## 3 Conclusions and Future Perspectives
is expected to continue, but, in the future, the total manufacturing cost of RNAi-based products, including these delivery formulations, will have to be reduced to a level where they can be used commercially. We have mentioned that high productivity of dsRNA by the microbe is crucial for economic efficiency of the production of target dsRNA for RNAi pesticides. An ideal production strain might be a dsRNA-producing microbe that enables the RNAi effect to be effectively demonstrated against pests when dsRNA-containing killed-mic probes are fed to the target pest. RNA molecules are inherently chemically labile and are susceptible to degradation by the ribonucleases of various organisms. In addition, when naked dsRNA is topically applied to plants by foliar spraying, the RNA does not persist on the leaf surface and the period during which the dsRNA functions effectively in RNAi may be short. To address this issue, it has been demonstrated that by loading the target dsRNA onto a special layered double hydroxide (LDH) clay nanosheet, the stability of the dsRNA on the leaf surface is maintained, and sustained dsRNA release from the clay complex is achieved [16; 105; 106]. Thus, LDH can extend the effective period of topically applied dsRNA for inducing RNAi against plant viruses. Such innovations for more effective application of dsRNAs will contribute greatly to reduction of the total costs of RNAi-based products. RNAi-based pesticides are characterized by the principle that the dsRNA only works on mRNA with the identical nucleotide sequence. Therefore, when a mutation occurs in the target gene of a target pest (but substantial functional expression of the mutated gene is retained), the originally designed dsRNA may no longer be effective against the pest. To cope with such a situation, it is essentially sufficient to change the dsRNA sequence such that RNAi can be performed on the altered sequence, although the issue of off-target effects should be revisited. However, a case was reported in which genetic mutation in more fundamental aspects of the target pest rendered RNA-based pesticides ineffective; the occurrence of such mutations had been predicted. A study of acquired resistance to RNAi-based pesticides by [PERSON] et al. [107] using WCR indicated that the resistance was due to a defect in dsRNA uptake function in gut cells of the pest. It was also suggested that dsRNA uptake into cells other than gut cells and systemic spread of RNAi silencing signals may also have been impaired. In addition, it is conceivable that mutations in genes related to the core RNAi pathway leading to defects in dsRNA processing and/or RNA-induced silencing complex formation could contribute to the acquisition of resistance to RNAi-based pesticides. To address these issues, further elucidation of the mechanism of action of RNAi in pests will be crucial. In the meantime, it will be important to delay the emergence of dsRNA-resistant pests as much as possible by developing appropriate Insecticide Resistance Management strategies, including the use of other compounds with different modes of action (e.g., _Bacillus thuringiensis_ (_Bt_)-derived agents and some existing biological pesticides) and physical crop segregation. Indeed, to enhance the insecticidal activity of _Bt_-based biopesticides, [PERSON] et al. exploited the use of _E. coli_ as a delivery vector for dsRNA that targeted the immune system of a pest of interest, the notcuid moth _Spodoptera litterorialis_[108]; dsRNA-induced immunosuppression was shown to enhance the insecticidal effect of the _Bt_-based biopesticides. Another issue in the application of RNAi-based products is the possibility of off-target effects of siRNA. This is when short dsRNAs generated from a long dsRNA silence genes other than the target gene within the target pest [109]. As the dsRNAs for RNAi action are only approximately 21 to 23 bp long, the possibility of off-target binding sites in genomic DNA is real.