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MaltAtt-1 is an antimicrobial peptide isolated from Monochamus alternatus with nematocidal activity against pine wood nematode. In this study, a eukaryotic expression system based on Komagataella phaffii GS115 was established, and its secretory expression of MaltAtt-1 was realized. The basic properties and secondary and tertiary structures of the antimicrobial peptide MaltAtt-1 were identified by bioinformatics analysis. MaltAtt-1 is a hydrophilic stable protein, mainly composed of an α-helix (Hh), β-folds (Ee), and irregular curls (Cc). The optimal fermentation conditions for MaltAtt-1 were determined by a single-factor test and the Box–Behnken response surface method, including an induction time of 72 h, induction temperature of 30 °C, culture medium of pH 7.6, methanol volume fraction of 2.0%, and an initial glycerol concentration of 1%. The stability of MaltAtt-1 indicated its resistant to UV irradiation and repeated freezing and thawing, but the antibacterial activity decreased significantly under the influence of high temperature and a strong acid and base, and it decreased significantly to 1.1 cm and 0.83 cm at pH 2.0 and pH 10.0, respectively. The corrected mortality of B. xylophilus achieved 71.94% in 3 h at a concentration of 300 mg·L−1 MaltAtt-1 exposure. The results provide a theoretical basis for the antimicrobial peptide MaltAtt-1 to become a new green and efficient nematicide.

Pine wood nematode disease is currently the most deadly forest disease in China, and the Monochamus alternatus is its primary vector. Controlling the M. alternatus is crucial for managing pine wood nematode disease. This study, based on the selected HasA (pGHKW4) secretory expression vector, used electroporation to combine the genetically modified high-toxicity toxin Cry3Aa-T with the entomopathogenic bacterium Yersinia entomophaga isolated from the gut of the M. alternatus. The SDS-PAGE and Western blotting techniques were employed to confirm the toxin protein’s secretion capability. The engineered bacteria’s genetic stability and effectiveness in controlling M. alternatus were assessed for their insecticidal activity. The results of the SDS-PAGE and Western blotting analyses indicate that the HasA system effectively expresses toxin protein secretion, demonstrates certain genetic stability, and exhibits high insecticidal activity against M. alternatus. This study constructed a highly toxic entomopathogenic engineered bacterial strain against M. alternatus larvae, which holds significant implications for controlling M. alternatus, laying the foundation for subsequent research and application of this strain.

Lymantria xylina Swinhoe (Lepidoptera: Erebidae) is a potentially invasive pest, similar to Lymantria dispar asiatica Vnukovskij and Lymantria dispar japonica Motschulsky (Lepidoptera: Erebidae). To evaluate its potential for spread and flight distance related to egg deposition on vessels at ports, we employed a flight mill to assess the flight capabilities of its adults under varying conditions. Our findings revealed that females primarily flew short distances and ceased flying after 3:00 AM, whereas males covered much longer distances throughout the day. Sex, age, and flight duration significantly influenced flight ability. Females exhibited weaker flight capability than males, and their ability declined with increasing age or flight duration. Notably, 1-day-old moths displayed the strongest flight ability, with average flight distances of up to 3.975 km for females and 8.441 km for males. By the fifth day, females no longer flew, and males experienced reduced flight ability. After continuous hanging for 16 h, females lost most of their flight capacity, while males remained capable of flight even after 32 h. Additionally, female flight ability decreased significantly after mating, possibly due to factors such as egg-carrying capacity, weight, and load ratio. This study provides a foundation for assessing the risk of long-distance dispersal of L. xylina via ocean-going freighters, considering female moths’ phototactic flight and oviposition.

