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Aquatic Toxicology 93 (2009) 61–69
Contents lists available at ScienceDirect
Aquatic Toxicology
journa l homepage: www.e lsev ier .com/ locate /aquatox
Impact of glyphosate and benzo[a]pyrene on the tolerance of mosquito larvae to
chemical insecticides. Role of detoxification genes in response to xenobiotics
Muhammad Asam Riaza, Rodolphe Poupardina, Stéphane Reynauda, Clare Strodeb,
Hilary Ransonb, Jean-Philippe Davida,∗
a Laboratoire d’Ecologie Alpine (LECA, UMR CNRS 5553), équipe Perturbation Environnementales et Xenobiotiques, France
b Vector Research Group, Liverpool School of Tropical Medicine, United Kingdom
a r t i c l e i n f o
Article history:
Received 21 December 2008
Received in revised form 19 March 2009
Accepted 20 March 2009
Keywords:
Mosquitoes
Aedes aegypti
Induction
Resistance
Insecticides
Xenobiotics
Detoxification
Cytochrome P450 monooxygenases
Glutathione S-transferases
Esterases
Oxidative stress
a b s t r a c t
The effect of exposure of Aedes aegypti larvae for 72 h to sub-lethal concentrations of the herbicide
glyphosate and the polycyclic aromatic hydrocarbon benzo[a]pyrene on their subsequent tolerance to
the chemical insecticides imidacloprid, permethrin and propoxur, detoxification enzyme activities and
transcription of detoxification genes was investigated. Bioassays revealed a significant increase in larval
tolerance to imidacloprid and permethrin following exposure to benzo[a]pyrene and glyphosate. Larval
tolerance to propoxur increased moderately after exposure to benzo[a]pyrene while a minor increased
tolerance was observed after exposure to glyphosate. Cytochrome P450 monooxygenases activities were
strongly induced in larvae exposed to benzo[a]pyrene and moderately induced in larvae exposed to imida-
cloprid and glyphosate. Larval glutathione S-transferases activities were strongly induced after exposure
to propoxur and moderately induced after exposure to benzo[a]pyrene and glyphosate. Larval esterase
activities were considerably induced after exposure to propoxur but only slightly induced by other xeno-
biotics. Microarray screening of 290 detoxification genes following exposure to each xenobiotic with the
DNA microarray Aedes Detox Chip identified multiple detoxification and red/ox genes induced by xeno-
biotics and insecticides. Further transcription studies using real-time quantitative RT-PCR confirmed the
induction of multiple P450 genes, 1 carboxy/cholinelesterase gene and 2 red/ox genes by insecticides
and xenobiotics. Overall, this study reveals the potential of benzo[a]pyrene and glyphosate to affect the
tolerance of mosquito larvae to chemical insecticides, possibly through the cross-induction of particular
genes encoding detoxification enzymes.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Mosquitoes transmit numerous human and animal pathogens
and chemical insecticides are widely employed in their control.
However the success of control programs is now threatened as the
repeated exposure of mosquito populations to chemical insecti-
cides has led to the selection of mutations conferring an increased
resistance to these insecticides (Hemingway et al., 2004). Inherited
resistance to chemical insecticides is usually caused by mutations
 Data deposition: The description of the microarray ‘Aedes Detox Chip’ can be
accessed at http://www.ebi.ac.uk/arrayexpress. Experimental microarray data have
been deposited at VectorBase.org and can be accessed at: http://funcgen.vectorbase.
org/ExpressionData/experiment/Larval%20response%20to%202%20pollutants%20
and%203%20insecticides%20(Riaz%20et%20al.,%202009).
∗ Corresponding author at: Laboratoire d’Ecologie Alpine (LECA), UMR CNRS-
Université 5553, Unit Perturbations Environnementales et Xénobiotiques, Domaine
Universitaire de Saint-Martin d’Hères, 2233, rue de la piscine Bât D Biologie, BP 53,
38041 Grenoble Cedex 9, France. Tel.: +33 476 51 44 59; fax: +33 476 51 44 63.
E-mail address: jean-philippe.david@ujf-grenoble.fr (J.-P. David).
in the protein targeted by the insecticide (target-site resistance)
or the increases in the rate of bio-degradation of the insecticide
(metabolic resistance). Considerable research efforts are focused
on elucidating the molecular basis of these resistance mechanisms
but less attention has been paid to the short-term effect of exposure
to insecticides or other xenobiotics on the mosquitoes’ tolerance to
insecticides and yet this could also have a significant impact on
the efficacy of mosquito control. More precisely, it can be hypothe-
sized that in polluted environments, xenobiotics found in mosquito
habitats may induce particular enzymes involved in the degrada-
tion of chemical insecticides, leading to an increased tolerance of
mosquitoes to insecticides. This is supported by the capacity of
detoxification enzymes such as cytochrome P450 monooxygenases
(P450s or CYP for genes), glutathione S-transferases (GSTs) and car-
boxy/cholinelesterases (CCEs), to be induced by various chemicals
(Hemingway et al., 2002, 2004; Feyereisen, 2005).
