Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors
The Oriental Armyworm Mythimna separata Walker (Lepidoptera: Noctuidae) is one of the most important pests of cereal crops including corn, wheat and rice. In recent years, outbreaks of M. separata are severely threatening corn production in Northern China. To date, control strategy toward this insect pest heavily relies on the usage of chemical pesticides [1, 2]. Besides the environmental pollution and non-specific toxicity of chemical pesticide, insect resistance toward the chemicals is also becoming a serious problem in majority of the insect pests including M. separata. Bt (Bacillus thuringiensis) toxin engineered to be expressed in transgenes is an alternative for the chemicals, but it still encountered the problem of broad-spectrum toxicity and evolution of insect resistance. In fact, development of resistance against Bt toxin was also reported for M. separata [3, 4].
RNAi is a natural gene regulation and antiviral defense system of eukaryotic cells. RNAi has been exploited as a technology for silencing of target genes via sequence-specific manner [5, 6]. There are already many examples and even practical implementation of RNAi-based technologies for development of anti-pathogenic crops. RNAi-based strategies have been used for development of genetically modified plants resistant to variety of disease agents such as bacteria, nematode and virus [7–9]. In 2007, two research groups reported the development of transgenic corn and cotton resistant to insect herbivores using RNAi technology, providing a species-specific and environmentally sound anti-insect strategy [10, 11]. Since then, successful RNAi experiments have also been reported in different insect species including some lepidopteran insects [12, 13]. To screen large-scale insect genes to identify an optimal candidate target which is effective and species-specific, there is a need to develop high-throughput method for future RNAi-based control strategy in each insect species of interest.
In this study, we propose to determine the possibility of using RNAi technology to knockdown the expression of M. separata genes. To date, the sources of interfering RNAs (dsRNA or siRNA) commonly utilized in insect RNAi study include in vitro synthesized dsRNA, virus-expressed dsRNA or siRNA, or transgenically expressed hairpin RNA, all of which are costly synthetic molecules or are produced from time-consuming laborious procedures. To overcome the shortages of these methods, we used here an oral delivery of bacterially derived sequence specific dsRNAs to silence M. separata genes. We chose chitinase encoding genes as targets in this preliminary effort. Chitin (C8H13O5N)n is a long-chain polymer of a N-acetylglucosamine linked by ?-1,4-glycosidic bond. In insects, chitin is a main structural component which lines the cuticle of foregut, hindgut, trachea, and PM (peritrophic matrix) of the midgut, and is playing structural and protective roles in insect. Chitinases, also known as chitin synthases, are family 18 glycosyl hydrolases that break down glycosidic bonds in chitin and are responsible for the hydrolysis and synthesis of chitin [14–16]. Chitinase gene has long been studied and used as the biocontrol molecule agent toward biotic stresses including fungi and insects [17, 18]. Functions of chitinase genes were heavily investigated in different insect species. Several lines of evidence suggested that RNAi silencing of chitinase genes led to strong phenotypic effects in different insect species including Ostrinia nubilalis (lepidopteran), Tribolium castaneum (Coleopteran), Anopheles gambiae (Dipteran), and Locusta migratoria (Neopteran) [19–22]. Two chitinase homologs were identified in M. separata. We utilized L4440 vector to prepare dsRNAs in the E.coli cells for these chitinase genes, and directly fed them to recipient insects by mixing with artificial diet. Molecular evidence suggested that oral delivery of bacterial dsRNA caused the knockdown of target chitinase gene expression, as was also evidenced by the appearance of sequence-specific siRNA in the recipient insect. Mortality increase and body weight decline were also observed in recipient insects. Our data provided both theoretical and applicational direction toward the use of RNAi as a reverse genetic tool for studying the function of specific genes and also as a biocontrol method for the control of this insect species.