RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

The N-glycosylation-related genes as potential targets for RNAi-mediated pest control of the Colorado potato beetle (Leptinotarsa decemlineata).

BACKGROUND: N-glycosylation is one of the most common and important post-translational modifications in the eukaryotic cell. The study of protein N-glycosylation in several model insects confirmed the importance of this process in insect development, immunity, survival and fertility. The Colorado potato beetle (Leptinotarsa decemlineata) (CPB) is a common pest of Solanaceae crops. With the infamous title of champion of insecticide resistance, novel pest control strategies for this insect are needed. Luckily this pest insect is reported as very sensitive for the post-genomic technology of RNA interference (RNAi). RESULTS: In this project, we investigated the importance of N-glycosylation in the survival and development of CPB using RNAi-mediated gene silencing of N-glycosylation-related genes (NGRGs) during the different transition steps from the larva, through the pupa to the adult stage. High mortality was observed in the larval stage with the silencing of early NGRGs, as STT3a, DAD1 and GCS1. With dsRNA against middle NGRGs, abnormal phenotypes at the ecdysis process and adult formation were observed, while the silencing of late NGRGs did not cause mortality. CONCLUSION: The lethal phenotypes observed on silencing of the genes involved in the early processing steps of the N-glycosylation pathway suggest these genes are good candidates for RNAi-mediated control of CPB. Next to the gene-specific mechanism of RNAi for biosafety and possible implementation in integrated pest management, we believe these early NGRGs provide a possible alternative to the well-known target genes Snf7 and vacuolar ATPases that are now used in the first commercial RNAi-based products and thus they may be useful in the context of proactive resistance management. © 2021 Society of Chemical Industry.

Protection of rice against Nilaparvata lugens by direct toxicity of sodium selenate.

Nilaparvata lugens is one of the most notorious pest insects of cultured rice, and outbreaks of N. lugens cause high economic losses each year. While pest control by chemical pesticides is still the standard procedure for treating N. lugens infections, excessive use of these insecticides has led to the emergence of resistant strains and high pesticide residues in plants for human consumption and the environment. Therefore, novel and environment-friendly pest control strategies are needed. In previous studies, selenium was shown to protect selenium-accumulating plants from biotic stress. However, studies on nonaccumulator (crop) plants are lacking. In this study, rice plants (Oryza sativa, Nipponbare) were treated with sodium selenate by seed priming and foliar spray and then infested with N. lugens. Brown planthoppers feeding on these plants showed increased mortality compared to those feeding on control plants. Treatment of the plants with sodium selenate did not affect the enzymes involved in the biosynthesis of the plant stress hormones jasmonic acid and salicylic acid, suggesting that the observed insect mortality cannot be attributed to the activation of these hormonal plant defenses. Feeding assays using an artificial diet supplemented with sodium selenate revealed direct toxicity toward N. lugens. With a low concentration of 6.5 ± 1.5 µM sodium selenate, half of the insects were killed after 3 days. In summary, sodium selenate treatment of plants can be used as a potential alternative pest management strategy to protect rice against N. lugens infestation through direct toxicity.