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Background: Global climate change is causing an increase in extreme high temperatures (EHTs), which subject insects to unprecedented stress. Behavior plasticity in response to EHTs, particularly oviposition behavior, is important for the persistence and outbreak of insect populations. Investigating the plasticity of oviposition behavior and its underlying mechanisms has theoretical importance to pest management, but knowledge gaps still remain. Results: Herein, we characterized the reproductive traits of Monochamus alternatus, a dominant insect vector of the destructive pine wilt disease, including oviposition behavioral patterns, fecundity, offspring fitness and sperm viability, under simulated heatwave conditions in the laboratory. The results showed that (i) EHTs induced a novel oviposition behavior, whereby females deposited multiple eggs into a single groove rather than laying one egg per groove under normal condition; (ii) EHTs exerted stage‐ and sex‐specific effects on fecundity, offspring fitness and sperm viability; and (iii) there was a significant correlation between frequency of the novel oviposition strategy and sperm viability. Conclusion: We hypothesized that this beetle pest has the ability to flexibly shift towards a low‐cost oviposition strategy to counteract the fitness costs caused by heat stress. Taken together, these findings provide a theoretical foundation for personalized pest management strategies in the context of climate change. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Euwallacea interjectus, a recently discovered pest in poplar plantations, poses a significant economic threat due to its role in causing widespread tree mortality. This pest's cryptic behaviour has hindered research and control efforts, making laboratory rearing a valuable tool for studying its development and biology. We investigated the development period and biological characteristics of E. interjectus using artificial diets and fungal medium. Our findings revealed that the development time for eggs, larvae, and pupae averages approximately 6, 18, and 6 days, respectively. Notably, first and second instar larvae displayed peak moulting periods at 3.45 ± 0.64 SD and 7.92 ± 1.77 SD days, respectively. Furthermore, we measured head capsule widths of postmolt larvae, yielding values of 318.02 ± 7.38 SD μ m for first-instar larvae, 403.01 ± 11.08 SD μ m for second-instar larvae, and 549.54 ± 20.74 SD μ m for third-instar larvae. Our research also uncovered a positive correlation between the number of progeny (eggs, larvae, pupae, and adults) and the mean length of the gallery system. Interestingly, the haplodiploid reproductive strategy did not significantly affect the number of offspring produced by the foundress. Additionally, we observed that foundresses displayed higher fecundity when subjected to nutrient-rich diets as compared to nutrient-poor diets. Our results will deepen our understanding of the biology of E. interjectus and provide criteria for larval instar classification. Additionally, managing nutrient availability within the colony could be considered a viable approach to regulating population size. [ABSTRACT FROM AUTHOR]

Conifers have evolved sophisticated terpenoid defenses for protection against herbivores and pathogens. Pinus massoniana Lamb. is the most widely distributed pioneer afforestation and resin tree species in China, but is seriously harmed by pine wilt disease. Monochamus alternatus is the main vector of pine wilt disease in China. Monoterpenes, sesquiterpenes and diterpenes, the main secondary defensive compounds of P. massoniana, are catalyzed by different terpene synthases (TPSs), which participate in the important defense pathways against external biotic and abiotic stresses. Here, we aimed to identify the terpene synthases (TPSs) in P. massoniana, responding to the feeding of M. alternatus, and to characterize the functions and products of the mono-TPSs. We identified six differentially expressed TPS genes in the P. massoniana fed upon by M. alternatus, including four mono-TPS and two sesqui-TPS genes. The functions of the four mono-TPSs were verified by analysis of the main product and by-products of these mono-TPSs. (+)-α-Pinene, (−)-α-pinene, and limonene were the major products of TPS (+)-α-pinene, TPS (−)-α-pinene, and TPS limonene, respectively, but TPS (−)-β-pinene only catalyzed a trace amount of (−)-β-pinene in the products. Our findings shed light on the potential relationships between the structure of terpene synthases and their corresponding products. [ABSTRACT FROM AUTHOR]

