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Dendrolimus houi Lajonquière is one of the most prevalent pine caterpillars in southern China, causing overwhelming forest infestation. It can overcome the negative impact of host shift from the original host, Cryptomeria fortune, to other tree species to complete its life cycle. In this work, D. houi larvae (1st to 3rd instar), originally feeding on C. fortunei needles, were used to determine the changes in the growth and development after they moved to needles of Cupressus funebris and Pinus yunnanensis during their 4th to 5th instar. Metabonomics and transcriptomics were conducted to evaluate the effects of the host shift on metabolite accumulation and gene expression of D. houi larvae. The results showed that the host shift significantly inhibited the pest growth and development by extending developmental duration and minifying the body length, body weight and head shell width. Besides, we found that the host shift influenced steroid hormone biosynthesis in D. houi larvae, which might lead to their abnormal development. The results may help to understand the response of D. houi larvae to host shift, and their adaptation mechanism to different hosts during multiple life cycles, providing a new plantation mode for mixed forests to suppress D. houi as well.

Monochamus alternatus Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars in economic losses, is a serious pest of Pinus massoniana in many Asian countries. Clarifying the eye pigment gene and its knockdown phenotype of M. alternatus can provide functional gene identification and a marker for screening of gene editing, as well as help develop new control ideas. In this study, we first screened the transcriptome and found one homologous gene of tryptophan 2,3-dioxygenase (TDO) and one of kynurenine-3-monooxygenase (KMO). By measuring the expression levels of TDO and KMO in different developmental periods, it was indicated that TDO and KMO were expressed in various stages of M. alternatus. The gene expression of MaKMO was higher than MaTDO, showing high expression after pupation and decreasing at the beginning of eclosion. MaTDO and MaKMO were knocked down using RNA interference technology in different periods of expression, and the temporal expression changes were obtained using RT-qPCR technology. The results showed that the expressions of MaTDO and MaKMO were significantly inhibited by the injection of dsRNA; the expressions of MaTDO at 48 h, 96 h and after pupation were 21.9%, 32.3% and 59.2%, respectively, meanwhile, those of KMO were 23.4%, 25.0% and 69.7%, respectively. There was a significant change in eye color, and the beetles were able to pupate normally without their activity being affected. Therefore, both MaTDO and MaKMO can be used as tag genes for M. alternatus. A dominant marker system based on eye color can be developed for the genetic manipulation and control of M. alternatus.

IntroductionBeauveria spp. and Dastarcus helophoroides Fairmaire adults were simultaneously released to attack elder larvae or pupae of Monochamus alternatus in pine forests in China. However, little is known about the pathogenicity virulence and biosafety of Beauveria spp. on beneficial adults of D. helophoroides, and specific Beauveria bassiana (Bb) strains should be selected for synthetic release together with D. helophoroides.MethodsA total of 17 strains of Beauveria spp. were collected, isolated, and purified, and then their mortality, cadaver rate, LT50, spore production, spore germination rate, and growth rate of D. helophoroide adults were calculated based on 0–20 days data after spore suspension and powder contact.Results and discussionThe lethality rate of BbMQ, BbFD, and BbMH-03 strains to D. helophoroides exceeded 50%, and the cadaver rate reached 70.6%, among which the mortality rate (82.22%), cadaver rate (47.78%), spore production (1.32 × 109 spores/ml), spore germination rate (94.71%), colony dimension (49.15 mm2), and LT50 (10.62 d) of the BbMQ strain were significantly higher than those of other strains (P < 0.01), and the mortality of D. helophoroides adults increased significantly with increased spore suspension concentration, with the highest mortality reaching 92.22%. This strain was identified as Beauveria bassiana by morphological and molecular methods, while the BbWYS strain had a minimum lethality of only 5.56%, which was safer compared to other strains of adult D. helophoroide. Consequently, the biological characteristics and pathogenicity of different Beauveria bassiana strains varied significantly in their effects on D. helophoroide adults, and the safety of different strains should be assessed when they are released or sprayed to control multiple pests in the forest. The BbMQ strain should not be simultaneously sprayed with releasing D. helophoroide adults in the same forest, while the BbWYS strain can be used in concert with D. helophoroide to synergize their effect.

Fluopyram is a novel nematicide compound with systemic activity, and it is a potential chemical agent for controlling pine wilt disease, which is devastating pine trees worldwide. Here, a fluopyram suspension concentrate (SC) was used to determine its efficacy in controlling pine wilt disease, its distribution in pine tree tissues, and its environmental degradation. Fluopyram SC had an evident inhibitory effect on Pestalotiopsis sp. and showed a good preventive effect on pine wilt disease in healthy pine seedlings. In the field application, after spraying onto the canopy of pine trees, fluopyram was absorbed and translocated downward to all parts of the tree via the branches and needles, and its distribution was stable at 30 days post-application (all samples’ mean residual concentration = 1.166 mg/kg). In addition, the maximum concentration of fluopyram residue in soil and water was 1.139 and 0.099 mg/kg, respectively, at 30 days post-application when fluopyram was significantly degraded. The control efficiency of fluopyram SC in the forest reached 90.48% when the formulation was sprayed by agricultural unmanned aerial vehicles. The half-life of fluopyram SC was 346.6 days, with a persistence of 3 years. These results indicate that spraying fluopyram SC could effectively reduce the number of pine trees dying from pine wilt disease.