Kriechbaumerella dendrolimi (Hymenoptera, Chalcididae) is a dominant pupal parasitoid species of various significant pine caterpillars, including Dendrolimus houi Lajonquiere (Lepidoptera, Lasiocampidae), with great potential for utilization. So far, the mass rearing of K. dendrolimi has been successfully established using Antheraea pernyi (Lepidoptera, Saturniidae) pupae as alternative hosts and released in the forest to suppress D. houi populations. However, the outcome is still expected to be improved due to lower parasitism rates, which might be related to the autonomous immune function of A. pernyi pupae. In our study, we investigated the effects of K. dendrolimi parasitization on the immune responses of A. pernyi pupae by measuring the expression of key immune factors: superoxide dismutase (SOD), polyphenol oxidases (PPOs), Attacin, Lysozymes (LYSs), and serine proteases (PRSSs). Our results show that parasitization significantly upregulated these immune factors, with distinct temporal patterns observable between 4 and 48 h post-parasitization. This upregulation highlights a robust immune response, adapting over time to the parasitic challenge. These findings suggest that specific immune mechanisms in A. pernyi pupae are activated in response to K. dendrolimi, shedding light on potential targets for enhancing host resistance. Metabolomic analyses complemented these findings by illustrating the broader metabolic shifts associated with the immune response. Specifically, Attacin was significantly upregulated twice, hypothesizing that the parasitoid’s venom contains at least two parasitic factors. Metabolomics analysis revealed a significant metabolite difference within parasitized A. pernyi pupae. The highest number of differential expression metabolites (DEMs) was observed at 16 h post-parasitism (1184 metabolites), with fewer DEMs at 8 h (568 metabolites) and 32 h (693 metabolites), suggesting a close relationship between parasitism duration and the number of DEMs. These fluctuations reflected the fundamental process of immune interaction. KEGG enrichment results showed that the DEMs were mainly enriched in energy metabolism and immune-related pathways, indicating that parasitism is a process of continuous consumption and immune interaction in the host. These DEMs could also become future targets for regulating the immune functions of A. pernyi pupae and could provide reference data for optimizing mass-rearing techniques.

The Japanese pine sawyer Monochamus alternatus serves as the primary vector for pine wilt disease, a devastating pine disease that poses a significant threat to the sustainable development of forestry in the Eurasian region. Currently, trap devices based on informational compounds have played a crucial role in monitoring and controlling the M. alternatus population. However, the specific proteins within M. alternatus involved in recognizing the aforementioned informational compounds remain largely unclear. To elucidate the spatiotemporal distribution of M. alternatus chemosensory-related genes, this study conducted neural transcriptome analyses to investigate gene expression patterns in different body parts during the feeding and mating stages of both male and female beetles. The results revealed that 15 genes in the gustatory receptor (GR) gene family exhibited high expression in the mouthparts, most genes in the odorant binding protein (OBP) gene family exhibited high expression across all body parts, 22 genes in the odorant receptor (OR) gene family exhibited high expression in the antennae, a significant number of genes in the chemosensory protein (CSP) and sensory neuron membrane protein (SNMP) gene families exhibited high expression in both the mouthparts and antennae, and 30 genes in the ionotropic receptors (IR) gene family were expressed in the antennae. Through co-expression analyses, it was observed that 34 genes in the IR gene family were co-expressed across the four developmental stages. The Antenna IR subfamily and IR8a/Ir25a subfamily exhibited relatively high expression levels in the antennae, while the Kainate subfamily, NMDA subfamily, and Divergent subfamily exhibited predominantly high expression in the facial region. MalIR33 is expressed only during the feeding stage of M. alternatus, the MalIR37 gene exhibits specific expression in male beetles, the MalIR34 gene exhibits specific expression during the feeding stage in male beetles, the MalIR8 and MalIR39 genes exhibit specific expression during the feeding stage in female beetles, and MalIR8 is expressed only during two developmental stages in male beetles and during the mating stage in female beetles. The IR gene family exhibits gene-specific expression in different spatiotemporal contexts, laying the foundation for the subsequent selection of functional genes and facilitating the full utilization of host plant volatiles and insect sex pheromones, thereby enabling the development of more efficient attractants.

