Anoplophora glabripennis, an invasive longhorned beetle, has the potential to damage fruit trees in Japan.

Invasive Anoplophora glabripennis recently became established in Japan and has caused heavy damage to several street-tree species. Overseas, A. glabripennis infests trees of the genera Acer and Populus as common host plants, and Malus, Pyrus, and Prunus (Rosaceae), including apple, pear, and plum trees; it therefore poses a potential risk to the production of economically valuable fruits in Japan. Fruit farms in areas already invaded by A. glabripennis are now threatened with tree infestation. We aimed to determine the potential damage to major fruit species in Japan. In the laboratory, we determined if the adult beetle is attracted to the odor of each of these tree species' branches; two confirmed host plant species and five Rosaceae fruit species, as well as its feeding preferences among branches of one host plant and the five fruit trees and its oviposition preferences among them. Among the fruit species, cherry branch had the highest rate of odor orientation by males. The feeding-preference assay showed that, besides the host plant, Japanese pear was the most consumed among the fruit trees. The potential risk of A. glabripennis laying eggs on fruit-tree branches was high for Japanese pear and above zero for plum, apple, and cherry branches.

Is there hybridization between 2 species of the same genus in sympatry?-The genetic relationships between Anoplophora glabripennis, Anoplophora chinensis, and putative hybrids.

Anoplophora glabripennis (Asian longhorn beetle, ALB) and Anoplophora chinensis (Citrus longhorn beetle, CLB) are native forest pests in China; they have become important international quarantine pests. They are found using the same Salix aureo-pendula host tree of Cixi, Zhejiang province, China. On this host tree, we collected additional beetles that appeared to be morphologically intermediate between ALB and CLB. By using a stereoscope, we observed that there were several bumps on the base of the elytra, which was inconsistent with ALB, which typically has a smooth elytral base, but was more like CLB, which has numerous short tubercles on the elytral base. Given their sympatry and intermediate morphology, we hypothesized that these may represent ALB × CLB hybrids. We studied the genomic profiles for 46 samples (ALB, CLB, and putative hybrids) using genotyping-by-sequencing (GBS) providing a reduced representation of the entire genome. Employing principal component analyses on the 163 GBS-derived single nucleotide polymorphism data, we found putative hybrids tightly clustered with ALB, but genetically distinct from the CLB individuals. Therefore, our initial hybrid hypothesis was not supported by genomic data. Further, while mating experiments between adult ALB and CLB were successful in 4 separate years (2017, 2018, 2020, and 2021), and oviposition behavior was observed, no progeny was produced. Having employed population genomic analysis and biological hybridization experiments, we conclude that the putative hybrids represent newly discovered morphological variants within ALB. Our approach further confirmed the advantage of genome-wide information for Anoplophora species assignment in certain ambiguous classification cases.

Effects of Fusarium solani on the Growth and Development of Anoplophora glabripennis Larvae.

Anoplophora glabripennis (Asian longhorned beetle) is a wood-boring pest that can inhabit a wide range of healthy deciduous host trees in native and non-native habitats. Lignocellulose degradation plays a major role in the acquisition of nutrients during the growth and development of A. glabripennis larvae. In this study, the lignocellulose degradation capacity of Fusarium solani, a fungal symbiont of A. glabripennis, was investigated in fermentation culture and in four host tree species. The impact of F. solani on larval growth and survival parameters was assessed. Fermentation culture demonstrated continuous and stable production of lignocellulolytic enzymes over the cultivation period. Furthermore, F. solani was able to degrade host tree lignocellulose, as shown by decreased soluble sugar and cellulose contents and an increase in protein content. No significant differences in larval survival were observed in larvae fed with or without F. solani. However, weight and head capsule width were higher in larvae fed on F. solani, and gut lignocellulose activities were elevated in fed larvae. Our results indicate a role for F. solani in the predigestion of lignocellulose during the colonization and parasitic stages of A. glabripennis larval development, and also the F. solani an important symbiotic partner to A. glabripennis, lowering barriers to colonization and development in a range of habitats.

Distinct gut bacterial composition in Anoplophora glabripennis reared on two host plants.