To date, few studies have investigated molecular interac-
tions between other environmental xenobiotics and insecticides
in aquatic insects. Exposure of Ae. albopictus larvae to benzoth-
iazole (a major leachate compound of automobile tires) and
0166-445X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2009.03.005

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62 M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69
pentachlorophenol (a wood-protecting agent) increased their tol-
erance to different types of insecticides such as carbaryl, rotenone
and temephos (Suwanchaichinda and Brattsten, 2001, 2002). This
increased tolerance was correlated with an induction of P450
activity. Recently, microarray-based approaches have been used to
investigate the effect of xenobiotic exposure on the transcription
of detoxification genes in Drosophila. The barbiturate phenobarbi-
tal and the herbicide atrazine induced the transcription of multiple
CYPs and GSTs in adult flies including genes previously linked to
insecticide resistance (Le Goff et al., 2006). In mammals, a causal
link between the induction of particular detoxification enzymes
by xenobiotics and their ability to metabolize them has been
demonstrated and successfully utilized to identify drug metabo-
lizing enzymes (Waxman, 1999; Luo et al., 2004). This approach
was also used to identify two CYP6 genes in Papilio polyxenes
metabolizing furanocoumarins, toxins produced by their host plant
(Petersen et al., 2001; Wen et al., 2003). Hence, studying the induc-
tion profile of insect detoxification enzymes has been suggested
as a mean to identify the major enzymes involved in insecticide
detoxification. In Drosophila, exposure to high concentrations of
insecticides induced the transcription of few detoxification genes
while two known inducers (phenobarbital and caffeine) and piper-
onyl butoxide induced multiple detoxification genes, including
those involved in insecticide metabolism (Willoughby et al., 2006,
2007). In mosquitoes, insecticides have also been shown to induce
detoxification enzymes. By using a microarray representing more
than 11,000 unique ESTs, Vontas et al. (2005) identified Anopheles
gambiae detoxification genes induced by the insecticide perme-
thrin. Recently, we used Ae. aegypti larvae to study the interactions
between three environmental pollutants and three chemical insec-
ticides (Poupardin et al., 2008). This study revealed that exposing
mosquito larvae to sub-lethal concentrations of the herbicide
atrazine, copper sulfate and fluoranthene increased their tolerance
to the pyrethroid insecticide permethrin and the organophosphate
insecticide temephos. In these experiments, increased tolerance
was correlated to an elevation of detoxification enzyme activities
and, by using a DNA microarray approach, specific detoxification
genes induced by these xenobiotics were identified (Poupardin et
al., 2008).
The objective of the current study was to determine whether
other environmental xenobiotics found in polluted mosquito
breeding sites also impacted on the mosquitoes’ tolerance to chem-
ical insecticides. Glyphosate (N-(phosphonomethyl)glycine, trade
name Roundup) is a soluble systemic herbicide. It is used massively
on crops genetically engineered to resist its effects (Roy, 2004;
Young, 2006). Although glyphosate does not seem to generate a sig-
nificant toxicity on most arthropods (Haughton et al., 2001; Jackson
and Pitre, 2004), its indirect potential effects on insect ability to
resist insecticides have not yet been investigated. Concentrations of
glyphosateup to1 mg/Lhavebeen recorded inpoolsor streamsnear
agricultural areas (Wanetal., 2006) suggesting thatmosquito larvae
near treated areas can be temporarily exposed to high concentra-
tions of this herbicide and its metabolites. The polycyclic aromatic
hydrocarbon (PAH) benzo[a]pyrene is a common product of incom-
plete combustion of fossil fuels such as coal, diesel and gasoline
(Bostrom et al., 2002; Pengchai et al., 2003). This hydrophobic pol-
lutant has been found at concentrations up to 5 ppm adsorbed on
particles from various ecosystems (Lewis et al., 1999; Lambert and
Lane, 2004) and is likely to be in contact with mosquito larvae,
commonly feeding on small particles, in breeding sites located in
proximity of industrial or urban areas (Hassanien and Abdel-Latif,
2008). In vertebrates, planar aromatic hydrocarbons can trigger
the induction of CYP genes via the intracellular aryl hydrocar-
bon receptor (AhR) (Goksoyr and Husoy, 1998). As these genes
have been frequently involved in metabolic resistance to chemical
insecticides in insects, it can be hypothesized that benzo[a]pyrene
has an impact on the tolerance of mosquito larvae to chemical
insecticides.
In the present study, we investigate the capacity of glyphosate
and benzo[a]pyrene to modify the tolerance of Ae. aegypti lar-
vae to three different chemical insecticides used worldwide for
controlling mosquito populations (permethrin, imidacloprid and
propoxur). We exposed mosquito larvae for 72 h to sub-lethal
concentrationsof eachchemical before comparing their larval toler-
ance to each insecticide and their detoxification enzyme activities.
Transcription pattern of 290 detoxification genes following expo-
sure to xenobiotics and insecticides were compared by using the
microarray ‘Aedes Detox Chip’ (Strode et al., 2008) and validated
by real-time quantitative RT-PCR. Overall, our work suggests that
the induction of detoxification enzymes involved in insecticide
metabolism by benzo[a]pyrene and glyphosate may enhance the
tolerance of mosquito larvae to chemical insecticides.
2. Materials and methods
2.1. Mosquitoes and xenobiotics
A laboratory strain of Ae. aegypti (Bora–Bora strain, susceptible
to insecticides) was reared in standard insectary conditions (26 ◦C,
8 h/12 h light/dark period, tap water) and used for all experiments.
This mosquito species is an important vector of human pathogens
such as dengue hemorrhagic fever and is often found in close prox-
imity to urban, sub-urban and industrial areas (Dutta et al., 1999).
Larvae were reared in insectary conditions with controlled amount
of larval food (hay pellets) for 3 days before exposure for 72 h to two
different xenobiotics likely to be found in highly polluted mosquito
larvae habitats: the herbicide glyphosate (trade name Roundup,
Monsanto,Belgium)and thepolycyclic aromatichydrocarbon (PAH)
benzo[a]pyrene (Fluka, USA).
