The Neurotranscriptome of Monochamus alternatus.

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.

Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

Double-strand RNAs targeting MaltRelish and MaltSpz reveals potential targets for pest management of Monochamus alternatus.

Overcoming the innate immunity of insects is a key process to improve the efficiency of biological control. Antimicrobial peptides (AMPs) are important effectors in insect innate immunity, usually mediating resistance to pathogenic microorganisms through Toll and IMD signaling pathways. This study investigated the effect of key genes on upstream immune recognition receptor (GNBP3) and downstream effectors (AMPs) by RNAi technology. The transcriptome KEGG enrichment analysis and differential gene annotation results showed that the immune response genes MaltSpz and MaltRelish are important regulators of Toll and IMD signaling pathways, respectively. Both dsSpz and dsRelish could affect AMP gene expression and increase the expression of the immune recognition receptor MaltGNBP3. Moreover, they significantly reduce the survival rate of Monochamus alternatus and promote hyphal growth after Beauveria bassiana infection. This helps to improve the biological control effect of B. bassiana, control the population of vector insects and cut off the transmission route of pine wood nematode. The combined MaltSpz and MaltRelish knockdown increased the infection rate of M. alternatus larvae from 20.69% to 83.93%, achieving the best efficiency in synergistic B. bassiana infection. Our results showed important roles of MaltRelish- and MaltSpz-mediated regulation of AMP genes function in insect entomopathogenic fungi tolerance and induced significant mortality in larvae. Based on this study, MaltSpz and MaltRelish could represent candidate gene targets for the biological control of M. alternatus by RNAi.

Antibacterial peptides from Monochamus alternatus induced oxidative stress and reproductive defects in pine wood nematode through the ERK/MAPK signaling pathway.

Pine wilt disease is a devastating disease of pine caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Long-term use of chemical nematicides leads to the development of resistance in nematodes and harms the environment. Evaluations for green environmental protection agents, identified the antibacterial peptide, MaltDef1, from Monochamus alternatus which had nematicidal effect. We studied its nematicidal activity and action against PWN. In this study, the antibacterial peptide S-defensin was synthesized from M. alternatus. The results showed that S-defensin caused mortality to the PWN, causing shrinkage, pore, cell membrane dissolution and muscle atrophy. In addition, PWN reproduction was also affected by S-defensin; it decreased in a concentration dependent manner with increasing treatment concentration. By contrast, reactive oxygen species (ROS) in vivo increased in a concentration-dependent manner. We applied transcriptome to analyze the changes in gene expressions in S-defensin treated PWN, and found that the most significantly enriched pathway was the ERK/MAPK signaling pathway. RNAi was used to validate the functions of four differential genes (Let-23, Let-60, Mek-2 and Lin-1) in this pathway. The results showed that knockdown of these genes significantly decreased the survival rate and reproductive yield of, and also increased ROS in PWN. The antibacterial peptide S-defensin had a significant inhibitory effect on the survival and reproduction of PWN, shown by cell membrane damage and intracellular biological oxidative stress via regulating the ERK/MAPK signaling pathway. This indicates that S-defensin has a target in B. xylophilus, against which new green target pesticides can be developed.

Heterologous Expression and Bioactivity Determination of Monochamus alternatus Antibacterial Peptide Gene in Komagataella phaffii (Pichia pastoris).

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.

Discovery and Characterization of MaK: A Novel Knottin Antimicrobial Peptide from Monochamus alternatus.

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.

Bacillus thuringiensis toxins with nematocidal activity against the pinewood nematode Bursaphelenchus xylophilus.

The pine wilt disease is caused by the pinewood nematode Bursaphelenchus xylophilus and it results in serious ecological and economic losses. Therefore, effective prevention and control methods for the pinewood nematode are urgently required. Bacillus thuringiensis (Bt), a widely used microbial insecticide, produces toxins that are toxic to several species of parasitic nematodes, however, its effects on B. xylophilus have not been determined. In this study, Cry5Ba3, App6Aa2, Cry12Aa1, Cry13Aa1, Cry14Aa1, Cry21Aa3, Cry21Fa1, Xpp55Aa1, and Cyt8Aa1 toxins' nematocidal activity against B. xylophilus was evaluated, six toxins with high toxicity were identified: App6Aa2 (LC50 = 49.71 µg/mL), Cry13Aa1 (LC50 = 53.17 µg/mL), Cry12Aa1 (LC50 = 58.88 µg/mL), Cry5Ba3 (LC50 = 63.99 µg/mL), Xpp55Aa1 (LC50 = 65.14 µg/mL), and Cyt8Aa1 (LC50 = 96.50 µg/mL). The six toxins caused shrinkage and thinning of the intestinal cells, contraction of the intestine from the body wall, vacuolization, and degenerated appearance of the pinewood nematodes. The results of this study provide basic information to study the action mechanism of nematocidal toxins on the pinewood nematode and direction for the use of nematocidal toxins in the biological control of B. xylophilus.

