prof Ladi Semali for believing in me and for his continuous support.

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A special Thank you goes to Dr. David Baker for 1) serving on my committee; 2) helping me immensely in my job search; and 3) including me in his team and giving me the opportunity of being a co-author on a report published by OECD in March 2009.

I would also like to express my sincere gratitude to Dr. Aijun Zhang, for providing the pheromones that were at the base of this project, and Drs. Tom Baker, Jim Tumlinson, Lawrence

Hanks, and Ann Hajek for scientific support and advice. I thank Dr. Katalin Borocsky for help with chemical analyses, Jacob Wickham, Zhichun Xu and Wenchao Xu for their help with the field work in China, and the Hoover lab, Scott Geib, Jim McNeil, Randa Jabbour and Erin Scully,

for their scientific contributions and friendly support.

My gratitude goes also to Deanna Behring and the staff of the International Programs

office of the College of Agricutlural, for giving me the opportunity of going to Europe on the

SUSPROT exchange program; and Dr. Negar Davis and the staff of the International Programs at

Penn State University, for making my international experience at Penn State memorable and

productive

A large part of this work was funded by Alphawood foundation and by USDA-APHIS-

PPQ. On this note, I’d like to thank Vic Mastro, Dave Lance, Joe Francese, Christine Markham,

Michael Stefan, from USDA-APHIS, for their continuous support and for facilitating the travel to

China.

I look back at the three last years that I spent in State College, and I cannot count the

beautiful memories and enjoyable moments I had, and they’re all due to the amazing group of

friends I have met here. To all my friends and fellow graduate students at the Entomology

department, the CIED program, the Lebanese Student Association, the Euro Club, and other

groups on campus, Thank you for all you’ve done!

To my wonderful family and all my friends in Lebanon: being away from you was hard!

Thank you for understanding and for always supporting and encouraging me! I Love you!

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Chapter 1

Literature review

Introduction

Among wood-boring insects, the Asian longhorned beetle (ALB), Anoplophora

glabripennis Motschulsky (Coleoptera: Cerambycidae), is considered a high-risk invasive species

in North America and Europe (USDA-APHIS 1998, EPPO 2004). Since the late 1970’s, it has

become a major forest pest in mainland China. It is endemic to China and Korea (Cavey et al.

1998, Nowak et al. 2001, Williams et al. 2001). In China, ALB was reported as early as the Qing

Dynasty (1644-1912) and is currently found in 25 provinces extending throughout a vast

geographic range, 21ºN to 43ºN latitude and 100ºE to 127ºE longitude (Yan 1985).

According to the United States Department of Agriculture’s Animal and Plant Health

Inspection Service (USDA- APHIS), pest risk analysis indicates that ALB was introduced into the

U.S. in solid wood packing materials (SWPM) from Asia (USDA-APHIS 2005b). Field infestations were first discovered in Brooklyn, New York in 1996 (Smith 2000a). This infestation spread to Long Island, Queens and Manhattan. In 1998, a separate introduction of ALB was found in the Ravenswood area of Chicago, as well as two suburban areas, Bloomingdale and

Summit. Beetles were then detected at two separate locations in New Jersey: Jersey City in 2002 and Middlesex/Union counties in 2004 (APHIS 2005). In 2006 and 2007, new infestations were also found on Prall’s Island, an uninhabited island lying close to Staten Island between New York and New Jersey (USDA-APHIS 2007) and in Eastern Linden in New Jersey (NJDA 2006).

Although the infestation in Chicago was thought to be eradicated in 2007 after four years of negative surveys, beetles were observed again in Deerfield, Cook County, Illinois in August 2008

(Bech 2008a). At the same time, a new infestation was reported in Worcester County,

Massachusetts. As of April 18th, 2009, in the Worcester area, MA, more than 20,000 trees have

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been removed, including over 8,000 infested trees and more than 12,000 high-risk trees (trees in the vicinity of infested trees, removed in order to ensure that the ALB population is controlled). A total of more than 635,000 trees are awaiting survey (MA Introduced Pests Outreach Project

2009).

In September 2003, a resident of Woodbridge, Toronto, Ontario reported beetles to the

Canadian Food Inspection Agency (CFIA) that were identified as ALB. Surveys led to the discovery of a localized infestation in an industrial park (Bell 2005). Latest records show additional infestations found in October 2007 within the city’s regulated area, on Jane Street and

Sheppard/Finch Avenue (CFIA 2007).

In Europe, ALB was first discovered in 2001 in the urban area of Braunau, Nothern

Austria, feeding on Norway maple, Acer platanoides L.. Additional infestations were found in

Montpellier, France in 2003 and Sainte-Anne-sur-Brivet in 2004 (Herard et al. 2006). In

Germany, ALB was first found in 2004 in Neukirchen, then in Bornheim in 2005 (EPPO 2008) and in 2007, a small outbreak was found in Corbetta, Italy (Maspero et al. 2007). An introduced population of ALB was also found in Yokohama, Japan in 2003 (Takahashi and Ito 2005).

Economic Impact

ALB larval feeding can cause widespread mortality to more than 24 deciduous tree species, starting as canopy dieback and resulting in tree death when girdling occurs, all of which cause economic damage to lumber, nursery and tourism industries. In addition, trees weakened by insect tunneling pose a serious threat to pedestrians and vehicles from falling limbs and trees.

ALB’s preference for maple species (Haack et al. 1996, Li et al. 1999b) can have a serious impact on maple forests managed for lumber and maple-dependent industries, such as maple syrup production.

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In China, A. glabripennis became a serious pest after the launching of the “Green Wall” campaign by the Chinese government through the Three-North Shelterbelt Programme in 1978.

The campaign called for large tree plantations, most of which were poplar species susceptible to

ALB (Hu et al. 2008). Since the early 1980’s, ALB infestations have caused more than 10 billion

Chinese Yuan (roughly $1.47 billion US) in losses, accounting for approximately 12% of total economic losses by forest pests (Lingafelter and Hoebeke 2002b).

Thousands of infested shade trees have been cut down and chipped in suburban neighborhoods in the Northeast and North Central United States (U.S.). Two highly prized and well-known urban parks in the United States ⎯ New York City’s Central Park and Chicago’s

Lincoln Park ⎯ are thus presently at risk (Becker 2000b). The estimated potential urban impact of ALB in the U.S. is a loss of nearly 12-61% of tree population in the cities, based on field data from nine U.S. cities, with an estimated value of $72 million- $23 billion per city, a corresponding estimated maximum potential impact of 34.9% of total canopy cover, 30.3% tree mortality, and a compensatory value loss of $669 billion (Nowak et al. 2001).

Several models have been developed to predict the survival and spread of ALB in different regions. Models were based on either temperature, such as in the Pest Risk assessment model CLIMEX (MacLeod et al. 2002), or ecological niches and presence of hosts/potential hosts such as in the Genetic Algorithm for Rule-set Prediction model (Peterson et al. 2004), among others. Studies on beetle survival, longevity and fecundity at different temperatures were also used for predictions (Keena 2006). A common conclusion among all was that A. glabripennis has the potential to invade most of North Eastern America and some regions in the west, as well as several places in Europe and Asia.

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Need for monitoring techniques

USDA-APHIS, together with various federal and state agencies and scientific institutions,

are currently attempting to eradicate ALB, as well as to detect and intercept new introductions.

Research efforts include developing detection and control methods, gathering information about

the dispersal potential of adults to be used in establishing quarantine boundaries, and defining

host preference and susceptibility indices of the beetle, which will help focus survey and

detection efforts and assist in the choice of tree species for re-planting in affected areas. Long-

term efficient management practices are needed. The implementation of such measures requires

the development of an adequate knowledge-base on the beetle’s biology and behavior and

definition of the factors regulating this behavior (Smith 2000a) .

Monitoring of ALB populations is still restricted to scouting by climbing individual

trees and searching for oviposition scars, sap flow, larval frass or dime-sized adult emergence

holes. Large bucket trucks are some times used to aid in detection (Solter et al. 2001, Dubois et

al. 2004b, Fallon et al. 2004, APHIS 2005, Hajek et al. 2006, Poland et al. 2006). This

approach is very expensive and time consuming. Rapid detection of this serious, invasive

species is necessary to prevent the spread of these beetles and intercept new introductions

before they establish.

This thesis addresses the need for an economically and environmentally sound monitoring tool to detect new infestations at ports of entry and in the field, and to monitor established and/or eradicated infestations in the quarantined areas. The purpose of this project is to develop an attractive lure for early detection and monitoring of ALB, coupled with the most attractive trap design. It also aims at improving our understanding of the interactions and mechanisms regulating ALB host location and mate finding.

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Anoplophora glabripennis

Taxonomy

Anoplophora glabripennis is one of 36 Anoplophora species, all grouped under the

common name, Asian longhorned beetles. The genus Anoplophora (Coleoptera: Cerambycidae:

Lamiinae) is widely distributed throughout Asia, mainly in tropical and sub-tropical regions.

Beside Anoplophora spp., the tribe Lamiini comprises eight genera: Plectrodera, Monochamus,

Lamia, Hebestola, Goes, Microgoes, Neoptychodes and Plagiohammus (Lingafelter and Hoebeke

2002a). Striking similarities between different species of Anoplophora and other closely related genera have often led to confusion and misidentification. Anoplophora glabripennis was first placed in the genus Cerosterna (Cerosterna glabripennis (Motschulsky) in 1853 and Cerosterna laevigator (Thomson) in 1857), then moved to the genus Melanauster (M. nobilis (Ganglebauer) in 1890; M. luteonotatus and M. angustatus in 1925, M. nankineus in 1926 and M. glabripennis var. laglaisei in 1953) (Lingafelter and Hoebeke 2002a). Recently, the two species A. glabripennis and A .nobilis were recognized as being two forms of the same species, following a series of morphological, physiological and genetic studies (Hu et al. 2008). Anoplophora chinensis in its two forms, A. chinensis and the former A. malasiaca, have also been confused in some references with A. glabripennis. However, the clear difference in elytral base texture helped provide a good method of distinguishing them (Lingafelter and Hoebeke 2002b). The A. malasiaca form occurs only in Japan and Korea and attacks primarily willow and fruit trees (Hu et al. 2008).

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Host range

A. glabripennis attacks both healthy and stressed trees. Tree age does not seem to affect

beetle preference; young and old trees are attacked indiscriminately (Lingafelter and Hoebeke

2002a). ALB outbreaks in northern China occur mostly in poplar stands suffering from drought

stress for several years (Hu et al. 2008).

In China, ALB feeds predominately on poplar and willow, but also on maples (Acer), alder (Alnus), crab apple (Malus), chinaberry (Melia), mulberry (Morus), sycamore (Platanus), plum (Prunus), pear (Pyrus), black locust (Robinia pseudoacacia L.), roses (Rosa), and necklacepod (Sophora). It also attacks and oviposits on a number of urban trees, including ash

(Fraxinus), rose of Sharon (Hibiscus), horsechestnut (Aesculus) and birch (Betula). A survey conducted in Yinchuan, Ningxia (north-western China) reported ALB on 34 tree species, belonging to 14 genera and 10 families (Li et al. 1999b). In South Korea, ALB feeds primarily on

Acer mono (Maximowicz) and A. truncatum Bunge (Williams et al. 2004a).

In North America, ALB was mainly found on maples (Haack et al. 1997). More than

24 deciduous tree species have been recorded as actual or potential hosts for ALB, among which

Acer, Aesculus, Salix and Ulmus spp. are particularly threatened in the U.S.(Lingafelter and

Hoebeke 2002a, Sawyer 2003).

In Austria, ALB was found on Acer spp., Betula spp., Aesculus hippocastanum L. and

Fagus sylvatica L., in order of abundance (Tomiczek and Hoyer-Tomiczek 2007). In France, it was mostly observed on Betula, Acer and Populus spp. (Tomiczek and Hoyer-Tomiczek 2007), and on Betula pendula Roth and Acer pseudoplatanus L. in Italy (Maspero et al. 2007).

The larva is the most damaging stage, feeding and tunneling in the inner wood of the tree.

Adult emergence also affects tree vigor when adults chew emergence holes, causing heavy sap flow (Becker 2000a).Based on observations in China, new generations of beetles re-infest the

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same tree they emerged from by laying eggs further down toward the base of the crown, until exhaustion of the host (Hu et al. 2008).

Host preference experiments have also revealed the presence of tree species totally repellent and/or resistant to ALB. Callery pear, Pyrus calleryana (Decne.), is an example of such

non-hosts. Feeding on callery pear decreased adult longevity and female fecundity and increased

larval mortality to 100% (Morewood et al. 2004b).

Life cycle

In China, ALB may take one or two years to complete its life cycle depending on the

time of adult emergence. Late emerging adults lay eggs that may not hatch until the following

spring (Keena 2002a). In Liaoning province, over 80% of the population is univoltine, with first

to third instar larvae overwintering, while another 11-20% of the population is bivoltine with

overwintering mature larvae (Hua et al. 1992). These percentages vary between different Chinese

provinces according to the host and climatic conditions, but the majority of the population is

univoltine.

In the spring, mature overwintering larvae pupate, while other larvae feed until late

March and pupate from late April to early May. The prepupal stage lasts 9-39 days. Pupation

takes another 13-24 days (Fig. 1-1). At 25°C, newly formed adults remain in the pupal chamber

for ~5 days to complete sclerotization and then bore out of the tree in ~4 days (Keena and

Sanchez 2005), forming characteristic round holes approximately 1 cm in diameter. The time

required by beetles to chew their way out of the wood differed significantly according to temperature. Beetles chewed slower at lower temperatures (around 13 days for both sclerotization and chewing at 20°C) and faster around 30°C (a total of ~8 days for both) (Keena and Sanchez

2007b). In China, adults’ emergence starts in early April, with peak emergence from mid-June to early July. They stay active till October (Lingafelter and Hoebeke 2002b) Emergence is believed

7

to be influenced by annual temperature (Hu et al. 2008). In New York City and Chicago, beetles have been recorded from July to November (Haack et al. 1996).

Figure 1-1. Life cycle of Anoplophora glabripennis.

Newly emerged adults feed on the tender bark of the twigs and occasionally on leaves

and leaf petioles. A period of 7 to 14 days of “maturation feeding” is required for females to

develop an egg load. Li and Liu (1997) suggest that the females’ ovaries develop in the first 10

days of her life and that mating improves ovary maturation. Males may require approximately 11

days to produce viable sperm (Keena and Sanchez 2005). Males live from 3-50 days and females

from 14-66 days (Li and Wu 1993). Adult beetle longevity is optimized at around 18 °C. Females

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can survive temperatures between 39 and -3 °C and males between 38 and -2 °C (Keena 2002b).

Mating occurs on large branches and trunks. Mating occurs repeatedly for both sexes. Males guard females even during oviposition.

After mating, females chew pits in the bark and insert a single egg through the pit, underneath the inner bark. Egg-laying can take up to 50-72 minutes. Females are usually careful about placing their eggs, chewing many pits before deciding on one placement. Little is known about the parameters affecting female choice of oviposition sites but common insect evolutionary biology theory base female choice of oviposition sites on two major factors, safety and quality of the host (Keena and Sanchez 2007). Zhao et al. (1997) suggested that bark thickness might be the most important factor affecting female choice of oviposition sites. A female can lay between 30 and 178 viable eggs during her lifespan, with highest fecundity recorded at 25°C yielding on average 67 eggs. Female fecundity is positively correlated with body size and negatively correlated with age (Keena 2002b, 2006). Females do not oviposit at temperatures < 10 and >

35°C and days to oviposition are shortened as temperature increased, with a minimum of 16d at

30°C (Keena 2006).

Eggs are typically laid in the upper trunk and along major branches where the bark is thin, smooth and tender (Haack et al. 2006). Egg hatch requires temperatures between 10°C and

32°C, with the fastest hatch occurring around 29°C and the highest percentage at around 23 °C.

On average, eggs hatch in one to two weeks, depending on temperature, and small white larvae feed exclusively on the cambium for the first two instars. Third instar larvae enter the sapwood and bore their way into the heartwood where they spend most of their life boring tunnels in the trunk and branches. When they’re close to pupation, larvae migrate slowly back toward the sapwood and form a cavity in the wood where they pupate. (Smith 2000b, Lingafelter and

Hoebeke 2002a, b, APHIS 2005).

Duration of different stages of the life cycle, as well as insect fecundity, flight, egg hatch and larval survival seem to be highly affected by temperature. Beetles live longer at lower

9

temperatures but fecundity, egg hatch and flight propensity increase with increased temperature, until reaching the lethal threshold of 32°C. Temperature thresholds and duration of life stages seem to differ slightly between populations, suggesting the ability of some populations of ALB to adapt to different environmental conditions (Keena 2006).

Management of invasive species

Invasive species are plant or animal species that are not native to the ecosystem under consideration and whose introduction causes or is likely to cause economic or environmental harm or harm to human health (NICS 2001). In this new ecological niche, introduced organisms might develop new traits, potentially favoring their establishment and population outbreaks.

Major causes of outbreaks could be the availability of suitable hosts, adaptation to new hosts, and absence of co-evolving natural enemies to regulate the pest population. Modern classical biological control that started in 1889 with Albert Koebele intentionally introducing the

Australian Vedalia lady beetle from New Zealand to California initiated a distinct group of invasives, those that were introduced on purpose for the management of invasive, or even sometimes native, pests (Howarth 1991).

Management of Anoplophora glabripennis

Prevention

Preventing new introductions is a major step in the management of invasive species,

especially when the potential for establishment in the field is high. ALB was first introduced in

Solid Wood Packing Material (SWPM). Preventing further introductions required more stringent

regulations of the treatment of wood materials involved in international trade. In September 1998,

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the U.S. government issued the Solid Wood Packing Material from China Interim Rule which stated that all “wooden pallets, crating, dunnage, and other wooden packing material imported into the United States from China will have to be heat treated, fumigated, or treated with preservatives prior to departure from China”(USDA-APHIS 1998). In September 16, 2005, a final rule was issued again requiring all wood packing material used in foreign trade to be heat- treated or fumigated prior to arrival in the U.S. (USDA-APHIS 2005). European countries and

Canada have also enforced their requirements for SWPM treatment of wood introduced from

China (Bell 2005, Schröder et al. 2005).

Heat treatment and fumigation by methyl bromide (MeBr) are currently the only worldwide approved methods for treatment of SWPM under ISPM-15 (International Standards for Phytosanitary Measures) set by the International Plant Protection Convention (IPPC).

Recently, IPPC has been seeking alternatives for treatment of SWPM to be included in ISPM-28, and the International Forest Quarantine Research Group (IFQRG), a panel composed of scientists from different countries, is exploring possibilities. These include the use of ethanedinitrile (Ren et al. 2006) or sulfuryl fluoride (Barak et al. 2006a) for fumigation, and the use of microwave and radio-frequency as alternatives to the conventional heat treatment (Fleming et al. 2004).

Prevention can also be partially accomplished through thorough visual inspection of

SWPM on ports of entry and in warehouses. ALB has been intercepted in warehouses in more than 14 states in the U.S. Visual inspection and wood treatment have helped in preventing its establishment in most of those states, along with other natural factors such as the lack of nearby hosts and environmental conditions not favorable for establishment (Hoebeke 2007). The

Canadian Food Inspection Agency (CFIA) has been involved in extensive inspection since the first interception of ALB in Burnaby, BC in 1992 (Bell 2005).

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Physical control and eradication efforts

Since the first discovery of ALB in New York, USDA-APHIS has been implementing intensive surveys and eradication programs. Eradication efforts include the removal, chipping and incineration of all infested material, followed by extensive survey of all trees and soil injection of imidacloprid on all potential hosts within an 800m-radius (USDA-APHIS 2006). Tree removal helped limit sources of infestation and chipping has proven to kill 100% of larvae and pupae remaining inside the wood (Wang et al. 2000). Until August 2008, eradication efforts were thought to be successful in Chicago, Illinois, after four years of negative survey. However, the recent discovery of ALB on a car in Deer County in August 2008 required the authorities to rethink the process, although no infestations were found in the field (USDA-APHIS 2008a). In the U.S., thousands of trees have been cut in the infested areas. A total of more than 7000 trees in

Illinois, more than 6000 in New York and 600 in New Jersey by were cut by the end of 2005

(USDA-APHIS 2006).

In Canada, similar eradication programs were applied. Over 15,000 trees were removed in Greater Toronto between November 2003 and March 2004 (Bell 2005). In Germany, 16 infested trees were destroyed and quarantine regulations were applied for a radius of 2 km around the center of the infestation in 2004 (Schröder et al. 2005). In Japan, eradication efforts were declared successful in 2005 after chemical treatment and removal of infested hosts (Takahashi and Ito 2005).

Most of the implemented eradication programs included planting resistant tree species after removal of infested trees. In China, white poplar has been used to replace susceptible poplar varieties as wind breaks between vineyards and field crops. In November 2004, the U.S. Forest

Service in New Jersey published a “Recommended Tree Planting List for the ALB Quarantine

Zone” consisting of 42 tree species with descriptions and recommendations for location and time

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of planting (See Annex I). Similar lists were developed by each of the states where ALB infestations have been found.

