The vital hormone 20-hydroxyecdysone controls ATP production by upregulating binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera.

Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.

Trehalase is required for sex pheromone biosynthesis in Helicoverpa armigera.

Trehalase (Treh) hydrolyzes trehalose to generate glucose and it plays important role in many physiological processes. Acetyl-CoA, the precursor of sex pheromone biosynthesis in the pheromone gland (PG) of Helicoverpa armigera, originates from glucose during glycolysis. However, the function of Treh in sex pheromone biosynthesis remains elusive. In the present study, H. armigera was used as a model to investigate the function of two Trehs (Treh1 and Treh2) in sex pheromone biosynthesis. Results demonstrated that knockdown of HaTreh1 or HaTreh2 in female PGs led to significant decreases in Z11-16:Ald production, female ability to attract males, and successful mating proportions. Pheromone biosynthesis activating neuropeptide (PBAN) treatment triggered HaTreh1 and HaTreh2 activities in the isolated PGs and Sf9 cells. However, the activities of HaTreh1 and HaTreh2 triggered by PBAN were offset by H-89, the specific inhibitor of protein kinase A (PKA). Furthermore, the H-89 treatment significantly decreased the phosphorylation level of Trhe2, which was induced by PBAN. In addition, sugar feeding (5% sugar) increased the enzyme activities of Treh1 and Treh2. In summary, our findings confirmed that PBAN activates Treh1/2 activities by recruiting cAMP/PKA signalling, promotes glycolysis to ensure the supply of acetyl-CoA, and ultimately facilitates sex pheromone biosynthesis and mating behaviour.

Insight into maize gene expression profiles responses to symbiotic bacteria derived from Helicoverpa armigera and Ostrinia furnacalis.

The insects of Ostrinia furnacalis and Helicoverpa armigera are the two main pests that affect maize growth, which significantly decrease the yield. Plants induce various immune-related pathways to antagonize insect feeding during insect-plant interactions. Moreover, different insect elicitors or effectors participate in the interactions via releasing into plants. While there are many bacteria during insect regurgitation, their roles in insect-plant interaction are unknown. In this study, four bacterial strains were isolated from regurgitation fluid of O. furnacalis and H. armigera, and their cultures were inoculated on maize leaves for response analysis. All the four bacterial strains altered gene expression profiles in maize, and these altered expression profiles included phytohormones, secondary metabolic pathways, transcription factors, MAPK, and plant-pathogen interaction-related genes. A total of 210 genes, such as WRKY54, WRKY62, PIF5, argonaute 1, Xa21, NRR, ubiquitin-proteasome system genes, were co-changed in response to bacterial inoculation. These changes were similar with maize gene profile changes after insect feeding. Symbiotic insect bacteria participate in insect-plant interactions by changing maize gene expression profiles, which might be used to develop anti-pest microbial agents by activating plant defense system with identified microbes. In future, understanding the roles of symbiotic insect bacteria on plant-insect interaction might provide a promising and novel strategy for pest biocontrol using microbes.

New insights on the effects of spinosad on the development of Helicoverpa armigera.

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

Complete genome sequence of a novel rhabdo-like virus from the Chinese black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

This work identified a novel rhabdo-like virus in a Chinese black cutworm (Agrotis ipsilon), which we tentatively named "Agrotis ipsilon virus" (AIpsV). The complete genome of AIpsV is 15,454 nucleotides in length and contains seven open reading frames, collectively encoding more than 160 amino acids. The AIpsV genome is predicted to encode three structural proteins, nucleoprotein (N), glycoprotein (G), and large polymerase protein (L), and four unknown proteins. Phylogenetic analysis revealed that the AIpsV clusters with Wuhan ant virus and Hubei rhabdo-like virus 1 within the rhabdo-like virus clade. The level of expression of AIpsV genes was found to be higher in the pupal and adult stages than in the egg and larval stages.

Comparative analysis of dsRNA-induced lncRNAs in three kinds of insect species.

