Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 +/- 11.1%) compared to control groups (88.9 +/- 11.1% and 100.0 +/- 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.