Resource Sharing between the Invasive Sirex noctilio and Native Woodborers and Beetles in Pinus Plantations.

Sirex noctilio, a European woodwasp, occasionally shares resources with the native S. nitobei and other colonizers in northeast China. The impact of its coexistence on individual species remains unclear. Random sampling was conducted to assess the patterns and extent of insect co-colonization across various spatial scales. Additionally, we analyzed wood sections to determine the density, adult size, and distribution of the two Sirex species. Spatial scales revealed negative associations (Asemum striatum and Phaenops sp.) and neutral ones (Ips acuminatus) between woodwasps and other co-colonizers. Clustering of woodwasps and Phaenops sp. occurred at a small scale (0-7.3 m). Regression analysis showed a positive correlation between the chance of woodwasp attacks and past attacks on the same host, with little impact from other colonization factors. The distribution and body size of S. noctilio within the tree appeared unaffected by S. nitobei's presence. In the presence of S. noctilio, S. nitobei tended to lay eggs in damaged sections. At the stand level, the overall impact of S. noctilio on S. nitobei population density is likely positive because S. nitobei prefer weaker trees, a preference potentially influenced by initial attacks from S. noctilio on healthier hosts.

Different genotypes and species of symbiotic fungi mediate the behavioral response of invasive Sirex noctilio fabricius (Hymenoptera: Siricidae).

In northeast China, the invasive woodwasp., Sirex noctilio, attacks Pinus sylvestris var. mongolica Litv and often shares habitat with native Sirex nitobei. Previous research showed that S. noctilio can utilize the volatiles from its symbiotic fungus (A. areolatum IGS-BD) to locate host trees. Consequently, symbiotic fungi (A. areolatum IGS-D and A. chailletii) carried by S. nitobei may influence the behavioral selection of S. noctilio. This study aimed to investigate the impact of fungal odor sources on S. noctilio's behavior in laboratory and field experiments. Our observations revealed that female woodwasps exhibited greater attraction toward the fungal volatiles of 14-day-old Amylostereum IGS-D in a "Y"-tube olfactometer and wind tunnel. When woodwasps were released into bolts inoculated separately with three strains in the field, females of S. noctilio exhibited a preference for those bolts pre-inoculated with A. areolatum IGS-BD. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the volatiles emitted by the two genotypes of A. areolatum were similar yet significantly distinct from those of Ampelopsis chailletii. Hence, we postulate that the existence of native A. areolatum IGS-D could potentially facilitate the colonization of S. noctilio in scenarios with minimal or no A. areolatum IGS-BD present in the host.

Physiological Measurements and Transcriptomics Reveal the Fitness Costs of Monochamus saltuarius to Bursaphelenchus xylophilus.

The pine wood nematode (PWN) uses several Monochamus species as vehicles, through a temporary hitchhiking process known as phoresy, enabling it to access new host plant resources. Monochamus saltuarius acts as a new and major vector of the PWN in Northeastern China, showing lower PWN carrying capacity and a shorter transmission cycle compared to established vectors. The apparently altered symbiotic relationship offers an interesting area for researching the costs and adaptions involved in nematode-beetle, a specialized phoresy. We analyzed the response and fitness costs of M. saltuarius through physiological measurements and transcriptomics. The PWN exerted adverse repercussions on the growth and development of M. saltuarius. The PWN accelerated larval development into pupae, while beetle adults carrying the PWN exhibited an elevated abnormality rate and mortality, and reduced starvation resistance. During the pupal stage, the expression of growth-related genes, including ecdysone-inducible genes (E74EA), cuticle proteins, and chitin genes (CHTs), markedly increased. Meanwhile, the induced immune response, mainly by the IMD and Toll signaling pathways, could be a contributing factor to adult abnormality and mortality. Adult gonads and trachea exhibited enrichment in pathways related to fatty acid elongation, biosynthesis, and metabolism. FASN, ELOVL, and SCD possibly contributed to resistance against PWN. Our research indicated that phoretic interactions between vector beetles and PWN vary throughout the vector's lifespan, particularly before and after entry into the trachea. This study highlighted the fitness costs of immunity and metabolism on the vector beetle, indicating the adaptation mechanisms and evolutionary trade-offs to PWN.

