evidence of insecticide resistance to pyrethroids and bendiocarb...

Research ArticleEvidence of Insecticide Resistance to Pyrethroidsand Bendiocarb in Anopheles funestus from TsararanoMarovoay District Madagascar

Tsiriniaina Rakotondranaivo12 Solohery Fanomezana Randriamanarivo1

Mihajarilala Rakotoniaina Tanjona1 Inegraves Vigan-Womas3

Milijaona Randrianarivelojosia45 andMamadou Ousmane Ndiath 1

1 Institut Pasteur de Madagascar G4 Malaria Group Ambatofotsikely Antananarivo 101 Madagascar2Ecole Doctorale Genie du Vivant et Modelisation Universite de Mahajanga Mahajanga 401 Madagascar3Institut Pasteur de Madagascar Unite dImmunologie des Maladies Infectieuses Ambatofotsikely Antananarivo 101 Madagascar4Institut Pasteur de Madagascar Unite de Recherche sur le Paludisme Ambatofotsikely Antananarivo 101 Madagascar5Faculte des Sciences Universite de Toliara Toliara 601 Madagascar

Correspondence should be addressed to Mamadou Ousmane Ndiath ousmanendiathgmailcom

Received 5 March 2018 Revised 17 May 2018 Accepted 20 September 2018 Published 8 October 2018

Guest Editor Ademir J Martins

Copyright copy 2018 Tsiriniaina Rakotondranaivo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Introduction In Madagascar malaria control relies on the countrywide use of long lasting insecticide treated bed nets (LLINs) andon indoor residual spraying (IRS) in the central highland area as well as a small area on the eastern coast We tested insecticideresistance mechanisms of Anopheles funestus from Tsararano a malaria endemic village in the coastal health district of MarovoayMethods Insecticide susceptibility bioassays were done in July 2017 on first-generation Anopheles funestus (F1) to assess (i) thesusceptibility to permethrin (005) deltamethrin (005) DDT (4) malathion (5) fenitrothion (1) and bendiocarb (01)(ii) the effect of preexposure to the piperonyl butoxide (PBO) synergist and (iii) the enzymatic activities of cytochrome P450esterases and glutathione S-transferases (GST) Results Our results demonstrated thatAn funestus was phenotypically resistant topyrethroids and bendiocarb with a mortality rate (MR) of 336 (95CI 245-437) and 86 (95CI 776-921) respectivelyIn contrast An funestus were 100 susceptible to DDT and organophosphates (malathion and fenitrothion) Preexposure of Anfunestus to PBO synergist significantly restored the susceptibility to bendiocarb (MR=100) and increased theMR in the pyrethroidgroup from96 (95CI 900-989) to 100 for deltamethrin andpermethrin respectively (1205942 =43 df = 3Plt 00001) Enzymaticactivities of cytochromeP450 and120572-esteraseswere significantly elevated amongAn funestus comparedwith the IPM reference strain(Mann-WhitneyU= 30 Plt00001U = 1455 P lt00001 respectively) No significant differences of 120573-esterases activities comparedto the IPM reference strain were observed (Mann-Whitney U = 3925 P = 008) Conclusion In Tsararano despite the absenceof an IRS programme there is evidence of high levels of insecticide resistance to pyrethroids and bendiocarb in An funestusBiochemical data indicated that a metabolic resistance mechanism through the cytochrome P450 genes is operating in the Anfunestus population

1 Background

Malaria remains amajor public health burden on the island ofMadagascar Today despite the fight against malaria which ismainly based on the use of rapid diagnostic test (RDT) theadministration of artemisinin-based combination therapies(ACTs) as first-line treatment and the implementation of

vector control measures malaria remains the third leadingcause of morbidity and mortality in Madagascar behindrespiratory infections and diarrhea [1] In 2015 malariamorbidity was 101 for all ages and 186 for children under5 years of age The malaria-related mortality ranged from123 to 257 and incidence from 31 to 67 amongthose under 5 years of age [2 3] Thus malaria still hampers

HindawiBioMed Research InternationalVolume 2018 Article ID 5806179 9 pageshttpsdoiorg10115520185806179

2 BioMed Research International

the prosperity and economic development of the country asthe economic cost of malaria is estimated at more than 50millionUSDper year [4] Consequently inMadagascar thesemalaria control measures must be increased to achieve theglobal malaria action plan (GMAP) [5]

