Development of RNAi method for screening candidate genes to control emerald ash borer, Agrilus planipennis

Abstract

The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, β subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.

Oral RNAi of diap1 in a pest results in rapid reduction of crop damage

“…Similar to the induction of the lethal effect of diap1 f-RNAi in previous studies 21,23,24,25 , we showed that in H. vigintioctopunctata , the effect of diap1 f-RNAi on feeding disorder has dose-sensitivity for dsRNA (Fig. 2d).…”

“…3). This lethal effect is rapid compared to that of the previous study in A. planipennis (10 days for 78% mortality) 24 . Since all of the diap1 f-RNAi larvae were dead within 1-4 days after the feeding cessation, this rapid lethality may have occurred as a result of the acute feeding cessation, either indirectly or directly.…”

“…For these reasons, diap1 is one of the candidate target gene for RNAi pesticides and has been investigated in various pests. So far, RNAi of the diap1 gene by injecting the dsRNA into the hemocoel of pest has been shown to induce over 70% mortality in T. castaneum (Coleoptera) 14 , Anoplophora glabripennis (Coleoptera) 20 , Musca domestica (Diptera) 21 , Delia radicum (Diptera) 21 , Heliothis virescens (Lepidoptera) 22 , Lygus lineolaris (Hemiptera) 23 and Apolygus lucorum (Hemiptera) 24 . Diap1 silencing by f-RNAi has also been reported to induce 30– 78% of mortality in Agrilus planipennis 24 , T. castaneum 14 , and Diaphorina citri 25 .…”

“…This lethal effect of diap1 by f-RNAi is also dose-dependent. For example, in A. planipennis , the mortality of diap1 f-RNAi using 1, 6 and 10 µg/µl dsRNA is 30, 35 and 78%, respectively 24 . In M. domestica and D. radicum , it was reported that diap1 gene silencing by injection of the dsRNA induced lethality but the f-RNAi assay did not 21 .…”

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

“…11,49,50 The level of sensitivity to RNAi by microinjection and oral exposure can be variable among insects within the same order. For example, in the Coleoptera order, D. virgifera virgifera, T. castaneum, Leptinotarsa decemlineata, Meligethes aeneus, Rhynchophorus ferrugineus, C. puncticollis, C. brunneus and A. planipennis are sensitive to both methods of dsRNA delivery, 29,30,49,[51][52][53][54][55] whereas Anthonomus grandis, Anthonomus eugenii and Aethina tumida are recalcitrant to RNAi by oral exposure. [56][57][58] These studies demonstrate the importance of evaluating the RNAi sensitivity by microinjection as well as by feeding in the target insect species since it is not possible to predict the RNAi effectiveness based on the RNAi response found in other insects, even in closely related species.…”