2.2. Pre-exposure of mosquito larvae to xenobiotics
Pre-exposures to xenobiotics were performed in triplicate with
100 homogenous 2nd stage larvae in 200 mL of tap water con-
taining 50 mg of ground larval food (hay pellets). Concentrations
of xenobiotics used for larval pre-exposure were chosen accord-
ing to the concentrations likely to be found in highly polluted
mosquito breeding sites (INERIS, http://www.ineris.fr/rsde/). Prior
to bioassays with insecticides, larvae were exposed for 72 h to 0.1
or 1M benzo[a]pyrene and glyphosate separately. After 72 h, 4th
stage larvae were collected, rinsed twice in tap water and immedi-
ately used for bioassays. Biochemical and molecular analysis were
performed on the mosquitoes pre-exposed in the same manner
but in addition to benzo[a]pyrene and glyphosate, the effect of
pre-exposure to three chemical insecticides on enzyme activity
and gene transcription was also investigated. Three insecticides
massively employed worldwide for mosquito control, belong-
ing to different chemical classes and having different modes of
action were used: the neonicotinoid imidacloprid (Sigma–Aldrich,
Germany), the pyrethroid permethrin (Chem Service, USA) and
the carbamate propoxur (Sigma–Aldrich, Germany). For insecti-
cide pre-exposures, a concentration resulting in 10–15% larval
mortality after 72 h exposure was selected. This low mortality
threshold was chosen in order to minimize the effect of the arti-
ficial selection of particular phenotypes more resistant to the
insecticide during pre-exposure. Concentrations of xenobiotics
used for pre-exposure were: 1M (169.1g/L) glyphosate, 1M
(252.3g/L) benzo[a]pyrene, 25g/L imidacloprid, 1g/L per-
methrin and 200g/L propoxur. For benzo[a]pyrene, the water
solubility limit (∼10g/L) was exceeded in order to mimic an
aquatic environment highly contaminated with benzo[a]pyrene
where mosquito larvae can ingest high dose of this pollutant

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M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69 63
together with food particles or as micro-crystals. After 72 h, 4th
stage larvae were collected, rinsed twice in tap water and immedi-
ately used for the determination of detoxification enzyme activities
and RNA extractions. All larval pre-exposures were repeated three
times with egg batches from different generations.
2.3. Bioassays with insecticides
Larval bioassays were conducted comparatively on larvae
exposed to glyphosate or benzo[a]pyrene and unexposed larvae
(controls) with the 3 chemical insecticides imidacloprid, perme-
thrin and propoxur. Bioassays were performed in triplicate with 25
larvae in 50 mL insecticide solution and repeated 3 times with lar-
vae from different xenobiotic exposure experiments (see above).
Four different insecticide concentrations leading to larval mortal-
ity ranging from 5% to 95% were used. Imidacloprid, permethrin
and propoxur were used at 300–2750, 2.5–10 and 400–1000g/L,
respectively. Larval mortality was monitored after 24 h contact
with insecticide and further analyzed using the Log-Probit software
developed by Raymond (1993). For each insecticide, the mean LC50
was determined and tolerance ratios for larvae exposed to each
xenobiotic comparatively with unexposed larvae were calculated
and expressed as fold increased tolerance. Because comparison of
LC50 values may not well represent differential tolerance across
all concentrations of insecticide used for bioassays, differential
insecticide tolerance between larvae exposed to each xenobiotics
and controls was further analyzed as described in Poupardin et al.
(2008) by generating a Generalized Linear Model (GLM) from mor-
tality data followed by a likelihood ratio test using R software (R
Development Core Team, 2007).
2.4. Glutathione S-transferase activities
Glutathione S-transferase (GST) activities were measured
on cytosolic fractions using 1-chloro-2,4-dinitrobenzene (CDNB;
Sigma–Aldrich,Germany) as substrate (Habig et al., 1974).Onegram
of fresh larvae were homogenised in 0.05 M phosphate buffer (pH
7.2) containing 0.5 mM DTT, 2 mM EDTA and 0.8 mM PMSF. The
homogenate was centrifuged at 10,000 × g for 20 min at 4 ◦C and
the resulting supernatant was ultracentrifuged at 100,000× g for
1 hat4 ◦C. Protein contentof the cytosolic fraction (100,000 g super-
natant) was determined by the Bradford method before measuring
GST activities. The reaction mixture contained 200g protein,
2.5 mL of 0.1 M phosphate buffer 1.5 mM reduced glutathione
(Sigma) and 1.5 mM CDNB. The absorbance of the reaction was mea-
sured after 1 min at 340 nm with a UVIKON 930 spectrophotometer.
Results were expressed as median nanomoles of conjugated CDNB
per mg of protein per minute ± interquartile ranges (IQR). Three
biological replicates per treatment were made and each measure-
ment was repeated 6 times. Statistical comparison of GST activities
between controls and pre-exposed larvae was performed by using
a Mann and Whitney test (N = 3).
2.5. Cytochrome P450 monooxygenase activities
P450 monooxygenase activities were comparatively evaluated
by measuring ethoxycoumarin-O-deethylase (ECOD) activities on
microsomal fractions based on the microfluorimetric method of
De Sousa et al. (1995). For each sample, the microsomal fraction
was obtained from 100,000 g pellet (see above) and resuspended
in 0.05 M phosphate buffer before measuring microsomal protein
content by the Bradford method. Twenty micrograms microso-
mal proteins were then added to 0.05 M phosphate buffer (pH
7.2) containing 0.4 mM 7-ethoxycoumarin (7-Ec, Fluka) and 0.1 mM
NADPH for a total reaction volume of 100l and incubated at
30 ◦C. After 15 min, the reaction was stopped and the production
of 7-hydroxycoumarin (7-OH) was evaluated by measuring the flu-
orescence of each well (380 nm excitation, 460 nm emission) with
a Fluoroskan Ascent spectrofluorimeter (Labsystems, Helsinki, Fin-
land) in comparison with a scale of 7-OH (Sigma). P450 activities
were expressed as median picomoles of 7-OH per mg of microsomal
protein per minute ± IQR. Three biological replicates per treatment
were made and each measure was repeated 8 times. Statistical com-
parison of P450 activities between controls and pre-exposed larvae
was performed by using a Mann and Whitney test (N = 3).
