Bark beetles depend on detoxifying enzymes to counteract the defensive terpenoids produced by host trees. Insect ABC transporters play a critical role in the detoxification of insecticides and plant secondary metabolites. However, the specific functions of ABC genes in the metabolism of host allelochemicals remain unclear in D. armandi. In this study, we observed that verapamil significantly enhanced the mortality of host allelochemicals in beetles, indicating that ABC transporter genes are involved in the metabolism of monoterpenes by D. armandi. We then sequenced and characterized the full-length cDNAs of three ABCH subfamily genes (DaABCH1–DaABCH3) from D. armandi. Spatiotemporal expression profiling revealed that all three genes were upregulated during developmental transitions (egg to larva and pupa to adult) and tissue-specific enrichment in detoxification-related organs (Malpighian tubules, fat body, and midgut). Additionally, DaABCH3 expression was detected in the hindgut and brain. Furthermore, DaABCH1 and DaABCH2 were significantly induced by treatment with α-pinene and limonene, whereas DaABCH3 was induced by β-pinene and limonene. Importantly, silencing DaABCH1 significantly increased mortality in adults fumigated with α-pinene and limonene. These results strongly suggest that DaABCH1 acts as a key regulator modulating D. armandi’s sensitivity to host plant allelochemicals. This finding provides a conceptual basis for developing novel control strategies against this economically significant forest pest.

Low temperature and cold damage are natural factors that seriously reduce wheat yield. Thus, how to improve the cold resistance of wheat has been the focus of wheat breeders and geneticists. However, the genetic improvement for this trait has been slow, mainly because cold resistance is a complex quantitative trait and field phenotypic identification is relatively difficult. Therefore, the discovery, mapping, and cloning of the cold resistance genes of wheat provide a theoretical basis for the genetic improvement of wheat against cold resistance and facilitate the analysis of the molecular mechanisms of cold resistance in wheat. This study used the wheat line H261 and its EMS mutants LF2099 and XiNong 239 as materials. Cold trait segregation occurred in the F 2 generation of mutants LF2099 and XiNong 239 at a 15:1 separation ratio. Genetic analysis showed that two dominant overlapping genes, temporarily named Wcr-3 and Wcr-4 , control cold resistance in wheat. Furthermore, a combined BSA and SNP array established that Wcr-3 is between BU100519 (SSR marker) and AX-94843669 (SNP marker). The markers are 1.32 cM apart, corresponding to the 5.41 Mb physical interval on the Chinese Spring 2B chromosome with 67 functionally annotated genes. Wcr-4 is located between AX-94657955 (SNP marker) and LC-23 (SSR marker), which are 1.79 cM apart, corresponding to a 2.35 Mb physical interval on the Chinese Spring 2D chromosome, which contains 66 functionally annotated genes. Wcr-3 and Wcr-4 are two new cold resistance genes, laying the foundation for their fine mapping and cloning.

Insulin - including insulin-like peptides (ILPs), relaxins and insulin-like growth factors (IGFs) - is an evolu-tionarily conserved hormone in all metazoans. It is involved in various physiological processes, such as meta-bolism, growth, reproduction, lifespan and stress resistance. However, there are no reports on the functional role of ILPs in the Chinese white pine beetle, Dendroctonus armandi. In this study, we have cloned and identified two ILP cDNAs in D. armandi. The expression levels of DaILP1 and DaILP2 were significantly changed in different developmental stages. Both ILPs were expressed mostly in the head and fat body. Moreover, starvation induces the reduction of ILP1 mRNA level in adults and larvae, while ILP2 only in larvae of D. armandi, respectively. Additionally, RNA-interference (RNAi) using double stranded RNA to knock down ILP1 and ILP2 reduced the mRNA levels of the target genes, and caused a significant reduction in body weight of D. armandi. Moreover, silencing ILP1 led to an increase of trehalose and glycogen and significantly enhanced starvation resistance in both adults and larvae. The results show that the ILP signaling pathway plays a significant role in growth and carbohydrate metabolism of D. armandi and may provide a potential molecular target for pest control.

