High nitrogen application in maize enhances insecticide tolerance of the polyphagous herbivore Spodoptera litura by induction of detoxification enzymes and intensification of cuticle.

Nitrogen (N) is one of the most intensively used fertilizers in cropping system and could exert a variety of bottom-up effects on the ecological fitness of herbivores. However, the effects of increased N inputs on insect pesticide tolerance have not been comprehensively understood. Bioassays showed that high N (HN) applied to maize plants significantly increased larval tolerance of Spodoptera litura to multiple insecticides. Activities of detoxification enzymes were significantly higher in the larvae fed on maize plants supplied with HN. RNA-seq analysis showed that numerous GST and cuticle-related genes were induced in the larvae fed on HN maize. RT-qPCR analysis further confirmed four GST genes and larval-specific cuticle gene LCP167. Furthermore, when injected with dsRNA specific to GSTe1, GSTs5, and LCP167, the mortality of larvae treated with methomyl was about 3-fold higher than that of dsGFP-injected larvae. Electron microscope observation showed that cuticle of the larvae fed on HN maize was thicker than the medium level of N. These findings suggest that increased application of N fertilizer enhances insecticide tolerance of lepidopteran pests via induction of detoxification enzymes and intensification of cuticle. Thus, overuse of N fertilizer may increase pest insecticide tolerance and usage of chemical insecticides.

Comparative study of the gut microbial community structure of Spodoptera frugiperda and Spodoptera literal (Lepidoptera).

Background: Spodoptera frugiperda, the fall armyworm is a destructive invasive pest, and S. litura the tobacco cutworm, is a native species closely related to S. frugiperda. The gut microbiota plays a vital role in insect growth, development, metabolism and immune system. Research on the competition between invasive species and closely related native species has focused on differences in the adaptability of insects to the environment. Little is known about gut symbiotic microbe composition and its role in influencing competitive differences between these two insects. Methods: We used a culture-independent approach targeting the 16S rRNA gene of gut bacteria of 5th instar larvae of S. frugiperda and S. litura. Larvae were reared continuously on maize leaves for five generations. We analyzed the composition, abundance, diversity, and metabolic function of gut microbiomes of S. frugiperda and S. litura larvae. Results: Firmicutes, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in both species. Enterococcus, ZOR0006, Escherichia, Bacteroides, and Lactobacillus were the genera with the highest abundance in S. frugiperda. Enterococcus, Erysipelatoclostridium, ZOR0006, Enterobacter, and Bacteroides had the highest abundance in S. litura. According to α-diversity analysis, the gut bacterial diversity of S. frugiperda was significantly higher than that of S. litura. KEGG analysis showed 15 significant differences in metabolic pathways between S. frugiperda and S. litura gut bacteria, including transcription, cell growth and death, excretory system and circulatory system pathways. Conclusion: In the same habitat, the larvae of S. frugiperda and S. litura showed significant differences in gut bacterial diversity and community composition. Regarding the composition and function of gut bacteria, the invasive species S. frugiperda may have a competitive advantage over S. litura. This study provides a foundation for developing control strategies for S. frugiperda and S. litura.

Dual-crosslinking gelatin-hyaluronic acid methacrylate based biomimetic PDAC desmoplastic niche enhances tumor-associated macrophages recruitment and M2-like polarization.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by deposition of desmoplastic matrix (including collagen and hyaluronic acid). And the interactions between tumor-associated macrophages (TAMs) and tumor cells play a crucial role in progression of PDAC. Hence, the appropriate model of tumor cell-macrophage interaction within the unique PDAC TME is of significantly important. To this end, a 3D tumor niche based on dual-crosslinking gelatin methacrylate and hyaluronic acid methacrylate hydrogels was constructed to simulate the desmoplastic tumor matrix with matching compressive modulus and composition. The bionic 3D tumor niche creates an immunosuppressive microenvironment characterized by the downregulation of M1 markers and upregulation of M2 markers in TAMs. Mechanistically, RNA-seq analysis revealed that the PI3K-AKT signaling pathway might modulate the phenotypic balance and recruitment of macrophages through regulating SELE and VCAM-1. Furthermore, GO and GSEA revealed the biological process of leukocyte migration and the activation of cytokine-associated signaling were involved. Finally, the 3D tumor-macrophage niches with three different ratios were fabricated which displayed increased M2-like polarization and stemness. The utilization of the 3D tumor niche has the potential to provide a more accurate investigation of the interplay between PDAC tumor cells and macrophages within an in vivo setting.

