CncC-Keap1-P450s pathway is involved in the detoxification of emamectin benzoate in the spongy moth Lymantria dispar.

The detoxification of insecticides in insects is dependent on the expression and activity of multiple detoxification enzymes. As an important modulator of detoxification enzymes, the CncC-Keap1 pathway was involved in the detoxification of various pesticides. However, whether the CncC-Keap1 pathway is involved in the detoxification of emamectin benzoate (EMB) is unclear. In this study, we cloned the LdCncC and LdKeap1 from spongy moths (Lymantria dispar). Our results showed that EMB exposure induced oxidative stress, and activated the CncC-Keap1 pathway at mRNA and protein levels. Removing ROS by N-acetylcysteine remarkably decreased H2O2 levels and restored the expression of LdCncC and LdKeap1. The silencing LdCncC, not LdKeap1, by dsRNA significantly decreased the cytochrome P450 activities, and increased the sensitivity of larvae to EMB. Besides, the expression of CYP6B7v1, CYP321A7 and CYP4S4v1 were significantly decreased after silencing LdCncC. Notably, the knockdown of CYP6B7v1, CYP321A7 or CYP4S4v1 significantly increased the mortality induced by EMB exposure. Therefore, we proposed that activation of CncC-Keap1 pathway induced by ROS increased the detoxification of EMB in spongy moths by regulating the expression of CYP6B7v1, CYP321A7 and CYP4S4v1. Our study strengthened the understanding of the detoxification of EMB from the perspective of CncC-Keap1-P450s pathway.

Gut bacterial microbiota of Lymantria dispar asiatica and its involvement in Beauveria bassiana infection.

The gut bacterial microbiota of insects has been shown to play essential roles in processes related to physiology, metabolism, and innate immunity. In this study, we firstly performed a broad analysis of the gut bacteria in Lymantria dispar asiatica, one of the most devastating forestry defoliators. We analyzed the bacterial composition among different individuals from lab-reared or wild-collected using 16 s rRNA-sequencing, revealing that the gut bacteria of wild-collected larvae were highly diverse, while lab-reared larvae were only associated with a few genera. We found Lactobacillus sp. present in all the gut samples, which indicates that it is part of the core microbiome in the caterpillar. Further Beauveria bassiana infection-based assays showed that the mortality of non-axenic L. dispar asiatica larvae was significantly higher than that of axenic larvae at 72 h. Moreover, we isolated several bacteria from the hemolymph of the non-axenic larvae infected by B. bassiana, which may be caused by the translocation of gut bacteria from the gut to the hemocoel. Reintroduction of Enterococcus sp., Pseudomonas sp., Enterobacter sp., and Microbacterium sp. into axenic larvae recurred the larval mortality in their non-axenic counterpart. Taken together, our study demonstrates that the gut bacteria of L. dispar asiatica are highly volatile, and different bacteria taxa can promote host infection by entomopathogenic fungus, providing a new strategy for the pest management.

Mutation of glucan synthase catalytic subunit in Beauveria bassiana affects fungal growth and virulence.

Beauveria bassiana is a well-known entomopathogenic fungus that parasitizes on a variety of insect species. Glucan in the cell wall of B. bassiana plays a crucial role in its structure and growth and is also involved in the activation of the host insect's immune system. Glucan biosynthesis is primarily regulated by glucan synthase, however, it is unclear if the glucan synthase catalytic subunit gene (GluS) affects the growth and virulence of B. bassiana. In this study, we constructed the mutant of the B. bassiana glucan synthase catalytic subunit (ΔGluS) by homologous recombination and observed that glucan synthase knockout affects both spore germination and cell area. Further enzyme-based assays along with gene expression analysis of glucan synthase revealed a significant downregulation in the mutant strains compared to the wild type of B. bassiana. Moreover, the virulence of ΔGluS strains against gypsy moth (Lymantria dispar) showed no significant difference compared to the wild-type strains when injected, while the spraying gypsy moths with the conidia of ΔGluS was significantly more lethal than spraying the conidia of the wild type. Altogether, our study constructed a new, highly efficient B. bassiana mutant that can be used for pest control and provides a readily transferable method for other insect-entomopathogenic interaction studies.

Comparative analysis of the immune system and expression profiling of Lymantria dispar infected by Beauveria bassiana.

