Agrilus mali Matsumara (Coleoptera: Buprestidae), a new invasive pest of wild apple in western China: DNA barcoding and life cycle

Abstract Agrilus mali Matsumara (Coleoptera: Buprestidae) is a wood‐boring beetle distributed to eastern China that occasionally injures apple species. However, this wood‐boring beetle is new to the wild apple forests (Malus sieversii) of the Tianshan Mountains (western China) and has caused extensive tree mortality. The development of a biological control program for these wild apple forests is a high priority that requires exploration of the life cycle, DNA barcoding and taxonomic status of A. mali. In this study, to determine the diversity of invasive beetles, a fragment of the mitochondrial cytochrome oxidase gene was analyzed. Based on the results, beetles from Gongliu and Xinyuan counties of Xinjiang were identical but differed from those in the apple nursery of Gongliu by a single‐nucleotide substitution. We summarize the taxonomic status, relationships, and genetic distances of A. mali among other Agrilus species using the Tajima‐Nei model in maximum likelihood phylogeny. Analysis revealed that A. mali was closely related to Agrilus mendax and both belong to the Sinuatiagrulus subgenus. The life cycle of A. mali was investigated based on a monthly regular inspection in the wild apple forests of Tianshan. Similar to congeneric species, hosts are injured by larvae of A. mali feeding on phloem tissue, resulting in serpentine galleries constructed between bark and xylem that prevent nutrient transport and leading to tree mortality. Future studies will focus on plant physiological responses to the invasive beetles and include surveys of natural enemies for a potential classical biological control program.

