Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus.

Abstract

Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCq(G0) and MAmCq(G0)) and their permethrin selected offspring (HAmCq(G8) and MAmCq(G6)). While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCq(G8) and MAmCq(G6) were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCq(G8) and MAmCq(G6) mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCq(G8) and MAmCq(G6); of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment.

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Genome Analysis of Cytochrome P450s and Their
Expression Profiles in Insecticide Resistant Mosquitoes,
Culex quinquefasciatus
Ting Yang, Nannan Liu*
Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, United States of America
Abstract
Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus
mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito
populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCqG0
and MAmCqG0) and their permethrin selected offspring (HAmCqG8and MAmCqG6). While the majority of the P450 genes
were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-
regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental
strains and the susceptible S-Lab strain, HAmCqG8and MAmCqG6were found to up-regulate 11 and 6% of total P450 genes
in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults.
Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were
either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or
down-regulated in the HAmCqG8and MAmCqG6mosquito populations in response to insecticide selection. Several genes
were identified as being up- or down-regulated in either the larvae or adults for both HAmCqG8and MAmCqG6; of these,
CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of
mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this
study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450
genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of
mosquitoes to changes in cellular environment.
Citation: Yang T, Liu N (2011) Genome Analysis of Cytochrome P450s and Their Expression Profiles in Insecticide Resistant Mosquitoes, Culex
quinquefasciatus. PLoS ONE 6(12): e29418. doi:10.1371/journal.pone.0029418
Editor: Immo A. Hansen, New Mexico State University, United States of America
Received September 2, 2011; Accepted November 28, 2011; Published December 29, 2011
Copyright: ? 2011 Yang, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The project described was supported by award number R21AI076893 to N.L. from the National Institute of Allergy and Infectious Diseases, AAES Hatch/
Multistate Grants ALA08-045 and ALA015-1-10026 to N.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript. No additional external funding was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: liunann@auburn.edu
Introduction
Cytochrome P450s have long been of particular interest as they
are critical for the detoxification and/or activation of xenobiotics
such as drugs, pesticides, plant toxins, chemical carcinogens and
mutagens. They are also involved in metabolizing endogenous
compounds such as hormones, fatty acids, and steroids. Basal and
up-regulation of P450 gene expression can significantly affect the
disposition of xenobiotics or endogenous compounds in the tissues
of organisms, thus altering their pharmacological/toxicological
effects[1]. Insect cytochrome P450sareknown toplayanimportant
role in detoxifying exogenous compounds such as insecticides [2–4]
and plant toxins [5,6]. While all insects probably possess some
capacity to detoxify insecticides and xenobiotics, the degree to
which they can metabolize and detoxify these toxic chemicals is of
considerable importance to their survival in a chemically unfriendly
environment [7] and to the development of resistance. A significant
characteristic of insect P450s is their transcriptional up-regulation,
resulting inincreased P450 protein levelsand P450 activities, which,
in turn, cause enhanced metabolic detoxification of insecticides and
plant toxins in insects, leading to the development of insecticide
resistance [4,8–16] and a higher tolerance to plant toxins [17,18].
Insect P450s are also known to be an important part of the
biosynthesis and degradation pathways of endogenous compounds
such as pheromones, 20-hydroxyecdysone, and juvenile hormone
(JH) [19–23] and thus play important roles in insect growth,
development, and reproduction.
Cytochrome P450s are a superfamily that can take a number of
related forms that frequently co-exist in the same cell type [24].
The rate at which a particular substrate is oxidized differs from
one P450 to another, so that the overall metabolism of a specific
substrate depends on the different forms present and varies
between tissues, life stages, and sexes [25]. Because of the multiple
cytochrome P450s expressed in each organism and the broad
substrate specificity of some of these isoforms, P450s are capable of
oxidizing a bewildering array of xenobiotics [25]. While the
importance of P450s in insect physiology and toxicology is widely
recognized, it is not yet clear how many P450 genes precisely are
involved in insecticide resistance in a single insect such as the
mosquito.
