Anoplophora Glabripennis Research Articles

PM 7/149 (1) Anoplophora glabripennis and Anoplophora chinensis

Specific scopeThis Standard describes a diagnostic protocol for Anoplophora glabripennis and Anoplophora chinensis.1This Standard should be used in conjunction with PM 7/76 Use of EPPO Diagnostic Protocols.Specific approval and amendmentFirst approved in 2020–10.Authors and contributors are given in the Acknowledgements section.

Anoplophora glabripennis: Host choice, oviposition and performance of new hatched larvae on ‘resistant’ poplar species

AbstractForests in north‐western China are comprised mainly of poplar trees, predominantly Populus alba var. pyramidalis (PAP). Both Populus deltoides cl. Beikang (PDB) and PAP are attacked by the Asian long‐horned beetle (ALB), but vary in their resistance to attack. To investigate performance of ALB on PDB and PAP, we evaluated: host‐locating time, feeding and oviposition; and larval survival rate and weight in single‐choice and two‐choice bioassays; and undertook a field single‐choice test. Results showed that there was no significant difference in the time that female ALB took to locate PDB and PAP in a cage bioassay, but males located PDB faster than PAP. Although not statistically significant, the mean weight of frass produced by adults feeding on PDB was less than on PAP in both the two‐choice and single‐choice experiments. The mean numbers of oviposition notch grooves on PDB and PAP in the single‐choice experiment were similar, but there were more oviposition notch grooves on PDB than on PAP in the two‐choice experiment. Survival rates of young larvae on PDB were significantly lower than on PAP. The average weights of larvae on PAP were significantly higher than on PDB in both two‐choice and single‐choice experiments. In the field experiment, mean numbers of oviposition notch grooves were significantly higher on PDB than on PAP, but mean weights and survival rates of larvae were significantly lower on PDB than on PAP, which suggested that PDB attracted ALB oviposition but showed strong resistant to developing ALB offspring. In conclusion, although PDB was more attractive for oviposition by ALB, it was still more resistant than PAP, providing a useful reference for selection of resistant varieties.

Defensive responses of Populus deltoides cl. Beikang to mechanical injury and Anoplophora glabripennis infection.

Poplar, as the main tree species of the protection forest in the "Three-North" area of China, has been seriously damaged by Anoplophora glabripennis since 1970s. Populus deltoides cl. Beikang (PDB) shows strong resistance to A. glabripennis, but the biochemical mechanism of resistance is unclear. In this study, the quantities of secondary metabolites and the activity of defense enzymes in bark and xylem from undamaged, mechanically damaged, and A. glabripennis infected PDB were detected by kit and HPLC techniques. The results showed different responses of PDB to mechanical damage and A. glabripennis infection. Secondary metabolites, the quantities of D-(-)-salicin and aspen significantly increased after mechanical injury compared with the undamaged materials, but the quantities of quercitrin decreased. The quantities of D-(-)-salicin and quercitrin significantly increased after insect infection, but without difference for aspen. There was no significant difference for the total quantities of phenolics between insect infected and uninjured xylem, but both were significantly lower than that in the mechanical damaged xylem. The quantities of aspen and flax lignans in the insect infected xylem was higher than those in the mechanical damaged xylem and uninjured xylem, while the quantities of total phenolic glycosides in the mechanical damaged xylem and the insect infected xylem were both significantly higher than that in the uninjured xylem. PAL activity was not different between mechanical damage and insect infection, whereas both were significantly higher than that of uninjured bark. SOD activity in mechanical damaged bark was significantly higher than that in insect infected bark, which were both higher than that of uninjured bark. POD activity had no significant difference between mechanical damage and insect infected xylem, but were higher than that of uninjured xylem. Compared with the uninjured PDB tree, the secondary metabolites and defense enzymes in the mechanical damaged and A. glabripennis infected PDB increased in varied degrees, which would be related with the resistance of PDB to external injury.

Successful Eradication of the Asian Longhorn Beetle, Anoplophora glabripennis, from North-Eastern Italy: Protocol, Techniques and Results.

