Mutations in the voltage-gated sodium channel gene of anophelines and their association with ...

14 Pages·2014·0.46 MB·English

Silvaetal.Parasites&Vectors2014,7:450
http://www.parasitesandvectors.com/content/7/1/450
REVIEW Open Access
Mutations in the voltage-gated sodium channel
gene of anophelines and their association with
–
resistance to pyrethroids a review
Ana Paula B Silva1, Joselita Maria M Santos1 and Ademir J Martins2,3*
Abstract
Constant and extensive use of chemical insecticides has createda selection pressure and favored resistance
development inmany insect species worldwide. One of the mostimportant pyrethroid resistance mechanisms is
classified as target site insensitivity, due to conformational changes in thetarget site that impair a proper binding
ofthe insecticidemolecule. The voltage-gated sodium channel (Na ) is the target of pyrethroids and DDT
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insecticides, used to control insects ofmedical, agricultural and veterinary importance, such as anophelines. It has
beenreportedthatthepresenceofafewnon-silentpointmutationsintheNa geneareassociatedwithpyrethroid
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resistance, termed as ‘kdr’(knockdown resistance) for preventing theknockdowneffect oftheseinsecticides. The
presence of thesemutations,as well as their effects, has been thoroughly studied inAnopheles mosquitoes. So far,
kdrmutationshave already been detected inat least 13 species (Anopheles gambiae, Anopheles arabiensis, Anopheles
sinensis, Anopheles stephensi, Anopheles subpictus, Anopheles sacharovi, Anopheles culicifacies, Anopheles sundaicus,
Anopheles aconitus, Anopheles vagus, Anopheles paraliae, Anopheles peditaeniatus and Anopheles albimanus) from
populations ofAfrican,Asian and, more recently, American continents. Seven mutational variants (L1014F, L1014S,
L1014C, L1014W, N1013S, N1575Y and V1010L) were described, withthe highest prevalenceof L1014F, which
occursatthe1014siteinNa IIS6domain. Theincreaseoffrequencyanddistributionofkdrmutationsclearlyshows
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theimportanceof this mechanism inthe process of pyrethroid resistance. Inthis sense, several species-specific and
highly sensitive methods have been designedin order to genotype individualmosquitoes for kdrin large scale,
which may serve as importanttolls for monitoring thedynamics ofpyrethroid resistance innatural populations. We
also briefly discuss investigations concerningthe course of Plasmodium infection inkdr individuals.Considering the
limitationofinsecticidesavailableforemploymentinpublichealthcampaignsandtheabsenceofavaccineableto
brakethelifecycleofthemalariaparasites,theuseofpyrethroidsislikelytoremainasthemainstrategyagainst
mosquitoesbyeitherindoorresidualspraying(IR)andinsecticidetreatednets(ITN).Therefore,monitoringinsecticide
resistanceprogramsisacrucialneedinmalariaendemiccountries.
Keywords:Anopheles,Sodiumchannel,Malaria,Pyrethroids,Resistance,kdr
Introduction and subtropical regions, specially given the decrease in
Theglobalsituationofmalariaanditsvectors workcapacityoftheaffectedvictims[1].Accordingtothe
Malaria is one of the most serious and complex health World Health Organization (WHO), approximately 207
problemsfacedbyhumanity.Besidesthat,ithasbecomea millioncasesofmalariawerereportedin2012,withanes-
threat for social and economical development in tropical timate of 627,000 deaths, with the highest incidence rates
observed in Africa (80%), Asia (15%) and the Americas
(14%)[2].Amongthefactorscontributingtothisscenario,
*Correspondence:[email protected] it is possible to highlight the absence of an effective
2LaboratóriodeFisiologiaeControledeArtrópodesVetores,Instituto
antimalarial vaccine, the distribution of drug-resistant
OswaldoCruz,FIOCRUZ,RiodeJaneiro,Brazil
3InstitutoNacionaldeCiênciaeTecnologiaemEntomologiaMolecular,Rio Plasmodium, the development of insecticide resistance in
deJaneiro,Brazil vector mosquitoes, as well as ecological, socio-economic
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Silvaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
unlessotherwisestated.