Characterization of bacterial communities associated with the pinewood nematode insect vector Monochamus alternatus Hope and the host tree Pinus massoniana.

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.

Characterization of Bacterial Communities Associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) and its Host Phoenix sylvestris.

This study measured the changes of microorganisms in the midgut and habitat niche of Rhynchophorus ferrugineus Olivier, an invasive quarantine pest, by Illumina sequencing. The bacterial diversity in the R. ferrugineus larvae midgut and their habitat niche was compared to the uninfected P. sylvestris. The Proteobacteria and Firmicutes occupied a dominant position in the R. ferrugineus midgut and infected P. sylvestris, while in the uninfected P. sylvestris the predominant bacterial phylum was the Cyanobacteria. Enterobacter, Dysgonomonas, and Entomoplasma were the dominant bacterial genera in R. ferrugineus midgut and also within the infected trees and uninfected trees with low relative abundance. These bacteria could be exploited as the biopesticide vector to control R. ferrugineus population. Besides, Sphingobacterium, Shinella, and Rhodobacter genera had the same distribution pattern in the infected and uninfected P. sylvestris, and these bacteria were not found in the midgut of R. ferrugineus. Interestingly, Paludibacter and Parabacteroides were only distributed in the wood fiber of the infected P. sylvestris, which could be used as potential microbial markers to detect if the palm plants are damaged by the R. ferrugineus. The results of this study will be beneficial to the development of control strategies for R. ferrugineus.

Function of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity.

Galectins are a family of ß-galactoside binding proteins, and insect galectins play a role in immune responses and may also affect Cry toxin activity. In this study, we aimed to further understand the function and molecular mechanism of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity. A. aegypti galectin-6 was cloned, and the recombinant galectin-6 was expressed and purified. Bioassays indicated that galectin-6 could reduce the toxicity of Cry11Aa, protecting A. aegypti larvae. To determine interactions among galectin-6, Cry11Aa and putative toxin receptors, Octet Red System, western blotting, far-western blotting and ELISA assays were performed. Octet Red System showed that galectin-6 bound to BBMVs of A. aegypti larvae with lower affinity than that of Cry11Aa. Western blotting and far-western blotting analyses demonstrated that galectin-6 bound to A. aegypti ALP1 and APN2 as well as to BBMVs, consistent with the results of ELISA and protein docking simulations. However, galectin-6 did not bind to Cadherin in far-western blotting or ELISA assay, though the protein docking simulations suggested their binding potential. These findings support the conclusion that galectin-6 may block Cry11Aa from binding to ALP1 and APN2 due to structural similarity, which might decrease the mosquitocidal toxicity of Cry11Aa.

Overexpression of PtrMYB121 Positively Regulates the Formation of Secondary Cell Wall in Arabidopsis thaliana.

MYB transcription factors have a wide range of functions in plant growth, hormone signaling, salt, and drought tolerances. In this study, two homologous transcription factors, PtrMYB55 and PtrMYB121, were isolated and their functions were elucidated. Tissue expression analysis revealed that PtrMYB55 and PtrMYB121 had a similar expression pattern, which had the highest expression in stems. Their expression continuously increased with the growth of poplar, and the expression of PtrMYB121 was significantly upregulated in the process. The full length of PtrMYB121 was 1395 bp, and encoded protein contained 464 amino acids including conserved R2 and R3 MYB domains. We overexpressed PtrMYB121 in Arabidopsis thaliana, and the transgenic lines had the wider xylem as compared with wild-type Arabidopsis. The contents of cellulose and lignin were obviously higher than those in wild-type materials, but there was no significant change in hemicellulose. Quantitative real-time PCR demonstrated that the key enzyme genes regulating the synthesis of lignin and cellulose were significantly upregulated in the transgenic lines. Furthermore, the effector-reporter experiment confirmed that PtrMYB121 bound directly to the promoters of genes relating to the synthesis of lignin and cellulose. These results suggest that PtrMYB121 may positively regulate the formation of secondary cell wall by promoting the synthesis of lignin and cellulose.