Simple SummaryAnoplophora glabripennis (Coleoptera: Cerambycidae) is an extremely polyphagus Asian wood-boring beetle accidentally introduced into North America and Europe. In 2009, an infestation was found in the municipality of Cornuda (Veneto Region, Italy). In order to eradicate the pest, several actions were immediately undertaken in the delimitated infested and buffer zones: tree visual inspections twice a year, felling and chipping of infested and suspected trees, trapping protocols, mitigation plans based on substitution of felled trees with new plants, and citizen alerts. The program lasted 11 years, after which the species was declared eradicated from the region. During the eradication program more than 36,000 trees were surveyed and more than 2000 trees were felled. Trees most affected by the pest were birches, elms, maples, and willows. This paper describes all the actions undertaken during the eradication program, providing a protocol that can also be used for future eradications of the species.The Asian Longhorn Beetle (ALB), Anoplophora glabripennis (Coleoptera: Cerambycidae), is an important and extremely polyphagous wood-boring beetle native to Asia. In the 1990s, ALB was accidentally introduced into North America and Europe. In 2009, a large ALB infestation was found in the Veneto Region (north-eastern Italy), in the municipality of Cornuda (Treviso province). Eradication actions were immediately undertaken, based on delimitation of infested and buffer zones, tree visual inspections, felling and chipping of infested trees, trapping protocols, and citizen alerts. A total of 36,361 trees, belonging to 16 genera, were surveyed twice a year over an area of 7594 hectares. In 2020, after 11 years of eradication measures, the ALB population of Cornuda was declared eradicated. Overall, 2361 trees belonging to 8 genera were felled and destroyed, of which 1157 were found to be infested by ALB. This paper describes all the actions carried out and the procedures applied in order to eradicate ALB from north-eastern Italy, providing a useful example for current and future ALB eradication programs.

Preventing invasions of Asian longhorn beetle and citrus longhorn beetle: are we on the right track?

Two Asian longhorn beetles, Anoplophora glabripennis and Anoplophora chinensis, are among the most serious alien invasive species attacking forest and urban trees, both in North America and Europe. Major efforts have been put into preventing further entry and establishment of the two species as well as promoting their successful eradication. Here, we review these efforts, their progress and outcome, and scientific advancements in monitoring and control methods. The combined international activities and harmonizing legislative changes in detection and eradication methods have proven worthwhile, with more than 45% of eradication programmes successful in the last 12 years. Some countries were able to completely eradicate all populations and others managed to reduce the area affected. Although the costs of the eradication programmes can be very high, the benefits outweigh inaction. Attempts to eradicate A. chinensis have been more challenging in comparison with those targeting A. glabripennis. For both species, efforts are hampered by the ongoing arrival of new beetles, both from their native regions in Asia and from other invaded regions via bridgehead effects. The methods used for eradication have not changed much during the last decade, and host removal is still the method most commonly used. On the other hand, detection methods have diversified during the last decade with advances in semiochemical research and use of detection dogs. The next decade will determine if eradications continue to be successful, particularly in the case of A. chinensis, which has been targeted in some countries for containment instead of eradication.

Pest detection dogs for wood boring longhorn beetles

Invasive alien species are increasing due to globalization. Their spread has resulted in global economic losses. Asian [Anoplophora glabripennis (Motschulsky)] (ALB) and citrus [A. chinensis (Forster)] (CLB) longhorn beetles are two introduced wood borers which contribute to these economic losses e.g. the destruction of tree plantations. Early detection is key to reduce the ecological influence alongside the detrimental and expensive eradication. Dogs (Canis lupus familiaris) can detect these insects, potentially at an early stage. We trained two privately owned dogs to investigate their use as detection tools. We tested the dog’s ability to discriminate ALB and CLB from native wood borers by carrying out double-blind and randomized experiments in three search conditions; (1) laboratory, (2) semi-field and (3) standardized field. For condition one, a mean sensitivity of 80%, specificity of 95% and accuracy of 92% were achieved. For condition two and three, a mean sensitivity of 88% and 95%, specificity of 94% and 92% and accuracy of 92% and 93% were achieved. We conclude that dogs can detect all types of traces and remains of ALB and CLB and discriminate them from native wood borers and uninfested wood, but further tests on live insects should be initiated.

Molecular identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) and detection from frass samples based on real-time quantitative PCR

Anoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach.