Long noncoding RNAs (lncRNAs) that have immune responses to various stimuli have been identified in some insects. One type of pathogen-associated molecular pattern, double-stranded RNA (dsRNA), can trigger the RNA interference (RNAi) pathway and immune response. Interestingly, there has been no research into characterizing the relationship between lncRNA and dsRNA-induced RNAi pathways. In this study, dsRNA-induced lncRNAs were investigated in two species of lepidopteran insects, Helicoverpa armigera and Plutella xylostella, and one species of coleopteran insects, Tribolium castaneum. Between untreated group and dsRNA-induced group; 3,463 H. armigera, 6,245 P. xylostella, and 3,067 T. castaneum differentially expressed lncRNAs were identified while 156 H. armigera, 247 P. xylostella, 415 T. castaneum lncRNAs and their putative target genes showed consistent changes in gene expression. In T. castaneum, most target genes of the differentially expressed lncRNAs are enriched in the cyclic adenosine monophosphate signaling pathway, ABC transporters, and Janus kinase-signal transducers and activators of the transcription signaling pathway. Conversely, in H. armigera and P. xylostella, the differentially expressed lncRNAs were mainly enriched in the metabolic, digestive, and synthetic signaling pathways. This result indicates that dsRNA-induced lncRNA is species-dependent. We also found that both Dicer-2 and the lncRNA that targets Dicer-2 were significantly upregulated after dsRNA treatment in P. xylostella, indicating that some lncRNAs may be involved in the regulation of the core RNAi pathway in insects. Our results are the first to identify a relationship between lncRNAs and dsRNA in various insect species with different RNAi efficiencies. These results provide a reference for future study of the dsRNA-induced RNAi pathway and different RNAi efficiencies among insect species.

A nuclease specific to lepidopteran insects suppresses RNAi.

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer Ostrinia furnacalis, we identified a gene, termed up56, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that up56 encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in O. furnacalis strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo Of note, we found that this up-regulation of up56 expression is faster than that of the gene encoding the key RNAi-associated nuclease Dicer. up56 knockdown in O. furnacalis significantly enhanced RNAi efficiency. Moreover, up56 overexpression in Drosophila melanogaster suppressed RNAi efficiency. Finally, up56 knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.

New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests.

Insect double-stranded (ds)RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. Arabidopsis Mob1A is required for organ growth and reproduction. When Arabidopsis roots were soaked in dsMob1A, the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty-four hours after rice roots were immersed in fluorescent-labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of Ago and Dicer in rice and planthoppers were induced by dsEYFP. When rice roots were soaked in dsActin, their growth was also significantly suppressed. When planthoppers or Asian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing-sucking and stem-borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.

Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

Since the discovery of the RNA interference (RNAi) mechanism, it has been widely used in the fields of gene function, biomedicine, and crop pest control. In the direction of agricultural application, this technology is highly expected, especially in the field of pest control, which is called "the third revolution in the history of pesticides". Currently, RNA biopesticides are developing rapidly all over the world. A genetically modified product (MON87411) has been approved for marketing, and a large number of agricultural companies are developing products based on direct spraying RNA biopesticides and submitting them for regulatory approval. The biggest problem that has emerged for spray RNA biopesticides is the technology for large-scale and low-cost production of dsRNA. At present, the bacterial fermentation production technology can realize large-scale dsRNA production with a yield of 4.23~182 mg/L bacterial solution. This article describes the experimental protocol for dsRNA production technology based on bacterial fermentation.

Identification of differential expression genes associated with host selection and adaptation between two sibling insect species by transcriptional profile analysis.