Identification of transcripts involved in digestion, detoxification and immune response from transcriptome of Empoasca vitis (Hemiptera: Cicadellidae) nymphs.

Tea production has been significantly impacted by the false-eye leafhopper, Empoasca vitis (Göthe), around Asia. To identify the key genes which are responsible for nutrition absorption, xenobiotic metabolism and immune response, the transcriptome of either alimentary tracts or bodies minus alimentary tract of E. vitis was sequenced and analyzed. Over 31 million reads were obtained from Illumina sequencing. De novo sequence assembly resulted in 52,182 unigenes with a mean size of 848nt. The assembled unigenes were then annotated using various databases. Transcripts of at least 566 digestion-, 224 detoxification-, and 288 immune-related putative genes in E. vitis were identified. In addition, relative expression of highly abundant transcripts was verified through quantitative real-time PCR. Results from this investigation provide genomic information about E. vitis, which will be helpful in further study of E. vitis biology and in the development of novel strategies to control this devastating pest.

Molecular Detection and Genetic Diversity of Casuarina Moth, Lymantria xylina (Lepidoptera: Erebidae).

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.

Isolation and characterization of a novel native Bacillus thuringiensis strain BRC-HZM2 capable of degrading chlorpyrifos.

Studies were carried out to isolate chlorpyrifos degrading Bacillus thuringiensis (Bt) strains from chlorpyrifos-contaminated samples. Six Bt strains (isolation rate 2.7%) were isolated by modified sodium acetate antibiotic heat treatment, and one novel strain (BRC-HZM2) was selected for further analysis. Phenotype and phylogeny analysis of this strain was conducted on the basis of biochemical reactions, antibiotic sensitivity, 16s rRNA genes, plasmid profile, insecticidal crystal protein profiles, and PCR-RFLP for cry and cyt genes. The degradation rate of chlorpyrifos in liquid culture was estimated during 48 h of incubation for the isolate BRC-HZM2. More than 50% of the initial chlorpyrifos concentration degraded within 12 h, 88.9% after 48 h. These results highlight the potential of the Bt strain for biological control and the bioremediation of environments contaminated with chlorpyrifos.

Use of spent mushroom substrate for production of Bacillus thuringiensis by solid-state fermentation.

The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As a locally available agroindustrial byproduct, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32 degrees C, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3, and inoculum volume 0.5 ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1,487 international toxic units/milligram) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides. The cost of production of 1 kg of Bt was approximately US$0.075.

Calcium stress reduces the reproductive capacity and pathogenicity of the pine wood nematode (Bursaphelenchus xylophilus) by inhibiting oxidative phosphorylation reaction.

The continuous use of chemical pesticides to control nematodes could result in the developing of pesticide-resistant nematodes. Novel nucleic acid pesticides are becoming the focus of pesticide research due to their strong specificity, high efficiency, and environmental friendliness. However, the limited known biochemical targets restrict the development of target pesticides for nematodes. The calcium stress experiments on pine wood nematodes (PWN) showed that 100 mmol/L Ca2+ resulted in longitudinal depression on the PWN body wall, reduced oviposition, and increased corrected mortality. To enrich the biological targets of nematode pesticides, we further investigated the response mechanism of PWN to calcium stress at the molecular level. Differentially expressed gene analysis showed that genes involved in the oxidative phosphorylation (OXPHOS) pathway were significantly enriched. RNA interference results of 6 key genes belonging to four mitochondrial complex I (BXNDUFA2), III (BXQCR8), IV (BXCOX17), V (BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε) in non-stressed nematodes showed reduction in PWN oviposition, population size, feeding ability, and pathogenicity. The BXNDUFA2 gene interference had the highest inhibitory impact by decreasing the oviposition from 31.00 eggs to 6.75 eggs and PWN population size from 8.27 × 103 nematodes to 1.64 × 103 nematodes, respectively. Interestingly, RNA interference of these 6 key genes in calcium-stressed nematodes also led to increased mortality and decreased oviposition of PWN. In summary, calcium stress inhibited the reproductive capacity of PWN by down-regulating key genes BXNDUFA2, BXQCR8, BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε, and BXCOX17, thereby reducing the pathogenicity. The current results enrich the RNAi targets in PWN and provide a scientific basis for developing novel nucleic nematicides.

[Study on tolerability for safflower peony ointment in clinical trial phase I].

