Aquaculture industries, and in particular the farming of fish and crustaceans, are major contributors to the economy of many countries and an increasingly important component in global food supply. However, the severe impact of aquatic microbial diseases on production performance remains a challenge to these industries. This article considers the potential applications of microalgal technology in the control of such diseases. At the simplest level, microalgae offer health-promoting benefits as a nutritional supplement in feed meal because of their digestibility and high content of proteins, lipids and essential nutrients. Furthermore, some microalgal species possess natural anti-microbial compounds or contain biomolecules that can serve as immunostimulants. In addition, emerging genetic engineering technologies in microalgae offer the possibility of producing ‘functional feed additives’ in which novel and specific bioactives, such as fish growth hormones, anti-bacterials, subunit vaccines, and virus-targeted interfering RNAs, are components of the algal supplement. The evaluation of such technologies for farm applications is an important step in the future development of sustainable aquaculture.

RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners. For a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge. The present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

RNA interference has been proposed to be a promising tool for combating shrimp viruses. Antiviral double-stranded (ds)RNA has been mostly produced in Escherichia coli-expression system because of its high efficiency and inexpensive operations. However, overusing the bacteria may raise concerns regarding public health and environmental contamination, and seeking for a new dsRNA production platform would be alternative for future molecular farming. In this study, we exploited the green microalgae Chlamydomonas reinhardtii to produce dsRNA targeting the lethal shrimp yellow head virus (YHV). The expression plasmid pSL18 for C. reinhardtii was constructed to contain YHV-specific hairpin RNA expression cassette, and the successful assembly of pSL18-YHV was confirmed by PCR and enzymatic digestions. Glass bead method was employed for transformation of C. reinhardtii nuclear genome with pSL18-YHV. Microalgal expression of dsRNA-YHV, approximately 45ng from 100-mL culture, was detected by qRT-PCR. Oral feeding experiment on postlarval shrimp revealed that the formulated feed with C. reinhardtii expressing dsRNA-YHV, at the ratio of 1×108 transformants per gram feed, improved 22% survival rate after YHV challenge. The present study suggests that C. reinhardtii can be bioengineered to produce viral-specific dsRNA for shrimp viral disease control, and the developed qRT-PCR could detect microalgal dsRNA with detection limit of subpicogram