Physical control measures are the primary control method applied in China. The Forestry offices in Ningxia require agricultural laborers working in fields with susceptible poplar varieties to conduct mass collections and destruction of adult beetles during the summer. They also encourage people to help in decreasing the beetle population by physically catching and killing beetles, eggs and larvae, or blocking frass holes (Hu et al. 2008).

Biological control

Anoplophora glabripennis biocontrol studies investigated the potential effect of entomopathogenic fungi, nematodes, parasitoids and predators. Fungi seem by far the most prevalent and virulent entomopathogens infecting ALB in quarantine colonies and in field surveys. A fungus-based insecticidal product, consisting of non-woven fabric bands impregnated with cultures of Beauveria brongniartii (Sacc.), commonly used for control of cerambycids including Anoplophora malasiaca and the pinewood nematode vector, Monochamus alternatus

(Hope) in Japan, was tested on ALB adults and showed a significant decrease in female oviposition as well as shortening of adult longevity (USDA 2001, Dubois et al. 2004a). Strains of

Beauveria bassiana (Bals.)Vuill., B. brongniartii and Metarhizium anisopliae (Metsch.) were tested for biocontrol of ALB, all of which decreased adult longevity. In the U.S., research has focused more on a virulent strain of M. anisopliae that is already registered for pest control for safety purposes (Dubois et al. 2004b). Bands impregnated with M. anisopliae and B. bassiana were effective in field conditions for as long as 63 days, reducing both adult longevity and female fecundity. Infections were also observed in offspring, probably transmitted vertically from an infected mother (Hajek et al. 2003, Hajek et al. 2006).

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Other entomopathogens of ALB include microsporidia and nematodes. Microsporidia

are obligate intracellular protozoans that cause chronic, debilitating diseases and mortality in the

field, and severely compromise laboratory animal colonies once contaminated. A new species of

microsporidium was found in China but studies were inconclusive as to its potential use as a

biocontrol agent (USDA 2001). Bacillus thuringiensis var. tenebrionis and B.th. toxins were

tested against larvae and adults of ALB but were not found to be significantly effective (D'Amico

et al. 2004).

A study conducted by Solter et al. (2001) compared the effect of four species of nematodes, namely Steinernema carpocapsae (All), Heterorhabditis bacteriophora, H. indica and H. marelatus (Liu and Berry) and showed a positive effect of both S. carpocapsae and H. marelatus on larval mortality and production of infective larvae (Solter et al. 2001).

However, another comparison between isolates of Steinernema feltiae (Filipjev), S.

glaseri, S. riobrave, S. carpocapsae, and H. marelatus showed that S. feltiae and S. carpocapsae

were the most effective with 100% mortality for first and third instar larvae. S. feltiae was even

effective against older larvae (Fallon et al. 2004). Application of nematodes in the field proved

however to be difficult since an average of 7,500 nematodes per beetle were required to achieve

around 85% mortality, and requirements for moisture and distance from the host were difficult to

achieve (Hu et al. 2008)

The Chinese woodpecker Dendrocopos major (Beicki) has been observed feeding on

ALB larvae and adults. Studies on the ecology and potential effect of D. major in regulating ALB populations are ongoing (Hu et al. 2008).

In Ningxia, China, an ecto-parasitoid beetle, Dastarcus helophoroides Fairmaire was found parasitizing A. glabripennis larvae and pupae, and showed promising results when tested in the field. Scleroderma guani (Xiao et Wu) (Hymenoptera: Bethylidae) is another ecto-parasitoid that has been tested in China for the control of ALB and yielded promising results. A similar

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species, S. sichuanensis Xiao was successfully used as a biological control agent against A. chinensis (Hu et al. 2008).

In Italy, Aprostocetus anoplophorae Delvare (Hymenoptera: Eulophidae) was reared from eggs of A. chinensis and identified as a new species (Delvare et al. 2004). The potential of

A. anoplophorae to parasitize ALB is still under investigation (Herard et al. 2006).

Chemical control

Since ALB larvae spend most of their time inside the heartwood where they are protected from external factors, application of pesticides for the management of this pest has proven to be a challenge. In China, three chemical control strategies are used in the infested areas. In high infestation areas, tree canopies are sprayed with chemical pesticides to control adults. Larvae are targeted by inserting bamboo or wooden sticks containing aluminum phosphide through the frass holes into the tunnels. The third strategy uses bands impregnated with microcapsules of a cypermethrin-based pesticide that are wrapped around the trees. Beetles walking on the trunk will come in contact with the microcapsules, triggering the release of the active ingredient.

Cypermethrin microcapsules are also sprayed on trunks and leaves. In both cases, results using the microcapsules are promising (Hu et al. 2008).

In the U.S., there have been several studies that investigated the use of pesticides to control ALB. The neonicotinoids, imidacloprid and azadirachtin, were found to have antifeedant and toxic effects against ALB larvae. Imidacloprid was particularly effective due to its rather fast translocation within the tree and its persistence (Poland et al. 2006). However, concerns were raised over the antifeedant effect of imidacloprid for ALB adults, which might increase dispersal.

Wang et al. (2005) compared the efficacy of five different neonicotinoid insecticides in controlling ALB larvae. The LC50 ranged from 1 to 2.9ppm at 72 hours, with imidacloprid ranked

after thiamethoxam and clothianidin. The antifeeding effect of imidacloprid, coupled with its

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ingestion and contact toxicity made it the chemical of preference in the management programs

(Wang et al. 2005). Currently, soil injections with imidacloprid are the most commonly used chemical management strategy in the U.S., with over 600,000 trees treated between the years

2000 and 2005.

Use of pheromones in management

Examples of effective monitoring, mating disruption and population control through the use of pheromones are abundant in the literature (Burkholder and Ma 1985, Bjostad et al. 1993,

Shea 1995, Boake et al. 1996, Rajendran 1999, Schmidt et al. 2003, Boo and Park 2005,

Brockerhoff et al. 2006, Ibeas et al. 2007). Pheromone composition and concentrations are also critical parameters to be considered. Monitoring using insect pheromones decreases the use of pesticides by eliminating the need for preventive applications. It is also a tool for studying population dynamics and for modeling population dispersal/spread. For invasive and/or alien insect management, trapping methods that provide early detection are critical to identify new introductions or spread before these species become established (Burkholder and Ma 1985,

Bjostad et al. 1993, Shea 1995, Boake et al. 1996, Howse et al. 1998, Rajendran 1999, Schmidt et al. 2003, Brockerhoff et al. 2006, Ibeas et al. 2007). If possible, development of pheromone- baited traps is ideal for this purpose because of their specificity and distances over which they can operate.

Pheromones of Cerambycids

Although the taxonomy, host range and distribution of cerambycids have been well studied, little is known about their reproductive behavior (Linsley and Chemsak 1997, Hanks

1999, Allison et al. 2004). Recent studies revealed taxonomic patterns in pheromone production

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within subfamilies of the Cerambycidae, with two major types of pheromones: volatile and contact pheromones (Hanks 1999, Allison et al. 2004, Ray et al. 2006).

Volatile pheromones are short chain chemicals often used for mate finding. They vary in their volatility, range of action and source. In the subfamily Cerambycinae, volatile pheromones were found in at least nine species, Anaglyptus subfasciatus Pic. of the tribe Anaglyptini (Leal et al. 1995), Xylotrechus pyrrhoderus Bates, Xylotrechus quadripes Chevrolat, Xylotrechus chinensis Chevrolat, Neoclytus mucronatus mucronatus (F.) and Neoclytus acuminatus acuminatus (F.) of the tribe Clytini (Sakai et al.1984; Kuwahara et al. 1987; Lacey et al. 2004;

Hall et al. 2006; Ray et al. 2006), Hylotrupes bajulus (L.) and Pyrrhidium sanguineum (L.) of the tribe Callidiini (Fettköther et al.1995; Schröder et al. 1994), and Anelaphus pumilus (Newman) of the tribe Elaphidiini (Ray et al. 2006). In these species, pheromones were produced by males and were structurally similar. Pheromone release was positively correlated with the presence of pores on the males’ prothorax and setae on the female (Ray et al. 2006). Some of these pheromones, referred to as aggregation pheromones, attracted both males and females (Lacey et al. 2004,

Hanks et al. 2007, Lacey et al. 2007). Ray et al. (2006) made the observation that most of the species shown to have male-produced volatile pheromone had rather short antennae. However, species with long antennae were not less likely to produce volatile pheromones. The correlation between antennal length and presence of prothoracic pores was low for the species studied in the subfamily Cerambycinae. Male-produced pheromones were recently identified in the subfamilies

Aseminae (for Tetropium fuscum (F.) and T. cinnamopterum (Kirby)) (Sweeney et al. 2008) and

Lamiinae (for Hedypathes betulinus (Klug)) (Zarbin et al. 2008).

Female- produced volatile pheromones were recorded in only three species of cerambycids so far. These are the primitive prionines, Prionus californicus (Subfamily:

Prioninae) (Cervantes et al. 2006) and Mallodon dasystomus (Say) (Spikes et al. 2008a) in which females attract males over a long distance using their volatile pheromones; and Styloxus bicolor

(Champlain and Knull) (subfamily Bombinae) (Itami and Craig 1989).

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While volatile pheromones are used for mate location, contact cues found on the elytra

of females of several cerambycid species in the genera Xylotrechus (Rhainds et al. 2001,

Ginzel et al. 2003), Megacyllene (Ginzel et al. 2006) , Anoplophora (Li et al. 1999a, Zhang et

al. 2003, Fukaya et al. 2004) , Dectes (Crook et al. 2004), Plectrodera (Ginzel and Hanks 2003)

and others play a major role in mate recognition. In these species, males were observed to

approach the female and mount quickly after touching her elytra with their antennae. A contact

sex pheromone was recently characterized in Anoplophora chinensis and is composed of three

gomadalactones, A, B and C (Mori 2007, Hu et al. 2008).

ALB Adult behavior and semiochemical-based communication

In the field, adults fly occasionally, in early morning or afternoon. Although they rarely do so, adults have the capacity to fly for very long distances (>2 miles) (Smith et al. 2004) and they are prone to flying when the host quality degrades(Keena and Major 2001). ALB adults, similar to other members of the subfamily Lamiinae, are diurnally active. They spend most of their time scouting tree trunks (Keena 2002a, Lingafelter and Hoebeke 2002a, Smith et al.

2002).

Once a male’s antennae touch the female elytra, mounting occurs in a matter of seconds

(Zhang et al. 2003). Mating occurs repeatedly for both sexes. Individual mating events last around 2.8 minutes on average and 1-10 copulations can occur in one bout, followed by a rest period of 60-95 minutes. Males guard females even during oviposition (Keena and Sanchez

2007). However, little is known about mate location and communication between males and female in this species.

The presence of a volatile pheromone in A. glabripennis has remained a mystery for the

last several decades. Li et al. (1999) suggested that this species does not communicate through

volatile compounds but rather uses vision and contact cues for mate finding and recognition.

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Male-produced pheromone of Anoplophora glabripennis

Zhang et al. (2002) identified two chemicals in aeration extracts of males that are unique to males; i.e. not found in females aerations. These putative semiochemicals are two functionalized dialkyl ethers, one alcohol (4-(n-heptyloxy)butan-1-ol) and one aldehyde (4-(n- heptyloxy)butanal). Antennal sensory neurons of both male and female ALB responded to the blend (1:1 (v/v)) of this male-produced pheromone. Preliminary olfactometer bioassays showed that these compounds were equally attractive to both sexes, but they only produced a moderate behavioral response (Zhang et al. 2002). More bioassays and field tests were needed to verify the attractiveness of this pheromone to adult Anoplophora glabripennis. In Chapter 2 of this thesis, the results of laboratory behavioral assays using this male-produced pheromone are presented, comparing the attractiveness of the two components of this pheromone to each other and to the blend. Chapter 3 illustrates trapping experiments conducted in the field in Ningxia province, China in order to assess the efficacy of ALB male-produced pheromone as a lure in monitoring traps.

Female-produced pheromones of Anoplophora glabripennis

ALB females appear to produce different types of pheromones. Li et al. (1999) suggested the presence of a contact pheromone on the elytra of ALB females. They also stated that males follow odor residues of females on the tree trunk. Further investigation of females’ cuticular hydrocarbons revealed five male-active compounds, namely (Z)-9-tricosene, (Z)-7- pentacosene, (Z)-9-pentacosene, (Z)-7-heptacosene, and (Z)-9-heptacosene, (in a relative ratio of 1:2:2:1:8). These five compounds are used by males as contact cues for recognition of conspecifics (Zhang et al. 2003). Wickham et al. (2007) have also identified female produced

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volatile compounds that are believed to affect males’ behavior. The chemical structure of these compounds is still unknown.

Synergism between insect pheromones and plant volatiles

As suggested by Hanks et al. (1996a), longhorned cerambycids probably locate their hosts

by using host volatiles. Once on the same tree, the diameter of antennal spread of the males

improves their chances of finding a mate. Mate recognition in these species is believed to depend

largely on female-produced contact pheromones.

Saint-Germain et al (2007) offered the “primary attraction and random landing”

hypothesis to explain why some insects use long-range pheromones while others rely on short-

range pheromones in combination with plant volatiles. According to their model, wood-feeding

insects may locate their hosts from long distances using plant stress-related or other plant odors,

but once inside the host canopy, landing is no longer mediated by plant volatiles (Saint-

Germain et al. 2007). This hypothesis is supported by numerous examples of cerambycids that

use plant cues instead of long-range pheromones to increase their chances of finding a mate.

Among these are flower-visiting cerambycids that depend largely on floral scents (Hanks

1999).

These findings have been highly exploited for pest management. Traps baited with

plant volatiles have been used to monitor several species of cerambycids and other wood-

boring insects (Linsley 1959, Hanks 1999, Allison et al. 2004, Sweeney et al. 2004, Ginzel and

Hanks 2005, Ibeas et al. 2006b, Miller 2006, Sweeney et al. 2007). Trap catches can be further

enhanced by adding plant volatiles to the insect pheromones. Since the 1970’s, it has been

known that bark beetles can be captured in greater numbers in traps baited with a combination

of plant stress odors and bark beetle aggregation pheromones (Pitman et al. 1975, Byers et al.

1988b). Combining the male sex pheromone of Hylotrupes bajulus (Coleoptera: Cerambycidae)

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with four different monoterpenes was found to significantly improve trap catches (Reddy et al.

2005). To date, a long range pheromone for A. glabripennis has not been reported. It is possible that these beetles follow the model posed by Saint-Germain (2007) in that ALB respond to plant volatiles to find a host tree, but then use short range cues to find a mate. If this hypothesis holds true, then we would expect to find synergism between the ALB male-produced relatively short-range pheromone and host tree volatiles.

Volatiles of Anoplophora glabripennis host trees

Anoplophora glabripennis feeds primarily on poplars in China and maples in the U.S.

Studies have been conducted in both countries on how ALB locates its host trees, including

detection of plant volatiles, and the possibility of using host volatiles in baited traps for

monitoring. Numerous studies suggested that ALB uses plant volatiles to find their host (Li et

al. 1999b, Hu et al. 2008). Volatiles of ashleaf maple, Acer negundo L., were studied

extensively following the results of an attractiveness study in which 66% of beetles within 100

m of the trees flew preferentially to this species. More than 32 compounds were identified from

the volatile blend produced by ashleaf maple. Conclusions as to which of the 32 compounds

affected the behavior of ALB were controversial. According to Fan et al.(2007), the most

effective antennal stimulants were trans-2-hexen-1-al, decyl aldehyde and trans-2-hexen-1-ol.

In Y-tube olfactometer bioassays, these three compounds were significantly attractive to adults.

Li et al. (2003) argued that (Z)-3-hexen-1-ol, 1-butanol, 1-pentanol and 2-pentanol are

significantly more attractive than other ashleaf maple’s volatile organic compounds (VOCs).

Volatiles of drought stressed Acer negundo were also investigated. Nine volatile compounds

(Acetophenone, butyl alcohol, pentyl alcohol, trans-2-hexenol, (Z)-3-hexenol, pentanal, pentoic

acid, hexanol and hexanoic acid) increased in response to stress, while longifolene decreased.

Butyl alcohol, pentyl alcohol and (Z)-3-hexenol elicited the strongest antennal responses using

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GC-EAD (gas chromatography coupled with electroantennographic detection) ((Hu et al.

2008)).

Wickham et al. (2007) extracted volatiles from a range of ALB host trees in the U.S. and found several volatiles that are common across the host range of ALB (and absent in non- hosts), including (Z)-3-hexen-1-ol, trans-caryophyllene, delta-3-carene, linalool, and camphene.

Public outreach and management of ALB

Recent history of invasive species management has witnessed a great contribution from citizens and local organizations. In the U.S., a vast majority of ALB infestations were found by residents who reported their observations to local authorities. The first discovery of ALB in 1996 was reported by a landowner who thought someone was drilling holes in his trees. This also occurred in IL and MA where infestations were reported by people seeing the beetles, or their damage. A recent study in New Jersey showed that reports of infestations increased with time after implementation of outreach campaigns. Since most of infestations were found in people’s backyards and private properties, cooperation from landowners was required to optimize management. This cooperation depended largely on people’s awareness of the extent of the problem and the impact of ALB on the environment, including the various tree species present on their properties.

In Yinchuan, Ningxia province (north-western China), a public campaign organized by local students led to the collection and destruction of 500,000 beetles in the summer of 2007 (Hu et al. 2008).

The National Management Plan: Appendix II – Summary of Federal Roles and

Responsibility states the responsibility of federal agencies for spreading public awareness about

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national issues. U.S. Department of Agriculture agencies have branches that deal specifically with public information and awareness campaigns. APHIS has a “public affairs effort” that responds to emergency situations and eradication programs. The Forest Service (FS) “administers a number of cooperative programs that assist partnerships and encourage forest stewardship for non- industrial forest landowners, including control of invasive species.” The Cooperative State

Research, Education, and Extension Service (CSREES) is heavily involved in outreach through its Land Grant University System. The Extension Service provides a vital link between USDA, stakeholders and citizens, through on-campus and county offices across the U.S. The Agricultural

Research Service (ARS) has Area-wide Pest Management Programs that provide support to transfer technology within five years for the management of invasive species.

Since the first ALB infestation was found in New York City in 1996, the government agencies listed above mobilized federal and state resources and personnel to impose regulated boundaries and conduct survey and eradication measures. In 2005, a strategic plan was developed for the Asian longhorned beetle Cooperative Eradication Program in New York, New Jersey and

Illinois. The purpose of the program is to protect the forest products industry, biodiversity of forests and park-lands in the U.S. and the quality of the urban environment through complete elimination of all reproducing populations of ALB in the U.S. The strategic plan recognized the importance of public support and cooperation for the success of the eradication and re-plantation efforts. According to section VIII of the strategic plan, training more people to recognize and report signs of the beetle will largely improve detection and eradication (USDA-APHIS 2005b).

Outreach efforts include among others a broad awareness campaign during the flight season in the summer, when people are more likely to see the beetles, and continuous warning and information broadcasting on damage and symptoms throughout the year. Campaigns include advertisements in newspapers and on billboards; pictures and information of beetles with hotline numbers for reporting on gadgets such as key chains, bookmarks, door hangers, magnets, tattoos

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etc.; pamphlets with pictures and warning signs such as the “Wanted” posters; “Help save our maple trees” cards; ALB identification cards with hotline phone numbers; and pest alerts among others. A great amount of information is posted on the web by federal and state agencies and extension offices at universities. Easy to access and understand material has been made available to the general public on the net; the “Beetle Busting Toolbox” is a slide show displaying beetle pictures, damage symptoms, Identification cards, pest alerts, citizens guides and “Wanted” posters. Most of public outreach material can be accessed through the USDA-APHIS or Forest

Service websites and university extensions websites. The University of Vermont’s Asian longhorned beetle website features most of those materials at http://www.uvm.edu/albeetle/.

Since the discovery of ALB in MA in August 1, 2008, the State of Massachusetts developed an Introduced Pests Outreach Project for ALB. The project involved training sessions for business owners during October and November 2008, community meetings in Worcester to discuss eradication strategies, and ALB “compliance training” for businesses working with host wood materials within the regulated area. The Massachusetts Tree Wardens and Foresters

Association holds free ALB information sessions for professionals and tree growers. Toll free numbers were established for the ALB Eradication program and notices were sent to homeowners and residents of the regulated areas (MA ALB CEP 2008).

Public involvement was key to the success of the eradication program in Illinois, according to the report developed by Antipin and Dilley (2004) in the Chicago regulated area.

During interviews conducted with residents, public outreach and media coverage were the most frequently cited factors affecting the success of the eradication program. Citizens felt involved and compelled to help when they saw the city, state and government authorities devoting time and resources to solve problems and especially when they were informed that the eradication program will not affect their businesses or add additional costs to their households (Antipin and Dilley

2004).

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Just as public support improved detection of Asian longhorned beetle new introductions and infestations through visual observations of insects and damage, involvement of regulated areas’ residents in any new detection procedure is expected to improve the efficacy and reduce the cost of detection. Developing a new detection tool will not be sufficient unless outreach materials and training sessions explaining the use and potential benefits of such tools are provided. Chapter 5 of this thesis presents a tentative design for an outreach program allowing homeowners, manufacturers and owners of businesses dealing with SWPM and authorities at ports of entry as well as USDA personnel participating in the eradication program, to participate in the detection of ALB infestations and new introductions using the developed monitoring lures and traps.