Bark beetles mainly rely on detoxification enzymes to overcome the defensive oleoresin terpenes of their host. Insect Carboxylesterases (CarEs) play an important role in the detoxification of plant allelochemicals and insecticides. Nevertheless, the functions of specific CarE genes in metabolization of exogenous substances are still unclear in the Chinese white pine beetle, Dendroctonus armandi . In this study, we sequenced and characterized the full-length cDNAs of six CarE ( DaEST1 - DaEST6) genes from D. armandi . Spatiotemporal expression profiling showed that the expression of DaEST1, DaEST3 and DaEST4 in D. armandi was higher in the larval and adult stages, and these genes mainly expressed in fat body, Malpighian tubules and midgut. Moreover, the expression of DaEST1, DaEST3 and DaEST4 was significantly induced after treatment with four terpenoids. Importantly, disruption of DaEST3 significantly inhibited the CarE activity and reduced the tolerance of adults to host allelochemicals in D. armandi . Additionally, treatment with triphenyl phosphate to adults also reduced the tolerance after terpenoids exposure, which resulted in a significant decrease of CarE activity, leading to a significant improvement of mortality in D. armandi . These results indicate that DaEST3 plays an important role in determining the tolerance to host allelochemicals in D. armandi , which might be of great significance in developing new therapeutic methods to pest control.

The sulfakinin (SK) is an important signal molecule. As a neuromodulator, it mediates a variety of behavioral processes and physiological functions in invertebrates through the interaction with G-protein-coupled receptors (GPCRs). However, there is no report on the functional role of SK in the Chinese white pine beetle, Dendroctonus armandi. We have cloned and characterized SK and SKR genes in the D. armandi and carried out bioinformatics predictions on the basis of the deduced amino acid sequences, which are very similar to those from Dendroctonus ponderosa. The expression levels of the two genes were different between male and female adults, and there were significant changes in different developmental stages, tissues, and between starvation and following re-feeding states. Additionally, RNA-interference (RNAi) using double-stranded RNA to knock down SK and SKR reduced the transcription levels of the target genes and increased their body weight. In parallel, injection of SK caused a significant reduction in body weight and increase in mortality of D. armandi and also led to an increase in trehalose and a decrease in glycogen and free fatty acid. The results show that the SK signal pathway plays a positive and significant role in feeding regulation and provides a potential molecular target for the control of this pest.

Bark beetles rely on detoxifying enzymes to resist the defensive oleoresin terpenes of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of plant allelochemicals and pesticides. CYP6 family is unique to Insecta, and its biochemical function is basically related to catabolize heterologous substances. In this study, two Dendroctonus armandi CYP6 genes, CYP6DF1 and CYP6DJ2, were characterized. Spatiotemporal expression profiling revealed that CYP6DF1 and CYP6DJ2 expressions were higher in larvae and adult stages of D. armandi than in egg and pupae stages, and that two genes predominantly expressed in brain, midgut, fat body, or Malpighian tubules. Moreover, CYP6DF1 and CYP6DJ2 expressions were significantly induced after exposure to (+)-α-pinene. Importantly, silencing CYP6DF1 and CYP6DJ2 significantly inhibited the CYP activity and increased the mortality in the adults fumigated with (+)-α-pinene. Additionally, piperonyl butoxide exposure to adults also increase the sensitivity after treatment with (+)-α-pinene, which led to a significant reduction of the CYP activity, resulting a significant increase in adult mortality. These results suggest that the CYP6 family plays a key role in determining the susceptibility of D. armandi to (+)-α-pinene, which may have implications for the development of novel therapeutics to control this important pest.

Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of pesticides and plant allelochemicals. CYP6 family is unique to Insecta, and its biochemical function is basically related to the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of two CYP6 genes from Chinese white pine beetle, Dendroctonus armandi. Spatiotemporal expression profiling revealed that the expression of CYP6CR2 and CYP6DE5 was higher in larval and adult stages of D. armandi than that in other developmental stages, and that two genes predominantly expressed in brain, midgut, fat body, Malpighian tubules or hemolymph. The expression of CYP6CR2 and CYP6DE5 was significantly induced after feeding on the phloem of Pinus armandii and exposure to six stimuli [(±)- α -pinene, (-)-α-pinene, (-)-β-pinene, (+)-3-carene, (±)-limonene and turpentine]. Importantly, silencing CYP6CR2 and CYP6DE5 separately could increase the sensitivity, led to a significant reduction of the activity of P450, resulting a significant increase in adult mortality after treatment with terpenoids. The comprehensive results of this study showed that in the process of host selection and colonization, the functions of CYPs were mainly to hydrolyze the chemical defense of the host and degrade odor molecules. These findings may help to develop new treatments to control this important pest.

