Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, <i>Myllocerus undecimpustulatus undatus</i> Marshall

Pinheiro, Daniele H.; Taylor, Caitlin; Wu, Ke; Siegfried, Blair D.

doi:10.1002/ps.5601

Cited by 19 publications

(13 citation statements)

References 57 publications

(91 reference statements)

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“… Wu et al (2019) observed no effect on survival of pepper weevil ( Anthonomus eugenii ) after dietary exposure to pepper coated with dsRNA (target genes Snf7 and vATPase A ) at a concentration of 0.3 μg dsRNA/μl. Pinheiro et al (2020) observed between 43 and 93% mortality in the Sri Lanka weevil ( Myllocerus undecimpustulatus ), depending on the target gene. However, as mortality was only monitored after 26 days (dsRNA treatments were administered for 20 days), there remain critical unknowns with regard to timeliness of RNAi-induced mortality.…”

Section: Observed Rnai Sensitivities In Coleopteramentioning

confidence: 99%

Highly Variable Dietary RNAi Sensitivity Among Coleoptera

Willow

Veromann

2021

Front. Plant Sci.

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Many herbivorous beetles (Order Coleoptera) contribute to serious losses in crop yields and forest trees, and plant biotechnology solutions are being developed with the hope of limiting these losses. Due to the unprecedented target-specificity of double-stranded RNA (dsRNA), and its utility in inducing RNA interference (RNAi) when consumed by target pest species, dsRNA-based plant biotechnology approaches represent the cutting edge of current pesticide research and development. We review dietary RNAi studies in coleopterans and discuss prospects and future directions regarding RNAi-based management of coleopteran plant pests. Herein, we also provide a balanced overview of existing studies in order to provide an accurate re-assessment of dietary RNAi sensitivity in coleopterans, despite the limitations to the existing body of scientific literature. We further discuss impediments to our understanding of RNAi sensitivity in this important insect order and identify critical future directions for research in this area, with an emphasis on using plant biotechnology approaches.

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Section: Observed Rnai Sensitivities In Coleopteramentioning

confidence: 99%

Highly Variable Dietary RNAi Sensitivity Among Coleoptera

Willow

Veromann

2021

Front. Plant Sci.

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“…All these methods are useful, but there are still some shortcomings. As a new molecular biological technology, RNAi has been applied in some insects of coleoptera (Ke et al, 2018;Haller et al, 2019;Pinheiro et al, 2020). In this study, five effective primers of visual and olfactory target genes for RNAi of EAB were designed, providing a basis for RNAi-based new controlling method development of A. planipennis at adult stage.…”

Section: Discussionmentioning

confidence: 99%

Five Visual and Olfactory Target Genes for RNAi in Agrilus Planipennis

Fan

Zhang

et al. 2022

Front. Genet.

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RNA interference (RNAi) is a widely used technique for gene function researches and recently pest controls. It had been applied in emerald ash borer (EAB Agrilus planipennis) larvae and adults, and achieved significant interference effects, whether by ingesting or microinjecting. Feeding in the phloem and cambial regions, the larvae of A. planipennis are difficult to be controlled by conventional insecticides, so adult stage is the critical stage for EAB control. However, the target genes of adult stage of A. planipennis need to be further screened. Here, we preliminarily screened five potential target genes of vision and olfaction for RNAi in A. planipennis. Three odorant binding proteins (OBPs) and three opsins, which expressed significantly different between newly emerged and sexually mature EABs (OBP5, OBP7, OBP10, LW opsin 1 and UV opsin 2) or highly in sexually mature male EAB (UV opsin 3), were selected as targets to design primers for gene silencing. After dsRNA injection, the gene expression levels were determined by real-time quantitative PCR. We found that the expression levels of five genes were significantly down-regulated, during the 4 days after dsRNA injection. Among these genes, the expression of LW opsin 1 was down-regulated the most, causing a reduction of 99.1% compared with the control treated with EGFP dsRNA, followed by UV opsin 3 (97.4%), UV opsin 2 (97.0%), OBP7 (96.2%), and OBP10 (88.7%). This study provides a basis for further RNAi-based new controlling method development of A. planipennis at adult stage.

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“…Degenerate primers for RPS23, 18S, 28S and RpII140 were manually designed based on conserved nucleotide sequence among other Coleoptera species. Primers for ArgK, EF1-α, RPL12, α-TUB and Prosα-2 were obtained from previous works 30,47 ( Supplementary Table S2). PCR amplifications consisted of 5 µL of 10x PCR buffer, 8 µL of MgCl 2 (50 mM), 5 µL of dNTPs mix (10 mM of each nucleotide), 8 µL of each primer (10 µM), 1.25 µL of Taq DNA Polymerase (5U/µL) (ThermoFisher Scientific), 2 µL of cDNA (diluted 10×) and 12.75 µL of nuclease-free water.…”

Section: Reference Gene Selection Gene Fragment Cloning and Primer Dmentioning

confidence: 99%

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Pinheiro

Siegfried

2020

Sci Rep

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The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. careful selection of adequate reference genes to normalize Rt-qpcR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. the lack of studies on validation of reference genes for Rt-qpcR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and normfinder) were used to analyze the expression stability of candidate reference genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by Rt-qpcR in biological experiments involving A. eugenii.Reverse-transcription quantitative PCR (RT-qPCR) is widely used in gene expression studies due to its simplicity, reproducibility, high sensitivity, accuracy and cost-effectiveness 1,2 . Although RT-qPCR is considered a highly accurate technique, several experimental factors can lead to results that are not reliable measurements of gene expression. These factors include purity and integrity of RNA, quantity of starting RNA and cDNA, reverse transcription and PCR efficiency, and pipetting errors 3 . Thus, in RT-qPCR analysis is necessary to use reference genes to normalize the data in order to eliminate or at least reduce the technical variation among the tested samples and precisely estimate the expression of the target genes 4 .Usually, housekeeping genes related to basic cellular functions are used as reference genes in the normalization strategy because these genes are supposed to have constitutive and stable expression under a variety of physiological conditions and experimental treatments. However, several studies have demonstrated that the expression of housekeeping genes is not always stable and can be influenced by developmental stage, tissue, sex, and biotic or abiotic stresses that the organism is subjected 5-9 . Therefore, the selection of suitable reference genes according to the specific experimental conditions is essential to ensure accurate results.The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the most economica...

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Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

Cited by 19 publications

References 57 publications

Highly Variable Dietary RNAi Sensitivity Among Coleoptera

Highly Variable Dietary RNAi Sensitivity Among Coleoptera

Five Visual and Olfactory Target Genes for RNAi in Agrilus Planipennis

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

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