The authors hope that the findings summarized in this thesis will be used for improvement of ALB detection in regulated areas and in ports of entry.

25

Chapter 2

Attractiveness of Anoplophora glabripennis to male-produced pheromone and plant volatiles

M. E. Nehme1, M. A. Keena2, A. Zhang3, T. C. Baker1 and K. Hoover1*

1 Department of Entomology, The Pennsylvania State University, University Park, PA 16802

2 U.S. Forest Service Northern Research Station, Hamden, CT 06514

3 USDA-ARS, Invasive Insect Biocontrol and Behavior Laboratory, BARC-West, Beltsville, MD

20705

In review- Journal of Environmental Entomology

26

Abstract

The male-produced pheromone of Anoplophora glabripennis (Motschulsky) (Coleoptera:

Cerambycidae), which is an equal blend of 4-(n-heptyloxy)butan-1-ol and 4-(n- heptyloxy)butanal, was used in laboratory bioassays and in the greenhouse to determine its potential for attracting A. glabripennis adults. In modified ‘walking wind tunnels’, virgin females were most attracted to the alcohol component and to the pheromone blend. Virgin males were repelled by the pheromone at the lowest and highest amounts offered. Y-tube olfactometer bioassays also showed that females were significantly more attracted to the pheromone and its components than males were. However, males were more attracted to plant volatiles than females.

Out of 12 plant volatiles tested, (-)-linalool, (Z)-3-hexen-1-ol and linalool oxide were attractive to both genders, while 3-carene and (E)-caryophyllene were only attractive to males. Combining the male pheromone blend with (-)-linalool alone or with (Z)-3-hexen-1-ol attracted significantly more males than did the pheromone alone. We tested four trap designs in our quarantine greenhouse with 8 different lures. The InterceptTM Panel traps and the hand-made screen sleeve traps caught more beetles than the Plum Curculio traps and Lindgren funnel traps. Intercept traps worked best when baited with (Z)-3-hexen-1-ol, while screen sleeve traps were most attractive when baited with (-)-linalool. Our findings provide evidence of the attractiveness of the A. glabripennis male-produced pheromone and suggest that it has a role in mate-finding. It is also a first step towards the development of an efficient trap design and lure combination to monitor A. glabripennis infestations in the field.

Key Words: Anoplophora glabripennis; male-produced pheromone; mate-finding; trap design;

Cerambycidae

27

Introduction

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae, subfamily

Lamiinae), commonly known as the Asian longhorned beetle, is a high-risk invasive species

(USDA-APHIS 1998, EPPO 2004). It was introduced into the U.S. in solid wood packing materials from Asia and was first discovered in New York in 1996 (USDA-APHIS 2008).

Anoplophora glabripennis larvae feed in the wood of deciduous trees. Similar to other lamiines,

A. glabripennis is a diurnal species. Adults emerge in early summer and feed on small twigs and leaves. They spend most of their time in shaded areas on the trunk of the same tree they emerged from (Williams et al. 2004). After resource depletion, flight to a healthy host is believed to be guided by olfactory cues from the host trees (Lance et al. 2000).

In the U.S., monitoring of A. glabripennis infestations is restricted to scouting by climbing individual trees and searching for oviposition scars, sap flow, larval frass or dime-sized adult emergence holes. Large bucket trucks or tree climbers are sometimes used to aid in detection (USDA-APHIS 2005a). This approach is expensive and time consuming. Rapid detection of this serious, invasive species is necessary to intercept new introductions before they establish and verify success (or failure) of eradication efforts. Development of an attractive lure to be used in monitoring traps is a first major step towards the improvement of detection techniques for A. glabripennis.

Recent studies revealed taxonomic patterns in pheromone production within subfamilies of the Cerambycidae (Hanks 1999, Allison et al. 2004, Ray et al. 2006). In the Cerambycinae, volatile pheromones were found in most of the species studied so far (Sakai et al. 1984,

Kuwahara et al. 1987, Lacey et al. 2004, Hall et al. 2006, Ray et al. 2006). These pheromones are produced by males to attract females and share a similar structural motif. Some of these compounds also attract males and thus likely play a role in aggregation (Lacey et al. 2004, Hanks et al. 2007, Lacey et al. 2007). Pheromone production was correlated positively with the presence

28

of pores on the prothorax of male cerambycids (Ray et al. 2006). However, prothoracic pores are not as common in the lamiines (Ray et al. 2006). In fact, electron microscopy of A. glabripennis

(Coleoptera: Cerambycidae: Lamiinae) adults showed no difference in prothoracic pores between

males and females (L. Hanks, personal communication). However, consistent with the

cerambycines, male-produced pheromones were identified in two Aseminae, Tetropium fuscum

(L.) and Tetropium cinnamopterum (Kirby) (Silk et al. 2007), and two lamiines, Hedypathes

betulinus (Klug) (Vidal et al. 2008) and A. glabripennis (Zhang et al. 2002).

Putative pheromone components produced by A. glabripennis males consist of two

functionalized dialkyl ethers, one alcohol (4-(n-heptyloxy)butan-1-ol) and one aldehyde (4-(n-

heptyloxy)butanal) (Zhang et al. 2002). Antennae of both males and females responded to the

blend (1:1 (v/v)) of these two compounds using gas chromatography-electroantennographic

detection (GC-EAD). Preliminary olfactometer bioassays showed that these compounds were

equally ‘stimulatory’ to both genders, but only moderately so (Zhang et al. 2002).

Several species of cerambycids use plant compounds rather than long-range pheromones

as mediators for mate location. Such is the case for most flower-visiting cerambycids that depend

largely on floral scents (Hanks 1999). Attraction to plant volatiles has been exploited for pest

management and in monitoring traps, alone or in combination with insect pheromones (Linsley

1959, Byers et al. 1988, Hanks 1999, Allison et al. 2004, Sweeney et al. 2004, Ginzel and Hanks

2005, Ibeas et al. 2006, Miller 2006, Sweeney et al. 2007).

Anoplophora glabripennis feeds primarily on poplars in China and maples in the U.S.

Studies have been conducted in both countries on host location, including detection of plant

volatiles, and the possibility of using host volatiles in baited traps for monitoring. Volatiles of

ashleaf maple, Acer negundo, were studied extensively. Li et al. (2003) found that, in ashleaf maple, (Z)-3-hexen-1-ol, 1-butanol, 1-pentanol and 2-pentanol were significantly attractive to adults. Wickham and Teale (2009) extracted volatiles from a range of A.

29

glabripennis host trees in the U.S. and found several volatiles that are common across the host

range (and absent in non-hosts), including (Z)-3-hexen-1-ol, (E)-caryophyllene, delta-3-carene, (-

)-linalool, linalool oxide and camphene. Our knowledge about A. glabripennis attraction to plant

volatiles, however, is still in its infancy.

The purpose of this study was to develop an attractive lure to be used for monitoring A.

glabripennis populations in the field. This involved testing a series of hypotheses followed by

empirical testing of various trap designs and lure combinations. First, we tested the hypothesis,

suggested by preliminary GC-EAD results, that the two male-produced compounds, 4-(n-

heptyloxy)butan-1-ol and 4-(n-heptyloxy)butanal, either together or alone, were attractive to

females and play a role in sexual communication in this species. A corollary to this hypothesis

included determining if there is a difference in attractiveness of the two putative pheromone

components.

The second hypothesis was that host plant volatiles enhance performance of the putative

pheromone components, and we chose a list of volatiles and essential oils to test based on

previous studies with A. glabripennis (Chenier et al. 1989, Allison et al. 2001, Ping et al. 2001,

Francese 2005, Wickham and Teale 2009). These plant volatiles were evaluated alone, in

mixtures and/or in combination with the male-produced pheromone to identify the most effective

combination as well as to detect any possible synergism or antagonism between different

chemicals.

Our third objective was to determine the best trap design in combination with the best

lure. Francese et al. (2004) tested five different trap designs without lures and found the screen

sleeve trap to be the most effective by far. However, semiochemical-based trapping for A.

glabripennis has been done almost exclusively with InterceptTM panel traps (Lund et al. 2004,

Wickham and Teale 2009). In this study, we tested four different trap designs with eight different

lures in a quarantine greenhouse to determine the most attractive combination of trap design and

lures to be further tested as a monitoring tool for A. glabripennis in the field.

30

Materials and Methods

Insects.

Most A. glabripennis adults were reared in the USDA-Forest Service Northern Research

Station Quarantine laboratory in Ansonia, CT. These beetles came from two colonies: one established from adults that emerged from infested wood obtained from the Ravenswood area,

Chicago, IL infestation (UIC- reared for 9 generations) and the other from large larvae imported from Inner Mongolia, China (IMC- reared for 6 generations). Insects used in the greenhouse trapping trials were from the A. glabripennis colony at Penn State University, which is of mixed origins (field collected from New York and lab reared). All A. glabripennis were transported and reared under USDA permits at each quarantine facility. Voucher specimens of the Ansonia A. glabripennis colonies were deposited at the Entomology Division, Yale Peabody Museum of

Natural History, New Haven, CT and those of the Penn State colony were deposited at the Frost

Museum, The Pennsylvania State University, University Park Campus, PA. All insects were reared on artificial diet (Keena 2005) until pupation. All life stages were kept at 25ºC, 60% humidity, L:D 16:8 h with constant ventilation. Males and females were kept individually in 750 ml glass jars following emergence and supplied with Norway maple twigs (Acer platanoides L.)

for feeding. For mating, one male and one female A. glabripennis were placed in a 3.75 L glass

jar and provided with a ~5 cm diameter x 20 cm long Norway maple bolt and twigs for

oviposition and feeding, respectively. Healthy, 15-30 day-old mated insects were only used in this

study after their first oviposition event to ensure mating compatibility and sexual maturity (Keena

2002b). Beetles used in the pheromone experiments were well-fed, while those used in plant

volatiles experiments were starved 5-7 hours to enhance attraction.

31

Response of A. glabripennis adults to the two putative male-produced pheromone components.

Wind Tunnel bioassays.

To test the hypothesis that the two male-produced compounds are attractive to females

and play a role in sexual communication, we offered each of 4-(n-heptyloxy)butan-1-ol and 4-(n-

heptyloxy)butanal alone or in an equal-ratio blend to A. glabripennis males and females, before

and after mating, in modified wind tunnels. The alcohol, 4-(n-heptyloxy)butan-1-ol, and aldehyde, 4-(n-heptyloxy)butanal, components of the putative pheromone were synthesized following the protocol described by Zhang et al. (2002). One μg/μl solutions of each were prepared in hexane (mixture of isomers, 98.5 %, HPLC Grade, EMD Chemicals Inc., Gibbstown,

NJ). Blends were created by mixing equal amounts of pure chemicals then diluting in hexane.

The modified ‘walking’ wind tunnels consisted of (1.5m long x 1mm thick x 10cm diameter) glass tubes (Chemglass, Vineland, NJ). For each tube, one plastic beaker (VWR

International, West Chester, PA) was connected tightly to one end. Part of the base of the beaker was removed to allow the insertion of a 12 V computer fan. The fan was placed face out and manually adjusted with the aid of a flow meter such that it provided unidirectional air flow at

0.5L/min through the beaker to the inside of the tube. Chemicals were applied to a sterile cotton wick (TIDI Brand, Banta Healthcare group Ltd., Boston, MA) and inserted in front of the fan through a small covered opening in the beaker (Fig. 2.1). The advantage of this method is that, unlike commonly used olfactometers where there is a greater risk that the test individual would smell a mixture of the treatments offered rather than each alone, and where air turbulence is likely to affect the directionality of the odor plume, the modified wind tunnels limit air turbulence and provide a homogeneous unidirectional flow, ensuring purity of the chemical offered.

32

Figure 2.1. The modified walking wind tunnel, composed of a 1.5 m long glass tube connected to a plastic beaker closed by a 12 V fan at the end. Chemicals were applied on the cotton wick and beetles introduced at the free end of the tube and allowed to walk inside for 20 minutes. Tubes were virtually divided into three compartments: I (initial), M (middle) and NS (near source).

Four identical tunnels were used for each replicate, placed parallel to each other, ~30 cm apart, on a flat surface directly under the room’s ambient fluorescent lighting. The four treatments: 4-(n-heptyloxy)butan-1-ol alone; 4-(n-heptyloxy)butanal alone; blend of the two; and hexane control, were randomly distributed in the four tunnels. Four insects, of the same gender/mating status, were introduced simultaneously to the four tunnels to correct for possible temporal variation in insect response to the different treatments. All experiments were conducted

in the late morning – early afternoon period (11 am -12pm and 2- 4pm), when beetles were

observed to be active in their feeding jars. Cotton wicks were replaced and tunnels cleaned with

hexane between replicates. Relative positions of the test compounds in the four parallel tunnels

were alternated every other replicate to randomize potential environmental factors. Tunnels were

virtually divided lengthwise into three equal compartments: “initial”, “middle” and “near source”

(Fig. 2.1). One insect was introduced at the free end of each tunnel at the same time (opposite end

33

to the fan). Beetles were allowed to walk inside the tunnel for 20 minutes. The time spent by each insect in each compartment was recorded, as well as the number of times each insect touched the cotton wick. Four different amounts of chemicals were tested: 0.05, 0.5, 1, and 2 μg. Ten each of virgin males, virgin females, mated males and mated females were tested separately for each treatment and each amount. All tests were conducted in the quarantine facility in Ansonia, CT under conditions suitable for A. glabripennis (25ºC, 60% humidity) (Keena 2006).

Y-olfactometer bioassays.

After determining optimal amounts of the putative pheromone to be used in the wind tunnels, we conducted further bioassays in a Y-olfactometer for verification purposes. Bioassays were conducted to test attractiveness of the male-produced compounds, each alone or in a 1:1 blend, at the 1 μg level, when offered against a hexane control. The Y-olfactometer used was built following the description by Ginzel and Hanks (2005) (6 cm diameter, main tube 26 cm long, arm length 22 cm, angle between arms 70°, Penn State University Glass Shop). Chemicals were applied on 1 x 1 cm2 pieces of filter paper (Filter paper 413; 9.0 cm OD; VWR Brand; VWR

International Inc. West Chester, PA). Clean air was pushed into the two arms using two air pumps

(‘Push-Pull’ pump, Bryan Banks, Penn State University) at ~0.5 L/min using 90 cm of Teflon®

tubing (6.35 x 9.52 mm, VWR brand, VWR Scientific Products Corp., San Francisco, CA) and

air was pulled from the main tube using another ‘Push-Pull’ pump calibrated at ~1 L/min. The

olfactometer was washed with hexane between replicates to avoid residual odors. Treatment and

control arms were alternated every other replicate. Fifteen to 20 replicates each of virgin males

and virgin females were tested in each experiment. Choice and time to choice were recorded

when beetles had moved forward at least 10 cm within the arm. All tests were conducted in the

quarantine facilities in Ansonia, CT or Penn State University, PA under A. glabripennis rearing

conditions.

34

Response of A. glabripennis adults to plant volatiles.

Based on previous studies with A. glabripennis (Chenier et al. 1989, Allison et al. 2001,

Francese 2005, Wickham and Teale 2009), we chose a list of plant volatiles and essential oils to test to determine the most attractive plant volatiles that could be used as lures in monitoring traps for this species. Two essential oils were tested: the commonly used eucalyptus oil and manuka oil (Silky Scents Inc., Riverside, CA), which has been recently shown to be moderately attractive to the emerald ash borer (Crook et al. 2008) and other Coleoptera (Hanula and Sullivan 2008) .

Twelve plant volatiles were also purchased from VWR International Inc. and tested. These were:

(-)-verbenone (Fluka; purity 99%), and trans-pinocarveol (Fluka; purity 96%), which are attractants used to trap bark beetles and other wood-boring species (Reddy et al. 2005, Ibeas et al.

2006a); (Z)-3-hexen-1-ol (Fluka; purity 98%), (-)-linalool (Fluka; purity 98.5%), linalool oxide

(Fluka; purity >97%; GC grade), camphene (Chem Service Inc.; purity >96%), delta-3-carene

(Fluka; purity 98.5%), trans-caryophyllene (MP Biomedicals; purity 98%), myrcene (Alfa Aesar; purity 95%), (+/-)-2-pentanol (Alfa Aesar; purity 99%), (Z)-3-hexenyl acetate (Alfa Aesar; purity

99%), and benzenyl acetate (Fluka; purity 99.7%), which are produced either by healthy or stressed Acer mono and A. negundo and/or have been shown in different studies to attract A. glabripennis and other cerambycid adults (Chenier et al. 1989, Allison et al. 2001, Ping et al.

2001, Francese 2005, Wickham and Teale 2009).

Ten virgin males and 10 virgin females were tested. The rest of the plant volatiles and essential oils were tested in the Y-olfactometer against a hexane control. In all cases, 50 μg of chemicals were used for each replicate. Fifteen to 20 replicates each of virgin males and virgin females were tested for each chemical.

35

Response of A. glabripennis adults to a combination of the male-produced compounds and plant volatiles in the Y-olfactometer.

Previous studies showed that combining plant volatiles with insect pheromones enhances beetle trap catches significantly (Byers et al. 1988). To verify whether this would apply to A. glabripennis, we tested combinations of the putative male-produced pheromone blend with each

of (-)-linalool alone, (-)-linalool + (Z)-3-hexen-1-ol, delta-3-carene + (Z)-3-hexenyl acetate,

offered against the putative pheromone blend in the Y-olfactometer. The choice of plant volatiles

to be used in these combinations was based on previous Y-olfactometer and greenhouse trapping

results, where linalool and (Z)-3-hexen-1-ol were equally attractive to both males and females A.

glabripennis, while delta-3-carene was more attractive to males than females and (Z)-3-hexenyl-

acetate was more attractive to females then males. Ten to twenty virgin males and virgin females

were tested for each combination. Choice and time to choice were recorded and analyzed for

comparisons.

Comparison of trap designs and lures in the greenhouse.

Baited trap catches were compared among four trap designs in the quarantine greenhouse at Penn State University. The four traps tested were the InterceptTM Panel Trap (or Intercept PT;

APTIV Inc., Portland, OR), which was designed for bark beetles and other wood borers, and is

commonly used in research work with A. glabripennis and Sirex noctilio F. (Joseph Francese

personal communication); a circle trunk trap (or curculio trap; Great Lakes IPM Inc., Vestaburg,

MI) used for plum curculio, pecan and acorn weevils; the Lindgren funnel trap (12-funnel size,

Contech Inc., Delta, BC), a widely used trap for bark beetles and other small beetles; and a screen

sleeve trap, designed by Victor Mastro and Dave Lance (USDA-APHIS- PPQ, Otis, MA) for A.

36

glabripennis following the limited success of the commonly available trap types (Vic Mastro personal communication), and manually made from metal screen (Fig. 2.2).

The quarantine greenhouse contains two screen cages of equal dimensions (~3 x 3 x 3.9 m). In each cage, four different traps were placed in the four corners. The two walk-in trap designs (circle trunk and screen sleeve traps) were placed around the trunk of (~3m high, 10-15 cm DBH) sugar maple trees (Acer saccharum), while the flight traps (the InterceptTM Panel and the Lindgren funnel) were attached to the screen wall of the cage. Because of limited time and limited number of beetles, we combined trap design and lure testing in one experiment. The purpose was to acquire preliminary information about what lures and trap designs to use for field testing in China the subsequent summer. For this, 8 lure treatments were tested and compared to empty control traps (see Table 2.1 for lure information). All lures were dispensed on rubber septa

(Stopper sleeve 5 x 11, VWR Scientific, West Chester, PA), widely used dispensers for insect pheromones (Butler and McDonough 1981, Weatherston 1989) . Each lure and empty controls were tested in three temporal replicates, each replicate lasting for one week using the same lure treatment in all four traps so that in a given test, trap design was the only variable. Relative placement of traps and greenhouse cage used were re-randomized for each replicate. To correct for time and potential microclimate influences, lure treatments and empty controls were completely randomized over the weeks. Five A. glabripennis males and five females were released in the middle of each of the two greenhouse cages and allowed to move freely within each cage. Traps were checked daily. Trapped beetles were recorded, marked and released back in the middle of the same cage. Dead beetles were continuously replaced. Greenhouse temperature was maintained between 24 and 28°C with 50-70% humidity.

37

Table 2.1. Lure treatments used in the greenhouse trapping experiments.

Abbreviation Lure Treatment Dosage (μg) Replicates

MP Male pheromone* 1 3

L Linalool (98.5%; Fluka) 50 3

LO Linalool oxide (97%; Fluka) 50 3

Hex (Z)-3-hexen-1-ol (98%; Fluka) 50 3

HA (Z)-3-hexenyl acetate (99%; Alfa Aesar) 50 3

MPL Linalool (98.5%; Fluka) + Male pheromone 1 MP + 50 L 3

MPHex (Z)-3-hexen-1-ol (98%; Fluka) + Male 1 MP + 50 Hex 3

pheromone

MPHA (Z)-3-hexenyl acetate (99%; Alfa Aesar) + 1 MP + 50 HA 3

Male pheromone

Lindgren funnel Screen sleeve Intercept PT Circle trunk

Figure 2.2. Trap designs used in the greenhouse trapping experiment. From left to right: the

Lindgren funnel trap, the hand-made screen sleeve trap, the InterceptTM Panel trap, and the circle trunk curculio trap.