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and medical-sanitary factors [3,4]. Mosquito resistance to and non-cumulative, they have disadvantages, like
at least one insecticide used for malaria control has been chemical instability and high toxicity for vertebrates
identified in 64 countries [5]. Malaria vectors are part of [18]. The carbamates, also referred to as methylcarba-
theAnophelesgenus,includingnearly484species,distrib- mates for deriving from the methylcarbamic acid [19], are
utedinsevensubgenera[6],70ofwhichshowingvectorial compounds used as insecticides, nematicides and acari-
competenceforhumanmalaria[7],with41ofthembeing cides.Theyhavelowenvironmentalpersistenceandareless
consideredasdominantvectorspecies[8](Table1). toxictolivingorganismsthanorganochlorines.Duetotheir
wide use in agriculture, they were incriminated as food,
Useofinsecticidesagainstmalariavectors water and air contaminant agents, with adverse effects in
The strategic tools to fight malaria are oriented towards humansandotheranimals[20].Around1970,syntheticpy-
two principal directions: (i) prevention, by means of con- rethroidswerereleasedasaclassofinsecticidesconsidered
trolling vector mosquitoes; and (ii) case management, more efficient and less toxic. These insecticides raised the
through malaria diagnosis and treatment with effective attention for presenting higher lethal capacity against in-
medicines, being the former considered as the most effect- sects,requiringonlysmalldosesoftheproductforsatisfac-
ive [12]. The techniques for controlling vector mosquitoes tory effects [21]. Consequently, pyrethroids virtually
are didactically classified as: mechanical (elimination of substituted/supplemented the use of other classes in many
breeding sites), biological (use of predators or parasitoids) pestcontrolareas,representingnearly23%ofthe chemical
or chemical (application of synthetic insecticides) [13,14]. insecticides market, more than one fourth of the world
The development of chemical insecticides that remain market[22].
activeforlongperiodsoftimewasoneofthemostrelevant Pyrethroids are synthetic analogues of the chrysanthe-
breakthroughs of the 20th century [15] and nowadays they mic acid (pyrethrins I) and pyrethric acid (pyrethrins II)
stillplayanimportantroleinthecontrolofdiseasevectors esterinsecticides,naturallyfoundinleavesofChrysanthe-
andplaguesinagriculture. mumcinerafolis.Theyarechemicallydistinguishedastype
There are four main groups of neurotoxic insecticides I, compounds that lack an alpha-cyano group, like per-
permittedtobeusedforpublichealthpurposes,classified methrin,andtype2,withanalpha-cyanogroup,likedelta-
according to their chemical nature and mode of action: methrin [23]. They are biodegradable, non-cumulative
organochlorines,organophosphates,carbamatesandpyre- insecticides that rarely cause acute intoxication in birds
throids.Thefirstinsecticideusedagainst anophelineswas and mammals [24]. Currently, malaria control basically
theDDT,anorganochlorinefirstlyusedinNaplesin1944 dependsuponthisinsecticideclass,whichhasbeenwidely
against a typhus epidemic [15]. In 1995, WHO proposed employed in indoor residual spraying (IRS) and also to
the global eradication of malaria based on the spraying of controlagriculturalpestsworldwide.Besides,pyrethroidis
DDT inside the houses. Highly efficient and inexpensive, theonlyclassapprovedbytheWorldHealthOrganization
itwasabletodecimatepopulations of vectorson a global PesticideScheme(WHOPES)formosquitonetimpregna-
scale. However, the development of environmental and tion(InsecticideTreatedNet–ITN;LongLastingInsecti-
sanitary problems, coupled with the emergence of resist- cideTreatedNet-LLIN)[1,25,26].
ance, resulted in the prohibition of the product in many IRSisamethodinwhichresidualinsecticidesareap-
countries [16]. In spite of that, after the “Stockholm Con- plied on the surface of walls and ceilings of houses
vention on Persistent Organic Pollutants” in 2007, DDT [27]. Based on this strategy it is expected that the mos-
was reestablished in restricted areas with high malaria quitoes, after feeding on blood, rest on these surfaces
transmission,suchasinAfricanlocations[17]. and remain long enough to absorb a lethal dose of the
The organophosphates (malathion, temephos, feni- insecticide.ITNisamosquitonetthatrepels,incapaci-
trothion etc.) were developed in the 1940s and have tates or kills mosquitoes that come into contact with
been used ever since as insecticides, herbicides and the insecticide impregnated in the net material, being
plant growth regulators. Despite being biodegradable both a chemical and a physical barrier against insects.
Table1Listof41dominantvectorspeciesbyarea
Continent Anophelinespecies
Africa Anophelesarabiensis,Anophelesfunestus,Anophelesgambiae,Anophelesmelas,Anophelesmerus,AnophelesmouchetieAnophelesnili
Asia Anophelesbarbirostris,Anopheleslesteri,Anophelessinensis,Anophelesaconitus,Anophelesannularis,Anophelesbalabacensis,Anopheles
culicifacies,Anophelesdirus,Anophelesfarauti,Anophelesflavirostris,Anophelesfluviatilis,Anopheleskoliensis,Anophelesleucosphyrus,
Anophelesmaculatus,Anophelesminimus,Anophelespunctulatus,Anophelesstephensi,AnophelessubpictuseAnophelessundaicus
Americas Anophelesfreeborni,Anophelespseudopunctipennis,Anophelesquadrimaculatus,Anophelesalbimanus,Anophelesalbitarsis,Anopheles
aquasalis,Anophelesdarlingi,AnophelesmarajoaraeAnophelesnuneztovari
CompiledfromSinkaetal.[9-11].
