Neuronal signalling molecules as targets for green peach aphid (Myzus persicae) control via RNA interference

The Green peach aphid (GPA) (Myzus persicae) is an important insect pest which causes substantial economic losses to many glasshouse and field crops. Alarmingly, GPAs are becoming resistant to many conventional insecticides, and this trend indicates that there is a real need to develop alternative strategies to protect crops from this insect pest. The aim of this research project was to investigate the potential of RNA interference (RNAi) technology as a strategy to control GPAs. Genes involved in insect neuronal signalling pathways were selected as RNAi targets. Bioinformatic analysis tools were used to identify ESTs putatively encoding sixty-three Neuronal Signalling Molecules (NSMs) from publicly available sequences and from GPA transcriptome data generated in-house. The NSMs included 30 Neuropeptides (NPs), 24 Neuropeptide Receptors (NPRs), and 9 Biogenic Amine Receptors (BARs). From these, transcripts for 24 NSMs were selected for in vitro RNAi assays to determine their suitability as targets for host-induced gene silencing (HIGS). Successful ingestion of dsRNA of target genes by nymphs was confirmed using the presence of a neutral red dye in the body of aphids, incorporated in the dsRNA+30% sucrose diet. Silencing effects of nine genes, e.g. Ecdysis triggering Hormone (eth), Capability (capa), Juvenille hormone binding protein (jhbp), Leucokinin (lk), Crustacean Cardioactive Peptide (ccap), Octopamine beta 3R (octβ3r), Muscarinic acetylcholine receptor 3 (mAChrM3), Short NPF (snpf ) and Insulin-related peptide 2/3 (irp2/3) were obvious 24 hours after feeding on the dsRNA diet. RNAi phenotypes included incomplete moulting, uncoordinated movement, lethargy, paralysis and lethality, whereas the control GPAs exposed to no-dsRNA and dsRNA of the green fluorescent protein (GFP) gene of the jellyfish, Aequorea victoria moved normally, showing no obvious effects of the treatment. For GPAs treated with dsRNAs of six of these genes (ccap, capa, mAChrM3, lk, octβ3r and irp2/3), silencing also significantly affected survival and fecundity when the aphids were later transferred to tobacco plants for 12 days. Silencing of ccap, capa, irp2/3, lk and octβ3r resulted in 100% lethal phenotypes on the tobacco plants. Knockdown of dscapar1 and dsnplp1 also affected GPA reproduction although no visible effects were observed 24 hours after ingestion of dsRNA. The effectiveness of nine of the 24 genes (ccap, jhbp, nplp1, capar1, irp5, lk, octβ3r, snpf and opsin) as targets for RNAi control of GPAs were evaluated using HIGS, in which two model plants, tobacco and Arabidopsis thaliana were used. Transgenic tobacco plants carrying hairpins (hp) of all nine GPA genes were developed of which those for six genes (except for lk, octβ3r and snpf), were advanced to the T2 generation, and used for GPA bioassays. In T1 tobacco, the mean population was reduced by 97% for hpoctβ3r event 2 and event 5, while significantly lower GPA populations were recorded for all the lines expressing hpccap, hpnplp1 and hplk after 12 days (p<0.05). As for the T1 generation, most of the T2 transgenic events also supported significantly fewer GPA nymphs, with reductions in numbers ranging from 3% to 69%. GPAs feeding on events of hpccap, hpnplp1 and hplk produced fewest nymphs, as was observed for T1 generation. In addition, an 80% to 100% reduction in GPAs was evident for T2 transgenic Arabidopsis plants expressing dsccap, dsjhbp and dsnplp1, and complete mortality was recorded for the hpccap event 3. The results obtained from two transgenic generations and two model plants therefore indicate that the genes studied were vital for the GPA life cycle and knocking down of these genes affects their fecundity or survival. An in vitro study was also conducted to evaluate the effects of silencing five different lengths of dsRNA from different regions of the same EST putatively encoding the JHBP protein, as well as siRNAs of the gene generated in vitro from digestion with an RNAseIII enzyme. The longest dsRNA (284 bp) was the most effective in inducing RNAi effects on treated nymphs, since there were more restricted movements in aphids 24 hours after exposure, and the fewest offspring were produced in the longer-term. One of the shorter dsRNAs (86 bp long, not the shortest,70 bp), also significantly reduced GPA movement, survival and reproduction at levels similar to that of the longest dsRNA. These results show that RNAi effects can vary with the target region from which the hp dsRNA is derived and in this case silencing was more effective for one of the sequences derived from the 3´ region. This study indicates that both the length of the dsRNA and the specific sequence chosen can influence the effectiveness of RNAi. This project provides new information on GPA neuronal genes as novel candidates for its control via gene silencing. It also offers additional data to achieve better RNAi effects related to the target sequence selected. The in planta RNAi study also demonstrated that RNAi can be used as a new strategy to control this important crop pest, and its use, either alone or in combination with other gene targets, is discussed.