Prediction of the potential global distribution of the Asian longhorned beetle Anoplophora glabripennis (Coleoptera: Cerambycidae) under climate change

AbstractThe Asian longhorned beetle (ALB) Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) is an important wood‐boring pest that has caused substantial damage to broadleaf trees in Asia, North America, and Europe. We used the modelling software CLIMEX to project the potential global distribution of ALB based on both historical (1987–2016) and future (2021–2050) climate conditions. ALB has possible hosts in 37 genera, and their known distributions were incorporated into the model to assess their effect on pest distribution. Suitable regions for ALB are predicted to be widely distributed under both historical and future climate conditions, and across all continents except Antarctica. With climate change, climate suitability would increase in the regions north of 30°N and decline in most regions south of 30°N. The area of most climate‐suitable regions would be covered by potential hosts, and optimum hosts would dominate. The possibility of ALB outbreaks in the Northern Hemisphere is much higher than in the Southern Hemisphere, owing to the richer abundance of hosts. These results provide theoretical guidance for developing effective ALB monitoring and mitigation measures.

Combining WV-2 images and tree physiological factors to detect damage stages of Populus gansuensis by Asian longhorned beetle (Anoplophora glabripennis) at the tree level

BackgroundAnoplophora glabripennis (Motschulsky), commonly known as Asian longhorned beetle (ALB), is a wood-boring insect that can cause lethal infestation to multiple borer leaf trees. In Gansu Province, northwest China, ALB has caused a large number of deaths of a local tree species Populus gansuensis. The damaged area belongs to Gobi desert where every single tree is artificially planted and is extremely difficult to cultivate. Therefore, the monitoring of the ALB infestation at the individual tree level in the landscape is necessary. Moreover, the determination of an abnormal phenotype that can be obtained directly from remote-sensing images to predict the damage degree can greatly reduce the cost of field investigation and management.MethodsMultispectral WorldView-2 (WV-2) images and 5 tree physiological factors were collected as experimental materials. One-way ANOVA of the tree’s physiological factors helped in determining the phenotype to predict damage degrees. The original bands of WV-2 and derived vegetation indices were used as reference data to construct the dataset of a prediction model. Variance inflation factor and stepwise regression analyses were used to eliminate collinearity and redundancy. Finally, three machine learning algorithms, i.e., Random Forest (RF), Support Vector Machine (SVM), Classification And Regression Tree (CART), were applied and compared to find the best classifier for predicting the damage stage of individual P. gansuensis.ResultsThe confusion matrix of RF achieved the highest overall classification accuracy (86.2%) and the highest Kappa index value (0.804), indicating the potential of using WV-2 imaging to accurately detect damage stages of individual trees. In addition, the canopy color was found to be positively correlated with P. gansuensis’ damage stages.ConclusionsA novel method was developed by combining WV-2 and tree physiological index for semi-automatic classification of three damage stages of P. gansuensis infested with ALB. The canopy color was determined as an abnormal phenotype that could be directly assessed using remote-sensing images at the tree level to predict the damage degree. These tools are highly applicable for driving quick and effective measures to reduce damage to pure poplar forests in Gansu Province, China.

Chemical Ecology of the Asian Longhorn Beetle, Anoplophora glabripennis.

The Asian longhorn beetle (ALB), Anoplophora glabripennis (Motschulsky), is a destructive forest pest in its native range, East Asia, or a high-risk invasive species in many other parts of the world. Extensive research has been directed toward the development of ALB management strategies. However, semiochemical-based trap lures, which are one of the effective tools for detecting, monitoring, and potentially assisting in eradicating cerambycids, have not reached operational efficacy for ALB to date, which is probably due to a grossly incomplete understanding of its chemical ecology. Here, we summarize the current progress in ALB chemical ecology including host selection and location, pheromone identification, trapping techniques, olfactory system, and related biology and behavior. We also briefly review the known semiochemicals in the subfamily Lamiinae, particularly the ALB congener, A. chinensis. Based on this knowledge, we highlight a potentially important role of some host-original chemicals, such as sesquiterpenes, in ALB host and mate location, and emphasize the basic studies on the biology and behavior of adult ALB. Last, we formulate suggestions for further research directions that may contribute to a better understanding of ALB chemical ecology and improved lure efficacy.

