Expression of a mosquito larvicidal gene in chloroplast and nuclear compartments of Chlamydomonas reinhardtii

As a part of the search for environment-friendly biocontrol of mosquito-borne diseases, mosquito larvicidal potential of Bacillus thuringiensis subsp. jegathesan (Btj) Cry toxins is explored for toxins with increased toxicity. Safe delivery of the Cry toxins to mosquito larvae in aquatic habitats is a major concern. This is because in water bodies Bacillus thuringiensis (Bt) protein formulations degrade by sunlight, can sink down and get adsorbed by the silt. So, because of its short persistence the toxin requires repeated applications at the given site. Therefore, an upcoming approach is incorporating the Bt toxins in Chlamydomonas reinhardtii (C. reinhardtii) because it is a food of mosquito larvae in water and its molecular toolkit is well investigated for foreign gene expression. The present work aimed to compare the feasibility of C. reinhardtii chloroplast and nuclear compartments for stable expression of Cry11Ba toxin as this is the most toxic Btj protein to date, lethal to different mosquito species. With chloroplast expression of cry11Ba gene we were able to generate marker-free C. reinhardtii strain stably expressing Cry11Ba protein and demonstrating mortality against Aedes aegypti larvae. Moreover, for nuclear expression linking the cry11Ba gene to zeocin via foot and mouth disease virus (FMDV) 2A peptide resulted in the selection of transformants with increased cry11Ba mRNA expression levels by semi-quantitative reverse transcriptase PCR. Obtained results lay a foundation for the C. reinhardtii chloroplast expression system to be used for genetic engineering with Bt toxins which possess enhanced toxicity^.