Functional and Compositional Changes in Sirex noctilio Gut Microbiome in Different Habitats: Unraveling the Complexity of Invasive Adaptation.

The mutualistic symbiosis relationship between the gut microbiome and their insect hosts has attracted much scientific attention. The native woodwasp, Sirex nitobei, and the invasive European woodwasp, Sirex noctilio, are two pests that infest pines in northeastern China. Following its encounter with the native species, however, there is a lack of research on whether the gut microbiome of S. noctilio changed, what causes contributed to these alterations, and whether these changes were more conducive to invasive colonization. We used high-throughput and metatranscriptomic sequencing to investigate S. noctilio larval gut and frass from four sites where only S. noctilio and both two Sirex species and investigated the effects of environmental factors, biological interactions, and ecological processes on S. noctilio gut microbial community assembly. Amplicon sequencing of two Sirex species revealed differential patterns of bacterial and fungal composition and functional prediction. S. noctilio larval gut bacterial and fungal diversity was essentially higher in coexistence sites than in separate existence sites, and most of the larval gut bacterial and fungal community functional predictions were significantly different as well. Moreover, temperature and precipitation positively correlate with most of the highly abundant bacterial and fungal genera. Source-tracking analysis showed that S. noctilio larvae at coexistence sites remain dependent on adult gut transmission (vertical transmission) or recruitment to frass (horizontal transmission). Meanwhile, stochastic processes of drift and dispersal limitation also have important impacts on the assembly of S. noctilio larval gut microbiome, especially at coexistence sites. In summary, our results reveal the potential role of changes in S. noctilio larval gut microbiome in the successful colonization and better adaptation of the environment.

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.

Predicting the current and future distribution of Monochamus carolinensis (Coleoptera: Cerambycidae) based on the maximum entropy model.

BACKGROUND: Monochamus carolinensis is an important vector of pinewood nematodes in North America that is under quarantine in several countries worldwide. The distribution of M. carolinensis was previously thought to be limited to North America; however, we discovered it during trapping in China in 2022. Using this discovery and information regarding the area of origin, we applied a machine-learning algorithm based on the maximum entropy principle to predict the current and future (2050s, 2070s) potential distribution areas of M. carolinensis using bioclimatic variables. RESULTS: The biological suitability of M. carolinensis was mainly driven by precipitation factors (BIO18, BIO15, BIO19), with 87.18% of the potential distribution areas located in South America, Asia, North America and Africa. Future potential distribution areas of M. carolinensis are predicted to expand to high latitudes, with an average increase of 10 245 874.88 km2 , and only 6.89% of the current suitable areas will become unsuitable. The potential distribution areas in 2070 are largest under the SSP585 scenario, with a 41.40% predicted increase (52 309 803.61 km2 ) above the current distribution, mainly reflecting an increase of the marginally and highly suitable areas. CONCLUSION: The determination of dominant climatic factors and potential distribution areas will help provide an early warning for an M. carolinensis invasion, as well as provide a scientific basis for the spread and outbreak, facilitating development of effective governmental prevention and control measures. © 2023 Society of Chemical Industry.

The interpretability of the activity signal detection model for wood-boring pests Semanotus bifasciatus in the larval stage.

