Transgenic microalgae expressing double-stranded RNA as potential feed supplements for controlling white spot syndrome in shrimp aquaculture

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga, Chlamydomonas reinhardtii and used to control disease in shrimp. Here we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high level transcription of both strands of the WSSV gene element in the chloroplast. New vectors were designed that allow rapid Golden Gate-mediated assembly of a transformation plasmid in which the dual promoter-WSSV DNA cassette is targeted into the chloroplast genome, with selection based on the restoration of photosynthesis. PCR analysis of transformant lines confirmed the integration of the cassette and homoplasmy of the polyploid genome. Transcribed sense and antisense VP28-RNA were hypothesised to form an RNA duplex in the chloroplast stroma, and quantitative RT-PCR indicated that [~]100 g dsRNA is produced per litre of transgenic microalgae culture. This represents an [~]10,000-fold increase in dsRNA relative to previous reports using convergent psaA promoters. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. Survival of shrimp fed with dsRNA-expressing C. reinhardtii was significantly enhanced (68.3%) relative to the negative control. The study suggests that this new dsRNA production platform is significantly more efficient than that reported previously, and merits further scale-up and downstream processing studies.