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the bHLH-PAS (basic Helix–Loop–Helix–Period/ARNT/Single-minded) family of transcription factors, which participate in the sensing and transmitting stimuli of exogenous and endogenous chemical substances, and subsequently activates genes transcription involved in various detoxification and physiological functions. However, they have not been identified in Dendroctonus armandi, and their roles in the detoxification metabolism are unclear. In the present study, AhR and ARNT of D. armandi were characterized. Spatiotemporal expression profiling indicated that DaAhR and DaARNT were highly expressed in the adult and larval stages of D. armandi and mainly expressed in the midgut and Malpighian tubules of adults. Additionally, the expression of DaAhR and DaARNT significantly increased after exposure to (−)-𝛽-pinene, (+)-3-carene, and (±)-limonene. Silencing DaAhR and DaARNT increased the susceptibility of D. armandi to (−)-𝛽-pinene, (+)-3-carene, and (±)-limonene, and the activities of detoxification enzyme were also remarkably reduced. Moreover, DaCYP6DF1 and DaGSTs2 were significantly down-regulated after injections of dsAhR and dsARNT in the male and female adults, with the expression of DaCYP6DF1 decreasing by higher than 70%. The present study revealed that the transcription factors AhR and ARNT of D. armandi were induced by terpenoids and participated in the regulation of DaCYP6DF1 expression, which was associated with D. armandi’s susceptibility to (−)-𝛽-pinene and (±)-limonene. These results may provide a theoretical basis for the integrated control of D. armandi and improve our comprehension of insect toxicology.

Pinus armandii phloem is the key tissue in which Dendroctonus armandii live and breed.Although previous research examined the volatiles in P. armandii phloem, the dynamic changes and functions of these volatiles were never revealed.This study detected the changes in P. armandii phloem volatiles at each stage of infestation from healthy to dead trees and tested the toxicity of these volatiles against D. armandii.It revealed that (1) the weight of females was significantly greater than that of males, and females were more tolerant to host volatiles than males.(2) Limonene and myrtanol of P. armandii phloem volatiles played the key roles in resisting the invasion of D. armandii.The first increased the percentage of limonene together with other volatiles killing D. armandii in the resistant period of P. armandii, and the second synthesised myrtanol to further resist the invasion of D. armandii in the retreat period of P. armandii.These observations highlight the differences in the resistance of males and females to toxicity from the volatiles and the difference in the toxicity of different volatiles to D. armandii, revealing the defensive system of P. armandii phloem to provide a theoretical basis for the control and management of D. armandii.

Bark beetles mainly rely on detoxification enzymes to resist the host tree's defense against oleoresin terpenes. Cytochrome P450 enzymes (CYPs) play an important role in the detoxification of plant allelochemicals and pesticides in insect. One P450 gene (DaCYP4BQ1) is associated with the response of (+)-α-pinene in Dendroctonus armandi. However, the regulatory mechanism of this P450 gene response to (+)-α-pinene is still unknown. In this study, spatiotemporal expression profiling indicated that CYP4BQ1 was highly expressed in adult and larval stages of D. armandi, and it was predominantly expressed in fat body, midgut, and Malpighian tubules of adults. Moreover, the expression of CYP4BQ1 significantly increased after exposure to (+)-α-pinene, and depletion of it decreased the tolerance of adults to (+)-α-pinene. In addition, (+)-α-pinene treatment induced the expression of the transcription factors cap 'n' collar isoform C (CncC) and its binding factor muscle aponeurosis fibromatosis (Maf), elevated the level of hydrogen peroxide (H2O2), and increased the activities of antioxidant enzymes. Silencing CncC suppressed CYP4BQ1 expression and enhanced the susceptibility of beetles to (+)-α-pinene. Similarly, application of the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the production and accumulation of H2O2, suppressed the expression of CncC, Maf, and CYP4BQ1 and led to decreased tolerance of adults to (+)-α-pinene. In contrast, ingestion of the CncC agonist curcumin elevated CYP4BQ1 expression and enhanced (+)-α-pinene tolerance. The results demonstrate that, in D. armandi, (+)-α-pinene induces CYP4BQ1 via activation of the ROS/CncC signaling pathway.