Alpha-pinene is one of the main defensive components in conifers. Monochamus alternatus (Coleoptera: Cerambycidae), a wood borer feeding on Pinaceae plants, relies on its detoxifying enzymes to resist the defensive terpenoids. Here, we assayed the peroxide level and GST activity of M. alternatus larvae treated with different concentrations of α-pinene. Meanwhile, a gst gene (MaGSTe3) was isolated and analyzed. We determined its expression level and verified its function. The results showed that α-pinene treatment led to membrane lipid peroxidation and thus increased the GST activity. Expression of MaGSTe3 was significantly upregulated in guts following exposure to α-pinene, which has a similar pattern with the malonaldehyde level. In vitro expression and disk diffusion assay showed that the MaGSTe3 protein had high antioxidant capacity. However, RNAi treatment of MaGSTe3 did not reduce the hydrogen peroxide and malonaldehyde levels, while GST activity was significantly reduced. These results suggested MaGSTe3 takes part in α-pinene adaptation, but it does not play a great role in the resistance of M. alternatus larvae to α-pinene. [ABSTRACT FROM AUTHOR]

Hyphantria cunea (Drury), a destructive polyphagous pest, has been spreading southward after invading northern China, which indicates that this insect species is facing a huge thermal challenge. Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones that protect insects from heat stress damage. In order to explore the role of sHSPs in the thermotolerance of H. cunea, five novel sHSP genes of H. cunea were cloned, including an orthologous gene (HcHSP21.4) and four species-specific sHSP genes (HcHSP18.9, HcHSP20.1, HcHSP21.5, and HcHSP29.8). Bioinformatics analysis showed that the proteins encoded by these five HcHSPs contained typical α-crystallin domains. Quantitative real-time PCR analysis revealed the ubiquitous expression of all HcHSPs across all developmental stages of H. cunea, with the highest expression levels in pupae and adults. Four species-specific HcHSPs were sensitive to high temperatures. The expression levels of HcHSPs were significantly up-regulated under heat stress and increased with increasing temperature. The expression levels of HcHSPs in eggs exhibited an initial up-regulation in response to a temperature of 40 °C. In other developmental stages, the transcription of HcHSPs was immediately up-regulated at 30 °C or 35 °C. HcHSPs transcripts were abundant in the cuticle before and after heat shock. The expression of HcHSP21.4 showed weak responses to heat stress and constitutive expression in the tissues tested. These results suggest that most of the HcHSPs are involved in high-temperature response and may also have functions in the normal development and reproduction of H. cunea. [ABSTRACT FROM AUTHOR]

Background: Commensal microorganisms are widely distributed in insect gut tissues and play important roles in host nutrition, metabolism, reproductive regulation, and especially immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial‐based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. Results: We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that re‐introduction of the HcM7 strain to germfree larvae preactivated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont‐induced gene in host defenses against pathogenic infections. Conclusion: These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Hyphantria cunea Drury (Lepidoptera: Erebidae) is a quarantine pest in China that can cause damage to hundreds of plants. As biological control agents, Nuclear Polyhedrosis Virus (NPV) and Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt) are commonly used to inhibit the prevalence of H. cunea. To investigate the role of midgut bacteria in the infection of NPV and Bt in H. cunea , we performed a series of tests, including isolating the dominant culturable bacteria in the midgut, eliminating intestinal bacteria, and respectively inoculating the dominant strains with NPV and Bt for bioassay. Two dominant bacteria, Klebsiella oxytoca Lautrop (Enterobacterales: Enterobacteriaceae) and Enterococcus mundtii Collins (Lactobacillales: Enterococcaceae), in the midgut of H. cunea were identified, and a strain of H. cunea larvae without intestinal bacteria was successfully established. In the bioassays of entomopathogen infection, K. oxytoca showed significant synergistic effects with both NPV and Bt on the death of H. cunea. In contrast, E. mundtii played antagonistic effects. This phenomenon may be attributed to the differences in the physico-chemical properties of the two gut bacteria and the alkaline environment required for NPV and Bt to infect the host. It is worth noting that the enhanced insecticidal activity of K. oxytoca on NPV and Bt provides a reference for future biological control of H. cunea by intestinal bacteria. [ABSTRACT FROM AUTHOR]