2009; Jendek & Grebennikov, 2009). A fourth species, the East Asian buprestid Agrilus smaragdifrons Ganglbauer has been reported for the first time in the Western Hemisphere and suggests establishment of this metallic wood-boring beetle in the northeastern US (Hoebeke et al., 2017). A fifth species, Agrilus ribesi Schaefer is an invasive that has established in North America that is mostly native to the Eurasian continent (Jendek, Grebennikov, & Bocak, 2015). All these species are intercontinental invaders with cross-continental invasions. However, Agrilus species can also invade within a continent or locally. Emerald ash borer has been expanding to European Russia and likely to most of Europe (Orlova-Bienkowskaja, 2014).
Another example is the gold-spotted oak borer, Agrilus auroguttatus Schaeffer, is an invasive oak borer in California, but is native to southern Arizona of the USA (Lopez, Rugman-Jones, Coleman, Hoddle, & Stouthamer, 2014). The establishment of introduced invasive species has become problematic on a large scale causing very high levels of ecological and economic (Lodge et al., 2016). Most invasive wood-boring insects cause mortality of trees that results in direct or indirect effects on forest ecosystem processes (Gandhi & Herms, 2010a;Herms & McCullough, 2014). As an example, mortality of ash trees affects several insects and arthropod species that feed on ash trees and may be at risk of co-extirpation (Gandhi & Herms, 2010b;Herms & McCullough, 2014). Increasing international or domestic exports/imports of timber, firewood, and seedlings without quarantine assessment accelerates the distribution of wood-boring beetles to non-native habitats (Haack, Herard, Sun, & Turgeon, 2010;Poland & McCullough, 2006;Sydnor et al., 2007).
Among the Agrilus species, an invasive Agrilus mali Matsumara injures Malus species (Matsumura, 1924), including their endangered wild relatives (Ji, Ji, & Huang, 2004 (Jendek & Poláková, 2014;Ji et al., 2004). Agrilus mali is native to the eastern part of the Eurasian continent, primarily northeastern China and Amurskaya oblast, Khabarovsk and Primorskiy Kray of the Russian Federation and the Korean Peninsula and Japan (Chebanov, 1977;Cui, Liu, & Liu, 2015;Matsumura, 1924;Nikritin, 1994). Agrilus mali sporadically (i.e., non-epidemically) injures Malus (M. domestica and M. pumila) and Pyrus species (Chebanov, 1977;Matsumura, 1924;Nikritin, 1994). The first record of A. mali in western China was in 1993 due to the introduction of domesticated apple seedlings from Shangdong province to Xinyuan orchard in Ili Valley, Xinjiang for breeding purpose (Ji et al., 2004). Within 10 years after introduction, the beetle had escaped and rapidly distributed in the wild apple forests of Xinyuan located in the middle of Tianshan Mountains killing thousands of trees. Notably, this beetle only sporadically injures other species of apple tree, in addition to M. sieversii, that grow in native habitats of eastern regions of China (Cui et al., 2015); however, the lack of a co-evolutionary history likely led to the extensive mortality in the wild apple forests of Tianshan.
Forests of the Tianshan Mountains are rich in wild fruit relatives of domesticated species. The forest composition of wild fruits in the Chinese Tianshan (eastern Tianshan) is estimated at 38%, whereas in the central Asian portion, the estimate is 62% (Lin & Lin, 2000).
Malus sieversii (Ledeb.) Roem. is a wild apple species native to central Asia found in Kazakhstan, Kyrgyzstan, Tajikistan, Uzbekistan, and northeastern Afghanistan, and in western China, the species is widely distributed in the Tianshan sub-mountain area (Lin & Lin, 2000;Sokolov, Svjazeva, & Kubly, 1980). Although wild apple forests occupy an estimated 93% among the wild fruit forests in Chinese Tianshan, compared with 78% for the central Asian portion (Lin & Lin, 2000), the wild apple forests are not well studied. The wild apple M. sieversii is the primary progenitor of all cultivated domesticated apple species (Duan et al., 2017;Harris, Robinson, & Juniper, 2002;Hokanson et al., 1998;Richards et al., 2009;Zhang, Zhang, & Wang, 2015) and therefore is a globally important resource for apple breeding because of the rich genetic diversity. Thus, the ex and in situ preservation of this species is globally important (Hokanson et al., 1998;Yang et al., 2016).
Unfortunately, in the past two decades, A. mali has heavily attacked this species, which has caused extremely high mortality in the apple forests of the Tianshan Mountains since the first detection in 1993 (Ji et al., 2004). Since then the estimate is that 40% (3,866.67 hm 2 ) of the area of wild apple forest in Tianshan has been damaged, with the death of 666.67 hm 2 of forest (Wang, 2013).
Moreover, the vulnerability of wild apple increases under A. mali attack because fungi are also promoted. Among the fungi, Valsa canker, Valsa mali var. mali (Vmm), has been one of the important threatening factors for the wild apple forests (Wang, Li, et al., 2014a;Wang, Zang, Yin, Kang, & Huang, 2014b). With the attack of wild apple branches by A. mali, the vulnerability to V. mali invasion increases, which can accelerate tree mortality (Wang, Li, et al., 2014a;Wang, Wei, Huang, & Kang, 2011;Wang, Zang, et al., 2014b). Currently, A. mali is the primary threat to wild apple in the Tianshan region of Xinjiang-Uyghur Autonomous Province and is considered as a quarantine pest. Thus, pest management and biological control on A. mali are quite an urgent task in ecological recovery of the Tianshan wild apple forest.
Development of pest management programs that use chemical, biological, or cultural (i.e., pruning of infested branches) control options are essential. However, application of pesticides often does not provide expected results. Treatment of an infested tree may continue canopy decline during the first year and if treatment is effective, the canopy will usually begin to improve in the second year of treatment. Probably as the tree repairs its vasculature system after infestation has been reduced (Herms et al., 2009). Early in the discovery of the EAB invasion in North America, a survey was begun to seek natural enemies, parasitoids, and pathogens that parasitized EAB (Cappaert, McCullough, Poland, & Siegert, 2005;Liu et al., 2007). A classical biological control approach might efficiently eradicate an invasive insect species in a new habitat, or manage providing long-term, widespread control of the pest without application of chemicals or other strategies that require human intervention (Paine et al., 2015;Van Driesche et al., 2010). For instance, introduced encyrtid egg parasitoid Avetianella longoi (Siscaro) from Australia to California resulted in complete biological control of wood borer Phoracantha semipunctata throughout the state (Hanks, Gould, Paine, Millar, & Wang, 1995). Currently, several natural enemies have been found that prey on EAB in different life stages, which co-evolved in Asia with EAB (Duan, Bauer, Abell, Ulyshen, & Driesche, 2015;Gould, Ayer, & Fraser, 2011). Unfortunately, little is known about the natural enemies that could be used to control invasive A. mali. Liu et al. (2010) discussed preliminary research on the control of A. mali and proposed that Atanycolus species could be a dominant natural enemy for these invasive insects.
The establishment and behavioral ecology of the invasive woodboring insect A. mali in the forests of Tianshan must be investigated because of the significance for in situ conservation of wild apple populations in their natural habitat. First, the taxonomic traits and systematics position of the A. mali should be made clear. In this study, we explored the diversity among A. mali specimens in the forests of the Tianshan Mountains with partial cytochrome oxidase (COI) sequences using DNA barcoding. The mitochondrial COI gene is widely used to separate species that are morphologically indistinguishable in early stages and also in insect inter-and intra-population comparative analyses (Havill, Montgomery, Yu, Shiyake, & Caccone, 2006;Rugman-Jones, Hoddle, & Stouthamer, 2010;Wilson, 2012). Using maximum likelihood tree analysis following, a phylogenetic analysis determined taxonomic status among Agrilus species. Additionally, we report on insect description, the life cycle of the invasive beetles and described the taxonomic traits of A. mali. This research provides basic information that is fundamental for initiating the search for natural enemies of A. mali in order to use in a classical biological control program. Among the Rosaceae, wild apple is major species fruit forest, but also a few wild pear species can be found. Insect specimens were collected between 2014 and 2016 from infested wild apple trees in Xinyuan and Gongliu counties, including a wild apple nursery in Gongliu (Table 1, Figure 1). Ten beetle specimens were randomly collected from leaves of infested trees from different sites of forest. All collected specimens were immediately preserved in 95% ethanol and stored at −20°C before use for morphological description and DNA barcoding. Individual A. mali adult specimens were removed from tubes and air-dried on filter paper for homogenization. Beetles were ground under liquid nitrogen with mortar and pestle. Fine-powdered tissue was transferred to 2 ml Eppendorf tubes. Next, we used a Blood and Tissue kit for DNA isolation (Qiagen, Germany) following the manufacturer's protocols. Following 0.8% agarose (Invitrogen, USA) gel electrophoresis, total DNA was visualized using a Tanon 2500R Gel Documentation System (Tanon Science and Technology, China).

