## 2 Production of Recombinant dsRNA in Microorganisms
### Chlamydomonasomonas reinhardtii  
_Chl. reinhardtii_, a eukaryotic unicellular green microcalga, is a potential industrial bioproduction platform for various products including recombinant proteins, enzymes, and biofuels [76; 77; 78]. Recently, the expression of recombinant dsRNA in _Chl. reinhardtii_ has also been investigated. Since _Chl. reinhardtii_ typically inhabits freshwater environments such as ponds and lakes, research has been conducted to use this microbe producing target dsRNAs to confer resistance to viral diseases in edible shrimp in aquaculture. Viral infection such as by WSSV or YHV results in extensive production losses in shrimp farming [79]. The expression of target recombinant genes in the microcalga can be achieved by incorporating the desired dsRNA expression unit into the nuclear DNA, or into the chloroplast DNA [76; 80]. For the control of YHV, an RNA virus that infects peneaid shrimp including _Panaeu svanamei_, [PERSON] et al. produced dsRNA (hpRNA) that can act specifically on the expression of RNA-dependent RNA polymerase (RdRp) of YHV, through integration of the dsRNA expression unit into the nuclear genome of _Chl. reinhardtii_[81]. The expression of hpRNA-YHV was approximately 45 ng per 100 mL of culture (\(\sim\)1 \(\times\) 10\({}^{8}\) cells of the engineered _Chl. reinhardtii_ strain). A feed containing _Chl. reinhardtii_ cells expressing YHV-dsRNA (1 \(\times\) 10\({}^{8}\) cells per gram of feed) was formulated; oral administration of the feed to post-larval _P. vannamei_ improved the survival rate after YHV infection. Specifically, at 9 days post-treatment, all shrimp fed the normal feed had died, while approximately 22% of the shrimp fed the feed containing the dsRNA-containing cells survived [81]. This showed the potential effectiveness of dsRNA produced from the nuclear genome of _Chl. reinhardtii_ in protecting shrimp from YHV infection. However, because the nucleus of the microcalga is equipped with RNAi machinery for dsRNA processing, which could have degraded the target dsRNA produced, the researchers then tried to produce the target dsRNA within the chloroplast, which lacks any such functions [82]. Target dsRNA for the RdRp-encoding gene of YHV was produced in the chloroplast using a convergent transcription mode with the promoter of the _psaA_ gene (which encodes the PsaA subunit of photosystem I in the chloroplast). In this transformation system, a selection system eliminating the need for antibiotic resistance markers was used. The engineered _Chl. reinhardtii_ produced a low level of dsRNA (approximately 16 ng-dsRNA per liter of culture medium). However, when the generated strain was used in a shrimp feeding trial to evaluate the efficacy of the microalgal RNAi against YHV, the mortality rate of shrimp pretreated with dsRNA-expressing microalgae was approximately 50% at 8 days post-YHV infection, compared with approximately 84% mortality in the control group, suggesting that the use of dsRNA-producing microalgae may be a promising strategy to reduce mortality caused by YHV in shrimp farming [82]. _Chlamydomonas_ was also used as a host to produce hpRNA as an RNAi-based microalgal larvicide for mosquito control. Mosquitoes transmit many life-threatening parasitic and viral diseases, including filaria, yellow fever, dengue fever, and malaria. Many chemical pesticides and biological control agents have been developed and used to control mosquitoes, but there is a need for control methods that are safer for humans and ecosystems. Furthermore, the long-term use of these conventional chemical pesticides has resulted in the development of pesticide-resistant mosquito species. Microalgae are a major source of nutrition for mosquito larvae in water bodies that serve as breeding grounds for mosquitoes. Hence, [PERSON] et al. [83] designed an hpRNA expression system in _Chlamydomonas_ chloroplasts to suppress expression of the gene encoding 3-hydroxykynuree transaminase (3-HKT), which is involved in tryptophan catabolism and is essential for mosquito growth, by RNAi.