Madagascar deployed traditional vector control methodsas early as 1949 [6 7] The global malaria eradicationcampaign in the 1950s and 1960 which was based on thelarge-scale use of dichlorodiphenyltrichloroethane (DDT)was successful in Madagascar and there was a significantdrop in national malaria transmission from 1959 until theearly 1970s [8] Unfortunately these effortswere not sustainedand recurring malaria outbreaks were observed in the centralhighlands in the 1980s [9 10]The upsurge of the disease wasdue to several factors such as the abandonment of indoorresidual spraying (IRS) insufficient funding for malariacontrol the erosion of the public health system and the poorknowledge of the malaria vector biology (including that ofAnopheles funestus the main vector involved in the malariaepidemics in the central highlands) as well as the Anophelesresistance to insecticides [11ndash13]

The diversity of Madagascarrsquos ecosystems with 5 epi-demiological zones several vectors 2 major parasites (Pfalciparum and P vivax) and the changes they experienceas a result of human activities (eg the use of insecticidesfor agriculture and vector control) among others rendersthe malaria vector system extremely complex and constantlyevolving Today it is widely accepted that vector controlstrategies will only be effective in the long term if there is athorough knowledge of vector biology and particularly thestatus of insecticide resistance [14 15] The failure to takeinto account insecticide susceptibility of the potential vectorsinvolved in malaria transmission has contributed to failuresin malaria control throughout Africa [5]

The most effective way to prevent malaria transmissionis to avoid human-vector contact hence the importance ofvector control [5] However in many countries where vectorcontrol strategies including IRS and long lasting insecticidetreated bed nets (LLINs) have been implemented on alarge scale profound vector changes have been observedThese range from behavioral changes to insecticide resis-tance which greatly undermine the success of current vectorcontrol [16 17] This situation is all the more compellingwhen considering that Anopheles exhibits a strong resistanceto pyrethroids [5] the only insecticide recommended andapproved by the WHO for bed net impregnation [18] Inseveral countries resistance to pyrethroids has been reportedin major malaria vectors including An gambiae sl andAn funestus [19 20] This has become an almost universalproblem and may seriously impair the progress noticed infighting malaria [5]

In Madagascar IRS using DDT has been implementedsince the 1950s whereas LLINs were widely distributed inthe last decade [9 21] Malaria control still relies on the useof LLINs throughout the country and on IRS in the centralhighland area and in a small area on the eastern coast [22]

In malaria endemic regions mosquito resistance canincrease rapidly following the implementation of vectorcontrol [17] Therefore it is crucial to monitor the resistance

Figure 1 Map of Marovoay district (Madagascar) showing studyTsararano area

Figure 2 Photograph showing the vast rice fields at TsararanoMarovoay constituting potential larval breeding site for Anophelesfunestus

to insecticides particularly in countries where the policyof universal bed net coverage is applied Recent studiesdemonstrated the occurrence of the resistance to pyrethroidsand DDT in An arabiensis population in several districtsof Madagascar [23] However little information is availableon the status and insecticide resistance mechanisms in Anfunestus one of the main malaria vectors [24] In this paperwe report the insecticide susceptibility in wild An funestuspopulation fromTsararano and the involved resistancemech-anisms

2 Methods

21 Study Site This study was carried out in the village ofTsararano (S 16∘10101584042410158401015840 E 046∘40101584013610158401015840) located in the dis-trict ofMarovoay in the Boeny region approximately 540 kmnorthwest of Antananarivo the capital city of Madagascar(Figure 1) The village has about 1200 inhabitants It has atropical climate and rainfall occurs from October to AprilThe average annual temperature is 29∘C and the averagerainfall is 1500mm Tsararano is situated on themarshy bankof a permanent stream river the Betsiboka In this secondlargest rice granary region of Madagascar agriculture is themain activity (Figure 2) Anopheles larval sites are presentall year round and malaria is endemic No IRS has beenimplemented in the district since the 1950s however LLINswere distributed for the fifth round in December 2015

BioMed Research International 3

22 Mosquito Collection Blood fed female mosquitoes werecollected in the morning between 6 and 10 am from ten(10) households during the dry season in July 2017 usinga mouth aspirator and transferred at the insectary of CSB2Andriba platform After morphological identification [25]one hundred and eighty-six (186) blood fed An funestuswere individually transferred into a tube (Eppendorf) forforced oviposition according to the method described byNepomichene et al [26] In brief female mosquitoes weremaintained at 28∘C in 75-80 relative humidity with freeaccess to 10 sucrose until they became fully gravid (aboutfour days) and individually placed inside a tube with a filterpaper placed at the bottom Mosquitoes were introducedinside the tube one by one from within the cage Ovipositedeggs from each female were counted and pooled and weresubsequently placed into a rearing pan containing well-water Pooled larvae of different stages L1 L2 L3 andL4 were fed daily with Tetramin baby fish food Pupaewere collected and placed in 2-L plastic buckets whichwere covered with mosquito gauze with a cotton sleevefor introducing 10 glucose on filter paper and allowed toemerge locally Unfed 2-5-day females on first-generationAn funestus (F1) were used for insecticide susceptibilitybioassay