CYP321A Subfamily P450s Contribute to the Detoxification of Phytochemicals and Pyrethroids in Spodoptera litura.

Although the induction of cytochrome P450 monooxygenases involved in insect detoxification has been well documented, the underlying regulatory mechanisms remain obscure. In Spodoptera litura, CYP321A subfamily members were effectively induced by exposure to flavone, xanthotoxin, curcumin, and λ-cyhalothrin, while knockdown of the CYP321A genes increased larval susceptibility to these xenobiotics. Homology modeling and molecular docking analyses showed that these four xenobiotics could stably bind to the CYP321A enzymes. Furthermore, two transcription factor genes, CncC and MafK, were significantly induced by the xenobiotics. Knockdown of CncC or MafK reduced the expression of four CYP321A genes and increased larval susceptibility to the xenobiotics. Dual-luciferase reporter assays showed that cotransfection of reporter plasmids carrying the CYP321A promoter with CncC and/or MafK-expressing constructs significantly magnified the promoter activity. These results indicate that the induction of CYP321A subfamily members conferring larval detoxification capability to xenobiotics is mediated by the activation of CncC and MafK.

Biomimetic Microenvironmental Stiffness Boosts Stemness of Pancreatic Ductal Adenocarcinoma via Augmented Autophagy.

Pancreatic ductal adenocarcinoma (PDAC) features high recurrence rates and intensified lethality, accompanied by stiffening of the extracellular matrix (ECM) microenvironment, which is mainly due to the deposition, remodeling, and cross-linking of collagen. Boosted stemness plays an essential role during occurrence and progression, which indicates a poor prognosis. Therefore, it is of great importance to understand the effect of the underlying interaction of matrix stiffness and stemness on PDAC. For this purpose, a methacrylated gelatin (GelMA) hydrogel with tunable stiffness was applied for incubating MIA PaCa-2 and PANC-1 cells. The results demonstrated that compared to the soft group (5% GelMA, w/v), the expression of stemness-related genes (SOX2, OCT4, and NANOG) in the stiff group (10% GelMA, w/v) displayed pronounced elevation as well as sphere formation. Intriguingly, we also observed that matrix stiffness regulated autophagy of PDAC, which played a momentous role in stemness promotion. In order to clarify the underlying relationship between matrix stiffness-mediated cell autophagy and stemness, rescue experiments with rapamycin and chloroquine were conducted with transmission electron microscopy, immunofluorescence staining, sphere formation, and qRT-PCR assays to evaluate the level of stemness and autophagy. For exploring the molecular mechanism in depth, RNA-seq and differential expression of miRNAs were carried out, which may sensor and respond to matrix stiffness during the regulation of stemness and autophagy. In conclusion, we validated that blocking autophagy repressed the stemness induced by matrix stiffness in PDAC and provided a potential therapeutic strategy for this aggressive cancer.

Effects of Septoglomus constrictum and Bacillus cereus on the competitive growth of Ageratina adenophora.

Beneficial microorganisms play a pivotal role in the invasion process of exotic plants, including arbuscular mycorrhizal fungi (AMF) and Bacillus. However, limited research exists on the synergistic influence of AMF and Bacillus on the competition between both invasive and native plants. In this study, pot cultures of Ageratina adenophora monoculture, Rabdosia amethystoides monoculture, and A. adenophora and R. amethystoides mixture were used to investigate the effects of dominant AMF (Septoglomus constrictum, SC) and Bacillus cereus (BC), and the co-inoculation of BC and SC on the competitive growth of A. adenophora. The results showed that inoculation with BC, SC, and BC + SC significantly increased the biomass of A. adenophora by 14.77, 112.07, and 197.74%, respectively, in the competitive growth between A. adenophora and R. amethystoides. Additionally, inoculation with BC increased the biomass of R. amethystoides by 185.07%, while inoculation with SC or BC + SC decreased R. amethystoides biomass by 37.31 and 59.70% compared to the uninoculated treatment. Inoculation with BC significantly increased the nutrient contents in the rhizosphere soil of both plants and promoted their growth. Inoculation with SC or SC + BC notably increased the nitrogen and phosphorus contents of A. adenophora, therefore enhancing its competitiveness. Compared with single inoculation, dual inoculation with SC and BC increased AMF colonization rate and Bacillus density, indicating that SC and BC can form a synergistic effect to further enhance the growth and competitiveness of A. adenophora. This study reveals the distinct role of S. constrictum and B. cereus during the invasion of A. adenophora, and provide new clues to the underlying mechanisms of interaction between invasive plant, AMF and Bacillus.