Insect intestinal bacteria play an important role in resisting defensive substances of host plants. Pagiophloeus tsushimanus (Coleoptera: Curculionidae) feeds exclusively on camphor trees (Cinnamomum camphora , Laurales: Lauraceae) in China, causing substantial economic and ecological losses. It is unclear how the larvae of P. tsushimanus outcome the main secondary metabolites of C. camphora such as D -camphor, eucalyptol, and linalool. In this study, we isolated terpenoid-degrading bacteria from the gut of P. tsushimanus larvae by using selective culture medium. Maximum likelihood phylogenetic analyses were performed with 16S rDNA sequences to identify the bacteria, and results showed ten strains belonged to four genera, including Pseudomonas , Enterobacter , Serratia , and Corynebacterium. Then, gas chromatography was employed to determine the degradability of D -camphor, eucalyptol, and linalool by the isolated strains, results showed that Z5 strain (i.e. Corynebacterium variabile , Actinomycetales: Corynebacteriaceae), F1 strain (i.e. Pseudomonas aeruginosa , Pseudomonadales: Pseudomonaceae), and A3 strain (i.e. Serratia marcescens , Enterobacterales: Enterobacteriaceae) had the highest degradation rates of D -camphor, linalool, and eucalyptol, respectively. The intestinal bacteria were capable of terpenoid degradation in vitro, which suggested that these gut bacteria associated with P. tsushimanus play an important role in overcoming host plant secondary metabolite defense, thereby facilitating the host specialization of this pest. [ABSTRACT FROM AUTHOR]

Understanding the mechanism of insect resistance to toxic effects of phytochemicals can provide an insight into plant–herbivore interactions. Monochamus alternatus Hope, a main vector of pine wood nematode (Bursaphelenchus xylophilus), prefers to infest masson pine (Pinus massoniana Lamb) and causes huge economic and environmental losses. α‐Pinene is the primary component of resin produced by masson pine and plays a crucial role in defending against herbivorous insects, but its defensive effects on M. alternatus larvae are barely known. Here, we explored the physiological metabolism in M. alternatus larvae that were fumigated with (+)‐α‐pinene, (‐)‐α‐pinene, β‐pinene, (R)‐(+)‐limonene, myrcene and (‐)‐α‐phellandrene. (+)‐α‐Pinene showed the highest fumigant toxicity to M. alternatus larvae among these monoterpenes, and the fourth instar larvae possessed the highest resistance to this compound at the larval stage. Comparative transcriptome analysis detected 423 up‐regulated and 311 down‐regulated genes in the larvae fumigated with (+)‐α‐pinene, which were mainly related to detoxification, energy and protein metabolism. Among the differentially expressed genes encoding for detoxification enzymes, 14 P450s, 13 UGTs, 1GSTs and 3ABCs were dramatically up‐regulated in the larvae fumigated with (+)‐α‐pinene. Silencing of P450s via RNA interference led to an increased mortality in the larval fumigated with (+)‐α‐pinene. Piperonyl butoxide exposure significantly increased the mortality and inhibited the expressions of P450s, UGTs and ABCs in the larvae fumigated with (+)‐α‐pinene. These results suggested an important role of P450s in the resistance of M. alternatus larvae to (+)‐α‐pinene fumigation, which may facilitate the understanding of terpenoid detoxification in M. alternatus larvae. [ABSTRACT FROM AUTHOR]

Monochamus alternatus , the dominant vector of Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), has caused immense damage to forest resources. In China, this vector was native to the southern regions but has spread northward recently. To adapt to more challenging environments in the northern winter, M. alternatus has evolved an intricate strategy for overwintering, which remains largely unknown. Herein, we compared the transcriptome data of the overwintering and non-overwintering larvae of M. alternatus larvae to investigate the molecular mechanisms in overwintering. A total of 53.10 GB clean bases and 28, 245 unigenes were obtained by RNA-seq. Analysis of 2597 upregulated and 2429 downregulated unigenes, as well as the enrichment of DEGs showed that many genes and pathways were jointly involved in the overwintering period. Besides, the accuracy of the RNA-seq data was tested by using qPCR experiment involving 13 selected genes. The results revealed that the overwintering process relied largely on the energy allocation trade-off. Specifically, overwintering M. alternatus inhibited energy-intensive activities, such as growth and molting, detoxification, and trehalose transport, and the reserved energy was skewed towards the synthesis of antifreeze compounds and immune response to cope with the deleterious effects of winter. [ABSTRACT FROM AUTHOR]