23 Susceptibility Assays Insecticide susceptibility bioas-says following WHO protocol [27] were done on F1 Anfunestus generation at the insectary of Andriba platformSix insecticides of technical grade quality were tested twopyrethroids (permethrin 075 deltamethrin 005 ) twoorganophosphates (malathion 5 fenitrothion 1) oneorganochlorine (DDT 4) and one carbamate (bendiocarb01) Impregnated papers were obtained from the WHOreference center (Vector Control Research Unit UniversitySains Malaysia Penang Malaysia) Tests were performedwith batches of 25 unfed females aged 2-5 days fourfoldwith each insecticide Mosquitoes were exposed for 1 hour at28∘C and 80 relative humidity After exposure mosquitoeswere kept in observation tubes and supplied with a 10sugar solution The number of knockdown (KD) mosquitoeswas recorded at 10 15 20 30 40 50 and 60min Mortalityrate (MR) was recorded after 24 hours Two batches of 25females exposed to untreated papers were used as negativecontrol All batches of insecticide paper used were pretestedon the strain of An arabiensis from the Institut Pasteurde Madagascar known to be susceptible to all insecticides[28]

24 PBO Synergist Assays In the event of confirmed insecti-cides resistance additional tests were conducted by using thesynergist piperonyl butoxide (PBO) an inhibitor of oxidasesThree combinations were used PBO + deltamethrin PBO +permethrin and PBO + bendiocarb For each combinationtwo exposed batches and one negative control batch exposedonly to PBO were used Each batch was constituted by 25unfed female mosquitoes aged 2-5 days old Mosquitoeswere first exposed for one hour to 4 PBO and second toimpregnated papers KD mosquitoes and mortality rate wererecorded as described above

25 Biochemical Enzyme Assays A subset of F1 An funestusgeneration (nonexposed to insecticide) stored at -80∘C untiluse was used for biochemical enzyme assays Enzymaticactivities of cytochrome P450 esterases (120572 and 120573) andglutathione S- transferases (GST) were measured accordingthe protocol described by Brogdon et al [29] and byHemingway et al [30] and slightly modified by Sangba et al[31] Absorbance was measured using a spectrophotometertype ldquoMultiskan GO and SkanIt SoftwareSerial number 1510-05171rdquo (httpswwwthermofishercomusenhomelife-sci-encelab-plasticware-supplieslab-plasticware-supplies-learn-ing-centerlab-plasticware-supplies-resource-libraryskanIt-software-protocols-multiskan-gohtml) In the absence ofAn funestus susceptible strain the An arabiensis strain fromthe Institut Pasteur de Madagascar known to be susceptibleto all insecticides was used as control [28] All experimentswere conducted at the laboratory of the Institut Pasteur deMadagascar

26 Species Molecular Identification DNA was extractedfrom all F0 individual mosquitoes by DNAzol (InvitrogenCA USA) according to the manufacturerrsquos recommenda-tions Anophelesmosquitoes were identified by the PCRusingthe method described by Santolamazza et al forAn gambiaeand the technique described byWilkins et al forAn funestus[32] The PCR for An gambiae sl discrimination is basedon the amplification of a ribosomal DNA fragment smalleramplified by PCR by using the following primers IGS441(F) [TGG TCT GGG GAC CAC GTC GAC ACA GG]IGS783 (R) [CGT TTC TCA CAT CAA GAC AAT CAAGTC] while the PCR forAn funestus discrimination is basedon species-specific single nucleotide polymorphism (SNPs)in the second internal transcribed spacer region (ITS2) byusing seven primers UV (F) [CCG ATG CAC ACA TTCTTG AGT GCC TA] FUN (R) [CTC GGG CAT CGA TGGGTT AAT CAT G] VAN (R) [AAC TCT GTC GAC TTGGTA GCC GAA C] RIV (R) [AAT CAG GGT CGA ACGGCT TGC CG] PAR (R) [GCC CTG CGG TCC CAA GCTAGA TT] RIVLIKE (R) [CTC CCG TGG AGT GGG GGATC] LEES (R) [GAC GGC ATC ATG GCG AGC AGC] Allprimers were provided by the Center for Diseases Control(CDC) and the experiments were done at the laboratory ofInstitut Pasteur de Madagascar

