Mosquito-borne pathogens cause the deadliest diseases around the globe causing more than 700,000 deaths annually (Ursino et al., 2020). As no vaccines are currently present to prevent a majority of them so in this scenario an effective strategy can be cutting off the pathogen transmission through vector control (Takken and Knols, 2009). Usage of chemical insecticides in this facet brings about poor human health, environmental pollution and provokes resistance build-up in mosquito species (Benelli and Beier, 2017, Moyes et al., 2017). Therefore, biological control of mosquitoes has gained increased attention, as this is an environment-friendly approach to combat the spread of mosquito-borne diseases.

In this context, the insecticidal potential of Cry (Crystal) toxins of the bacterium Bacillus thuringiensis (Bt) is up and coming tool for the biological control of mosquitoes. Search for new toxins or genetic modifications of Cry toxins by creating chimeric toxins for increased toxicity against a broad range of insects is an actively researched area (Rao et al., 2021). However, Bt formulations of Cry toxins are rendered less efficacious and require repeated applications because of their degradation due to sunlight and Bt spores do not produce well in aquatic habitats (Moustafa et al., 2018, Poopathi and Abidha, 2010).

A successful way out can be by carrying out the safe delivery of the toxin to the larvae. This can be achieved by genetically manipulating the microbiota on which the mosquito larvae feed and which carry a well-known genetic background, allowing the expression of Bt Cry toxins (Juntadech et al., 2012, Kang et al., 2017, Kumar, 2010). Around 200 species of microalgae can be found on mosquito breeding habitats and larvae feed on them extensively (Ranasinghe and Amarasinghe, 2020). The fact that mosquito larvae feed on microalgae and it is widely used for the expression of recombinant proteins (Esland et al., 2018, Schroda, 2019), has made it possible to genetically modify green algae (Chlamydomonas and Chlorella) and cyanobacteria (Anabaena and Synechococcus), resulting in transgenic strains lethal to mosquito larvae (Borovsky et al., 2016, Fei et al., 2020, Kang et al., 2018, Soltes-Rak et al., 1995, Zaritsky et al., 2010).

Chlamydomonas reinhardtii (C. reinhardtii), classified as GRAS (Generally Regarded as Safe) organism, demonstrates a promising platform for recombinant protein production. It has served as an expression host for heterologous genes and a model organism for genetic manipulation studies (Rasala et al., 2014, Wijffels et al., 2013). Foreign gene expression in chloroplast and nuclear compartments of C. reinhardtii is a thoroughly investigated area (Larrea-Alvarez and Purton, 2020, Schroda, 2019). Transformation protocols are well established for both the hosts (Hallmann, 2007, Purton, 2007). Chloroplast expression of foreign genes in C. reinhardtii offers several benefits like (a) specific integration of the gene of interest (GOI) via homologous recombination, (b) higher levels of gene expression and protein accumulation because of a variety of endogenous cis-elements, (c) selection of transformants without using antibiotic resistance markers and (d) no risk of cytotoxic effects to the cell posed by the foreign gene (Esland et al., 2018, Purton, 2007, Tran et al., 2013).

On the other hand, nuclear expression of transgenes is considered low as compared to chloroplast expression, but it offers benefits like protein targeting to specific organelles, protein glycosylation, and other post-translational modifications (León-Bañares et al., 2004). The choice of a strong promoter directly affects nuclear transgene expression therefore, endogenous promoters are recommended for successful transgene expression (Díaz-Santos et al., 2013). Moreover, the inclusion of other regulatory elements, such as endogenous intronic sequences in transgenes can enhance their expression (Lumbreras et al., 1998). Furthermore, nuclear gene expression can be improved if the transcriptional fusion of the heterologous gene and selectable marker can be made by adding a self-cleavable peptide (Rasala et al., 2013, Rasala et al., 2012).

Therefore, for the expression of Bt Cry toxins, C. reinhardtii dominates the other microbial expression hosts because of its ability to swim and multiply easily in water habitats. Moreover, Bt toxin inside the algal cell will not be affected by polluted waters (Kang et al., 2017).

Bacillus thuringiensis subsp. jegathesan (Btj) is well known for its mosquito larvicidal potential (Kawalek et al., 1995). The bacterium’s larvicidal ability can be attributed to the presence of Cry and Cyt toxins in its parasporal inclusion produced during the sporulation phase. Cry toxins are produced by the bacterium as protoxins. Once they enter the mosquito larva midgut, proteolytic cleavage activates them and they disrupt the midgut’s membrane, causing the death of larvae. These toxins are very target specific and pose no harm to non-specific species and humans (Achari et al., 2020, Bravo et al., 2017, Rao et al., 2021). The Cry11Ba (81 kDa) toxin of Btj is 58% similar, in amino acid sequence, to Cry11Aa (72 kDa) toxin of Bacillus thuringiensis subsp. israelensis (Bti). It is reported that to date Cry11Ba is the most toxic Bt protein against Aedes, Anopheles, and Culex mosquito larvae (Delecluse et al., 1995, Florez et al., 2018, Valtierra-de-Luis et al., 2020).

Genetically incorporating Cry11Ba protein in an algal host can be quite challenging because (a) it has a very large size (81 kDa) and (b) Cry11Ba has the highest reported toxicity, it can pose toxic effects to the host cell. However, with the knowledge of C. reinhardtii expression systems, we have attempted to transform the chloroplast and nuclear expression hosts of C. reinhardtii with Btj Cry11Ba toxin. This allowed us to compare the feasibility of chloroplast and nuclear compartments of C. reinhardtii for the generation of recombinant C. reinhardtii strains harboring this very toxic protein.