| PCR and sequencing
A degenerative primer pair COIFor-1 (5′-GGAAAYCCHG GDGCWTTAATTGG-3′) and REVCOI-3 (5′-TCTCCCCCYCCTGCYG GGTCAAA-3′) was designed from aligned COI sequences from Agrilus species to amplify a 530 bp fragment of the COI gene of A. mali. For designing primers, the most conserved region among the aligned congeneric COI sequences was selected. Then, nucleotide mixes were included into the primer sequence to be most complementary to the COI region. Amplifications were performed in a total volume of 20 µl containing 10 µl of PrimeSTAR HS (Premix) (Takara, Japan) that contained an appropriate concentration of dNTP and Taq-polymerase, 1 µl of (0.2 µM of each primer), and 4 µl of DNA template. Amplification was performed on a Veriti thermocycler (Applied Biosystems, USA). PCR conditions were as follow: 5 min at 95°C for the initial step, 35 cycles of 15 s at 94°C for denaturation, 30 s at 52°C for annealing, and 1 min at 72°C for an elongation step, and 5 min at 72°C for final elongation. PCR products were visualized on 1.5% agarose gel. PCR products were ligated into TA cloning vector pMD20-T (Takara) and then transformed into Escherichia coli

| Phylogenetic analysis
To determine the similarities of A. mali COI sequence with other Agrilus species, a BLAST search was performed and partial COI gene sequences of congeneric species were obtained from GenBank databases (NCBI and BOLD). Thirty-five COI sequences of Agrilus species were retrieved from NCBI and BOLD systems using a BLASTn search. Multiple sequence alignment was performed with MEGA7 using the CLUSTALW tool. The bootstrap method with 1,000 replicates and the Van Driesche,-Nei method in MEGA7 (Felsenstein, 1985;Kumar, Stecher, & Tamura, 2016;Tajima & Nei, 1984) were used for pairwise analysis of sequences.
The number of base substitutions per site was analyzed for all sequences. Nucleotide percentage of 1st + 2nd + 3rd codon positions was calculated using MEGA7. All positions containing gaps and missing data were eliminated from the data set. The neighbor-joining (NJ) (Saitou & Nei, 1987) tree was built using MEGA7 to resolve similarity.
The evolutionary history was inferred by using maximum likelihood method based on Hasegawa-Kishino-Yano (HKY) model with the rapid bootstrap method (500 replicates) with MEGA7.
Initial tree(s) for the heuristic search were obtained by applying the BioNJ method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach by applying uniform rates. We conducted parallel Bayesian MCMC analysis based on HKY model with BEAST V.1.8.4 (http://beast.community) which was used to run 1,000,000 generations (Drummond, Suchard, Xie, & Rambaut, 2012).

