RNAi by Soaking Aedes aegypti Pupae in dsRNA

Arshad, Fiza; Sharma, Arvind; Lu, Charleen; Gulia-Nuss, Monika

doi:10.3390/insects12070634

Cited by 7 publications

(6 citation statements)

References 30 publications

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“…Initial assessment of the impact of laccase 2 suppression on pupal mortality revealed rates of 29.4–19.17% and 12.5% for the target and control groups, respectively ( Figure 3 A,B). These rates were lower than those described by Arshad and co-authors [ 20 ] in their assay (56.92% and 56.92%) of dsRNA soaked (treatment and control) Ae. aegypti pupae.…”

Section: Resultscontrasting

confidence: 81%

“…Furthermore, our approach reduced the manipulation and handling of pupae to improve survivorship. As a result, our pupal mortality rates (17–44% in the dsLac2 groups and 16–20% in the dsGFP group) were much lower than documented in previous studies [ 20 ]. Our technique eliminated or minimized the risk of damage to pupae and reduced undesirable stress-related effects on mosquito fitness and development [ 38 ].…”

Section: Discussioncontrasting

confidence: 63%

“…We used freshly molted (0–2 h) pupae hours before any signs of cuticle tanning appeared. It had been previously demonstrated that soaking efficacy was reduced when applied to older pupae [ 20 ]. We also did not observe any phenotypic changes when 12–20 h old pupae were soaked in the same concentrations of dsRNA used in our assays.…”

Section: Discussionmentioning

confidence: 99%

“…aegypti pupae simply by soaking them in water with added dsRNA. This effect lasted into early adulthood [ 20 ]. To our knowledge, no studies have yet used soaking for dsRNA delivery to study gene function in Culex pupae.…”

Section: Introductionmentioning

confidence: 99%

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RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

Naumenko,

Fritz

2024

Insects

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Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

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Section: Resultscontrasting

confidence: 81%

Section: Discussioncontrasting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

Naumenko,

Fritz

2024

Insects

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“…dsRNA can be used to silence specific genes in insects, leading to reduced expression of target proteins and ultimately insect mortality. Soaking is a method of delivering dsRNA by immersing insects or plant tissues in a solution containing the dsRNA (Arshad et al 2021).…”

Section: Delivery Of Dsrna Through Soakingmentioning

confidence: 99%

RNAi FOR THE MANAGEMENT OF INSECT PESTS: A REVIEW

Munawar¹

2023

Agrobiol. Rec.

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RNA interference (RNAi) is a biological mechanism that involves the inhibition of gene expression by the introduction of double-stranded RNA (dsRNA) molecules that are complementary to specific target genes. This technique has emerged as a promising strategy for the management of insect pests in crops. The application of RNAi to insect pest management involves the use of dsRNA molecules that are specific to essential genes in the target insect species. When these dsRNA molecules are ingested by the pests, they trigger a silencing mechanism that inhibits the expression of the target genes, leading to reduced pest populations and damage to crops. RNAi-based insect pest management has several advantages over traditional chemical-based methods, including its specificity, effectiveness, and environmental safety. However, there are still some challenges associated with the use of RNAi, such as the potential for offtarget effects and the need for effective delivery methods. Nonetheless, RNAi technology holds great promise for sustainable and effective management of insect pests in agriculture.

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Lab-scale method for plant-mediated delivery of dsRNAs to phloem-feeding leafhoppers

et al. 2023

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RNA interference (RNAi) regulates gene expression in eukaryotes, and it is an emerging tool in crop protection by exogenous applications of double-stranded RNAs (dsRNAs) to silence the expression of essential pest genes. Nevertheless, delivery of dsRNAs to sap-sucking insects is a major challenge for RNAi applications. The present work aimed at verifying whether in leafhopper species, RNAi can be triggered by plant-mediated delivery, and providing a proof of concept towards field applications. Two phytoplasma vectors species, Euscelidius variegatus and Scaphoideus titanus (Hemiptera: Cicadellidae), were used as case study. Gene silencing can be achieved efficiently in both species through microinjection of dsRNAs, despite the technique being time consuming and inapplicable on large scale. This protocol was set as gold standard for the development of a higher throughput approach. Soaking of nymphs in a solution with co-adjuvant and dsRNAs as well as insect feeding on whole plants or detached leaves immersed in a dsRNA solution were assayed as alternative delivery strategies. Nymph soaking did not induce specific gene silencing, while plant absorption proved to be suitable to deliver both a coloured solution and control dsRNAs targeting green fluorescent protein gene. Insect feeding on detached leaves immersed in dsRNA solution was selected to test silencing of two gut-specific (legumain and natterin) and one ubiquitous (ATP synthase β) genes. The expression of the three genes significantly decreased in E. variegatus insects fed on dsRNA-treated plants. Similarly, a significant reduction of ATP synthase β transcript was measured in S. titanus fed on dsRNA-treated plants.

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RNAi by Soaking Aedes aegypti Pupae in dsRNA

Cited by 7 publications

References 30 publications

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

RNAi FOR THE MANAGEMENT OF INSECT PESTS: A REVIEW

Lab-scale method for plant-mediated delivery of dsRNAs to phloem-feeding leafhoppers

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