Permethrin induces overexpression of multiple genes in Aedes aegypti.

Abstract

Using the polymerase chain reaction (PCR)-select subtractive cDNA hybridization technique, 18 different genes were isolated from a permethrin-treated versus acetone-treated Aedes aegypti subtractive library. Quantitative PCR (QPCR) results showed that 8 of the 18 gene's transcriptional levels in permethrin-treated Ae. aegypti were at least two-fold higher (ranging from 2.6 +/- 0.5 to 4.8 +/- 0.2) than that in acetone-treated Ae. aegypti. These eight genes include three functionally known genes (cytochrome c oxidase subunit III, NADH2 dehydrogenase, deltamethrin resistance associated protein), three functionally unknown genes (Ae. aegypti putative 16.9-kDa secreted protein, Anopheles gambiae ENSANGP00000019508, Cryptococcus neoformans hypothetical protein CNE05340), and two novel genes. Transcriptional levels for 11 of the 18 genes were induced significantly higher by permethrin than by fipronil (P < 0.05). Our results suggest that subtractive cDNA hybridization and QPCR are powerful techniques to identify differentially expressed genes in response to pesticide treatment.

Full text

Page 1

VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Permethrin Induces Overexpression of Multiple Genes in
Aedes aegypti
JULIA W. PRIDGEON,1JAMES J. BECNEL, GARY G. CLARK, AND KENNETH J. LINTHICUM
Center for Medical, Agricultural, and Veterinary Entomology, USDAÐARS, 1600 SW, 23rd Dr., Gainesville, FL 32608
J. Med. Entomol. 46(3): 580Ð587 (2009)
Using the polymerase chain reaction (PCR)-select subtractive cDNA hybridization
technique, 18 different genes were isolated from a permethrin-treated versus acetone-treated Aedes
aegypti subtractive library. Quantitative PCR (QPCR) results showed that 8 of the 18 geneÕs tran-
scriptional levels in permethrin-treated Ae. aegypti were at least two-fold higher (ranging from 2.6 ?
0.5 to 4.8 ? 0.2) than that in acetone-treated Ae. aegypti. These eight genes include three functionally
known genes (cytochrome c oxidase subunit III, NADH2 dehydrogenase, deltamethrin resistance
associated protein), three functionally unknown genes (Ae. aegypti putative 16.9-kDa secreted pro-
tein, Anopheles gambiae ENSANGP00000019508, Cryptococcus neoformans hypothetical protein
CNE05340), and two novel genes. Transcriptional levels for 11 of the 18 genes were induced
signiÞcantly higher by permethrin than by Þpronil (P ? 0.05). Our results suggest that subtractive
cDNA hybridization and QPCR are powerful techniques to identify differentially expressed genes in
response to pesticide treatment.
ABSTRACT
KEY WORDS
permethrin, Þpronil, overexpression, gene, Aedes aegypti
Aedes aegypti L. (Diptera: Culicidae) transmits viral
pathogens of humans, including yellow fever (Gillett
and Ross 1955, Aitken et al. 1977) and dengue (Mat-
tingly 1967, Degallier et al. 1988), both of which can
cause severe human morbidity and mortality. Al-
though there is a safe and effective vaccine for yellow
fever virus, epidemic transmission still occurs in Af-
rica, with sporadic cases in South America (Vascon-
celos et al. 2001; de Filippis et al. 2002; Valero 2003;
Onyangoetal.2004a,b).Dengueisthemostimportant
arboviral disease in the world and can cause an un-
differentiated fever, dengue fever, dengue hemor-
rhagic fever, or dengue shock syndrome (Malavige et
al. 2004). Annually, dengue epidemics account for
several million cases and thousands of deaths world-
wide (Teixeira et al. 2005).
Mosquitocontrolinmanycountriesreliesprimarily
on insecticides and pyrethroid insecticides have been
widely used because of their high effectiveness and
low mammalian toxicity. However, frequent use of
pyrethroids such as permethrin has resulted in resis-
tance development in Þeld populations (Yaicharoen
et al. 2005, Cui et al. 2006, Flores et al. 2006, Jirakan-
janakit et al. 2007). Furthermore, mosquitoes seem to
develop resistance to permethrin at a faster rate than
to malathion or temephos (Hamdan et al. 2005).
