Preventing the Arrival of New Agrilus Jewel Beetles Into Canada–The

Preventing the Arrival of New Agrilus Jewel Beetles into Canada – The Potential Invasive Species Agrilus dureli Jendek

Meichen Wu

Submitted in conformity with the requirements for the degree of Master of Forest Conservation

University of Toronto Supervisor: Professor Sandy Smith

Preventing the Arrival of New Agrilus Jewel Beetles into Canada – The Potential Invasive Species Agrilus dureli Jendek

Meichen Wu Master of Forest Conservation Faculty of Architecture, University of Toronto

Abstract

Agrilus dureli Jendek is a jewel beetle recently discovered in Beijing, China. Willow trees are severely infected with this wood borer in this area, and thus the purpose of this study was to gather information to determine whether Agrilus dureli would be harmful to Canada and warrants a risk assessment by CFIA (Canadian Food Inspection Agency). Here, research is presented on the spread of Agrilus dureli in the Beijing area, its effects on trees, and description of its main biological characters. The extent of spread by Agrilus dureli was relatively local with all infected sites located near the river. Based on the current results, it appears that Agrilus dureli tends to infect short, thin willow trees with the larval survival higher on the southern side of the tree than other directions. The density of larval galleries showed a relationship to tree crown health. The biological life history of Agrilus dureli was similar to that of other Agrilus species, in particular the emerald ash borer (EAB). Although limited by sample size, this study is the first to describe the biology and impact of Agrilus dureli, and suggest more research is needed to assess its overall trend and potential for environmental conditions to affect its growth in the Beijing area.

Acknowledgments

I would like to thank Dr. Sandy Smith at the University of Toronto for her supervision and guidance throughout my capstone project and the MFC program. Without her knowledgeable guidance, I would not be able to write this paper. I would also like to thank Troy Kimoto from the Canadian Food Inspection Agency as my external supervisor for providing this opportunity and who did not hesitate to answer my questions and to guide me every step of the way during the work. Thanks to Dr. Jacob Wickham from the Chinese Academy of Sciences who helped with my work in Beijing and to Dr. Jay Malcolm (University of Toronto) for assisting in data organization. Additional thanks to Jozef Ric, Josh McMeekin, and Christine Orchard from the City of Toronto for graciously training me during May 2019 before my field work in China.

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Table of Contents

Introduction ...... 1

Objectives ...... 4 Methodology ...... 5

Surveys in the Beijing...... 5 a) Study sites ...... 5 b) Surveying for infested trees ...... 7

Factors of infested trees and biology of Agrilus dureli ...... 9 a) Describing Agrilus dureli life signs ...... 9 b) Biological measurements ...... 10 Results ...... 11

Survey in Beijing area ...... 11 a) Study sites ...... 11 b) External sign of infested trees ...... 13

Factors of infested trees and biology of Agrilus dureli ...... 14 a) Larvae and adult stages ...... 14 b) Internal signs of infected trees ...... 17

Assessing the site-level impact of Agrilus dureli ...... 18 Discussion ...... 22

Recommendations ...... 25

References ...... 27

Appendices ...... 30

List of Tables

Table 1. The 19 study sites were arranged into five different forest types...... 12

Table 2. P-values for infested tree's external factors on different environmental conditions...... 14

List of Figures

Figure 1. Four main work procedures in all 19 study sites...... 5

Figure 2. Three main work procedures for surveying external factors of infested trees ...... 7

Figure 3. Ash canopy health condition rating system ...... 7

Figure 4. Three main work procedures for examining internal signs of infested trees ...... 9

Figure 5. Willow trees gallery density rating scale ...... 9

Figure 6. D-shape exit hole, Agrilus dureli larvae and adult...... 10

Figure 7. Measurements used for distinguishing between larval instars of Agrilis species...... 10

Figure 8. Map of Agrilus dureli infested area in Mentougou, Beijing, China...... 11

Figure 9. Agrilus dureli adult and larval specimen number and date of collection...... 15

Figure 10. Distribution of measurements of sclerotized parts of larvae of Agrilus dureli...... 16

Figure 11. Frequency distribution for observed Agrilus feeding galleries...... 17

Figure 12. Number of infested trees found damaged by A. drueili in either Category ...... 18

Figure 13. The location of all 19 study sites in Beijing, China...... 19

Figure 14. Top view of drones on site 0,1 and 2 ...... 20

Figure 15. Infested trees comparing with uninfested tress on height and DBH ...... 21

Figure 16. The mean height of infested trees and infested rate in forest types ...... 21

List of Appendices

Appendix 1．The image of crown dieback of site 0...... 30

Appendix 2．The location of study sites and the distance to breakout site 0...... 31

Appendix 3． Atanycolus species in willow tree, site 9, Yanchizhen, Mentougou, Beijing...... 32

Appendix 4． Linear relationship between gallery density and crown dieback for all willow trees...... 33

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Introduction

With increasing transportation, there is more trade of products and communication for tourism between countries. However, with this comes the potential threat of invasive species that can cause great concern to the environment. In order to reduce the biological problems of transactions between countries, there are corresponding management organizations. The World Trade Organization (WTO) is a global organization that establishes international standards and tackles safety rules between countries under which the North American Plant Protection Organization (NAPPO) in North America operates to prevent the introduction and spread of regulated pests. NAPPO has established an international black list of unwanted pests in Canada, that is regulated by the Canadian Food Inspection Agency (CFIA) (CFIA, 2019). If invasive organisms are allowed to spread, then they can result in severe environmental and economic losses. Therefore, it is important to know which invasive has the potential for detrimental impact. To assess this risk, evidence-based blacklist must be developed to enhance customs, national vigilance, and avoid environmental disasters.