With the availability of the whole genome sequence for the
mosquito Culex quinquefasciatus [26], we are now able to
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characterize the expression profiles of P450s in insecticide resistant
mosquitoes and thus improve our understanding of the P450 gene
interactions that play a role in the physiological and toxicological
processes of insects. The current study focused on characterizing
the expression profiles of these P450 genes from mosquito
populations of Cx. quinquefasciatus bearing different phenotypes in
response to permethrin (susceptible, intermediate and highly
resistant) in order to pinpoint the key P450 genes involved in
insecticide resistance.
Materials and Methods
Mosquito strains
Five strains of the mosquito Cx. quinquefasciatus were studied.
HAmCqG0and MAmCqG0were field resistant strains collected
from Huntsville and Mobile, respectively, from sites located
.600 km apart in the state of Alabama, USA in 2002; the
locations were not privately-owned or protected in any way, no
specific permissions were required for these locations/activities,
and the study did not involve endangered or protected species.
Because Cx. quinquefasciatus is an important urban pest in Alabama,
it has been a major target for several insecticides, including Bti,
malathion, resmethrin, and permethrin, and control difficulties
have been reported before the collection [27]. Both Field strains
had the similar levels (10-fold compared with susceptible S-Lab) of
resistance to permethirn [28] and did not exposure to insecticides
after established as colonies in the laboratory. HAmCqG8was the
8thgeneration of permethrin-selected HAmCqG0offspring with a
2,700-fold level of resistance and MAmCqG6was the 6th
generation of permethrin-selected MAmCqG0offspring with a
570-fold level of resistance [29]. The permethrin selections for
both HAmCqG8and MAmCqG6were performed at the 4thinstar
larval stage [28,29]. S-Lab was an insecticide susceptible strain
provided by Dr. Laura Harrington (Cornell University).
All the mosquitoes were reared at 2562uC under a photoperiod
of 12:12 (L:D) h and fed blood samples from horses (Large Animal
Teaching Hospital, College of Veterinary Medicine, Auburn
University).
Quantitative real-time PCR (qRT-PCR)
The 4thinstar larvae and 2–3 day-old adults (before blood
deeding) of each mosquito population had their RNA extracted for
each experiment using the acidic guanidine thiocyanate-phenol-
chloroform method [8]. Total RNA (0.5 mg/sample) from each
mosquito sample was reverse-transcribed using SuperScript II
reverse transcriptase (Stratagene) in a total volume of 20 ml. The
quantity of cDNAs was measured using a spectrophotometer prior
to qRT-PCR, which was performed with the SYBR Green master
mix Kit and ABI 7500 Real Time PCR system (Applied
Biosystems). Each qRT-PCR reaction (25 ml final volume)
contained 16 SYBR Green master mix, 1 ml of cDNA, and a
P450 gene specific primer pair designed according to each of the
P450 gene sequences (http://cquinquefasciatus.vectorbase.org/),
Table S1 with accession number for each of P450 genes) at a final
concentration of 3–5 mM. All samples, including the A ‘no-
template’ negative control, were performed in triplicate. The
reaction cycle consisted of a melting step of 50uC for 2 min then
95uC for 10 min, followed by 40 cycles of 95uC for 15 sec and
60uC for 1 min. Specificity of the PCR reactions was assessed by a
melting curve analysis for each PCR reaction using Dissociation
Curves software [30]. Relative expression levels for the P450 genes
were calculated by the 22DDCTmethod using SDS RQ software
[31]. The 18 S ribosome RNA gene, an endogenous control, was
used to normalize the expression of target genes [15,32,33].