BACKGROUND: Cotton bollworm (Helicoverpa armigera) and oriental tobacco budworm (Helicoverpa assulta) are noctuid sibling species. Under artificial manipulation, they can mate and produce fertile offspring. As serious agricultural insect pests, cotton bollworms are euryphagous insects, but oriental tobacco budworms are oligophagous insects. To identify the differentially expressed genes that affect host recognition and host adaptation between the two species, we constructed digital gene expression tag profiles for four developmental stages of the two species. High-throughput sequencing results indicated that we have got more than 23 million 17nt clean tags from both species, respectively. The number of unique clean tags was nearly same in both species (approximately 357,000). RESULTS: According to the gene annotation results, we identified 83 and 68 olfaction related transcripts from H. armigera and H. assulta, respectively. At the same time, 1137 and 1138 transcripts of digestion enzymes were identified from the two species. Among the olfaction related transcripts, more odorant binding protein and G protein-coupled receptor were identified in H. armigera than in H. assulta. Among the digestion enzymes, there are more detoxification enzyme, e.g. P450, carboxypeptidase and ATPase in H. assulta than in H. armigera. These differences partially explain that because of the narrow host plant range of H. assulta, more detoxification enzymes would help them increase the food detoxification and utilization efficiency. CONCLUSIONS: This study supplied some differentially expressed genes affecting host selection and adaptation between the two sibling species. These genes will be useful information for studying on the evolution of host plant selection. It also provides some important target genes for insect species-specific control by RNAi technology.

A TIL-Type Serine Protease Inhibitor Involved in Humoral Immune Response of Asian Corn Borer Ostrinia furnaculis.

To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response.

Advances in the Development of Microbial Double-Stranded RNA Production Systems for Application of RNA Interference in Agricultural Pest Control.

RNA interference (RNAi) is a valuable and revolutionary technology that has been widely applied in medicine and agriculture. The application of RNAi in various industries requires large amounts of low-cost double-stranded RNA (dsRNA). Chemical synthesis can only produce short dsRNAs; long dsRNAs need to be synthesized biologically. Several microbial chassis cells, such as Escherichia coli, Saccharomyces cerevisiae, and Bacillus species, have been used for dsRNA synthesis. However, the titer, rate of production, and yield of dsRNA obtained by these microorganism-based strategies is still low. In this review, we summarize advances in microbial dsRNA production, and analyze the merits and faults of different microbial dsRNA production systems. This review provides a guide for dsRNA production system selection. Future development of efficient microbial dsRNA production systems is also discussed.

The Novel Agrotis ipsilon Nora Virus Confers Deleterious Effects to the Fitness of Spodoptera frugiperda (Lepidoptera: Noctuidae).

In the present study, we identified a novel, positive-sense single-stranded RNA virus in the Chinese black cutworm, Agrotis ipsilon. It has a genome length of 11,312 nucleotides, excluding the poly(A) tails, and contains five open reading frames. The ORF2 encodes the conserved domains of RNA helicase and RNA-dependent RNA polymerase, while ORF4 and 5 encode three viral proteins. Herein, the A. ipsilon virus was clustered with a Helicoverpa armigera Nora virus and was thus provisionally named "Agrotis ipsilon Nora virus" (AINV). AINV was successfully transmitted into a novel host, Spodoptera frugiperda, through injection, causing a stable infection. This found the possibility of horizontal AINV transmission among moths belonging to the same taxonomic family. Nonetheless, AINV infection was deleterious to S. frugiperda and mainly mediated by antiviral and amino acid metabolism-related pathways. Furthermore, the infection significantly increased the S. frugiperda larval period but significantly reduced its moth eclosion rate. It suggests that AINV is probably to be a parasitic virus of S. frugiperda.

Metabolic Engineering for Glycyrrhetinic Acid Production in Saccharomyces cerevisiae.

Glycyrrhetinic acid (GA) is one of the main bioactive components of licorice, and it is widely used in traditional Chinese medicine due to its hepatoprotective, immunomodulatory, anti-inflammatory and anti-viral functions. Currently, GA is mainly extracted from the roots of cultivated licorice. However, licorice only contains low amounts of GA, and the amount of licorice that can be planted is limited. GA supplies are therefore limited and cannot meet the demands of growing markets. GA has a complex chemical structure, and its chemical synthesis is difficult, therefore, new strategies to produce large amounts of GA are needed. The development of metabolic engineering and emerging synthetic biology provide the opportunity to produce GA using microbial cell factories. In this review, current advances in the metabolic engineering of Saccharomyces cerevisiae for GA biosynthesis and various metabolic engineering strategies that can improve GA production are summarized. Furthermore, the advances and challenges of yeast GA production are also discussed. In summary, GA biosynthesis using metabolically engineered S. cerevisiae serves as one possible strategy for sustainable GA supply and reasonable use of traditional Chinese medical plants.