Accurately segmenting an insect from its original ecological image is the core technology restricting the accuracy and efficiency of automatic recognition. However, the performance of existing segmentation methods is unsatisfactory in insect images shot in wild backgrounds on account of challenges: various sizes, similar colors or textures to the surroundings, transparent body parts and vague outlines. These challenges of image segmentation are accentuated when dealing with camouflaged insects. Here, we developed an insect image segmentation method based on deep learning termed the progressive refinement network (PRNet), especially for camouflaged insects. Unlike existing insect segmentation methods, PRNet captures the possible scale and location of insects by extracting the contextual information of the image, and fuses comprehensive features to suppress distractors, thereby clearly segmenting insect outlines. Experimental results based on 1900 camouflaged insect images demonstrated that PRNet could effectively segment the camouflaged insects and achieved superior detection performance, with a mean absolute error of 3.2%, pixel-matching degree of 89.7%, structural similarity of 83.6%, and precision and recall error of 72%, which achieved improvements of 8.1%, 25.9%, 19.5%, and 35.8%, respectively, when compared to the recent salient object detection methods. As a foundational technology for insect detection, PRNet provides new opportunities for understanding insect camouflage, and also has the potential to lead to a step progress in the accuracy of the intelligent identification of general insects, and even being an ultimate insect detector.

To identify microorganisms that can colonise pine trees and have a toxic effect on Monochamus alternatus (Coleoptera: Cerambycidae), we isolated the CSHH2 bacterial strain from the phloem of nemato...

The rapid spread of pine wilt disease has become a major crisis with regard to forest ecological security. Against the background of China’s concerted effort to achieve carbon neutrality by 2060, balancing economic and environmental costs during the environmentally sound treatment of nematode-infected pine wood is an issue worthy of attention. In this study, we constructed an evolutionary game model of the central government, local governments, and infected wood management-related enterprises and analyzed the evolutionary process of the three parties with stable strategies based on a full consideration of actual circumstances. To verify the theoretical results, we conducted numerical simulations using MATLAB. The results of the study are as follows. 1) The central government plays a decisive role in how infected wood is handled. The greater the degree of regulation is, the greater the local government favors low carbon emissions and the more companies will choose the pulverization method. 2) Local governments bear the main responsibility in the processing of infected wood, and only when local governments are sufficiently penalized will enterprises choose to pulverize the wood and dispose of it. 3) Policy subsidies are an effective measure that will help the three parties in the model reach a balance more quickly. Overall, this study clarified the realization path of low-carbon treatment of nematode-infected pine wood from a policy perspective. The study results should help promote the low-carbon treatment of nematode-infected pine wood and provide a basis for the formulation of relevant forestry policies in the context of carbon neutrality.

Culm rhomboid rot is a new disease of Moso bamboo (Phyllostachys edulis) and mainly occurs on Moso bamboo grown at an altitude above 800 m. The typical symptoms start with black spots and irregular shapes, which expand vertically into an elongated, fusiform, or rhomboid shape up to 15 cm in length. Eleven fungal isolates were isolated from infected tissue, and the isolate BBB1 was confirmed as the pathogen following Koch’s postulates in vitro and in vivo. Regarding morphology, BBB1 was identified as Arthrinium sp.–conidia single-celled, brown to dark, smooth, globose to subglobose, with an equatorial slit, 5.0–9.3 × 3.1–7.3 μm in size. The conidiophores were reduced to conidiogenous cells, pale brown to dark, smooth, and ampulliform. Phylogenetic analysis based on ITS-TEF1-α-TUB2 concatenate sequences identified BBB1 as A. arundinis. Furthermore, the sensitivity of the pathogen against six common fungicides was investigated with mycelial growth assays. Prochloraz exhibited the most potent inhibition with an EC50 value of 0.019 mg/L; another effective fungicide was difenoconazole, with an EC50 value of 0.195 mg/L. This is the first report of A. arundinis causing culm rhomboid rot of Moso bamboo in China; the results provide a basis for managing Moso bamboo culm rhomboid rot.

In this study, we sequenced the complete mitochondrial genome of

Diapause allows insects to survive harsh environments, and its termination is crucial for their normal development after diapause. However, little is known about the regulatory pathways and signals involved in insect diapause termination.We discovered that high temperature (25 °C) influenced larval diapause termination in Monochamus alternatus. Likewise, metal ions (CaThe results indicate that genes CYP314a1, CYP18a1, FOHSDR-1 and JHEH mediated 20E and JH biosynthesis and degradation to regulate diapause termination in M. alternatus. We elucidated the molecular mechanism underlying the regulation of diapause termination and provided a basis for the prevention and control of M. alternatus infestation. © 2022 Society of Chemical Industry.