Preliminary qRT-PCR experiments with the primer pair (Table
S1) for the 18 S ribosome RNA gene designed according to the
sequences of the 18 S ribosome RNA gene had revealed that the
18 S ribosome RNA gene expression remained constant among all
3 mosquito strains, so the 18 S ribosome RNA gene was used for
internal normalization in the qRT-PCR assays. Each experiment
was repeated three to four times with different preparations of
RNA samples. The statistical significance of the gene expressions
was calculated using a Student’s t-test for all 2-sample comparisons
and a one-way analysis of variance (ANOVA) for multiple sample
comparisons (SAS v9.1 software); a value of P#0.05 was
considered statistically significant. Significant overexpression was
determined using a cut-off value of a $2-fold change in expression
[34].
Results
Cytochrome P450 genes in Cx. quinquefasciatus
The Cx. quinquefasciatus genome sequence has revealed 204
putative P450 (CYP) genes (including 8 pseudogenes) in Cx.
quinquefasciatus
mosquitoes[26,35],
vectorbase.org/). The Cx. quinquefasciatus P450s fall into four
major clans of CYP2, CYP3, CYP4, and mitochondrial (Fig. 1), as
do those identified in other insects [36]. Of the 204 Cx.
quinquefasciatus P450s, the majority assemble in clans 3 and 4: 89
P450s were found in the clan CYP3, with 24 in the CYP9 family,
64 in the CYP 6 family and 1 in the CYP329 family, and 82 in the
clan CYP4, with 34 in the CYP4 family, 47 in the CYP325 family,
and 1 in the CYP326 family. Sixteen P450 genes were found in
clan 2, with CYP families of 303 to 307, 18 and 15. The remaining
12 P450 genes were found in the mitochondrial clan with 6 P450
families of CYP12, CYP49, CYP301, CYP302, CYP314 and
CYP315. Comparing this distribution with those of other insect
species, Cx. quinquefasciatus showed a clear expansion of P450s in
clans 3 and 4. This expanded P450 supergene family in the Cx.
quinquefasciatus genome may provide a clue to the mechanisms that
permit Culex mosquitoes to adapt to polluted larval habitats [26].
(http://cquinquefasciatus.
Dynamic changes of P450 gene expression in the
mosquito populations of Culex quinquefasciatus
following permethrin selection
To understand how the P450 gene expression profile changes
following permethrin selection, we compared the gene expression
of 204 P450 genes [26], (http://cquinquefasciatus.vectorbase.org/
, http://drnelson.utmem.edu/CytochromeP450.html) in both
larvae and adults between susceptible and resistant Culex mosquito
populations, two different field populations of mosquitoes, and
field parental mosquitoes and their permethrin selected offspring
using qRT-PCR. The accession numbers of the P450 genes were
listed in Table S1. Mosquito populations bearing 3 different
resistance phenotypes in response to permethrin were used,
ranging from susceptible (S-Lab), through intermediate resistant
(HAmCqG0,fieldparental population)
(HAmCqG8, 8thgeneration permethrin selected offspring of
HAmCqG0). Comparing the P450 gene expression profiles in
both larvae and adults of permethrin selected HAmCqG8
mosquitoes with those of their field parental population revealed
that 69% of genes were expressed at a similar level in both
HAmCqG8and HAmCqG0(Fig. 2A), 11% were up-regulated in
HAmCqG8larvae compared to HAmCqG0, 7% were up-regulated
in HAmCqG8adults, 2 gene were up-regulated in both larvae and
adults, 5% were down-regulated in HAmCqG8larvae, 4% were
down-regulated in HAmCqG8adults, and 2% were down-
regulated in both larvae and adults of HAmCqG8. Applying a
to highlyresistant
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cut off level of 2 [34], among the up-regulated P450 genes in
larvae and adults of HAmCqG8, the majority were expressed at 2-
to 4-fold elevated levels compared with HAmCqG0and only 32%
and 12% in larvae and adults, respectively, had .5-fold
overexpression (Fig. 2A).