The Chinese white pine beetle (Dendroctonus armandi Tsai and Li) is a significant pest of pine forests in the Qinling and Bashan Mountains of China. Adult males commonly produce frontalin using precursors synthesized through the mevalonate pathway, which is regulated by juvenile hormone III (JHIII). In this study, the expression levels of mevalonate pathway genes were quantified after phloem feeding and topical application of the JHIII solution. The frontalin was quantified by gas chromatography-mass spectrometry. Both the phloem feeding and JHIII treatments produced an evident upregulation in the male gut, mainly in 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). Moreover, HMGS, HMGR, isopentenyl diphosphate isomerase, and geranyl diphosphate synthase/farnesyl diphosphate synthase were upregulated in fed and JHIII-stimulated males of D. armandi under both conditions (solitary and paired). The expression levels were higher in paired compared to solitary males. Males had higher expression levels compared with females. Correspondingly, the phloem-feeding males produced more frontalin than JHIII-treated males, and the production of frontalin was higher in paired males than in solitary males. The knockdown of mevalonate pathway genes using RNAi in vivo effectively reduced the messenger RNA level of these genes and inhibited the production of frontalin. Among them, the silencing of HMGR or HMGS genes reduced the synthesis of frontalin most significantly.

The short neuropeptide F (sNPF) is an essential signaling molecule that is evolutionarily conserved and involved in a broad range of physiological functions in the invertebrates, by interacting with sNPF receptors, which belong to G protein-coupled receptors (GPCR). However, the function of sNPF in regulating the food intake of Dendroctonus armandi has been unclear. In this study, we cloned and characterized cDNAs encoding sNPF and sNPF receptor in the D. armandi and made bioinformatics predictions on the deduced amino acid sequences. They had a high degree of similarity to that of Dendroctonus ponderosa. Quantitative real-time reverse transcription PCR (qRT-PCR) revealed that the transcript levels of both sNPF and sNPFR varied across developmental stages and body parts. In addition, the sNPF and sNPFR expression levels were upregulated in starved beetles, and the expression levels recovered after re-feeding. Furthermore, RNAi knockdown by the injection of sNPF and sNPFR dsRNA into beetles significantly increased mortality and reduced their food intake and body weight, and also caused decrease of glycogen and free fatty acid and increase of trehalose. These results indicate that sNPF signaling pathway plays an important role in the regulation of food intake and provides a potential molecular target for the eco-friendly control strategies of this pest.

Bark beetle anti-aggregation is important for tree protection due to its high efficiency and fewer potential negative environmental impacts.Densitometric variables of Pinus armandii were investigated in the case of healthy and attacked trees.The range of the ecological niche and attack density of Dendroctonus armandii in infested P. armandii trunk section were surveyed to provide a reference for positioning the anti-aggregation pheromone verbenone on healthy P. armandii trees.2, 4, 6, and 8 weeks after the application of verbenone, the mean attack density was significantly lower in the treatment group than in the control group (P < 0.01).At twelve months after anti-aggregation pheromone application, the mortality rate was evaluated.There was a significant difference between the control and treatment groups (chi-square test, P < 0.05).These results provide insight into the characteristics of infected P. armandii and demonstrate that anti-aggregation treatment of D. armandii can improve the protection of healthy P. armandii.

Capa peptides are of great importance for both understanding the control of salt balance by neuropeptides and revealing the mechanisms of response to cold stress. Presently, research on the physiological function of neuropeptides typically includes identifying and analysing specific receptors and their interactions. Given their role in osmotic balance and the importance of cold tolerance, this study attempted to identify the sequence and function of Capa peptide receptors (CapaRs) in and to prove that CapaRs play an important role in the cold stress responses. In this study, two full-length cDNAs encoding more than 400 amino acids of Capa peptide receptor genes (DaCapaRs) and their sequence features were identified using bioinformatic analysis methods. The alignment of DaCapaRs with homologous proteins from other species indicated that the most similar are Capa peptide receptors. The expression of DaCapaRs was high in December and in the larvae, adults and head and midgut of . In addition, with decreasing temperatures and long-term exposure to cold conditions, the expression of DaCapaRs increased markedly when the temperature was 4 to –10°C. RNA interference analysis demonstrated that DaCapaRs play an important role in the cold tolerance of overwintering larvae and indicated that a reverse interaction occurs between DaCapaRs and aquaporins. The results showed that DaCapaRs play an essential role in understanding the neuropeptide regulation of cold tolerance in overwintering larvae and that it can assist in understanding adaptive evolution of and its relationship with the ecological environment of the Qinling Mountains.