38

Statistical analyses.

In the wind tunnel experiment #1, two responses of A. glabripennis adults were recorded: time spent near source and number of visits to source. Time spent near source varied between 0 and 1200 seconds. General Linear Models (GLM) for both data sets were performed by chemical amount, insect gender/mating status and pheromone treatment for the three highest chemical amounts, since mated adults were not tested at the lowest amount. Separate GLMs were then performed for each of the four chemical amounts tested by pheromone treatments and insect categories (combinations of gender and mating status) because of significant interactions among these variables. Orthogonal contrasts (OC) were performed after each GLM for specific mean comparisons, for both pair-wise and multiple mean comparisons.

For all insects used in the wind tunnel experiment #1, age was divided into classes 1 to 6, with each class consisting of 3 consecutive days, starting at 15 days old, with class 6 grouping those individuals 30 days old or more. Spearman’s correlation test was used to determine the effect of age class and insect weight on the time spent near source by A. glabripennis adults and

the number of visits to source in the wind tunnel.

All Y-olfactometer results followed a binomial distribution and were analyzed using

frequency analysis followed by a probability test, which allowed comparison of the real means to

hypothetical means of 50%, (i.e., no choice was made) at the 95% confidence level using

Pearson’s test of significance. Comparisons between males and females were performed using

Fisher’s Exact test. Spearman’s correlations were used to determine the relationship between

choice and time to choice.

Greenhouse trapping data were compared by trap design and lure using a GLM followed

by orthogonal contrasts. All data were analyzed using JMP 7.0 (SAS Institute Inc. 2007).

39

Results

Response of A. glabripennis adults to the two putative male-produced pheromone components.

Wind Tunnel bioassays.

The response of A. glabripennis adults to the putative male-produced pheromone and its

two components differed significantly by gender and mating status (see Table 2.2). Overall,

mated males and mated females showed no significant difference in the time spent near source or

the number of visits to source across chemical treatments and amounts (data not shown).

Because of significant interactions of chemical amount with chemical treatment and

gender/mating status (see Table 2.2), we analyzed the time spent near source and number of visits

to source data for each chemical amount separately to determine the effect of pheromone

treatment and gender on insect response.

Overall, time spent near source was highest at the 1 μg level (OC: χ2 = 18.2; df = 2; P =

0.0002). At this level, virgin females spent more time near the source of the alcohol treatment compared to the remaining three treatments (OC: χ2 = 5.26; df = 3; P = 0.021) and visited the source of the alcohol treatment significantly more often than the blend (OC: χ2 = 5.54; df = 1; P =

0.018) (Fig.3). Virgin males visited the source of the alcohol/aldehyde blend and the aldehyde alone more than the control and alcohol sources (OC: χ2 = 4.74; df = 3; P = 0.029) (Fig. 2.3).

At the lowest chemical amount (0.05 μg), the number of visits to the source was different by gender/mating status (GLM: χ2 = 19.78; df = 3; P = 0.0195). Virgin males visited the source in control tunnels significantly more times than the alcohol (OC: χ2 = 4.15; df = 1; P = 0.04) (Fig.

2.3) and spent more time near the source in control wind tunnels compared to the three chemical

40

treatments (OC: χ2 = 16.13; df = 3; P <0.0001) (data not shown). Virgin females did not show any

significant response at this level.

A. glabripennis adults showed no significant difference in their response to the alcohol,

aldehyde, or blend treatments at the 0.5 μg and 2 μg levels (Fig. 2.3). At the 2 μg level, the

2 number of visits to source was only significantly different by gender (GLM: χ = 32.9; df = 3; P =

0.0098) and virgin males visited the source more frequently (OC: χ2 =7.95; df = 3; P = 0.0047) in the control tunnels than any of the putative pheromone component treatments (Fig. 2.3).

Table 2.2. General linear model results: Effects tests for time spent near source and number of visits to source over the three highest chemical amounts (0.5, 1, and 2 μg).

Time spent near source Number of visits to source GLM effects Chi-Square df P-value Chi-Square df P-value

Whole Model 329 20 <0.0001* 78.8 47 0.002*

Treatment 39.3 3 0.024* 4.31 3 0.229

Amount 25.2 2 <0.0001* 0.0001 2 0.999

Gender/mating 35.2 3 0.002* 9.89 3 0.019* status

Treatment*Amount 55.6 6 0.003* 2.92 6 0.819

Treatment*Gender/ 39.6 9 0.121 3.24 6 0.840 mating status

Amount*Gender/ 115 6 <0.0001* 4.93 9 0.778 mating status

Treatment*Amount* 129 18 <0.0001* 29.9 18 0.0383* gender/mating status

* represents significant P values at the 95% confidence level

41

Figure 2.3. Mean number of visits of A. glabripennis adults to the source in the walking wind tunnel at 4 different chemical amounts (0.05, 0.5, 1, and 2 μg) during 20 minutes. Treatments include the male pheromone (A. glabripennis male-produced pheromone blend), alcohol (4-(n-heptyloxy) butan-1-ol), and aldehyde (4-(n-heptyloxy) butanal) offered against a hexane control. Each pheromone treatment and

chemical amount combination was tested separately for virgin males and virgin females (n=10 for each).

Error bars represent standard errors of the mean. Letters over bars represent significantly different means

within chemical amount and gender/mating status at the 95% confidence level.

42

Y-olfactometer bioassays.

There was not a statistically significant difference in attraction to the alcohol or aldehyde

or the blend of the two between males and females as a main effect (Fisher’s exact test: df = 1; P

2 >0.5) (Table 2.3). However, females preferred the blend (Pearson: χ = 5.4; df = 14; P = 0.02) over the control, whereas males did not make a significant choice in any of the three tests. In no case did time to choice correlate significantly with quality of choice, suggesting that response times did not differ among any of the treatments.

Table 2.3. Percentage of A. glabripennis virgin males and virgin females that chose the male- produced blend, the alcohol alone, or the aldehyde alone when offered 1 μg of each against a hexane control in a Y-olfactometer (Mean percentage ± SEM).

Male-produced Blend Alcohol Aldehyde

Virgin males 46.7 ± 13.3 % 33.0 ± 12.5 % 60.0 ± 13.1 %

Virgin females 80.0 ± 10.7 % * 46.7 ± 13.3 % 73.3 ± 11.8 %

* Values with an asterix represent a significant choice compared to the control.

Effect of age and weight on A. glabripennis response to pheromones.

Insect age and weight did not affect the capacity of A. glabripennis adults to walk

towards the end of the wind tunnel. No correlation was found between insect weight or age and

any of the response data (Spearman: P >0.5), even when analyzed separately for each insect

gender/mating status group.

43

Response of A. glabripennis adults to plant volatiles in the Y-olfactometer.

Of the 12 plant volatiles tested, seven elicited more than a 50% response in virgin males while only four of them attracted more than 50% of virgin females (see Figure 4). The difference

2 between males’ and females’ attraction was significant for both 3-carene (Pearson: χ = 7.5;

2 n=20; P = 0.0062) and trans-caryophyllene (Pearson: χ = 4.46; n= 14; P = 0.0346). 3- Carene was highly attractive for males, with 90.0% of virgin males choosing the treatment arm in the Y-

2 olfactometer (Pearson: χ = 6.4; df = 1; P = 0.0114) compared to 22.2% of females.

When (Z)-3-hexenyl acetate or myrcene were offered against a hexane control, males

2 chose the control arm of the olfactometer significantly more often (Pearson: χ = 3.8; df = 1; P =

2 0.029 and χ = 3.85; df = 1; P = 0.04, respectively). Females preferred the control significantly

2 more frequently when offered against camphene (Pearson: χ = 3.85; df = 1; P = 0.04) (Fig. 2.4).

44

Figure 2.4. Percentage of virgin males and virgin females that chose plant volatiles when offered against a hexane control in a Y-olfactometer. Error bars represent standard errors of the mean. Letters over bars represent significantly different responses between males and females at the 95% confidence level.

Percentages that are significantly higher or lower than 50% are labeled with an asterix. Numbers under plant volatiles names represent the number of beetles of each gender tested.

45

Response of A. glabripennis adults to a combination of the male-produced compounds and plant volatiles in the Y-olfactometer.

When linalool was combined with the blend of these two compounds, twice as many adults of both genders chose the combination compared with the male-produced blend alone (Fig.

2 2.5). This difference was especially significant for males (Pearson: χ = 4; df = 1; P = 0.04). When

(Z)-3-hexen-1-ol was added to the mixture above, 90% of males chose the combination (Pearson:

2 χ = 4.5; df = 1; P = 0.034) while only 10% chose the male-produced blend alone; in contrast,

females were equally attracted to both plant volatiles.

We then chose 3-carene as one host volatile that is only attractive to males and (Z)-3-

hexenyl acetate that was more attractive to females, to test whether the combination of both with

the male-produced blend would attract both genders of A. glabripennis. In this case, 75% of

females preferred the male blend alone, while males were equally attracted to both treatments

(Fig. 2.5).

46

Figure 2.5. Response of A. glabripennis virgin males and virgin females to combinations of the male-produced pheromone blend with plant volatiles offered against the pheromone alone in a Y- olfactometer. Error bars represent standard errors of the mean. Letters over bars represent significantly different means between treatments at the 95% confidence level.

47

Comparison of trap designs and lures in the greenhouse.

When the four trap designs were tested in the greenhouse, the Intercept panel traps and the screen sleeve traps caught equal numbers of beetles per week on average across all lures.

Mean trap catches per week were significantly lower for the circle trunk and Lindgren funnel traps compared to the Intercept panel and screen sleeve traps (GLM: χ2 = 17.5; df = 3; P =

0.0006) (Fig. 2.6). Although overall mean trap catches were equal between the intercept panel

and screen sleeve traps, mean trap catches per week differed by lure (GLM: χ2 = 27.2; df = 8; P =

0.0007) and by the interaction of trap design and lure (GLM: χ2 = 20.5; df = 8; P = 0.0087).

Screen sleeve traps baited with linalool caught the highest number of beetles per week, while

Intercept panel traps baited with the same plant volatile did not catch any beetles. Intercept traps baited with (Z)-3-hexen-1-ol caught twice as many beetles on average than screen sleeve traps.

The combination of the male-produced blend with (Z)-3-hexen-1-ol attracted higher numbers of beetles in both trap designs. This combination caught significantly more beetles than (Z)-3-hexen-

1-ol in screen sleeve traps (OC: χ2 = 4.15; df =1; P=0.04), the (Z)-3-hexenyl acetate alone (OC: χ2

= 10.4; df =1; P =0.0012) or in combination with the male blend (OC: χ2 = 10.4; df =1; P

=0.0012) (Fig. 2.7). (Z)-3-hexenyl acetate had the lowest overall trap catches when offered alone

or in combination with the male-produced blend. Although some trap designs x lure treatments

were significantly more attractive than others, none was significantly different from empty

control traps in the greenhouse.

48

Figure 2.6. Mean trap catches per week for the four trap designs tested in the greenhouse: the screen sleeve trap, Intercept Panel trap (or Intercept PT), Circle trunk trap (or Curculio trap) and

Lindgren funnel trap. Error bars represent standard errors of the mean. Letters over bars represent significantly different means at the 95% confidence level.

49

Figure 2.7. Mean trap catches per week per lure for the Intercept panel (top) and screen sleeve

(bottom) traps. Error bars represent standard errors of the mean. Letters over bars represent significantly different means within lure treatments at the 95% confidence level. (MP= A. glabripennis male pheromone blend; L= linalool; LO = linalool oxide; Hex= (Z)-3-hexen-1-ol;

HA= (Z)-3-hexenyl acetate; MPL = male pheromone blend + linalool; MPHex = male pheromone blend + (Z)-3-hexen-1-ol; MPHA= male pheromone blend + (Z)-3-hexenyl acetate; ref. Table 1 for amount

50

Discussion

The A. glabripennis male-produced compounds 4-(n-heptyloxy)butan-1-ol and 4-(n- heptyloxy)butanal showed potential as attractive lures to be used in monitoring this species.

Based on the trends observed in both the wind tunnel and the Y-olfactometer, females responded behaviorally to both compounds, which suggests that 4-(n-heptyloxy)butan-1-ol and 4-(n- heptyloxy)butanal (1:1) are both required components for attraction. This result is consistent with findings for several members of the Cerambycidae where males produce volatile pheromones to attract females (Sakai et al.1984; Kuwahara et al. 1987; Lacey et al. 2004; Hall et al. 2006; Ray et al. 2006; Fettköther et al.1995; Schröder et al. 1994). More recently, the male-produced pheromone of another member of the subfamily Lamiinae (to which A. glabripennis belongs),

Hedypathes betulinus, was also identified (Vidal et al. 2008). The more moderate attraction of males to the pheromone blend, mainly guided by their ability to cue in on the aldehyde component, is also consistent with other examples from the family (Lacey et al. 2004, Hanks et al. 2007, Lacey et al. 2007) and may explain the EAD response of both genders to the pheromone found by Zhang et al. (2002). Cases of males responding to pheromones produced by other males of the same species are abundant in the literature, and are not limited to cerambycids but seem to be common for wood-boring beetles (e.g. bark beetles, see Byers et al. 1983). A. glabripennis males’ ability to respond behaviorally to other males’ pheromone might help such ‘opportunistic’ males increase their chances of finding females through competition and interception, as males are known to do in many other insect groups. However, the lower attraction of males compared to females and the repellency observed at the lowest and highest amounts also suggest that a certain proportion of A. glabripennis males might prefer to avoid competition by staying far from pheromone sources, especially at high beetle densities, and that they may use the concentration of this pheromone as a spacing/dispersal signal.

51

In the case of bark beetles, the ability of males to cue in on other males’ pheromones helps them overcome tree defenses through aggregation (Raffa and Berryman 1983). In the case of A. glabripennis, there is no evidence of plant defenses limiting the beetles’ development in preferred hosts. However, A. glabripennis is known to re-colonize the natal host or adjacent trees.

Reported observations of A. glabripennis dispersal propose a higher flight propensity when the host is exhausted (Williams et al. 2004). In mark-release-recapture studies, beetle dispersal from release trees was positively associated with the abundance of beetles at the release tree (Bancroft and Smith 2005). We suggest that higher flight propensities could also be correlated with a higher number of males on the host; males have been observed to fly more than females (Keena and

Major 2001). This can be translated into males moving to new hosts to avoid competition and to draw females to new healthy trees where resources are abundant.

Besides their smaller size and higher flight propensity, the greater attraction of males to plant volatiles compared to females provides additional support for their role in host location and later mate attraction to new resources. The higher attraction of males toward delta-3-carene and trans-caryophyllene suggests that these two terpenes might play a role in males’ host choice. Li et al. (2003) and Wickham and Teale (2009) found linalool and (Z)-3-hexen-1-ol to be attractive to both genders of A. glabripennis. We have seen a similar trend in our Y-olfactometer results, although the attraction was not significant. Greenhouse results further suggest that these two plant volatiles might be used in lures for field trapping, especially with screen sleeve traps. (Z)-3- hexenyl acetate, a stress-induced volatile in A. negundo (Li et al. 2003), repelled males in the Y- olfactometer. Traps baited with this plant volatile did not catch any beetles. This correlates well with the known preference of A. glabripennis for slightly weakened but not highly stressed hosts

(Hanks 1999).

Some of the volatiles we tested are commonly used lures for cerambycids and other wood-boring insects. For example, (-)-verbenone was found in earlier studies to enhance

52

attraction of Monochamus galloprovincialis Olivier (Coleoptera: Cerambycidae) to a blend of

alpha-pinene, ipsenol and methyl butenol (Ibeas et al. 2006a). The monoterpenes (+)-alpha-

pinene, (-)-verbenone, (-)-trans-pinocarveol and (+)-terpenen-4-ol were significantly attractive to

Hylotrupes bajulus (L.) (Reddy et al. 2005). A. glabripennis adults, however, were not attracted to any of these commonly available lures, which excludes the possibility of their use for

monitoring. We also tested alpha-pinene and ethanol, which were found to be attractive to eight

species of Cerambycidae and some Buprestidae, Elateridae and Curculionidae (Miller 2006), but

were not attractive to A. glabripennis (unpublished data).

Further studies of the quantity and quality of pheromone produced by the male at

different times might reveal some interesting information as to the sequence of chemical

communication events in this species. The difference in response to the aldehyde component

between the wind tunnel and the olfactometer experiments might be an artifact of the differential

speed of oxidation of alcohols versus aldehydes, and to differences in distance travelled by the

chemical and exposure time to ambient oxygen in both settings. Also, insects, and more

particularly A. glabripennis, are generally less active on artificial substrates than on natural ones.

This might have contributed to the lower response in the wind tunnels where beetles had to walk for a much longer distance inside a glass tube. The fact that these beetles walked more than 1 m toward the odor source suggests that they are attracted to it; the majority of the beetles that did not show a response stayed in their introduction point without any movement for 20 minutes or circled the inside of the first 0.5 m of the tunnel.

An interesting finding was that many beetles spent a relatively long time near the treatment source but never visited the source itself. Although this might be a means of selecting a pheromone concentration level that is most attractive to them, our observations of courting behavior of A. glabripennis in the field in Ningxia, China, introduce another possible interpretation. In the field, three out of three times when a male and a female were observed in

53 proximity to one another, A. glabripennis males were observed standing on the upper surface of a leaf at the top of the host tree during early morning. Shortly after, females walked upward on branches connected to the leaves on which males were standing. However, the females did not walk all the way up to the leaves. Instead, they stopped a few centimeters lower on the same branch and waited for the male to walk down. Both the male and the female then walked toward each other in what appeared to be a series of ‘courtship’ movements on adjacent small branches.

Mating occurred only after the male’s antennae touched the female’s elytra, probably guided by the contact pheromone identified by Zhang et al. (2003). These observations suggest that, by basing our analyses on beetles that visited and spent time near the source, it is possible that the walking wind tunnel test was a highly conservative response variable. In the Y-olfactometer, choices were recorded when beetles were at least 10 cm within the arm, which ensured they crossed the potential turbulence area created by the merging flows from the two arms, but were still 16 cm behind the actual chemical source, located at the far end of the arm. Thus, the wind tunnel bioassay was useful for determining optimal chemical amount and for comparing the four treatments together. However, the magnitude of the response was better observed in the Y- olfactometer.

The greenhouse study provided needed information for further field studies. The equal trap catches in both Intercept panel traps and screen sleeve traps suggests that both trap types could be used for monitoring purposes. However, the significant interaction between lures and trap design showed that the quality of the lure used will affect success in trapping. Based on these results, we decided to test screen sleeve traps baited with (-)-linalool and Intercept traps baited with a combination of the male-produced pheromone blend and (Z)-3-hexen-1-ol in the field for confirmation. This was especially important given that the greenhouse studies contained inherent limitations; the traps were touching the screen wall of the greenhouse cages and the number of

54 beetles released per cage was relatively low, both of which may be responsible for the small variation found between treatments,

This study was a first step toward developing an attractive lure for use in traps to monitor

A. glabripennis populations and detect new introductions in the field. Results of these laboratory and greenhouse experiments will be used as a basis for further field trapping experiments in

China.

55

Acknowledgments

We thank the Statistics Department consultation services at Penn State University for

their assistance with the statistical analyses. We also thank Larry Hanks, Scott Geib, Erin Scully,

Katalin Borocsky and the staff of the US Forest Service Northern Research Station Quarantine laboratory in Ansonia, CT for their scientific and technical support, and June Nie of the Invasive

Insect Biocontrol and Behavior Laboratory in Beltsville for assistance with chemical synthesis.

This work was supported by grants to KH from the Alphawood Foundation, the Horticultural

Research Institute, and cooperative agreements with the USDA, APHIS, PPQ.

56

Chapter 3

Field testing of Anoplophora glabripennis male-produced pheromone and

plant volatiles

M. E. Nehme1, M. A. Keena2, A. Zhang3, T. C. Baker1, Z. Xu4 and K. Hoover1*

1Department of Entomology, The Pennsylvania State University, University Park, PA 16802

2 USDA Forest Service, Northern Research Station, Hamden, CT 06514

3 USDA-ARS, Invasive Insect Biocontrol and Behavior Laboratory, BARC-West, Beltsville, MD

20705

4 Beijing Forestry University, Beijing, China 100083

In review - Journal of Environmental Entomology.

