Molecular mechanisms and monitoring of permethrin resistance in human head lice

Pesticide Biochemistry and Physiology 97 (2010) 109–114

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Pesticide Biochemistry and Physiology

journal homepage: www.elsevier .com/locate /pest

Molecular mechanisms and monitoring of permethrin resistance in human head lice

Si Hyeock Lee a,*, J. Marshall Clark b, Young Joon Ahn a, Won-Ja Lee c, Kyong Sup Yoon b, Deok Ho Kwon a,Keon Mook Seong a

a Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Koreab Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USAc Division of Zoonoses, Korea National Institute of Health, Seoul 122-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 September 2008Accepted 30 April 2009Available online 15 May 2009

Keywords:Head licePermethrin resistanceMolecular monitoringQuantitative sequencing

0048-3575/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.pestbp.2009.04.017

* Corresponding author. Fax: +82 2 873 2319.E-mail address: [email protected] (S.H. Lee).

Head lice resistance to permethrin is mainly conferred by the knockdown resistance (kdr) trait, a voltage-sensitive sodium channel (VSSC) insensitivity factor. Three VSSC mutations (M815I, T917I and L920F)have been identified. Functional analysis of the mutations using the house fly VSSC expressed in Xenopusoocytes revealed that the permethrin sensitivity is reduced by the M827I (M815I) and L932F (L920F)mutations when expressed alone but virtually abolished by the T929I (T917I) mutation, either alone orin combination. Thus, the T917I mutation is primarily responsible for permethrin resistance in head lice.Comparison of the expression rates of channel variants indicates that the M815I mutation may play a rolein rescuing the decreased expression of channels containing T917I. A step-wise resistance monitoringsystem has been established based on molecular resistance detection techniques. Quantitative sequenc-ing (QS) has been developed to predict the VSSC mutation frequency in head lice at a population basis.The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance mon-itoring tool to screen a large number of wild louse populations as an alternative to conventional bioassay.As a secondary monitoring method, real-time PASA (rtPASA) has been devised for more precise determi-nation of low resistance allele frequencies. To obtain more detailed information on resistance allelezygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) has been devel-oped as an individual genotyping method. Our approach of using three tiers of molecular resistancedetection should facilitate large-scale routine resistance monitoring of permethrin resistance in head liceusing field-collected samples.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Pediculosis, caused by the human head louse, Pediculus hum-anus capitis De Geer, is the most prevalent ectoparasitic infestationof humans, particularly in school-aged children, worldwide [1].Unlike body lice, P. h. humanus, head lice do not transmit diseasesand their infestation symptoms are relatively mild. Nevertheless,the social, mental, and economic impacts of pediculosis are sub-stantial, considering the loss of school day by infested childrendue to the ‘No nit policy’ and the overall expenses of social carefor the infested children. The pyrethrins and pyrethroids have beenwidely used as over-the-counter (OTC) pediculicides. However,extensive use of these pediculicides, particularly permethrin, inev-itably resulted in resistance problems. Permethrin resistance inhead louse populations appears widespread worldwide but itsintensity and distribution vary geographically [2], highlightingthe necessity of proactive resistance management system prior tothe complete saturation of resistance.

ll rights reserved.

For the establishment of an efficient resistance managementsystem, understanding the molecular and genetic basis of resis-tance is imperative. Early studies by Clark’s group demonstratedthat knockdown resistance (kdr)1 is a major factor in all permeth-rin-resistant lice worldwide and supports the claim that treatmentfailure is largely due to resistance [3]. Three point mutations(M815I, T917I and L920F) in the voltage-sensitive sodium channel(VSSC) a-subunit gene identified in permethrin-resistant head licewere suggested to be responsible for kdr-type resistance [3–5].

Detection of the early phase of resistance is a crucial element inany long-term resistance management system designed to sup-press the resistance. However, early resistance detection by con-ventional bioassay-based monitoring methods is very difficult,particularly when resistance is recessive. In addition, collectinglarge numbers of live specimens, as in the case of lice, is oftenimpractical and always difficult. To circumvent these limits, vari-ous individual genotyping techniques for the detection of resis-

1 Abbreviations used: kdr, knockdown resistance; QS, quantitative sequencing;rtPASA, real-time PCR amplification of specific allele; SISAR, serial invasive signalamplification reaction; VSSC, voltage-sensitive sodium channel a-subunit.