BACKGROUND: The acoustic detection model of activity signals based on deep learning could detect wood-boring pests accurately and reliably. However, the black-box characteristics of the deep learning model have limited the credibility of the results and hindered its application. Aiming to address the reliability and interpretability of the model, this paper designed an active interpretable model called Dynamic Acoustic Larvae Prototype Network (DalPNet), which used the prototype to assist model decisions and achieve more flexible model explanation through dynamic feature patch computation. RESULTS: In the experiments, the average recognition accuracy of the DalPNet on the simple test set and anti-noise test set for Semanotus bifasciatus larval activity signals reached 99.3% and 98.5%, respectively. The quantitative evaluation of interpretability was measured by the relative area under the curve (RAUC) and the cumulative slope (CS) of the accuracy change curve in this paper. In the experiments, the RAUC and the CS of DalPNet were 0.2923 and -2.0105, respectively. Additionally, according to the visualization results, the explanation results of DalPNet were more accurate in locating the bite pulses of the larvae and could better focus on multiple bite pulses in one signal, which showed better performance compared to the baseline model. CONCLUSION: The experimental results demonstrated that the proposed DalPNet had better explanation while ensuring recognition accuracy. In view of that, it could improve the trust of forestry custodians in the activity signals detection model and aid in the practical application of the model in the forestry field. © 2023 Society of Chemical Industry.

Factors Affecting the Flight Capacity of Two Woodwasp Species, Sirex noctilio F. (Hymenoptera: Siricidae) and Sirex nitobei M. (Hymenoptera: Siricidae).

Sirex noctilio F. is an invasive woodwasp that causes pine mortality in plantations in China. Sirex nitobei M. is a native woodwasp in large areas of China. In this study, the flight capacity of the two woodwasps was studied and compared using a tethered-flight mill system to find individual factors affecting the flight capacity. After flight bioassays, woodwasps were dissected to determine nematode infestation. Post-eclosion-day (PED) age significantly influenced the flight capacity of S. noctilio females and males; as woodwasps become older, their flight capacity decreased. For S. nitobei, PED age did not significantly affect their flight capacity. In general, the flight capacity of S. noctilio was greater than that of S. nitobei. Females flew further and for longer than males for both Sirex species. The Deladenus spp. parasitism status of the two Sirex species did not significantly affect their flight performance parameters. PED age and body mass were key individual factors significantly affecting the flight capacity of the two Sirex species. In this study, detailed and accurate tethered-flight parameters of S. noctilio and S. nitobei were obtained. Although this is different from natural flight, it also provides us substantial laboratory data on their flight capacity, and facilitates risk analysis of the two woodwasp species.

Identification and expression profile analysis of chemosensory genes in pine needle gall midge, Thecodiplosis japonensis (Diptera: Cecidomyiidae).

Insects have highly specialized and sensitive olfactory systems involving several chemosensory genes to locate their mates and hosts or escape from predators. Pine needle gall midge, Thecodiplosis japonensis (Diptera: Cecidomyiidae), has invaded China since 2016 and caused serious damage. Till now, there is no environmentally friendly measure to control this gall midge. Screening molecules with high affinity to target odorant-binding protein to develop highly efficient attractants is a potential pest management method. However, the chemosensory genes in T. japonensis are still unclear. We identified 67 chemosensory-related genes in the transcriptomes of antennae, including 26 OBPs, 2 CSPs, 17 ORs, 3 SNMPs, 6 GRs, and 13 IRs, using high throughput sequencing. Phylogenetic analysis of these six chemosensory gene families among Dipteran was performed to classify and predict the functions. The expression profiles of OBPs, CSPs and ORs were validated by quantitative real-time PCR. 16 of the 26 OBPs were biased expressed in antennae. TjapORco and TjapOR5 were highly expressed in the antenna of unmated male and female adults. The functions of related OBPs and ORs genes were also discussed. These results provide a basis for the functional research on chemosensory genes at the molecular level.

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.

Impact of stand- and landscape-level variables on pine wilt disease-caused tree mortality in pine forests.

BACKGROUND: Pine wilt disease (PWD) outbreaks have affected extensive areas of South China's forests, but the factors explaining landscape patterns of pine mortality are poorly understood. The objective of this study was to determine the relative importance of stand structure, topography, landscape context, and beetle pressure in explaining PWD severity. During 2020-2021, we identified 66 plots based on mapped PWD infestation severity. We built PWD infestation maps for 2019-2021 through field surveys. Stand structure and topography were obtained from Forest Resources Management 'One Map' and elevation raster data. We then used 'One Map' and PWD infestation maps to determine landscape context and beetle pressure variables at different spatial scales. The relative importance of 12 explanatory variables was analyzed using multi-model inference. RESULTS: In this study, we show that: (i) 1 km was the best spatial scale related to pine mortality, and (ii) models including landscape context and beetle pressure were much better at predicting pine mortality than models using only stand-level variables. CONCLUSION: Landscape-level variables, particularly beetle pressure, were the most consistent predictors of subsequent pine mortality within susceptible stands. These results may help forest managers identify locations vulnerable to PWD and improve existing strategies for outbreak control. © 2023 Society of Chemical Industry.

