Environmental Engineering Reference
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the baseline value of 1 (equal gene transcription in both samples). Genes showing a
transcription ratio > 1.5-fold in either direction and a t-test P value lower than 0.01
after Benjamini and Hochberg multiple testing correction [67] were considered signifi -
cantly differentially transcribed between the two strains.
Real-Time Quantitative RT-PCR Validation
Transcription profiles of 10 detoxification genes in 4th-stage larvae and adults were
validated by reverse transcription followed by real-time quantitative RT-PCR on the
same RNA samples used for microarray experiments. Four μg total RNAs were treated
with DNAse I (Invitrogen) and used for cDNA synthesis with superscript III (Invit-
rogen) and oligo-dT
20
primer for 60 min at 50°C according to manufacturer's instruc-
tions. Resulting cDNAs were diluted 125 times for PCR reactions. Real-time quantita-
tive PCR reactions of 25 μL were performed in triplicate on an iQ5 system (BioRad)
using iQ SYBR Green supermix (BioRad), 0.3 μM of each primer and 5 μL of diluted
cDNAs according to manufacturer's instructions. For each gene analyzed, a cDNA
dilution scale from 5 to 50,000 times was performed in order to assess efficiency of
PCR. Data analysis was performed according to the ΔΔCT method taking into account
PCR efficiency [68] and using the genes encoding the ribosomal protein L8 [GenBank
DQ440262] and the ribosomal protein S7 [GenBank EAT38624.1] for a dual gene nor-
malization. For each life-stage, results were expressed as mean transcription ratios (±
SE) between the insecticide-resistant strain Vauclin and the susceptible strain Bora-Bora.
Only genes showing more than 2-fold over- or under-transcription in the Vauclin strain
were considered significantly differentially expressed.
CONCLUSION
We have identified multiple insecticide resistance mechanisms in
Ae. aegypti
mos-
quitoes from Martinique (French West Indies) significantly reducing the insecticidal
activity of insecticides used for their control. Microarray screening identified mul-
tiple detoxification genes over-transcribed at both life-stages in resistant mosquitoes,
suggesting their possible involvement in insecticide-resistance. Further experimental
validation by using enzyme characterization and RNA interference will allow con-
firming the role of these genes in the resistance phenotype. As previously shown in
mosquitoes [57], the epistasis between the
kdr
mutation and particular P450s genes is
likely to contribute to the high level of resistance to pyrethroids in
Ae. aegypti
from
Martinique and might seriously threatens the control of dengue vectors in the future.
A better understanding of the genetic basis of insecticide resistance is an essential step
to implement more effective vector control strategies in the field in order to minimize
dengue outbreaks.
AVAILABILITY
Data Deposition
The description of the microarray “
Aedes Detox Chip
” can be accessed at ArrayEx-
press http://www.ebi.ac.uk/arrayexpress acc. No. A-MEXP-623.
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