110 S.H. Lee et al. / Pesticide Biochemistry and Physiology 97 (2010) 109–114

tance allele frequencies have been developed [6,7]. In this review,we discuss on the current knowledge on molecular mechanisms ofhead louse resistance to permethrin and the molecular tools thatcan be used as alternatives for conventional bioassay-based resis-tance monitoring.

2. Status and mechanisms of permethrin resistance

Permethrin has been used to control pediculosis since the 1970sand its use intensified following the introduction of OTC productsin the 1980s. Despite the introductions of effective pediculicides,pediculosis has increased noticeably since the mid-1990s due, inpart, to the development of insecticide resistance. Resistance tothe pyrethroid pediculicides was first reported in France in 1994,followed by several other countries worldwide [3]. Early reportsindicated that permethrin resistance was mostly conferred by thekdr trait from the fact that permethrin-resistant head lice are toler-ant to knockdown in behavioral bioassays, cross-resistant to DDT,and only showed low levels of metabolic synergism [3].

To elucidate the molecular mechanisms of permethrin resis-tance in the head louse, cDNA fragments that spanned theIIS4 � IIS6 region of para-orthologous head louse VSSC a-subunitgene, where most of the mutations associated with kdr are located,were cloned and their sequences determined [8]. Sequence com-parison between the permethrin-resistant and -susceptible strainsidentified two point mutations (T917I and L920F), located in theIIS5 transmembrane segment, as putatively responsible for resis-tance (Fig. 1). Correlation study also revealed that frequencies ofthe T917I and L920F mutations are closely related with actual lev-els of permethrin resistance in field populations of head lice [2]. Se-quence comparisons of the complete open reading frame identifiedone additional novel mutation (M815I), which was located in theIIS1-2 extracellular loop of the a-subunit, to be associated withpermethrin-resistance [4,5] (Fig. 1). All three mutations weredetermined to exist en bloc as a resistant haplotype through se-quence analyses of cloned cDNA and genomic DNA fragments fromindividual louse samples, both containing the three mutation sites.

Functional analysis of the mutations was conducted by usingthe house fly para-orthologous VSSC a-subunit as a surrogatechannel. The three mutations were introduced into the house flyVSSC a-subunit cDNA individually or in combination, and eachchannel variant was heterologously expressed in Xenopus oocytes[9]. Two-electrode voltage clamp analysis of the sodium channelvariants with different combinations of the mutations revealed

Fig. 1. Transmembrane topology of voltage-sensitive sodium channel a-subunit showinlouse.

that the M827I (M815I) and L932F (L920F) mutations reduced per-methrin sensitivity 2–3-fold when expressed alone but the T929I(T917I) mutation, either alone or in combination, virtually abol-ished permethrin sensitivity (Fig. 2). Thus, the T917I mutationplays a major role in permethrin resistance via a kdr-type nerveinsensitivity mechanism, and can be used as a molecular markerfor resistance detection. Comparison of the expression rates ofchannel variants indicates that the M815I mutation may have afunction of rescuing the decreased expression of channels contain-ing T917I.

3. Resistance monitoring by bioassay

A contact bioassay based on insecticide-impregnated filter pa-per disks was developed to evaluate head lice resistance to variouspediculicides [10]. In this method, newly hatched first instars weregiven a blood meal and placed onto the insecticide-impregnatedfilter paper disk (35 mm diameter, Whatman No. 1). Mortalitywas determined at regular time intervals for 4 h and log time ver-sus logit percent mortality regressions were generated to deter-mine median lethal time (LT50) values. Contact bioassay usingseveral field populations of head lice revealed that permethrinresistance is widespread in the US (MA, FL, TX, and CA) at a levelof �4–8-fold more than the susceptible strain [11]. Additionally,the lice strains from Florida and California showed cross-resistanceto pyrethrins and to PBO-synergized pyrethrins (�3-fold) and toDDT (�3-fold). These findings indicate that target site insensitivityis the main cause of pyrethrin/permethrin resistance to date andthat oxidative metabolism, which is likely, has not yet occurredwidely at this point. The contact bioassay is easy and simple torun but the assay time is limited to a relatively short period be-cause lice cannot survive without feeding every 10–12 h. In addi-tion, only insecticidal active ingredients can be used for thecontact bioassay.

