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

Abstract

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.

Advanced Applications for Protein and Compounds from Microalgae

“…Microalgae represent an important biotechnology resource for the production of recombinant proteins for pharmacological applications. Successful results were reported by the genetic manipulation of microalgae for the production of antigens for vaccines, antibodies, immunotoxins, hormones, and antimicrobial agents [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ]. Strategies and techniques have been developed for the exploitation of microalgae as expression systems for a number of advanced genetic toolkits [ 62 , 63 , 64 , 65 ].…”

“…Antigens, antibiotics, and other bioactive molecules against animal pathogens have been produced. Examples include swine fever virus [ 74 ], white spot syndrome virus [ 64 , 72 , 73 ], lethal shrimp yellow head virus [ 77 , 78 ], Aeromonas hydrophila bacteria [ 79 ], and vibriosis disease [ 80 ].…”

Evaluation of Microalgae as Immunostimulants and Recombinant Vaccines for Diseases Prevention and Control in Aquaculture

“… Somchai et al (2016) constructed a shrimp yellow head virus (YHV) RNAi vector in the C. reinhardtii system, which improved survival by 22% after oral administration to prawn larvae. In the same way, Charoonnart et al (2019) engineered the chloroplast genome of C. reinhardtii to express double-stranded RNA (dsRNA) designed to knock down key viral genes, Shrimps fed with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection, whereas only 15.9% survival rate was observed in control groups, and RT-PCR results revealed a lower infection rate in dsRNA-expressing algae treated shrimp compared to control groups. In all cases, aquatic animals were direct protected by feeding genetic modified microalgae.…”

Recent Advances in Microalgal Bioactives for Food, Feed, and Healthcare Products: Commercial Potential, Market Space, and Sustainability

“…In a recent study, a C. reinhardtii transformant was constructed, which expressed dsRNA that is specific to the pathogen yellow head virus (YHV), a shrimp virus. These transformants were fed to the shrimps for disease control as a result of which 50% survival rates were observed (Charoonnart et al., ).…”