Lymantria dispar is one of the most devastating forest pests worldwide. Fungal biopesticides have great potential as alternatives owing to their high lethality to pests and eco-friendly feature, which is, however, often severely compromised by the pests' innate immunity. A better understanding of the antifungal immune system in L. dispar would significantly facilitate the development of the biopesticide. Here, we investigated phylogenetic characteristics of immunity-related genes as well as the tissue expression patterns in L. dispar after the infection of an entomopathogen Beauveria bassiana using RNA-sequencing data. Results showed most immune genes remain at a low level of response after 24 h post-infection (HPI). Almost all genes in the Toll pathway were significantly up-regulated at 48 HPI, and SPH1, SPN6, Toll6, Toll12, Myd88, pelle, and Drosal were significantly down-regulated at 72 HPI. Immunoblotting analysis revealed that the protein levels of ßGRP3 and PPO1 were significantly upregulated at 24 and 48 HPI, while Myd88 was downregulated at 24 HPI, which was further confirmed by Quantitative real-time PCR experiments. Moreover, the relative content of H2O2, a potent reactive oxygen species (ROS), was significantly increased with the decrease of the total antioxidant capacity, indicating that oxidative stress system positively participates in the clearance of the pathogenic fungus. Together, our study provides detailed genetic characteristics of antifungal immunity as well as profiling of the host defense against entomopathogenic infection, and comprehensive insight into molecular interaction between L. dispar and the entomopathogen.

Endosymbiont Tremblaya phenacola influences the reproduction of cotton mealybugs by regulating the mechanistic target of rapamycin pathway.

The intricate evolutionary dynamics of endosymbiotic relationships result in unique characteristics among the genomes of symbionts, which profoundly influence host insect phenotypes. Here, we investigated an endosymbiotic system in Phenacoccus solenopsis, a notorious pest of the subfamily Phenacoccinae. The endosymbiont, "Candidatus Tremblaya phenacola" (T. phenacola PSOL), persisted throughout the complete life cycle of female hosts and was more active during oviposition, whereas there was a significant decline in abundance after pupation in males. Genome sequencing yielded an endosymbiont genome of 221.1 kb in size, comprising seven contigs and originating from a chimeric arrangement between betaproteobacteria and gammaproteobacteria. A comprehensive analysis of amino acid metabolic pathways demonstrated complementarity between the host and endosymbiont metabolism. Elimination of T. phenacola PSOL through antibiotic treatment significantly decreased P. solenopsis fecundity. Weighted gene coexpression network analysis demonstrated a correlation between genes associated with essential amino acid synthesis and those associated with host meiosis and oocyte maturation. Moreover, altering endosymbiont abundance activated the host mechanistic target of rapamycin pathway, suggesting that changes in the amino acid abundance affected the host reproductive capabilities via this signal pathway. Taken together, these findings demonstrate a mechanism by which the endosymbiont T. phenacola PSOL contributed to high fecundity in P. solenopsis and provide new insights into nutritional compensation and coevolution of the endosymbiotic system.

Chromosome-level genome assembly of the Asian spongy moths Lymantria dispar asiatica.

The Asian spongy moth, Lymantria dispar asiatica, is one of the most devastating forestry defoliators. The absence of a high-quality genome limited the understanding of its adaptive evolution. Here, we conducted the first chromosome-level genome assembly of L. dispar asiatica using PacBio HIFI long reads, Hi-C sequencing reads and transcriptomic data. The total assembly size is 997.59 Mb, containing 32 chromosomes with a GC content of 38.91% and a scaffold N50 length of 35.42 Mb. The BUSCO assessment indicated a completeness estimate of 99.4% for this assembly. A total of 19,532 protein-coding genes was predicted. Our study provides a valuable genomics resource for studying the mechanisms of adaptive evolution and facilitate an efficient control of L. dispar asiatica.

Transcriptome analysis of emamectin benzoate caused midgut damage by inducing oxidative stress, energy metabolism disorder and apoptosis in gypsy moth (Lymantria dispar).

BACKGROUND: Emamectin benzoate (EMB) is a semisynthetic bioinsecticide, which has been widely used in the control of forestry and agricultural pests. However, the mechanism of its toxic effects on the non-neural tissues has been rarely reported. Here, we explored the mechanism of the midgut damage induced by EMB in gypsy moth (Lymantria dispar) in order to better understand the toxicological mechanism of EMB. RESULTS: Our results confirmed that EMB caused damage to the midgut of gypsy moth by inducing apoptosis. Transcriptome showed that 1469, 650 and 950 genes were significantly differentially expressed in the midgut of gypsy moth after 24, 48 and 72 h of EMB exposure, and oxidative stress, energy metabolism disorder and apoptosis may be related to the toxic effects of EMB. The indicators related to oxidative stress, energy metabolism and apoptosis were further examined. The results showed that EMB could cause oxidative stress by increasing ROS level and inhibiting antioxidant enzymes (P < 0.05), such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), which in turn causes mitochondria injury. Subsequently, energy metabolism was inhibited by downregulating the activities and mRNA level of energy metabolism enzymes. Furthermore, the mitochondrial apoptosis pathway was activated, triggering apoptosis, and eventually causing midgut injury in gypsy moth. CONCLUSION: Our results indicated that EMB caused damage to midgut by inducing oxidative stress, energy metabolism disorder and apoptosis in gypsy moth. Our findings shed new light on the toxicological mechanism of EMB on non-neural tissues from oxidative stress, energy metabolism and apoptosis perspectives. © 2022 Society of Chemical Industry.

Transcriptome Analysis of Antennal Chemosensory Genes in Curculio Dieckmanni Faust. (Coleoptera: Curculionidae).