Agrilus dureli Jendek (Coleoptera: Buprestidae), is a new native wood-boring beetle recently been discovered attacking willow near Beijing, China (Appendix 1). It was first described in 2011 based on a few museum specimens, but nothing was known about its biology until 2016 when a CFIA project inadvertently discovered an infestation in willows (Salix spp.) just outside Beijing. A key question that arose was whether this insect could become invasive in Canada given the absence of any biological data. Only one paper was published on this species before its discovery although it was known to be closely related the emerald ash borer (EAB, Agrilus planipennis Fairmaire), an invasive species also from eastern Asia (Haack et al., 2002). Agrilus dureli and EAB both are buprestid beetles from the same genus Agrilus and their larvae and adults are similar, although they attack different host plants (Jendek & Grebennikov, 2011). In 2002, EAB was first discovered in North America where it caused extensive ash mortality (Haack et al., 2002). EAB infestations have been transmitted through natural, short-range propagation to two provinces in Canada although human transport has led to the more common, long-distance propagation (Aubin et al., 2015). EAB has now been added to the NAPPO

quarantine pest alert list (Orlova-Bienkowskaja & Bieńkowski, 2016) and there is similar concern about the potential impact of Agrilus dureli if it was to arrive in NA.

Canada’s government concerned about invasive species, as several invasive species previously had a severe impact on Canada's environment (Humble & Allen, 2016). The main cause of the environmental impacts of invasive species relies on Canada’s forest structure (Humble & Allen, 2016) and human-caused (Aubin et al., 2015). Forest structures in China are different from Canada. In Canada, there are > 4000 native plant species (Flora of Canada, 2019) compared to China where there are 30,000 native plant species. Besides the less number of plant species, the number of insects’ species in Canada less than China either (Biological Survey of Canada, 2014). In Beijing, willows and poplars (Populus spp.) are widely distributed and abundant, like parks, street trees and riverbanks (Su, Zhang & Qiu, 2010). In order to understand the spread of Agrilus dureli and the spread of infested areas, it is required to extensive biological knowledge, including their life history. Basic information about Agrilus dureli activity, generation time and behaviour is needed in this survey. The biology of Agrilus dureli has not been studied, but its life history is likely similar to EAB (Jendek & Grebennikov, 2011). Agrilus species’ adults feed on leaves until they approach sexual maturity when females and males mate in the canopy. Females usually produce eggs on the surface of the bark and larvae enter the interface between the outer bark and sapwood. They then develop and feed on sugar and overwinter for one or two years (Orlova- Bienkowskaja & Bieńkowski, 2016). Adults appear at the end of May and reach peak flight in early June (Mozolevskaya & Izhevskiy, 2007). The speed of adult feeding is likely due to their size and/or gender. Besides understanding its life cycle, basic information on the interaction of natural enemies with Agrilus dureli would also be helpful to predict its potential impact.

Agrilus species can cause tree decline and death (Aubin et al., 2015); they are able to attack healthy trees and kill them at all ages, from saplings to mature trees (Haack et al. 2002). With larger trees, gallery infestation usually begins in the canopy or on the trunk after several years (Ryall et al. 2011). Observing the host tree is critical in order to understand the beetle's behaviour, and determining the host tree characteristics of beetles survival based on beetle's signs of life is essential also. Since willows and poplars are closely related trees, wood-boring insects that breed in them may be propagated in either (Jendek & Grebennikov, 2011). It is vital to

identify the biology of insect species and to study the spatial scales about the relationship between trees and insects (Sperry et al., 2001). In particular, their mechanism for locating hosts might be important for the shape and orientation of this relationship (Knight, Brown & Long, 2012).

Investigating the relationship between cities and biodiversity is important to understand the potentially harmful effects on local wildlife. Besides study field sites, other parts of Beijing also need to know whether they have Agrilus dureli and we need to increase our understanding of the distribution of Agrilus dureli in this area. Willows and poplars are commonly planted in Beijing (Su, Zhang & Qiu, 2010). Canada has many willows as well (Tree Canada, 2019). The trunk represents a unique biotic environment, and it is necessary to know what kind of environment and conditions Agrilus dureli lives in. In order to suppose the impact of Agrilus dureli to Canada, it is essential to determine the spatial distribution pattern of Agrilus dureli on the urbanization gradient in Beijing. As China has millions of hectares of hybrid poplar and willow plantations (Su, Zhang & Qiu, 2010), there are potential which Agrilus dureli could be exported to Canada via solid wood packaging material, but whether this insect could become invasive is uncertain due to the absence of biological data. In Canada, willows and poplar are common in biodiversity. If Agrilus dureli becomes an invasive species in Canada someday, they may bring significant threat and consequences of effect on ecological and economic. Hence, my study aims to analyze the data collected in Beijing to determine whether Agrilus dureli should be included on the CFIA blacklist or not.

The only known Agrilus dureli infestation is just east of Yanchizen, which is northwest of Beijing. Poplars were found there in conjunction with willows; most willows are found along the riverbank of the Yongding River in Yanchizhen. In the summer of 2019, the Guanting Reservoir was discharged into the Yongding River with the approval of the Beijing government, and it was the first time that the water supply has been achieved in the past 40 years. The Yongding River, which was > 100 km in Mentougou area, now ran through the water (People.cn, 2019). Because the amount of water released was heavy, the water level was much higher than the original watercourse. Unfortunately, the infestation site that was confirmed in 2016 which also flooded, and the majority of the infected trees were in water making it difficult to complete this survey.