BACKGROUND plant viral diseases are difficult to control and have caused serious damage to agricultural industry. Nowadays, botanical biopesticide characterized by environment friendly, safe to non-target organism and not as susceptible to produce drug resistance, have exhibited great potential to be developed as antiviral agents. To screen the natural products with antiviral effect, three alkaloids possessed anti-TMV activity were isolated from Chelidonium majus. And the modes of action were investigated. RESULT The anti-TMV effect of crude extracts at 10 mg ml-1 was 51.73%. Bioassay-guided fractionation and isolation of the compounds with anti-TMV activity were performed on the methanol extract of C. majus yielding three bioactive alkaloids namely: chelerythrine (1), chelidonine (2), and sanguinarine (3). The results of bioassay showed that chelerythrine exhibited great inactivation, proliferation inhibition and protection effects against TMV at 0.5 mg ml-1 with the efficiency of 72.67%, 77.52% and 59.34%, respectively. Chelidonine at 0.1 mg ml-1 can provide 54.90% and 64.45% inhibitions on TMV through inducing resistance in two kinds of tobacco. Sanguinarine showed a weaker protection for resisting TMV in comparison to chelerythrine and chelidonine. CONCLUSION Chelerythrine and chelidonine displayed significant inhibitions on TMV with different modes of action. These results provided important evidence that the extracts in C. majus might be a potential source of new drugs in controlling virus disease agriculturally. This article is protected by copyright. All rights reserved.

Background Systemic acquired resistance (SAR) induced by elicitors is a highly satisfying form of resistance that protects plants against invading pathogens. Exploration and development of new elicitors is a promising alternative to conventional biocides in resistant pest management. In our previous broad screening, fruit peel extract ofCitrus reticulataBlanco exhibited the ability to induce SAR in tobacco. Result A new potent elicitor reticine A was isolated from the fruit peel extract of industrial cropC. reticulateand its structure was well elucidated.In vivoassays showed that reticine A had considerable control efficacies at 100 and 500 mu g mL(-1), being superior to commercial elicitor benzothiadiazole (BTH) (100 mu g mL(-1)). Reticine A had no significant impact on the virulence of tobacco mosaic virus (TMV) particles underin vitroconditions. Application of reticine A induced a local hypersensitive reaction (HR), systemic accumulation of H(2)O(2)and salicylic acid (SA), systemic increase in defensive enzyme activities and systemic upregulated expression of pathogenesis-related (PR) proteins, suggesting its induction of SAR in tobacco. The expression ofNPR1and SA biosynthesis genesICSandPALwere systemically upregulated. Conclusion SAR induced by reticine A against TMV in tobacco was demonstrated and the mechanism might be attributed to activating the expression of several defensive genes mediated by an SA signal. This study highlights the potential of reticine A which is recommended to be applied directly or as an active ingredient in the crude extract formulation ahead of time in the field, as well as being a potential lead compound for further optimization.

In order to prevent any further spread of Dendroctonus armandi (Coleoptera: Curculionidae: Scolytidae), it is important to clarify its geographic distribution in China. Species Distribution Models were used to identify the variables influencing the distribution of D. armandi in China, and to create maps of its distribution. D. armandi almost exclusively attacked Pinus armandi Franch (IP (frequency of its incidence) = 98.2%), and its distribution is focused on the Qinling Mountains and the Ta-pa Mountains. The current distribution of P. armandi does not limit the distribution of D. armandi, despite the host occurring in in northern and southwestern China. Temperature and precipitation limit the current distribution of this beetle. The mean temperature of coldest quarter (−5 °C) does not guarantee that D. armandi larvae can overwinter in northern China, and the precipitation of wettest quarter plays an important role in the dispersal and colonization of D. armandi adults in southwestern China. Therefore, the ecological niche of this beetle is relatively narrow when it comes to these environmental variables. The climatic conditions where this beetle inhabit are different from the prevalent climate in the Qinling Mountains and the Ta-pa Mountains. At the meso- and micro-scale levels, terrain variables create habitat selection preferences for D. armandi. D. armandi predominately colonizes trees on the southern slopes of valleys and canyons with elevations between 1300 m a.s.l and 2400 m a.s.l.