2.6. Esterase activities
Esterases activities were comparatively measured on cytosolic
fractions from the 100,000 g supernatant (see above) accord-
ing to the method described by Van Asperen (1962) with
-naphthylacetate and-naphthylacetateusedas substrates (-NA
and -NA, Sigma–Aldrich, Germany). Thirty micrograms cytosolic
proteins were added to 0.025 mM phosphate buffer (pH 6.5) with
0.5 mM of -NA or -NA for a total volume reaction of 180L and
incubated at 30 ◦C. After 15 min, the reaction was stopped by the
addition of 20L 10 mM Fast Garnett (Sigma) and 0.1 M sodium
dodecyl sulfate (SDS, Sigma–Aldrich, Germany). The production of
- or-naphthol was measured at 550 nm with a 960 microplate
reader (Metertech, Taipei, Taiwan) in comparison with a scale of
-naphthol or -naphthol and expressed as median moles of -
or -naphthol per mg of cytosolic protein per minute ± IQR. Three
biological replicates per treatment were made and each measure
was repeated 8 times. Statistical comparison of esterases activities
between controls and pre-exposed larvae was performed by using
a Mann and Whitney test (N = 3).
2.7. Microarray screening of detoxification genes induced after
xenobiotic exposure
The ‘Aedes detox chip’ DNA-microarray developed by Strode et al.
(2008) was used to monitor changes in the transcription of multiple
detoxification genes in larvae exposed to each xenobiotic compared
to unexposed larvae. This microarray contains 318 70-mer probes
representing 290 detoxification genes including all cytochrome
P450 monooxygenases (P450s), glutathione S-transferases (GSTs),
carboxy/cholinesterases (CCEs) and additional enzymes potentially
involved in response to oxidative stress from the mosquito Ae.
aegypti. Each 70-mer probe, plus 6 housekeeping genes and 23 arti-
ficial control genes (Universal Lucidea Scorecard, G.E. Health Care,
Bucks, UK) were spotted four times on each array.
RNA extractions, cDNA synthesis and labelling reactions were
performed independently for each biological replicate. Total RNA
was extracted from batches of thirty 4th stage larvae using the
PicoPureTM RNA isolation kit (Molecular Devices, Sunnyvale, CA,
USA) according to manufacturer’s instructions. Genomic DNA was
removed by digesting total RNA samples with DNase I by using the
RNase-free DNase Set (Qiagen). Total RNA quantity and quality were
assessedbyspectrophotometrybefore furtheruse.MessengerRNAs
were amplified using a RiboAmpTM RNA amplification kit (Molec-
ular Devices) according to manufacturer’s instructions. Amplified
RNAs were checked for quantity and quality by spectrophotometry.
For each hybridisation, 8g of amplified RNAs were reverse tran-
scribed into labelled cDNA and hybridised to the array as previously
described by David et al. (2005). Each comparison was repeated
three times with different biological samples. For each biological
replicate, 2 hybridisations were performed in which the Cy3 and
Cy5 labels were swapped between samples for a total of 6 hybridi-
sations per comparison. All hybridisations were performed against
a global reference sample obtained from a pool of amplified RNAs
from un-exposed larvae obtained from each biological replicate.

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64 M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69
Spot finding, signal quantification and spot superimposition for
both dye channels were performed using Genepix 5.1 software
(Axon Instruments, Molecular Devices, Union City, CA, USA). For
each data set, any spot satisfying one of the following conditions
for any channel was removed from the analysis: (i) intensity val-
ues less than 300 or more than 65,000, (ii) signal to noise ratio less
than 3, (iii) less than 60% of pixel intensity superior to the median
of the local background ±2. Normalization and statistic analysis
were performed on R software (R Development Core Team, 2008)
with limma package available on www.bioconductor.org according
to Muller et al. (2007). First, background intensities were sub-
tracted to the foreground intensities for both Cy3 (G) and Cy5 (R)
intensities. Then, corrected intensities were transformed to inten-
sity log-ratios, M = log2 R/G, and their corresponding geometrical
means, A = (log2 R + log2 G)/2. Data were then normalized using the
local intensity-dependent algorithm Lowess (Cleveland and Devlin,
1988). For each comparison, only genes detected in at least 2 of 6
hybridisations were used for further statistical analysis. To assess
the data significance, M values were then submitted to a one sample
Student’s t-test against the baseline value of 1 (equal gene tran-
scription in both samples). Genes showing an transcription ratio
>1.5-fold in either direction and a corrected P-value lower than
0.01 (Benjamini and Hochberg’s multiple testing correction) were
considered significantly differentially expressed after xenobiotic
exposure. In Table 2, M values were transformed into transcription
ratios.
2.8. Quantitative real-time RT-PCR
Transcription profiles of 8 particular genes found induced by dif-
ferentxenobiotics in larvaewerevalidatedby real-timequantitative
RT-PCR using the same RNA samples as used for microarray exper-
iments. Four micrograms of total RNA were treated with DNase I
(Invitrogen) and used for cDNA synthesis with superscript III and
oligo-dT20 primer for 60 min at 50 ◦C according to manufacturer’s
instructions. Resulting cDNAs were diluted 100 times for real-time
quantitative PCR reactions. All primer pairs used for quantitative
PCR were tested for generating a unique amplification product
by melt curve analysis. Real-time quantitative PCR reactions of
25L were performed in triplicate on an iQ5 system (BioRad)
using iQ SYBR Green supermix (BioRad), 0.3M of each primer
and 5L of diluted cDNAs according to manufacturer’s instruc-
tions. For each gene analysed, a cDNA dilution scale from 10 to
100,000 times was performed in order to assess efficiency of PCR.
Data analysis was performed according to the CT method taking
into account PCR efficiency (Pfaffl, 2001) and using the two genes
encoding the ribosomal protein L8 (AeRPL8 GenBank accession no.
DQ440262) and the ribosomal protein S7 (AeRPS7 GenBank acces-
sion no. EAT38624.1) for normalisation. Results were expressed as
mean transcription ratios (±SE) between larvae exposed to each
xenobiotic or insecticide and unexposed larvae (controls). Only
genes showing more than 1.5-fold over-transcription were consid-
ered induced.