Objectives

The overall goal of the study was to survey for Agrilus dureli in Beijing in order to better understand its biology, attack pattern on host trees, and to make recommendations for CFIA and the Beijing Municipal Forestry Bureau. Specifically, the aim of this paper was to:

1) Survey for Agrilus dureli in the Beijing area; 2) Describe factors of infested trees and the biology of Agrilus dureli; 3) Assess the impact of the beetle on infested stands; and 4) Provide recommendations for CFIA and the Beijing Municipal Forestry Bureau.

Methodology

Surveys in the Beijing area

a) Study sites

The first fieldwork started in the Yanchizhen area (40°02’32.5’’N; 115°83’76.2’’W), the first confirmed site of Agrilus dureli in 2016. Infected willows were first checked carefully here, and marked this place as a study site. Because Agrilus dureli can fly, willows near Yanchizhen were more susceptible to this insects. To determine the area of the infestation, I drove from Yanchizhen to find willows and poplars that might be infested along the road. Talking to locals also helped me with its search. Wherever I visited, looking for signs of Agrilus dureli, which would attack in declining willows, is my aim, such as the crown dieback, branch dieback, epicormic shoots and woodpecker damage. After finding the declining willows, poplars, approaching, and examining were the next procedure. If there are D-shaped exit holes, woodpecker feeding, removing some bark to check whether S-shaped galleries shown on the sapwood. Once it is determined that the tree is infected, marked it with spray paint, and recorded the location with GPS. Established a new study site, started testing this site until the examining completed and then left to find the next infected site. In this way, driving around and observing, the site number was compiled gradually by the of detection. The infected trees found in each site were painted and numbered. These marks helped to find the trees when they need to be accessed again.

After selecting a site, I conducted a series of surveys on the site, included determining site type, recorded site information, and counted tree number by crown rating and measurement (Fig. 1). First of all, I needed to determine the type of site and record the site information like tree regeneration (nature or man-made) and disturbance history (flooding and soil compaction). In order to assess the structure of the forest, live and dead trees of all willows and poplars need to be identified. And then, count the number of uninfested and infested trees by crown rating. Next, count the amount of the infested trees. If the amount is greater than twenty, only examine the

first 20 infested trees in detail. Otherwise, examine all infested trees. Finally, randomly choose twenty trees including health and infested trees, and measure their height and DBH. All

Figure 1. Four main work procedures in all 19 study sites, including determining site type, recording site information, counting tree number by crown rating and measuring trees. All study sites were found in the Beijing area during the summer of 2019. information record in site forms. The infestation rate was calculated by the amount of infested willows divide by uninfected willows. All research sites and infestation levels displayed in maps using ArcGIS online. Addressing objective 3, the height and DBH of the infested tree and uninfected tree used One-way ANOVA (SAS Institute 2018) to analyze the external difference between the infected tree and the uninfected tree. The subsequently collected data also combined the information of the site to comparison and analysis within sites and sites. b) Surveying for infested trees

After finish surveying the study site, each infested willow trees that chose into the 20 trees need to be examined, and work procedures include identifying, observation, assessment and measurement (Fig. 2). First of all, identify the host tree species. Walk around and observe the tree from crown to ground. Classify the canopy rating scale by 5 categories (Fig. 3), record the position of present epicormic shoots (present = 1, absent = 0)in the tree in the lower, middle, upper trunk and crown total of 6 positions in tree height. Similar to epicormic shoots, the sign of

woodpecker feeding needs to be assessed by three different levels (light = 1, moderate = 2 and heavy = 3) in 6 different tree height positions. The position and approximate height of exposed

Figure 2. Three main work procedures for surveying external factors of infested trees, including identify tree species, observation and assessment, and tree measurement. All infested trees were discovered in the Beijing area during the summer of 2019.

1 2 3 4 5

Figure 3. Ash canopy health condition rating system is defined as follows: 1. Full canopy; 2. <30% decline; 3. 30-70% decline; 4. >70% canopy thinning; 5. Dead canopy (Smith, 2006; photos by Daniel A. Herms; Smith et al., 2015). This rating system normally use for measuring ash trees canopy decline by EAB infestation. Since Agrilus dureli is a newly studied species, this study used the criteria of the Ash tree's canopy health condition rating system to detect willow trees canopy health.

galleries were recorded. After observation, the height and DBH (diameter at breast height) of the infested tree were mearsued using the clinometer and diameter type. All data were tested for normality using residual plots. I used analysis of variance (One-way ANOVA) to analyze tree height, DBH, and crown dieback in infested trees as dependent variables in different environment condition categories. Tukey’s honestly significant difference (HSD) multiple comparison procedure was applied for the height of infested trees in five different forest type. Because the data source of woodpecker feeding and epicormic shoots disperse in six positions on the tree, transforming these data from grading to sum percentage was necessary. For woodpecker feeding, the highest damage recorded is 3 in each position. Thus, full damage 100% of woodpecker feeding counted as 18 (3 x 6 positions = 18). The sum of all height record numbers divided by 18 and then times 100 were the final woodpecker feeding damage percentage for each tree. Epicormic shoots data recorded by present or absent (1 or 0) in six different height positions. The percentage of epicormic shoots was calculated by the sum of all positions and divide by six. Because of many zeros shown into woodpecker and epicormic data, non- parametric One-Way ANOVA median test was used.

