## 2 Production of Recombinant dsRNA in Microorganisms
### Other Microorganisms for dsRNA Production  
There have been studies on the use of the probiotic bacterium _Lactobacillus plantarum_ and the oleaginous yeast _Yarrovia lipolytica_ as production hosts for dsRNAs that can be used to help alleviate viral and bacterial diseases of marine shrimp [84, 85]. Saksmerprome's research group expressed in _L. plantarum_ a hairpin-type dsRNA to function against shrimp YHV. They had already orally administered YHV-dsRNA expressed in _E. coli_ to shrimp and confirmed its effectiveness as a potential antiviral agent [86], but lactic acid bacterium are more suitable for application in the shrimp farming environment. _L. plantarum_ is a plant-derived lactobacillus that is found in many fermented food products and is nonpathogenic to humans. Lactobacillus have already been applied to farmed shrimp to enhance the immunity of shrimp toward _Vibrio_ and other pathogenic bacteria. The target RNA was expressed in _L. plantarum_ using the xylose-induced _xylIA_ promoter; the production was approximately 1.66 ng per 7 \(\times\) 10\({}^{11}\) colony-forming units of the lactobacillus [84], although the bacterial strain used for the dsRNA production was not mutated in dsRNA-degrading enzyme genes. Thus, it was shown that oral administration of _L. plantarum_ containing YHV-specific dsRNA in shrimp improved their survival and reduced YHV propagation. Although this dsRNA accumulation was estimated to be lower than that of the production of similar hairpin-type RNAs in _E. coli_, it is still a promising method for disease prevention in cultured shrimp because the lactobacilli themselves, which are administered with the produced RNA, are expected to have a probiotic effect on the shrimp. Another host microorganism for the production of hpRNA for disease prevention in cultured shrimp is _Y. lipolytica_, a GRAS microorganism that does not produce endotoxins and can be grown on low-cost substrates. In addition, the high protein content of _Y. lipolytica_ is expected to contribute to the growth of shrimp that feed on the yeast cells. Furthermore, yeasts including _Y. lipolytica_ have been used as immunostimulants to enhance the immune response of aquaculture organisms to pathogenic bacteria and viruses. [PERSON] al. produced hpRNA in _Y. lipolytica_ to suppress the virulent activity of WSSV in the white leg shrimp _Litopenaeus vannamei_[85]. The selected target was the _orf89_ gene of WSSV, a region of approximately 0.4 kbp, of which was used as the dsRNA portion to be expressed. The promoter for the hpRNA expression was the constitutive promoter of _Y. lipolytica XPR2_, a gene encoding an extracellular alkaline protease (the major protein secreted by this yeast), and the hpRNA expression system was constructed on a plasmid. The RNase III gene (_Rnt1_) of the host _Y. lipolytica_ strain remained intact. In the end, the maximum amount of hpRNA produced was 182 ng/L, but when the total RNA extracted from the producing strain was injected intramuscularly into shrimp, the WSSV-dependent mortality rate improved by approximately 25%. Furthermore, there is research where microorganisms symbiotic in the gut of a target pest were made to produce dsRNA for RNAi action. [PERSON] et al. [87] selected the blood-sucking bug _Rhodrius prolixus_ and the globally invasive polyphagous agricultural pest Western thrips (_Franklinella occidentalis_) as target pests, expressed dsRNAs within symbiotic bacteria of each pest, and administered the symbiotic bacteria to the corresponding pests. For the former, _Rhodococcus rhodii_ strain LMG5362, which has evolved to survive in the midgut of _Rhodrius prolixus_, was employed as the host microbe, and dsRNA targeting the vittellogenin (_Vg_) gene of the pest was expressed in the strain lacking the RNase III gene. For the latter pest, an RNase III-deficient strain of the Gram-negative facultative symbiotic bacterium, named Bfo2 [88; 89], was created to produce dsRNA for RNAi of the \(\alpha\)-tubulin gene (_Tub_) of _F. occidentalis_.