57

Abstract

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), commonly known as the Asian longhorned beetle, is a wood-boring invasive species introduced from Asia to

North America and Europe in solid wood packing material. Efficient monitoring traps are needed to assess population density and dispersal in the field and to detect new introductions at ports of entry. For this purpose, we conducted field trapping experiments in China in the summers of 2007 and 2008. In 2007, we tested Intercept™ panel traps hung on poplar trees. In 2008, we used

Intercept™ panel traps hung on poplar trees, screen sleeve traps wrapped around poplar trunks, as well as Intercept™ panel traps hung on bamboo polls 20 m away from host trees. Traps were baited with A. glabripennis male-produced pheromone alone or in different combinations with plant volatiles. Traps baited with the male-produced pheromone alone caught significantly more females than control traps in both years. The addition of a mixture of (-)-linalool, (Z)-3-hexen-1- ol, linalool oxide, trans-caryophyllene and trans-pinocarveol to the pheromone significantly increased trap catches of females, 85% of which were virgin. Screen sleeve traps baited with a combination of (-)-linalool and the pheromone caught the highest number of beetles overall in

2008, while traps placed on bamboo polls caught the lowest number. Although the logistics for the most effective implementation of a trapping program using a mixture of the pheromone and plant volatiles require additional studies, these results indicate that this pheromone has considerable promise as a monitoring tool for A. glabripennis in the field.

Key Words- Anoplophora glabripennis; male-produced pheromone; plant volatiles; monitoring

traps; field trapping experiments.

58

Introduction

Recent globalization trends and international trade movements have greatly strengthened the links between continents and countries, which have tremendously accelerated introduction of non-native pests (Liebhold et al. 1995). The Asian longhorned beetle, Anoplophora glabripennis

(Motschulsky) (Coleoptera: Cerambycidae: Lamiinae), is a wood-boring invasive species introduced from Asia to North America and Europe in solid wood packing material. In the United

States, A. glabripennis was first discovered in New York City, New York in 1996. More field infestations have since been found in New York, New Jersey and Illinois. Most recently, a new infestation was reported in Worcester County, Massachusetts (USDA-APHIS 2008). As of April

18th, 2009, in the Worcester area, MA, more than 20,000 trees have been removed, including over

8,000 infested trees and more than 12,000 host trees found near the infested trees. More than

635,000 trees are awaiting survey (MA Introduced Pests Outreach Project 2009).

The host range of A. glabripennis includes more than 24 deciduous tree species. Tree infestations result in canopy dieback. Trees weakened by insect tunneling pose a serious threat to pedestrians and vehicles from falling limbs and trees. Insect tunneling thus can cause serious economic damage to the lumber, nursery and tourism industries. (USDA-APHIS 2005b).

Preference of A. glabripennis for maple species (Haack et al. 1996, Li et al. 1999) can have a serious impact on maple forests managed for lumber and maple-dependent industries, such as maple syrup production. The estimated potential urban impact of A. glabripennis in the US is a loss of nearly 35% of total canopy cover, 30.3% tree mortality, and a compensatory value loss of

$669 billion (Nowak et al. 2001).

The United States Department of Agriculture- Animal and Plant Health Inspection

Service (APHIS) is currently attempting to eradicate A. glabripennis, as well as to detect

potential outlier populations. Monitoring of A. glabripennis populations is restricted to

59

scouting by climbing individual trees and searching for oviposition scars, sap flow, larval

frass or dime-sized adult emergence holes (USDA-APHIS 2005a). This approach is highly

labor intensive and time consuming. Efficient monitoring traps are needed to assess

population density and dispersal in the field as well as to detect new introductions at ports of

entry before establishment.

Anoplophora glabripennis males produce a pheromone that consists of a blend of two dialkylethers, (4-(n-heptyloxy)butan-1-ol and 4-(n-heptyloxy)butanal) (Zhang et al. 2002), which was shown to attract females, especially virgins, in laboratory assays. Attractiveness of the male-produced pheromone to A. glabripennis adults in a Y-olfactometer was further enhanced when coupled with plant volatiles such as (-)-linalool and (Z)-3-hexen-1-ol (Nehme et al. revised version in review). Greenhouse trapping studies also provided additional evidence for the attractiveness of this pheromone and its potential use for monitoring. In the greenhouse, both Intercept™ panel traps and screen sleeve traps were found to effectively trap A. glabripennis adults. However, efficacy of each trap design depended largely on the lure used

(Nehme et al. revised version in review).

Using the male-produced pheromone described in the preceding paragraph, we conducted field trapping experiments in China for two consecutive years, 2007 and 2008, to determine the most effective combination of lure and trap design for catching A. glabripennis

adults in the field. We also investigated the effect of adding different combinations of plant

volatiles to the male-produce pheromone on trap catches of males and females.

60

Materials and Methods

Comparison of lure treatments for Intercept panel traps in 2007

In July 2007, trapping studies were conducted for three weeks in Qing Tong Xia, Ningxia province, China (hereafter referred to as Site A; 38°06’82.40” N; 105°91’60.60” E), to determine the attractiveness of the male-produced pheromone and two plant volatiles to A. glabripennis adults in the field and its potential for use in monitoring. The site consists of a vineyard with poplars planted as wind breaks between the 20 m-wide grapevine plots. The poplar trees included in the study were ~10 and 20 cm in diameter (measured ~ 1 m above ground). Fourty-nine

InterceptTM Panel Traps (or InterceptTM PT; APTIV Inc., Portland, OR) were hung on host trees

so that the bottoms were 1.5 - 2 m aboveground. Seven traps were placed, 7 to 10 m apart, in each

of 7 rows of poplar trees. Treatments consisted of traps baited with 5 live males in cages; 5 live

females in cages; 10 μg of 4-(n-heptyloxy)butan-1-ol; 10 μg of 4-(n-heptyloxy)butanal; 10 μg of the pheromone blend (equal ratio of the two components); and 10 μg of the pheromone blend

with 100 μg of each of (-)-linalool (Fluka; purity 98.5%) and trans-pinocarveol (Fluka; purity

96%). The alcohol (4-(n-heptyloxy)butan-1-ol) and aldehyde (4-(n-heptyloxy)butanal) components of the male-produced pheromone were synthesized by Zhang et al. (2002). Solutions of both chemicals were prepared in hexane (mixture of isomers, >98.5 %, HPLC Grade, EMD

Chemicals Inc., Gibbstown, NJ). The blend was created by mixing equal volumes (1:1) of each pure pheromone component in hexanes.

Treatments were compared to controls (unbaited) traps and randomized within rows.

Live insect baits were replenished as needed. All chemicals were applied on rubber septa

(Stopper sleeve 5 x 11, VWR Scientific, West Chester, PA), which are commonly used dispensers

61

for insect pheromones (Butler and McDonough 1981, Weatherston 1989). Septa were hung in the

empty space in the middle of the trap and changed every four to five days. Since septa were not

touching the trap itself, we were able to re-randomize lure treatments in traps with every change

of lure. Trap cups were filled with a solution of ~5% propylene glycol (or antifreeze; purchased

locally). Traps were checked daily, and beetles caught were preserved individually in alcohol.

Comparison of lure treatments, trap design and placement in 2008

In July and August 2008, trapping studies were conducted again at Site A and in a second field site in the same town (hereafter referred to as Site B; 38°08’50.80” N; 105°91’82.10” E);

Site B was planted in corn and alfalfa. The purpose of this study was to determine: a) the most attractive lure combination for monitoring A. glabripennis adults in the field, b) the best trap design and c) the best trap placement. Trap design and placement treatments included (1)

Intercept TM panel traps hung on host trees, (2) Intercept TM panel traps hung on a row of bamboo

poles 20 m away from host trees and (3) screen sleeve traps (handmade from metal screen,

designed by V. Mastro and D. Lance; USDA-APHIS- PPQ, Buzzards Bay, MA) (Fig. 3.1)

wrapped around host tree trunks, 1.5 – 2 m aboveground. All Intercept TM panel traps were hung

so that the bottoms were 1.5 - 2 m aboveground. The three trap design/placement treatments were

randomized within both of the field sites. Six to seven traps were placed, 7- 10 m apart, in each of

12 rows of poplar trees in Site A and 7 rows in Site B. Rows of poplar were ~ 20 m apart in both

sites. Five lure treatments were tested against control (unbaited) traps and randomized within

each trap design/placement (see Table 3.1).

62

Figure 3.1. Intercept TM PT traps (left) and screen sleeve traps (right).

The alcohol (4-(n-heptyloxy)butan-1-ol) and aldehyde (4-(n-heptyloxy)butanal) were prepared as described above for the 2007 field study. In addition to (-)-linalool and trans- pinocarveol, (Z)-3-hexen-1-ol (Fluka; purity 98%), linalool oxide (Fluka; purity >97%), and trans-caryophyllene (MP Biomedicals; purity 98%) were purchased and used. Our choice of plant volatiles was based on previous Y-olfactometer bioassays in which (-)-linalool, linalool oxide and

(Z)-3-hexen-1-ol were moderately attractive to both sexes of A. glabripennis (Nehme et al. revised version in review). Trans-caryophyllene and trans-pinocarveol were mainly attractive to males and were added to the mixture to enhance attraction of the blend to both males and females.

All chemicals were again applied on rubber septa. Septa carrying plant volatiles were placed in amber polyethylene bags (VWR reclosable UV- protectant amber bags 2 x 3 cm; VWR Scientific,

West Chester, PA) for protection from photo-oxidation and for slower release.

63

Table 3.1. Lure treatments used in field experiments in China in 2008.

Abbreviation Lure treatment components Dosage (μg) Application

MP Male-produced pheromone* 10 Rubber septa

L (-)-linalool 100 Rubber septa

LMP Male-produced pheromone 10 Rubber septa

(-)-linalool 100 Rubber septa

Mix (-)-linalool 100 Rubber septa

(Z)-3-hexen-1-ol 100 in amber

(-)-trans-pinocarveol 100 bags

linalool oxide 100

trans-caryophyllene 100

MM Male-produced pheromone 10 Rubber septa

(-)-linalool 100 Rubber septa

(Z)-3-hexen-1-ol 100 in amber

(-)-trans-pinocarveol 100 bags

linalool oxide 100

trans-caryophyllene 100

*A. glabripennis male-produced pheromone; consists of an equal blend of 4-(n-heptyloxy)butan-1-ol and 4-

(n-heptyloxy)butanal

For the 2008 field experiments, the number of host and non-host trees and the number of beetles observed per row of poplar trees at each site were recorded on three different dates and then averaged. Host trees at each site were classified into four categories according to their level of infestation determined by the number of exit holes and dead branches on a tree (Table 3.2).

The number of traps and beetles caught in traps per row were calculated at the end of the season.

64

This information was used to estimate the proportion of the observable population trapped and the

relationship between level of infestation and trap catches. Temperature and precipitation

information were collected from the local weather station.

Table 3. 2. Categories of host trees assigned according to infestation level.

Infestation level # of exit holes # dead branches Overall appearance

1 < 10 0 Healthy

2 10 -20 < 2; large Weak

Dying but with few 3 > 20 > 2; large green branches

dead > 20 All Dead

Field observations of mate-finding, flight and dispersal behaviors of A. glabripennis.

In 2007, a series of observations and small-sample-size-experiments on behavior of A. glabripennis were performed to better understand mate-finding, flight direction, flight propensity, and dispersal behavior of the species. All experiments were conducted on the edge of the study

Site A in Qing Tong Xia, Ningxia, China, in rows of poplar trees that were not included in the trapping study.

On July 7, 2007, 5 females and 5 males were collected, marked on the prothorax using nail polish and re-released on a poplar tree. The release tree and the trees adjacent to it were checked daily until July 18, 2007 and the number and sex of marked beetles found on these trees were recorded in an attempt to determine beetle dispersal by sex.

65

On July 18, 2007, beetle behavior was observed, photographed, and video-taped between

8 and 10 pm within the field site to study beetle activity at night. On the afternoon of July 20,

2007, flight experiments were conducted during the afternoon to investigate flight direction and

propensity. Ten males and 10 females were collected from the field, kept in separate cages by

sex, and then released individually by allowing each of them to walk upward to an opening in the

top of the cage. The release point was on a road section running East-West between two

grapevine plots, perpendicular to four poplar rows located North and South of each plot. Release

cages were placed in the middle of that road section, so that beetles were released at equal

distances (~10 m) from the ends of the four poplar rows. Beetle flight was video-taped. The

number of beetles that flew and the direction of their flight were recorded.

On July 23, 2007, the same flight experiment was conducted at night, 2 m away from a

light trap set up by the Qingtong Xia Forestry Bureau, using a total of 45 males and females,

collected earlier during the day and transported to the light trap location in cages. Flight

occurrence and direction were recorded.

Since most beetle activity occurred in the upper part of the canopy, we could only

observe males and females in the pre-mating phase on three occasions during the summer field

experiments of 2007 and 2008. Behaviors were video-taped and analyzed based on movement

initiation and direction. Other noted behaviors included grooming, arresting and antennal

movements.

Statistical analyses.

Traps catches were recorded daily then summed at each lure change (every 4-5 days).

Comparisons among treatments in 2007 and among treatments and trap design/placement in 2008 were conducted using Generalized Linear Models (GLM) followed by orthogonal contrasts (OC)

66

for mean comparisons. Correlation analyses between treatments and tree infestation levels, and

beetle and tree counts per row were conducted using Spearman’s correlation analysis. All

statistical analyses were performed using JMP v7.0 software (SAS Institute Inc. 2007).

Results

Comparison of lure treatments for Intercept panel traps in 2007

Total trap catches per lure change data fit a Poisson distribution (Goodness-of-fit test: χ2

= 3.06; P = 0.22). The pheromone-baited Intercept TM traps caught 9 beetles over the three week trapping period (5 females and 4 males), which was the highest number over all lure treatments.

Significantly more females were caught in pheromone-baited traps than the control, live male- baited traps and aldehyde-baited traps (OC: χ2 = 5.54; df = 3; P = 0.018) (Fig. 3.2). Pheromone- baited traps also caught significantly more males than the alcohol and aldehyde-baited traps (OC:

χ2 = 11.0; df = 2; P = 0.0009), but not significantly more than the control, live insects, or the

combination of the pheromone, (-)-linalool and trans-pinocarveol. The alcohol component of the

pheromone attracted only females, while the aldehyde alone did not attract any beetles of either

sex.

67

Figure 3.2. Mean number of A. glabripennis adults caught in Intercept TM PT traps in China

(July 2007), based on total caught per lure change. Controls consisted of unbaited traps. Live

female and live male traps contained 5 live females or males, respectively, in small cages

attached to the trap. The pheromone treatment consisted of 10 µg of the male-produced

pheromone blend; the alcohol treatment of 10 µg of 4-(n-heptyloxy) butan-1-ol; the aldehyde

treatment of 10 µg of 4-(n-heptyloxy) butanal; and MPPL of 10 µg of the pheromone and 100 µg

of each of linalool and (-)-trans-pinocarveol. All chemicals were applied on rubber septa. Error

bars represent standard errors of the mean. Letters over bars represent significantly different

means at the 95% confidence level among lure treatments for each gender (small letters for males

and capital letters for females).

Comparison of lure treatments for Intercept panel traps in 2008

Temperature and precipitation averages were quite similar throughout the time of the study at the two sites, but Site B was more prone to flooding. The average low temperature from

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July 1 to August 7, 2008 was 17-18 °C, with an average high of 28 °C and an average

precipitation of 0.05 to 0.1 cm per day.

Total trap catches per lure change data in 2008 also fit a Poisson distribution (Goodness-

of-fit test: χ2= 4.53; P = 0.21). Intercept TM panel traps baited with the pheromone caught more females at both sites. At Site A, traps baited with the pheromone blend and the full mix of five plant volatiles (MM traps) had the highest overall female trap catches (GLM: χ2 = 15.6; df = 5; P

<0.0001), and caught significantly more females than males (GLM: χ2 = 6.69; df = 1; P =

0.0097). In fact, MM traps caught 15 of the 44 total beetles trapped in Intercept panel traps on

host trees at this site (ca. 34 %), and 12 of these 15 beetles were females. The number of males

trapped did not differ among lure treatments (Fig. 3..3A).

Trap catches were significantly lower at Site B (17 beetles in total) compared with Site A

(44 beetles in total) for Intercept TM panel traps hung on host trees (GLM: χ2 = 29.4; df = 1; P

<0.0001). No significant difference was found among lure treatments at Site B. However, females were caught only in traps baited with the pheromone alone or in combination with plant volatiles; no beetles were caught in control traps and more males were caught in traps baited with plant volatiles than in those baited with the pheromone alone (Fig.3.4).

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Figure 3.3. Mean number of male and female Anoplophora glabripennis caught in Intercept TM

PT traps hung on host trees (Graph A) and screen sleeve traps (Graph B) at Site A in July and

August 2008, based on total caught per lure change. L=linalool; MP=male-produced pheromone;

MPL= male-produced pheromone + linalool; Mix= linalool+ (Z)-3 + (-)-trans-pinocarveol + linalool oxide + trans-caryophyllene; MM= male-produced pheromone + linalool+ (Z)-3 + (-)- trans-pinocarveol + linalool oxide + trans-caryophyllene. Error bars represent standard errors of the mean. Letters over bars represent significantly different means at the 95% confidence level across lure treatments for each gender. An asterisk over the bar represents significantly different means between males and females. Male trap catches were not significantly different among lures

(no letters over bars).

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Figure 3.4. Mean numbers of male and female Anoplophora glabripennis caught in Intercept TM

PT traps hung on host trees (trap type 1) at Site B, China in July and August 2008, based on total

caught per lure change. L=linalool; MP=male-produced pheromone; MPL= male-produced

pheromone + linalool; Mix= linalool+ (Z)-3 + (-)-trans-pinocarveol + linalool oxide + trans-

caryophyllene; MM= male-produced pheromone + linalool+ (Z)-3 + (-)-trans-pinocarveol +

linalool oxide + trans-caryophyllene (see Table 3-2). No significant differences were found

among treatments or between genders.

Comparison of trap designs and placement in 2008

In 2008, relative effectiveness of trap designs and trap placement were compared at both sites. At Site B, no screen sleeve traps were set up due to lack of space. InterceptTM panel traps on host trees caught a total of 13 beetles, compared to only 4 beetles caught in traps hung on bamboo poles.

At Site A, screen sleeve traps had the highest mean trap catch per trap (GLM: χ2 = 41.6;

df = 2; P <0.0001), followed by Intercept TM panel traps hung on host trees (Table 3.3). Intercept

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TM panel traps hung on bamboo poles caught only 3 beetles in total, which were found in one trap baited with the pheromone alone and two traps baited with a combination of the pheromone and the mix of five plant volatiles. Consequently, only Intercept TM panel traps hung on host trees were used in further comparisons with screen sleeve traps. Trap catches increased toward the end of July; peak trap catches occurred on July 26, 2008 for the screen sleeve traps and on Aug 1,

2008 for the Intercept panel traps (data not shown).

Table 3.3. Mean total trap catches of A. glabripennis for each trap design/placement at Sites A and B (Qing Tong Xia, Ningxia, China) for all lures for the entire trapping season of July 11 –

Aug 7, 2008.

Trap design/ Site A Site B placement No. of Mean no. of No. of Mean no. of replicates** beetles caught ± replicates beetles caught ± SE* SE* Intercept TM PT on 36 1.22 ± 0.09 ab 36 0.36 ± 0.12a host tree Intercept TM PT on 18 0.16 ± 0.22 b 12 0.33 ± 0.25a bamboo poles Screen sleeve trap 18 2.38 ± 0.85 a 0 NA on poplar tree * Represents mean number of beetles caught per lure change. Means with different superscript

letters within a column are significantly different at the 95% confidence level.

** Number of replicates = Number of lures (= 6 for all) x Number of traps baited with each lure

(different across trap design/placement)

NA= not applicable

Female trap catches at Site A differed significantly by lure treatment and by the interaction of lure treatment with trap design, while male trap catches were not significantly different across lure treatments or trap designs (Table 3.4). Significantly more females were 72

caught in screen sleeve traps baited with the male-produced pheromone and (-)-linalool than in

Intercept TM panel traps baited with the same lure (OC: χ2 = 4.23; df = 1; P = 0.03). More females were also caught in control screen sleeve traps than in control Intercept TM panel traps (OC: χ2 =

13.1; df = 1; P = 0.0002). Trap catches with the remaining lures did not differ significantly between the two trap designs.

Table 3.4. Generalized linear model results from analysis of trapping data to test main effects of lure treatments (6 total) and trap designs (hung on host trees) on total, female, and male trap catches at Site A.

Total trap catch Female trap catch Male trap catch

Chi- Chi- P- Chi- P- df P-value df df Square Square value Square value

Trap design 2.71 1 0.099 2.65 1 0.1029 3.17 1 0.075

Lure treatment 14.1 5 0.014 20.4 5 0.0011 1.01 5 0.962

Trap design x 26.1 5 < 0.0001 18.5 5 0.0024 7.61 5 0.178 lure treatment

Trap catches of males in screen sleeve traps were significantly different across lure treatments (GLM; χ2 = 15.3; df = 5; P = 0.009), while females’ were not. Screen sleeve traps

baited with a combination of the pheromone and (-)-linalool caught significantly more males than

control screen sleeve traps (OC: χ2 = 6.85; df = 1; P = 0.008), and traps baited with (-)-linalool

(OC: χ2 = 6.85; df = 1; P = 0.008), the mix of five plant volatiles (OC: χ2 = 9.75; df = 1; P =

0.001) and the combination of the pheromone with the mix of five plant volatiles (OC: χ2 = 6.85;

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df = 1; P = 0.008), but was not significantly different from the male pheromone alone (OC: χ2 =

2.3; df = 1; P = 0.12) (Fig. 3.3B).

