Produção de dsRNAs direcionados a patógenos virais e bacterianos de camarões utilizando a microalga Chlamydomonas reinhardtii

General Objective: • Induce the silencing of the Rab7 and Ftz-F1H genes in L. vannamei by oral administration of dsRNA-LvRab7-LvFtz-F1H produced in C. reinhardtii. In particularly, it is intended to accomplish the following specific objectives: i) Build a LvRab7-LvFtz-F1H transgene in the dsRNA chloroplast expression vector pLY118 developed in the Purton’s Lab 1 and generation of a C. reinhardtii transformant line expressing dsRNA-LvRab7-LvFtz-F1H using the TN72 2 recipient strain (also generated at Purton’s Lab) . The genotype of this strain will be confirmed by molecular analysis (PCR, sequencing and RT-qPCR) to confirm dsRNA-LvRab7-LvFtz-F1H cassette integration, homoplasmic state, and quantification of dsRNA levels 3. These analyses will be done and validated at CIIMAR and UCL. ii) Scale-up the culture of C. reinhardtii expressing dsRNA-LvRab7-LvFtz-F1H to 50 liters using the hanging bag system 4, cell harvest and product recovery 5, including flocculation using chitosan and drying of the biomass of microalgae (spray drying 6) to be used as an oral treatment by mixing it with food (pellet coating). These analyses will be done and validated at UCL. iii) Carry out shrimp feeding and pathogen-challenge tests to verify the silencing of LvRab7 and LvFtz-F1H genes, and the inhibition of replication of V. parahaemolyticus and WSSV (including expression analyses of Argonaute and Dicer shrimp genes by RT-qPCR). These analyses will be done and validated at CIIMAR and CENTEX, respectively. 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. These analyses will be done at CIIMAR.