Factors of infested trees and biology of Agrilus dureli a) Describing Agrilus dureli life signs

Trees were not only visually examined for typical signs, but also need to be worked with symptoms associated with Agrilus species (Fig. 4). Typically D-shaped exit holes and exposure meandering larval galleries on the trunk are internal signs. Measure the height (mm) and width (mm) of D-shape exit holes by a caliper (Fig. 6). Using axe and chisel remove of bark with D- shaped exit holes, woodpeckers foraging damage or epicormic shoots. After this, a 20 x 20 cm standard bark window needs to be cut between 1 and 1.5 m height on the trunk. Classify the gallery density scale by five categories within that sampling window area (Fig. 5). If there were any adults and larvae apparent during examining (Fig. 6), collect them into vials with ethanol and record the host tree information. D-shape exit hole size (mm2) equal to pi times width and times height (S=π × width × height). Gallery density and D-shape exit hole size data tested by One-Way ANOVA in different environment condition categories. Simple linear regression (PROC REG, SAS Institute 2018) was used to evaluate the relationship between

Figure 4. Three main work procedures for examining internal signs of infested trees. Finding living sign of Agrilus dureli, measuring exit holes, cutting bark and specimens collection. All infested trees was discovered in the Beijing area during the summer of 2019.

1 2 3 4 5

Figure 5. Willow trees gallery density rating scale, 1. <20% gallery area; 2.20-40% gallery area; 3. 40-60% gallery area; 4. 60-80% gallery area; 5. 80-100% gallery area. All gallery windows were cut in Beijing area during summer in 2019.

crown dieback and the density of larvae gallery. All analyses were conducted at P < 0.05 level of significance using SAS statistical software (SAS Institute 2018).

A B

C D

Figure 6. A) The D-shape exit hole and the measurement method; B) Agrilus dureli larvae in inner bark; C) Agrilus dureli adult in sapwood; D) Alive Agrilus dureli adult on trunk. All photos were taken in Beijing, China (2019).

b) Biological measurements After finishing collection of larvae and adults, measure the width of epistome (Fig. 7, A, 1), the length of urogomphi (Fig. 7, B, 4) and the length of Agrilus dureli adult by microscope. All adult specimens will send to Eduard Jendek who named Agrilus dureli to identify.

Figure 7. Measurements used for distinguishing between larval instars of Agrilis species. A) Anterior part of the body. B) Posterior part of the body. 1, width of epistome; 2, width of head; 3, width of last segment; 4, length of urogomphi (Orlova-Bienkowskaja & Bieńkowski, 2016). Since Agrilus dureli is a newly studied species, this study used the measurement method of EAB to measure the larvae of Agrilus dureli.

Results

Survey in Beijing area a) Study sites

After two and a half months of searching, I found that willows are everywhere in Beijing, like parks with small lakes, wild streets in urban areas, and even willow trees that can be seen in residential areas. The life sign of Agrilus dureli only found in Mentougou, not shown in other areas in Beijing (Fig. 8). However, the willows in other areas are not very healthy either,

Figure 8. Map of Agrilus dureli infested area in Mentougou, Beijing, China (2019). All sites category by three different infested levels, 0-30%, 30-60% and 60-100% in yellow, orange and red points.

and most of the willow was attacked by wood boring moths (Order: Lepidoptera ) and Apriona species (Coleoptera: Cerambycidae). The infestation described in this article only targets Agrilus dureli. A total of 19 sites were found in Mentougou area (Appendix 2), where the willows were currently or previously infected by Agrilus dureli. Although each affected area is small in size, there is enough willow and poplar density (living or dead). All selected sites were discovered along the Yongding River or nearby (Fig. 8). These sites distribute between the country and the suburb, and signs of life history about Agrilus dureli were found in each site. Study sites can be classified into parks (8 sites), riparian forests (5 sites), street trees (4 sites), residential (1 site) and windrow (1 site) in Mentougou District (Table 1). The number of infested willows in site 0 is uncertain because it is the original infestation location and suffering flooding lately. I was not able to approach majority willows in there but I could visually tell the number of actually infested trees way more than 7 infested trees (Appendix 1). Site 0 is the most serious infestation site. Besides site 0, the greatest amount of infested trees found is in site 2, where is 100 meters far away site 0. The infested rate is 87% in site 2, the death rate in infested willows is 31%. Following site 2, the site of 3,4,7,9,11 and 15 have death rates. Especially, the infested rate in site 11 is a hundred percentage, but the number of infested willow is only one. For site 3, only two infested trees in the forest with a 5% infested rate, one of infested willow dead. The same number of infested willow trees with site 3, the infested rate of site 7 is 40%. Site 4 and 9 shown similar infested and death rates in willows. All infested trees were identified as willows, include Salix matsudana (N=61) and Salix babylonica (N=7).

b) External sign of infested trees

External factors of infected trees under different environmental conditions. The height of the infected tree showed significant differences among the 5 different site types (P < 0.001); the predation and epidermal bud percentage of the woodpecker of the infected tree also showed a difference (P < 0.001; P = 0.003). The DBH and crown dieback of the infected tree did not show differences in different forest type environments. Agrilus dureli adult and non-adult tree data classification, the DBH and height of infested trees, crown dieback, woodpecker predation

Table 1. The 19 study sites were arranged into five different forest types. Each study site is numbered, and the infection rate of each site is calculated from the total number of willows and the number of infected willows. Mortality rate of infested willows is calculated from the number of infected dead willows and total infected willows.

