Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae

Cooper, Anastasia M.W.; Yu, Zujiang; Biondi, Marie; Song, Huifang; Silver, Kristopher; Zhang, Jianzhen; Zhu, Kun Yan

doi:10.1016/j.pestbp.2020.104672

Cited by 28 publications

(35 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ECB used in this study originated from French Agricultural Research (Lumberton, MN, USA) and were continuously reared in the laboratory at Kansas State University, Manhattan, KS, as described in Cooper et al 20 …”

Section: Methodsmentioning

confidence: 99%

“…To obtain dsRNA for stability enhancement experiments and RNAi assays, dsRNA targeting either an endogenous gene encoding lethal giant larvae protein ( OnLgl ; MT467568) from the ECB or an exogenous gene encoding enhanced green fluorescent protein ( GFP ; LC336974.1) were synthesized, purified, and assessed as described in Cooper et al 20 . The primer sequences for dsRNA synthesis are listed in Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…To assess which of the candidate reagents could enhance dsRNA stability in ECB tissues, ex vivo incubation experiments were performed using HE and GC harvested from fifth‐instar larvae following the detailed protocols described in Cooper et al 20 . In brief, after harvesting tissues from the ECB larvae, PBS was used to normalize the total protein content between biological replicates (each containing tissue pooled from 15 individuals).…”

Section: Methodsmentioning

confidence: 99%

“…Cycle threshold (Ct) values were converted to the amount of ds GFP in nanograms based on standard curves (Fig. S1), as previously described 20 . Experiments followed a two‐way factorial treatment design to allow for assessment of significant effects on dsRNA stability due to reagent (present or absent), incubation fluid (tissue extract or PBS), and/or interaction between both factors.…”

Section: Methodsmentioning

confidence: 99%

“…Ct values > 32 were considered non‐detectible, and a fold change of zero was used for analysis. NormFinder, 26 geNorm, 27 BestKeper, 28 and RefFinder (http://150.216.56.64/referencegene.php) were used to verify the stability of reference genes across ECB tissues and developmental stages 20 . Percent suppression of the target gene was calculated as [(control − target)/control) × 100].…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

Cooper

Song

et al. 2020

Pest Management Science

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

Cooper

Song

et al. 2020

Pest Management Science

Self Cite

View full text Add to dashboard Cite

show abstract

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults

Cooper

Song

Shi

et al. 2021

Arch Insect Biochem Physiol

Self Cite

View full text Add to dashboard Cite

show abstract

RNAi efficiency is enhanced through knockdown of double‐stranded RNA‐degrading enzymes in butterfly Papilio xuthus

Shu,

Liu,

et al. 2024

Arch Insect Biochem Physiol

View full text Add to dashboard Cite

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double‐stranded RNA‐degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1–6) were identified in P. xuthus genome, and their dsRNA‐degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA‐degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi‐of‐RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

show abstract

Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae

Cited by 28 publications

References 40 publications

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults

RNAi efficiency is enhanced through knockdown of double‐stranded RNA‐degrading enzymes in butterfly Papilio xuthus

Contact Info

Product

Resources

About