The olfactory system plays a key role in regulating insect behaviors, such as locating host plants, spawning sites, and mating partners and avoiding predators. Chemosensory genes are required for olfactory recognition in insects. Curculio dieckmanni Faust. (Coleoptera: Curculionidae) damages hazelnuts and causes severe economic losses. There are no effective control measures, but understanding the olfaction mechanisms of this insect could lead to a new approach for population management. However, the genes that perform chemosensory functions in C. dieckmanni are still unclear. Using high-throughput sequencing, we assembled the antennal transcriptome of C. dieckmanni and annotated the major chemosensory gene families. Of the chemosensory gene families, we found 23 odorant-binding proteins, 15 chemosensory proteins, 2 sensory neuron membrane proteins, 15 odorant receptors, 23 ionotropic receptors, and nine gustatory receptors. Using Blast sequence alignment and phylogenetic analysis, the sequences of these proteins were identified. Male- and female-specific chemosensory genes involved in odorant detection and recognition were validated by qRT-PCR. Among the chemosensory genes, we found significant differences in the expression of CdieOBP8, CdieOBP9, CdieOBP19, CdieOBP20, CdieOBP21, CdieCSP15, CdieOR13, and CdieOR15 between adult male and female C. dieckmanni. A total of 87 expressed chemosensory proteins were found in C. dieckmanni. Investigating these proteins will help reveal the molecular mechanism of odorant recognition in C. dieckmanni and may aid the development of novel control strategies for this species.

Sublethal concentration of emamectin benzoate inhibits the growth of gypsy moth by inducing digestive dysfunction and nutrient metabolism disorder.

BACKGROUND: Gypsy moth (Lymantria dispar) is one of the most important pests in the world. Emamectin benzoate (EMB) is widely used in the control of agricultural and forestry pests. Here, we explored the sublethal effects of EMB on gypsy moths in order to better understand the toxicological mechanism of EMB. RESULTS: The sublethal concentration of EMB exposure significantly decreased the larvae body weight. To further explore the mechanism, indicators related to digestion and nutrient metabolism were detected. The results showed that EMB exposure caused midgut damage, reduced the activities of digestive enzymes and changed the content of sugar and amino acids. Moreover, the expression of insulin/phosphoinositide-3-kinase (PI3K)/forkhead box protein O (FoxO) pathway and sugar metabolism-related genes was abnormal. The expression of insulin receptor (InR), chico, PI3K, and protein kinase B (Akt) significantly reduced, and that of phosphatase and tensin homologue (PTEN) and FoxO increased. The expression of glycogen phosphorylase (GP) was upregulation and that of glycogen synthase (GS), trehalase (TRE) and trehalose-phosphate synthase (TPS) were downregulation. All results indicated that EMB inhibits the growth of gypsy moth by inducing midgut injury, digestive dysfunction and nutrient metabolism disorder. In addition, EMB caused midgut injury may be related to apoptosis or a collateral effect of the damage in other tissues, and more extensive and deeper research is still needed to investigate the detailed mechanism. CONCLUSION: Our finding strengthens the understanding of the sublethal effect of EMB, and provides a theoretical basis for the application of EMB in the prevention and control of gypsy moth.

Genome-Wide Analysis of Major Facilitator Superfamily and Its Expression in Response of Poplar to Fusarium oxysporum.

The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.

Temporospatial modulation of Lymantria dispar immune system against an entomopathogenic fungal infection.

BACKGROUND: Lymantria dispar is an economically impactful forest pest worldwide. The entomopathogenic fungi Beauveria bassiana shows great promise in pest management due to its high lethality in Lymantria dispar. A complete understanding of the immune interactions between the pest and the pathogenic fungus is essential to actualizing biological pest management. RESULTS: Following the infection of Lymantria dispar by Beauveria bassiana spores, we performed a time-course analysis of transcriptome in Lymantria dispar fat bodies and hemocytes to explore host immune response. A total of 244 immunity-related genes including pattern recognition receptors, extracellular signal modulators, immune pathways (Toll, IMD, JNK and JAK/STAT), and response effectors were identified. We observed contrasting tissue and time-specific differences in the expression of immune genes. At the early stage of infection, several recognition receptors and effector genes were activated, while the signal modulation and effector genes were suppressed at later stages. Further enzyme activity-based assays coupled with gene expression analysis of prophenoloxidase revealed a significant upregulation of phenoloxidase activity at 48- and 72-h post-infection. Moreover, fungal infection led to dysbiosis in gut microbiota that seems to be partially attributed to reduced gut hydrogen peroxide (H2 O2 ) amount, which indicates a significant impact of fungal infection on host gut microbes. CONCLUSION: Our study provides a comprehensive sequence resource and crucial new insights about an economically important forest pest. Specifically, we elucidate the complicated multipartite interaction between host and fungal pathogen and contribute to a better understanding of Lymantria dispar anti-fungal immunity, resulting in better tools for biological pest control. © 2020 Society of Chemical Industry.