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There are two ITN categories: conventional nets and genetic composition of a given population, in response to
LLIN[27,28]. the selection pressure. This is a typical case of Natural
The initial success of insecticide based strategies Selection,whichconsistsintheincreaseoftherelativefre-
caused the optimistic sensation that the elimination of quencies of some “pre-adapted” individuals present in a
malaria as a public health concern would be possible population, resulting from the constant application of the
through the elimination of its vectors. However, these samechemicalproduct[35].Intensiveandextensiveuseof
strategies are threatened today, due to the emergence chemical insecticides has selected populations resistant to
of vector populations resistant to insecticides. Since these compounds [36]. The resistance phenomenon has
new classes of alternative, equally interesting insecti- beenobservedinmorethan500insect speciesaroundthe
cides are not yet available on the market, the selection world, among which more than 50 are anophelines [37].
for resistance tends to continue increasing, unless ef- According to WHO [5], resistance to at least one insecti-
fectivemanagementstrategiesareimplemented[29]. cide had been identified in 64 malaria-endemic countries.
Resistancetopyrethroidsseemstobethemostwidespread.
Review Twomainmechanismsareincriminatedasresponsiblefor
Modeofactionofpyrethroids the pyrethroid resistance: metabolic resistance and target-
Pyrethroids, such as DDT and its analogues, belong to a siteinsensitivity[38,39].
group of neurotoxins that share a similar mode of action. Metabolicresistanceoccurswhenhighactivityofoneor
Theyall target Na ,which is present in cellsofthe central moreenzymesresultsina sufficientportionofinsecticide
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and peripheral nervous systems (neurons, myocytes, being sequestered or detoxified before reaching its target
endocrine cells and ovaries), changing the kinetics of andpromotingthedesiredeffect[38].Itoccursduetothe
propagation of nerve impulses [22]. Structurally, Na is increase in the number of available molecules (genetic
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an integral transmembrane protein, composed of four amplificationorhyperactivationofthegeneexpression)or
homologous domains (I-IV), each of them composed of through mutations in the coding gene portion of the
six helices (S1-S6) connected by loops. The segments enzyme, producing the more efficient metabolization of
S5, S6 and the S5-S6 P-loops form a central aqueous the insecticide [37,40,41]. This mechanism is highly com-
pore, and the S1-S4 helices of each domain unite to plex, although recent advances have been characterizing
form four independent voltage-sensitive domains [30,31]. the main enzyme genes responsible for the detoxification,
The A. gambiae Na alpha subunit gene comprises an pavingthewayforthedevelopmentofmolecularmarkers
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ORF (Open Reading Frame) with 6,417 nucleotides that for the resistance [42]. Three main enzyme superfamilies
encodes 2,139 amino acids, resulting in a protein with a are involved in the detoxification process: Esterases,
molecularmassof240kDa.Thisgene,locatedatthepara Mixed Function Oxidases (MFO, or simply P450) and
(paralysis) loco of the X chromosome, is composed of 35 Glutathione S-Transferases (GST) [37]. Colorimetric
exons, including two duplicated exons, and 32 introns, biochemical trials are widely employed to detect changes
which transcribes for different messenger RNAs (mRNA) in the activity of detoxification enzymes. In this test, the
throughalternatesplicing[31]. enzymatic activity of a natural population is compared
The effects of pyrethroids are stereospecific and two with the control lineages ones, using specific substrates
different Na binding sites were identified. The first was foreachenzymaticfamily[43].