Therefore, there is an urgent need to develop insec-
ticides with novel modes of action and/or understand
the molecular mechanisms involved in pyrethroid re-
sistance to facilitate resistance management in the
Þeld.
Extensive research has been conducted on the mo-
lecular basis of pyrethroid resistance. Different resis-
tance mechanisms, such as decreased target site sen-
sitivity and increased metabolic detoxiÞcation have
been identiÞed in pyrethroid-resistant populations of
Ae. aegypti (Kumar et al. 1991; Brengues et al. 2003;
Lumjuan et al. 2005; Rodriguez et al. 2005; Saavedra-
Rodriguez et al. 2007, 2008; Rajatileka et al. 2008;
Strodeetal.2008).However,onlylimitedresearchhas
been aimed at elucidating the molecular responses of
mosquitotoshort-termexposureofpyrethroidandyet
thiscouldprovideimportantinformationontheinitial
stepsintheprocessofresistancedevelopment.There-
fore,thegoalofthisstudywastoisolategenesthatare
induced by short exposure to sublethal dose of per-
methrinwithoutanypreconceptionoftheiridentities.
Materials and Methods
Aedes aegypti Mosquito. The Orlando strain of Ae.
aegypti was reared in the insectary of the Mosquito
and Fly Research Unit at Center for Medical, Agri-
cultural, and Veterinary Entomology(CMAVE)Ð
USDAÐARS. The Orlando strain of Ae. aegypti has
beenestablishedinCMAVEsince1952.Femaleadults
The use of trade, Þrm, or corporate names in this publication is for
the information and convenience of the reader. Such use does not
constituteanofÞcialendorsementorapprovalbytheU.S.Department
of Agriculture or the Agricultural Research Service of any product or
service to the exclusion of others that may be suitable.
1Corresponding author, e-mail: Julia.Pridgeon@ars.usda.gov.

Page 2

were used for all experiments because only females
take bloodmeals and are of concern to the general
public. Eggs were hatched by placing a square of a
paper towel with eggs in a ßask Þlled with 1,000 ml of
distilled water containing 40 mg of larval diet (3:2
brewerÕs yeast:liver powder; MP Biomedicals, Irvine,
CA). The hatched larvae were held overnight in the
ßask,and200larvaeweretransferredtoa4-literplastic
tray containing 2 liters of distilled water. Larval diet
was added to each tray according to the following
schedule: day 1, 80 mg; day 3, 40 mg; day 4, 80 mg; day
5,120mg;day6,150mg.Mosquitoeswererearedinan
environmentalchambersetwithatemperatureproÞle
representing a simulated summer day regimen (rang-
ing from 22 to 30?C) and 80% RH. Incandescent lights
weresettoacrepuscularproÞlewithaphotoperiodof
14:10(L:D)h,including2hofsimulateddawnand2h
ofsimulateddusk.Adultswereheldinascreenedcage
and provided 10% sucrose ad libitum.
Chemical Treatment of Adult Ae. aegypti. Before
insecticide application, 3- to 5-d-old females were
brießy anesthetized for 30 s with carbon dioxide and
placedona4?Cchilltable(BioQuipProducts,Rancho
Dominguez,CA).Adropletof0.5?lofsublethaldose
of permethrin (2.5 ?10?5?g/mosquito) or Þpronil
(2 ?10?7?g/mosquito) determined from a previous
study (Pridgeon et al. 2008a) and diluted in acetone
was topically applied to the dorsal thorax of female
mosquito using a 700 series syringe and a PB 600
repeating dispenser (Hamilton, Reno, NV). The LD50
value of permethrin was 1.4 ? 10?4?g/mosquito and
theLD50valueofÞpronilwas1.4?10?6?g/mosquito.
After treatment, mosquitoes were kept in plastic cups
andsuppliedwith10%sucrosesolutionfor6h.Forthe
control, a droplet of 0.5 ?l of acetone was topically
applied to the dorsal thorax of female mosquitoes.
Temperature and humidity were maintained at 25 ?