Cold tolerance of laboratory-reared Asian longhorned beetles

Low winter temperatures in temperate climates can limit the success of non-native species. The Asian longhorned beetle, Anoplophora glabripennis, is an invasive wood-boring pest of hardwood trees in North America and Europe. Native A. glabripennis populations are spread across several climate zones in China and the Korean Peninsula and are likely to encounter low temperatures in at least some of this range. Understanding the lethal limits of the overwintering life stages of A. glabripennis is essential for accurately modeling the risk that invasive populations pose to non-native environments. In this study, we provide the first systematic characterization of the cold tolerance strategy and lower lethal limits of A. glabripennis eggs, larvae, and pupae. In diapausing larvae, the most common overwintering stage in this species, we measure hemolymph glycerol and osmolality and identify the effects of prolonged low temperature exposure. In developing pupae, we identify sublethal effects caused by low temperature exposure before freezing. Eggs and larvae were the most cold-tolerant life stages; eggs were freeze-avoidant with an average supercooling point of −25.8 °C and larvae were freeze tolerant with an LT90 of −25 °C. Hemolymph osmolality of freeze-tolerant larvae, on average, increased to 811 mOsm during chilling. This increase was primarily driven by a concurrent, average increase of 232 mM hemolymph glycerol. Pupae died upon exposure to freezing temperatures, but accumulate strong sublethal effects prior to freezing, indicating that they are chill susceptible. Taken together, these data will be useful to inform species distribution modeling in A. glabripennis.

Improved Power Normalized Cepstrum Coefficient Based on Wavelet Packet Decomposition for Trunk Borer Detection in Harsh Acoustic Environment

The sound-detection method of trunk borer is a very promising method in the field of forestry prevention and control of trunk borers. However, the detection accuracy of commonly used algorithms often decreases sharply in the case of noise reverberation interference. In practical applications, the sound monitoring of trunk borers often takes place in a harsh acoustic environment. To solve this problem, we intend to introduce methods which are effective in other related acoustic fields. Unfortunately, most of the methods are not suitable for acoustic detection of trunk borers and perform extremely poorly. After trying various methods, we found that Power-Normalized Cepstral Coefficients (PNCC) performed well in some cases, while it did not in others. This is due to the difference between speech and trunk borer sound. Therefore, an improved anti-noise PNCC based on wavelet package is proposed. The dmey wavlet system always obtains the best performance. We collected the audio of the following five dry borer pests for testing. They are red palm weevil, mountain pine beetle, red necked longicorn, Asian longhorn beetle and citrus longhorn beetle. In the experimental part, we used genetic algorithm-support vector machine (GA-SVM) as a classifier to compare Mel Cepstral Coefficients (MFCC), which are the most common methods in the field of audio detection of trunk borer, PNCC and improved PNCC in a variety of noise environments. The results showed that, compared with other methods, the newly proposed method can often achieve better results. The above experiments take the audio clips made of clear pest sound mixed noise. In order to further verify the effectiveness of the method, we designed another experiment with a harsh outdoor acoustic environment. We found that the proposed method achieved 88% accuracy and the traditional PNCC achieved 78% accuracy. However, the Mel cepstrum coefficient completely lost its ability to distinguish. In sum, the proposed PNCC based on wavelet packet decomposition can be used as a detection method for trunk borer in the harsh acoustic environment. This method has many advantages, including simple extraction and strong robustness to noise. Combined with cheap audio acquisition equipment, this method can effectively improve the early warning ability of forestry borer pests.

From mycelia to mastodons – A general approach for simulating biotic disturbances in forest ecosystems

Forest disturbance regimes are changing around the globe. Of particular concern are biotic disturbance agents, as they respond strongly to climate warming and invade new ecosystems as alien pests and pathogens. To date, biotic disturbances are either ignored in simulations of vegetation dynamics or only a small number of common agents are considered explicitly. Here we present BITE, a general, process-based approach to simulate biotic forest disturbance agents from fungi to large mammals. BITE considers the processes of agent introduction, dispersal, colonization, population dynamics, and vegetation impact explicitly. Here we parameterize the model for six widely different biotic disturbance agents (Heterobasidion annosum, Hymenoscyphus fraxineus, Lymanthia dispar, Anoplophora glabripennis, Capreolus capreolus, Mammut americanum) and evaluate it using pattern-oriented modeling. BITE enables the inclusion of both established and novel biotic disturbance agents in vegetation models, and is a step towards the comprehensive simulation of forest disturbance regimes in a changing world.