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proposed by O’Reilly et al. [32], in which IIS5 and IIIS6 On a transcriptional level, more recently microarray as-
helices would play an important role in the interaction says have gained prominence in the investigation of meta-
with the insecticide molecule and the additional link in bolic resistance. In this technique, the detoxification chips
the IIS4-S5 linker would explain the higher potency of (or detox chips) compare the expression of virtually all
pyrethroids compared with DDT. The second was genes of the families related to the metabolism of insecti-
suggestedbyDuet al.[33],wherethebindingsitewould cides (GSTs, MFOs, Esterases), between resistant and sus-
be a type of “pocket” formed by the IS4-S5 linker and ceptiblemosquitoes.Inadditiontothesemainfamilies,the
the helices IS5 and IIS6. For both models, the selective expression of other genes are evaluated, such as some
effect of the insecticide would be explained by the non- related to redox metabolism, involved in the protection
conservation of the amino acids of these regions be- against free radicals [44]. The analysis of the gene expres-
tweenarthropodsandotheranimals. sionthroughdetoxchipinA.gambiaeshowedhighactivity
of GST genes (GSTE2), P450 (CYP6Z1 and CYP325) and
Pyrethroidresistancemechanisms peroxidases in DDT resistant mosquitoes [44]. Genes with
Insecticide resistance can be defined as the ability of indi- anti-oxidizingfunction(Superoxidedismutase,GST,Perox-
vidualsofa speciestowithstanddosesoftoxicsubstances, idaseandP450)weredifferentlyexpressedindeltamethrin-
that would be lethal for most individuals of a population resistant populations of A. arabiensis in Cameroon
[34]. It is, therefore, a milestone in the change of the [45]. High expression of CYP6P3, a gene of the P450
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family, was observed in permethrin-resistant popula- the amino acid asparagine to serine [63,64]. In Indian A.
tions of A. gambiae [46]. Differential expression was culicifacies populations, also in addition to the L1014F/S
also observed in A. funestus, whose P450 genes substitutions, a new mutation in the site 1010 was
(CYP6P9, CYP6M7) and COI (from the redox system) described,substituting valine byleucine (V1010L)[65].
were more expressive in resistant individuals [47]. AnothermutationintheNa ofMuscadomestica,which
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Resistancebasedontarget-siteinsensitivityoccurswhen substitutesmethioninebythreoninein918position,corre-
thereisanalterationinthemoleculesthatdirectlyinteract spondingtotheloopbetweenIIS4-S5segments,synergicto
with the insecticide, making it less toxic or inefficient the classical L1014F mutation, was associated with high
[42,43,48]. Since insecticide targets are structural mole- levelsofDDTandpyrethroidresistance,thusbeingreferred
cules of the nervous system, highly conserved throughout to as super kdr [66]. An analogous situation was observed
evolution, few alterations are permissive without the loss inotherinsectspecies,suchasinthehornflyHaematobia
of their physiological functions. Thus, it is common that irritans [67], green peach aphid Myzus persicae [68,69],
themutationsselectedforresistanceoccurathomologous onionthripsThripstabaci[70]andinthemothTutaabso-
sitesamongdifferentinsectspecies[49].Target-siteinsensi- luta[71].However,therearestillnorecordsofhomologous
tivity is the most understood mechanism, and in many substitutionsinanophelines.
casesisthecharacteristicattributedtothehigherportionof Based on the current molecular techniques, it was pos-
thegeneticvariationrelatedtoresistance[50].Inthissense, sible to identify and map the distribution of kdr mutations
molecular diagnoses for detection target-site mutations are amongawiderangeofAnophelesspeciesaroundtheworld.
part of the strategies to monitor insecticide resistance in Since it was first described in 1998 [53], the identification
manymalariacontrolprograms[51]. of changes in the Na gene in the Anopheles genus has
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been widely monitored, in a way that we were able to
recorded about 98 references published until the end of
Kdrmutationsasaresistancemechanism 2013(Table2).
Manystudiesshowedthatresistancetotheknockdownef- Sofar,Na mutationsweredescribedinatleast13differ-
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fect ofseveral insect species isassociatedwith point muta- ent anophelines. A. gambiae was the most studied (62 re-
tions in the Na gene. By definition, the knockdown effect cords), showing three mutational variants (L1014F, L1014S
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is the loss of coordination and paralysis caused by the in- and N1575Y), detected in 19 out of 54 countries in Africa
secticide, which are often accompanied by spasms and (Figure 1). Following, the African A. arabiensis presented
tremors[22].Thisresistancemechanismwasfirstobserved 17 records, showing two variants (L1014F and L1014S)
in the housefly Musca domestica [52], where later it detected in seven countries. A. sinensis was the third one,
was suggested that the substitution of one amino acid withsixrecords.Surprisingly,itshowedthehighestnumber
leucine by phenylalanine in the hydrophobic segment of kdr variants (L1014F, L1014S, L1014C, L1014W and
IIS6 (L1014F) resulted in a moderate increase of DDT N1013S), distributed in five Asian countries, mostly
resistance, termed as the kdr mutation (knockdown detected in China. According to Kang et al. [142], this fact
resistance). In Anopheles the homologous L1014F kdr is related to the high population size and to the wide
mutation was first identified in lineages of A. gambiae geographical distribution of the species, which tends to
resistant to pyrethroids [53] and since then it has also increasethegeneticvariability.
