Cutting-Edge Mosquito Management

Engineering Yeast Strains for Eco-Friendly Insecticide Production

Despite advancements in modern medicine and pest control, mosquitoes remain the deadliest animal in the world, causing more than 700,000 human deaths each year¹. There were 249 million cases of malaria alone in 2022, illustrating the importance of developing effective malaria prevention strategies and treatments². Malaria together with other common mosquito-borne diseases such as Zika virus, West Nile virus, Chikungunya virus, Dengue, and Yellow Fever cause infections in over 700 million people annually, with most cases concentrated in tropical and subtropical regions where medical resources are limited³.

Taking appropriate measures to prevent mosquito bites, particularly in areas where mosquito-borne diseases are prevalent, is critical to controlling these illnesses and reducing their health burden. However, the global deployment of affordable vector-borne disease control programs remains challenging and growing insecticide resistance hinders progress⁴. Thus, there is an acute need for species-specific, environmentally friendly insecticides that can protect vulnerable populations against mosquito-borne illnesses.

In a 2023 study, scientists with Demeetra Ag Bio, Indiana University School of Medicine, and the University of Notre Dame applied genome editing technology in the budding yeast Saccharomyces cerevisiae to develop a new class of eco-friendly insecticides based on elevated shRNA expression⁵. Cas-CLOVER, an RNA-guided system similar to the original CRISPR/Cas9 technique, was used to generate auxotrophic yeast strains with URA3 and LEU2 gene deletions. These engineered strains were used in combination with sPB transposase/transposon technology, which was employed to prepare plasmids containing copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. The mutant S. cerevisiae strains were transformed with the transposon “cargo” plasmid using Zymo Research’s Frozen-EZ Yeast Transformation II Kit for simple and efficient transformation in less than one hour.

Sh.463 expression levels in the selected clones of genetically modified S. cerevisiae were assessed using RT-qPCR. Yeast cells were pelleted and then resuspended in Zymo Research’s YR Digestion Buffer and Zymolyase, a yeast lytic enzyme optimized for maximal digestion of the fungal cell wall. After forming spheroplasts, highly pure total RNA was extracted using the YeaStar RNA Kit from Zymo Research. Sh.463 expression levels were quantified using RT-qPCR and strains with the highest expression were further characterized. Whole genome sequencing was used to verify that select S. cerevisiae strains retained multiple integrated copies of the Sh.463 insecticide expression cassette in the genome.

The second-generation S. cerevisiae strains were then used in laboratory and field trials to assess their insecticidal activity against A. gambiae, A. aegypti, A. albopictus, C. quinquesfasciatus, and C. pipiens mosquito species. Laboratory trials demonstrated that the S. cerevisiae strains effectively killed mosquito larvae in the third or fourth instar of larval development prior to adult emergence. The same genetically modified yeast strains were also evaluated for insecticidal efficacy in adult mosquitoes. Strains with the highest Sh.463 expression levels were suspended in a sucrose solution that was used as bait and fed to adult female mosquitoes. The sugar bait solution exhibited a median lethal dose of just 0.0192 mg/µL in adult mosquitoes, which is tenfold less than other insecticides prepared using similar methods. This suggests that the genome editing technology developed in this study can be used as a cost-effective alternative for controlling mosquitoes.

Pilot fermentations were then initiated and confirmed that the modified S. cerevisiae strains could be cultured with high growth levels and robust expression of insecticidal Sh.463 shRNA. This suggests that genetically engineered insecticidal yeast strains may be feasible to deploy in larger scale fermentations as malaria prevention strategies. While additional field studies and regulatory approval is still required, this research represents a significant step toward eco-friendly and targeted mosquito management, with potential applications in pesticide distribution and vector-borne disease control worldwide.

Discover innovative technologies for yeast strain engineering

References

1. Deadliest animals worldwide by annual number of human deaths as of 2022. Statista (2022). Deadliest animals to humans 2022 | Statista.
2. Malaria. World Health Organization (2023). Malaria (who.int).
3. Vector-borne diseases. World Health Organization (2020). Vector-borne diseases (who.int).
4. Fighting the World’s Deadliest Animal. Centers for Disease Control and Prevention (2023). Fighting the World’s Deadliest Animal | Global Health | CDC.
5. Brizzee, C., Mysore, K., Njoroge, T. M., McConnell, S., Hamid-Adiamoh, M., Stewart, A. T. M., Kinder, J. T., Crawford, J., and Duman-Scheel, M. (2023). Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. Journal of Fungi, 9(11), 1056. https://doi.org/10.3390/jof9111056.