Figure 3.5. Total trap catches per day per trap design/placement at Site A (Qing Tong Xia,

Ningxia, China) from July 11 – Aug 7, 2008 over all lures. Treatments included differences in trap design and placement as follows: Intercept panel traps hung on host trees (n = 36); Intercept panel traps hung on bamboo poles 20 m far from host trees (n =18); and screen sleeve traps wrapped around host tree trunks (n = 18).

All females caught were dissected and the spermatheca of each female was observed to determine mating status. Of the females caught in traps baited with the combination of the pheromone with the mix of five plant volatiles, 85% were virgins, while most females caught in the remaining treatments had mated, as evidenced by the presence of sperm in the spermatheca

(Fig. 3.5).

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Influence of tree infestation level on trap catches

Site information on beetle abundance and infestation level of host trees in the 2008 summer study sites are given in Table 3.5. At Site A, beetles were more abundant in rows with a higher number of host trees (Spearman: χ2 = 0.67; P = 0.033). Trap catches for both Intercept TM

panel traps hung on host trees and screen sleeve traps correlated positively with the number of

hosts ranked at infestation level 3 (Spearman: χ2 = 0.07; P = 0.0221). At this site, the two trap designs together caught ≈ 22% of the observed beetle population (Table 3.5).

At Site B, trap catches were not correlated with any of the parameters evaluated. Site B was characterized by a low insect population (mean number of beetles observed per row = 4.20 ±

2.33). Intercept TM traps hung on poplar trees at this site caught ≈ 67% of the observed beetle population, with trap catches per row averaging 2.80 ± 0.66 beetles (Table 3.5).

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Table 3.5. Site details and correlation with trap catches at both sites in 2008.

Site A Site B Mean ± SE IPT* on host IPT on IPT on host IPT on (P- value) trees + screen bamboo poles trees bamboo poles sleeve traps Row length (m) 81.30 ± 8.47 51.50 ± 6.99 148.20 ± 28.32 97.50 ± 1.50 (0.36) (0.74) (0.08) NA Number of host 58.10 ± 7.32 0.00 ± 0.00 174.80 ± 36.05 0.00 ± 0.00 trees (0.13) NA (0.55) NA Number of non- 4.30 ± 0.93 0.00 ± 0.00 48.60 ± 7.47 121.50 ± 13.50 host trees (0.007) NA (0.21) NA Number of beetles 36.80 ± 6.61 14.00 ± 14.00 4.20 ± 2.33 0.00 ± 0.00 observed per row (0.44) (0.66) (1.00) NA Number of traps 5.10 ± 0.50 5.00 ± 1.00 7.20 ± 1.66 6.00 ± 0.00 per row (0.38) (0.66) (0.74) NA Number of dead 11.50 ± 2.95 0.00 ± 0.00 8.60 ± 2.25 0.00 ± 0.00 trees (0.05) NA (0.93) NA Number of trees at 30.00 ± 4.26 0.00 ± 0.00 22.80 ± 6.49 0.00 ± 0.00 infestation level 3 (0.022) NA (0.74) NA Number of trees at 22.50 ± 4.49 0.00 ± 0.00 44.60 ± 14.67 0.00 ± 0.00 infestation level 2 (0.60) NA (0.21) NA Number of trees at 4.20 ± 1.27 0.00 ± 0.00 107.40 ± 37.97 0.00 ± 0.00 infestation level 1 (0.028) NA (0.55) NA Number of beetles 8.10 ± 1.77 0.75 ± 0.25 2.80 ± 0.66 2.00 ± 2.00 caught per row

P values (values between parentheses under the means) are for the results of a Spearmans non-parametric correlation test (JMP7.0, (SAS Institute Inc. 2007). NA = correlation analysis not applicable due to non- positive values or small sample size.

* IPT = Intercept TM panel traps

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Field observations of mate-finding, flight and dispersal behaviors of A. glabripennis.

We observed that A. glabripennis adults were primarily active during daylight hours. Between 8 and 10 a.m., beetles were usually in the tree canopy, feeding on leaf edges and petioles. Males (n=59 observed) tended to walk on the top branches of the tree and stand on the upper surface of leaves, while females walked more on main branches in the middle part of the canopy. Around noon, beetles were observed walking toward shaded areas of the trees. Beetles moved less as temperatures increased. As temperatures began to fall in the mid-afternoon (4-6 pm), activity increased again, and more feeding, mating and oviposition were observed. At night, beetles (n >50 observed) migrated to the lower part of tree trunks (within ≈ 1 m above-ground), the lower branches of the tree, or the litter layer under the tree. When disturbed at night (8-10 pm;

July 18, 2007), beetles rarely moved. Mate guarding was observed at different times of the day.

Mating pairs spent most of their time on shaded sections of trunks and larger branches.

Beetles moved slowly to adjacent trees and within the field. On July 18, 2008, roughly

60% of the beetles marked and released on July 7 were found on the same tree they were released on, and about 80- 90% of these were females.

Eighteen of the 20 beetles used in the flight experiment on July 20, 2007 took flight immediately after leaving the cage. As soon as they reached the cage opening, beetles walked on the top of the cage, alternately raised and lowered their antennae several times, appeared to balance themselves, opened their wings and took flight. Fifteen out of the 18 beetles released flew the same general direction toward the south-west, went to the back of the first row of poplar trees

South-West of the release site and landed on the sunny side at the top of one of the first 10 poplar trees in that row.

In contrast, none of the beetles released at night on July 23, 2007, 2 m away from a light trap, attempted to fly. Instead, beetles walked down towards the ground or fell to the ground,

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walked a short distance, and then stopped. Although the light trap was placed close to highly

infested poplar plots, no A. glabripennis were caught in the light traps.

In 2007 and 2008, we made three observations of pre-mating sequences. In these three

cases, A. glabripennis males stood on the upper surface of a leaf at the top of a poplar tree during

early morning. Thirty minutes to one hour later, females walked upward on branches connected to

the leaves on which males stood. However, in no case did the females walk all the way to the

males. Instead, they stopped a few centimeters lower on the same branch and waited for the male

to walk down. Both the male and the female then walked toward each other on small adjacent

branches, sometimes even on both sides of the same branch. After the first approach, females

stopped and started grooming and moving their antennae. These activities stopped when the male

turned and walked down the branch toward the female. Males moved slowly and felt the bark

with their labial palpi almost continuously. Mounting occurred only after the male’s antennae

touched the female’s elytra. Unfortunately, we were unable to observe additional pre-mating

behaviors due to the location of the beetles in the upper canopy during morning hours and the

length of the process (1 hour on average).

Discussion

Trapping results indicate that the male-produced pheromone of A. glabripennis, especially when combined with specific plant volatiles, shows promise as a lure for monitoring of this exotic species. Previous laboratory and greenhouse bioassays showed significant attraction of females to the male-produced pheromone, in addition to moderate attraction of opportunistic males (Nehme et al. revised version in review). In the field, trapping results provided additional evidence for the attractiveness of the pheromone blend and for its role in mate-finding. More females were caught in Intercept TM panel traps baited with the pheromone blend compared to

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control traps both in 2007 and 2008 and at both field sites. Although pheromone-baited Intercept

TM panel traps caught some males, the number of males caught was not significantly different from control traps, which suggests that the males might be attracted more to the shape and color of the trap than to the pheromone itself.

Between the two components of the male-produced pheromone, the alcohol attracted only females to Intercept TM panel traps, while the aldehyde-baited traps did not catch any beetles.

Although some attraction to the aldehyde component was observed in the laboratory experiments, especially for males, the absence of beetles in aldehyde-baited traps in the field might be due to conversion of the aldehyde into the acid under ambient oxygen conditions and high temperatures in the field. Alternatively, this could also be due to a difference in beetle behavior between laboratory and field environments.

Anoplophora glabripennis female trap catches were greatly enhanced by the addition of (-)-linalool, (Z)-3-hexen-1-ol, linalool oxide, trans-caryophyllene and trans-pinocarveol to the pheromone. Most of the females caught in traps baited with this combination were virgins.

Enhancement of trap catches of cerambycids and other wood-boring insects through the addition of plant volatiles to insect pheromones is a common practice. Since the 1970’s, it has been known that bark beetles can be captured in greater numbers in traps baited with a combination of plant stress odors and bark beetle pheromones (Pitman et al. 1975, Byers et al.

1988b). Combining the male sex pheromone of Hylotrupes bajulus L. (Coleoptera:

Cerambycidae) with four monoterpenes was found to significantly improve trap catches (Reddy et al. 2005). Ginzel and Hanks (2005) also provided evidence for the role of host volatiles in mate finding for three species of longhorned beetles, Xylotrechus colonus (Fabricius),

Megacyllene caryae (Gahan) and Neoclytus mucronatus mucronatus (Fabricius). Plant volatiles seem to play a similar role for A. glabripennis virgin females searching for a mate.

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These trapping results, combined with previous laboratory results showing beetle

preference to a combination of the male-produced pheromone with plant volatiles over the

pheromone alone in a Y-olfactometer (Nehme et al. revised version in review), suggest that A.

glabripennis follows the model posed by Saint-Germain et al. (2007) where males respond to

plant volatiles to find a host tree and females respond to the combination of plant volatiles and

the male-produced pheromone blend to find a mate. This is also supported by our field

observations where females were observed walking toward the males, but stopping short. The

male then walked to meet the female, which could also be guided by additional, unknown

short-range cues, including vision. Contact pheromones, which occur on the elytra of females,

likely provide the final cue for male recognition of con-specific females (Zhang et al. 2003).

Our field observations also support earlier descriptions of the behavior of this species.

Observations of beetle activity support previous descriptions by Li et al. (2003) and William et

al. (2004a) of A. glabripennis as a diurnal species. Also, greenhouse observations by

Morewood et al. (2004a) indicated that beetles were more active during the early and late hours

of the day in summer. Beetle activity was reported to decrease at night and in the middle of the

day when beetles preferred shaded areas during high temperature hours. Effects of temperature

on beetle activity were described by Zhou et al. (1984) and in controlled experiments conducted

by Keena (2006) showing that temperature strongly impacts beetle longevity and fecundity.

The average temperature at our field site was ≈ 28 °C, with mid-day temperature exceeding the

30- 32 °C threshold above which fecundity and longevity are negatively affected (Keena 2006).

Flight occurrence in the field is rare. In our flight experiments performed during the

day, we found that beetles flew more often in the direction of the sun. In fact, the three beetles

that did not fly to the west (direction of the sun at this time of day) were the last three released;

the time of release of these last beetles coincided with the occurrence of clouds covering the

sun in the southwest. Landing after flight occurred almost exclusively on host poplar trees.

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Beetles hovered for a few minutes before landing and landed securely on leaves in the upper

canopy. The results of our small release experiment are consistent with larger dispersal studies

showing that A. glabripennis prefer to stay on the same tree and disperse slowly in circles

around the source (Smith et al. 2004, Bancroft and Smith 2005).

In terms of trap design, as we observed earlier in the greenhouse (Nehme et al. revised

version in review), Intercept TM panel traps and screen sleeve traps were equally successful in trapping A. glabripennis adults in the field. The significant interaction observed between trap design and lures in the greenhouse (Nehme et al. revised version in review) also persisted in the field where numbers of beetles captured by the two trap types depended on the lure used.

Screen sleeve traps caught the highest number of beetles when baited with a combination of the male-produced pheromone and (-)-linalool, while Intercept TM traps had the highest catches when baited with the male-produced pheromone and a mix of five plant volatiles: (-)-linalool,

(Z)-3-hexen-1-ol, linalool oxide, trans-caryophyllene and trans-pinocarveol. Implementation of either of the trap types will depend on availability, feasibility, location, tree type and shape, accessibility, and other economic factors beyond the scope of this paper. Although we believe the combination of plant volatiles used was successful in trapping A. glabripennis in the field when combined with the male-produced pheromone, additional field studies using variations of the combination of the plant volatiles used in 2008 and other plant volatiles shown to be attractive in Y-olfactometer bioassays, are needed. For example, 3- carene was found to be highly attractive to males (Nehme et al. revised version in review) and may be useful when combined with the pheromone blend.

Our trapping results from Site B (2008), although not statistically significant, are promising given the low density of A. glabripennis observed at this site. In the US, quarantine regulations require eradication of known infestations. Unfortunately, too often infestations are not discovered until a large number of trees are infested (e.g. New York and Worcester, MA). This,

81 the need for monitoring traps to detect outlier populations or the presence of low numbers of beetles remaining in a quarantine zone is critical at places such as ports of entry, adjacent to known infestation areas, and other high risk sites such as those released from quarantine when eradication efforts have been completed. If beetles are present at these types of locations, they will usually be at low to very low population densities, more similar to what we observed at Site

B than at Site A in this study. The finding that, at Site B, InterceptTM panel traps caught more than

50% of the observed beetle population, including similar numbers of males and females, is a highly promising result given the likely similarity to A. glabripennis populations in the U.S.

Being able to trap both sexes is very important at ports of entry where there is an equal chance of introducing males and females. At the same time, the female’s perceived ability to discriminate between lures that contained the male-produced pheromone and those that did not is also critical for monitoring purposes in the field. Trapping a good proportion of females at low densities may reduce the likelihood of establishment at that site, both by reducing the number of females for reproduction, which may produce an Allee effect, and by reducing the number of ovipositing females and thus limiting future generations.

In conclusion, the A. glabripennis male-produced pheromone is a promising lure for monitoring when combined with (-)-linalool, (Z)-3-hexen-1-ol, linalool oxide, trans- caryophyllene and trans-pinocarveol and placed in Intercept TM panel traps. In addition, screen sleeve traps, baited with this pheromone and (-)-linalool, appear to provide a potential alternative.

Challenges to the success of this monitoring technique in the U.S include differences in host tree species composition, including height, shape and location of host trees, compared with

China. These factors are likely to affect beetle behavior and attractiveness to specific plant volatiles. Consequently, trap designs, trap placement, lure combinations, and the type of emitters used in China should be tested and adapted as needed to field conditions in the US to optimize their efficacy.

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Acknowledgments

The authors extend their gratitude to Prof. Luo of the Beijing Forestry University, the

Ningxia Forestry Bureau direction and staff, and Victor Mastro, Dave Lance and Boade Wang at

USDA-APHIS-PPQ, Otis, MA for facilitating collaborations in China. We also thank Wenchao

Xu and Jacob Wickham for technical support during field studies and June Nie of the Invasive

Insect Biocontrol and Behavior Laboratory in Beltsville for assistance with chemical synthesis.

This work was supported by grants to KH from the Alphawood Foundation, the Horticultural

Research Institute, cooperative agreements with the USDA, APHIS, PPQ and USDA NRI

Arthropod and Nematode Biology and Management: Suborganismal Biology (Grant No. 2008-

03986).

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Chapter 4

Find the beetles/ fā jué jiă chóng!: A Plan for Participatory Action Research

to detect and manage Asian Longhorned Beetle in China and the U.S.

Introduction

History and description of the Asian longhorned beetle problem

The Asian longhorned beetle (ALB) is a wood-boring insect species native to

China and Korea. ALB was introduced to the U.S., Canada and several countries in

Europe through solid wood packaging material. The insect is a serious pest both in its native and introduced range.

According to the United States Department of Agriculture’s Animal and Plant Health

Inspection Service (USDA- APHIS), pest risk analysis indicates that ALB was introduced into the

U.S. in solid wood packing materials (SWPM) from Asia (USDA-APHIS 2005b). Field infestations were first discovered in Brooklyn, New York in 1996 (Smith 2000b). This infestation spread to Long Island, Queens and Manhattan. In 1998, a separate introduction of ALB was found in the Ravenswood area of Chicago, as well as two suburban areas, Bloomingdale and

Summit. Beetles were then detected at two separate locations in New Jersey: Jersey City in 2002 and Middlesex/Union counties in 2004 (APHIS 2005). In 2006 and 2007, new infestations were also found on Prall’s Island, an uninhabited island lying close to Staten Island between New York and New Jersey (USDA-APHIS 2007) and in Eastern Linden in New Jersey (NJDA 2006).

Although the infestation in Chicago was thought to be eradicated in 2007 after four years of

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negative surveys, beetles were observed again in Deerfield, Cook County, Illinois in August 2008

(Bech 2008a). At the same time, a new infestation was reported in Worcester County,

Massachusetts. Sixty-two square miles were currently under quarantine and scouting is still

ongoing (USDA-APHIS 2008a, Bech 2008b).

In September 2003, a resident of Woodbridge, Toronto, Ontario reported beetles to the

Canadian Food Inspection Agency (CFIA) that were identified as ALB. Surveys led to the

discovery of a localized infestation in an industrial park (Bell 2005). Latest records show

additional infestations found in October 2007 within the city’s regulated area, on Jane Street and

Sheppard/Finch Avenue (CFIA 2007).

In Europe, ALB was first discovered in 2001 in the urban area of Braunau, Nothern

Austria, feeding on Norway maple, Acer platanoides. Additional infestations were found in

Montpellier, France in 2003 and Sainte-Anne-sur-Brivet in 2004 (Herard et al. 2006). In

Germany, ALB was first found in 2004 in Neukirchen, then in Bornheim in 2005 (EPPO 2008)

and in 2007, a small outbreak was found in Corbetta, Italy (Maspero et al. 2007). An introduced

population of ALB was also found in Yokohama, Japan in 2003 (Takahashi and Ito 2005).

Insect tunneling inside the wood causes weakening of trees, and poses a serious threat to

pedestrians and vehicles from falling limbs and trees. ALB’s preference for maple species in the

U.S. (Haack et al. 1996, Li et al. 1999b) can have a serious impact on maple forests managed for

lumber and maple-dependent industries, such as maple syrup production. The estimated potential

urban impact of ALB in the U.S. is a loss of nearly 12-61% of tree population in the cities, based

on field data from nine U.S. cities, with an estimated value of $72 million- $23 billion per city, a

corresponding estimated maximum potential impact of 34.9% of total canopy cover, 30.3% tree mortality, and a compensatory value loss of $669 billion (Nowak et al. 2001).

In China, ALB was reported as early as the Qing Dynasty (1644-1912) and is currently found in 25 provinces extending throughout a vast geographic range, 21 ºN to 43 ºN latitude and

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100 ºE to 127 ºE longitude (Yan 1985). The abundance of susceptible hosts turned this local non-

pest insect into an outbreaking pest problem. Since the early 1980’s, ALB infestations have

caused more than 10 billion Chinese Yuan (roughly $1.47 billion US) in losses, accounting for

approximately 12% of total economic losses by forest pests (Lingafelter and Hoebeke 2002a).

Management of this pest is mainly done through cutting host trees in a certain radius

around the center of infestation. If detected early enough, infestations can be eradicated.

Detecting these rare infestations in time enough to eradicate nascent populations is time

consuming and expensive. An integrated approach involving technical advances in technical tools

and enlisting and organizing large numbers of observers would make early detection more

probable.

A major challenge to the success of ALB management has been the lack of an efficient

monitoring tool to detect new infestations and monitor the development and spread of established

ones. New research studies have shown promising results for potentially successful monitoring

trap designs and lures (Nehme et al. 2009). These traps, using pheromone technology, attract

beetles from a wide area making young populations more apparent. Traps can be placed in

potential ALB habitat and periodically monitored. However, implementation of this monitoring

strategy requires a certain level of collaboration between different governmental agencies, non-

governmental organizations, landowners and land-managers, and individuals in both China and the U.S. to ensure success of monitoring. The success of this integrated approach is rooted in the awareness and knowledge of all of these collaborators.

Hereafter, I propose a Participatory Action Research approach to engaging stakeholders and the public in the detection and management of ALB in both China and the U.S. The history, government, culture and economy of these two countries are quite different. Existing government structures offer different sets of resources to support such an effort. Our PAR offers approaches to

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accomplish the dual tasks of preventing ALB outbreaks in the expanding forestry industry in

China and preventing the invasion of ALB, an exotic pest in the U.S., from establishing

permanent populations.

Specific aims of the campaign

The goal of Find the beetles / fā jué jiă chóng! program is to improve monitoring strategies for the Asian longhorned beetle (ALB) in the U.S. and China through collaborative efforts from local communities, governmental agencies, and public-private partnerships. By implementing a participatory action research approach, the campaign will provide a model and ground base for farmers and local communities to build upon for further participation and empowerment in the forestry, pest management and landscape protection fields.

The specific aims of the Find the beetles / fā jué jiă chóng! program are as follows:

• To identify best practices in monitoring Asian longhorned beetles in China and the U.S.

• To create a network of scientific researchers at universities and research centers working

on Asian longhorned beetle management, tree growers, wood manufacturers, and

government agencies involved in pest management and quarantine.

• To engage the public as partners and stakeholders in ALB detection and management.

• To develop Popular Education material and user-friendly protocols to transfer recent

research findings to the local communities and stakeholders.

• To identify supportive policies in the forestry, tree growing or wood production sectors

that will facilitate effective involvement of members of the community.

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• To initiate public-private and public-government partnerships that would benefit other

wood pest management efforts as well.