Early Monitoring of Forest Wood-Boring Pests with Remote Sensing.

Wood-boring pests (WBPs) pose an enormous threat to global forest ecosystems because their early stage infestations show no visible symptoms and can result in rapid and widespread infestations at later stages, leading to large-scale tree death. Therefore, early-stage WBP detection is crucial for prompt management response. Early detection of WBPs requires advanced and effective methods like remote sensing. This review summarizes the applications of various remote sensing sensors, platforms, and detection methods for monitoring WBP infestations. The current capabilities, gaps in capabilities, and future potential for the accurate and rapid detection of WBPs are highlighted.

Composition and Diversity of the Endobacteria and Ectobacteria of the Invasive Bark Beetle Hylurgus ligniperda (Fabricius) (Curculionidae: Scolytinae) in Newly Colonized Areas.

Hylurgus ligniperda (Fabricius) (Curculionidae: Scolytinae) is a new invasive pest beetle in China, which colonized the Shandong province, causing devastating damage. Originating in Europe, it has spread to Oceania, Asia, North and South America. Bacterial associates have been frequently reported to play a vital role in strengthening the ecological adaptations of bark and ambrosia beetles. The environmental adaptability of H. ligniperda may be supported by their associated bacteria. Bacterial communities colonizing different body parts of insects may have different functions. However, little is known about the bacteria associated with H. ligniperda and their potential involvement in facilitating the adaptation and invasion of the beetles into new environments. In this study, we employed high-throughput sequencing technology to analyze the bacterial communities associated with male and female adults of H. ligniperda by comparing those colonizing the elytra, prothorax, and gut. Results showed that the bacterial communities of male and female adults were similar, and the elytra samples had the highest bacterial diversity and richness, followed by the gut, while the prothorax had the lowest. The dominant phyla were Proteobacteria, Firmicutes, and Actinobacteriota, while the dominant genera were Serratia, Lactococcus, Rhodococcus, unclassified Enterobacteriaceae, and Gordonia. Among these, Rhodococcus and Gordonia were the specific genera of endobacteria and ectobacteria, respectively. Differences in the distribution of associated bacteria may suggest that they have different ecological functions for H. ligniperda. The results of functional prediction showed that bacteria were enriched in terpenoid backbone biosynthesis, degradation of aromatic compounds, limonene and pinene degradation, neomycin, kanamycin and gentamicin biosynthesis, indicating that they may assist their beetles in synthesizing pheromones, degrading toxic secondary metabolites of host trees, and antagonizing pathogenic fungi. These results help us understand the interaction between H. ligniperda and bacteria and highlight possible contributions to the invasion process.

Early detection of pine wilt disease tree candidates using time-series of spectral signatures.