Effects of Elevated CO2 Concentration on Host Adaptability and Chlorantraniliprole Susceptibility in Spodoptera frugiperda.

Elevated atmospheric carbon dioxide concentrations (eCO2) can affect both herbivorous insects and their host plants. The fall armyworm (FAW), Spodoptera frugiperda, is a highly polyphagous agricultural pest that may attack more than 350 host plant species and has developed resistance to both conventional and novel-action insecticides. However, the effects of eCO2 on host adaptability and insecticide resistance of FAW are unclear. We hypothesized that eCO2 might affect insecticide resistance of FAW by affecting its host plants. To test this hypothesis, we investigated the effect of eCO2 on (1) FAW's susceptibility to chlorantraniliprole after feeding on wheat, (2) FAW's population performance traits (including the growth and reproduction), and (3) changes in gene expression in the FAW by transcriptome sequencing. The toxicity of chlorantraniliprole against the FAW under eCO2 (800 µL/L) stress showed that the LC50 values were 2.40, 2.06, and 1.46 times the values at the ambient CO2 concentration (400 µL/L, aCO2) for the three generations, respectively. Under eCO2, the life span of pupae and adults and the total number of generations were significantly shorter than the FAW under aCO2. Compared to the aCO2 treatment, the weights of the 3rd and 4th instar larvae and pupae of FAW under eCO2 were significantly heavier. Transcriptome sequencing results showed that more than 79 detoxification enzyme genes in FAW were upregulated under eCO2 treatment, including 40 P450, 5 CarE, 17 ABC, and 7 UGT genes. Our results showed that eCO2 increased the population performance of FAW on wheat and reduced its susceptibility to chlorantraniliprole by inducing the expression of detoxification enzyme genes. This study has important implications for assessing the damage of FAW in the future under the environment of increasing atmospheric CO2 concentration.

Effect of arbuscular mycorrhizal fungi on the responses of Ageratina adenophora to Aphis gossypii herbivory.

The invasive weed Ageratina adenophora can form a positive symbiotic relationship with native arbuscular mycorrhizal fungi (AMF) to promote its invasion ability. However, the function of AMF during the feeding of Aphis gossypii in A. adenophora was poorly understand. This study aimed to investigate the effects of two dominant AMF (Claroideoglomus etunicatum and Septoglomus constrictum) on A. adenophora in response to the feeding of the generalist herbivore A. gossypii. The results showed that A. gossypii infestation could significantly reduce the biomass, nutrient and proline contents of A. adenophora, and increase the antioxidant enzyme activities, defense hormone and secondary metabolite contents of the weed. Compared with the A. gossypii infested A. adenophora, inoculation C. etunicatum and S. constrictum could significantly promote the growth ability and enhanced the resistance of A. adenophora to A. gossypii infestation, and the aboveground biomass of A. adenophora increased by 317.21% and 114.73%, the root biomass increased by 347.33% and 120.58%, the polyphenol oxidase activity heightened by 57.85% and 12.62%, the jasmonic acid content raised by 13.49% and 4.92%, the flavonoid content increased by 27.29% and 11.92%, respectively. The survival rate of A. gossypii and density of nymphs were significantly inhibited by AMF inoculation, and the effect of C. etunicatum was significantly greater than that of S. constrictum. This study provides clarified evidence that AMF in the rhizosphere of A. adenophora are effective in the development of tolerance and chemical defense under the feeding pressure of insect herbivory, and offer references for the management of the A. adenophora from the perspective of soil microorganisms.

Rhizospheric Bacillus-Facilitated Effects on the Growth and Competitive Ability of the Invasive Plant Ageratina adenophora.

The rhizospheric microbial community affects the population establishment of invasive plants in introduced areas, among which Bacillus has numerous functions in promoting plant growth. This study isolated and enriched the Bacillus community in the rhizospheric soil of the invasive plant Ageratina adenophora and the native accompanying plant Rabdosia amethystoides. The effects of these rhizospheric Bacillus communities on the growth and competition of A. adenophora and R. amethystoides were evaluated in pot experiments. The results showed that the number and diversity of Bacillus in the rhizospheric soil of A. adenophora were higher than those of R. amethystoides (A. adenophora: 122 strains in soil, 16 Bacillus taxa; R. amethystoides: 88 strains in soil, 9 Bacillus taxa). After Bacillus inoculation of A. adenophora in a pot experiment, Bacillus idriensis, Bacillus toyonensis and Bacillus cereus were accumulated in the rhizospheric of A. adenophora, which significantly increased the nitrate nitrogen (NO3 --N) content in the soil and the total carbon and nitrogen concentrations in A. adenophora in the mixed treatment. The selective accumulation of Bacillus enhanced the competitive advantage of A. adenophora over the native accompanying plant; the corrected index of relative competition intensity of A. adenophora-inoculated Bacillus reached double that of the uninoculated treatment, and the growth of native plants was greatly suppressed under mixed planting. Our study confirmed that invasion of A. adenophora can lead to the accumulation of specific Bacillus taxa in the rhizospheric soil, which in turn can increase the competitive advantage of A. adenophora.

