It is estimated that viral and bacterial pathogens annually cause billion-dollar losses to the shrimp industry around the world. According to FAO, the crustacean farming subsector represents about 30% of the total profits generated by the aquaculture industry, and the production of the Pacific white shrimp (Litopenaeus vannamei) generates a value of about 25 billion (USD) per year. According to the OIE, the main health problem facing the sector is the incidence of viral etiology diseases. Of the 10 pathogens listed by the OIE, 7 are of viral origin. Among the bacterial pathogens listed, the highlighted bacterium is Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND). Periodic epizootics of these diseases have caused large economic losses that have been estimated in more than 15 billion USD in the last three decades. The challenge in developing sustainable shrimp farming has been put to the test due to the periodic appearance of new viral and bacterial pathogens or the presence of more lethal strains of those already known.
Taking this context into consideration, the ChlamyR7 project would initiate studies on whole-cell therapeutic strategies using RNAi effector molecules expressed in microalgae. ChlamyR7 is based on the synergistic combination between different approaches framed in the synthetic biology paradigm. The first is about the expression of double-stranded RNA (dsRNA) in the microalgal species Chlamydomonas reinhardtii, which makes it an ideal platform (biological chassis used as whole-cell therapeutic) for oral delivery of RNA interference (RNAi) effector molecules. Moreover, the Generally Recognized as Safe (GRAS) status of C. reinhardtii, and the lack of production of endotoxins and infectious agents make it particularly attractive. In addition, due to the selection method of the C. reinhardtii strain used in this case (TN72), which does not involve the use of antibiotics; this approach is considered as environmentally friendly.
The second is based on the simultaneous silencing (RNAi) of the shrimp endogenous Rab7 (therapeutic target 1) and Fushi tarazu (FTZ) transcription factor (Ftz-F1H) (therapeutic target 2) genes. Rab7 works as an important intracellular traffic regulatory factor, and its silencing inhibits both the infection of DNA viruses such as the white spot syndrome virus (WSSV), and also RNA viruses such as yellow head virus (YHV), Taura syndrome virus (TSV), and Laem-Singh virus (LSNV). In addition, the silencing of Ftz-F1H, which is an orphan nuclear receptor, significantly decreased the mortality rate of V. parahaemolyticus infected shrimp as well as the bacterial load. Thus, the silencing of both target genes simultaneously produces an anti-multi-viral and anti-V. parahaemolyticus effect.
Therefore, our main objective is to exploit the platform C. reinhardtii TN72 for the simultaneous production of dsRNA shrimp anti-multi-viral and anti-V. parahaemolyticus (dsRNA-LvRab7-Ftz-F1H) in the chloroplast, without the use of antibiotics as a selection mechanism. In particularly, it is intended to: i) Construct a dsRNA expression plasmid using a vector developed by the Purton group and generate by chloroplast transformation a C. reinhardtii strain expressing dsRNA-LvRab7-LvFtz-F1H. Transformants would be genotyped by PCR and sequencing, and specific dsRNA-LvRab7-LvFtz-F1H detected and quantified using RT-PCR and RT-qPCR; ii) Scale-up, cell harvest and product recovery, including drying of the microalgal biomass to be used as an oral delivered treatment by mixing with food (pellet coating). Also, it is intended to iii) carry out shrimp feeding test to verify the silencing of LvRab7 and LvFtz-F1H genes, and also the inhibition of replication of WSSV and V. parahaemolyticus, respectively. Finally, the present study expects to iv) develop a preliminary risk assessment for the generation of the genetically modified microalgae. Based on the current approach to case-by-case evaluation, this project has also its main objective in identifying the possible risks associated with the use of this technology, both at the level of laboratory tests (off-target gene silencing), as well as making use of bioinformatics tools for the determination of possible non-target organisms in the environment.
The ChlamyR7 project could contribute to the creation of new oral anti-multi-viral and anti-bacterial therapy to improve shrimp immune response and resistance. It is essential to highlight that the treatment generated could inhibit at least 4 of the 10 pathogens listed by the OIE simultaneously (WSSV, YHV, TSV, and V. parahaemolyticus). The ChlamyR7 team is composed of researchers with important knowledge and know-how on the modulation of shrimp immune responses (A2S-CIIMAR, Portugal), and world class experts in biotechnology of microalgae (Purton’s Lab-UCL, UK), and RNAi mechanisms applied to shrimp aquaculture (AFST-CENTEX, Thailand).