Camphor, Cinnamomum camphora (L.) Presl, is used for mothproofing material due to its content of secondary metabolites. However, the phloem‐feeding weevil, Pagiophloeus tsushimanus (Coleoptera: Curculionidae), has been observed to feed exclusively on this plant without incurring apparent fitness costs. This phenomenon remains to be elucidated.Gas chromatography–mass spectrometry analysis of essential oil extracted from the phloem of camphor tree was performed. The top three components, all monoterpenoids, were eucalyptol (31.9%), D‐camphor (19.2%) and linalool (10.2%), respectively.In headspace bioassays, both eggs and larvae showed the greatest sensitivity to eucalyptol, and there was a bell‐shaped relationship between the concentrations used and developmental time for eggs when exposed to D‐camphor volatile.Exposure to essential oil mixture and D‐camphor notably increased the activity of glutathione S‐transferase in the weevil. A corresponding short‐term induction of gene expression was also observed according to quantitative real‐time polymerase chain reaction analysis.In general, P. tsushimanus possessed various levels of tolerance to different host monoterpenoids. Over‐expression of glutathione S‐transferase in vivo probably relates to detoxification metabolism of these monoterpenoids. Our findings provide a valuable clue to understanding the mechanism underlying resistance to host‐specific chemical defences in this weevil pest. [ABSTRACT FROM AUTHOR]

A substantial increase in the occurrence of extremely high‐ or low‐temperature events caused by global climate change has imposed pressure on the survival and reproduction of invertebrates. Hyphantria cunea (H. cunea; Lepidoptera: Arctiidae) is a worldwide defoliator and has become a major forestry and agricultural pest during the past three decades in China. The thermal and cold tolerance has directly affected the population and geographical distribution of H. cunea, which remains poorly understood. Here, we examined the thermal tolerance of H. cunea larvae and discovered that they could endure extremely high temperatures, as demonstrated by median lethal times (Ltim50) of 103.3 hr at 37°C, 62.0 hr at 40°C and even 14.5 hr at 43°C. To further explore the molecular evidence of the physiological response to heat and cold, we cloned and characterized six small heat shock proteins (sHSPs). These proteins possessed the conserved α‐domain of the sHSP family at the C‐terminus and various N‐terminal domains. Phylogenetic analysis showed that the HCsHSPs presented a close evolutionary relationship with sHSP genes in noctuidae species. Four sHSPs in H. cunea were sensitive to heat stress, and only HCsHSP28.7 was induced by cold stress. The expression level of HCsHSPs peaked at 40°C and decreased in treatment at 45°C. HCsHSP20.0 transcripts were abundant in the cuticle after heat shock, and HCsHSP28.7 was most enriched in the fat body after cold shock. In addition, an equal molecular weight of the HCsHSP20.0 protein could completely protect malate dehydrogenase (MDH) from thermal aggregation. Recombinant HCsHSP28.7 E. coli cells exhibited tolerance to cold stress. Overall, our results demonstrate that HCsHSPs, especially HCsHSP20.0 and HCsHSP28.7, are vital genes in the adaptation of H. cunea larvae to extreme conditions in the context of global climate change. [ABSTRACT FROM AUTHOR]

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35–50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2–3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25–40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity. [ABSTRACT FROM AUTHOR]

BACKGROUND Symptoms of pine wilt disease (PWD) are caused when pathogenic pine wood nematodes (PWN) invade healthy host trees via wounds created by adult Monochamus alternatus. Exogenous methyl jasmonate (MeJA) can trigger terpene‐based induced defense in conifers, which is a core part of the conifer defense system. Herein, we hypothesized that the terpene‐based plant defense induced by MeJA could negatively affect the feeding behavior of M. alternatus adults, and may contribute to a new strategy in the control of PWD. RESULTS: The feeding area for M. alternatus adults feeding on MeJA‐treated seedlings was significantly smaller compared with control seedlings. However, no significant difference was detected in the enzyme activities in the midgut of beetles that had fed on these seedlings. Terpenoids were mainly accumulated in traumatic resin duct, whereas the constitutive resin duct accumulated only diterpenoids. Correspondingly, large‐scaled responses at the transcriptional level mainly focused on terpenoid and phenolic biosynthesis in the defending trees. CONCLUSIONS: Breeding tree species with a high resin yield may contribute to control of the spread of PWD by suppressing the feeding of M. alternatus adults. Transcriptome sequencing results provided abundant information for further breeding of highly resistant trees. Based on these findings, a potential push–pull strategy for the control of M. alternatus was discussed. [ABSTRACT FROM AUTHOR]