Similar expression patterns were also identified in another
permethrin selected mosquito strain, Here, MAmCqG6, the 6th
generation of permethrin selected field strain of MAmCqG0, were
compared with their parental strain of MAmCqG0, which was
collected at a location 600 km south of the collection site for the
HAmCqG0mosquitoes (Fig. 2B). In MAmCqG0, 6% of genes were
found to be up-regulated in the larvae of MAmCqG6compared
with those of MAmCqG0and S-Lab (Fig. 2B), 4% were up-
regulated in MAmCqG6adults, 3 genes were up-regulated in both
larvae and adults, 11% were down-regulated in larvae, 2% were
down-regulated in adults, 2% were down-regulated in both larvae
and adults, and 2% were down-regulated in larvae but up-
regulated in adults. Taken together, these results revealed equally
dynamic changes in abundance in both increased and decreased
P450 gene expression in the two field mosquito strains of Culex
quinquefasciatus following permethrin selection. Applying a cut off
level of 2 [34], among the up-regulated P450 genes in larvae and
adults of MAmCqG6the majority exhibited 2- to 4-fold elevated
levels compared with MAmCqG0and only 7% and 27% in larvae
and adults, respectively, of MAmCqG6had .5-fold overexpres-
sion (Fig. 2B).
P450 genes involved in up- and down-regulation in the
larvae of resistant Cx. quinquefasciatus
Twenty five P450 genes were found to be up-regulated in the
larvae stage (4thlarval instar) of HAmCqG8mosquitoes. The
expression levels of these P450 genes were $2-fold higher in
HAmCqG8than that in both S-Lab and HAmCqG0mosquito
strains (Table 1). The genes were distributed in clans CYP3,CYP4,
and mitochondria with 7 genes in family 9, 7 in family 6, 5 in
family 4, 3 in family 325, 2 in mitochondria, and 1 without
anotation. Except the six P450 genes CYP6AG12, CYP6AA7,
CYP4C38, CYP9J35, CYP6BZ2, and CYP9M10 whose expression
levels in parental HAmCqG0mosquitoes were 2.2-, 2.8-,2.1-, 11-,
2.0- and 5-fold higher than in susceptible S-Lab mosquitoes, the
expression levels of other genes were similar or lower in
HAmCqG0compared with the susceptible S-Lab strain (Table 1).
Similar patterns were observed when comparing the changes in
P450 expression in the larvae of MAmCqG6with those of both the
S-Lab and MAmCqG0mosquito strains. Fifteen P450 genes were
found to be up-regulated in the larvae of MAmCqG6mosquitoes.
The expression levels of these P450 genes in MAmCqG6were $2-
fold higher than those in both the S-Lab and MAmCqG0mosquito
strains (Table 1). These genes were distributed in clans CYP2,
CYP3, and CYP4 with 7 genes in family 9, 5 in family 6, and 1 in
each of families 4, 306, and 307. The expression of these genes was
similar or lower in MAmCqG0compared with the susceptible S-
Lab strain (Table 1) except for CYP9M10 and CYP6AA7, whose
expression levels in the parental MAmCqG0mosquitoes were 8.9-
and 4.5-fold higher, respectively.
Beside the up-regulation of P450 genes identified in the larvae of
Cx. quinquefasciatus following permethrin selection, a number of
P450 genes were found to be down-regulated in larvae of
permethrin selected Cx. quinquefasciatus. Sixteen P450 genes were
down-regulated in the larvae (4thlarval instar) of HAmCqG8
mosquitoes. The expression levels of these P450 genes in
HAmCqG8were #2-fold lower than that in HAmCqG0mosqui-
Figure 1. Number, family and clan distribution of cytochrome P450 genes in mosquitoes, Culex quinquefasciatus. The number shown
along each column represents the P450 family and the number in parenthesis is the number of individual genes in the corresponding family. The
P450 gene sequence information generated is from the vectorbase of the Cx. quinquefasciatus genome sequence (http://cquinquefasciatus.
vectorbase.org/).