OBJECTIVE: To evaluate the tolerability for safflower peony ointment and determine its safe dosage, by selecting health volunteers and testing from the initial safety dosage, in order to provide basis for formulating administration scheme of the drug in clinical trial phase II. METHOD: Forty-six healthy volunteers were included in the open, random, dose escalation, self control clinical trial on tolerability for single dosage scheme or multi-dosage. In the single dosage scheme, dosages of test drugs were 2.16 g (four people), 4.32 g (6 people), 6.48 g (6 people), 8.62 g (6 people), 11.46 g (6 people), 15.24 g (including crude drug) for 24 hours, once everyday. In the multi-dosage scheme, dosages of test drugs were 8.62 g (6 people), 11.46 g (including crude drug) once everyday for 7 days. RESULT: The maximum safe dosage of single administration was 15.24 g (including crude drug) , while that of multiple administration 8.62 g (including crude drug). The occurrence rate of side effect was as low as 2.17%, which was recovered by medicines, without severe adverse event. CONCLUSION: The study proves the safe application of single administration and multiple administration of safflower peony ointment in human bodies, which lays a foundation for the application in the clinical trial phase II.

The complete mitochondrial genome of Batocera rubus Linnaeus, 1785 (Coleoptera: Cerambycidae).

Batocera rubus severely impacts on the health of banyan trees. In this study, the whole mitochondrial genome for B. rubus was found to be 16,158 bp with a GC content of 23.9%, including 39.1% A, 37.0% T, 14.8% C, and 9.1% G. This genome contains 13 protein-coding genes, 22 tRNAs, and two rRNAs. Phylogenetic analysis revealed that B. rubus is close to Batocera celebiana. This study provides valuable information that can help improve the classification and phylogeny of B. rubus and facilitate further evolutionary studies.

Spatiotemporal dynamics of fluopyram trunk-injection in Pinus massoniana and its efficacy against pine wilt disease.

BACKGROUND: Pine wilt disease (PWD) is a destructive disease of pine trees caused by the pinewood nematode, Bursaphelenchus xylophilus. Fluopyram, a novel nematicide compound with systemic activity, is a prospective trunk-injection agent against pinewood nematodes. The disadvantage of current trunk-injection agents is that they were not evenly distributed in tree tissues and were poor in the persistence of effect and efficiency. Therefore, we investigated the spatiotemporal transport pattern and residue behavior of fluopyram following its injection into the trunk of Pinus massoniana. RESULTS: Fluopyram transport in the trunk occurred through radial diffusion and vertical uptake within 1 week of the injection, reaching all tissues of P. massoniana, including apical branches and needles. Three years after the field test, the infection of PWD declined substantially with treatment using the fluopyram trunk-injection agent, which demonstrated 100% efficacy in both the mild and moderate occurrence areas, and 71.1% efficacy in the severe occurrence area. Fluopyram as trunk-injection agent exerted substantial control over PWD, with its efficacy being influenced by the infection time of PWD. The half-life of 10% fluopyram in treated pine trees was 346.6 days with 3-year persistence. CONCLUSION: The advantages of overall distribution and long persistence of fluopyram in the tree after injection help explain its evident efficacy against PWN. Overall, fluopyram trunk-injection has potential to prevent PWD. © 2023 Society of Chemical Industry.

Temperature and metal ions regulate larval diapause termination via the 20-hydroxyecdysone and juvenile hormone pathways in Monochamus alternatus.

BACKGROUND: 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. RESULTS: We discovered that high temperature (25 °C) influenced larval diapause termination in Monochamus alternatus. Likewise, metal ions (Ca2+ ) promoted diapause termination by reducing diapause duration. We combined transcriptomic and metabolomic analyses to investigate changes in gene expression and metabolism in diapause-terminated larvae treated with high temperature (MaHt) and metal ions (MaCa). Hormone biosynthesis and metabolism contained the highest proportion of significant differentially expressed genes (DEGs) in the two groups. 20-hydroxyecdysone (20E) and juvenile hormone (JH) were closely related to diapause termination in M. alternatus. RNA interference (RNAi) experiments verified that 20E biosynthesis (CYP314a1) and degradation (CYP18a1), JH biosynthesis (FOHSDR-1) and degradation (JHEH) genes affected the larval diapause duration significantly. In addition, dysfunction of CYP314a1 resulted in increased larval mortality (P < 0.01), reduced pupation rate and emergence rate (P < 0.05). Enzyme-linked immunosorbent assay (ELISA) analysis showed that the ecdysone content decreased after dsCYP314a1 injection and JH content increased after dsJHEH injection. CONCLUSION: The 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.