• Suitable reference genes should be screened out under different experimental conditions. • Three stable reference genes combination are necessary for quantitative analysis. • RPS3 , RPL10 , and UBQ are for developmental stages in Pagiophloeus tsushimanus. • RPL10 , RPS3 , and 18S rRNA are for different tissues in P. tsushimanus. • 18S rRNA , TBP , and RPS3 are for P. tsushimanus reared on different plants. Pagiophloeus tsushimanus is a newly and specialist wood-boring beetle of Cinnamomum camphora in China. RT-qPCR is an accurate quantitative method to quantify target genes expression, which relies on suitable reference genes for data normalization. Reference genes must to be stably expressed under specific experimental conditions. No suitable reference genes of P. tsushimanus have been reported so far. Therefore, it is necessary to identify and evaluate suitable reference genes for the study of functional genes of this pest. In this research, the expression stability of eight candidate reference genes (RPS3 , 18S rRNA , GAPDH , TBP , RPL10 , UBQ , GST , and RPS27A) were systematically evaluated in P. tsushimanus by five algorithms (geNorm, BestKeeper, NormFinder, delta C q , and RefFinder) under different developmental stages, various tissues, and insects reared on different plants, and validated by the olfactory key gene odorant binding protein 33 (PtsuOBP33). The results showed that three stable reference genes combination were necessary for quantitative analysis of target gene. RPS3 , RPL10 , and UBQ were the optimal reference genes combination for gene expression analysis of developmental stages, while RPL10 , RPS3 , and 18S rRNA were recommended for different tissues, and 18S rRNA , TBP , and RPS3 were recommended for insects reared on different plants. The results indicated that suitable reference genes should be screened out for gene expression analysis under different conditions. This paper systematically analyzed and obtained suitable reference genes in P. tsushimanus for the first time, which would contribute to the functional analysis of genes and the in-depth mining of genetic resources in it. [ABSTRACT FROM AUTHOR]

Temperature is a critical factor of insect population abundance and distribution. Monochamus alternatus Hope (Coleoptera: Cerambycidae) is a significant concern since it is transmitted vector of the pinewood nematode posing enormous economic and environmental losses. This pest shows tolerance to heat stress, especially extremely high temperatures. Exposing for 6, 12, 24, 48, or 96 h, the 50% median lethal temperatures (Ltem50) for fourth-instar larvae were 47.5, 45.5, 43.9, 43.4, and 42.3°C, respectively. A total of 63,360 unigenes were obtained from complementary DNA libraries of M. alternatus fourth-instar larvae (kept at 25°C and exposed to 40°C for 3 h) and annotated with six databases. Five hundred sixty-one genes were significantly upregulated, and 245 genes were downregulated after heat stress. The Gene Ontology enrichment analysis showed that most different expression genes are categorized into "protein folding" and "unfold protein binding" terms. In addition, "Longevity regulating pathway-multiple species," "Antigen processing and presentation" as well as "MAPK signaling pathway" were significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways. Further analysis of different expression genes showed that metabolism processes were suppressed, while ubiquitin proteolytic system, heat shock proteins, immune response, superoxide dismutase, cytochrome P450s, and aldehyde dehydrogenase were induced after heat shock. The stress signaling transduction pathways such as MAPK, Hippo, and JAK-STAT might be central convergence points in M. alternatus heat tolerance mechanism. The expression levels from quantitative real-time PCR of 13 randomly selected genes were consistent with the transcriptome results. These results showed that M. alternatus possessed strong heat tolerance and genes related to protein activity, immune response, and signal transduction composed of a complicated heat tolerance mechanism of M. alternatus. This research provided new insights into the mechanisms of thermal tolerance in other insects and aided in exploring the function of heat resistance-related genes. [ABSTRACT FROM AUTHOR]