Salicinoid phenolics reduce adult Anoplophora glabripennis (Cerambicidae: Lamiinae) feeding and egg production

Plant chemistry plays an important role in mediating resistance against insect herbivores. Depending on insect species and feeding strategies, plant defenses can prevent insect attacks, impact digestive physiology and influence reproductive output. The Asian longhorned beetle (Anoplophora glabripennis) is an invasive, tree-killing insect that exploits multiple host plant species. Anoplophora glabripennis is documented to attack Populus spp., but the ability for adults to consume Populus varies between beetle populations. In this study, we explored the influence of salicinoid phenolic glycosides, known to be potent chemical defenses in Populus against generalist insects, on A. glabripennis adult behavior and fecundity. We performed a series of choice- and no-choice experiments using extracts of salicinoids applied to plants that do not produce these compounds. Salicinoids affected A. glabripennis performance in a dose-dependent manner, with higher concentrations significantly reducing feeding. Egg development was reduced by > 90% in females fed salicinoids at ~ 1.3% dry weight. We then compared salicinoids present in feeding material vs. frass and found both a decrease in concentration and a shift in composition of these compounds. However, some of the more complex salicinoids were unchanged in the excrement, suggesting minimal detoxification had occurred. Our results show that salicinoids disrupt egg development in A. glabripennis, thereby reducing fitness. These results have implications for how A. glabripennis metabolizes plant defense chemicals and how tolerance may vary among populations.

Green-colored paperboard enhances the Asian longhorned beetle response to host plant odor cues

The Asian longhorned beetle (Anoplophora glabripennis) attacks apparently healthy trees, and more effective tools for surveying and monitoring them are needed. To better understand how the beetle recognizes and locates its hosts, we compared adult feeding on and orientation to cut branches of Acer negundo (a preferred host), Salix matsudana, Ailanthus altissima and Pinus bungeana, and found that adults preferred Acer negundo to others. We then evaluated whether color cues enhanced host (A. negundo) odor attractiveness for A. glabripennis. We assessed preference of adult A. glabripennis for colors that mimicked the bark of cut branches and found that adults were significantly attracted by forest green. We examined the effect of plant odors alone and found no significant difference among tested plants; however, the combination of forest green and A. negundo odors was more attractive to adults than combinations of other colors with A. altissima and P. bungeana odors. Finally, we studied the interaction between color and odor to determine their potential influence on A. glabripennis behavior. We found that combinations of cues that included forest green and host odor were more attractive to adults than others, but these preference of adults for forest green was disrupted by A. altissima odors.

First Recorded Asian Longhorned Beetle (Coleoptera: Cerambycidae) Infestation in the Southern United States

Abstract The Asian longhorned beetle [Anoplophora glabripennis (Motschulsky), ALB] was found in Hollywood, SC, in May 2020. This infestation is at least 7-yr old and represents the southernmost infestation in North America. A federally regulated invasive forest and tree pest, ALB primarily attacks maples but is known to attack and reproduce in trees in at least 12 different genera. Damage observed in South Carolina is typical of that seen in other ALB infestations, including oviposition sites, splitting bark around larval feeding sites, and round holes made by emerging adults. Copious weeping from oviposition sites was commonly observed in the summer of 2020, and characteristic frass and wood shavings were commonly exuded from attacked trees. Federal and state regulatory officials established a 152 km2 (58.6 mi2) regulated area in October—which the state increased to 188.0 km2 (72.6 mi2) in January—and to date >4,000 trees (98% Acer spp.) infested with ALB have been identified. Several uncertainties regarding ALB’s biology and ecology exist due to this area’s subtropical climate, including impacts to the beetle’s life cycle. Questions also surround potential biological control agents in this area and whether new host trees would be utilized. While the standard eradication strategy of tree removal and chipping is being performed, there are several forested wetlands and areas otherwise inaccessible to typical removal equipment where alternate eradication strategies may need to be developed and deployed. Because this ALB infestation is located within a novel environment, it provides an opportunity to research new strategies that will help improve future detection and eradication efforts.