No. No. infested No. Infested Death rate in Forest Type site willow willow rate infested willows 7 2 5 40% 50% 10 3 17 18% 0 11 1 10 10% 100% 12 1 14 7% 0 Park 14 1 2 50% 0 15 6 9 67% 67% 17 3 7 43% 0 18 2 13 15% 0 0 7+ 40+ Unknown Unknown 3 2 43 5% 50% Riparian Forest 4 9 19 47% 22% 5 1 4 25% 0 9 4 10 40% 25% 1 3 6 50% 0 2 13 15 87% 31% Street Tree 13 1 1 100% 0 8 2 18 11% 0 Residential 16 2 4 50% 0 Windrow 6 5 8 63% 0

and epicormic shoots did not show any difference (Table 2). The DBH of infested trees were different between naturally grown and human-planted trees (P = 0.020), and height of infested trees is significantly different (P <0.001). The percentage of woodpecker feeding on infected trees was also significantly different between natural growth and man-made planting (P <0.001). The data analysis results of the trees affected by the flood and those not affected by the flood are similar to those of the classification of the infestation tree generation data. The DBH factor showed a difference between the flooded and uninfected trees (P = 0.035); both the tree height and the woodpecker showed significant differences under this classification (P <0.001). What's more,

epicormic shoots also showeD differences between infected trees under flooding and not undergoing classification (P = 0.042). Among the last group of infected trees that were soil compacted and not soil compacted, only data on crown dieback rate and percentage of woodpecker damage showed differences (P = 0.003; P = 0.028).

Table 2. P-values for infested tree's DBH, height, crown dieback, the percentage of woodpecker predation and epicormic shoots present on trees, all five different factors have an effect on the tree under different environmental conditions. The category of the different growth environments of the trees are: under 5 forest types (park, street tree, residential, windrow, and riparian forest); trees with Agrilus dureli adults and the ones without adults; natural growth and artificial planting; the trees affected by floods and none flooding trees; trees affected by soil compaction and trees without soil compaction. All bold P values in this table are significant (P <0.05).

Condition categories Infested tree Infested tree Crown Woodpecker Epicormic DBH Height dieback Predation Shoots (cm) Height(m) (rating) (%) (%) 5 Different Site Type 0.474 <0.001 0.691 <0.001 0.003 Adult/ Non-adult 0.216 0.203 0.431 0.339 0.792 Natural/ Man-made 0.020 <0.001 0.063 <0.001 0.163 Flooding/ None 0.035 <0.001 0.081 <0.001 0.042 Soil compaction/None 0.274 0.752 0.003 0.0281 1.000

Factors of infested trees and biology of Agrilus dureli

a) Larvae and adult stages

A total of 47 larvae and 62 adult specimens were collected in Beijing. The appearance of Agrilus dureli is dark emerald green, with 4 white dots on the wings and 2 white stripes on the sides of the body (Fig. 6-D). The average length of Agrilus dureli is 12.65 mm in total 62 specimens. The majority of adults were collected alive and a few of them be found under the bark. The adult of Agrilus dureli has been discovered since May 29, and the peak period of the adult was in mid- June (Fig. 9). A maximum of 15 adult specimens can be found a day. From late June to mid-July, the number of Agrilus dureli adults found gradually decreased. No Agrilus dureli adults found

after July 20. Unlike the appearing frequency of Agrilus dureli adults, larvae were found after July 9th, and 39 samples were collected on that day (Fig. 9). Larvae are found in small numbers after mid-July, and they are very short in length. It was difficult to find during debarking the tree.

30 collected 25

specimens 15 of

Number 5

2-Jul 4-Jul 6-Jul 8-Jul

2-Jun 4-Jun 6-Jun 8-Jun

12-Jul 10-Jul 14-Jul 16-Jul 18-Jul 20-Jul 22-Jul 24-Jul 26-Jul

14-Jun 28-Jun 10-Jun 12-Jun 16-Jun 18-Jun 20-Jun 22-Jun 24-Jun 26-Jun 30-Jun

31-May 29-May Adults Larvae

Figure 9. Agrilus dureli adult and larval specimen number and date of collection. All specimens collected in Mentougou, Beijing, China (2019).

The distribution of measurements of width of the epistome and length of the urogomphi (Fig. 7) indicated that there are four different size categories in a total of 47 specimens.(Fig. 10). All four instars are different and it did not indicate clear curves between these instars (Fig. 10). In particular, there is only one specimen of the second instar in the width of the epistome graph, less than the number of the second instar larvae in length of urogomohi (6 specimens). The number of specimens in the first instar is similar in two measurements (4 specimens in Fig. 10-A; 5 specimens in Fig. 10-B). However, the number of specimens in the third and fourth instar distribution is different in two measurements. The number of larvae in third and fourth instar is quite even (20 specimens; 22 specimens) in epistome measurement, but the quantity of fourth instar larvae in measurements of urogomphi is only 3 specimens comparing with 33 specimens of third instars (Fig. 10-B).

7 1st instar 2nd instar 3rd instar (A) 6 4th instar

4 specimens

of 3

2 Number 1

Width of epistome(mm)

(B) 7 1st instar 2nd instar 6 3rd instar 4th instar 5

specimens 4 of 3

2 Number 1 0

Length of urogomphi(mm)

Figure 10. (A–B) Distribution of measurements of epistome and urogomphi parts of larvae of Agrilus dureli. A) The number of larvae in the standard width of epistome within four instars. B) The number of larvae in the standard length of urogomphi within four instars (Orlova- Bienkowskaja, 2016). Since Agrilus dureli is a newly studied species, this study used the EAB standard length range of epistome and urogomphi to distinguish the larvae instars of Agrilus dureli. All larvae were collected in the Beijing area during the summer of 2019.

b) Internal signs of infected trees

Before cutting the windows, the naturally exposed galleries need to be recorded, viewing and estimating height and orientation from the ground to the canopy. Exposed galleries are unevenly distributed in height (Fig. 11). Within the height range of lower, middle, and higher trunk, exposed galleries were found in all four directions. Lower trunk showed gallery frequency is average in all directions (3 or 4 in 1-meter height). In the middle trunk, most galleries were found on the south and east sides with an average height of 1.7 m and 1.8 m, their gallery frequency was the highest (Fig. 11). North was outstanding than other directions in the upper trunk, gallery frequency was 6. Within the height range of lower, middle, and higher crown, the number of exposed galleries that can be observed was much less than the trunk. In the lower crown, the east side showed a more exposed gallery than the other direction. The middle crown and upper crown did not show any interesting data. In research, it was found that some dead willows can have galleries in all directions.