3. Results
Exposing Ae. aegypti larvae to sub-lethal concentrations of the
herbicide glyphosate and the PAH benzo[a]pyrene for 72 h affected
their subsequent tolerance to insecticides. Overall, exposing larvae
to these xenobiotics increased larval tolerance to insecticides with
a more pronounced effect observed with higher concentrations of
xenobiotics (Table 1). Larval tolerance to the neonicotinoid insecti-
cide imidacloprid increased after exposure to 1M benzo[a]pyrene
and glyphosate (3.51-fold and 1.98-fold increase in LC50, respec-
tively) and also, to a lesser extent, after exposure to 0.1M
benzo[a]pyrene and glyphosate (1.83-fold and 1.70-fold, respec- Ta
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M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69 65
Fig. 1. Differential GST activities of Ae. aegypti larvae exposed for 72 h to sub-
lethal concentrations of glyphosate, benzo[a]pyrene, imidacloprid, permethrin and
propoxur. Larval GST activities were measured with the CDNB method (Habig et al.,
1974) on 200g cytosolic proteins during 1 min and expressed as median nmol of
conjugated CDNB/mg protein/min ± interquartile ranges (IQR). For each treatment,
statistical comparison of larval GST activities between xenobiotic-exposed larvae
and controls were performed with a Mann and Whitney’s test (N = 3, *P < 0.05).
tively). Larval tolerance to the pyrethroid insecticide permethrin
increased after exposure to 1M benzo[a]pyrene or glyphosate
(1.78-fold and 1.72-fold, respectively). This increased tolerance to
permethrin remainsevenwhenusing0.1Mbenzo[a]pyrene (1.72-
fold) but decreased when using 0.1M glyphosate (1.39-fold).
Larval tolerance to the carbamate insecticide propoxur was only
slightly enhanced after exposure to the highest concentration of
benzo[a]pyrene and glyphosate (1.39-fold and 1.14-fold, respec-
tively).
Larval exposure to xenobiotics and insecticides led to signifi-
cant modifications of their GST, P450 and esterases activities, as
measured using model substrates. GST activity with CDNB (Fig. 1)
was strongly induced after exposure to propoxur (2.04-fold with
P < 0.05). Exposureof larvae tobenzo[a]pyrenealso slightly induced
GST activity (1.37-fold and P < 0.05) while exposure to glyphosate,
imidacloprid and permethrin did not significantly affect larval GST
activities. Microsomal P450 activities (Fig. 2) were significantly
induced after exposing larvae to benzo[a]pyrene (2.09-fold with
P < 0.05) while no significant changes were observed after exposure
to other xenobiotics. Significant modifications of esterase activities
were observed in larvae exposed to xenobiotics and insecticides
(Fig. 3). Alpha-esterase activities were highly induced in larvae
Fig. 2. Differential microsomal P450 activities of Ae. aegypti larvae exposed for 72 h
to sub-lethal concentrations of glyphosate, benzo[a]pyrene, imidacloprid, perme-
thrin and propoxur. Larval P450 activities were measured with the ECOD method
(De Sousa et al., 1995) on 20g microsomal proteins after 15 min and expressed
as median pmol of 7-OH/mg microsomal protein/minute ± interquartile ranges
(IQR). For each treatment, statistical comparison of larval P450 activities between
xenobiotic-exposed larvae and controls were performed with a Mann and Whitney’s
test (N = 3, *P < 0.05).
Fig. 3. Differential esterase activities of Ae. aegypti larvae exposed for 72 h to
sub-lethal concentrations of five different xenobiotics (glyphosate, benzo[a]pyrene,
imidacloprid, permethrin and propoxur). Larval-esterase and-esterase activities
were measured with -naphthyl-acetate and -naphthyl-acetate as substrates on
30g cytosolic proteins during 15 min and expressed as median mol of -or -
naphtol/mg protein/min ± interquartile ranges (IQR). Statistical comparison of larval
esterasesactivitiesbetweenxenobiotic-exposed larvaeandcontrolswereperformed
with a Mann and Whitney’s test (N = 3, *P < 0.05).
exposed to propoxur (2.20-fold with P < 0.05), slightly significantly
elevated after exposure to glyphosate (1.10-fold with P < 0.05) while
no significant induction was observed with other xenobiotics. Sim-
ilarly, -esterase activities were highly induced in larvae after
exposure to propoxur (2.40-fold with P < 0.05) but no significant
induction was observed with other xenobiotics.
By using the microarray ‘Aedes Detox Chip’ representing 290 Ae.
aegypti genes encoding detoxification and red/ox enzymes (Strode
et al., 2008), 23 detoxification genes significantly induced in 4th
stage larvae following a 72 h exposure to a sub-lethal concentration
of xenobiotics or insecticides were identified (Table 2 and Suppl.
Table 1). Among them, 9 genes encode P450s (CYPs), 4 encode
GSTs, 3 encode carboxy/cholinelesterases (CCEs) and 7 encode
enzymes putatively involved in response to oxidative stress (red/ox
enzymes). Larvaeexposed to theherbicideglyphosate showedasig-
nificant induction of 5 CYPs (CYP6N11, CYP6N12, CYP6Z6, CYP6AG7
and CYP325AA1), 3 GSTs (AaGSTe4, AaGSTe7, AaGSTi1 and AaGSTs1-2)
and 1 glutathione peroxidase. Exposing larvae to benzo[a]pyrene
significantly induced 3 CYP genes (CYP6Z6, CYP6Z8 and CYP9M5),
2 GSTs (AaGSTi1 and AaGSTs1-2) and 2 red/ox genes (1 superoxide
dismutase and 1 reductase). Exposure to imidacloprid significantly
induced 2 CYPs (CYP4G36 and CYP6CC1), 1 GST (AaGSTs1-2), 3 CCEs
(CCEae1o, CCEae2o and CCEae3o) and 6 red/ox genes including a
superoxide dismutase, 4 peroxidases and 1 reductase. Exposure to
a sub-lethal concentration of the pyrethroid insecticide permethrin
significantly induced only one CCE (CCEae3o). Propoxur expo-
sure revealed a significant over-transcription of 1 GST (AaGSTi1),
1 CCE (CCEae3o) and 1 superoxide dismutase. Finally, microar-
ray screening revealed that different chemicals can significantly
induce identical genes such as CYP6Z6 induced by glyphosate and
benzo[a]pyrene, AaGSTi1 induced by glyphosate, benzo[a]pyrene
and propoxur and CCEae3o induced by the insecticides imidaclo-
prid, permethrin and propoxur.