To overcome these limitations, the Hair Tuft Mortality Bioassayhas been developed [12]. In this method, a louse-infested humanhair tuft is directly treated with formulated pediculicide productsand placed back on the in vitro rearing system for assessment. Thisapproach provides substantial improvements over existing bioas-say formats in that the pediculicide toxicity can be assessed in anenvironment that simulates the human scalp in many ways, allow-ing normal feeding and development of lice, and the actual situa-tion of pediculicide treatment. Hair tuft bioassay along with thein vitro rearing system was conducted to evaluate the efficacies

g the location of three mutations associated with kdr-type resistance in the head

0.2 µA

0.2 µA

50 ms

50 msControlPermethrin (mM)

0.1 1

10 100 200

BA

Fig. 2. Comparative sodium current traces from the house fly VSSC variants with and without head louse mutations expressed in Xenopus oocytes before and after exposure toincreasing concentrations of permethrin. Modified with permission from Ref. [9]. Copyright 2008 Elsevier.

A C A A C A A C A A C A A T A A T A A T A

Nucleotide signal ratio

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)

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0 0.2 0.4 0.6 0.8 1.0

0 10 30 50 70 90 100%

0

20

4

0

60

80

10

0

Fig. 3. Sequencing chromatograms of the standard template DNA mixtures withdifferent resistance allele frequencies (A) and the plot of resistance sequence signalratio versus resistance allele frequency at the T917I mutation site (B). Theintensities of resistance allele signals in the sequence chromatograms are markedwith arrows in (A). Quadratic regression line is shown in a solid line with the upperand lower 95% prediction lines indicated by dotted lines in (B). Reproduced withpermission from Ref. [13]. Copyright 2008 ESA.

S.H. Lee et al. / Pesticide Biochemistry and Physiology 97 (2010) 109–114 111

of three commercial pediculicidal products based on pyrethrin orpermethrin (Nix�, Rid�, or Pronto Plus�) [12]. All products werehighly active (100% mortality) to the susceptible strain but showedreduced efficacies (62–84% mortality) to the permethrin-resistantstrain from south Florida (SF-HL) when examined 8 days post-treatment. These results confirm resistance to pyrethrin- and per-methrin-based pediculicidal formulations.

Abovementioned bioassay methods can be employed for resis-tance monitoring of field populations of lice when a sufficientnumber of live specimens are available. Considering that obtaininga large number of live lice for bioassay is always difficult, however,routine monitoring of head lice resistance based on bioassay is notpractical, requiring efficient alternative molecular monitoringtools. In addition, bioassay methods are not sensitive when resis-tance allele frequency is low and particularly when resistance isrecessive as in the case of kdr.

4. Molecular detection of head louse resistance to permethrin

Three methods for molecular monitoring of lice resistance havebeen developed as a tier system: quantitative sequencing (QS) [13],real-time PCR amplification of specific allele (rtPASA) [14] and se-rial invasive signal amplification reaction (SISAR) [15]. All themethods were developed to detect the VSSC mutation frequenciesas a resistance marker. Among these, QS and rtPASA are designedfor the prediction of resistance allele frequency on a population ba-sis at the initial stage of resistance monitoring whereas SISAR is anindividual genotyping method for the analysis of resistance allelezygosity and frequency at a later step.

4.1. QS

QS-based population genotyping can process a large number oflouse populations simultaneously for the evaluation of resistanceallele frequencies at the initial stage of resistance monitoring. Inthe QS protocol, a genomic DNA fragment of the VSSC a-subunitgene that encompass the three mutation (M815I, T917I andL920F) sites was amplified from individual genomic DNA samples[13]. After verification of genotype, the PCR products with oppositegenotypes were mixed together to generate the standard DNA mix-ture templates with resistant allele frequencies of 0, 10, 30, 50, 70,90 and 100%. The set of standard DNA template mixtures (10 ngeach) were sequenced by cycle sequencing (Fig. 3A). The nucleo-tide signal intensities of both resistant and susceptible alleles ateach mutation site were determined from the sequence chromato-

gram and the signal ratios were calculated by dividing resistantnucleotide signal by the sum of the resistant and susceptible nucle-otide signals. The signal ratios of template DNA mixtures were nor-malized by multiplying them with the normalization factor (signalratio of the heterozygous DNA template/signal ratio of the 5:5standard DNA template). A plot of the normalized signal ratios ver-sus corresponding resistance allele frequencies were produced andstandard regression equations were created for the estimation ofresistance allele frequencies of unknown samples and their predic-tion intervals at the 95% confidence level (Fig. 3B). Using the lower

112 S.H. Lee et al. / Pesticide Biochemistry and Physiology 97 (2010) 109–114

and upper 95% prediction equations, the average lower detectionlimits for the three mutations (M815I, T917I and L920F mutations)were determined as 7.4% at the 95% confidence level.