Pine wilt disease (PWD), caused by pine wood nematode (PWN), poses a tremendous threat to global pine forests because it can result in rapid and widespread infestations within months, leading to large-scale tree mortality. Therefore, the implementation of preventive measures relies on early detection of PWD. Unmanned aerial vehicle (UAV)-based hyperspectral images (HSI) can detect tree-level changes and are thus an effective tool for forest change detection. However, previous studies mainly used single-date UAV-based HSI data, which could not monitor the temporal changes of disease distribution and determine the optimal detection period. To achieve these purposes, multi-temporal data is required. In this study, Pinus koraiensis stands were surveyed in the field from May to October during an outbreak of PWD. Concurrently, multi-temporal UAV-based red, green, and blue bands (RGB) and HSI data were also obtained. During the survey, 59 trees were confirmed to be infested with PWD, and 59 non-infested trees were used as control. Spectral features of each tree crown, such as spectral reflectance, first and second-order spectral derivatives, and vegetation indices (VIs), were analyzed to identify those useful for early monitoring of PWD. The Random Forest (RF) classification algorithm was used to examine the separability between the two groups of trees (control and infested trees). The results showed that: (1) the responses of the tree crown spectral features to PWD infestation could be detected before symptoms were noticeable in RGB data and field surveys; (2) the spectral derivatives were the most discriminable variables, followed by spectral reflectance and VIs; (3) based on the HSI data from July to October, the two groups of trees were successfully separated using the RF classifier, with an overall classification accuracy of 0.75-0.95. Our results illustrate the potential of UAV-based HSI for PWD early monitoring.

Chromosome-level genome assembly of Monochamus saltuarius reveals its adaptation and interaction mechanism with pine wood nematode.

Monochamus saltuarius (Coleoptera: Cerambycidae) was reported as the vector beetle of the pine wood nematode (PWN, Bursaphelenchus xylophilus) in Japan and Europe. It was first reported to transmitted the PWN to native Pinus species in 2018 in Liaoning Province, China. However, the lack of genomic resources has limited the in-depth understanding of its interspecific relationship with PWN. Here, we obtained a chromosome-level reference genome of M. saltuarius combining Illumina, Nanopore and Hi-C sequencing technologies. We assembled the scaffolds into ten chromosomes (including an X chromosome) and obtained a 682.23 Mb chromosome-level genome with a N50 of 73.69 Mb. In total, 427.67 Mb (62.69 %) repeat sequences were identified and 14, 492 protein-coding genes were predicted, of which 93.06 % were annotated. We described the mth/mthl, P450, OBP and OR gene families associated with the vector beetle's development and resistance, as well as the host selection and adaptation, which serve as a valuable resource for understanding the host adaptation in insects during evolution. This high quality reference genome of M. saltuarius also provide new avenues for researching the mechanism of this synergistic damage between vector beetles and PWN.

A Waveform Mapping-Based Approach for Enhancement of Trunk Borers' Vibration Signals Using Deep Learning Model.

The larvae of some trunk-boring beetles barely leave traces on the outside of trunks when feeding within, rendering the detection of them rather difficult. One approach to solving this problem involves the use of a probe to pick up boring vibrations inside the trunk and distinguish larvae activity according to the vibrations. Clean boring vibration signals without noise are critical for accurate judgement. Unfortunately, these environments are filled with natural or artificial noise. To address this issue, we constructed a boring vibration enhancement model named VibDenoiser, which makes a significant contribution to this rarely studied domain. This model is built using the technology of deep learning-based speech enhancement. It consists of convolutional encoder and decoder layers with skip connections, and two layers of SRU++ for sequence modeling. The dataset constructed for study is made up of boring vibrations of Agrilus planipennis Fairmaire, 1888 (Coleoptera: Buprestidae) and environmental noise. Our VibDenoiser achieves an improvement of 18.57 in SNR, and it runs in real-time on a laptop CPU. The accuracy of the four classification models increased by a large margin using vibration clips enhanced by our model. The results demonstrate the great enhancement performance of our model, and the contribution of our work to better boring vibration detection.

Acoustic detection of the wood borer, Semanotus bifasciatus, as an early monitoring technology.