Background Pine wilt disease (PWD) is a destructive disease that endangers pine trees, resulting in the wilting, with yellowing and browning of the needles, and eventually the death of the trees. Previous studies showed that the Avr9/Cf-9 rapidly elicited (PmACRE1) gene was downregulated by Bursaphelenchus xylophilus infection, suggesting a correlation between PmACRE1 expression and pine tolerance. Here, we used the expression of PmACRE1 in Arabidopsis thaliana to evaluate the role of PmACRE1 in the regulation of host defence against B. xylophilus infection. Results Our results showed that the transformation of PmACRE1 into A. thaliana enhanced plant resistance to the pine wood nematode (PWN); that is, the leaves of the transgenic line remained healthy for a longer period than those of the blank vector group. Ascorbate peroxidase (APX) activity and total phenolic acid and total flavonoid contents were higher in the transgenic line than in the control line. Widely targeted metabolomics analysis of the global secondary metabolites in the transgenic line and the vector control line showed that the contents of 30 compounds were significantly different between these two lines; specifically, the levels of crotaline, neohesperidin, nobiletin, vestitol, and 11 other compounds were significantly increased in the transgenic line. The studies also showed that the ACRE1 protein interacted with serine hydroxymethyltransferase, catalase domain-containing protein, myrosinase, dihydrolipoyl dehydrogenase, ketol-acid reductoisomerase, geranylgeranyl diphosphate reductase, S-adenosylmethionine synthase, glutamine synthetase, and others to comprehensively regulate plant resistance. Conclusions Taken together, these results indicate that PmACRE1 has a potential role in the regulation of plant defence against PWNs.

Insects have evolved to form a variety of complex natural compounds to prevent pathogen infection in the process of a long-term attack and defense game with various pathogens in nature. Antimicrobial Peptides (AMPs) are important effector molecules of the insect immune response to the pathogen invasion involved in bacteria, fungi, viruses and nematodes. The discovery and creation of new nematicides from these natural compounds is a key path to pest control. A total of 11 AMPs from Monochamus alternatus were classified into 3 categories, including Attacin, Cecropin and Defensin. Four AMP genes were successfully expressed by Komagataella phaffii KM71. The bioassay results showed that the exogenous expressed AMPs represented antimicrobial activity against Serratia (G−), Bacillus thuringiensis (G+) and Beauveria bassiana and high nematicide activity against Bursaphelenchus xylophilus. All four purified AMPs’ protein against B. xylophilus reached LC50 at 3 h (LC50 = 0.19 mg·mL−1 of MaltAtt-1, LC50 = 0.20 mg·mL−1 of MaltAtt-2 and MaltCec-2, LC50 = 0.25 mg·mL−1 of MaltDef-1). Furthermore, the AMPs could cause significant reduction of the thrashing frequency and egg hatching rate, and the deformation or fracture of the body wall of B. xylophilus. Therefore, this study is a foundation for further study of insect biological control and provides a theoretical basis for the research and development of new insecticidal pesticides.

In order to improve the conversion efficiency of Spent JUNCAO SUBSTRATE (SJS) into reducing sugar and improve the utility value of SJS, a one-step method was studied in this study. Three treatment ...

Summary The bacterial communities in the wound tissues of Pinus massoniana were analysed by 16S rDNA amplicon sequencing. The results showed that the bacterial richness and diversity changed remarkably whether the wound was inoculated with pine wood nematode (PWN; Bursaphelenchus xylophilus) or not after 12 h. However, the predominant bacteria Stenotrophomonas, Burkholderiaceae, Pseudomonas, Serratia and Delftia, introduced by PWN in the wound tissues, changed within 6 h. After 6 h of PWN inoculation, the most abundant genus associating with PWN, Stenotrophomonas, failed to colonise the wound tissues, and the abundance of Delftia decreased, while the other representative bacteria, Burkholderiaceae, Pseudomonas and Serratia, from the PWN were markedly enriched. In addition, our study is the first to report the association of Serratia liquefaciens with PWN. Predicted functional analyses using the Tax4Fun tool showed that the alterations in bacterial composition also led to shifts in their functional pathways, especially after 12 h of PWN inoculation. These findings clarified that the bacteria carried by PWN were responsible for the alterations in bacterial communities in the wound tissues and will shed light on the invasion mechanism of PWN.

Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

The vectors of pinewood nematode of Bursaphelenchus xylophilus (Steiner &amp, Bührer, 1934) are mainly known as xylophagous beetles. Understanding the composition and distribution of these xylophagous beetles in host pine trees infected by PWN is critical to control the spread of PWN. In this study, we investigated the community structures of the xylophagous beetles in two main host trees in Fujian and Shandong, Pinus massoniana Lamb. and Pinus thunbergia Parl., in different stages of infection. All beetles were collected by dissecting the whole pine trees and then identified by their morphological characteristics and COI genes. The results showed that the diversity of xylophagous beetles was different not only between the two host pine trees but also among the different infection stages. The diversity of P. massoniana xylophagous beetles was significantly higher than that of P. thunbergii, and there were also significant differences in the different stages of PWN infection. In total, Scolytinae was the most common (53.70%), followed by Curculionidae (18.26%), Cerambycidae (16.31%), and Cleridae (6.04%). Monochamus alternatus, the most important vector of PWN, occupied a large niche and showed different aggregation positions during the three infection stages in both host trees. This result might be related to the resistance of bark beetles to host trees and competition with other xylophagous beetles. The community diversity of xylophagous beetles was jointly affected by both the infection stages of PWN and the spatial niche of xylophagous beetles. Knowledge of the diversity and competitive relationships among xylophagous beetles might help regulate the population dynamics of these beetles.

Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an important trunk borer of pine trees and a major vector of pine wilt disease. Although chemicals are widely used in forest pest control, new strategies based on insect biology are offering promising approaches to manage the disease. Although there have been important research advances in this respect, there has not yet been a deep sequence analysis of M. alternatus describing the transcriptome, and no information is available about the gene function of this insect vector. We used next generation sequencing technology to provide a full transcriptome from the four larval instars of M. alternatus and successfully built an M. alternatus transcriptome database. In total, 67,456 unigenes were obtained with trinity software, information for 11,858 classified unigenes was obtained with the Clusters of Orthologous Groups (COGs) database, and 13,007 unigenes matched predicted pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, genes related to lignocellulose, and putative Bt receptors and genes related to digestion are described. Additionally, the differential gene expression of these genes in different larval stages was analyzed. This study provides valuable information to underpin the development of new molecular tools for M. alternatus control strategies.

The gut microbiota of insects has a wide range of effects on host nutrition, physiology, and behavior. The structure of gut microbiota may also be shaped by their environment, causing them to adjust to their hosts; thus, the objective of this study was to examine variations in the morphological traits and gut microbiota of Lymantria xylina in response to natural and artificial diets using high-throughput sequencing. Regarding morphology, the head widths for larvae fed on a sterilized artificial diet were smaller than for larvae fed on a non-sterilized host-plant diet in the early instars. The gut microbiota diversity of L. xylina fed on different diets varied significantly, but did not change during different development periods. This seemed to indicate that vertical inheritance occurred in L. xylina mutualistic symbionts. Acinetobacter and Enterococcus were dominant in/on eggs. In the first instar larvae, Acinetobacter accounted for 33.52% of the sterilized artificial diet treatment, while Enterococcus (67.88%) was the predominant bacteria for the non-sterilized host-plant diet treatment. Gut microbe structures were adapted to both diets through vertical inheritance and self-regulation. This study clarified the impacts of microbial symbiosis on L. xylina and might provide new possibilities for improving the control of these bacteria.

Pine wilt disease (PWD) is a devastating disease affecting the growth of Pinus massoniana, often leading to withering and death. To reveal the changes involved during disease progression, we investigated the mRNA expression profile of P. massoniana infested by Bursaphelenchus xylophilus. The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene, CC-NBS-LRR resistance-like protein, and the gene encoding a putative nematode resistance protein. Increased infestation pressure (number of nematodes inoculated) caused a continuous decline in the gene expression of stem samples. An infestation of P. massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis, which in turn reduced the levels of total phenols and total flavonoids. A downregulation of auxin responsive family protein was observed in infested samples, which resulted in a suppression of plant growth. Thus, upon B. xylophilus infestation, a downregulation of genes associated with the recognition of pathogens, PWD resistance, and growth regulation was observed in P. massoniana, together with a decrease in the levels of phytoalexin-like secondary substances, all of which resulted in withering and ultimately death of P. massoniana.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver) is an important pest of palms that causes significant damage by boring into and feeding within palm stem tissues. Here, we studied the proteolytic process of Cry3Aa in the RPW to understand the mechanism of Cry toxicity. The bioassays showed that Cry3Aa toxin is weakly toxic to the RPW. Proteolytic activation assays indicated the Cry3Aa protein is digested into smaller fragments than the 55-kDa activated fragments under different conditions. In particular, at higher mass ratios of gut protease and Cry3Aa protein (5:1, 2:1, and 1:1, respectively), and at 36.9°C for 16 h in a solution of pH 8.6, the Cry3Aa protoxin is over-digested by the gut proteases of weevil larvae. Moreover, the zymogram analysis of the gut proteases revealed the RPW larvae harbors intestinal digestive enzymes mainly composed of serine proteases. This study describes the proteolytic activation process of Cry3Aa in the midgut of RPW larvae.

Epicauta ruficeps is widely distributed in China and some countries in Southeast Asia, and plays an important role in medicine and biological control. The complete mitochondria genome of E. ruficeps was 15,813 bp in length, with 37 genes, including 13 PCGs, 22 tRNA genes (tRNAs), and two rRNA genes (rRNAs). The positions and sequences of genes were consistent with those of known Meloidae species. The nucleotide composition was highly A + T biased, accounting for ∼65% of the whole mitogenome. The complete mitogenome of E. ruficeps would help understand Meloidae evolution.

