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.
Highlights
The emerald ash borer (EAB), Agrilus planipennis, is an invasive forest pest that has caused the death of hundreds of millions of urban and forested ash trees (Fraxinus spp.) in North America[1]
A distinct band corresponding to the size of small interference RNAs (siRNAs) was observed in the RNA isolated from EAB larvae and showed the same intensity as observed for the positive control Colorado Potato Beetle (CPB, Leptinotarsa decemlineata), known to possess efficient RNA interference (RNAi) machinery[22] (Fig. 1), demonstrating that EAB contains machinery to transport Double-stranded RNA (dsRNA) into cells and convert it to siRNA
We demonstrate the existence of functional RNAi machinery in EAB larvae and develop a bioassay for rapid screening of target genes for use in RNAi-based control of this pest
Summary
The emerald ash borer (EAB), Agrilus planipennis, is an invasive forest pest that has caused the death of hundreds of millions of urban and forested ash trees (Fraxinus spp.) in North America[1]. The availability of large larvae is limiting mainly because: 1) the feeding behavior of endophagous insects such as EAB, which remain beneath the bark and develop inside the tree during all larval stages and 2) the lack of an artificial diet, which make efficient rearing of larvae in the laboratory outside host trees problematic. A feeding assay using neonate larvae would be an efficient and useful method to deliver dsRNA and rapidly screen potential target genes. Developing an oral delivery method using neonate larvae allows evaluation of the ability of EAB to process dsRNA and evaluation of RNAi effects throughout larval, pupal and adult stages. The primary goals of our study were to evaluate the efficiency of RNAi machinery to process dsRNA into siRNAs and to develop an oral delivery method that would allow screening and selection of the most efficient candidate genes that cause larval mortality.
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