Mating and adult feeding behaviour influence pheromone production in female Asian longhorn beetle Anoplophora glabripennis (Coleoptera: Cerambycidae)

Abstract The Asian longhorn beetle (ALB) Anoplophora glabripennis Motschulsky is a destructive invasive species worldwide. Female ALB produce a pheromone component, α‐longipinene, in the genitalia. However, the origin and factors, such as age, mating, adult feeding and host plant, influencing the production of this compound are unclear. Our results showed that virgin female ALB consistently produced α‐longipinene at various ages after feeding for several days post eclosion, but unfed adult females never produced this compound. Mating significantly reduced the amount of α‐longipinene in female genitalia. α‐Longipinene was the most dominant compound in the volatiles emitted by females, but not in those emitted by males or host twigs (Acer pensylvanicum L.). The proportion of α‐longipinene among the beetle‐released sesquiterpenes that were known to be male antennally active was significantly greater than that emitted by ALB damaged, mechanically damaged or control twigs. These results suggest that ALB females probably derive α‐longipinene or a precursor from hosts via adult feeding, and release it at a significantly different ratio from that found in host volatiles. The various ratios of α‐longipinene produced by beetles and host twigs may encode information pertaining to multiple purposes such as aggregation, mate and host location.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis

An insect’s capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period – results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Identification of key genes associated with overwintering in Anoplophora glabripennis larva using gene co-expression network analysis.

Anoplophora glabripennis (Coleoptera: Cerambycidae) is a major quarantine pest in forestry. It is widely distributed throughout many regions such as Asia, Europe, and North America, and has enormous destructive potential for forests. The larvae of A. glabripennis overwinter in a dormant state with strong cold tolerance, and whether the larvae survive winter determines the population density in the following year. However, the molecular mechanisms of this process are not clear. RNA sequencing (RNA-Seq) analysis of A. glabripennis larvae at five overwintering stages identified 6876 differentially expressed genes (DEGs). Among these, 46 functional genes that might respond to low temperature were identified. Weighted gene co-expression network analysis revealed that the MEturquoise module was correlated with the overwintering process. The STPK, PP2A, DGAT, and HSF genes were identified as hub genes using visualization of gene network. In addition, four genes related to sugar transport, gluconeogenesis and glycosylation were screened, which may be involved in the metabolic regulation of overwintering larvae. The protein-protein interaction network indicated that ribosomal protein and ATP synthase may play an important role in connecting with other proteins. The expression levels of fifteen hub genes were further validated by quantitative RT-PCR, and the results were consistent with RNA-Seq. This study demonstrates key genes that may reveal the molecular mechanism of overwintering in A. glabripennis larvae. The genes may be the potential targets to prevent larvae from surviving the cold winter by developing new biological agents using genetic engineering.

Optimizing Application Rates of Metarhizium brunneum (Hypocreales: Clavicipitaceae) Microsclerotia for Infecting the Invasive Asian Longhorned Beetle (Coleoptera: Cerambycidae).

The Asian longhorned beetle (Anoplophora glabripennis [Motschulsky]) is an invasive wood-boring beetle that threatens urban trees and forests in North America and Europe. The entomopathogenic fungus Metarhizium brunneum Petch strain F52 can infect and kill A. glabripennis adults. Products containing this fungus were available for commercial use in the United States but not registered for Asian longhorned beetle. This study tested different formulations and application rates of M. brunneum F52 microsclerotial granules for their potential development for management of A. glabripennis adults. Three application rates of M. brunneum microsclerotial granules relative to a 1× formulation from previous experiments (0.03 g/cm2; 2× = 0.06 g/cm2 and 3× = 0.09 g/cm2) were exposed on tree trunks for 4-wk periods during May-September. Increased application rates had better retention (% of initial g applied) than the 1× rate, rather than greater weathering loss. Microsclerotia at the 2× application produced 5.05 × 106 conidia/cm2, which was 18 times more conidia than the 1× application. Since A. glabripennis is under active eradication, bioassays with adult beetles were carried out in a quarantine laboratory, using the formulation samples from field exposures. The 2× application resulted in faster beetle mortality. The 3× and 2× rates were not significantly different in retention of the formulation, conidial production, or mortality, but 2× produced the most conidia per gram applied (3.92 × 109 conidia/g). An augmented formulation containing 70% M. brunneum by weight, rather than 50%, produced significantly more conidia and faster beetle mortality than the 50% formulation.