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PhD offer | Characterization of genetic markers associated with resistance to chemical insecticides in Dengue vector mosquitoes – Toward novel molecular resistance diagnostic tools

par Webmaster - 17 février 2016 ( dernière mise à jour : 8 mars 2016 )

To apply contact jean-philippe.david@ujf-grenoble.fr before March 20th 2016

Aedes mosquitoes represent a major threat by transmitting severe human diseases. The re-emergence of dengue is becoming a major concern with 50–100 million dengue infections worldwide every year while other arboviruses are re-emerging (e.g. Chikungunya and Zika). Controlling mosquitoes is essential for decreasing transmission rate and preventing outbreaks. While chemical insecticides remain the corner stone of mosquito control, their efficacy is now threatened by resistance developed by mosquitoes. Resistance of mosquitoes to insecticides is mainly by mutations of the proteins targeted by the insecticide (target-site mutations) or an increased biodegradation of the insecticide through enhanced detoxification (metabolic resistance). Target site mutations have been well characterized in dengue vectors and can be detected by simple PCR assays. Conversely, metabolic resistance far less understood. For example, particular detoxification enzymes have been found over-expressed in resistant populations but only few of them have been validated as capable of metabolizing insecticides. In addition, the lack of robust DNA markers of metabolic resistance is impairing its monitoring in the field. In this context, the main objectives of this PhD project are to :

1 : Combine different high-throughput sequencing approaches for identifying novel DNA markers of insecticide resistance in dengue vector mosquitoes.
2 : Validate the role of these markers in resistance through genetic and functional studies.
3 (optional) : Develop novel diagnostic assays for monitoring resistance mechanisms in natura.

This PhD will focus on two mosquito species threatening public health worldwide by transmitting dengue, chikungunya and zika viruses, namely Aedes aegypti and Aedes albopictus (tiger mosquito) and will involve various activities :

A) Sample mosquitoes worldwide and characterize their resistance levels : mosquito populations will be sampled in various regions (e.g. South America, the Caribbean’s, South-East Asia and Europe). Sampling effort will be supported by an existing collaboration network developed by the LECA mosquito group. Resistance levels will be characterized by bioassays. Resistant populations from each region will be combined (meta-populations) and colonized in P2+ insectaries for further molecular work.

B) Use RNA sequencing (RNA-seq) for identifying genes differentially transcribed, alternative splicing events and transcript variations associated with resistance. Use targeted DNA sequencing (targeted DNA-seq) consisting in capturing thousands of genomic regions of interest and deep sequencing them for identifying genomic changes associated with resistance. Such analysis will allow screening for polymorphism variations (coding + promoters) together with copy number variations (CNVs) associated with resistance in hundreds of candidate genes. By also capturing thousand small genomic regions spread across mosquito genomes, such approach will also allow scanning for genomic regions under selection by insecticides. Finally, comparing data generated by RNA-seq and DNA-seq will allow identifying promoter variations and CNV associated with the expression of resistance genes.

C) Validate DNA markers of resistance by combining controlled genetic crosses, genotype-phenotype association studies and functional biology techniques (e.g. RNAi, CRISP/CAs9, in vitro metabolism studies, promoter assays, …).

D) Depending on time and budget constraints, high-throughput molecular diagnostic assays based on qPCR or LAMP technologies will be developed for validated markers.

Sous la tutelle de :

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