Real-time quantitative RT-PCR was used to validate the tran-
scription pattern of 8 genes selected from microarray experiments
(Fig. 4). Overall, the induction patterns obtained from microar-
ray screening and real-time quantitative RT-PCR were in good
agreement (Pearson correlation coefficient r = 0.745, P < 0.001). The
induction of CYP6Z6, CYP6Z8, CYP9M5 and superoxide dismutase
(AAEL006271-RA) by benzo[a]pyrene was confirmed (3.1-fold, 4.4-
fold, 3.4-fold and 2.6-fold, respectively). Likewise, the induction of
CCEae3o (3.0-fold) and TPx2 (2.0-fold) by imidacloprid was con-
firmed. High induction ratios were obtained for CYP6Z8 and CYP9M5
(benzo[a]pyrene 4.4-fold and 3.4-fold, respectively). Finally, the

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66 M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69
Table 2
Microarray analysis of the induction of detoxification genes in Ae. aegypti larvae after 72 h exposure to xenobiotics and insecticidesa.
Gene name/annotation Transcript ID Glyphosate Benzo[a]pyrene Imidacloprid Permethrin Propoxur
Ratio P value Ratio P value Ratio P value Ratio P value Ratio P value
Cytochrome P450 monooxygenases
CYP4G36 AAEL004054-RA ND ND ND ND 1.77 8.3E−08 1.00 9.8E−01 1.04 6.8E−01
CYP6N11 AAEL009138-RA 1.75 2.1E−14 1.30 1.2E−03 0.88 4.0E−01 0.95 4.4E−01 1.07 4.6E−01
CYP6N12 AAEL009124-RA 1.68 4.2E−13 1.42 3.4E−12 0.86 1.6E−04 0.68 4.0E−12 1.04 2.0E−01
CYP6Z6 AAEL009123-RA 1.52 4.6E−14 1.96 3.9E−19 0.95 5.9E−02 1.06 3.5E−02 1.26 4.3E−09
CYP6Z8 AAEL009131-RA 1.09 8.8E−03 2.08 4.1E−17 0.86 1.9E−04 0.90 9.7E−04 0.81 2.9E−03
CYP6AG7 AAEL006989-RA 1.58 6.1E−09 1.06 2.6E−01 1.10 9.2E−02 0.87 1.6E−01 0.83 1.2E−02
CYP6CC1 AAEL014890-RA 0.47 4.6E−14 0.70 1.5E−05 1.63 1.2E−10 1.10 1.9E−02 1.18 1.4E−01
CYP9M5 AAEL001288-RA 1.49 6.8E−12 3.08 3.0E−13 1.10 6.1E−02 0.94 2.5E−01 1.48 3.4E−05
CYP325AA1 AAEL004012-RA 2.03 2.9E−12 1.46 2.5E−03 1.00 1.0E+00 1.25 1.6E−04 1.85 1.8E−02
Glutathione S-transferases
AaGSTe4 AAEL007962-RA 1.61 2.0E−20 1.37 4.8E−11 1.42 2.1E−10 1.03 3.5E−01 1.10 1.9E−01
AaGSTe7 AAEL007948-RA 1.56 3.2E−15 1.18 7.2E−08 0.93 5.4E−02 0.85 8.5E−06 1.02 5.6E−01
AaGSTi1 AAEL011752-RA 2.74 1.0E−23 2.33 2.9E−13 0.76 4.0E−02 0.87 2.1E−01 3.10 4.8E−10
AaGSTs1-2 AAEL011741-RB ND ND 1.60 5.1E−04 3.98 6.3E−09 ND ND ND ND
Carboxylesterases
CCEae1o AAEL004341-RA ND ND 1.06 4.6E−01 2.59 2.2E−06 1.49 1.8E−01 1.49 1.2E−04
CCEae2o AAEL007486-RA 0.72 2.5E−11 0.96 2.2E−01 1.56 1.3E−09 1.16 3.2E−04 1.13 9.9E−03
CCEae3o AAEL011944-RA 0.27 4.8E−11 0.88 1.6E−03 4.34 1.4E−16 1.67 8.4E−10 1.75 2.6E−08
Red/ox enzymes
Superoxide dismutase AAEL006271-RA 1.19 1.5E−06 1.89 9.8E−18 2.51 7.8E−10 1.39 2.4E−07 1.50 2.3E−09
Peroxidasin AAEL000376-RA ND ND 1.21 6.5E−01 1.77 6.3E−04 ND ND ND ND
Peroxidase AAEL013171-RA 0.77 8.0E−07 0.94 5.3E−02 1.67 5.8E−14 1.29 1.8E−07 1.29 7.5E−07
Glutathione peroxidase AAEL000495-RA 1.76 4.2E−06 1.45 9.4E−04 2.05 1.2E−05 0.76 2.5E−01 1.22 3.3E−02
Thioredoxin peroxidase TpX2 AAEL004112-RA ND ND 1.27 2.3E−01 2.19 4.3E−04 ND ND 1.22 4.2E−01
Aldo-keto reductase AAEL007275-RA ND ND 0.76 3.2E−02 1.88 1.3E−05 0.93 8.1E−01 1.09 3.9E−02
Aldo-keto reductase AAEL015002-RA 1.03 8.4E−01 1.94 4.3E−04 1.35 3.6E−01 1.50 3.8E−03 1.59 2.4E−03
a Larvae were exposed for 72 h to sub-lethal concentrations of five different insecticides and xenobiotics (permethrin, imidacloprid, propoxur, benzo[a]pyrene and
glyphosate) before microarray analysis of the transcription of detoxification genes. Only genes showing a significant over-transcription (ratio > 1.5 and P value < 1.0E−03)
after a minimum of one treatment are shown. Transcription ratios between treated larvae and controls are indicated for each treatment. Transcription ratios and P values of
genes significantly induced are shown in bold. ND: Gene not detected in at least 3 hybridisations out of 6.