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toes (Table 2). These down-regulated genes were distributed in
clans CYP3 and CYP4, with 2 genes in family 9, 10 in family 6,
and 2 in each of families 4 and 325. The expression of the majority
of these genes in HAmCqG8was at similar or lower levels
compared with that in susceptible S-Lab mosquitoes, even though
most were expressed at higher levels in HAmCqG0than in S-Lab
(Table 2). Although the similar P450 down-regulation patterns
were also found in the larvae of MAmCqG6compared with both
S-Lab and MAmCqG0, we did notice extended numbers and
distribution of these genes in the CYP clans compared with
HAmCq mosquitoes. Thirty P450 genes were down-regulated in
the larvae (4thlarval instar) of MAmCqG6mosquitoes. The
expression levels of these P450 genes in MAmCqG6were #2-fold
lower than in that in MAmCqG0mosquitoes (Table 2). The genes
were distributed in clans CYP2, CYP3,CYP4, and mitochondria
with 3 gene in family 9, 2 in family 6, 11 in family 4, and 8 in
family 325, 1 in family 326, 2 in family 12, 1 in each of families
304 and 18, and 1 without annotation. The expression levels of
these genes were again similar or lower in MAmCqG6compared
with susceptible S-Lab mosquitoes, even though most were
expressed at higher levels in MAmCqG0than in S-Lab (Table 2).
P450 genes involved in up- and down-regulation in
resistant Cx. quinquefasciatus adults
The expression of 204 Culex P450 genes in the adults of the 5
mosquito populations was examined using qRT-PCR. Seventeen
P450 genes were found to be up-regulated in the adult stage (2–3
day old) of HAmCqG8mosquitoes. The expression levels of these
P450 genes in HAmCqG8were $2-fold higher than that in both S-
Lab and HAmCqG0mosquito strains (Table 3). The overexpres-
sion levels of the up-regulated P450 genes in all the mosquito
populations tested were closely correlated with their levels of
resistance and were higher in permethrin-selected mosquitoes than
in their parent field strain. These genes were mainly distributed in
clans CYP3 and CYP4, with 3 genes in family 9, 5 in family 6, 5 in
family 4, and 3 in family 325. One gene was in mitochondria clan,
family 12. The expression of all these genes in HAmCqG0was
similar or lower than in susceptible S-Lab mosquitoes (Table 3).
Similar changes in the P450 gene expression were also found in
MAmCqG6adults compared with their S-Lab and MAmCqG0
counterparts. Fifteen P450 genes were up-regulated in adult
MAmCqG6mosquitoes. The expression levels of these P450 genes
were $2-fold higher than those in both S-Lab and MAmCqG0
adults (Table 3). As in the HAmCqG8mosquitoes, the genes whose
expression changed in MAmCqG6mosquitoes following permeth-
rin selection were also distributed in clans CYP3 and CYP4, with 1
gene in family 9, 2 in family 6, 1 in family 4, and 11 in family 325.
The expression of these genes was similar or lower in MAmCqG0
compared with susceptibleS-Lab
CYP325BF1v2 and CYP325K3v1, which were 2.4- and 3-fold
higher, respectively, in MAmCqG0(Table 4).
mosquitoes exceptfor
Figure 2. Diagrammatic representation of the analysis of the P450 gene expression profiles in both larvae and adults of permethrin
selected mosquito populations HAmCqG8and MAmCqG6compared with their corresponding field parental populations HAmCqG0
and MAmCqG0. The statistical significance of the gene expressions was considered to be a p value #0.05. Significant overexpression was analyzed
using a cut-off value of a $2-fold change in expression [29]. A. P450 gene expression profiles in HAmCqG8. B. P450 gene expression profiles in
MAmCqG6.
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Table 1. Up-regulation of P450 genes in larvae of permethrin selected offspring of the field populations of Culex quinquefasciatus.
