Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata , but not in the desert locust, Schistocerca gregaria

BACKGROUND RNA interference (RNAi) is widely used in entomological research for functional analysis of genes and is being considered as a new tool for insect pest management. Sri Lanka weevil (SLW) is a highly polyphagous pest of agronomically important plants, but currently only a few control methods are available for this insect. RESULTS In the present study, we evaluated the stability of candidate reference genes β‐ACT, α‐TUB, EF1‐α, RPL12 and GAPDH, and identified EF1‐α as the most reliable for gene expression normalization. Four target genes involved in different cellular processes, including Prosα2, RPS13, Snf7 and V‐ATPase A were selected to evaluate whether RNAi response in SLW adults can be triggered by microinjection and oral feeding of their double‐stranded RNAs (dsRNAs). Three days after injection of the dsRNAs for the target genes, their transcript levels were significantly reduced (up to 91.4%) when compared to the control. Additionally, weevils fed with the target dsRNAs showed significant decreases in gene transcript levels and significant mortality was observed in insects treated with Prosα2 and Snf7 dsRNAs (78.6 to 92.7%). CONCLUSION Our data demonstrate that microinjection and feeding of dsRNA produce a strong RNAi response in SLW, indicating that RNAi‐based strategies could be explored to develop a selective and environmentally safe control method against SLW. © 2019 Society of Chemical Industry

Section: Discussionmentioning

confidence: 99%

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

Pinheiro

Taylor

et al. 2019

“…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%

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

Song

Fan

Zhang

et al. 2019

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

“…also reported successful KD of vATPase expression in T. castaneum when the dsRNA was orally delivered. However, in a more recent study, Spit et al . failed to observe any knockdown effects via oral delivery, despite using the same insect species.…”

Section: Discussionmentioning

confidence: 94%

“…However, the RNAi response is often reduced due to mechanisms of transporting the dsRNA from within the insect gut. The longevity of dsRNA in the midgut of the insect may be strain dependent and be subject to the action of gut nucleases on the ingested dsRNA . One reason for this strong response is that the genome of several coleopteran insects is known to include two or even three Sid‐1 ‐like genes, which are necessary for RNAi pathways .…”

Section: Discussionmentioning

confidence: 99%

Targeting the potassium ion channel genes SK and SH as a novel approach for control of insect pests: efficacy and biosafety

Alshukri

Astarita

Al‐Esawy

et al. 2019

BACKGROUND Potassium ion channels play a critical role in the generation of electrical signals and thus provide potential targets for control of insect pests by RNA interference. RESULTS Genes encoding the small conductance calcium‐activated potassium channel (SK) and the voltage‐gated potassium channel (SH) were knocked down in Tribolium castaneum by injection and oral delivery of dsRNA (dsTcSK and dsTcSH, respectively). Irrespective of the delivery mechanism a dose‐dependent effect was observed for knockdown (KD) of gene expression and insect mortality for both genes. Larvae fed a 400 ng dsRNA mg–1 diet showed significant gene (P < 0.05) knockdown (98% and 83%) for SK and SH, respectively, with corresponding mortalities of 100% and 98% after 7 days. When injected (248.4 ng larva–1), gene KD was 99% and 98% for SK and SH, causing 100% and 73.4% mortality, respectively. All developmental stages tested (larvae, early‐ and late‐stage pupae and adults) showed an RNAi‐sensitive response for both genes. LC50 values were lower for SK than SH, irrespective of delivery method, demonstrating that the knockdown of SK had a greater effect on larval mortality. Biosafety studies using adult honeybee Apis mellifera showed that there were no significant differences either in expression levels or mortality of honeybees orally dosed with dsTcSK and dsTcSH compared to control‐fed bees. Similarly, there was no significant difference in the titre of deformed wing virus, used as a measure of immune suppression, between experimental and control bees. CONCLUSION This study demonstrates the potential of using RNAi targeting neural receptors as a technology for the control of T. castaneum. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.