Figure 11. Frequency distribution by compass direction for observed Agrilus feeding galleries on 68 willow trees (Salix matsudana and Salix babylonica) in the Beijing area during May to August 2019. The six compass circles indicate the frequency of exposed gallery showing in six different positions (lower trunk, middle trunk, upper trunk, lower crown, middle crown and upper crown) and four aspect directions in infested trees. Samples were taken in the Beijing area during the summer of 2019 by observation of exposed galleries.

A total of 88 windows cut on 68 examined infested willows in this survey. Gallery density categories 3 and 4 demonstrate the first and second frequency, separately 25 and 24 (Fig. 12). Category 5 is the third high appearance with 16 in the total count. Categories 1 and 2 in the gallery density do not show the high frequency (5 in category 1; 10 in category). It also happened 8 times that after cutting windows in infested trees, there is no gallery shown on windows.

5 10

4 16

3 21 category 2 7

1 4 Damage 0 7

Unknown 3

0 5 10 15 20 25

Number of infested willows

Figure 12. Number of infested trees found damaged by Agrilus dureli in either Category 0 = no galleries; Category 1 = 20%; Category 2 = 40%; Category 3 = 60%; Category 4 = 80%; or Category 5 =100% when tree trunks were observed using a fixed window sample. Samples were taken in the Beijing area during the summer of 2019 by cutting one observation window into each tree at 1.5 m height above ground.

Assessing the site-level impact of Agrilus dureli

Most of the infected sites near site 0, area A and B were both moderate and highly infested rate sites (Fig. 13). Other discovered sites spread along the east and west directions of the Yongding River, and the infection rate also followed the distance decrease. The driving distance from site 0 to the most eastern site 16 is 36 km, and the driving distance from site 0 to the most western site 11 is 6 km. Site 16 is further east to the urban area, and no signs of Agrilus dureli growth have been found. But site 11 is along the Yongding River and northwest is Pearl Lake. Because of the renovation of Pearl Lake Park this summer, the car could not allow drive in this park and I did

not search this area. Since the source of the Yongding River is the Guanting Reservoir, I have also reached the easternmost part of the Guanting Reservoir. There were no signs of the growth of Agrilus dureli.

Figure 13. The location of all 19 study sites in Beijing, China. All sites category by three different infested levels, 0-30%, 30-60% and 60-100%. These three different infested rates present yellow, orange and red points in this map. The overlapping points in the figure are enlarged in A, B and C. Blueline means the Yongding river.

The majority of alive Agrilus dureli adult found in site 1 and 2 (Table 1), these two field sites have the same features that are away from the exploding field site 0 around 100 meters inland and all the trees have cut crown before (Fig. 14). Locals told me site 1 and 2 was tourism place before. The previous landowner wants to make the willows look short and pleasing to the eye. The willow in sites 1 and 2 are scattered because they are human-planting trees. The red dots represent trees that were initially identified as infested because of D-shaped exits on the bark. After 20 days, came to sites 1 and 2 again, and new D-shaped exit holes appeared in the blue dot trees. The uninfected tree in the detection area is close to the infected tree, shown yellow dots in Figure 14.

Figure 14. Top view of drones on sites 0,1 and 2. The red frame is the detected area. Blue and red dots indicate infested willows, and yellow dots indicate uninfested willows in the detection area. Photo was taken in Beijing, China (2019).

The height and DBH of infected and uninfected trees are significantly different (Fig. 15). The average height of uninfected trees is 15 meters, which is much higher than the average height of infected trees (9 meters). The average DBH of uninfected trees is 30 cm, which is also higher than the average DBH (25 cm) of infected trees. Among the infected trees in different forest types, the average height of the street tree was the lowest (5 meters) with the highest infested rate, 62% (Fig. 16). The infested trees of windrow and park were successively tall, 8 meters and 10 meters respectively, with 62% and 31% infested rate; the infected trees of the riparian forest were the highest around 11 meters but with lowest infested rate, 29% (Fig. 16).

16 35 (A) P < 0.001 (B) P = 0.004

14 30

(m) 12

(cm) 25 10

tree 20 of

ree height ree 8 T

DBH 15

6 The 10 4

2 5

0 0 Infested trees Uninfested trees Infested trees Uninfested trees

Figure 15. (A-B) Infested trees comparing with uninfested tress on height and DBH. A)The average height of infested trees and uninfested trees. B)The average DBH of infested trees and uninfested trees. All examined tree measured during 2019 summer in Beijing, China.

Mean of height Mean of infested rate 18 b 70% b 16 60% 14

50% (%) (M) 12 a a, b a, b 40%

10 RATE

HEIGHT 8 30% 6

20% TREE 4 INFESTED 2 10% 0 0% Street tree Residential Windrow Park Riparian Forest

Figure 16. The mean height of infested trees and mean of infested rate in five different forest types includes park, residential, riparian forest, street tree and windrow. Different letters on each bar represent significant difference among forest types at the o.o5 level. All examined infested trees measured during 2019 summer in Beijing, China.