been detected in a series of other anophelines [54-59]. Among other Asian species, A. stephensi showed three
Still in the 1014 site, another substitution, leucine by records of two variants (L1014F and L1014S), detected in
serine (L1014S), was identified in A. gambiae, also Dubai and India. A.subpictus (L1014F), A. culicifacies
associated with the kdr phenotype [60]. The mutations (L1014F, L1014S and V1010L) and A. vagus (L1014F)
L1014F and L1014S were first observed in populations showedtwo records;whileA. sacharovi (L1014F/L1014S),
of West and East Africa, respectively. Therefore, the A. sundaicus (L1014F), A. aconitus (L1014F), A. paraliae
former is sometimes referred to as kdr-w (kdr-west), (L1014S) and A. peditaeniatus (L1014F/L1014S) had just
and the latter, as kdr-e (kdr-east) [61]. In any case, it is onerecord.ThepresenceofkdrmutationsintheAmericas
noticeable that the distribution of these mutations is wasobservedonlyinA.albimanus,forthevariantsL1014F
strongly related to sibling species of the Anopheles and L1014C in populations from Mexico, Nicaragua and
gambiaecomplex[62]. CostaRica(Table2).
In Asian A. sinensis populations, in addition to the A survey on the geographical distribution of kdr muta-
L1014F/S substitutions, the mutations L1014C and tions in African populations of A. gambiae, conducted by
L1014W were reported, changing the amino acid leucine Pinto et al. [61], detected the presence of the variant
to cysteine and to tryptophan, respectively. Additionally, L1014Finwestcountries(kdr-w),fromNigeriatoSenegal,
in the site immediately before the one of the classical the presence of L1014S (kdr-e) in the East (Kenya), and
kdr mutation, an N1013S substitution occurs, changing both mutations occurring in the Midwest, comprising
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Table2Anophelinespecieswithkdrmutationsdetected
Species Locality Typeofmutation References
Anophelesgambiae
Ghana L1014F/N1575Y/L1014S [62,72-77]
Nigeria L1014F/L1014S [56,62,78-80]
BurkinaFaso L1014F/N1575Y/L1014S [53,57,62,72,77,81-87]
Cameroon L1014F/N1575/L1014S [54,62,77,88-96]
IvoryCoast L1014F/L1014S [53,55,62,83,97-99]
Kenya L1014S [60,100-106]
Angola L1014F/L1014S [62,107]
Benin L1014F/N1575Y/L1014S [62,77,108-113]
Mali L1014F/L1014S [114,115]
Chad L1014F [116]
Congo L1014F/L1014S [117,118]
EquatorialGuinea L1014F/L1014S [54,119]
Gabon L1014F/L1014S [62,120,121]
Senegal L1014F/L1014S [62,122]
Uganda L1014F/L1014S [123-126]
Tanzania L1014S [127]
Burundi L1014S [128]
Liberia L1014F [129]
Niger L1014F [130]
Anophelesarabiensis
Sudan L1014F/L1014S [131-134]
BurkinaFaso L1014F/L1014S [57,82,84,86,135,136]
Ethiopia L1014F [58,137]
Kenya L1014S [102,104]
Benin L1014S [112]
Tanzania L1014F [138]
Uganda L1014S [125]
Anophelessinensis
China L1014F/L1014S/L1014C/L1014W/N1013S [64,139-141]
Korea L1014F/L1014C [142]
Laos L1014S [143]
Cambodia L1014S [143]
Vietnam L1014S [143]
Anophelesstephensi
Dubai L1014F [144]
India L1014F/L1014S [145,146]
Anophelessubpictus
SriLanka L1014F [147]
Indonesia L1014F [148]
Anophelesalbimanus
Mexico L1014F [149]
Nicaragua L1014C [149]
CostaRica L1014C [149]
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Table2Anophelinespecieswithkdrmutationsdetected(Continued)
Anophelessacharovi
Turkey L1014F/L1014S [150]
Anophelesculicifacies
India L1014F/L1014S/V1010L [65,151]
Anophelessundaicus
Indonesia L1014F [148]
Anophelesaconitus
Indonésia L1014F [148]
Anophelesvagus
Indonesia L1014F [148]
Laos L1014S [143]
Cambodia L1014S [143]
Vietnam L1014S [143]
Anophelesparaliae
Laos L1014S [143]
Cambodia L1014S [143]
Vietnam L1014S [143]
Anophelespeditaeniatus
Laos L1014F/L1014S [143]
Cambodia L1014F/L1014S [143]
Vietnam L1014F/L1014S [143]
Angola, Gabon, Equatorial Guinea and Cameroon. This Despite15yearsofresearch,somedoubtsstillarisewith
same distribution pattern was reported one year later by respect to the kdr mutations, especially if they are indeed
Santolamazza et al. [62]. The occurrence of both muta- correlated with the resistant phenotype. One of the tech-
tions is currently found, sympatrically, in several African niques adopted to test this association is the employment
countries. Exceptions were Niger, Ethiopia, Chad and of bioassays with insecticides (WHO cones, bottle test,
Liberia, which reported the presence of L1014F only, and ITN, LLIN) followed by the genotyping of kdr mutation
BurundiandKenyawithL1014Sonly(Figure1). between dead and surviving mosquitoes after the test. In