2?C and 80 ? 8% RH, respectively. For each chemical
treatment, 10 female mosquitoes were used. Adult
mosquitoes were stored in ?80?C 6 h after treatment
for later RNA isolation.
AedesaegyptiRNAExtractionandcDNASynthesis.
After 6 h, 10 adults treated with or without chemicals
were used for total RNA extraction. Total RNA was
isolated using TRIzol Reagent (Invitrogen, Carlsbad,
CA) following the manufacturerÕs protocol. Total
RNA was resuspended in diethylene pyrocarbonate
(DEPC)-treatedwaterandstoredat?80?C.TheÞrst-
strand cDNAs used for quantitative polymerase chain
reaction (QPCR) were synthesized using AMV re-
verse transcriptase (Invitrogen). Brießy, a 5-?g ali-
quot of total RNA was reverse transcribed in a 20-?l
reaction volume using Clone AMV Þrst-strand cDNA
Synthesis kit (Invitrogen). The reaction was termi-
nated by heat inactivation at 95?C for 5 min. The
cDNA samples were diluted with 80 ?l ddH2O and
stored at ?20?C. For subtractive library construction,
Poly(A)?RNA was isolated using Oligotex-dT sus-
pension (Qiagen, Valencia, CA) from permethrin- or
acetone-treatedmosquitoes.cDNAsweresynthesized
from poly (A)??RNAs of permethrin-treated or ac-
etone-treated Ae. aegypti using the PCR-selected
cDNA Subtraction Kit (Clontech, Palo Alto, CA) as
described by the manufacturer. The cDNAs that con-
tain speciÞc transcripts are referred to as “testers” (i.e.,
frompermethrin-treated)andthereferencecDNAsare
referredtoas“drivers”(i.e.,fromacetone-treated).The
double-strandedcDNAsofbothtestersanddriverswere
digested with RsaI to create smaller blunt-ended frag-
ments to be used as testers or drivers according to the
manufacturerÕs instructions (Clontech). The tester
cDNAs were subdivided into two portions (A and B)
and modiÞed by ligating with cDNA adaptors 1 and 2
(provided by the kit), respectively.
Construction of Subtractive cDNA Library. Two-
step subtractive hybridizations were performed ac-
cording to procedures used previously (Pridgeon and
Liu 2003). Brießy, in the Þrst-step hybridization, two
primary hybridization reactions (A and B) were
formed by adding excess amounts of unmodiÞed
driver cDNA to separate portions A and B of tester
cDNA samples at a 50:1 ratio. The samples were de-
naturedfor2minat98?Candallowedtoannealfor8h
at 68?C. The remaining single-stranded, adaptor-
ligated tester cDNAs were dramatically enriched in
each hybridization reaction for overexpressed se-
quences because nontarget cDNAs present in the
tester and driver formed hybrids. For the second step
hybridization,AandBprimaryhybridizationreaction
solutions were mixed together without denaturing.
These new hybrids were double-stranded tester mol-
ecules with different 5? ends corresponding to the
sequences of two different adaptors. Freshly dena-
tured driver DNA was added to the reaction without
denaturing the subtraction mix to further enrich new
double-stranded tester molecules that are differen-
tially expressed. After Þlling in the adapter ends with
DNA polymerase, overexpressed sequences (tester
cDNA)haddifferentannealingsitesontheir3?and5?
ends. The molecules were subjected to suppression
subtraction PCR as described by the manufacturer
(Clontech).ThePCRproductswereclonedintopCR
2.1-TOPO vector using TOPO TA Cloning Kit (In-
vitrogen). Plasmids were transformed into One Shot
TOP10 competent cells (Invitrogen). Transformed
cells were plated out on Luria-Bertani (LB) plates
containing ampicillin (100 ?g/ml) and X-Gal (5-
bromo-4-chloro-3-indolyl- beta-D-galactopyranoside;
40 ?g/ml).
Plasmid DNA Isolation and Sequencing. From the
library, a total of 74 white colonies were present and
subsequently picked to grow overnight in LB broth in
the presence of ampicillin (100 ?g/ml) in the Innova
4000 Incubator Shaker (New Brunswick ScientiÞc,
Edison,NJ)at37?Cand235rpmsettings,respectively.