Anoplophora glabripennis (Coleoptera: Cerambycidae: Lamiinae) is an invasive wood borer pest that has caused considerable damage to forests. Gut bacteria are of great importance in the biology and ecology of herbivores, especially in growth and adaptation; however, change in the gut bacterial community of this pest feeding on different hosts is largely unknown. In this study, we investigated the gut bacterial communities of A. glabripennis larvae fed on different preferred hosts, Salix matsudana and Ulmus pumila, using 16S rDNA high-throughput sequencing technology. A total of 15 phyla, 25 classes, 65 orders, 114 families, 188 genera, and 170 species were annotated in the gut of A. glabripennis larvae fed on S. matsudana or U. pumila using a 97% similarity cutoff level. The dominant phyla were Firmicutes and Proteobacteria and the core dominant genera were Enterococcus, Gibbsiella, Citrobacter, Enterobacter, and Klebsiella. There was significantly higher alpha diversity in the U. pumila group than in the S. matsudana group, and principal co-ordinate analysis showed significant differences in gut bacterial communities between the two groups. The genera with significant abundance differences between the two groups were Gibbsiella, Enterobacter, Leuconostoc, Rhodobacter, TM7a, norank, Rhodobacter, and Aurantisolimonas, indicating that the abundance of larval gut bacteria was affected by feeding on different hosts. Further network diagrams showed that the complexity of the network structure and the modularity were higher in the U. pumila group than in the S. matsudana group, suggesting more diverse gut bacteria in the U. pumila group. The dominant role of most gut microbiota was related to fermentation and chemoheterotrophy, and specific OTUs positively correlated with different functions were reported. Our study provides an essential resource for the gut bacteria functional study of A. glabripennis associated with host diet.

Niche shifts and range expansions after invasions of two major pests: the Asian longhorned beetle and the citrus longhorned beetle.

BACKGROUND: In recent years, the quarantine forestry pests the Asian longhorned beetle (ALB) Anoplophora glabripennis and the citrus longhorned beetle (CLB) Anoplophora chinensis have spread across the Northern Hemisphere, triggering concern about their potential distribution. However, little is known about the niche shifts of the pests during the invasion, making it difficult to assess their potential ranges. We thus employed two distinct approaches (i.e., ordination-based and reciprocal model-based) to compare the native and invaded niches of ALB and CLB after their spread to new continents based on global occurrence records. We further constructed models with pooled occurrences from both the native and invaded ranges to analyze the effects of occurrence partitioning on predicted ranges. RESULTS: We detected expansions in the invaded niches of both pests, indicating that the niches shifted to varying extents after the invasion. Large shares of the native niches of ALB and CLB remained unfilled, revealing the potential for further invasion in new regions. The models calibrated with pooled occurrences clearly underestimated the potential ranges in invaded regions compared with the projections based on partitioned models considering native and invaded areas separately. CONCLUSIONS: These results emphasize the importance of elucidating the niche dynamics of invasive species for obtaining accurately predicted ranges, which may help identify risk areas masked by the assumption of niche conservatism. Furthermore, prevention and quarantine measures for ALB and CLB are clearly needed to avoid future serious damage to forest ecosystems. © 2023 Society of Chemical Industry.

A Review of the Host Plant Location and Recognition Mechanisms of Asian Longhorn Beetle.

The Asian longhorn beetle (ALB), Anoplophora glabripennis Motschulsky, is a polyphagous xylophage with dozens of reported host tree species. However, the mechanisms by which individuals locate and recognize host plants are still unknown. We summarize the current knowledge of the host plant list, host kairomones, odorant-binding proteins (OBPs) and microbial symbionts of this beetle and their practical applications, and finally discuss the host localization and recognition mechanisms. A total of 209 species (or cultivars) were reported as ALB host plants, including 101 species of higher sensitivity; host kairomones were preferentially bound to ALB recombinant OBPs, including cis-3-hexen-1-ol, δ-3-carene, nonanal, linalool, and ß-caryophyllene. In addition, microbial symbionts may help ALB degrade their host. Complementarity of tree species with different levels of resistance may reduce damage, but trapping effectiveness for adults was limited using a combination of host kairomones and sex pheromones in the field. Therefore, we discuss host location behavior from a new perspective and show that multiple cues are used by ALB to locate and recognize host plants. Further research into host resistance mechanisms and visual signal recognition, and the interaction of sex pheromone synthesis, symbiont microbiota, and host plants may help reveal the host recognition mechanisms of ALBs.