Pinus massoniana Lamb. is an economically important pioneer conifer in southern China. Monochamus alternatus is considered the vector insect of the Bursaphelenchus xylophilus, a nematode that can cause destructive pine wilt disease. Many phenolic compounds have been identified to play key roles in plant defense against biotic/abiotic damages in multiple ecosystems. In addition, the cytochrome p450 enzymes reportedly participate in various secondary metabolism pathways in plant. Surprisingly, however, no study has characterized putative phenolic defenses nor the P450 enzymes in P. massoniana inoculated by M. alternatus at the transcriptome level. Here, we compared changes in the whole transcriptome by RNA-Seq, finding that different expressed genes in phenolic biosynthetic related pathways were significantly enriched, indicating phenolic secondary metabolism in P. massoniana was activated after M. alternatus feeding. Using qRT-PCR, we then explored the changes of limiting enzyme genes in phenolic biosynthetic pathways during a series sampling time-point in both M. alternatus feeding treatment and control. Expression profile of target genes varied in their expression level, initial reaction time, peak time and reaction duration. This study provides a platform for better understanding molecular changes that could aid forest protection and breeding efforts to improve performance in the face of biotic damage. [ABSTRACT FROM AUTHOR]

Abstract Clostera restitura Walker (Lepidoptera: Notodontidae) is one of the most destructive defoliators of poplars in China. We constructed an antennal transcriptome using Illumina Hiseq 2500™ sequencing and characterized the expression profiles of odorant binding proteins for better understanding of the olfactory receptive system and the role of putative olfactory proteins in C. restitura. A total of 165 transcripts were identified, including 43 transcripts encoding putative odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 78 odorant receptors (ORs), 15 ionotropic receptors (IRs), 13 gustatory receptors (GRs), and 3 sensory neuron membrane proteins (SNMPs). Furthermore, we systematically analyzed expression patterns of eight OBPs from different tissues of both C. restitura sexes by using reverse transcription PCR and quantitative real time PCR (RT-qPCR). The expression level of CresGOBP2 in female antennae was approximately two times higher than in males, and two pheromone binding proteins PBPs (CresPBP1 and -PBP3) and three OBPs (CresOBP9, −10, and −16) were more highly enriched in male antennae than in female antennae. CresOBP10 showed a remarkably high expression in legs compared to other studied insects. Our results suggested that these proteins might play a key role in foraging, seeking mates, and host recognition in C. restitura. Our findings provided a foundation for future studies on the molecular mechanisms controlling the olfactory system in C. restitura and potential novel targets for pest control strategies. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

The Asian wood-boring beetle Anoplophora chinensis (Forster) (Coleoptera: Cerambycidae) is an important pest of hardwood trees in its native range, and has serious potential to invade other areas of the world through worldwide commerce in woody plants and wood products. This species already has been intercepted in North America, and is the subject of ongoing eradication efforts in several countries in Europe. Attractants such as pheromones would be immediately useful as baits in traps for its detection. Because long-range pheromones are frequently conserved among closely related species of cerambycids, we evaluated two components of the volatile pheromone produced by males of the congener A. glabripennis (Motschulsky), 4-(n-heptyloxy)butan-1-ol and 4-(n-heptyloxy)butanal, as potential pheromones of A. chinensis. Both compounds subsequently were detected in headspace volatiles from male A. chinensis, but not in volatiles from females. Only 4-(n-heptyloxy)butanol elicited responses from beetle antennae in coupled gas chromatography-electroantennogram analyses, and this compound attracted adult A. chinensis of both sexes in field bioassays. These data suggest that 4-(n-heptyloxy)butan-1-ol is an important component of the male-produced attractant pheromone of A. chinensis, which should find immediate use in quarantine monitoring for this pest. [ABSTRACT FROM AUTHOR]