Astrocytes promote the proliferation of oligodendrocyte precursor cells through connexin 47-mediated LAMB2 secretion in exosomes.

BACKGROUND: Oligodendrocyte precursor cells (OPCs) can proliferate and differentiate into oligodendrocytes, the only myelin-forming cells in the central nervous system. Proliferating OPCs promotes remyelination in neurodegenerative diseases. Astrocytes (ASTs) are the most widespread cells in the brain and play a beneficial role in the proliferation of OPCs. Connexin 47 (Cx47) is the main component of AST-OPC gap junctions to regulate OPC proliferation. Nonetheless, the specific mechanism remains unclear. METHODS AND RESULTS: This study investigates the proliferation mechanism of OPCs connected to ASTs via Cx47. Cx47 siRNA significantly inhibited OPCs from entering the proliferation cycle. Transcriptome sequencing of OPCs and gene ontology enrichment analysis revealed that ASTs enhanced the exosome secretion by OPCs via Cx47. Transmission electron microscopy, Western blot, and nanoparticle tracking analysis indicated that the OPC proliferation was related to extracellular exosomes. Cx47 siRNA decreased the OPC proliferation and exosome secretion in AST-OPC cocultures. Exogenous exosome supplementation alleviated the inhibitory effect of Cx47 siRNA and significantly improved OPC proliferation. Mass spectrometry revealed that LAMB2 was abundant in exosomes. The administration of exogenous LAMB2 induced DNA replication in the S phase in OPCs by activating cyclin D1. CONCLUSIONS: Collectively, ASTs induce the secretion of exosomes that carry LAMB2 by OPCs via Cx47 to upregulate cyclin D1 thereby accelerating OPC proliferation.

Effects of Host Plants on Bacterial Community Structure in Larvae Midgut of Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is one of the most important invasive species and causes great damage to various host crops in China. In this study, the diversity and function of gut bacteria in the 5th instar larvae of FAW fed on maize, wheat, potato and tobacco leaves were analyzed through 16S rRNA sequencing. A total of 1324.25 ± 199.73, 1313.5 ± 74.87, 1873.00 ± 190.66 and 1435.25 ± 139.87 operational taxonomic units (OTUs) from the gut of FAW fed on these four different host plants were detected, respectively. Firmicutes, Proteobacteria and Bacteroidetes were the most abundant bacterial phyla. Beta diversity analysis showed that the gut bacterial community structure of larvae fed on different host plants was significantly differentiated. At the genus level, the abundance of Enterococcus in larvae fed on wheat was significantly lower than those fed on the other three host plants. Enterobacter and ZOR0006 were dominant in FAW fed on tobacco leaves, and in low abundance in larvae fed on wheat. Interestingly, when fed on Solanaceae (tobacco and potato) leaves which contained relative higher levels of toxic secondary metabolites than Gramineae (wheat and maize), the genera Enterococcus, Enterobacter and Acinetobacter were significantly enriched. The results indicated that gut bacteria were related to the detoxification and adaptation of toxic secondary metabolites of host plants in FAW. Further analysis showed that replication, repair and nucleotide metabolism functions were enriched in the gut bacteria of larvae fed on tobacco and potato. In conclusion, the gut bacterial diversity and community composition in FAW larvae fed on different host plants showed significant differences, and the insect is likely to regulate their gut bacteria for adaptation to different host plants.

High nitrogen in maize enriches gut microbiota conferring insecticide tolerance in lepidopteran pest Spodoptera litura.

Abuse of chemical fertilizers and insecticides has created many environmental and human health hazards. We hypothesized that high nitrogen (N) in crops changes insect gut microbiota leading to enhanced insecticide tolerance. We investigated the effect of high N in maize on gut microbiota and insecticide tolerance of the polyphagous pest Spodoptera litura. Bioassays showed that high N applied in both maize plants and artificial diets significantly enhanced larval growth but reduced larval sensitivity to the insecticide methomyl. High N promoted the gut bacterial abundance in the genus Enterococcus. Inoculation with two strains (E. mundtii and E. casseliflavus) isolated from the larval guts increased larval tolerance to methomyl. Incorporation of antibiotics in a high-N diet increased the larval sensitivity to methomyl. These findings suggest that excessive application of N fertilizer to crops can increase insecticide tolerance of insect pests via changing gut microbiota, leading to increased use of insecticides worldwide.