PlasmidDNAswereisolatedwithQIAprepSpinMini-
prep kit (Qiagen). Plasmid DNAs were digested by
EcoRI at 37?C for 1 h and subjected to 1% agarose gel
electrophoresis. Plasmid DNAs that contained insert
sizes ?100 bp were sent to University of Florida DNA
sequencing facility. Sequences were analyzed using
the National Center for Biotechnology Information
(NCBI) BLAST program to search for sequence ho-
mologies.
May 2009PRIDGEON ET AL.: OVEREXPRESSION OF MULTIPLE GENES INDUCED BY PERMETHRIN
581

Page 3

Primer Design and QPCR. Sequencing results of
different clones were used to design gene-speciÞc
primers using the Primer3 program (http://frodo.wi.
mit.edu/cgi-bin/primer3/primer3_www.cgi). QPCR
was performed using Platinum SYBR Green qPCR
SuperMix-UDGwithROX(Invitrogen)inavolumeof
15 ?l on an Applied Biosystems 7300 Fast Real-Time
PCRSystem(ABI,FosterCity,CA).ThePCRmixture
consisted of 1 ?l of diluted cDNA, 0.5 ?l of 5 ?M
gene-speciÞc forward primer, 0.5 ?l of 5 ?M gene-
speciÞc reverse primer, and 13 ?l of 1? SYBR Green
SuperMix. For all cDNA samples, Ae. aegypti actin
(GenBank accession no. DQ440059) primers were in-
cluded as an internal control to normalize the varia-
tion of cDNA amount as described previously (Prid-
geon et al. 2008b). Primers used for the ampliÞcation
of the actin gene were actin-152 F (5?-AGGACTCG-
TACGTCGGTGAC-3?) and actin-590R (5?-CGT-
TCAGTCAGGATCTTC-3?). The PCR thermal cy-
clingparameterswere50?Cfor2min,95?Cfor10min,
followed by 40 cycles of 95?C for 15 s and 60?C for 1
min. A dissociation stage was performed at 95?C for
15 s, 60?C for 30 s, and 95?C for 15 s. All QPCR was
replicated three times. The relative transcriptional
level of a speciÞc clone/gene was determined by di-
viding the threshold cycle (Ct) of a sample by that of
the actin gene, the calibrator, or internal control, as
per the following formula: ?Ct ? Ct (sample) ? Ct
(calibrator).ThetranscriptionallevelsofthatspeciÞc
clone/gene compared with actin was calculated as
?Ct where ?Ct ? Ct (clone) ? Ct (actin). The rel-
ative expression level of the speciÞc clone/gene in a
pesticide-treated mosquito compared with that in an
acetone-treated mosquito was calculated by the for-
mula 2??Ctwhere ??Ct ? ?Ct (pesticide) ? ?Ct
(acetone).Datawereanalyzedbyanalysisofvariance
(ANOVA)usingSigmaStatstatisticalanalysissoftware
(Systat Software, San Jose, CA).
Results
Using the PCR-select subtractive cDNA hybridiza-
tion technique, a subtractive library was constructed
to isolate overexpressed genes in permethrin-treated
Ae. aegypti compared with that in acetone-treated
mosquitoes. From the subtractive library, a total of 74
clones were isolated. Of the 74 clones, only 18 unique
genes were identiÞed. Using both BlastN and BlastX
programs from NCBI, similarities of the 18 different
genes to the GenBank database were analyzed. The
results of BlastX search of the 18 genes are summa-
rized in Table 1. Two clones (1 and 63) were similar
to Culex pipiens pallens deltamethrin resistanceÐasso-
ciated proteins. Three clones (52, 64, and 74) were
related to mitochondria energy production (i.e., cy-
tochrome c oxidase subunit III and NADH2 dehydro-
genase). The 18 genes also include one carboxypep-
tidase (clone 60), one mortality factor-like protein
(clone69),oneDNAtopisomeraseprotein(clone66),
one ubiquitin-like domain containing CTD phospha-
tase(clone70),sevenhypotheticalproteins(clones2,
4,54,64,67,68,and72),andthreenovelgenes(clones
56, 58, and 71).