Pine wilt disease (PWD), Bursaphelenchus xylophilus, is an extremely threatening invasion forest disease throughout the world, especially in Asia. B. xylophilus is spread in Asia by vector beetles of Monochamus alternatus, which has long no effective control method. Understanding of landscape effects on the dispersal and outbreaks of forest pests is crucial to establishing effective ecological control strategies. Here, we analyzed the samples of M. alternatus collected at landscapes in order to estimate the effects of landscape types on the genetic structure and dispersal of M. alternatus. The landscapes included the geographical scales, forest types and land uses. The individuals of M. alternatus were genotyped by using whole-genome resequencing. Population genetic structures were clearly differentiated at the intermediate scale, suggesting the intermediate scale is an effective barrier against natural dispersal of M. alternatus. We used the least-coat distances, least-cost transect analysis, and distance-based redundancy analysis to estimate the effects of forest types and land uses within the fine scales. The results showed that the gene flow and genetic diversity were positively correlated with host and mixed forests, whereas negatively with non-host forests. Among land-use landscapes, the roads had the positive effect on gene flow and genetic diversity but farmland and urban uses had negative effects. This highlights that human-mediated transport via roads was likely to be the main factor leading to the long-distance invasion of M. alternatus, whereas non-host landscapes could suppress the spread of this species. These findings may be useful to control the PWD dispersed by M. alternatus.

Background Monochamus alternatus Hope is one of the insect vectors of pinewood nematode (Bursaphelenchus xylophilus), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche. Methods Total DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units. Results Pinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium, Burkholderia, Dyella, Mycobacterium, and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus, but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%). Conclusions Overall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus.

Background Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. Results Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. Conclusion This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry.

Background Monochamus alternatus Hope is one of the insect vectors of pinewood nematode ( Bursaphelenchus xylophilus ), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche. Methods Total DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units. Results Pinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium , Burkholderia , Dyella , Mycobacterium , and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus , but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%). Conclusions Overall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus .

Dendrolimus houi is one of the most common caterpillars infesting Gymnosperm trees, and widely distributed in several countries in Southeast Asia, and exists soley or coexists with several congeners and some Lasiocampidae species in various forest habitats. However, natural hybrids occasionally occur among some closely related species in the same habitat, and host preference, extreme climate stress, and geographic isolation probably lead to their uncertain taxonomic consensus. The mitochondrial DNA (mtDNA) of D. houi was extracted and sequenced by using high-throughput technology, and the mitogenome composition and characteristics were compared and analyzed of these species, then the phylogenetic relationship was constructed using the maximum likelihood method (ML) and the Bayesian method (BI) based on their 13 protein-coding genes (PCGs) dataset, which were combined and made available to download which were combined and made available to download among global Lasiocampidae species data. Mitogenome of D. houi was 15,373 bp in length, with 37 genes, including 13 PCGs, 22 tRNA genes (tRNAs) and 2 rRNA genes (rRNAs). The positions and sequences of genes were consistent with those of most known Lasiocampidae species. The nucleotide composition was highly A+T biased, accounting for ~80% of the whole mitogenome. All start codons of PCGs belonged to typical start codons ATN except for COI which used CGA, and most stop codons ended with standard TAA or TAG, while COI, COII, ND4 ended with incomplete T. Only tRNASer (AGN) lacked DHU arm, while the remainder formed a typical “clover-shaped” secondary structure. For Lasiocampidae species, their complete mitochondrial genomes ranged from 15,281 to 15,570 bp in length, and all first genes started from trnM in the same direction. And base composition was biased toward A and T. Finally, both two methods (ML and BI) separately revealed that the same phylogenetic relationship of D. spp. as ((((D. punctatus + D. tabulaeformis) + D. spectabilis) + D. superans) + (D. kikuchii of Hunan population + D. houi) as in previous research, but results were different in that D. kikuchii from a Yunnan population was included, indicating that different geographical populations of insects have differentiated. And the phylogenetic relationship among Lasiocampidae species was ((((Dendrolimus) + Kunugia) + Euthrix) + Trabala). This provides a better theoretical basis for Lasiocampidae evolution and classification for future research directions.

Additional file 1: Figure S1. Rarefaction curves analysis of samples from Monochamus alternatus and its habitat niche. Figure S2. Profiling barplot of bacterial phyla from Monochamus alternatus and its habitat niche. Figure S3. The tax tree of bacterial genera from infected Pinus massoniana. Figure S4. The tax tree of bacterial genera from Monochamus alternatus. Figure S5. The tax tree of bacterial genera in the process of instar II larvae of Monochamus alternatus feeding on the phloem. Figure S6. The tax tree of bacterial genera in the process of instar III larvae of Monochamus alternatus feeding on the xylem. Figure S7. The tax tree of bacterial genera in the process of Monochamus alternatus adults feeding on the bark. Figure S8. The heatmap of Spearman’s rank correlation coefficients of bacterial genera.

