Vacuolar (H+ )-ATPase subunit c is essential for the survival and systemic RNA interference response in Locusta migratoria.

BACKGROUND: Vacuolar (H+ )-ATPase (V-ATPase) is a multi-subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V-ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V-ATPase-c functions in insects. RESULTS: We identified and characterized the V-ATPase-c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV-ATPase-c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV-ATPase-c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV-ATPase-c. In addition, silencing of LmV-ATPase-c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double-stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV-ATPase-c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. CONCLUSION: Our results indicate that LmV-ATPase-c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV-ATPase-c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.

Strategies for enhancing the efficiency of RNA interference in insects.

Low RNA interference (RNAi) efficiency in many insect pests has significantly prevented its widespread application for insect pest management. This article provides a comprehensive review of recent research in developing various strategies for enhancing RNAi efficiency. Our review focuses on the strategies in target gene selection and double-stranded RNA (dsRNA) delivery technologies. For target gene selection, genome-wide or large-scale screening strategies have been used to identify most susceptible target genes for RNAi. Other strategies include the design of dsRNA constructs and manipulate the structure of dsRNA to maximize the RNA efficiency for a target gene. For dsRNA delivery strategies, much recent research has focused on the applications of complexed or encapsulated dsRNA using various reagents, polymers, or peptides to enhance dsRNA stability and cellular uptake. Other dsRNA delivery strategies include genetic engineering of microbes (e.g. fungi, bacteria, and viruses) and plants to produce insect-specific dsRNA. The ingestion of the dsRNA-producing organisms or tissues will have lethal or detrimental effects on the target insect pests. This article also identifies obstacles to further developing RNAi for insect pest management and suggests future avenues of research that will maximize the potential for using RNAi for insect pest management. © 2021 Society of Chemical Industry.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis.

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Molecular mechanisms influencing efficiency of RNA interference in insects.

RNA interference (RNAi) is an endogenous, sequence-specific gene-silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry.

Contributions of dsRNases to differential RNAi efficiencies between the injection and oral delivery of dsRNA in Locusta migratoria.

BACKGROUND: The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive. RESULTS: We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut. CONCLUSION: The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management. © 2018 Society of Chemical Industry.