It is noteworthy that the A. gambiae complex is com- other words, it is aimed to test whether the mutation
posedofsevensiblingspecies:A.gambiaes.s.,A.arabiensis, frequency is higher among resistant than the susceptible
A. melas, A. merus, Anopheles quadriannulatus species A, individuals. In our survey, out of the 98 studies here
A. quadriannulatus species B and Anopheles bwambae. considered, 63 (64.3%), conducted bioassay followed by
They are morphologically indistinguishable, however, they genotyping, correlating the mutation with insecticide
can be classified according to fixed and polymorphic resistance. Among them six detected the involvement of
chromosomal inversions [152]. The classical molecular more than one mechanism of resistance (target site
forms are Savannah, Mopti, Bamako, Forest and Bissau, and metabolic alterations) [78,82,88,100,139,140] and
accordingtoparacentricinversionsofthesecondchromo- two only suggested their occurrence [123,129]. On the
some of A. gambiae s.s. [153]. The mutation L1014F was other hand, six studies (9.5%) did not associate the oc-
firstly described in the Savannah form of A. gambiae currence of Na mutations with knockdown resistance
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populations,alsoknownasSform,anduntilmid-1999this [58,107,119,131,136,143]. In these cases, the lack of a
mutation had not occurred in sympatry with the Mopti “mutation versus resistance” association was suggested
form (M form) [154]. However, later studies identified its due to low sample size [107], mutation similarly dis-
presencealsointheMform,possiblyresultingfromgenetic tributed between dead and surviving individuals in the
introgression from the S form [81,155]. Introgression was insecticide bioassay [58,131,136,143] or mutation among
alsosuggestedbyTripetetal.[114]whenthekdrallelewas susceptible individuals [119]. Nevertheless, in this last
detected in the Bamako form. On the other hand, a new example the authors recognized that the bioassays were
independentmutationaleventcouldexplaintheemergence performed outside the WHO recommended standards.
ofthekdrmutationinA.arabiensis[135]. Lastly, in 28 studies (28.6%) only the genotyping of field
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Figure1DistributionofkdrmutationsinAnophelesmosquitoesaroundtheworld:(a)America,(b)Africa,(c)Asia.
samples was performed, considering the presence of the in areas with high kdr and ace-IR mutation frequencies.
mutationasenoughevidenceforresistance. This ace-1R mutant allele belongs to the acetylcholin-
esterase gene, conferring resistance to organophos-
AssociationbetweenITNandkdrmutation phates [162].
The use of ITNs/LLINs treated with pyrethroids is an
important tool to reduce morbidity and mortality caused AssociationbetweenPlasmodiuminfectionandinsecticide
by malaria [26]. According to a survey performed by resistance
Lengeler et al. [156], the implementation of this strategy Regardless of the extensive literature concerning kdr
in Sub-Saharan Africa, between 1986 and 2003, was able mutations and their association with resistance to insecti-
to reduce morbidity by 50% and the infant mortality by cides, few reports have presented their impact on malaria
17%. In Kenya, for instance, the employment of ITNs transmissiondynamics,i.e.,ontheabilityofmosquitoesto
was able to prevent infant mortality in an area with high transmitmalaria.Infectionrateandoocystburdenaretwo
malaria transmission [25]. However, the maintenance of of the five factors that determine the vectorial capacity of
this efficiency is still a controversial issue nowadays, given mosquitoes [163]. The response to Plasmodium exposure
the occurrence of highly resistant anopheline populations. in vectors is modulated by the mosquito’s innate immune
Thereareseveralrecordsindicatinggoodresultsofpyreth- system. In A. gambiae, for example, changes in its global
roidtreatedmaterialswherekdrmutationhadbeenidenti- geneexpressionpatternsareexpressed uponPlasmodium
fied, such as in Nigeria [157], where the LLINs were infection [164]. Exposure to pyrethroids, in turn, induces
efficient at killing or reducing the blood feeding of A. metabolic changes that alters the immune response [165]
gambiae, Mali [115], Benin [108,158] and Uganda [124]. and may therefore affect the outcome of Plasmodium
On the other hand, a reduction in the susceptibility of A. infection.