The purpose of this study was to characterize the endogenous cellulase gene MaCel1 of Monochamus alternatus, which is an important vector of Bursaphelenchus xylophilus, a pine wood nematode, which causes pine wilt disease (PWD). In this study, MaCel1 was cloned by rapid amplification of cDNA end (RACE), and its expression analyzed by RT-qPCR (real-time quantitative PCR detecting). A total of 1778 bp of cDNA was obtained. The encoding region of this gene was 1509 bp in length, encoding a protein containing 502 amino acids with a molecular weight of 58.66 kDa, and the isoelectric point of 5.46. Sequence similarity analysis showed that the amino acids sequence of MaCel1 had high similarity with the &beta, Glucosinolate of Anoplophoraglabripennis and slightly lower similarity with other insect cellulase genes (GH1). The &beta, D-Glucosidase activity of MaCel1 was 256.02 &plusmn, 43.14 U/L with no &beta, Glucosinolate activity. MaCel1 gene was widely expressed in the intestine of M. alternatus. The expression level of MaCel1 gene in male (3.46) and female (3.51) adults was significantly higher than that in other developmental stages, and the lowest was in pupal stage (0.15). The results will help reveal the digestive mechanism of M. alternatus and lay the foundation for controlling PWD by controlling M. alternatus.

The gut microbiota of insects has a wide range of effects on host nutrition, physiology, and behavior. The structure of gut microbiota may also be shaped by their environment, causing them to adjust to their hosts, thus, the objective of this study was to examine variations in the morphological traits and gut microbiota of Lymantria xylina in response to natural and artificial diets using high-throughput sequencing. Regarding morphology, the head widths for larvae fed on a sterilized artificial diet were smaller than for larvae fed on a non-sterilized host-plant diet in the early instars. The gut microbiota diversity of L. xylina fed on different diets varied significantly, but did not change during different development periods. This seemed to indicate that vertical inheritance occurred in L. xylina mutualistic symbionts. Acinetobacter and Enterococcus were dominant in/on eggs. In the first instar larvae, Acinetobacter accounted for 33.52% of the sterilized artificial diet treatment, while Enterococcus (67.88%) was the predominant bacteria for the non-sterilized host-plant diet treatment. Gut microbe structures were adapted to both diets through vertical inheritance and self-regulation. This study clarified the impacts of microbial symbiosis on L. xylina and might provide new possibilities for improving the control of these bacteria.

The complete chloroplast genome of Cryptomeria japonica var. sinensis Miquel was assembled and analyzed. The chloroplast genome of C. japonica var. sinensis Miquel did not have a typical quadripartite structure with the inverted repeats (IR) absent, and the size of C. japonica var. sinensis is 131,412 bp. The overall GC content was 35.4%. The genome encoded a set of 119 genes, containing 83 protein-coding genes, 32 tRNA genes, and 4 rRNA genes. Phylogenomic analysis indicated that C. japonica var. sinensis is sister to C. japonica (Thunb. ex L. f.) D. Don.

In this study, the first complete mitochondrial genome of Chrysolina aeruginosa Fald was assembled and analyzed. The total length of this mitochondrial genome is 16,335 base pairs. It consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and an AT-rich region. Phylogenomic analysis indicated that C. aeruginosa Fald is sister to Chrysodinopsis sp. This study provides new molecular data for the further taxonomic and phylogenetic studies of the Chrysomelidae of Coleoptera.

Epicauta ruficeps is widely distributed in China and some countries in Southeast Asia, and plays an important role in medicine and biological control. The complete mitochondria genome of E. ruficep...

In this study, we sequenced the first complete mitochondrial genome of Cryptalaus larvatus. The complete mitochondrial genome of C. larvatus was 15,876 bp with 29.80% GC containing 13 protein-codin...

Pine wilt disease seriously endangers pine tree growth and can lead to wilting and death. In this study, Masson pine (Pinus massoniana) was used as the donor plant to study miRNA expression by high-throughput sequencing in needle leaves of trees during the first 3 days after infection by Bursaphelenchus xylophilus. The results showed that the number of differentially expressed miRNAs first increased and then decreased in samples within 3 days of infection, with the highest expression in samples collected on day 2. Analysis of miRNA target genes in needles on different days after infection revealed enrichment in two pathways: plant hormone signal transduction and RNA transport. Further comparison of the samples indicated differential expression of 10 miRNAs on different days after infection, and the corresponding target genes, such as those participating in plant hormone signal transduction, were also significantly enriched. In addition, the concentrations of zeatin and indole acetic acid in needle tissues were significantly lower after infection than in the control sample. The above results indicated that B. xylophilus infection influenced the expression of miRNAs related to plant hormone signal transduction in P. massoniana, decreasing plant hormone synthesis and ultimately affecting the growth of Masson pine.