Knockout of the HaREase Gene Improves the Stability of dsRNA and Increases the Sensitivity of Helicoverpa armigera to Bacillus thuringiensis Toxin.

Double-stranded RNA (dsRNA)-induced genes are usually related to RNA interference (RNAi) mechanisms and are involved in immune-related pathways. In a previous study, we found a lepidopteran-specific nuclease gene REase that was up-regulated by dsRNA and that affected RNAi efficiency in Asian corn borer (Ostrinia furnacalis). In this study, to verify the function of REase, the homologous gene HaREase in cotton bollworm (Helicoverpa armigera) was knocked out using CRISPR/Cas9 system. We found that the midgut epithelium structure was apparently not affected in the ΔHaREase mutant [Knock out (KO)]. Transcript sequencing results showed that most of the known insect immune-related genes were up-regulated in KO. When second instar larvae were fed artificial diet with Cry1Ac, a protoxin from Bacillus thuringiensis (Bt), in sublethal doses (2.5 or 4 µg/g), the growth rate of KO was repressed significantly. The dsRNA stability was also enhanced in midgut extraction of KO; however, RNAi efficiency was not obviously improved compared with the wild type (WT). The KO and WT were injected with dsEGFP (Enhanced green fluorescent protein) and subjected to transcriptome sequencing. The results showed that the expression levels of 14 nuclease genes were enhanced in KO after the dsRNA treatment. These findings revealed that HaREase expression level was not only related with dsRNA stability, but also with Bt resistance in cotton bollworm. When HaREase was knocked out, other immune- or nuclease-related genes were enhanced significantly. These results remind us that insect immune system is complex and pest control for cotton bollworm is an arduous task.

Prediction of effective RNA interference targets and pathway-related genes in lepidopteran insects by RNA sequencing analysis.

When using RNA interference (RNAi) to study gene functions in Lepidoptera insects, we discovered that some genes could not be suppressed; instead, their expression levels could be up-regulated by double-stranded RNA (dsRNA). To predict which genes could be easily silenced, we treated the Asian corn borer (Ostrinia furnacalis) with dsGFP (green fluorescent protein) and dsMLP (muscle lim protein). A transcriptome sequence analysis was conducted using the cDNAs 6 h after treatment with dsRNA. The results indicated that 160 genes were up-regulated and 44 genes were down-regulated by the two dsRNAs. Then, 50 co-up-regulated, 25 co-down-regulated and 43 unaffected genes were selected to determine their RNAi responses. All the 25 down-regulated genes were knocked down by their corresponding dsRNA. However, several of the up-regulated and unaffected genes were up-regulated when treated with their corresponding dsRNAs instead of being knocked down. The genes up-regulated by the dsGFP treatment may be involved in insect immune responses or the RNAi pathway. When the immune-related genes were excluded, only seven genes were induced by dsGFP, including ago-2 and dicer-2. These results not only provide a reference for efficient RNAi target predications, but also provide some potential RNAi pathway-related genes for further study.

The in vivo dsRNA Cleavage Has Sequence Preference in Insects.

Exogenous dsRNA enters the insect body and can induce the RNAi effect only when it is cleaved into siRNA. However, what kinds of base composition are easier to cut and what kinds of siRNA will be produced in vivo is largely unknown. In this study, we found that dsRNA processing into siRNA has sequence preference and regularity in insects. We injected 0.04 mg/g dsRNA into Asian corn borers or cotton bollworms according to their body weight, and then the siRNAs produced in vivo were analyzed by RNA-Seq. We discovered that a large number of siRNAs were produced with GGU nucleotide residues at the 5'- and 3'-ends and produced a siRNA peak on the sequence. Once the GGU site is mutated, the number of siRNAs will decrease significantly and the siRNA peak will also lost. However, in the red flour beetle, a member of Coleoptera, dsRNA was cut at more diverse sites, such as AAG, GUG, and GUU; more importantly, these enzyme restriction sites have a high conservation base of A/U. Our discovery regarding dsRNA in vivo cleavage preference and regularity will help us understand the RNAi mechanism and its application.