slight induction of CYP6Z6, AaGSTe4 and AaGSTe7 by glyphosate,
CCEae3o by permethrin and propoxur and superoxide dismutase
(AAEL006271-RA) by imidacloprid and propoxur were confirmed
by real-time quantitative RT-PCR. The most important discrepan-
cies between the two techniques were obtained for CYP6Z8 with
benzo[a]pyrene (4.4-fold in qRT-PCR and only 2.0-fold in microar-
ray) and CCEae3O with imidacloprid (3.0-fold in qRT-PCR and
4.34-fold in microarray).
Comparison of the transcription levels of those 8 detoxification
genes in 4th stage larvae revealed differences in their basal tran-
scription level (Fig. 5). As expected, transcription of detoxification
genes was considerably lower than the transcription of the house-
Fig. 4. Comparative real-time quantitative RT-PCR analysis of the differential
transcription of 8 selected genes in Ae. aegypti larvae exposed for 72 h to sub-
lethal concentrations of glyphosate, benzo[a]pyrene, imidacloprid, permethrin and
propoxur. Gene transcription values are indicated as transcription ratios (±SE) in
larvae exposed to each xenobiotic comparatively to unexposed larvae (controls).
The housekeeping genes AeRPL8 and AeRPS7 were used as internal controls for nor-
malization. Horizontal broken line indicates a 1.5-fold over-transcription in treated
larvae as compared to controls.
Fig. 5. Constitutive transcription levels of 8 selected genes in Ae. aegypti larvae. Gene
transcription was measured by real-time quantitative RT-PCR in 4th-stage larvae in
absence of xenobiotics. transcription levels were normalized with the housekeeping
gene AeRPL8 and are shown as transcription ratios relative to CYP6Z8, the detoxifi-
cation gene showing the highest transcription level (mean ± SE). Fold transcription
is indicated above each bar.
keeping gene AeRPL8 (from 33 to >3200-fold reduction). Among
detoxification genes, larval basal transcription levels vary greatly,
with CYP6Z8 and GSTe7 showing the highest transcription levels,
GSTe4, TPx2, CCEae3O, CYP6Z6 and SOD being moderately tran-
scribed (2–11-fold reduction comparatively to CYP6Z8) and CYP9M5
being transcribed at very low level in 4th-stage larvae (95-fold
reduction comparatively to CYP6Z8).
4. Discussion
Lasting recent decades, the amount of anthropogenic xenobi-
otics released into natural ecosystems has dramatically increased.
Although the effect of these chemicals on human health is inten-

Author's personal copy
M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69 67
sively studied, their impact on insect metabolism and insecticide
resistance mechanisms remains poorly understood. Here we inves-
tigated the potential of the herbicide glyphosate and the PAH
benzo[a]pyrene, likely to be found in polluted mosquito breed-
ing sites, to modify the tolerance of mosquito larvae to 3 chemical
insecticides through the induction of detoxification enzymes.
We showed that the presence of these xenobiotics in the water
where mosquito larvae develop can significantly increase their
tolerance to insecticides, particularly the pyrethroid permethrin
and the neonicotinoid imidacloprid. Although the increases in
insecticide tolerance reported here are lower than inherited
resistance levels obtained after many generations of selection
with insecticides, our results show that the presence of these
xenobiotics may contribute to insecticide tolerance in mosquito
larvae. This phenomenon might be more pronounced in highly
polluted mosquito breeding sites or following a temporary dra-
matic pollution event. Recently, we also showed that exposing
Ae. aegypti larvae for 24 h to low concentrations of the herbicide
atrazine and the PAH fluoranthene increase their tolerance to the
insecticide permethrin and temephos (Poupardin et al., 2008).
Suwanchaichinda and Brattsten (2001) exposed Ae. albopictus
larvae for 48 h to various herbicides and fungicides before mea-
suring their tolerance to the insecticide carbaryl. Interestingly,
no significant effect was observed with atrazine, simazine and
2,4-dichlorphenoxyacetic acid (2,4-D) while a 70% reduced mor-
tality to carbaryl and a significant increase of P450 activities were
observed after exposing larvae to pentachlorophenol.
Many studies have revealed the capacity of insect detoxifica-
tion enzymes to be induced by xenobiotics and the relationship
between elevated detoxifying enzyme levels and tolerance to
chemical insecticides (Yu, 1996; Hemingway et al., 2004; Enayati
et al., 2005; Feyereisen, 2005). Our work demonstrates that larval
GST activities were strongly induced by the insecticide propoxur
and to a lesser extent by benzo[a]pyrene. Esterase activities were
strongly induced by propoxur but very low effect was observed
after exposure to glyphosate, suggesting a limited impact of this
pollutant on esterase-related insecticide metabolism. P450 activ-
ities appeared strongly induced by benzo[a]pyrene. Overall, our
work also suggests that insecticides may not always be the most
potent inducers of detoxifying enzymes able to metabolize them.
This hypothesis is supported by results obtained in Drosophila by
Willoughby et al. (2006) showing that short exposures to high
lethal concentrations of insecticides only induce few detoxification
genes comparatively to other inducers. Benzo[a]pyrene exposure
led to the highest increase of larvae tolerance to permethrin and
imidacloprid and was also the best inducers of P450 activities.
