ARE DETOXIFICATION GENE MUTATIONS ASSOCIATED WITH INSECTICIDE RESISTANCE IN AEDES AEGYPTIMOSQUITOES?

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?

✓

Deltamethrin1.5 µg1 hourn = 248

Activen = 107

Inactiven = 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 five detox gene SNPs in Viva Caucel

0

0.2

0.4

0.6

0.8

1

kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

Alle

lic F

requ

ency

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 two detox gene SNPs in 5 de Febrero

0

0.2

0.4

0.6

0.8

1

kdr V1016I kdr C1534F kdr V410L Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

Alle

lic F

requ

ency

* **

*

kdr mutations in vgsc

kdr V1016I kdr C1534F kdr V410L

00.05

0.10.15

0.20.25

0.30.35

0.4

Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

0

0.1

0.2

0.3

0.4

0.5

0.6

Aldox 10391 CCEAE2B CYP4H33 CYP325M2 CYP325G3 CYP325L1

Alle

lic F

requ

ency

*

*

**

*

*

*

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