• To determine how social capital and political structures in the U.S. and China affect the

efficacy of pest management strategies and participatory approaches.

Community members will be involved in all steps of the project, starting in defining the research questions to complete evaluation of the project. The positive outcomes of community participation and Popular education programs has been shown in earlier research studies, mainly in the public health sector (CCPH 2008). In this proposal, we suggest combining popular education methodology with participatory action research principles to develop a solid, context- adaptable monitoring program for the Asian longhorned beetle. Our approach is very similar to the Community Health Workers (CHW) programs applied in Latino and African American

Communities in the U.S. (NY State Department of Health 2009). Trained community members will be the “agents of change” who will be responsible for the first stages of monitoring and will transmit their knowledge to others in later stages.

To ensure the achievement of these goals, qualitative and quantitative measures will be conducted for evaluation at different steps of the program. Evaluation will be conducted at the personal, organizational, community and policy levels.

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Background

Popular Education and Participatory Action Research Principles

The notion of popular education comes from the work of the Brazilian educator, Paulo

Freire. According to Freire, everyone learns from his/her own life experience, and as a consequence, develops a valuable knowledge base. The role of researchers and educators is to facilitate the sharing of this knowledge among individuals and communities (Freire 1973).

Participatory action research (PAR) relies on the participation of all stakeholders in all stages of the project, ranging from the first inquiry or diagnosis phase to evaluation and implementation. Researchers in this case are insiders to the setting, rather than observing outsiders as in conventional action research projects. PAR involves a spiral of steps that feed from each other, each of which includes diagnosis, prescription or planning, implementation and evaluation (McTaggart 1997, Herr and Anderson 2005).

Combining popular education and participatory action research is easy in the sense that

no basic differences are found in the two concepts. In fact, popular education ensures that PAR

principles are followed and vice versa. In the diagnosis phase of our proposed PAR, members of

the community will be invited to meet and share their knowledge about the beetle, its host trees

and optimal conditions for survival, with each other and with the team of research staff involved

in the project. Needs assessment and asset mapping will be based on this shared knowledge, as

well as the identification of best practices.

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Social Capital and Participatory Action Research

Social capital is defined by Robert Putnam as “the process and conditions among people and organizations that lead to accomplishing a goal of mutual social benefit, usually characterized by four interrelated constructs: trust, cooperation, civic engagement, and reciprocity” (Putnam 2000). Social capital also has been defined as the driver of success of groups, as compared to individuals, through collaboration and mutual help (Coleman 1990).

Forestry and urban plantations are both community-oriented since changes or benefits to each affect a large group of individuals in the central community or even the neighboring ones.

Consequently, solving issues of invasive and pest insects in forest and urban trees requires a deep and efficient collaboration between the different affected groups. Based on the historical, social and structural analysis of ALB detection and management between China and the U.S. described above, I am proposing a PAR approach to monitoring ALB for each country. Particular attention will be paid to the differences in social capital and how community-based approaches can be used to improve detection and management.

Overview of forestry and pest management in China

China has undergone a major transition since the agricultural reforms in 1978, which increased decentralization, as manifested first by the Household Responsibility System (HRS) that transferred agricultural land-use rights to individual households, then by the CRS (Contract

Responsibility System) in the Southern Region in 1981 that did the same for forested land. Prior to the reform, agricultural production and forested lands were managed by collectives, agricultural production teams and state-owned forestry bureaus, and was suffering from major

90 issues such as decrease in soil quality and low return. Per capita annual income was as low as 63

Yuan (~ US $25 at the time) (Hyde et al. 2003). Forests covered only 8.6% of China’s total land area. Following the establishment of HRS for agricultural producers, China saw a tremendous increase in production and profits. By 1984, 3 years after the implementation of CRS, 57 million households were managing around 30 million ha of forested land. However, forestry reform was not as efficient as agricultural reforms. Households managing forested areas were still required to sell their production back to state-owned wood-processing operations, and most of China’s forests are still under the authority of a central forest ministry. In 2003, China joined the World

Trade Organization. Since then, roundwood harvest has increases by 182% in the first two years, while domestic timber harvest has declined (Hyde et al. 2003).

The Northern plains of China were important for agricultural production but forestry did not play a major role in that part of the country early on, compared to the Southern regions where forests were more important and forested areas increased from 1977 to 1984 by more than 20%.

An important lesson learned from the agricultural reform in China is the value of ownership and local management. While supporters of state management systems argued that farmers do not value long term management of forest land, forests are regarded as major social assets that should be managed by an authority that could be held accountable to the whole society. However, the predominance of corruption and illegal logging practices on state-managed land and the statistics showing an increase in forested areas in China after implementation of CRS reform strategies clearly contradict these arguments. The same farmers who were not willing to invest in forestry practices before 1978 were the ones responsible for the 20% increase in forested land in China in the following 7 years. This change carries two important messages; one is the value of ownership and its effect on people’s willingness to put energy and effort into a certain practice, and the second is the usually underestimated flexibility of human nature, openness to learn and tendency toward innovation (Hyde et al. 2003).

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China’s Northern plains, where Asian longhorned beetle infestations are widely spread, and where we propose to commence the implementation of our participatory action research project, are predominantly agricultural lands that had very little forested areas. Flooding and erosion of unstable agricultural soils in the Yangtze River Basin in 1998 and periodically in the

Yellow River Basin have led to serious rethinking of the agricultural programs in the regions of the two basins, and the subsequent implementation of the Natural Forest Protection Program by the government. Actual forest protection and reforestation efforts were however driven mainly by private investment (Hyde et al. 2003).

Although the Green Great Wall Campaign, launched in 1978 by the Chinese government through the Three-North Sheltherbelt Programme, did increase forested areas to a certain extent, the failure of government and state-owned management failed to consider all of the implications of this strategey (Hu et al. 2008). When large plantations were implemented, local pest problems were not taken in consideration. Almost all tree species planted during that campaign were susceptible to an insect native to the region, namely the Asian longhorned beetle (‘Tian Niu’ in pinyon Chinese), Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae). In fact, although the 1981 forestry reform through CRS slightly shifted the ownership from government and collectives to households, decision-making about global issues such as landscape-scale pest management and species selection for planting is still in its majority controlled by state-owned forestry bureaus. Bureau officials from different states meet at the regional or national level to make decisions. Attendance at these meetings is usually restricted to directors and co-directors who return to their state-located offices and communicate the decisions and new regulations to their employees who then transmit them to farmers.

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The Chinese Community

In the province of Ningxia where research was done on monitoring Asian longhorned beetles by scientists from the U.S. (Nehme et al. 2009) and European countries, collaboration projects were established with Chinese universities and the local Forestry bureau of Ningxia.

Research was done on state-owned land and communication with farmers working that land was done through bureau officials. Those farmers employed by the state to cultivate the state-owned land were required at the end of the season, when production-related work decreases, to go out in the fields and collect Asian longhorned beetles and destroy them.

However, even under such a controlled environment, the voluntary role of the local community emerged to help fight ALB. For example, in Yinchuan, the capital of Ningxia province (north-western China), local students organized a public campaign to control ALB. The campaign led to the collection and destruction of 500,000 beetles in the summer of 2007 (Hu et al. 2008).

Invasive pest detection, management and quarantine in the U.S.

The U.S. National Management Plan: Appendix II – Summary of Federal Roles and

Responsibility states the responsibility of federal agencies for spreading public awareness about national issues. U.S. Department of Agriculture agencies have branches that deal specifically with public information and awareness campaigns. APHIS has a “public affairs effort” that responds to emergency situations and eradication programs. The Forest Service (FS) “administers a number of cooperative programs that assist partnerships and encourage forest stewardship for non- industrial forest landowners, including control of invasive species.” The Cooperative State

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Research, Education, and Extension Service (CSREES) is heavily involved in outreach through its Land Grant University System. The Extension Service provides a vital link between USDA, stakeholders and citizens, through on-campus and county offices across the U.S. The Agricultural

Research Service (ARS) has Area-wide Pest Management Programs that provide support to transfer technology within five years for the management of invasive species.

Since the first ALB infestation was found in New York City in 1996, the government agencies listed above mobilized federal and state resources and personnel to impose regulated boundaries and conduct survey and eradication measures. In 2005, a strategic plan was developed for the Asian longhorned beetle Cooperative Eradication Program (ALB CEP) in New York, New

Jersey and Illinois. The purpose of the program is to protect the forest products industry, biodiversity of forests and park-lands in the U.S. and the quality of the urban environment through complete elimination of all reproducing populations of ALB in the U.S. The strategic plan recognized the importance of public support and cooperation for the success of the eradication and re-plantation efforts. According to section VIII of the strategic plan, training more people to recognize and report signs of the beetle will largely improve detection and eradication

(USDA-APHIS 2005b). In 2008, a new strategic plan was developed for the ALB CEP in

Worcester, MA. The CEP involved USDA-APHIS-PPQ, DCR (Department of Conservation and

Recreation) Massachusetts, the Massachusetts Department of Agricultural Resources and the City of Worcester.

Outreach efforts have been mostly handled by the USDA outreach office in Washington

D.C. and included among others a broad awareness campaign during the flight season in the summer, when people are more likely to see the beetles, and continuous warning and information broadcasting on damage and symptoms throughout the year. Campaigns included advertisements in newspapers and on billboards; pictures and information of beetles with hotline numbers for

94 reporting on gadgets such as key chains, bookmarks, door hangers, magnets, tattoos etc.; pamphlets with pictures and warning signs such as the “Wanted” posters; “Help save our maple trees” cards; ALB identification cards with hotline phone numbers; and pest alerts among others.

A huge amount of information is posted on the web by federal and state agencies and extension offices at universities. Easy to access and understand material has been made available to the general public on the net; the “Beetle Busting Toolbox” is a slide show displaying beetle pictures, damage symptoms, Identification cards, pest alerts, citizens guides and “Wanted” posters. Most of public outreach material can be accessed through the USDA-APHIS or Forest Service websites and university extensions websites. The University of Vermont’s Asian longhorned beetle website features most of those materials at http://www.uvm.edu/albeetle/.

Since the discovery of ALB in Worcester, MA in August 1, 2008, the State of

Massachusetts developed an Introduced Pests Outreach Project for ALB. The project involved training sessions for business owners during October and November 2008, community meetings in Worcester to discuss eradication strategies, and ALB “compliance training” for businesses working with host wood materials within the regulated area. The Massachusetts Tree Wardens and Foresters Association held free ALB information sessions for professionals and tree growers.

Toll free numbers were established for the ALB Eradication program and notices were sent to homeowners and residents of the regulated areas (MA ALB CEP 2008).

The U.S. Community

Public involvement was key to the success of the eradication program in the Chicago regulated area in Illinois (Antipin and Dilley 2004). According to interviews conducted with residents, public outreach and media coverage were the most frequently cited factors affecting the

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success of the eradication program. Citizens felt involved and compelled to help when they saw

the city, state and government authorities devoting time and resources to solve problems and

especially when they were informed that the eradication program will not affect their businesses

or add additional costs to their households.

Recent history of invasive species management in the U.S. has witnessed a great

contribution from citizens and local organizations. Most, if not all, ALB infestations in the U.S.

were found by residents who reported their observations to local authorities. The first discovery

of ALB in 1996 was reported by a landowner who thought someone was drilling holes in his

trees. This also occurred in IL and MA where infestations were reported by people seeing the

beetles, or their damage. A recent study in New Jersey showed that reports on infestations

increased with time after implementation of outreach campaigns. Since most of infestations were

found in people’s backyards and private properties, cooperation from landowners was required to

optimize detection. This cooperation depended largely on people’s awareness of the extent of the

problem and the impact of ALB on the environment, including the various tree species present on

their properties. Just as public support improved detection of Asian longhorned beetle new

introductions and infestations through visual observations of insects and damage, involvement of

regulated areas’ residents in any new detection procedure is expected to improve the efficacy and

reduce the cost of detection.

Significance of the project

Findings from this study will contribute to the development of efficient monitoring techniques for the Asian longhorned beetle both in the U.S. and China, and could even be valuable for regions in Europe and Canada where ALB infestations are found. Efficient

96 monitoring will allow early detection of infestations before complete establishment of insect population and prior to severe irreversible tree damage. This will benefit tree growers in protecting their trees from dieback, field crop growers in Northern China by protecting poplar wind breaks from destruction and consequently protecting their crops and their land from wind damage and erosion. In the U.S., efficient monitoring largely reduces the costs inflicted by current monitoring practices. This approach helps define the quarantine boundaries and localizes efforts to achieve efficient eradication. Detection at ports of entry largely reduces risks of infestations in the field.

Indirect outcomes of the project involve the improvement of social capital within affected communities in both countries, as well as an increased participation of the general public in solving issues of similar impact and description, such as other cases of invasive species in the

U.S. (e.g, Emerald ash borer, Gypsy moth, and others). From a regulatory point of view, involvement of the community in monitoring efforts facilitates the tasks of government agents who are currently facing the challenge of requesting landowners’ approval for monitoring private properties. The forestry sector in China and the U.S. can benefit from the information collected on tree species, distribution, level of infestations, etc. Introducing communities to the concept of participation and popular education in the context of this project is expected to be repeated in future projects in different sectors, helping in the empowerment of previously marginalized groups such as local farmers in Ningxia province in China, or wood manufacturing workers in the

U.S., and enhancing collaboration among government agencies, research institutes and local communities for the development of other forestry-related programs.

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Preliminary studies in China and a comparison with the U.S. and other countries

During our visits to China in July 2007 and July/August 2008, we conducted a

series of interviews with the forestry bureau director and employees, university students and local

farmers. The interviews yielded the following information:

The role of the Forestry Bureau in China

The Forestry Bureau in Ningxia Province follows the national Ministry of Forestry in all

major production and protection decisions. The Bureau owns a majority of the forested land, as

well as some fields planted with grapevine or corn and in which poplar trees are planted as

windbreaks and it overlooks all forest-related activities, including research, production and

protection work.

Forestry Bureau employees hire local workers as wage payrolls to work the land under

their direct supervision. All details of the work are set by the Forestry Bureau officials, and

signed by the director. Workers do not participate in the decision-making process and are

expected to apply the orders as given to them.

To our knowledge, the only cooperative projects between the Forestry Bureau in Ningxia

and Beijing Forestry University are those established by USDA for research purposes. The

Bureau conserves its authority over the land even within these cooperative projects. University

researchers, both American and Chinese, work under the supervision and upon the approval of the

Forestry Bureau, on land specified by the latter, and are expected to pay field rental fees to the

Forestry Bureau.

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Except for personal friendships, there is no direct communication between researchers and the local community or hired wage payroll workers. All communication is channeled through the Forestry Bureau.

The role of universities and researchers in China

Unlike the land grant university system in the U.S. where education, research and outreach are integrated within the institution, such integration is very limited, if ever present, in

China.

During my visit to Beijing Forestry University in July 2008, I had the chance to interview three graduate students in the Forest Entomology group. All three students worked on practical research projects for which the outcome should benefit Chinese forestry. Two of them were finishing their degree and writing their research findings. However, none of them had experiences during the course of their program any sort of direct communication with local communities, forestry group or any primary beneficiaries of their findings. When asked how would they convey their research findings to the people who would most benefit from them, students answered that all their findings will be written in a thesis that will be kept in the university library and people are welcome to visit their library and request to read their theses. The dissemination of their knowledge, as well as their future career, depends on the coincidence of the ‘right people’ walking in the university library and reading their thesis. If they’re lucky, they said, the CEO of a major company or a Forestry Bureau high-rank employee will come across their thesis when searching for information and would hire them. When asked about publications in refereed journals, the students explained that they’re all encouraged to publish in local scientific journals, which they do in most cases, but that it is very rare for students to publish outside the country, mainly due to language barriers. In fact, lot of research has been done on Asian longhorned beetle

99 in China, most of which was published in known Chinese journals, in Mandarin Chinese. It is only through personal efforts for translation that researchers in the U.S. interested in ALB had access to these papers.

Chinese students were unfamiliar with the concept of national and international scientific meetings or conferences. There are only few scientific meetings students are allowed to attend while the rest of the meetings are attended by professors only.

Also in the summer of 2008, I interviewed workers in the field about hteir experience working in fields where research on Asian Longhorned beetle was conducted. Questions addressed decision-making, their freedom to make choices, their personal opinion about the problem and management strategies, and the depth of their knowledge about the problem. Only few workers overcame their shyness and answered my questions. Not only they’re not used to see foreigners and talk to them, in some cases, they were not allowed to address us and ordered not to interfere with our activities. They clearly had no role in the decision-making. Some of them were illiterate, others with limited reading and writing literacy. They came from the poorest section and worked the fields for long hours for small wages. They received detailed orders from the Forestry

Bureau officials, to which they abided with no arguments. These orders included instructions on cultivation, pesticide use, calendar of applications, etc… They knew little about the negative impact of the Asian longhorned beetle. Obviously, they saw and understood that U.S. researchers were interested in the beetle because it is a problem in the U.S. For them, ALB was part of the ecosystem and most of them were quite unaware of the losses it is causing in their own country.

Besides the Forestry Bureau and the University, China has a network of regional agricultural research stations (government-owned research centers) favorably looked upon by locals and more respected by government officials than universities. These research stations

100 collaborate with Forestry Bureaus to conduct applied research related to major pest and production problems. On my last day in Ningxia, while I was removing my traps from the trees at the end of the field season, I met a lady from the local agricultural research station, who was collecting ALB adults from the field, along with a group of employees. The collection was meant to be a control measure for ALB. When I inquired why collection wasn’t done earlier in the season when it would have been more efficient in limiting the population, the answer was the lack of personnel since workers were busy with other tasks in the field.

Chinese society and farming systems

In Ningxia, I saw mainly two farming systems: small farms family-owned and maintained by all family members, elderly and toddlers included; and large farms owned by cooperatives or rich families and worked by hired labour, housed in small building on the farm. In both cases, field crops yield harvested at the end of the season is sold to a ‘cooperative center’, managed by the government. Prices are set by the government based on the total production and market price and are usually uncontestable. Irrigation is also managed by the government and follows a strict schedule.

In most of the farms where poplar trees are planted as windbreaks, owners realize the damage caused by the Asian longhorned beetles to their trees but are either too poor or too ignorant to deal with the problem. Since poplar trees are not the major source of production for them, they prefer to ignore the problem.

Men and women work equally in the field. However, there is a clear distribution of tasks in the household and in the society. Women are expected to cook, clean, raise children, garden and take care of the livestock. They are rarely seen in social gatherings. They stay in the kitchen when people are invited for dinner and they do not join dinner invitations in restaurants with their

101 husbands. Smoking and drinking are very common for men but not for women. However, there are some clear differences in the gender trends in the new generation. Especially in families that followed the one-child policy, girls and boys are equally empowered and outgoing. Most of the teenagers I met in Ningxia and Beijing do not care for farming or forestry and are more attracted to recent technology, computer programs and languages.

Comparison with U.S. system

Invasive pest management in the U.S., unlike pest management in China, has always been approached from a collaborative perspective. Parallel to the Ministry of Forestry in China, the US department of Agriculture is responsible for managing pest infetations. However, in all known cases of invasive species, local communities and the private sector were involved through extension and outreach, as well as direct partnerships and collaborations. The development of the land-grant and county extension system allows communication of information between universities and farmers through local extension agents within a state. Communication between the states is allowed through regional and nation-wide meetings and forums, such as the USDA

Interagency Forum on Invasive Species held every year in January in Annapolis, MD.

Networking between graduate students, researchers and government employees across the country is facilitated by international, national and regional meetings of different scientific societies. Cooperative projects between USDA agencies and universities are common and allow a continuous exchange of ideas and practices.

Although the U.S. system favors more collaboration and communications, it still faces some major challenges. First of all, USDA agencies are constantly facing financial challenges that prohibit complete participation of USDA scientists in forums and meetings that involve lot of travel expenses. It also limits the amount of eradication work that could be done in a certain time

102 and space. Inspection, in its current form, is particularly expensive and budget restrictions have always limited the areas covered by inspection in the infested regions and delayed detection of some rather important infestations.

Another challenge resides in the structure of the USDA agencies. For example, USDA-

APHIS is a regulatory agency in which employees are not required to conduct research and consequently no publication requirements are imposed. However, management of invasive species is the responsibility of USDA-APHIS and thus a large amount of practical research on invasive species is conducted by USDA-APHIS employees but rarely published, which renders access to the findings from these studies very limited.

Also, unlike in China where ALB occurs in forested land or windbreak rows, ALB infestations in the U.S. are mostly in urban areas and private properties. Proper management and application of monitoring practices is subject to property right and other regulatory challenges.

Community awareness is much more crucial. And since ALB is an invasive in the U.S., the tolerance threshold for beetles in the field is zero, compared to a higher tolerance in the native regions in China.

Another major challenge to proper management of ALB in the U.S. is the number of stakeholders involved. Because the U.S. system allows for more freedom and calls for consensus in decision-making, the reality of getting a large number of organizations and individuals to agree on a certain strategy becomes more difficult. However, the presence of established connections in the U.S. makes the establishment of a PAR easier by working with already established cooperatives, such as the ALB CEP, farmers’ cooperatives, community/neighborhood groups, etc.