Members of the large and complex genus Xyleborus (Coleoptera: Scolytinae: Xyleborini) are the most commonly intercepted beetles associated with solid wood-packing materials at ports of entry in China. The accurate identification of species is critical in preventing the invasion of exotic insects. Considering the difficulties in morphological identification, genetic divergences of mitochondrial cytochrome c oxidase subunit I (COI) genes have been used in insect species identification. In this study, 32 Xyleborus and 2 outgroup species were collected from Jiangsu ports and selected to evaluate the effectiveness of DNA barcoding for Xyleborus species. The results showed that the mean interspecific divergence values (23.6%) were 15-fold higher than the observed intraspecific divergence (1.6%), except Xyleborus affinis. The results supported the inference that the barcode variation within species of insects is somewhat higher than interspecific ones. Thus, this study validated the effectiveness of barcoding for the identification of Xyleborus species. [ABSTRACT FROM AUTHOR]

Simple Summary: Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most accessible and widely-applied method for quantifying gene transcription. Stable reference genes need to be normalized to facilitate gene expression studies and obtain more accurate qRT-PCR data. In this research, eight reference genes from different functional categories and gene families were used to assess the stability level of gene expression under different conditions in H. cunea. Finally, the expression pattern of the target gene serine proteases (HcSP1) was evaluated under different conditions to verify the precision and reliability of the results. This study provides a comprehensive list of suitable reference genes for analyses and optional reference genes for other gene function research in H. cunea. The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species. [ABSTRACT FROM AUTHOR]

Monochamus alternatus is an important insect pest in pine forests of southern China and the dispersing vector of the pine wood nematode, Bursaphelenchus xylophilus, which leads to pine wilt disease (PWD). Microbiome of M. alternatus may contribute to survival of larvae in the host pine trees. In order to investigate the intestinal bacterial structure of M. alternatus during the larvae and pupae stages in host trees, and infer the function of symbiotic bacteria, we used 16S rRNA gene Illumina sequencing to obtain and compare the bacterial community composition in the foregut, midgut, and hindgut of larvae, pupal intestines, larval galleries, and pupal chambers of M. alternatus. The diversity of the bacterial community in larval intestines and pupal intestines were similar, as well as was significantly greater in larval galleries and pupal chambers. Although there were differences in bacterial compositions in different samples, similar components were also found. Proteobacteria and Firmicutes were the two most dominant phyla in all samples, and genera Enterobacter, Raoultella, Serratia, Lactococcus, and Pseudomonas were dominant in both the intestinal samples and plant tissue samples. Enterobacter was the most abundant genus in larval intestines, and Serratia was dominant in pupal intestine. The functions of these dominant and specific bacteria were also predicted through metagenomic analyses. These bacteria may help M. alternatus degrade cellulose and pinene. The specific role of symbiotic bacteria in the infection cycle of PWD also warrants further study in the future. [ABSTRACT FROM AUTHOR]

Two sibling weevil species, Pissodes strobi Peck and P. nemorensis Germar (Coleoptera: Curculionidae), can form reduced-fitness hybrids in the laboratory, but neither their premating isolation mechanisms nor mating behaviors are well-understood. Cuticular hydrocarbons (CHCs) have been reported as crucial chemical cues in mating recognition in many insects, including weevils, and, thus, may also mediate the mating behavior of P. strobi and P. nemorensis. We conducted a series of behavioral observations, bioassays, and chemical analyses to investigate the role of CHCs in their mating behavior. Copulation behavior of both species followed similar steps: approaching, mounting, tapping, aedeagus extrusion, and copulation. In P. strobi, hexane extraction significantly reduced the number of successful male copulations compared with freeze-killed females. Conversely, significantly fewer P. nemorensis males copulated with dead females compared with live females. No significant differences were detected among hexane-extracted, freeze-killed or recoated female carcasses to P. nemorensis. These findings suggested that female cuticular extracts contain important cues in mate recognition in P. strobi but not in P. nemorensis. We identified 21 CHCs from both species with variation in abundances between sexes and seasons. Discriminant analysis revealed incomplete overlap of CHC compositions in females of the two species in summer, when hybridization potentially occurs. [ABSTRACT FROM AUTHOR]

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori , using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori , we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis -jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control. [ABSTRACT FROM AUTHOR]