Deficiency in Silicon Transporter Lsi1 Compromises Inducibility of Anti-herbivore Defense in Rice Plants.

Silicon (Si) application can significantly enhance rice resistance against herbivorous insects. However, the underlying mechanism is elusive. In this study, silicon transporter mutant OsLsi1 and corresponding wild-type rice (WT) were treated with and without Si to determine Si effects on rice resistance to leaffolder (LF), Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae). Si application on WT plants significantly promoted rice plant growth, upregulated expression level of OsLsi1 and increased Si accumulation in the leaves and roots, as well as effectively reduced LF weight gain, while it showed only marginal or no effect on the mutant plants. Furthermore, upon LF infestation, transcript levels of OsLOX, OsAOS2, OsCOI1a, OsCOI1b, and OsBBPI, and activity of catalase, superoxide dismutase, peroxidase, and polyphenol oxidase were significantly higher in Si-treated than untreated WT plants. However, OsLsi1 mutant plants displayed higher susceptibility to LF, and minimal response of defense-related enzymes and jasmonate dependent genes to Si application. These results suggest that induced defense plays a vital role in Si-enhanced resistance and deficiency in silicon transporter Lsi1 compromises inducibility of anti-herbivore defense in rice plants.

Identification of a cytochrome P450 CYP6AB60 gene associated with tolerance to multi-plant allelochemicals from a polyphagous caterpillar tobacco cutworm (Spodoptera litura).

Generalist phytophagous insects adapt to adventurous chemical environment in a wide variety of host plants by extraordinary detoxifying metabolic abilities. However, how polyphagous insect cope with the diversity of plant defenses remains largely unknown and only a few counter-defense genes detoxifying a wide range of toxic secondary metabolites have been well characterized. Here, we identify a cytochrome P450 gene (CYP6AB60) from tobacco cutworm (Spodoptera litura) in response to three different plant's defense metabolites. After being exposed to artificial diet supplemented with coumarin (COU), xanthotoxin (XAN) or tomatine (TOM), activities of P450 and CYP6AB60 transcript levels in both midgut and fat body tissues were significantly increased. Developmental expression analysis revealed that CYP6AB60 was expressed highly during the larval stages, and tissue distribution analysis showed that CYP6AB60 was expressed extremely high in the midgut, which correspond to the physiological role of CYP6AB60 from S. litura larvae in response to plant allelochemicals. Furthermore, when larvae are injected with double-stranded RNA (dsRNA) specific to CYP6AB60, levels of this transcript in the midgut and fatbody decrease and the negative effect of plant's defense metabolites on larval growth is magnified. These data demonstrate that the generalist insect S. litura might take advantage of an individual detoxificative gene CYP6AB60 to toxic secondary metabolites from different host plants. The CYP6AB60 can be a potential gene to carry out RNAi-mediated crop protection against the major polyphagous pest S. litura in the future.

Activation of the TOR Signalling Pathway by Glutamine Regulates Insect Fecundity.

The target of rapamycin (TOR) positively controls cell growth in response to nutrients such as amino acids. However, research on the specific nutrients sensed by TOR is limited. Glutamine (Gln), a particularly important amino acid involved in metabolism in organisms, is synthesised and catalysed exclusively by glutamine synthetase (GS), and our previous studies have shown that Gln may regulate fecundity in vivo levels of the brown planthopper (BPH) Nilaparvata lugens. Until now, it has remained unclear whether Gln activates or inhibits the TOR signalling pathway. Here, we performed the combined analyses of iTRAQ (isobaric tags for relative and absolute quantification) and DGE (tag-based digital gene expression) data in N. lugens at the protein and transcript levels after GS RNAi, and we found that 52 pathways overlap, including the TOR pathway. We further experimentally demonstrate that Gln activates the TOR pathway by promoting the serine/threonine protein kinase AKT and inhibiting the 5'AMP-activated protein kinase AMPK phosphorylation activity in the pest. Furthermore, TOR regulates the fecundity of N. lugens probably by mediating vitellogenin (Vg) expression. This work is the first report that Gln activates the TOR pathway in vivo.