Totestwhetherthe18geneswereindeedinducedin
permethrin-treated Ae. aegypti, gene-speciÞc primers
were designed (Table 2), and relative QPCR experi-
ments were performed. Our QPCR results showed that
therelativetranscriptionallevelsofthe18geneswereall
higherinpermethrin-treatedAe.aegypticomparedwith
thatinacetone-treatedAe.aegypti(Table3).Transcrip-
tional levels of cDNA 1, 4, 52, 54, 56, 67, 71, and 74 were
at least two-fold higher (ranging from 2.6 ? 0.5 to 4.8 ?
0.2) in permethrin-treated Ae. aegypti compared with
that in acetone-treated Ae. aegypti (Fig. 1).
Totestwhetherapesticidewithadifferentmodeof
action also induced the overexpression of the same 18
genes,Ae.aegyptifemalesweresubjectedtosublethal
Table 1. List of genes isolated from the permethrin- versus acetone-treated subtractive cDNA library of Ae. aegypti
Clone
Homology
Accession no.Protein nameIdentities Region
1 AAP59419.1 Culex pipiens pallens deltamethrin resistance-associated NYD-OP5
protein, length ? 371
Anopheles gambiae ENSANGP00000016852 protein, length ? 458
Aedes aegypti putative 16.9-kDa secreted protein, length ? 148
Culex pipiens cytochrome c oxidase subunit III protein, length ? 262
Anopheles gambiae ENSANGP00000019508 protein, length ? 1,324
Unknown
Unknown
Aedes aegypti carboxypeptidase protein, length ? 423
Culex pipiens pallens deltamethrin resistance-associated NYD-OP7
protein, length ? 371
Thermobia domestica hypothetical protein, length ? 2327
Aedes aegypti prokaryotic DNA topoisomerase protein, length ? 856
Cryptococcus neoformans var. grubii hypothetical protein CNE05340,
length ? 107
Aedes aegypti conserved hypothetical protein, length ? 215
Tribolium castaneum similar to mortality factor 4 like 1, length ? 322
Danio rerio ubiquitin-like domain containing CTD phosphatase 1,
length ? 318
Unknown
Tribolium castaneum hypothetical protein, length ? 523
Leishmania tarentolae mitochondrion NADH2 dehydrogenase
(ubiquinone) (EC 1.6.5.3) chain 5, length ? 502
39/40 (97%) 295Ð334
2
4
52
54
56
58
60
63
EAA07773.3
AAX54868.1
ABD62340.1
EAA04820.3
Unknown
Unknown
EAT37214.1
AAU93633.1
19/54 (35%)
135/139 (97%)
99/116 (85)
16/49 (32%)
Unknown
Unknown
241/244 (98%)
108/125 (86%)
101Ð154
9Ð147
143Ð258
1169Ð1217
Unknown
Unknown
1Ð244
76Ð200
64
66
67
CAM36311.1
EAT36302.1
ABD97233.1
26/32 (81%)
129/130 (99%)
22/74 (29%)
1Ð32
727Ð856
4Ð77
68
69
70
EAT37811.1
XP_969099.1
AAH75753.1
104/104 (100%)
117/179 (65%)
95/128 (74%)
112Ð215
135Ð310
146Ð273
71
72
74
Unknown
XP_976402.1
I30010
Unknown
17/43 (39%)
18/49 (36%)
Unknown
317Ð356
69Ð108
582JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 46, no. 3

Page 4

dose of Þpronil treatment for 6 h, and the treated
mosquitoes were subjected to QPCR analysis (Table
4). Transcriptional levels of cDNA 2, 4, 52, 54, 64, 66,
67,69,70,72,and74werehigher,whereasthreegeneÕs
transcriptional levels (clones 58, 60, and 68) were
lower in Þpronil -treated Ae. aegypti compared with
that in acetone-treated Ae. aegypti (Table 3). How-
ever, the induction levels of the 11 genes by Þpronil
werealllessthantwo-fold(Fig.1),andthisdifference
was not statistically signiÞcant (P ? 0.05). When the
transcriptional levels induced by permethrin were
compared with that induced by Þpronil, 11 of the 18
genes were signiÞcantly (P ? 0.05) induced by per-
methrin(Fig.1).ThesewerecDNAsofcytochromec
oxidase subunit III (52), deltamethrin resistance-as-
sociated protein (63), DNA topoisomerase (66),
NADH2 dehydrogenase (74), and seven functionally
unknown genes (putative or hypothetical proteins).