Current and future control of the wood-boring pest Anoplophora glabripennis.

The Asian longhorn beetle (ALB) Anoplophora glabripennis is one of the most successful and most feared invasive insect species worldwide. This review covers recent research concerning the distribution of and damage caused by ALB, as well as major efforts to control and manage ALB in China. The distribution and destruction range of ALB have continued to expand over the past decade worldwide, and the number of interceptions has remained high. Detection and monitoring methods for the early discovery of ALB have diversified, with advances in semiochemical research and using satellite remote sensing in China. Ecological control of ALB in China involves planting mixtures of preferred and resistant tree species, and this practice can prevent outbreaks. In addition, strategies for chemical and biological control of ALB have achieved promising results during the last decade in China, especially the development of insecticides targeting different stages of ALB, and applying Dastarcus helophoroides and Dendrocopos major as biocontrol agents. Finally, we analyze recommendations for ALB prevention and management strategies based on native range and invasive area research. This information will hopefully help some invaded areas where the target is containment of ALB.

Genome-Wide Identification of the Odorant Receptor Gene Family and Revealing Key Genes Involved in Sexual Communication in Anoplophora glabripennis.

Insects use a powerful and complex olfactory recognition system to sense odor molecules in the external environment to guide behavior. A large family of odorant receptors (ORs) mediates the detection of pheromone compounds. Anoplophora glabripennis is a destructive pest that harms broad-leaved tree species. Although olfactory sensation is an important factor affecting the information exchange of A. glabripennis, little is known about the key ORs involved. Here, we identified ninety-eight AglaORs in the Agla2.0 genome and found that the AglaOR gene family had expanded with structural and functional diversity. RT-qPCR was used to analyze the expression of AglaORs in sex tissues and in adults at different developmental stages. Twenty-three AglaORs with antennal-biased expression were identified. Among these, eleven were male-biased and two were female-biased and were more significantly expressed in the sexual maturation stage than in the post-mating stage, suggesting that these genes play a role in sexual communication. Relatively, two female-biased AglaORs were overexpressed in females seeking spawning grounds after mating, indicating that these genes might be involved in the recognition of host plant volatiles that may regulate the selection of spawning grounds. Our study provides a theoretical basis for further studies into the molecular mechanism of A. glabripennis olfaction.

Cloning and Molecular Characterization of Hsp Genes from Anoplophora glabripennis and Their Responses to Cold Stress.

Anoplophora glabripennis (Agla) is an important global quarantine pest due to its highly destructive impacts on forests. It is widely distributed in many countries in Asia, Europe, and North America. The survival of A. glabripennis larvae has been facilitated by its high adaptability to low temperature. When insects are subjected to temperature stress, heat shock proteins (Hsps) limit cell damage and improve cell tolerance via their protein folding, localization, and degradation activities. However, the temperature adaptation mechanisms of A. glabripennis Hsps remain unclear. In this study, four A. glabripennis Hsp genes, AglaHsp20.43, AglaHsp71.18, AglaHsp82.09, and AglaHsp89.76, were cloned. Sequence analysis showed that all four Hsps had specific conserved domains. Phylogenetic analysis revealed that Hsps from different subfamilies were evolutionarily conserved, and that AglaHsps were highly similar to those of Coleoptera species. Protein expression vectors (pET30a-AglaHsps) were constructed and used to express AglaHsps in E. coli, where all four proteins were expressed in inclusion bodies. Western blot analysis showed that AglaHsps were expressed at a range of temperatures, from -10 °C to 25 °C. AglaHsp82.09 and AglaHsp89.76 showed high expressions with treatment at 0 °C. Our results will facilitate clarification of the molecular mechanisms underlying A. glabripennis responses to environmental stress.