Since QS is designed to use the DNA extracted and preparedfrom multiple louse specimens, it is suitable for processing a largenumber of louse populations. The use of DNA extracted from com-bined multiple lice for QS greatly reduces the overall cost and ef-fort as repetitive DNA extraction from individual lice is arduousand costly. QS for 90 different population samples can be com-pleted within 2 days in moderately equipped laboratories. Thetechnique dependency of QS is also relatively low compared toother population genotyping techniques such as rtPASA-TaqMan[16] and rtPASA. Thus, the speed, simplicity and moderate sensitiv-ity of QS make it an ideal candidate for a routine primary resistancemonitoring tool to screen a large number of wild louse populationsas an alternative to conventional bioassay. Since the sensitivity ofQS is ca. 7.4%, a small to medium-size sampling (7–14 lice) perlouse population would be sufficient, which appears very practicalconsidering the difficulty of collecting a large number of lice sam-ples. Taken together, prediction of resistance allele frequency byQS will greatly facilitate the initial resistance monitoring effortsin field populations of lice. The QS-based population genotypingprotocol should be readily applicable for other insects, includingother human louse species, as a routine resistance monitoring toolas long as the information on resistance-associated mutations inany target genes is available.

When several louse populations from different geographical re-gions were analyzed by QS, the T917I mutation frequencies variedsubstantially (Table 1) [13]. The T917I mutation frequencies in thelice from South Florida (USA), Onkaparinga (Australia), Bristol (UK),and Bobigny (France) were near 100%, suggesting that permethrinresistance is completely saturated or near saturation in these re-gions (Table 1). Lice from California and Texas were moderatelyresistant to pyrethroid as judged by their mean resistance allelefrequencies (86.2% and 60.4%, respectively). The lice from Ecuadorappeared to be only slightly resistant to pyrethroid by showing themutation frequency of 5.2%. No mutation was found in the licefrom Guatemala and Thailand. The resistance allele frequenciesin the head louse populations collected from several provinces ofKorea were also determined by QS (Table 1). Seven out of 10 pop-ulations examined showed 0% resistance allele frequency whereas

Table 1Predicted resistance allele frequencies in head louse populations from different global locagenotyping method. Global louse population data were reproduced with permission from

Lice population Stage used for QS (No. of lice used) Sign

Plantation and Homestead, FL, USA 2nd nymph (23) 1.0Onkaparinga, Australia 3rd nymph (17) 1.0Bristol, UK Male adult (20) 1.0Bobigny, France 3rd nymph (30) 1.0San Bernadino County, CA, USA 1st nymph (52) 0.73Jefferson and Orange Counties, TX, USA Female adult (30) 0.43Vilcabamba, Ecuador 1st nymph (30) 0.02Antigua, Guatemala Male adult (14) 0Northwestern Thailand Male adult (30) 0

KoreaSeoul 3rd nymph (10) 0ChungBuk B Female adult (8) 0GyeongGi 3rd nymph (11) 0DaeJeon Female adult (15) 0ChungNam A Male adult (8) 0ChungJu Female adult (17) 0GyeongGi GwangJu Male adult (7) 0ChungBuk C 3rd nymph (15) 0.29JeonNam Female adult (10) 0.17ChungNam B Female adult (15) 0.06

a If the signal ratio is 1.0 or 0, the resulting resistance allele frequency is considered

remaining three populations showed 9.8–36.7% frequencies. Previ-ous monitoring for the detection of the T917I mutation in localhead louse populations in Korea, which had been conducted in2004, did not identify any population with mutation. With this inmind and because pyrethrin-based pediculicides were not regis-tered in Korea until 2005, the sudden appearance of resistance al-lele in the Korean louse populations strongly demonstrates thatkdr-type resistance is rapidly selected and spread, thereby requir-ing an urgent resistance management. These findings are also ingood agreement with our original contention that head louse resis-tance to pyrethroid is wide spread worldwide but not yet homoge-neous [2].

