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:

• Dengue
• Zika
• Yellow Fever
• Chikungunya

Controlled primarily by:

Insecticides

But… insecticide resistance is widespread!

Ryan et al., 2019 & NPR

Insecticide Resistance Mechanisms

Mutations in vgsc → knockdown resistance (kdr) Metabolic detoxification enzymes

Saavedra-Rodriguez et al., 2018

✓ role in resistance clearly characterized

✘ not well understood

decreased pyrethroid binding

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 Using Allele- Specific PCR Melting Curves

TT AA TA

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?

✓

Deltamethrin 1.5 µg 1 hour

n = 248

Active

n = 107

Inactive

n = 141

4 h 4 h Alive = (Knockdown Resistant) 107 Inactive = Dead (Susceptible) 69 Genotype Genotype Resistant phenotype allelic frequencies Susceptible phenotype allelic frequencies

5 de Febrero population

Resistant and susceptible phenotypes have significantly different mutant allele frequencies in fiv five detox gene SNPs in Viva Caucel

0.2 0.4 0.6 0.8 1 kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1 Allelic Frequency

kdr mutations in vgsc detox gene SNPs * * * * * * * *

kdr V1016I kdr C1534F kdr V410L

detox gene SNPs

Resistant and susceptible phenotypes have significantly different mutant allele frequencies in tw two detox gene SNPs in 5 de Febrero

0.2 0.4 0.6 0.8 1 kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1 Allelic Frequency

* * * * kdr mutations in vgsc

kdr V1016I kdr C1534F kdr V410L

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

0.1 0.2 0.3 0.4 0.5 0.6

Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

Allelic Frequency * * * * * * *

Viva Caucel 5 de Febrero

Metabolic resistance is complex. We need more research!

Acknowledgments

• DR. KARLA

SAAVEDRA- RODRIGUEZ

• DR. EMILIE GRAY
• DR. BILL BLACK

CC ORGANISMAL BIOLOGY & ECOLOGY DEPARTMENT

• DR. SHARON

SMITH & JAMES ENDERSON RESEARCH FUND