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

RNA interference (RNAi) is an efective way of combating shrimp viruses by using sequence-specifc

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 ofering 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 efciency 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 specifc 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.