gambiae populations subjected to ITNs was observed in An insecticide susceptible strain of A. funestus showed
Uganda [159]. Besides that, increases in kdr frequency greater ability to become infected with Plasmodium
were evidenced for this same species after the distribu- bergheithanitsresistantcounterpart[166].InA.gambiae,
tion of LLINs in Kenya [101], Niger [130], Senegal infection with this same parasite increased the expression
[122] and Benin [160]. levelofCYP6M2,agenerelatedwithmetabolicresistance
ThemostrecentupdateofWHOPEScontinuesindicat- [164]. In relation to the possible impacts of kdr mutation
ing only pyrethroids (deltamethrin, alphacypermethrin, on vector competence, few records are available and
permethrin and a combination of deltamethrin or per- are sometimes conflicting. For instance, neither posi-
methrin and piperonyl butoxide – PBO) for LLINs [161]. tive or negative correlation was found between the
However, given the possibility of loss of effectiveness occurrence of kdr and ace-1R alleles with infection of
caused by resistance, the development of mosquito nets Plasmodium falciparum in A. gambiae natural popula-
impregnatedwithotherclassesofinsecticidesisapromis- tionsfromBenin[109].
ingalternative.Astudyconductedwithmosquitonetsim- Otherstudies,however,showedthatthepresenceofboth
pregnated with chlorpyrifos-methyl (organophosphate) resistant alleles could be associated with increased preva-
and lambdacyalothrin (pyrethroid), showed that, alone or lence of Plasmodium infection in an A. gambiae resistant
combined, they were efficient at killing or reducing strain. Additionally, individuals carrying the kdr mutation
blood feeding of A. gambiae from the Ivory Coast, even had increased prevalence of sporozoites, which is likely to
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Method Equipmentrequired Mutation References /1
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Allele-SpecificPolymeraseChainReaction(AS-PCR) PCRthermocycler,electrophoresisandimagingequipments L1014F/S/C [53,60] 5
0
HeatedOligonucleotideLigationAssay(HOLA) PCRthermocycler,ELISAplatereader L1014F/S [168]
Sequence-SpecificOligonucleotideProbe–Enzyme-LinkedImmunoSorbentAssay(SSOP-ELISA) PCRthermocycler,shakingincubatorandELISAplatereader L1014F/S [138]
PCRSequenceSpecificOligonucleotideProbeAssay(PCR-DotBlot) PCRthermocycler,shakingincubatorandnylonmembrane L1014F/S [169]
FluorescenceResonanceEnergyTransfer(FRET)/MeltCurveAnalysis(MCA) Real-TimePCRthermocycler L1014F/S [125]
PCRElongationwithFluorescence PCRthermocyclerandelectrophoresisequipments L1014F/S [170]
HighResolutionMelt(HRM) Real-TimePCRthermocycler L1014F/S [171]
Allele-SpecificLoop-MediatedIsothermalAmplification(AS-LAMP) Turbidimeterandwaterbath L1014F [172]
PolymeraseChainReaction-RestrictionFragmentLengthPolymorphismassay(PCR-RFLP) PCRthermocycler L1014F/C [141]
PrimerIntroducedRestrictionAnalysis-PCRassay(PIRA-PCR) PCRthermocycler,electrophoresisandimagingequipments L1014F/S [173]
MultiplexPrimerIntroducedRestrictionAnalysis-PCRassay(mPIRA-PCR) PCRthermocyclerandelectrophoresisequipments L1014F/S [174]
AmplificationRefractoryMutationSystem(ARMS) PCRthermocycler,electrophoresisandimagingequipments L1014F [151]
P
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1
4
Silvaetal.Parasites&Vectors2014,7:450 Page9of14
http://www.parasitesandvectors.com/content/7/1/450
impactonparasitetransmission[167].Giventhedissemin- survey conducted in populations of A. gambiae from
ationofkdrmutationinnaturalpopulations,similarstudies Burundi,wherehighfrequenciesofkdralleleweredetected
should be conducted in order to better understand the inunsprayedareas,duetomigration[128].
impactofinsecticideresistanceonvectorcompetence. Management by saturation involves methods that over-
come resistance mechanisms present in the insect, by the
useofhighratesofinsecticides,thatshouldkillevenresist-
MoleculartoolsforKDRmutationdiagnosis
ant individuals, or by the use of chemical synergists [21].