Background Leaf blight caused by Calonectria spp. is one of the most destructive diseases to affect Eucalyptus nurseries and plantations. These pathogens mainly attack Eucalyptus, a tree with a diversity of secondary metabolites employed as defense-related phytoalexins. To unravel the fungal adaptive mechanisms to various phytoalexins, we examined the genome of C. pseudoreteaudii, which is one of the most aggressive pathogens in southeast Asia. Results A 63.7 Mb genome with 14,355 coding genes of C. pseudoreteaudii were assembled. Genomic comparisons identified 1785 species-specific gene families in C. pseudoreteaudii. Most of them were not annotated and those annotated genes were enriched in peptidase activity, pathogenesis, oxidoreductase activity, etc. RNA-seq showed that 4425 genes were differentially expressed on the eucalyptus(the resistant cultivar E. grandis×E.camaldulensis M1) tissue induced medium. The annotation of GO term and KEGG pathway indicated that some of the differential expression genes were involved in detoxification and transportation, such as genes encoding ABC transporters, degrading enzymes of aromatic compounds and so on. Conclusions Potential genomic determinants of phytoalexin detoxification were identified in C. pseudoreteaudii by comparison with 13 other fungi. This pathogen seems to employ membrane transporters and degradation enzymes to detoxify Eucalyptus phytoalexins. Remarkably, the Calonectria genome possesses a surprising number of secondary metabolism backbone enzyme genes involving toxin biosynthesis. It is also especially suited for cutin and lignin degradation. This indicates that toxin and cell wall degrading enzymes may act important roles in the establishment of Calonectria leaf blight. This study provides further understanding on the mechanism of pathogenesis in Calonectria. Electronic supplementary material The online version of this article (10.1186/s12864-018-4739-1) contains supplementary material, which is available to authorized users.

We investigated the influence of larval instar on the structure of the gut bacterial community in the Japanese pine sawyer, Monochamus alternatus (Hope; Coleoptera: Cerambycidae). The diversity of the gut bacterial community in early, phloem-feeding larvae is significantly higher than in later, wood-feeding larvae. Many of these associates were assigned into a few taxonomic groups, of which Enterobacteriaceae was the most abundant order. The predominant bacterial genus varied during the five instars of larval development. Erwinia was the most abundant genus in the first and fifth instars, Enterobacter was predominant in the third and fourth instars, and the predominant genus in the second instars was in the Enterobacteriaceae (genus unclassified). Actinobacteria were reported in association with M. alternatus for the first time in this study. Cellulomonadaceae (Actinobacteria) was the second most abundant family in the first instar larvae (10.6%). These data contribute to our understanding of the relationships among gut bacteria and M. alternatus, and could aid the development of new pest control strategies.

Differences in composition of on-year, off-year and new leaves in Phyllostachys pubescens (moso bamboo) forests will affect the resistance of Pantana phyllostachysae larvae and the parasitic ability of Beauveria bassiana. Pantana phyllostachysae fed on mixed leaves of on-year, off-year and new bamboo leaves. The activities of acetylcholine esterase (AChE) and glutathione S-transferase (GSTs) in P. phyllostachysae larvae fed on mixed leaves were significantly higher, greatly reducing the pathogenicity of B. bassiana to P. phyllostachysae larvae compared with the treatments of off-year and new bamboo leaves. The phenoloxidase (PO) activity in P. phyllostachysae larvae feeding on mixed leaves varied within three days after parasitization by B. bassiana, which indicated higher and less changeable PO activity compared with treatments of off-year and new bamboo leaves. Inclusion of new leaves differed greatly due to its growth, in which, both AChE and GSTs activities were higher than the off-year leaf treatment, leading to a lower pathogenicity of B. bassiana to P. phyllostachysae larvae feeding on new leaves compared with off-year leaves. On the contrary, the pathogenicity of B. bassiana to P. phyllostachysae larvae feeding on on-year leaves was lower than other treatments. AChE and GSTs activities in P. phyllostachysae larvae were lower and PO activity was less changeable, which might be related to the disadvantage of the larval surface structure to B. bassiana infection in the on-year leaf treatment.

The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Erebidae), is an important pest in the Australian pine tree, Casuarina equisetifolia, forest in the coastal area of South China. At the same time, as a closely related species of Lymantria dispar L. (Lepidoptera: Erebidae), it is also a potential quarantine pest. In the present study, specific primers were designed for identification of L. xylina based on the COI barcoding sequence between L. xylina and four other common forest pests. A 569-bp fragment was successfully amplified from 40 L. xylina from five geographical populations in four Chinese provinces. In addition, even through the analysis came from five highly diverse populations of L. xylina, the genetic distances ranged from 0.001 to 0.031. The neighbor-joining tree showed that the species from Hubei and Chongqing were clustered within a distinct group.