Predicting Distribution of the Asian Longhorned Beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae) and Its Natural Enemies in China.

The Asian longhorned beetle, Anoplophora glabripennis, is a forestry pest found worldwide. A. glabripennis causes serious harm because of the lack of natural enemies in the invaded areas. Dastarcus helophoroides and Dendrocopos major are important natural enemies of A. glabripennis. MaxEnt was used to simulate the distribution of D. helophoroides and D. major in China, and their suitable areas were superimposed to pinpoint which regions are potentially appropriate to release or establish natural enemy populations under current and future conditions. The results showed that, with climate change, the suitable areas of D. helophoroides and D. major migrated northward; the centroid shift of A. glabripennis was greater than those of D. helophoroides and D. major. From current conditions to 2090, the suitable area of A. glabripennis, D. helophoroides, and D. major will increase by 1.44 × 104, 20.10 × 104, and 31.64 × 104 km2, respectively. Northern China (e.g., Xinjiang, Gansu, and Inner Mongolia), where A. glabripennis causes more serious damage, is also a potentially suitable area for D. helophoroides and D. major, and this provides a potential strategy for the management of A. glabripennis. Therefore, we suggest that natural enemies should be included in the model used for predicting suitable areas for invasive pests.

Specified host plants of the Asian longhorned beetle (Anoplophora glabripennis) - are lime trees and beech trees really at risk?

The Asian longhorned beetle Anoplophora glabripennis (ALB) is a dreaded quarantine pest that attacks a wide range of hardwood tree species. However, some of the specified host plants which have to be eradicated in infestation zones such as lime and beech trees almost never get infested or may even be resistant to the ALB. As a result, the specified host plant list needs to be revised based on the data from the infestation areas in the EU, in order to avoid further felling of whole lime avenues and major clear-cutting in parks with old growth. As large differences may exist between species of the same genus, exact species not merely genera should be specified on the host list.

Gut Lignocellulose Activity and Microbiota in Asian Longhorned Beetle and Their Predicted Contribution to Larval Nutrition.

Anoplophora glabripennis (Asian longhorned beetle) is a wood-boring pest that can inhabit a wide range of healthy deciduous host trees in native and invaded areas. The gut microbiota plays important roles in the acquisition of nutrients for the growth and development of A. glabripennis larvae. Herein, we investigated the larval gut structure and studied the lignocellulose activity and microbial communities of the larval gut following feeding on different host trees. The larval gut was divided into foregut, midgut, and hindgut, of which the midgut is the longest, forming a single loop under itself. Microbial community composition and lignocellulose activity in larval gut extracts were correlated with host tree species. A. glabripennis larvae fed on the preferred host (Populus gansuensis) had higher lignocellulose activity and microbial diversity than larvae reared on either a secondary host (Salix babylonica) or a resistant host (Populus alba var. pyramidalis). Wolbachia was the most dominant bacteria in the gut of larvae fed on S. babylonica and P. alba var. pyramidalis, while Enterococcus and Gibbsiella were the most dominant in larvae fed on P. gansuensis, followed by Wolbachia. The lignocellulose-degrading fungus Fusarium solani was dominant in the larval gut fed on different host trees. Functional predictions of microbial communities in the larval gut fed on different resistant host trees suggested that they all play a role in degrading lignocellulose, detoxification, and fixing nitrogen, which likely contribute to the ability of these larvae to thrive in a broad range of host tree species.

Genome-wide identification and expression analysis of the Hsp gene superfamily in Asian long-horned beetle (Anoplophora glabripennis).