| Insect description and life cycle
The format, style of the insect description and morphological terms were followed as described by Jendek and Grebennikov (2011). A binocular stereomicroscope Olympus SZX10 (https://www.olympusims.com) was used for morphological description of beetles. Larval instars were distinguished following Wang, Zhang, Yang, and Wang (2013). Briefly, binocular microscope with an eyepiece scale was used to measure peristoma width, urogomphus length, and larval length, and molting rate. Crosby ratio was determined to verify the accuracy in the grouping process of the larval instars (Craig, 1975). were observed for larval infestation, pupation, and D-shaped holes.
All count data from trees observed in different locations were combined and generated for an average value. Counting period was March to September and acquired data were used to determine borders of each developmental stage in life cycle.

| Statistical analysis
StatView software packages were used to perform ANOVA analysis (SAS Institute Inc., Cary, NC, USA).

| Analysis of the mitochondrial COI sequences of A. mali and hypothesized phylogenetic relationship
Multiple alignment analysis revealed that one nucleotide substitution (C/T) was found at position 27 of COI sequences among the specimens (Figure 2a) Figure   S2). In general, the divergence of COI sequences among congeneric species is over 2% (Hebert, Ratnasingham, & deWaard, 2003), which is consistent with our results of an overall distance among the Agrilus species of 0.223, whereas the average distance of A. mali with other congeneric species was 0.226.

F I G U R E 2
The neighbor-joining (NJ) tree showing sequence divergences of COI for collected specimens (a) and molecular phylogenetic analysis by maximum likelihood method among the Agrilus taxa (b). The bootstrap consensus tree inferred from 1,000 replicates is used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in <50% of bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The evolutionary distances were computed using the Tajima-Nei method and are in the units of the number of base substitutions per site (a). The evolutionary history was inferred by using the maximum likelihood method based on the Hasegawa-Kishino-Yano model. Initial tree(s) for the heuristic search were obtained automatically by applying NJ and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL). A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 1.0300]). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 27.3788% sites). The analysis involved 36 nucleotide sequences. All positions containing gaps and missing data were eliminated. Single-nucleotide substitution (C to T) is marked as "Y" in the Agrilus mali COI sequence, which was used in phylogenetic analysis.

| Insect description
Adults are medium-sized insects 7-10 mm long that are variable in color (dark brown or dark bronze) and shiny (metallic) (Figure 3a).