Relative Gene expression ± SEb
MosquitoesTranscript IDa
Gene Parental strainc
Resistant straind
Ratioe
HAmCq (25) CPIJ002538 CYP6AG122.260.3 4.761.82.1
CPIJ005959*#
CYP6AA72.860.9 5.960.6 2.1
CPIJ003082CYP9J42 1.360.22.961.52.2
CPIJ001810CYP4C382.160.6 5.160.32.4
CPIJ015957CYP325G4 1.260.2 3.360.32.8
CPIJ005957CYP6AA9 0.960.04 2.560.6 2.8
CPIJ007091CYP325Y6 1.160.33.161.22.8
CPIJ010546*CYP9J340.860.3 2.360.32.9
CPIJ000926"
- 0.760.12.260.23.1
CPIJ016847CYP6CQ10.960.22.860.9 3.1
CPIJ009478CYP4D42v1 1.360.14.160.93.2
CPIJ010540 CYP9J35 1162.0396103.5
CPIJ005956 CYP6BZ22.060.67.361.13.7
CPIJ010537*CYP9J450.660.32.361.33.8
CPIJ012470*CYP9AL10.660.22.360.43.8
CPIJ014218*CYP9M105.061.22164.04.2
CPIJ005954CYP6CC20.660.004 2.560.64.2
CPIJ010225 CPY12F70.560.06 2.660.55.2
CPIJ010227CYP12F13 0.560.06 2.660.55.2
CPIJ010543 CYP9J400.660.2 3.661.36.0
CPIJ018943*#
CYP4C52v1 0.560.04 3.161.86.2
CPIJ005955* CYP6P140.660.13.860.66.3
CPIJ001759 CYP4H400.360.09 2.160.67.0
CPIJ020229CYP4D42v2 0.460.1 2.862.47.0
CPIJ017021CYP325K3v10.260.022.160.211
MAmCq (15) CPIJ014218*CYP9M108.961.61463.91.6
CPIJ010548#
CYP9J390.960.42.060.32.2
CPIJ005958CYP6AA80.760.001 1.860.82.6
CPIJ001039CYP306A1 1.060.12.660.06 2.6
CPIJ005959*#
CYP6AA74.561.0 1264.22.7
CPIJ005332 CYP9J430.960.08 2.560.02 2.8
CPIJ004411CYP6Z121.460.24.061.92.9
CPIJ005955*CYP6P141.660.14.660.52.9
CPIJ008566 CYP6Z150.760.06 2.160.73.0
CPIJ010546*CYP9J341.160.43.461.1 3.1
CPIJ010537*CYP9J451.060.33.161.3 3.1
CPIJ012470*CYP9AL1 0.960.23.460.23.8
CPIJ010544 CYP9J330.660.12.961.04.8
CPIJ000989CYP307B10.560.22.561.25.0
CPIJ018943*#
CYP4C52v10.260.022.761.7 14
aThe transcript ID number from the vectorbase of the Cx. quinquefasciatus genome sequence (http://cquinquefasciatus.vectorbase.org/).
bThe relative level of gene expression represents the ratio of the gene expression in each permethrin selected strain compared with that in the susceptible S-Lab strain.
The relative level of gene expression for S-Lab is 1.
cParental strain for HAmCq population is HAmCqG0with a 10-fold level of resistance to permethrin compared with S-Lab and for MAmCq is MAmCqG0with a 10-fold
level of resistance to permethrin [28].
dPermethrin selected strain for HAmCq population is HAmCqG8with a 2700-fold level of resistance to permethrin and for MAmCq is MAmCqG6with a 570-fold level of
resistance to permethrin [28].
eThe ratio of the relative gene expression in each permethrin selected strain compared its parental strain.
*The genes that are up regulated in both larvae of HAmCqG8and MAmCqG6.
#The genes that are up regulated in both larvae and adults of HAmCqG8and/or MAmCqG6.
"No annotation in Dr. Nelson’s P450 homepage http://drnelson.utmem.edu/CytochromeP450.html.
doi:10.1371/journal.pone.0029418.t001
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