The resistance phenomenon can be studied on many
Forexample,theevaluationofthedosage-dependenteffect
levels, from biological assays in order to evaluate the sus-
of permethrin-treated nets in experimental hut trials from
ceptibility/resistance status to biochemical and molecular
Benin showed that nets treated with higher permethrin
characterizationsabletoinferthemechanismsandeffect-
concentrations provided better blood feeding prevention
ive genes selected for resistance. Currently, the develop-
against pyrethroid-resistant A. gambiae [158]. Similar effi-
mentoftools for genetic screening of natural populations
ciency against pyrethroid-resistant A. gambiae populations
onalargescale,areaimedtopredictthepredispositionof
were observed in a net impregnated with deltamethrin-
thosepopulationstodevelopinsecticideresistance.
pyperonilbutoxidecombination[157,178].
Thus,theidentificationofgeneticmarkersassociated
Finally, the management by multiple attacks involves
with resistance were included in the priorities of the
eithermixturesor rotationsofinsecticidestoavoid resist-
WHO Global Plan for Insecticide Resistance Manage-
ance. This method is based on the concept that insects
ment (GPIRM) [5]. In this sense, the identification of
resistanttooneinsecticidewillbekilledbytheothercom-
kdr genetic markers truly associated with pyrethroid
ponentofthemixtureandthatfewinsectswillberesistant
resistance,aswellastheimprovementofexistentdiag-
to the entire mixture [176]. A combination of IRS with
nostic assays are constantly in the course of studies in
chlorfenapyr and LLIN impregnated with deltamethrin, in
thisfield.DNAbasedgenotypingtechniqueshaveasmain
an experimental hut trial from Benin, was effective to pro-
advantages the high sensitivity and the capacity to distin-
vide additional level of transmission control and personal
guish between homo and heterozygous individuals [37].
protection against pyrethroid-resistant A. gambiae [108].
The principal methods employed in the detection of kdr
Similar results were obtained by the use of mosquito nets
mutations are listed in Table 3, with emphasis on the
impregnated with chlorpyrifos-methyl and lambdacyalo-
equipmentrequiredforeachtechnique.
thrinagainstA.gambiaefromIvoryCoast[162].
Strategiesformanagingresistance Conclusions
The evolution of insecticide resistance has become a After 15 years of intense research, kdr mutations were
great threat to chemical products-based malaria con- recorded in 13 anopheline species, in natural popula-
trol programs due to the strong selection pressure tionsfromthree continents,revealingthepreponderance
placedonresistancegenes [5]. Therefore, strategies for of this phenomenon in the process of resistance to pyr-
managing resistance to minimize operational obstacles ethroid insecticides, either alone or combined with other
to the use of a given product have gained prominence mechanisms (e.g., metabolic resistance). These alter-
on the world stage. The resistance management strat- ations emerged in different species as well as within
egies are divided into three groups: management by populations of the same species, and are spreading
moderation, management by saturation and manage- quickly, given the strong selection pressure exerted by
mentbymultipleattack[175]. the pyrethroids. Although compounds with new modes
Management by moderation aims to reduce the selec- ofaction,such asneonicotinoids andpyrroles,havebeen
tion pressure to conserve susceptible individuals of a introduced in public health, they are still not indicated
given population,bytheuseoflower dosages of insecti- for IRS and ITN, for instance. The availability of a new
cides, higher treatment thresholds, chemicals with generation of environmentally friendly compounds may
shorterresidualactivityandmaintainingunsprayedareasas takeaslongastheimplementationofadvancedstrategies,
refuges for susceptible individuals [176]. Even though, pe- likewise, the use of genetically modified mosquitoes.
culiarities have to be considered. For instance, a study Therefore,theuseofpyrethroidshastobeseverelymoni-
evaluatingtheeffectsofsublethaldosesofpermethrininan toredinordertotrytomaximizetheireffectiveness.
A. stephensi strain showed that lower concentrations were
Abbreviations
more efficient in increasing the mortality rates [177].
Na:Voltage-gatedsodiumchannel;DDT:Dichlorodiphenyltrichloroethane;
V
Concerning refuges, it isimportant to maintainsusceptible Kdr:Knockdownresistance;WHO:WorldHealthOrganization;IRS:Indoor
allelesinapopulation,mainlyinthecaseofresistantalleles, residualspraying;WHOPES:WorldHealthOrganizationpesticidescheme;
ITN:Insecticidetreatednet;LLIN:Longlastinginsecticidetreatednet;
whichcarryafitnesscost.However,resistantallelescanalso
MFO:Mixedfunctionoxidases;GST:GlutathioneS-Transferases;
invade untreated areas. This was the case observed in a PBO:Piperonil-butoxide.
Silvaetal.Parasites&Vectors2014,7:450 Page10of14
http://www.parasitesandvectors.com/content/7/1/450
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