The pine aphid Cinara pinitabulaeformis Zhang et Zhang is the main pine pest in China, it causes pine needles to produce dense dew (honeydew) which can lead to sooty mold (black filamentous saprophytic ascomycetes). Although common chemical and physical strategies are used to prevent the disease caused by C. pinitabulaeformis Zhang et Zhang, new strategies based on biological and/or genetic approaches are promising to control and eradicate the disease. However, there is no information about genomics, proteomics or transcriptomics to allow the design of new control strategies for this pine aphid. We used next generation sequencing technology to sequence the transcriptome of C. pinitabulaeformis Zhang et Zhang and built a transcriptome database. We identified 80,259 unigenes assigned for Gene Ontology (GO) terms and information for a total of 11,609 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs). A total of 10,806 annotated unigenes were analyzed to identify the represented biological pathways, among them 8,845 unigenes matched with 228 KEGG pathways. In addition, our data describe propagative viruses, nutrition-related genes, detoxification related molecules, olfactory related receptors, stressed-related protein, putative insecticide resistance genes and possible insecticide targets. Moreover, this study provides valuable information about putative insecticide resistance related genes and for the design of new genetic/biological based strategies to manage and control C. pinitabulaeformis Zhang et Zhang populations.

The beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an efficient vector of pine wood nematode, the causal pathogen of pine wilt disease, that has resulted in devastating losses of pines in much of Asia. We assessed the response of adult M. alternatus to 2-(undecyloxy)-ethanol, the male-produced pheromone of the congeneric M. galloprovincialis Dejean, in field experiments in Fujian Province, People's Republic of China. Both sexes of M. alternatus were attracted to lures consisting of 2-(undecyloxy)-ethanol combined with the host plant volatiles alpha-pinene and ethanol. A follow-up experiment showed that 2-(undecyloxy)-ethanol was synergized by both ethanol and alpha-pinene. Coupled gas-chromatography mass-spectrometry analyses of volatiles sampled from field-collected beetles of both sexes revealed that 2-(undecyloxy)-ethanol was a sex-specific pheromone component produced only by males. The combination of 2- (undecyloxy) -ethanol with ethanol and/or alpha-pinene will provide a valuable and badly needed tool for quarantine detection, monitoring, and management of M. alternatus.

We found that Pantana phyllostachysae, a dangerous pest of moso bamboo (Phyllostachys pubescens), showed differences in growth and development after feeding on diverse types of moso bamboo leaves. The mortality rate of Pa. phyllostachysae due to Beauveria bassiana, an entomopathogenic fungus, was also affected by the varied larval diet. Larval and pupal developmental duration of Pa. phyllostachysae was longer when feeding on “off-year” bamboo leaves. Pupal weight and adult fertility were higher when feeding on “on-year” bamboo leaves. Mortality due to B. bassiana was significantly lower in larvae fed on on-year bamboo leaves than in larvae fed on off-year bamboo leaves. Larvae fed on new bamboo leaves had a shorter development period and higher survival rate than those fed on off-year bamboo leaves. However, mixed feed (mixture of new, on-year, and off-year bamboo leaves) decreased the egg production of Pa. phyllostachysae. After infection by the second generation of B. bassiana, the survival time of Pa. phyllostachysae fed on mixed feed increased significantly compared with the first generation. We also fed Pa. phyllostachysae different proportion of new bamboo leaves in mixed feed to simulate natural conditions. We found that increasing the proportion of new bamboo leaves in the food promoted pupal development and increased egg production; it also increased the resistance of larvae to the first generation of B. bassiana. The pathogenicity of the second generation of B. bassiana declined in all mixed feed treatments.

This study investigated the influence of starvation on the structure of the gut bacterial community in the Chinese white pine beetle (Dendroctonus armandi). A total of 14 operational taxonomic units (OTUs0.03) clusters belonging to nine genera were identified. Denaturing gradient gel electrophoresis (DGGE) profiles of bacterial PCR-amplified 16S rRNA gene fragments from the guts of starved male and female adults revealed that the bacterial community diversity increased after starvation. The dominant genus Citrobacter decreased significantly, whereas the genus Serratia increased in both starved female and starved male adults. The most predominant bacterial genus in D. armandi adults was Citrobacter, except for starved male adults, in which Serratia was the most abundant genus (27%). Our findings reveal that starvation affects gut bacterial dynamics in D. armandi, as has been observed in other insect species.

Monochamus alternatus Hope is the main vector in China of the Pine Wilt Disease caused by the pine wood nematode Bursaphelenchus xylophilus. Although chemical control is traditionally used to prevent pine wilt disease, new strategies based in biological control are promising ways for the management of the disease. However, there is no deep sequence analysis of Monochamus alternatus Hope that describes the transcriptome and no information is available about gene function of this insect vector. We used next generation sequencing technology to sequence the whole fourth instar larva transcriptome of Monochamus alternatus Hope and successfully built a Monochamus alternatus Hope transcriptome database. In total, 105,612 unigenes were assigned for Gene Ontology (GO) terms, information for 16,730 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs) database, and 13,024 unigenes matched with 224 predicted pathways in the Kyoto Encyclopedia of Genes and Genome (KEGG). In addition, genes related to putative insecticide resistance-related genes, RNAi, the Bt receptor, intestinal digestive enzymes, possible future insect control targets and immune-related molecules are described. This study provides valuable basic information that can be used as a gateway to develop new molecular tools for Monochamus alternatus Hope control strategies.