Limitations in harnessing oral RNA interference as an antiviral strategy in Aedes aegypti

Romoli, Ottavia; Henrion-Lacritick, Annabelle; Blanc, Hervé; Frangeul, Lionel; Saleh, Maria-Carla

doi:10.1016/j.isci.2024.109261

Cited by 2 publications

(6 citation statements)

References 53 publications

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“…However, the amount could have been below a biologically meaningful threshold. This would be supported by observations made by Romoli et al [68], who could not detect siRNA enrichment in the tissues of Ae. aegypti females whose gut had been colonized by dsRNA-producing bacteria.…”

Section: Discussionsupporting

confidence: 75%

“…Several factors are critical for the successful induction of an RNAi response, including the stability of the RNA molecules in the environment and during delivery, the uptake from the gut lumen and intracellular release, the processing of the iRNA by the RNAi machinery, and the induction of systemic RNAi [4,17,18,56,62,68]. To troubleshoot the reasons for our failure to reproduce the published results, we independently started to test different RNAi-triggering molecules (siRNA, shRNA, and dsRNA) in combination with different delivery pathways (oral delivery via iRNA-producing microorganisms provided to the larvae in various formulations, soaking in pure iRNA solutions, and microinjections).…”

Section: Discussionmentioning

confidence: 99%

“…Another possibility for the lack of RNAi effects is the failed processing of the delivered dsRNA into siRNAs by the cellular RNAi machinery. While we did not experimentally assess this step, the assays described above from Romoli et al [68] show the processing of dsRNAs by the RNAi machinery. Based on this, we would have expected better RNAi efficiency when delivering naked dsRNA or chemically synthesized siRNAs against fez2, lrc, and sem-1a, via larval soaking or larval injections, using the published highly successful target sequences.…”

Section: Discussionmentioning

confidence: 99%

“…The effect of RNases in the gut and hemolymph remains a major mystery in mosquito RNAi. While dsRNA could be reextracted from mosquitoes at least in part up to two days post-oral delivery [25,68] and up to 7 days post-injection [71], the incubation of dsRNA with serial dilutions of gut juice led to the fast degradation of the dsRNA (Figure S2). Also, the pathway of dsRNA upon ingestion or injection is barely known.…”

Section: Discussionmentioning

confidence: 99%

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Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes

Figueiredo Prates,

Fiebig,

Schlosser

et al. 2024

IJMS

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In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi’s potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes

Figueiredo Prates,

Fiebig,

Schlosser

et al. 2024

IJMS

View full text Add to dashboard Cite

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“…Previous studies have tried to harness RNAi to control mosquito populations and curtail arbovirus transmission 49–54 , but dsRNA delivery by injection has a low throughput, and RNAi by dsRNA ingestion is also limited in mosquitoes 55 . Mosquito viruses like TMBTLV that can readily trigger dsRNA and siRNAs in mosquitoes and cells ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Global genomics ofAedes aegyptiunveils widespread and novel infectious viruses capable of triggering a small RNA response

Gupta,

Sharma,

Williams

et al. 2024

Preprint

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The mosquitoAedes aegyptiis a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200Ae. aegyptismall RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies ofAe. aegyptilack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus.Ae. aegyptifrom North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. AsianAe. aegyptismall RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. AfricanAe. aegyptialso contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes’ RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells withAe. aegyptihomogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

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Limitations in harnessing oral RNA interference as an antiviral strategy in Aedes aegypti

Cited by 2 publications

References 53 publications

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes

Global genomics ofAedes aegyptiunveils widespread and novel infectious viruses capable of triggering a small RNA response

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