ARE DETOXIFICATION GENE MUTATIONS ASSOCIATED WITH INSECTICIDE - PowerPoint PPT Presentation

ARE DETOXIFICATION GENE MUTATIONS ASSOCIATED WITH INSECTICIDE RESISTANCE IN AEDES AEGYPTI MOSQUITOES ? India Hilty Colorado College Organismal Biology & Ecology OBE Day, Spring 2020

Aedes aegypti : Vector of Deadly Human Diseases Transmits: Controlled primarily by: • Dengue Insecticides • Zika • Yellow Fever • Chikungunya But… insecticide resistance is widespread! Ryan et al., 2019 & NPR

Insecticide Resistance Mechanisms Metabolic detoxification Mutations in vgsc → enzymes knockdown resistance (kdr) decreased pyrethroid Saavedra-Rodriguez et al., 2018 binding ✓ role in resistance clearly characterized ✘ not well understood

2019 High Throughput Sequencing Study 268 detoxification genes Highly resistant Viva Caucel population >100 detoxification SNPs significantly associated with resistance

OBJECTIVES 1. Validate the accuracy of the HTS method by genotyping knockdown- resistant and susceptible individuals in the Viva Caucel population. ⇢ Selected 6 detoxification SNPs located in 1 esterase (CCE), 1 redox (Aldox) , and 4 cytochrome p450 enzymes (CYP) ⇢ Sanger sequencing to verify existence of SNPs

Genotyping TT Using Allele- AA Specific PCR TA Melting Curves

OBJECTIVES 1. Validate the accuracy of the HTS ✓ method by genotyping knockdown- resistant and susceptible individuals in the Viva Caucel population. 2. Are these detoxification SNPs associated with resistance in a second resistant A. aegypti population?

Alive = (Knockdown Active Resistant Resistant) 4 h Genotype phenotype allelic 107 n = 107 frequencies Deltamethrin 1.5 µg 1 hour n = 248 Inactive Inactive = Susceptible Dead 4 h Genotype n = 141 phenotype allelic (Susceptible) frequencies 69 5 de Febrero population

Resistant and susceptible phenotypes have significantly different mutant allele frequencies in five detox gene SNPs in Viva Caucel fiv * 1 * * 0.8 Allelic Frequency 0.6 * 0.4 * * 0.2 * * 0 kdr V1016I kdr C1534F kdr V410L kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1 kdr mutations in vgsc detox gene SNPs

Resistant and susceptible phenotypes have significantly different mutant allele frequencies in tw two detox gene SNPs in 5 de Febrero 1 0.8 Allelic Frequency 0.6 * * * * 0.4 0.2 0 kdr V1016I kdr C1534F kdr V410L kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1 detox gene SNPs kdr mutations in vgsc

Viva Caucel 0.4 * * 0.35 0.3 0.25 * * 0.2 0.15 * 0.1 Allelic Frequency 0.05 0 Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1 5 de Febrero 0.6 * 0.5 * 0.4 0.3 0.2 0.1 0 Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

Metabolic resistance is complex. We need more research!

DR. KARLA DR. EMILIE GRAY DR. BILL BLACK SAAVEDRA- RODRIGUEZ Acknowledgments CC ORGANISMAL DR. SHARON BIOLOGY & SMITH & JAMES ECOLOGY ENDERSON DEPARTMENT RESEARCH FUND