Expression Profile of Genes during Resistance Reversal in a Temephos Selected Strain of the Dengue Vector, Aedes aegypti

Background The mosquito Aedes aegypti is one of the most important disease vectors because it transmits two major arboviruses, dengue and yellow fever, which cause significant global morbidity and mortality. Chemical insecticides form the cornerstone of vector control. The organophosphate temephos a larvicide recommended by WHO for controlling Ae. aegypti, however, resistance to this compound has been reported in many countries, including Brazil. Methodology/Principal Findings The aim of this study was to identify genes implicated in metabolic resistance in an Ae. aegypti temephos resistant strain, named RecR, through microarray analysis. We utilized a custom ‘Ae. aegypti detox chip’ and validated microarray data through RT-PCR comparing susceptible and resistant individuals. In addition, we analyzed gene expression in 4th instar larvae from a reversed susceptible strain (RecRev), exposed and unexposed to temephos. The results obtained revealed a set of 13 and 6 genes significantly over expressed in resistant adult mosquitoes and larvae, respectively. One of these genes, the cytochrome P450 CYP6N12, was up-regulated in both stages. RT-PCR confirmed the microarray results and, additionally, showed no difference in gene expression between temephos exposed and unexposed RecRev mosquitoes. This suggested that the differences in the transcript profiles among the strains are heritable due to a selection process and are not caused by immediate insecticide exposure. Reversal of temephos resistance was demonstrated and, importantly, there was a positive correlation between a decrease in the resistance ratio and an accompanying decrease in the expression levels of previously over expressed genes. Some of the genes identified here have also been implicated in metabolic resistance in other mosquito species and insecticide resistant populations of Ae. aegypti. Conclusions/Significance The identification of gene expression signatures associated to insecticide resistance and their suppression could greatly aid the development of improved strategies of vector control

Differential expression of <i>Ae. aegypti</i> detoxification genes in 3 day old female mosquitoes of the parental RecL and RecR strains.

<p>Differences are indicated as a function of both expression ratio (X-axis) and significance, expressed as the negative log10 scale of the p-value of the t-test of the fold change between the groups (Y-axis). Vertical lines indicate two-fold expression differences in either direction. The horizontal line indicates the significance threshold of P<0.001 adopted for the one sample t-test.</p

Fold-change in <i>CYP6N12, Aldehyde oxidase 10382</i> (<i>ao10382</i>), <i>GSTi1, GSTo1, GSTx2</i>, and <i>CCEae3A</i> transcripts in RecR, RecRev1 and RecRev1 Exposed strains, compared to Rockefeller or RecL strains.

<p>Fold-change in <i>CYP6N12, Aldehyde oxidase 10382</i> (<i>ao10382</i>), <i>GSTi1, GSTo1, GSTx2</i>, and <i>CCEae3A</i> transcripts in RecR, RecRev1 and RecRev1 Exposed strains, compared to Rockefeller or RecL strains.</p

Microarray results of over expressed genes in 3 day old females of the RecR strain when compared with females from the parental RecL strain.

<p>Microarray results of over expressed genes in 3 day old females of the RecR strain when compared with females from the parental RecL strain.</p

Differential expression of <i>Ae. aegypti</i> detoxification genes in larvae of the parental RecL and RecR strains.

<p>Differences are indicated as a function of both expression ratio (X-axis) and significance, expressed as the negative log10 scale of the p-value of the t-test of the fold change between the groups (Y-axis). Vertical lines indicate two-fold expression differences in either direction. The horizontal line indicates the significance threshold of P<0.001 adopted for the one sample t-test.</p

Enzymatic activity associated with esterases (α, β and PNPA) acetylcholinesterase (ACE), glutathione-S-transferases (GST) and mixed function oxidases (MFO) observed in <i>Ae. aegypti</i> strains susceptible and resistant to temephos.

a<p>Rockefeller: susceptible laboratory strain; ^b RecR =  Brazilian resistant strain; ^c RecRev1_F13 =  sub colony of the RecR without temephos exposure; ^d RecL: Brazilian susceptible laboratory strain.^e 99 percentile for Rockefeller. ^f Number of RecR individuals with 99 percentile above than the 99 percentile for Rockefeller. ^g Percentage of individuals with 99 percentile above than the 99 percentile for Rockefeller. ^h Classification of enzyme activity compared to control (Rockefeller): unaltered (U); altered (A) and highly altered (HA). NT  =  not tested.</p

Microarray results of over expressed genes in the 4^th instar larvae of the RecR strain when compared with larvae from the parental RecL strain.

<p>Microarray results of over expressed genes in the 4^th instar larvae of the RecR strain when compared with larvae from the parental RecL strain.</p

Values of lethal concentration (LC) of temephos and resistance ratios (RR) for the <i>Ae. aegypti</i> strains.

<p>RecR =  Brazilian resistant strain; RecRev1_F13 =  sub colony of the RecR without temephos exposure; Rockefeller: susceptible laboratory strain; RecL: Brazilian susceptible laboratory strain.</p