West Nile virus (WNV) has become endemic to the US, with yearly infection peaks coinciding with the activity of its mosquito vector. 2012 was the worst year for WNV since 2003 with 286 deaths and estimates of 86000-200000 non-neuroinvasive cases. There is no specific treatment or vaccine, so mosquito control is the only approach available. Fortunately, controlling the vector also reduces the transmission of other diseases, such as Dengue. However, the chemical pesticides that have played an important role in mosquito management are losing their effectiveness due to increasing resistance. Moreover, there are growing concerns over damage to non-target organisms (such as honeybees), and possible links between pesticide exposure and neurogenerative diseases in people. We are developing new products for mosquito control that are based on the simple idea of turning a larval food source, the eukaryotic green alga Chlamydomonas , into a safe and effective biolarvacide. Chlamydomonas is an edible alga whose ability to swim and reproduce in aquatic habitats, and its development as a genetic research model make it an attractive platform for mosquito control. To this end, we are expressing genes based on the Cry and Cyt genes of Bacillus thuringiensis israelensis (Bti) in the chloroplast of Chlamydomonas . Bti is a safe biolarvacide that has not produced strong resistance in >20 yrs of use, but it does not recycle in aquatic habitats. In addition, by using the chloroplast genome, we avoid the use of bacterial antibiotic-resistance genes, and can restrict the possible transfer of Bti genes to other organisms. Our ultimate goal is robust strains that express 1 or 2 Cry genes and Cyt1Aa, which inhibits the development of strong resistance. Since we have demonstrated recently that Cry genes can be expressed in the organelle, our priorities for Phase I are to express Cyt1Aa in the chloroplast, and to identify elements that increase Cry gene expression using Cry11Aa as a model.
The Specific Aims for this proposal are to: (1) Synthesize a codon-adapted Cyt1Aa gene, and express it in the chloroplast using an inducible system, in order to optimize expression. (2) Use the gene from Aim 1 to establish wild-type strains that express the adapted Cyt1Aa gene. Wild-type, or constitutive Cyt1Aa expression is necessary for it to be used for mosquito control. (3) Identify cis-acting elements that increase the expression of Cry genes using Cry11Aa as a model. We will use our larvicidal strains that express Cry11Aa in a wild-type background as the starting point for optimization. Implications of the specific aims for Phase II: The successful chloroplast-expression of Cyt1Aa in the context of a stable wild-type strain will justify the notion that we can create Chlamydomonas strains that do not engender strong resistance. The increased expression of Cry11Aa will strengthen our argument that we can generate strains that are highly lethal to many mosquito species, including those that transmit WNV. We anticipate that Phase 2, the creation of strains that are lethal to larvae at ~104 cells/mL or lower (a mature culture of Chlamydomonas is ~107 cells/mL), can be completed for about $500,000 Direct. Validating the algal strains under field conditions, safety-testing, and getting them registered with EPA would likely occur after Phase 2.
This project will increase public health by innovating a safe, ecofriendly biological product for the control of mosquitoes that transmit West Nile virus, dengue, and encephalitis. Since there are no vaccines or effective therapeutics for these diseases, mosquito control is the only workable approach. However, there is growing concern over the chemical pesticides that make up most of the US market for mosquito control products, and many groups have called for new, safer products. We are developing strains of Chlamydomonas - a green alga that is normally a food source for mosquito larvae - that produce their own versions of naturally occurring larvicidal proteins from Bacillus thuringiensis israelenss (Bti). These proteins are known to kill mosquito larvae with great specificity, and formulations of them have been used around the world. However, they cannot recycle, and become inactivated in aquatic environments. Our algal strains will have the potential for safe, sustained control of mosquitoes, and should reduce the need for chemical pesticides.
| Kang, Seongjoon; Odom, Obed W; Thangamani, Saravanan et al. (2017) Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas. J Appl Phycol 29:1377-1389 |