| Insect life cycle
An expedition was conducted to

| D ISCUSS I ON
Molecular phylogeny provides insight into close level relationship in the genus Agrilus. In this molecular analysis, we explored the relationship of A. mali with other congeneric species. Since first reported in 1924 (Matsumura, 1924), detailed morphological traits and its phylogenetic status of A. mali have not been fully studied. The identical COI sequences from specimens collected in Gongliu and Xinyuan counties indicated that individuals were all one species, although one nucleotide differences were detected with individuals collected from the apple nursery in Gongliu. Genetic variation among Agrilus species in all ML groups was marginal. Clustering network analysis revealed that A. mali most closely grouped with A. mendax and was less related to A. ater, A. coxalis, and A. liragus. Generally, ML tree was consistent to morphologically classified species by subgenera.
F I G U R E 4 Average number of observed larvae, pupa and emergence holes in wild apple forests. Ten 50 cm branches per tree were observed for larvae, pupae and emergence holes. Data from each randomly chosen 10 trees combined together. Shown values are mean (± SE) of 100. Different letters show significant differences among months of each stage as determined by one-way ANOVA, followed by a Fisher PLSD post hoc test (p ≤ 0.05) Recent work by Kelnarova, Jendek, Grebennikov, and Bocak (2018) (Kelnarova et al., 2018). However, COI barcode and phylogenetic clustering with other congeneric species (Kelnarova et al., 2018) are enough to make a preliminary conclusion.
Thirty-two host-plant species are food sources for Agrilus species (Jendek & Poláková, 2014)  Understanding the life cycle of beetles allows using contact or systemic insecticides to treat plants in order to improve management. In addition, molecularly identified agricultural pests or invasive insects can provide a prerequisite to choose appropriate natural enemies in order to time releases during larvae stages or before emergency. The effectiveness of molecular methods has been referred to "who eats whom" questions in food-web ecology (Furlong, 2015;Hanner, Lima, & Floyd, 2009 A. planipennis which might be due to smaller diameters of branches.
Larval feeding behavior in the branch, particularly the formation of serpentine galleries, could interrupt upward nutrient movement to branches, resulting in dryness of the entire or partial trunk depending on the damaged area of the branch.
Overwintering period starts for larvae with decreasing temperature in September and continues through late March in Tianshan Mountains. During this period, larval instars did not grow much considerably but all larval instars could be found. However, adult insect overwintering were not observed and was not reported in literature. Climate of Tianshan Mountains is strongly continental based on Koppen climate classification (Peel & McMahon, 2007) with very cold winter and hot summer (Chen et al., 2013). The recent invasion of A. mali has caused more than 40% of apple forests to die (Wang, 2013). The consequence of the insect invasion can be clearly observed in the forests of Xinyuan County that led to huge mortality of wild apple population. In recent years, the invasion has expanded to other neighboring counties such as Gongliu and Yining (Figure 1b). Despite government efforts to prevent insect spread by pesticide application via air-spray or felling of trees, insects still escape to other locations (Liu, Zhang, Yue, & Wen, 2016). Our field investigations during the last 2 years showed limited numbers of larvae or adult beetles in Xinyuan apple populations that likely migrated to other places because of high tree mortality. Xinyuan could serve as the center for insect distribution to other locations like Gongliu. Apparently, adult beetles cannot fly far, and therefore distribution is not rapid (Sun, Liang, & Sun, 1979 Currently, wild apple populations in Gongliu suffer from insect attacks which might lead to an extensive decrease in apple forests. Moreover, A. mali invasion might not only restricted to wild apple forest of western China but could be also passed into western Tianshan mountains area, including Kazakhstan (Figure 1). During our expedition to the central Asian part of Tianshan, a field investigation was conducted in several M. sieversii populations of Ili-Altao, Jugar-Altao, and Chimkent (Kazakhstan) in 2017. We found that the apple populations had been seriously damaged and extensive tree mortality was observed (Figure 1c). However, Kazakh scientists and local gardeners did not establish causal agent of tree mortality. The wild apple forest in Central Asian Tianshan part could be potential area under high risk of insect invasion. Therefore, A. mali is an important pest that could be considered as an international threat.
Development of biological or chemical pest management programs for the whole Tianshan is challenging and requires developing action programs to stop insect distribution along with other central Asian countries.
In this study, we describe an invasive A. mali and its taxonomic status, including the relationship with other Agrilus species, DNA barcodes, and the life cycle in western China that will promote further research and assist the development of biological control.
Furthermore, based on field investigations, we considered that wild apple forest of Xinyuan as the center for A. mali invasion to other locations in western China which occurred for the last 20 years.
International cooperation is imminent to jointly develop a program to prevent A. mali expansion to other potential distribution area along Tianshan Mountains.

ACK N OWLED G M ENTS
We thank the Xinjiang Institute of Ecology and Geography for organizing the expedition to the Tianshan Mountains and Dr. Zhang YanLong from the Chinese Academy of Forestry for providing insect specimens. We also thank Dr. Lu Zhaozhi for discussion points about A. mali distribution. The National Key Research Project (2016YFC0501505) and a CAS PIFI fellowship (#2017PB0051) supported this research.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N
TAB performed the experimental work. TAB and DZ participated in the design of the study. TAB and ZL described insects. TAB, XL, and DZ collected insect larvae. TAB, ZL, and DZ studied life cycle in field.
TAB, XL, and DZ analyzed the data. TAB and DZ conceived of the study and edited the manuscript. TAB drafted the manuscript. DZ provided all of the fundings.

DATA ACCE SS I B I LIT Y
COI sequences in available in NCBI.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Bozorov TA, Luo Z, Li X, Zhang D.