Discussion

The life history of Agrilus dureli is similar to that of EAB because four larvale instars are clearly shown in Agrilus dureli based on the similar range of EAB larvale measurements. Chamorro et al. (2012) noted that four larval instars were showing four different stages of the size of larvae. Measuring the width of the head capsule and the terminal segments is generally considered to be the way to distinguish larvae instar (Wang et al., 2005). However, Orlova-Bienkowskaja and Bieńkowski (2016) mentioned that the instar distinction of the larvae is not obvious by measuring the head capsule and the terminal segments because of the soft parts of larvae gradually increasing over time. Measuring the sclerotized parts (the width of the epistome and length of the urogomphi) was more effective (Orlova-Bienkowskaja and Bieńkowski, 2016). Therefore, my research measured the width of the epistome and length of the urogomphi of larvae. However, the two sets of results did not show the same amount in four instars. The third and fourth instars shown differ in two measurements (Fig. 10). It means a total of 47 specimens are hard to distinguish larvae life stage clearly, or the size of urogomphi and epistome are might grows indifferent speed in late instars. In this study, no larvae were found in cutting windows until July. In addition, thirty-nine larvae of 47 total were found in one single tree, so it means intense competition barely happens between larvae. This sample size is limited in this research, and the development time and life stages of larvae cannot be determined. The peak of adult emergence was in June, and mating behaviour of Agrilus dureli was observed in mid-June and is similar to that of EAB where adults occur from early May to early July in Tianjin (Wang et al., 2010). The beginning of May, around 50% of the larvae of EAB were in the second instar (Orlova-Bienkowskaja & Bieńkowski, 2016). It is recommended that the collection time could be advanced to the beginning of May in future research.

Differences in the categories of Agrilus dureli gallery density were observed among the trees in the present study. Analyzing the data of infested trees, the gallery density shows more interesting results than the D-shaped exit hole. Although the height and bearing of the exposed gallery recorded, researchers can only approach the height below the middle of the trunk easily. Therefore, middle trunk data should be the most accurate. The south is the most frequent direction in the naturally exposed gallery in the middle trunk. The southern side of the tree is

warmer because of the sun's exposure. It can be inferred that Agrilus dureli larvae might prefer growing and feeding on one side of the higher temperature on the tree. However, larvae of Agrilus dureli usually move in S-shaped lines under bark. It seems that there are very few cases where the gallery starts S-shaped directly from the north to the south in this study. Therefore, we cannot conclude that larvae of Agrilus dureli prefer to grow on the south side. Timms et al. (2006) mentioned that females of the Agrilus species prefer to lay eggs on the sunny side of the tree because the temperature in this direction is conducive to the survival and development of larvae. In this study, the female Agrilus dureli could randomly ovulate on the tree in any direction, and the larvae on the south side were more likely to grow successfully.

Furthermore, the high density of galleries could affect tree health. There is a positive linear relationship between gallery density and crown dieback (Appendix 4), but the value of R-squared is small (Appendix 4) which means the percentage of the dependent variable variation is small. In addition, the P-value of the gallery density is not significant under the classification of each group of environments. Therefore, the external environment has little effect on the density of the gallery. Meanwhile, the crown health has an effect on soil compaction, which is also confirmed by Doick et al (2009). In addition to the inevitable environmental influence, the greater density of gallery density by Agrilus dureli might affect the more crown declining on the tree. Research on EAB confirms that the density of the gallery is related to canopy conditions (Jennings et al, 2013).

The mortality of infected trees may be related to the time of infection, and the time of infestation can be seen in the gallery density (Timms et al., 2006). Infested trees include larval galleries, which can determine the length of time that the trees were damaged (Timmset al., 2006). Gallery categories 3, 4 and 5 appear to have been infested by Agrilus dureli for at least two years or more (Fig. 5). More density gallery categories 3 and 4 found in trees (Fig. 5), it means that the majority of detected infested trees have been infested at least for 2 years. Another internal sign of Agrilus dureli is the D-shaped exit hole, but this sign cannot be shown any hint about the time of infestation. The data of the D-shaped exit hole size did not show any difference in environmental classification. This study also found that some trees were determined as uninfested trees since the crown ratio was good and no D-shaped exit hole on the bark, but there was D-shape exit

holes appearance after 20 days. These trees have actually been infested. External factors cannot be seen because it was newly-infested. DBH and height show that the infested and uninfested trees are significant differences. Wood boring insects usually need thick enough bark, because such a living environment can provide sufficient nutrition; the thick bark also protects against extreme temperature disturbances and predation or parasites (Smith et al., 2002). The host of Agrilus dureli is willow trees, and the bark of the willows is usually thick. Laying eggs on thick bark is not easy for female Agrilus dureli. It was found in research that Agrilus dureli larvae feeding gallery have traces in outer bark, inner bark, and sapwood. Data from the study also show that Agrilus dureli prefers trees with smaller DBH, and the bark thickness of trees with smaller DBH is usually thinner. This is different from the conclusion that woodboring prefers thicker bark proposed by Smith et al. (2002). Larger host trees may provide more living resources for larvae. More giant DBH trees may withstand certain natural enemies, offer more protection for larval, and increase larval survival (Hanks et al., 2005; Abell et al., 2012). Another external sign is the height of trees. The data shows that Agrilus dureli prefers shorter trees. Even in different forest types, this insect infestation rate of short trees is high. Therefore, Agrilus dureli is more inclined to infest young, thin and short willows.