Discussion
We performed this study to isolate genes that are
induced by short exposure to a sublethal dose of per-
methrin. Eighteen different genes were isolated from
thepermethrin-treatedversusacetone-treatedAe.ae-
gypti subtractive library. Eleven of the 18 genes were
signiÞcantly(P?0.05)inducedbypermethrinbutnot
byÞpronil(Fig.1).Oneofthe11signiÞcantlyinduced
genesbypermethrinwasacytochromecoxidasesub-
unit III gene. The Þnding of cytochrome c oxidase
subunit III induction in response to permethrin treat-
mentisveryinterestingbecauseoverexpressionofthe
cytochrome c oxidase subunit I gene has been linked
to pyrethroid resistance (Pridgeon and Liu 2003).
Cytochrome c oxidase is a key enzyme involved in
ATP generation in the mitochondria by oxidative
phosphorylation. In eukaryotes, the three subunits
(IÐIII) of cytochrome c oxidase are encoded by mi-
tochondrial DNA and form the functional catalytic
core of the enzyme (Wikstrom and Casey 1985). Cy-
Table 2.
the 18 genes
Gene-specific primers used in quantitative PCR for
ClonePrimer namePrimer sequence (5? to 3?)
1 1Ð9F
1Ð122R
2Ð513F
2Ð695R
4Ð93F
4Ð314R
52Ð151F
52Ð314R
54Ð54F
54Ð207R
56Ð18F
56Ð266R
58Ð55F
58Ð221R
60Ð402F
60Ð625R
63Ð65F
63Ð227R
64Ð9F
64Ð167R
66Ð82F
66Ð326R
67Ð72F
67Ð254R
68Ð199F
68Ð403R
69Ð382F
69Ð531R
70Ð327F
70Ð546R
71Ð48F
71Ð249R
72Ð217F
72Ð383R
74Ð59F
74Ð216R
GGATGGCTGATACCGTACACA
CTTCACCGGTATCTTCAAGG
TTATACGGCCAATGGCTTTC
GACTGGCCGTAGATCCAGAA
GCCGTCTTCAGTGTCGTGTA
AGAGCCAGCTTTCACCAAAA
GCTCCTTTTACAATTGCCGATA
GCTGCTTCGAATCCAAAATG
TCGATATCATTGGGGGTGAT
GTTACCGCTCAGGAATTTGC
GGTGTGATTTGGGGTCAGTC
AGGTACCTGCGATGGCTTC
GCTTTCCGTCGGGTAGACTT
CTGGCCGTAAATCCAAAAAC
CCGCCTAGACATCATTCGAT
ACAGCCAGCTGAAATTGCTT
AGCGTTCTCGGATTTCTTCA
GAACGCAATCATTGTCATGG
CGCGGCCGAGGTACTATAA
GCGACCTCGATGTTGGATTA
AATTGCGCCATCTTGTCTTT
GAATGTGGGGCTCAACAAGT
TTATACGGCCAATGGCTTTC
GACTGGCCGTAAATCCAAAA
TCCTTCTTGGCGGTACATTC
AAGTGGACAACGTGGAGGAC
TCGTGGACGGTTGTTTTACA
TAACGTGGAATCCGAAGACC
CCATCATGTTCGATGACCTG
AGTCGTTTCACGACGCTTCT
TGCACAAGATGTTGCTGACTC
TCGCGTCTTTACCGCTCTAT
TTATACGGCCAATGGCTTTC
CGAAACCGAGAAAAACCAAA
TCCGTCTCACATTTCCCTCT
ACGGTCGATGTTGATTGTGA
2
4
52
54
56
58
60
63
64
66
67
68
69
70
71
72
74
Table 3. Transcription levels of the 18 genes in permethrin- or acetone-treated Ae. aegypti
Gene/clone
Cycle threshold (Ct ? SD)
??Ct-1
Relative transcription level in permethrin
compared with acetone
(?SD)
Acetone Ct-1
Acetone
?Ct-1
Permethrin Ct-1
Permethrin
?Ct-1
2??Ct-1
2??Ct-2
2??Ct-3
2??Ct
Actin
1
2
4
52
54
56
58
60
63
64
66
67
68
69
70
71
72
74
14,105 ? 0,059
8,890 ? 0,108
5,330 ? 0,132
19,675 ? 0,018
9,175 ? 0,050
2,740 ? 0,017
2,760 ? 0,102
6,990 ? 0,283
14,960 ? 0,878
11,730 ? 0,027
24,625 ? 0,064