4.2. rtPASA

The rtPASA is another protocol based on real-time PCR (rtPCR)for the prediction of resistance allele frequency on a population ba-sis like QS [14]. The rtPASA protocol was developed to utilize thesame genomic DNA template used in the QS. If more precise deter-mination of resistance allele frequency below the QS detection lim-it is required, rtPASA can be employed as a supporting monitoringstep. The standard DNA mixture templates for rtPASA were pre-pared by using the same protocol as in the QS except that differentresistance allele frequencies were used in the standard mixes (0%,1%, 3%, 8% and 16%). Allele-specific primers were designed tomatch the T917I and L920F mutation sites simultaneously (Fig4A). rtPCR was conducted with resistant allele-specific primer setusing Chromo 4TM real-time detector (Bio-Rad, Hercules, CA), andthreshold cycle (Ct) values were determined from each amplifica-tion curve, normalized and plotted against respective resistance al-lele frequencies (Fig. 4B). Standard linear regression lines for theprediction of resistance allele frequency were generated by plot-ting the log scale of resistance allele frequency versus Ct value(Fig. 4C). Once the prediction equation established, resistance al-lele frequencies of unknown louse populations were estimatedby incorporating Ct values into the equation.

rtPASA enables the detection of the kdr allele frequency in thehead lice at the level as low as 1.13%. To detect the resistance allelefrequencies lower than ca. 1%, however, a large-size sampling(50 � 100 lice per population) would be required. In addition, thetechnical dependency of rtPASA is relatively high compared to

tions and local provinces in Korea at the T917I mutation site using the QS populationRef. [13]. Copyright 2008 Elsevier.

al ratioa Predicted resistance allele frequency,% (prediction interval at 95% CL)

100 (91.7 to 106.0)100 (91.7 to 106.0)100 (91.7 to 106.0)100 (91.7 to 106.0)86.2 (79.8 to 92.7)60.4 (54.2 to 66.6)5.2 (�1.3 to 11.7)0 (�5.0 to 8.1)0 (�5.0 to 8.1)

0 (�5.0 to 8.1)0 (�5.0 to 8.1)0 (�5.0 to 8.1)0 (�5.0 to 8.1)0 (�5.0 to 8.1)0 (�5.0 to 8.1)0 (�5.0 to 8.1)36.7 (30.7 to 42.6)22.9 (16.9 to 29.0)9.8 (3.6 to 16.0)

as 100% or 0%, respectively, without incorporating it into prediction equation.

A

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0.610 20 30 40 0.8 1.0 1.2 1.4 1.6 1.8

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0.25

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C

Fig. 4. rtPASA diagram (A), typical rtPCR amplification patterns using the DNA templates containing 0%, 1%, 3%, 8% and 16% resistant alleles (B) and the regression linegenerated from the plot of normalized Ct value versus the log of resistance allele frequency (C). Locations of the two mutations are marked with black circles and rtPASAprimers are indicated by horizontal arrows in (A). The regression line is indicated by a solid line with the upper and lower 95% prediction lines indicated by dotted lines in (C).Reproduced with permission from Ref. [14]. Copyright 2008 ACS.

S.H. Lee et al. / Pesticide Biochemistry and Physiology 97 (2010) 109–114 113

QS, requiring a well optimized protocol and experimental systemto guarantee an accurate prediction.

Head louse populations collected from Ecuador, Thailand andKorea (ChungNam B), of which resistance allele frequencies hadbeen determined by QS as 5.2%, 0% and 9.8%, respectively, wereanalyzed again by rtPASA [14]. The rtPCR reaction with 1 ng tem-plate DNA resulted in the Ct values of 7.4, 19.5 and 6.4, respec-tively. The Ct values were converted to actual allele frequenciesusing the equation generated by the set of the standard DNA mix-tures (y = �3.24x + 10.32, r2 = 0.999). The resistance allele frequen-cies were estimated as 8.0%, 0% and 16.2% in the Ecuador, Thailandand Korea populations, respectively, which were in good agree-ment with those determined by QS. These results suggest thatrtPASA protocol can be adapted as a secondary monitoring toolfor the prediction of resistance allele frequency if more preciseestimation is required.