BACKGROUND: Semanotus bifasciatus Motschulsky (Coleoptera: Cerambycidae) is one of the most destructive wood-boring pests of Platycladus trees in East Asia, threatening the protection of antique cypresses and urban ecological safety. Early identification of Semanotus bifasciatus attacks can help forest managers mitigate the infestation before it turns into an outbreak. Acoustic detection technology is a non-destructive and continuous monitoring method with the potential to early identify and accurately evaluate the wood-boring damage. However, few studies have focused on the detection timing and corresponding acoustic features. In this study, we employed a manipulated insect infestation experiment to identify time windows in which early instar Semanotus bifasciatus larvae are most actively boring and feeding within logs and to identify acoustic features that distinguish larval sounds from typical background noise. RESULTS: The Semanotus bifasciatus larvae produced sounds most frequently between 13:00 and 20:00 while sounds were detectable from the first to the third instar during the larval growth stage, indicating a suitable time window for early detection. The stepwise regression (SR) model was optimal for detecting the larval instar [coefficient of determination (R2 ) = 0.71, root mean squared error of prediction (RMSEp ) = 0.42, and relative percent deviation (RPD) = 3.38] while the best model for predicting larval population size was the partial least squares regression (PLSR) model (R2  = 0.97, RMSEp  = 61.96, and RPD = 28.87). CONCLUSION: This study developed an acoustic method for identifying the early attack of Semanotus bifasciatus (including detection time window, feature variables and models for larval instar prediction and population size estimation). This technology integrated with internet of things (IoT) framework can be of value in developing an automated monitoring system for forest wood borer, and provide necessary guidance for integrated pest management (IPM). © 2022 Society of Chemical Industry.

A LAMP Assay for the Detection of Thecodiplosis japonensis, an Alien Gall Midge Species Pest of Pine Trees.

Pine needle gall midge (T. japonensis), native to Japan, has become a serious invasive pest in South Korea and, more recently in 2006, in China. It was first discovered in Qingdao, Shandong Province, and has caused serious damage to local Pinus thunbergii. The insect's small size makes morphological-based identification difficult; therefore, molecular detection techniques are urgently needed for monitoring and preventing its further spread. At present, there is no simple and accurate field molecular identification tool. To solve this problem, a LAMP-based molecular diagnosis technology of T. japonensis was developed. Four LAMP primers were designed to specifically amplify T. japonensis DNA. Positive LAMP reactions usually produce amplification in one hour. The optimal incubation conditions for LAMP detection were determined with 4 LAMP primers for 60 min at 61 °C. The LAMP detection range of gDNA concentrations is wide, with a minimum detectable gDNA concentration of 300 fg. A non-destructive DNA-releasing procedure, HotSHOT "HS6", which could extract "crude DNA" for LAMP assay in 10 min, was used for larval and adult samples. Therefore, we established a LAMP-based rapid molecular identification method that can be applied in the monitoring and management of T. japonensis.

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment.

A special mutual relationship exists between the pine wood nematode (PWN) Bursaphelenchus xylophilus and its vector beetles of genus Monochamus, which enables PWN to spread, at the same time provides longhorned beetles with more weak hosts. PWN are attracted to the pupal chambers and then carried inside the trachea of beetle adults, which is a necessary part to complete the B. xylophilus infection cycle. The growth and immune responses of the vector beetle will affect this carrying process, however, they were rarely studied in Monochamus saltuarius. Real-time quantitative polymerase chain reaction (RT-qPCR), one of the most common methods for quantitative gene expression analysis, was performed to explore the key genes and pathways involved in the growth, development and immune responses of M. saltuarius at different developmental stages associated with infection of PWN and PWN treatment conditions. To enhance the accuracy of RT-qPCR data, the expression of target genes needs to be normalized with reference genes, which are stably expressed under varied experimental conditions. In our study, the stability of 14 candidate reference genes in M. saltuarius samples at different developmental stages associated with infection of PWN or PWN treatment conditions was evaluated using delta Ct, geNorm, NormFinder, BestKeeper and RefFinder algorithms. Moreover, KLF gene was used to validate the stability of the selected reference genes. Under experimental conditions of this study, RPL7 and TER were suitable reference genes at different developmental stages associated with infection of PWN. RPL7 and RPS5 were considered the most stable reference genes in the pupae treated with PWN. RPS5 and SNX6 could be used as reference genes in the adults treated with PWN. RPL7, EF1-γ, and RPS5 could be used as stable reference genes in all the samples. This work is the first to evaluate reference genes in M. saltuarius, laying a foundation for further gene expression experimental procedures and understanding the phoretic relationship between M. saltuarius and B. xylophilus.

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.