Fate of Ingested RNA in the Two-Spotted Spider Mite

RNA interference, or RNAi, is a gene regulation mechanism that uses small noncoding RNAs (sRNAs) to silence the expression of certain genes. The application of RNAi has been extended to insect pest control. The two-spotted spider mite Tetranychus urticae is a persistent agricultural pest that tends to develop pesticide resistance at an alarming rate, making it a perfect candidate for RNAi technology development. It was hypothesized that unique sRNAs could be isolated from RNA soaked spider mites, and new synthetic RNAs could be synthesized to elicit greater knockdown than was achieved in previous studies. To perform this research, a small RNA column filtration method that allows for enrichment of the organism’s endogenous functional RNAi effectors, which are small RNAs from 18 to 31 nucleotides long, was used on mites soaked in different RNA molecules and fed on four diverse species of plants. After sequencing the column-extracted RNA fraction, there was a predominance of 18-21 nt reads with a 1-2 nt offset. This is consistent with RNA processing by an enzyme called Dicer and is observed in other arthropods. However, antisense reads mapped to regions outside of the double-stranded RNA (dsRNA) showed a clear “T” at the 5’ end and an “A” at the 10th base from the 5’ end, which is suggestive of a different processing method of RNAi that is not dependent on Dicer and not seen in insects. It was determined that the majority of the recovered RNA fragments were derived from plant chloroplast RNA, which is slower to degrade during mite digestion of plant materials. Common reoccurring structures from the plant chloroplast RNA data collected from mite specimens were then curated. Synthetic RNA structures were created and fed to the mites in an attempt to elicit a superior inhibition of target gene expression. Most synthetic RNAs fed to mites exhibited knockdown equal to or greater than actin-targeted dsRNA fed to mites, indicating that standard RNAi strategies could be improved. This work highlights the changing nature of RNAi during animal evolution and the value of detailed analysis in a particular species to formulate an optimal strategy for controlling gene expression with this technology