Abstract 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.

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.

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease.

The Japanese fir, Cryptomeria japonica, is ecologically and commercially vital in China. However, infestations by Dendrolimus houi Lajonquière larvae cause widespread dieback, mimicking mechanical damage effects, with unclear differential impacts and mechanisms. To address this, 6th instar larvae of D. houi were introduced to three-year-old C. japonica seedlings to induce pest infestation, while mechanical damage and non-infested pests were controlled. Then, next-generation sequencing techniques were employed, and transcriptome sequencing was conducted to analyze the distinct damage mechanisms responding to C. japonica. This study revealed 10,412 DEGs between pest infestation and controls and 5535 DEGs comparing pest infestation to mechanical damage. Functional enrichment analysis highlighted the involvement of these DEGs in crucial processes such as photosynthesis, amino acid and nucleotide metabolism, flavonoid biosynthesis, and plant hormone signaling pathways. In particular, 786 unique DEGs were discerned in pest-infested samples. Key enriched pathways, such as pyruvate and propionate metabolism, were pinpointed, with flavonoid biosynthesis potentially closely linked to pest-feeding inductions. These findings provided valuable insights into the molecular responses of C. japonica to D. houi infestation, laying the foundation for future research aimed at developing pest-resistant varieties of Japanese fir.

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.

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.

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.

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.

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.

In this study, we sequenced the complete mitochondrial genome of Agriotes fuscicollis Miwa, 1928 (Coleoptera: Elateridae). The results showed that the length of complete mitochondrial genome was 15,866 bp with 26.8% GC content, containing 39.6% A, 33.5% T, 16.8% C, 10% G. There were 13 protein-coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes. Phylogenetic analysis showed that A. fuscicollis was closely related to Cryptalaus larvatus, Cryptalaus yamato, Pyrophorus divergens and Ignelater luminosus. The complete mitogenome of A. fuscicollis would contribute to the study of the phylogeny and evolution of Elateridae.

Abstract 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.

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.

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.

Abstract 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.

The vectors of pinewood nematode of Bursaphelenchus xylophilus (Steiner & 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.

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.

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.

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.

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-coding genes, 22 transfer RNA (tRNA), two ribosomal RNA (rRNA), as well as and an AT-rich region. Phylogenetic analysis showed that the C. larvatus, Pyrophprus divergens, Ignelater luminosus, Hapsodrilus ignifer, and Dicronychus cinereus were clustered together. This study provides important information for the identification of this species and the study of genetic evolution with other species of Elateridae.

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 β-Glucosinolate of Anoplophoraglabripennis and slightly lower similarity with other insect cellulase genes (GH1). The β-D-Glucosidase activity of MaCel1 was 256.02 ± 43.14 U/L with no β-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 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.

Eucalyptus leaf blight caused by Calonectria spp. is a serious disease in Eucalyptus seedling and plantations. However, the molecular mechanisms of the infection process and pathogenesis of Calonectria to Eucalyptus is not well-studied. In this study, we analyzed the transcriptomes of C. pseudoreteaudii at three stages of Eucalyptus leaf infection, and in mycelium grown in potato dextrose broth using Illumina RNA-Seq technology. We identified 161 differentially expressed genes between C. pseudoreteaudii from leaf and mycelium grown in potato dextrose broth. GO and KEGG enrichment analyses of these genes suggested that they were mainly involved in oxidoreductase activity, hydrolase activity, and transmembrane transporter activity. Most of the differentially expressed genes at the early infection stage were upregulated. These upregulated genes were mainly involved in cell wall hydrolysis and toxin synthesis, suggesting a role for toxin and cell wall hydrolases in the establishment of Calonectria leaf blight. Genes related to detoxification of phytoalexins were continually upregulated during infection. The candidate effectors and putative pathogenicity determinants identified in this study will help in the functional analysis of C. pseudoreteaudii virulence and pathogenicity.

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.

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.