A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Song, Huifang; Zhang, Jianqin; Li, Daqi; Cooper, Anastasia M.W.; Silver, Kristopher; Li, Tao; Liu, Xiaojian; Ma, Enbo; Zhu, Kun Yan; Zhang, Jianzhen

doi:10.1016/j.ibmb.2017.05.008

Cited by 83 publications

(112 citation statements)

References 52 publications

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“…The procedures for tissue‐specific and developmental expression analysis of LmdsRNases were adapted from those described by Song et al . In brief, ten different tissues were dissected from 3‐day‐old fifth‐instar nymphs (N5D3), including integument, fat bodies, foregut, gastric caeca, midgut, hindgut, Malpighian tubules, hemolymph, testis and ovary.…”

Section: Methodsmentioning

confidence: 99%

“…Rpl32 and Rp49 or EF1α were used as reference genes for tissue‐specific or developmental stage‐specific expression, respectively. As described by Song et al ., five candidate genes ( Rpl32 , Rp49 , EF1a , GAPDH and b‐actin ) were analyzed using geNorm and NormFinder software to determine the best reference genes for our use. Statistical significance was calculated using a one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test ( P < 0.05, SPSS software, SPSS Inc., Chicago, IL, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Western corn rootworm ( Diabrotica virgifera virgifera ) and Colorado potato beetle ( Leptinotarsa decemlineata ) are very susceptible to dsRNA delivery via both injection and oral approaches, whereas European corn borer ( Ostrinia nubilalis ) and tobacco hornworm ( Manduca sexta ) exhibit highly inefficient RNAi responses regardless of how dsRNA is delivered . By contrast, desert locust ( Schistocerca gregaria ), L. migratoria and German cockroach ( Blattella germanica ) are susceptible to administration of dsRNA by injection but not feeding . The inefficiency in response to dsRNA via feeding in these insect pests has severely limited the widespread implementation of RNAi technology in the field.…”

Section: Introductionmentioning

confidence: 99%

“…Studies in four representative species from different insect orders (Orthoptera, Blattaria, Lepidoptera and Coleoptera) were used for comparing dsRNA stability in hemolymph and midgut fluid, and researchers found a correlation between dsRNA stability and RNAi efficiency . There is also growing evidence to suggest that degradation of dsRNA prior to uptake is an important factor that limits the effectiveness of RNAi . Accordingly, an increasing number of research projects are focusing on dsRNA degradation by insect dsRNA nucleases (dsRNases).…”

Section: Introductionmentioning

confidence: 99%

“…In S. gregaria , four dsRNase genes have been identified that limit RNAi efficiency, all of them with high expression in midgut . Our group has previously identified two dsRNases expressed in midgut from L. migratoria , but only LmdsRNase2 was capable of degrading dsRNA quickly and limiting oral RNAi efficiency . Because S. gregaria has four dsRNases and is closely related to L. migratoria , we hypothesized that L. migratoria would also have four dsRNases and decided to use a genome‐wide search to identify the others.…”

Section: Introductionmentioning

confidence: 99%

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

Song

Fan

Zhang

et al. 2019

Pest Management Science

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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

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Song

Fan

Zhang

et al. 2019

Pest Management Science

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Identification and functional analysis of dsRNases in spotted‐wing drosophila, Drosophila suzukii

Yoon

Ahn

Flinn

et al. 2021

Arch Insect Biochem Physiol

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RNAi efficiency in insects is different from species to species; some species in Coleoptera are relatively more amenable to RNA interference (RNAi) than other species.One of the major factors is the presence of dsRNAdegrading enzymes, called dsRNases, in saliva, gut, or hemolymph in insects, which degrade the double-stranded RNA (dsRNA) introduced, resulting in the low efficacy of RNAi. In this study, we report a dsRNA-degrading activity in the gut homogenates from the spotted-wing drosophila, Drosophila suzukii, by ex vivo assay. Then, we identified two Drosophila suzukii dsRNase genes, named DrosudsRNase1 and DrosudsRNase2. In silico analysis shows that the gene structures are similar to dsRNases found in other insects.When dsRNases expressed in Sf9 cells were compared for their dsRNA degrading activities, dsRNase1 was more vital than dsRNase2. Both dsRNases were expressed highly and exclusively in the gut compared to the rest of body. Also, they were highly expressed during larval and adult stages but not in embryonic and pupal stages, suggesting the dsRNases protect foreign RNA molecules received during the feeding periods. DsRNase1 was expressed at a higher level in adults, whereas dsRNase2 showed more expression in early larvae. Our study on the tissue and development-This article has been contributed to by US Government employees and their work is in the public domain in the USA.

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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

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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.

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A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Cited by 83 publications

References 52 publications

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

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

Identification and functional analysis of dsRNases in spotted‐wing drosophila, Drosophila suzukii

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

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