## 2 Production of Recombinant dsRNA in Microorganisms
### Other Microorganisms for dsRNA Production
When the respective dsRNA-producing symbiotic strains were orally administered to the respective pests, a significant decrease in fecundity was observed in _R. prolixus_, and a significant increase in mortality was confirmed in _F. occidentalis_. This method of using symbiotic microorganisms producing a desired dsRNA may result in a continuous supply of dsRNA in the gut of that pest. This could be a very effective way to ensure RNAi action against the target pest. However, since this method also involves the release of recombinant bacteria into the environment, there are many issues from a biosafety perspective that will need to be addressed. The baker's yeast _Saccharomyces cerevisiae_ has also been used to produce target RNA for RNAi pesticides to combat mosquitoes. [PERSON] and colleagues studied the production in _S. cerevisiae_ of short hpRNA targeting the _Aedes aegypti_ synaptotagmin (_syt_) gene (a gene involved in the release of neurotransmitters in the larval neural synapse) [90; 91]. The yeast cells containing the produced target dsRNA were then heat-sterilized, dried, and used as a mosquito larvicide. Yeast acts as an oviposition attractant for gravid adult females of mosquitoes and _S. cerevisiae_ is also a source of nutrition and a strong odorant attractant for hatched mosquito larvae; therefore, the use of such formulations is expected to attract mosquito larvae and induce them to ingest the formulations, resulting in the death of the targeted mosquito larvae caused by the dsRNA contained in the yeast. The DNA region encoding the target hpRNA was placed under the control of the strong inducible _Gal1_ promoter, and a _cyc1_-terminator was used to ensure termination of transcription [90; 91]. To construct the hpRNA-producing strain, two copies of the hpRNA-expression unit were integrated into the genome of the strain _S. cerevisiae_ CEN.PK. Although _S. cerevisiae_ lacks endoribonuclease Dicer, which cleaves dsRNA into short dsRNA fragments, it has a gene encoding Rntp1, which shows RNase III-like activity; however, stem-loop type RNAs may not be recognized as substrates by Rntp1. The authors prepared yeast cells containing the expressed target hpRNA in a dried, inactivated tablet formulation and delivered it to mosquito larvae, which resulted in suppression of _syt_ gene expression, defects in the neural synapse, and high rates of larval _A. aegypti_ mortality. Interestingly, the sequence they employed for RNAi in _A. aegypti_ also induced RNAi effects in related mosquito larvae with the same sequence of the corresponding gene, leading to their death. This suggests that yeast expressing biorationally-designed hpRNAs may be used to combat multiple mosquito vectors of human diseases. The methanol-using yeast _Pichia pastoris_ has been employed as a host for the production of hpRNAs (ca. 120 bp) that induce RNAi effects against the expression of the juvenile hormone (JH) acid methyl transferase gene (_jmtA_) involved in JH biosynthesis in _A. aegypti_[92]. Although the productivity of the target hpRNA in the yeast was not specified, mosquito larvae orally administered heat-sterilized yeast cells containing the hpRNA showed delayed hatching into adults and high mortality. _P. pastoris_ is an excellent host for producing heterologous proteins, and it has already been reported that it is possible to express _Bacillus thuringiensis_ Cry toxin in this host, which made it toxic to _A. aegypti_ larvae [93]. Hence, _P. pastoris_ co-expressing Cry toxin and a dsRNA of interest is expected to be effective in killing target mosquito larvae.