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Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
Published on: February 27, 2012
Bacteria-mediated RNAi for managing fall webworm, Hyphantria cunea: screening target genes and analyzing lethal
Xun Zhang1, Zhizhi Fan1, Rong Zhang1
1Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China.
Background:
The fall webworm, Hyphantria cunea, an invasive forest pest found worldwide, causes serious ecological and economic damage. Currently, the application of chemical pesticides is the most widely used strategy for H. cunea management. However, long-term pesticide use leads to pest resistance, phytotoxicity, human poisoning, and environmental deterioration. RNA interference (RNAi) technology may provide an environmentally friendly and cost-effective option for H. cunea control. However, effective RNAi targets and application methods for H. cunea are lacking.
Results:
We screened and obtained two highly effective RNAi targets, vATPase A (V-type proton ATPase catalytic subunit A) and Rop (Ras opposite), from 23 candidate genes, using initial and repeat screening tests with the double-stranded RNA (dsRNA) injection method. RNAi against these two genes was effective in suppressing each target messenger RNA level and interfering with larval growth, leading to significant larval mortality and pupal abnormality. For massive production of dsRNA and practical application of RNAi technology in H. cunea, transformed bacteria expressing dsRNAs of these two genes were prepared using the L4440 expression vector and HT115 strain of Escherichia coli. Oral administration of bacterially expressed dsRNA targeting vATPase A and Rop genes showed high mortality and the same malformed phenotype as the injection treatment. To further investigate the lethal effects of targeting these two genes on larval development, transcriptome sequencing (RNA-seq) was performed on RNAi samples. The results demonstrated disorders in multiple metabolic pathways, and the expression levels of most genes related to insect cuticle metabolism were significantly different, which may directly threaten insect survival. In addition, some new findings were obtained via RNA-seq analysis; for example, the progesterone-mediated oocyte maturation and oocyte meiosis processes were significantly different after silencing vATPase A, and the insect olfactory protein-related genes were significantly downregulated after dsHcRop treatment.
Conclusion:
vATPase A and Rop are two highly effective RNAi-mediated lethal genes in H. cunea that regulate insect growth via multiple metabolic pathways. Oral delivery of bacterially expressed dsRNA specific to vATPase A and Rop can potentially be used for RNAi-based H. cunea management. This is the first study to apply bacteria-mediated RNAi for the control of this invasive pest, which is a major step forward in the application of the RNAi technology in H. cunea. © 2022 Society of Chemical Industry.