Agrilus dureli was first discovered in 2011. Eight years later, Agrilus dureli did not cause massive damage to Beijing's willows and attract local government notice. There are 19 study sites with uneven mortality rates, which may mean that the time of infection in the study sites was different. The area where Agrilus dureli was first discovered (site 0) was the hardest hit area and then spread along the banks of the Yongding River. Because the infection rate of sites on both sides of site 0 showed a gradient decline over distance, it is speculated that Agrilus spread from site 0. There may be four reasons why Agrilus dureli did not spread rapidly after 8 years. First, there are natural enemies of Agrilus dureli in Beijing area, like woodpecker and parasitoid wasp (Appendix 3). Various insects from region to region in Beijing, a natural enemy controls the wildlife balance. Second, the insect Agrilus dureli likes to reproduce on a host tree repeatedly. Since the bark of healthy trees is thick, it is difficult for female Agrilus dureli to lay eggs. Moreover, although Agrilus species are capable of flying, they naturally propagate through beetle flying. But they usually do not deviate from their vicinity when they appear (CFIA, 2019).

Third, after observation and inquiries, found the willow trees in the parks of Beijing's urban area are numbered, and management personnel regularly spray pesticides to willows. It is hard for Agrilus dureli to break through the long-managed willow. Fourth, because the willows have fewer commercial uses, the government does not allow the felling of timber, and few people cut away infected wooden branches with Agrilus dureli to move around, artificial transmission happens rarely. Therefore, Agrilus dureli has not spread across a large range in Beijing. These results are based solely on observations from a random search of selected sites, and it provides an interesting and important starting for further research.

Recommendations

This is the first study to show an understanding of Agrilus dureli, although the information collected here is based on a limited number of sites. Due to the small number of larvae collected, specifics around the life cycle of Agrilus dureli remain to be determined. For example, more work needs to be conducted on the east side of site 0. Populations of Agrilus dureli under a range of environmental conditions must also be studied to make sure if Agrilus dureli survived near Mentougou due to geographical conditions. It is suggested that more work needs to be done on the east side of site 0 to obtain a clear result to assess the risk rating of Agrilus dureli on trees and spread range.

The CFIA can now use the information collected here to start to determine whether Agrilus dureli should be added to their blacklist of unwanted pests. To date, the information collected shows that Agrilus dureli is unlikely to endanger large areas of forests in Beijing. Although Agrilus dureli has not much influence in Beijing, it remains possible that this species could arrive in Canada and cause damage. First, the environment and forest structure of Canada are different from Beijing. The willows planted in Canada are relatively scattered, there are few large willow forests. In Beijing, the closer to the city, the less sign of Agrilus dureli life is found. This does not rule out the existence of mutual restriction by natural enemies. However, Agrilus dureli is more suitable for living in forests near water sources, and there are many water sources in Canada. Second, the species of willows in China and Canada differ. Although Agrilus dureli is usually found in

willows, only Salix matsudana and Salix babylonica were found to be infected with Agrilus dureli in this study. Canada does only introduced Salix matsudana into Ontario, and Salix babylonica already extirpated in Canada. This can be assumed to reduce the risk of infestation. The identification of host-susceptible tree species also needs further investigation. However, a total of 54 native willow species covers the whole of Canada. Also, if Agrilus dureli has strong viability in a new environment, it is hard to say Agrilus dureli would not infest on Canada's native species willows. It must be mentioned that in this study, six poplar trees with corridors and D-shaped exit holes were found. It is uncertain whether these poplars were infected by Agrilus dureli, but three infected poplars were found near willows infected by Agrilus dureli. As a result, Agrilus dureli may not be too dangerous for Canada but still needs to be watched and avoided invasive.

In summary:

(1) The extent of damage caused by Agrilus dureli to trees and the characteristics of trees infested provides background information for future work in China and could be used by the CFIA to assess the future risk of introduction to Canada; (2) More data on the biology of this insect needs to be conducted under different conditions in the field; (3) Beijing managers who are concerned about the future of willows and poplars should be notified and encouraged to pay attention to the areas infested by Agrilus dureli; and (4) The local citizens shoud be informed that not transport the infested firewood and seedlings to avoid Agrilus dureli long-distance spread.

References

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Appendices

Appendix 1．The image of crown dieback of site 0. Photo was taken during 2019 summer in Beijing, China.

Appendix 2．The location of study sites and the distance to breakout site 0.

No. Site Forest Type Loation Latitude(N) Longitude(E) No. Adults No. Larvae 0 Riparian Forest Yanchizhen 40.02325 115.83762 4 0 1 Street Tree Yanchizhen 40.02366 115.83831 0 0 2 Street Tree Yanchizhen 40.02305 115.8386 51 0 3 Riparian Forest Yanchizhen 40.012552 115.822674 2 0 4 Riparian Forest Yanchizhen 40.01209 115.82532 0 6 5 Riparian Forest Yanchizhen 40.01403 115.83218 0 0 6 Windrow Yanchizhen 40.01362 115.83213 0 0 7 Park Yanchizhen 40.03237 115.85147 2 0 8 Street Tree Yanchizhen 40.03087 115.85292 0 0 9 Riparian Forest Yanchizhen 40.01254 115.82664 0 39 10 Park Yanchizhen 40.03284 115.83363 2 1 11 Park Yanchizhen 40.04877 115.8274 0 0 12 Park Yanchizhen 40.03027 115.8522 0 0 13 Street Tree Henantaicun 40.03501 115.89963 0 0 14 Park Anjiazhuangcun 40.01043 115.91921 0 0 15 Park Yanchizhen 40.01298 115.82731 0 0 16 Residential Longquanwucun 39.98092 116.08226 1 0 17 Park Datai Residential District 39.98394 115.96086 0 0 18 Park Wangpingzhen 39.97015 115.98209 0 1

Total 62 47

Appendix 3． Parasitoids wasp in willow tree, site 9, Yanchizhen, Mentougou, Beijing (2019).

Appendix 4． Linear relationship between gallery density and crown dieback for all willow trees (N=68) observed to have Agrilus dureli galleries during the summer of 2019 in the Beijing area. The blue area indicates 95% confidence limits.