This trend supports the central role of P450s in the tolerance of
mosquito larvae to these two insecticides. Poupardin et al. (2008)
revealed that fluoranthene, another PAH, strongly induced P450s in
mosquito larvae together with enhancing their tolerance to perme-
thrin. The capacity of PAHs to induce P450 activities is well known
in vertebrates. Many PAHs induce P450s by binding to the AhR
(aryl hydrocarbon receptor) in the cytosol. Upon binding, the trans-
formed receptor translocates to the nucleus where it dimerises
with the aryl hydrocarbon receptor nuclear translocator and then
binds to DNA sequences such as xenobiotic response elements
(XREs) located upstream of certain genes. This process increases
transcription of certain genes, followed by increased protein pro-
duction. Recently, XRE-like sequences have been found upstream
insect CYP genes involved in xenobiotic metabolism (McDonnell
et al., 2004; Brown et al., 2005). Putative XRE-like elements have
also been found upstream An. gambiae CYP genes induced by the
insecticide permethrin (David J.P., unpublished data). Recently,
we showed that XRE-like elements are also found upstream Ae.
aegypti CYP genes induced by fluoranthene (Poupardin et al.,
2008). The fact that exposure to different PAHs induce mosquito
larvae P450 activities together with increasing their tolerance to
permethrin and imidacloprid might indicate that PAHs have the
ability, through an AhR-like nuclear receptor, to induce P450s
involved in the degradation of these insecticides in mosquitoes.
We used the microarray Aedes Detox Chip (Strode et al.,
2008) to identify 23 genes encoding detoxification and
red/ox enzymes induced in 4th stage larvae after exposure
to benzo[a]pyrene, glyphosate, imidacloprid, permethrin and
propoxur. Benzo[a]pyrene induced a significant over-transcription
of CYP6Z8, CYP6Z6 and CYP9M5 (Fig. 4). Poupardin et al. (2008) also
found CYP6Z8 induced by fluoranthene, copper sulfate and the two
insecticides permethrin and temephos. In the malaria vector An.
gambiae, CYP6Z genes have been frequently found constitutively
over-transcribed in insecticide-resistant strains (Nikou et al., 2003;
David et al., 2005; Muller et al., 2007). Recent studies demonstrated
that the enzyme encoded by An. gambiae CYP6Z1 can metabolize the
insecticides carbaryl and DDT while CYP6Z2, with a narrower active
site, only metabolizes carbaryl (Chiu et al., 2008; McLaughlin et al.,
2008). The high transcription level of CYP6Z8 in larvae (Fig. 5) may
indicate that this particular P450 play a major role in xenobiotic
response during the aquatic larval stage. Although transcription
ratios were lower, glyphosate also induced several CYP6s and
epsilon GSTs, indicating that this chemical may have an impact on
insecticide tolerance through P450 or GST induction.
Epsilon GSTs have been widely implicated in resistance to DDT
and pyrethroid insecticides (Ding et al., 2003; Ortelli et al., 2003;
Lumjuanetal., 2005;Strodeet al., 2008). Therefore, the slight induc-
tion of GST activities by glyphosate including the specific induction
of two epsilon-class GST genes (GSTe4 and GSTe7) might contribute
to the improved insecticide tolerance of mosquito larvae exposed
to this herbicide.
Two P450s, 1 GST, 3 carboxy/cholinesterases and several genes
encoding for enzymes potentially involved in response to oxida-
tive stress were found induced in larvae exposed to imidacloprid.
Although esterases have been reported to be potentially involved
in cross-resistance between the pyrethroid fenvalerate and imi-
dacloprid in the cotton aphid Aphis gossypii (Wang et al., 2002),
the direct involvement of esterases in resistance to neonicotinoids
remains unclear. In human pulmonary and neuronal cultivated
cells, imidacloprid was showed to induce cell toxicity leading to
apoptosis (Skandrani et al., 2006). It is known that P450 func-
tioning can generates excess reactive oxygen species, leading to
oxidative stress (Zangar et al., 2004) and that P450s are likely to
be involved in metabolic resistance to imidacloprid in insects (Le
Goff et al., 2003). Therefore, the induction of several genes encod-
ing red/ox enzymes observed after exposing larvae to imidacloprid
might result from the generation of excess reactive oxygen species
from P450-mediated imidacloprid metabolism.
Overall, our study demonstrated that the herbicide glyphosate
and the PAH benzo[a]pyrene likely to be found in polluted mosquito
breeding sites were able to increase tolerance of mosquito lar-
vae to different classes of insecticides and suggested that this
is the consequence of an induction of particular detoxification
enzymes. Considering that only genes belonging to main detox-
ification and red/ox enzyme families are represented on the
‘Aedes detox Chip’, a whole transcriptome analysis will allow iden-
tifying additional genes and molecular mechanisms potentially
involved in mosquitoes’ response to pollutants and insecticides.
Our study was focused on the short-term effect of xenobiotics on
the phenotypic plasticity associated with the tolerance of mosquito
larvae to insecticides. Finally, considering the persistent contami-
nation of wetlands by anthropogenic chemicals and the potential
effect of phenotypic plasticity on the selection of particular genes
(Ghalambor et al., 2007), the question of the long-term impact of
environmental xenobiotics on inherited insecticide resistance also
represents an important future research direction.

Author's personal copy
68 M.A. Riaz et al. / Aquatic Toxicology 93 (2009) 61–69
Acknowledgments
The present research project was co-funded by the French
National Research Agency (ANR ‘Santé-Environnement Santé-
travail’ (SEST), grant MOSQUITO-ENV 07SEST014), and the
mosquito control unit ‘Démoustication Rhône-Alpes’. M.A. Riaz was
funded by the higher education commission (HEC) of Pakistan. We
are grateful to J. Patouraux and T. Gaude for technical help. We thank
Prof. A. Cossins, Dr. M. Hughes and the Liverpool Microarray User
Community for microarray printing. We thank Dr. P. Muller for use-
ful help with microarray analysis and Dr. B. Maccallum for help with
microarray data deposition. We are grateful to Prof. P. Ravanel for
useful comments on the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.aquatox.2009.03.005.
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