17,080 ? 0,042
15,615 ? 0,066
15,955 ? 0,104
19,675 ? 0,050
21,220 ? 0,438
3,020 ? 0,028
8,860 ? 0,270
21,655 ? 0,061
Ñ 17,165 ? 0,060
10,875 ? 0,124
7,700 ? 0,864
21,060 ? 0,043
10,705 ? 0,104
3,520 ? 0,071
3,610 ? 0,009
9,195 ? 0,647
17,940 ? 0,097
13,925 ? 0,031
27,235 ? 0,189
19,405 ? 0,021
17,305 ? 0,060
18,115 ? 0,039
22,365 ? 0,006
23,240 ? 0,440
4,100 ? 0,014
11,565 ? 0,091
17,245 ? 0,003
ÑÑÑÑÑÑ
?5,215
?8,775
5,570
?4,930
?11,365
?11,345
?7,115
0,855
?2,375
10,520
2,975
1,510
1,850
5,570
7,115
?11,085
?5,245
7,550
?6,290
?9,465
3,895
?6,460
?13,645
?13,555
?7,970
0,775
?3,240
10,070
2,240
0,140
0,950
5,200
6,075
?13,065
?5,600
5,880
1,075
0,690
1,675
1,530
2,280
2,210
0,855
0,080
0,865
0,450
0,735
1,370
0,900
0,370
1,040
1,980
0,355
1,670
2,107
1,613
3,193
2,888
4,857
4,627
1,809
1,057
1,821
1,366
1,664
2,585
1,866
1,292
2,056
3,945
1,279
3,182
2,630
1,537
3,555
2,732
4,579
3,745
2,258
1,815
1,784
3,000
1,597
3,784
1,505
1,310
2,007
3,042
3,084
3,215
3,171
4,258
5,260
3,824
4,993
4,857
1,993
1,873
1,979
3,618
1,659
3,937
1,828
1,485
2,594
4,611
2,704
4,773
2,636 ? 0,532
2,469 ? 1,549
4,003 ? 1,104
3,148 ? 0,591
4,810 ? 0,211
4,410 ? 0,587
2,020 ? 0,226
1,582 ? 0,455
1,861 ? 0,104
2,661 ? 1,164
1,640 ? 0,037
3,435 ? 0,740
1,733 ? 0,198
1,362 ? 0,107
2,219 ? 0,326
3,866 ? 0,787
2,356 ? 0,952
3,723 ? 0,909
May 2009PRIDGEON ET AL.: OVEREXPRESSION OF MULTIPLE GENES INDUCED BY PERMETHRIN
583

Page 5

tochrome c oxidase is a key oxidative enzyme for
neuronal oxidative capacity and, indirectly, for neu-
ronal activity (Wong-Riley et al. 1998). Functional
reduction in Na?K?ATPase (the sodium pump) ca-
pacity caused by the reduced level of cytochrome c
oxidaseactivityistheimportantfactorresponsiblefor
gradedneuronaldepolarization(orhyperexcitability)
and neuronal death in the mammalian brain (Wong-
Riley et al. 1998). Overexpression of cytochrome c
oxidase subunit I has been reported to be involved in
resistance of Schistosoma mansoni to praziquantel, a
drug for the control of schistosomiasis (Pereira et al.
1998). Overexpression of cytochrome c oxidase sub-
unitIIisalsoinvolvedinthedevelopmentofresistance
of Chinese hamster ovary cells to methotrexate, a
widely used drug in cancer chemotherapy (Alemany
et al. 2000). Furthermore, inhibition of cytochrome c
oxidase by mitochondrial antisense RNA induces cell
deathinhumancelllines(Shirafujietal.1997).Taken
together, these Þndings suggest that cytochrome c
oxidase might play an important role in the develop-
ment of pyrethroid resistance and might be an essen-
tial target for the development of novel molecular
insecticides through RNA interference technology
(Pridgeon et al. 2008c).
