## 3 Conclusions and Future Perspectives  
The RNAi phenomenon [2] was first reported in 1998 in _Caenorhabditis elegans_. Since then, RNAi technology has made great progress and is applied in basic research, such as for analysis of gene function, and in applied research in fields from medicine to agriculture. In 2007, a potential innovative pesticide, an RNAi-based pesticide that inhibited the growth of target agricultural pests, was developed [94]. However, a major barrier to the practical use of RNAi-based agents is the production cost. The amount of these pesticides required for use in practical situations has not yet been studied in detail, but recent estimates suggest that approximately 2-10 g of RNA per hectare may be needed [95; 96] (although this amount depends significantly on the efficiency of the RNAi action of the dsRNA in the target species and the stability of the formulated dsRNA product in the environment in which it is applied). Thus, it is also crucial to develop more effective application methods and formulations of dsRNA [6; 16; 97]. Recently, [PERSON] et al. [98] examined effective RNA doses for CPB from several regions of Europe to determine their differential susceptibility to RNAi action in foliar spray application. At the laboratory level, dsRNA targeting the actin-encoding gene of CPB resulted in almost 100% mortality with a dose equivalent to 0.96 g dsRNA/ha for the most susceptible sample species, although the least susceptible required approximately 7.5 times that amount of dsRNA. The production cost of RNAs has been decreasing year-by-year as manufacturing technology improves [17; 99]. A biotechnology company called RNAgri (formerly APSE) has developed a technology that can produce dsRNA at a cost of USD 1-2/g RNA. Their production system claims to be based on accumulation of target dsRNA in virus-like particles (VLPs) in _E. coli_. The coat protein (CP) of bacteriophage MS2 and the precursor of the target dsRNA containing packaging sites (PSs) are simultaneously expressed. The CP and PS bind and encapsulate the RNA in VLPs [100]. RNA production in GreenLight Biosciences' cell-free system for the synthesis of biomolecules is also attracting attention. In general, the main barrier to RNA production in cell-free systems is the high cost of the four ribonucleoside triphosphates (NTPs) that are the building blocks of RNA. This company has enabled low-cost production of target RNAs by degrading nucleic acids derived from yeast biomass into the constituent monomers using nucleases; phosphorylating and regenerating the NTPs, and then using them as substrates for RNA synthesis in the cell-free system [101]. The microbial production methods for dsRNAs described in this review can be used to mass-produce target dsRNAs from sustainable raw materials. Some representative reports describing the productivity of target dsRNAs are summarized in Table 1. The basic characteristics of the production methods of dsRNA by the representative microbes discussed here are summarized in Table 2. The methods applied must be tailored to specific needs; it will be necessary to establish production methods optimized for each RNAi-based product according to its use and practical requirements. Aspects such as using the producing microorganism as a capsule to protect the target dsRNA from unintended degradation, or using the microorganism itself as a supplemental nutrient for aquaculture, may add value to RNAi-based agents. RNAi-based products could contribute to solutions to various problems related to safe and reliable production of farm products now and in the future; in other words, the realization of \"sustainable farming\". RNAi-based pesticides will be helpful in conserving and maintaining natural ecosystems and will greatly assist in establishing \"sustainable agriculture\". In aquaculture, the antiviral effects of dsRNA species will reduce the extensive use of chemical antivirals, which have a wide spectrum of activity, helping with the establishment of \"sustainable aquaculture\".