The environmental adaptability of insects has been a key focus of ecological research. As molecular chaperones, Heat shock proteins (Hsps) play an important role in insect responses to environmental stress. Anoplophora glabripennis is a destructive pest of broad-leaved trees such as poplars. The ability to adapt to low temperature is an important factor for successful colonization of A. glabripennis in new diffusion area. However, the roles of Hsp in the stress responses in A. glabripennis have not been established. In this study, we identified 47 Hsp genes, including 3 Hsp90, 14 Hsp70, 9 Hsp60, and 21 sHsp genes. The Hsp gene family expanded substantially in A. glabripennis. The differences in expression patterns may be related to the type and intensity of stress. Larval overwintering transcriptomes showed that 13 Hsp genes were not induced during overwintering and 21 Hsp genes were involved in the regulation of life activities under non-stress conditions. In a quantitative RT-PCR analysis, AglaHsp90-2 responded more quickly under gradient cooling treatments; AglaHsp90-2 and AglaHsp90-3 were sensitive to treatment at 0 °C for 6 h under instantaneous cooling. Our results provide a theoretical basis for clarifying the molecular mechanism of Hsp genes in A. glabripennis in responsing to environmental stresses.

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.

Exogenous Gene Expression and Insect Resistance in Dual Bt Toxin Populus × euramericana 'Neva' Transgenic Plants.

Bacillus thuringiensis (Bt) insecticidal protein genes are important tools in efforts to develop insect resistance in poplar. In this study, the Cry1Ac and Cry3A Bt toxin genes were simultaneously transformed into the poplar variety Populus × euramericana 'Neva' by Agrobacterium-mediated transformation to explore the exogenous gene expression and insect resistance, and to examine the effects of Bt toxin on the growth and development of Anoplophora glabripennis larvae after feeding on the transgenic plant. Integration and expression of the transgenes were determined by molecular analyses and the insect resistance of transgenic lines was evaluated in feeding experiments. Sixteen transgenic dual Bt toxin genes Populus × euramericana 'Neva' lines were obtained. The dual Bt toxin genes were expressed at both the transcriptional and translational levels; however, Cry3A protein levels were much higher than those of Cry1Ac. Some of the transgenic lines exhibited high resistance to the first instar larvae of Hyphantria cunea and Micromelalopha troglodyta, and the first and second instar larvae and adults of Plagiodera versicolora. Six transgenic lines inhibited the growth and development of A. glabripennis larvae. The differences in the transcriptomes of A. glabripennis larvae fed transgenic lines or non-transgenic control by RNA-seq analyses were determined to reveal the mechanism by which Bt toxin regulates the growth and development of longicorn beetle larvae. The expression of genes related to Bt prototoxin activation, digestive enzymes, binding receptors, and detoxification and protective enzymes showed significant changes in A. glabripennis larvae fed Bt toxin, indicating that the larvae responded by regulating the expression of genes related to their growth and development. This study lay a theoretical foundation for developing resistance to A. glabripennis in poplar, and provide a foundation for exploring the mechanism of Bt toxin action on Cerambycidae insects.

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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Optimizing Parasitoid and Host Densities for Efficient Rearing of Ontsira mellipes (Hymenoptera: Braconidae) on Asian Longhorned Beetle (Coleoptera: Cerambycidae).

Ontsira mellipes Ashmead is a gregarious larval ectoparasitoid of woodboring cerambycids. It is native to North America but can readily attack the exotic Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). This study aimed to develop an efficient rearing system for this parasitoid, as a potential novel association biocontrol agent for the beetle, by investigating the effects of different densities of host (two, three, or four larvae) and parasitoid (one, two, three, four, five, six, seven, and eight female wasps) on Ontsira's parasitization efficiency and reproductive outcomes. Results showed that overall parasitism and total numbers of parasitized hosts or progeny produced increased with host and/or parasitoid densities, but the number of parasitized hosts or progeny produced per female parasitoid decreased with parasitoid density at each given host density. Nonlinear regression indicated a consistent pattern of mutual interference as parasitoid density increased. Additional experiments showed that superparasitism (indirect interference) did not occur probably because the parasitoid detects hosts through vibration cues from host feeding and attacked (thus paralyzed) hosts are no longer detectable. Thus, the interference probably results from direct or exploitative competition. Interestingly, female parasitoids responded to increased parasitoid density with a significant increase in clutch size. Overall, per capita parasitization efficiency or reproductive outcomes were optimized at a low parasitoid-host ratio but with large group size of hosts and parasitoids. Therefore, an optimal combination of exposing three or four parasitoids to four hosts is proposed for efficient mass-rearing of this parasitoid.