Three other genes signiÞcantly induced by per-
methrin were mitochondrial NADH2 dehydrogenase
(ubiquinone), DNA topoisomerase, and deltamethrin
resistanceÐassociated protein. Ubiquinone (CoQ) is a
component of the mitochondrial electron transport
chain, participating in cellular respiration and gener-
ating energy in the form of ATP. Ubiquinone overex-
pression has been induced by chemotherapy treat-
ment in all cancer cell lines tested (Brea-Calvo et al.
Fig. 1.
*There is a signiÞcant difference between the two (P ? 0.05).
Relative transcriptional levels of the 18 genes induced by permethrin compared with that induced by Þpronil.
Table 4.Expression levels of the 18 genes in fipronil- or acetone-treated Ae. aegypti
Gene/clone
Cycle threshold (Ct ? SD)
??Ct-1
Relative expression level in Þpronil compared
with acetone (?SD)
Acetone Ct-1
Acetone
?Ct-1
Fipronil
Ct-1
Fipronil
?Ct-1
2??Ct-1
2??Ct-2
2??Ct-3
2??Ct
Actin
1
2
4
52
54
56
58
60
63
64
66
67
68
69
70
71
72
74
13,580 ? 0,139
10,225 ? 0,052
7,055 ? 0,163
16,060 ? 0,084
8,180 ? 0,083
4,525 ? 0,124
3,750 ? 0,093
4,935 ? 0,473
17,165 ? 0,022
12,985 ? 0,125
10,565 ? 0,446
17,455 ? 0,025
12,060 ? 0,082
13,190 ? 0,071
20,125 ? 0,118
22,150 ? 0,033
3,730 ? 0,507
9,895 ? 0,037
19,175 ? 0,118
Ñ13,835 ? 0,140
10,475 ? 0,020
7,025 ? 0,559
15,945 ? 0,216
7,725 ? 0,021
4,450 ? 0,155
3,960 ? 0,061
5,535 ? 0,263
17,600 ? 0,037
13,180 ? 0,197
10,590 ? 0,410
17,530 ? 0,023
11,615 ? 0,672
13,585 ? 0,034
20,020 ? 0,003
21,540 ? 0,638
3,960 ? 0,044
9,175 ? 0,034
19,215 ? 0,047
ÑÑ
0,005
0,285
0,370
0,710
0,330
0,045
?0,345
?0,180
0,060
0,230
0,180
0,700
?0,140
0,360
0,865
0,025
0,975
0,215
ÑÑÑÑ
?3,355
?6,525
2,480
?5,400
?9,055
?9,830
?8,645
3,585
?0,595
?3,015
3,875
?1,520
?0,390
6,545
8,570
?9,850
?3,685
5,595
?3,360
?6,810
2,110
?6,110
?9,385
?9,875
?8,300
3,765
?0,655
?3,245
3,695
?2,220
?0,250
6,815
7,705
?9,875
?4,660
5,380
1,004
1,218
1,292
1,636
1,257
1,032
0,787
0,883
1,042
1,173
1,133
1,625
0,908
1,283
1,821
1,018
1,966
1,161
0,997
1,279
1,235
1,664
1,125
1,064
0,700
0,871
0,886
1,564
1,098
1,449
0,815
1,380
1,161
0,904
1,866
1,117
0,986
1,202
1,248
1,542
1,227
1,053
0,761
0,867
1,028
1,636
1,113
1,597
0,868
1,288
1,784
1,000
1,979
1,149
0,996 ? 0,009
1,233 ? 0,041
1,258 ? 0,030
1,614 ? 0,064
1,203 ? 0,069
1,050 ? 0,016
0,749 ? 0,045
0,874 ? 0,008
0,985 ? 0,086
1,458 ? 0,249
1,115 ? 0,018
1,557 ? 0,095
0,864 ? 0,047
1,317 ? 0,055
1,589 ? 0,371
0,974 ? 0,061
1,937 ? 0,062
1,142 ? 0,023
584JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 46, no. 3