More challenges are expected to emerge as the PAR project begins.

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Project Design and Methods

Overview

The research design for the proposed project will follow participatory action research and popular education principles. This requires, as a start, that each of the target groups listed in Table

5.1 name representatives to become members of two program steering committees, one in

Ningxia province, China and the second in Northeastern U.S. The program will be initiated in

Ningxia because of the already established collaboration between USDA and Ningxia Forestry

Bureau. The steering committees of the Find the beetles / fā jué jiă chóng! program, with the help of a research advisory board constituted of experts in social science, entomology and monitoring of invasive species, will then be responsible for following up on all steps of the program and conducting continuous evaluation.

Information will be solicited by the steering committee from and target group members community in popular education sessions and then used for needs assessment and asset mapping.

This step will then be followed by clear definition of the problem, the strengths and weaknesses, and best practices with the highest chance of success under local conditions in both countries. The committee will then decide on activities and programs and involve the target groups with the application steps. Evaluation will be conducted throughout the time of the project as a continuous process. The model presented in Figure 4-1 summarizes the major steps of the project.

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Table 4.1. List of groups to be involved in the PAR both in the U.S. and China

Country U.S. China

Government USDA-APHIS- PPQ China Ministry of Agriculture- Forestry Bureaus national direction USDA-APHIS ALB program Chinese Customs bureaus

US Forest Service- Northern Ningxia Forestry Bureau Research Station

Extension and Outreach CSREES

Land Grant and Counties Extension agents for Northeastern States Universities University researchers involved Collaborators from Beijing in ALB research Forestry University

Funding agencies Funding agencies supporting ALB research e.g. alphawood

Private sector Wood pellet manufacturing Wood pellet manufacturing industry industry

Local Communities Communities/neighborhoods Households owning forested living in infested or high risk land/farmers areas

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Figure 4-1. PAR model

The success of the PAR program depends largely on the first stage of the project, the diagnosis. Correct needs assessment and asset mapping is crucial to the success of any PAR

(Pilcher and Rajotte 2009). In our project, this translates into correct identification of monitoring sites, level of infestations in different sites, high risk versus low risk areas, and other information required to determine the need for implementing new monitoring strategies. It also translates into what equipment/materials are available, and what materials and strategies are more economically feasible and socially acceptable, while being environmentally-friendly as well. In the U.S., since monitoring is mostly required in sites where population levels are largely unknown, good knowledge of the risk levels of each site based on basic knowledge of the life cycle and host

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preferences of the insect and its dispersal is needed. This could be acquired from reviewing

research studies conducted on the subject both in China and the U.S. Ultimately, a collaboration

between the PAR steering committees in both countries would largely benefit both through

sharing of information and experience.

Goals and Objectives

There are objectives affecting each level of organization from individuals through policy makers.

Individual level objectives

• Increase the capacity of individuals to work in collaboration with people from their

community or other communities and sections of the society.

• Increase individual knowledge about ALB and the damage it inflicts.

• Increase leadership and communication skills.

• Empower individuals and improve their trust in their knowledge and abilities.

• Improve understanding of the value of trees in the ecosystem and the necessity to

preserve them, mainly in China.

• Improve the awareness of individuals in the U.S. of the extent of the risk posed by

invasive pest species.

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Organizational level objectives

• Increase collaboration among organizations and governmental agencies.

• Provide organizations with best practices and tools for self-evaluation and that could be

implemented for other similar projects.

• Improve social capital through the improvement of activities conducted by organizations

and mutual-benefit collaborations among organizations.

• Provide a channel for networking and information sharing among different organizations,

such as universities, government agencies, and local communities.

Community level objectives

• Identify strengths and resources in community that might not be discovered otherwise.

• Identify weaknesses and learn how to overcome them.

• Improve communication within and between communities, which could provide a bridge

for all sorts of exchange of ideas, information or even produce.

• Improve the value and empower local communities.

Policy level objectives

• Identify weaknesses and lacking policies needed to establish collaborations among

different social groups.

• Identify and change policies that pose a challenge to adequate monitoring of invasive

species in the U.S. and forest species in China.

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• Improve the link between research, development and policy by involving policy makers

in the project.

• Move more toward decentralization of forest management in China for better results.

• Learn how to influence bad/outdated policy through proper development of alternative

proposals and through the use of media.

Evaluation

Process and Impact evaluation

Process evaluation is continuous throughout the various steps of the project and involves evaluation of the strategies used, the level of collaboration between the different partners, the validity and completeness of information collected and documents available, and the gaps in knowledge, among others.

Impact evaluation is conducted at the end of each step, to evaluate the impact of the process and activities based on a comparison with the specific aims defined at the start of the program and the outcomes expected. In the diagnosis stage, impact evaluation addresses the following questions: Was the problem defined? Do we know the gaps in the knowledge that we need to fill? Do we realize the needs and resources? Did we collect all support information needed? Did participants learn anything new? Do they understand the severity of the problem?

Do they know what they need to learn more? Are they ready to learn it? Are all partners disposed to collaborate?

In the prescription phase, evaluation should address whether best practices are defined and whether the processed followed during this phase led to the development of a good plan of

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work. Implementation evaluation will then be more specific, addressing the actual quantitative

outcomes: Was the monitoring strategy implemented successful in improving monitoring of

ALB? Did we reduce costs of monitoring? Is it user-friendly? Are all participants satisfied with

the outcome? If not, why? Was the data analyzed correctly? Was the data shared with all affected

groups? What was done right and what was wrong?

The evaluation should also document political, social and ethical challenges and changes

inflicted by the program. A complete evaluation of the program should then be conducted at the

end of the program timeline and all results should be reported and documented to be used as

support documents for other programs or the following season of monitoring, since the process of

ALB monitoring has to be a continuous process repeated yearly during flight season.

Data Collection

Quantitative Data collection and measures of effectiveness

Quantitative data collection in this project pertains to three main topics:

1- Actual numerical data from trap catches that reflect the success or failure of the

monitoring strategy adopted. These data will be collected during the field season (June

through September in China and through October in the U.S.), and analyzed in proportion

to actual population counts obtained by means decided upon by the steering committee.

2- Survey data: representative samples of people from the different communities and groups

involved in and affected by the project (Table 5-1) will be chosen randomly. Another

smaller group of people from the community will be trained in basic survey techniques

and will be hired to survey the first group to assess the impact of the project on those

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individuals, ranging from personal impact on their skills and knowledge, to technical

impact in preservation of trees in their property.

3- Social Capital measurement: This involves the measurement of the social improvement in

the communities affected. In the U.S., this could mean better involvement of

neighborhoods and local communities in actual monitoring of ALB. In China, it could

translate into improved decentralization and empowerment of local communities.

Qualitative data Collection

Qualitative data collection includes in-depth interviews with key informants such as

community leaders and members, government employees in USDA-APHIS, US-FS, or the

Chinese Forestry Bureau in Ningxia, extension agents, landowners, and all other stakeholders.

More qualitative data can be obtained from field notes, steering committee meeting notes, process

and impact evaluation reports, training sessions notes, etc.

Data analysis

As process evaluation is ongoing throughout the program timeline, analysis of the qualitative data generated from evaluation forms is also an ongoing process. Fast analysis is a crucial step in the program since following steps of the program should be based on the results of the evaluations of earlier stages. Descriptive statistics (frequency analysis, distribution analysis, probability tests) will be used for qualitative data analysis. Generalized linear models will be used to analyze trap catches data and compare them between the different treatments/trap designs used, to determine best monitoring strategy.

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Data sharing

Sharing information is an important challenge for all development and scientific projects and is particularly difficult in large-scale monitoring projects both in the U.S. and China.

Ensuring correct sharing of the scientific results as well as feeding back information to participants in the project is important to the success of the program. Analyzed results should be documented and sent back to all participants for re-evaluation and information. In the U.S., a shared database on a secure website on the internet could serve such a goal. In China, where farmers have no access to the internet, information should be printed and sent as hard copies to all participants or verbally explained by community members. Other media alternatives could also be explored and decided on by the steering committee according to the results of resources assessment in specific locations.

Limitations and Challenges

First of all, we realize that this proposal is general and will be adapted to the two countries and regions within each country, as needed. But since it is a participatory action research project, adaptations should be suggested and implemented by the participants themselves.

Major challenges expected in China are:

The centralization of the system and current authority of Forestry Bureaus may limit the ability of community members to take decisions as well as their willingness to do so, for fear of this authority. Although Chinese decentralization efforts in forestry were not totally successful, decentralization in other sectors was much more pronounced and set the floor for more participatory work. An example is the village-level elections in late 1980’s that gave people the

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power of making their own choices in matters that affected their livelihood (Plummer and Taylor

2004). Parallel efforts at decentralization were also implemented in other countries and was

known as “community participation” in the U.S. and Canada, “joint forest management” in India,

“community forestry” or “community-based resource management” in Bolivia, Colombia,

Mexico, Nepal, the Philippines, Tanzania and Zambia (Hyde et al. 2003). After the fall of the

New Order government, Indonesia went through a rapid process of decentralization starting in

1999, in forestry as well as in other fields. The transfer of authority from the government to the

district level in Indonesia and the implementation of PAR programs to empower local

communities led to improved communication between the authorities and the public. Indonesia,

as China, is still suffering from a rigid institutional system and bureaucracy that is keeping PAR

practices from being well employed. However, change is expected to occur gradually through time (Komarudin et al. 2006).

Possible roadblocks are: the corruption in the government system and unwillingness of government employees to collaborate with the community, in fear of loosing their authority; lack of trust and respect among academics, government employees and farmers; the common followers’ behavior of farmers who are used to answer to official orders rather than take their own decisions; the lack of resources, including internet access and access to libraries which limit largely access to information, and lack of trapping material and chemicals used for the trapping; the stringent customs regulations in China that prohibit shipping of chemicals or valuable goods

(custom charges apply for shipment of more than $50 value); the lack of interest of the new generation and field crop farmers in the region in forestry; and the lack of successful cooperatives and need to establish all collaborations anew.

In the U.S., major identified challenges include: maintaining the trust and respect between participants; enhancing sharing of information; financial resources in USDA; and willingness of community members to invest time in this project.

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Project timeline

We propose to establish stage 0 of the project (see Figure 5-1) in the Fall of

2009. Stages 1 and 2 will then take place between Fall and Spring 2010 to allow for the start of implementation in June of the same year. After the field season, in October, quantitative data analysis would take place, as well as the final evaluation of the project. Final report will be due at the end of the Fall of 2010.

Suggested basic protocol for ALB monitoring

Following is a suggested protocol for ALB monitoring based on scientific research findings by Nehme et al. (2009).

1. Lures and trap designs proposed

Option Lure treatment components Dosage (μg) Trap design

1 Male-produced pheromone 10 Screen sleeve

(-)-linalool 100

2 Male-produced pheromone 10 Intercept™ panel

(-)-linalool 100 traps

cis-3-hexen-1-ol 100

delta-carene 100

linalool oxide 100

trans-caryophyllene 100

3 Male-produced pheromone 10 Intercept™ panel

(-)-linalool 100 traps

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cis-3-hexen-1-ol 100

(-)-trans-pinocarveol 100

linalool oxide 100

trans-caryophyllene 100

2. Timeline

• Hang traps at the start of the flight season, in early June in both U.S. and China.

• Remove traps at the end of the flight season: September in China and October in the U.S.

3. How to decide where to hang a trap?

ALB is known to spread slowly from its natal host. Areas with host trees most adjacent to previously detected infestations are high risk. Also, since ALB spread in the U.S. is correlated with human activity, places such as rest areas, sides of highways leading to infested areas and camping grounds are considered high risk if they carry host trees. In China, most of Chinese poplar and willow plantations are high risk. Forested areas planted with white poplar are low risk because white poplar is resistant to the insect.

4. How far traps should be from each other?

Traps should be at least 10 -20 m away from each other. Distance depends largely on the area to be monitored, its risk level, the number of traps available and the number and the distribution of host trees. Higher density of host trees will require higher density of traps. High risk areas should preferentially be covered by more traps, obeying as much as possible the 10-20 m rule.

5. Trap placement instructions

In China, traps should be set up on Chinese poplar trees.

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• Intercept™ panel traps are hung so that the bottom is 1.5 to 2 m above ground and

attached to a branch of the tree, on the shady side.

• Screen sleeve traps are wrapped around sections of poplar tree trunks that are clean

from side branches.

• Lures are attached to a twist tie and hung in the middle open section of the

Intercept™ panel traps through the hole in the trap panel. In Screen sleeve traps, lures

are attached to twist ties and inserted between the layers of the screen.

In the U.S., traps should be set up preferentially on maple trees or other host species.

• Intercept™ panel traps are hung so that the bottom is at least at same height of the

bottom of the tree canopy and attached to a branch of the tree, on the shady side.

• Screen sleeve traps are wrapped around sections of host tree trunks right under or

preferentially within tree canopy.

6. Trap checking protocol

• Check traps every two weeks: remove insects from traps, replenish liquid in cups of

Intercept ™ panel traps and replenish lures in all.

7. What to do with insects collected from traps?

Insects found in traps, ALB or others, should be counted, recorded and preserved either in Ethylene Glycol or 70% ethanol for further identification.

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Chapter 5

Conclusions

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), commonly known as the Asian longhorned beetle, is a serious wood-boring pest both in China, where it is endemic, and in the U.S., Europe and Canada, where it was introduced through wood-packaging material. A. glabripennis is a diurnal species. Adults re-infest their natal host and rarely fly

(Haack et al. 2006). While many studies addressed the dispersal, host range, and life cycle of the species (Lingafelter and Hoebeke 2002b), little was done on the semiochemicals affecting its behavior. To date, no long-range pheromone is known for A. glabripennis. Males recognize

females by a contact pheromone found on the female elytra (Zhang et al. 2003). Zhang et al.

(2002) identified a blend of 2 dialkylethers, (1:1) 4-(n-heptyloxy)butan-1-ol : 4-(n-

heptyloxy)butanal that elicited antennal response in both males and females. The blend was then

suggested as a putative aggregation pheromone for the species. Behavioral tests were needed to

determine the effect of this putative pheromone on adult behavior and to assess its potential for

use in monitoring traps.

Our observations of A. glabripennis behavior in the field and results of our choice tests

and field trapping experiments show that A. glabripennis males are highly attracted to plant

volatiles and females are attracted to the male-produced pheromone. Adding plant volatiles to the

male-produced pheromone enhances virgin female attraction and attracts males as well. Based on

these findings and on the fact that males are smaller in size and fly more than females (Keena and

Major 2001), we suggest that the male-produced blend is a sex pheromone produced by males to

attract females. This is consistent with findings for several members of the Cerambycidae where

males produce volatile pheromones to attract females (Sakai et al.1984; Kuwahara et al. 1987;

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Lacey et al. 2004; Hall et al. 2006; Ray et al. 2006; Fettköther et al.1995; Schröder et al.

1994(Zarbin et al. 2008).

Although males were attracted to combinations of the male-produced pheromone and plant volatiles and showed moderate attraction to the pheromone blend alone, we do not believe that the male blend plays a role in aggregation. The ability of males to cue on other males’ signals is consistent with other examples from the family (Lacey et al. 2004, Hanks et al. 2007, Lacey et al. 2007) and may explain the EAD response of both genders to the pheromone found by Zhang et al. (2002). However, beetle counts in the field showed a rather homogenous distribution of insects on trees, denying the presence of aggregation behavior. Also, aggregation is usually linked with insects need to overcome plant defenses. To our knowledge, there is no evidence to date on any effective tree defense mechanism acting against this species. A. glabripennis males’ ability to respond behaviorally to other males’ pheromone might help such ‘opportunistic’ males increase their chances of finding females through competition and interception, as males are known to do in many other insect groups.

A. glabripennis is known to re-colonize the natal host or adjacent trees. Reported observations of A. glabripennis dispersal propose a higher flight propensity when the host is exhausted (Williams et al. 2004). Based on observed repellency of males to higher doses of the pheromone, we suggest that higher flight propensities could be correlated with a higher number of males on the host. This can be translated into males moving to new hosts to avoid competition and to draw females to new healthy trees where resources are abundant. Males would thus be the founder or pioneer gender, flying to new healthy hosts to which they are chemically attracted.

Once on that host, the mixture of the pheromone they produce and volatiles produced by the tree attract virgin females to the vicinity of the tree and to close proximity of the males. Mate location is thus mediated by host location, following the model proposed by Saint-Germain (2007). Within a tree, the male-produced pheromone by itself acts as a short range signal. In fact, most of the

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pheromones reported within the Cerambycidae are short-range male-produced volatiles

pheromones or contact female-produced pheromone. Only two species in the primitive subfamily

Prioninae, Prionus californicus Motschulsky (Cervantes et al. 2006) and Mallodon dasystomus

(Say) (Spikes et al. 2008b), are known to have female-produced long-range pheromones.

Our field trapping results also indicate that the male-produced pheromone of A. glabripennis, especially when combined with specific plant volatiles, shows promise as a lure for monitoring of this exotic species. Based on these findings, we recommend the use of

Intercept TM panel traps baited with a combination of the male-produced pheromone blend, (-)- linalool, cis-3-hexen-1-ol, linalool oxide, trans-caryophyllene and trans-pinocarveol to catch virgin females. Another effective alternative is the use of screen sleeve traps baited with the male-produced pheromone and (-)-linalool which was found to attract mostly males. In the

U.S., trapping both sexes is very important at ports of entry where there is equal chance of introducing males and females. In such cases, both trap designs could be used in the same location.

Although we believe the combination of plant volatiles used was successful in trapping

A. glabripennis in the field when combined with the male-produced pheromone, additional field studies using variations of the combination of the plant volatiles used in 2008 and other plant volatiles shown to be attractive in Y-olfactometer bioassays, are needed. For example, 3- carene was found to be highly attractive to males (Nehme et al. 2009) and may be useful when combined with the pheromone blend. Developing a user-friendly version of the screen sleeve trap is also needed for large scale applications. Trap designs, trap placement, lure combinations, and the type of emitters used in China should be tested and adapted as needed to field conditions in the US to optimize their efficacy.

In Chapter 4 of this thesis, we went one step further into designing a

Participatory Action Research Program that allows the involvement of local communities,

119 governmental agencies, universities and research institutes, and the private sector (wood manufacturing industry), in full participatory programs that integrated popular education and participatory action research principles and aim at improving monitoring techniques for the Asian longhorned beetle (ALB). The PAR program designed would be adapted to the different conditions in China and the U.S.. Adaptation includes involving different stakeholders and taking in consideration the difference in the needs and assets between countries and even between communities within countries. In China, we suggest to start the first year of the program in

Ningxia province, the expand it to other provinces where ALB infestations are also found, due to the large variability in environmental, social, political and economic conditions between different provinces. Centralization of forestry authority in China might be the main challenge to the application of our program, since the major requirements for the success of the proposed program is equal participation of all parties involved and sharing of decision-making and authority. In the

U.S., limited budgets of government agencies dealing with invasive species and the occurrence of other invasive species that are equally or even more serious than ALB might pose a serious challenge.

In conclusion, our research provided needed evidence for the role of A. glabripennis male-produced volatile pheromone in mate-finding. We determined the most attractive trap designs and lures for monitoring A. glabripennis populations both in the U.S. and

China and provided a program design to guide in the first steps of the implementation of the suggested monitoring tools. Field testing of the suggested lures and trap designs in the U.S. is needed to ensure monitoring efficiency in U.S. field conditions. Further research studies are also needed to clarify the timing and production source of the male-produced pheromone used in this study.

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139 VITA

Maya E. Nehme

EDUCATION:

Dual-Title Ph.D. in Entomology and Comparative & International Education Jan 06 – Aug 09 The Pennsylvania State University University Park, PA

MS Plant Protection Feb 03 – Jan 05 The American University of Beirut Beirut, Lebanon

Diploma of Agricultural Engineering Oct 97- Dec 02 Lebanese University- Faculty of Agriculture Sin-El-Fil, Lebanon

WORK EXPERIENCE:

Graduate Research and Teaching Assistant Jan 06- Aug 09 The Pennsylvania State University University Park, PA

Research Assistant Feb 05- Feb 06 The American University of Beirut, Biochemistry Dept Beirut, Lebanon

Graduate Teaching Assistant Feb 03- Jan 05 The American University of Beirut- FAFS Beirut, Lebanon

Head of Household Pest Control Department Oct 01- Jan 03 FREELANCER Services & Insurance s.a.l. Hadath, Lebanon

PUBLICATIONS

Nehme, M., M. Keena, A. J. Zhang, T. C. Baker, and K. Hoover. In review. Attractiveness of Anoplophora glabripennis to the male-produce pheromone and plant volatiles, Environmental Entomology.

Nehme, M., M. Keena, A. J. Zhang, T. C. Baker, Z. Xu and K. Hoover. In review. Field testing of Anoplophora glabripennis male-produce pheromone and plant volatiles, Environmental Entomology.

OECD (Organization for Economic Co-operation and Development) 2009. Green at Fifteen? How 15-year-olds perform in environmental sciences and geosciences in PISA. Program for International Student Assessment. © OECD 2009. 116 pp. (Drafted in collaboration with David Baker and Juan Leon from Penn State University and OECD, funded by NSF).