4.3. SISAR

Although both QS and rtPASA enable the prediction of resistanceallele frequencies on a population basis, thereby allowing rapidscreening of resistant populations, they do not provide information

Genomic DNA extraction

Head louse samplecollection (10-100 lice/region)

PCRampof teDNA

Storage for later use

Stored in EtOH

Fig. 5. Tiered system for head lice pyrethroid resistan

on allele zygosity. If the information on resistance allele zygosity aswell as allele frequency in a population is required, individual geno-typing methods such as SISAR [15] can be conducted on a much re-duced number of populations as the secondary or tertiaryresistance monitoring step. The SISAR was originally developed forthe high throughput analysis of single nucleotide polymorphismsusing cleavase, a structure-specific endonuclease [17]. Informationon allele zygosity would be particularly useful for understandingthe resistance population dynamics at the early phase of resistancewhere resistance allele is present as heterozygous form in the popu-lation. SISAR requires, however, a large number of analyses(50 � 100 analyses of individual lice per population) to warrantaccurate estimation of resistance allele frequency, which limits itsapplicability for a routine resistance monitoring tool.

The three molecular tools aforementioned can be readily em-ployed for routine resistance monitoring of head louse populationsin a tiered system (Fig. 5). Although the proposed system only de-tects permethrin resistance mediated by sodium channel insensi-tivity, kdr is a major factor in all permethrin-resistant liceworldwide [3] and its detection should be useful for screening alarge number of wild louse populations as alternatives to conven-tional bioassay.

lification mplate

rrtPASAtPASA(population genotyping)

SISAR(Individualgenotyping)

QS(population genotyping)

Primaryresistance Monitoring

Secondaryresistance Monitoring

Tertiaryresistance monitoring

ce monitoring based on several molecular tools.

114 S.H. Lee et al. / Pesticide Biochemistry and Physiology 97 (2010) 109–114

5. Head louse resistance management and future prospects

Head lice resistance has occurred rapidly for several reasons: (1)head lice are exposed to pediculicides at all stages because mostinfestations are treated; (2) they have short generational timeand high fecundity, facilitating the selection by pediculicides; (3)there is only a small number of effective commercial pediculicides,the majority of which share common chemistry and elicit cross-resistance [11]. Louse resistance to most commercial pediculicideshas occurred and is increasing, particularly to DDT, the pyrethrins,the pyrethroids, and malathion. Current control and resistanceproblems highlight the need to understand the molecular mecha-nisms of insecticide resistance in human lice and to develop toolsfor effective and affordable monitoring. Based on the resistance al-lele frequencies estimated by these molecular techniques, differen-tial actions for resistance management should be implemented. Inregions where resistance allele frequency is saturated or near sat-uration, pyrethroids use should be curtailed and alternative pedi-culicides with different mode of actions used instead. In regionswhere the resistance allele frequencies are low or near zero, pyre-throids should be used cautiously and in conjunction with resis-tance monitoring program. This approach will extend theeffective life span for this valuable group of pediculicides.

Recent completion of the body louse (P. h. humanus) genomesequencing project allows us to acquire all the information on pedic-ulicide target site genes and defense genes associated with detoxifi-cation, and to use this information to effectively study the headlouse. Interestingly, body lice have the smallest number of defensegenes associated with metabolic resistance mechanisms (37P450s, 12 GSTs, 18 Ests, 40 ABC transporters, etc.) among insects[Body Louse Genome Sequencing Consortium, [18]]. The small num-ber of defense genes facilitates the construction of a minimal andefficient microarray for the identification and transcriptional profil-ing of a more complete set of genes that are differentially expressedin pesticide-resistant strains and involved in induced tolerance. Theidentification of such resistance mechanisms and novel target sitesmay allow the development of resistance-breaking compounds(e.g., negative cross-resistance compounds) for improved louse con-trol, more inclusive molecular diagnostics for effective and afford-able monitoring in resistance management and specific non-toxicsynergists useful in novel strategies to control pediculicide-resistantpopulations [11]. In addition to the aforementioned molecular toolsto detect the known mutations responsible for reduced sensitivity ofpediculicide target sites, complete understanding of detoxificationmechanism will enable to establish molecular methods to detectmetabolic resistance as well.

Acknowledgments

This work was supported by the NIH/NIAID (R01 AI045062-04A3). D.H. Kwon and K.M. Seong were supported in part by theBrain Korea 21 Program.

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