27 Data Analysis For each insecticide the percentage of KDandMRwas determined Since mortality in negative controlswas always under 5 no adjustment was performed fortreated batches TheWHO 2017 criteria were used to evaluatethe status of insecticide resistance (ie 98-100 mortalityindicates susceptibility and lt98 mortality indicates thatfurther investigation is required to confirm resistance) [27]Fifty and ninety-five percent KD times (KDT

50and KDT

95

respectively) were computed with probit regression modelsMortality rates expressed in () and 95 confidence inter-val were compared using Fisher exact test The enzymaticactivities of wild An funestus were compared with that of theAn arabiensis strain from the Institut Pasteur de Madagascarknown to be susceptible to all insecticides by Mann-Whitneytest Statistical analyses were performed using GraphPad


Table 1 Anophelines collected in Tsararano by indoor aspirator and molecular identification by PCR and circumsporozoite protein rate(CSP) Mosquitoes were captured in July 2017 during the dry season in ten (10) households at early morning between 6 and 10 am

Morphological identificationMethods Anopheles funestus sl Anopheles gambiae slIndoor aspirator mosquito catch 254 57

molecular identificationPCR species Anopheles funestus ss Anopheles arabiensis Anopheles gambiae

254 55 2Plasmodium falciparum circumsporozoite protein rate

Number of Positive ELISA 3 1 0CSP rate () 12 18 0

Table 2 Mortality rate (MR) 24 hours after exposition obtained after WHO bioassay first-generation An funestus population (F1) fromTsararanoMarovoay andknockdown time 50 (KDT

50) andknockdown time 95 (KDT

95)Mortality rate representsmeanwith 95confidence

intervals (CI) na not applicable

Mortality rate () Knockdown time 50 Knockdown time 95MR (n) 95CI KDT50 95CI KDT95 95CI

Permethrin 075 336 (101) [245-437] 7379 [704-771] na -Deltamethrin 005 39 (100) [294-492] 725 [681-769] na -Bendiocarb 01 86(100) [776-921] 381 [364-397] na -DDT 4 99 (101) - 344 [324-364] na -Malathion 5 100 (99) - 289 [256-322] 390 [369-420]Fenitrothion 1 100 (102) - 261 [227-296] 487 [447-527]

Prism software v50 (wwwgraphpadcom) A P value of 005or less was considered as significant

3 Results

31 Wild Anopheles Collections In July 2017 a total of311 malaria vectors were collected from 10 households inTsararano Among those 254 (817) were An funestus and57 (183)An gambiae sl (Table 1)Most of thesemosquitoeswere blood-fed From the 186 oviposition tubes set up withindividual gravid An funestus females 175 produced eggbatches The total number of eggs collected was 12011averaging 6457 eggs per female

32 Molecular Identifications and Infection Rates All F0 Anfunestus group mosquitoes (n=254) tested by PCR showedthat they all belonged to An funestus ss Of the 57 Angambiae sl tested 965 (n=55) were identified as Anarabiensis and 35 (n=2) as An gambiae (formerly Smolecular form) (Table 1)

The Plasmodium falciparum circumsporozoite protein(CSP) rate was determined by ELISA-CSP Of the 311Anophe-les specimens tested by ELISA-CSP 4 were positive for theCSP antigen resulting in a mean CSP rate of 13 (95CI035ndash32) with 12 and 18 for An funestus and Angambiae sl respectively (1205942 = 012 df = 1 P = 07)

33 Susceptibility of Anopheles funestus to Insecticides Table 2summarizes the MR of An funestus population induced byall insecticides Following 24 h observation MRs indicatedhigh resistance to pyrethroids and bendiocarb (Figure 3and Table 2) The mean MR induced by permethrin and

deltamethrin in the wild An funestus population was 336(95CI 245ndash437) and 39 (95CI 294ndash492) respec-tively No significant difference of theMRwas observed in thepyrethroid group (1205942 = 061 df = 1 P= 04) The mean MRinduced by bendiocarb in the wild An funestus populationwas 86 (95 CI 771 ndash 921)

An funestus was 100 susceptible to DDT andorganophosphate (malathion and fenitrothion) (Figure 3 andTable 2) Furthermore 100 of mosquitoes were knockdownafter 60min exposure to malathion fenitrothion and DDTwith a short KDT

50time lt289min (95CI 257-322)

(Table 2) For pyrethroids the KDT50

was much longer(higher than 72min) (Table 2) Meanwhile a low KDT

50was

observed after bendiocarb exposure despite the resistance(Table 2)

34 Effects of Synergist PBO on Anopheles funestus Resistant toPyrethroids and Bendiocarb For both pyrethroids and ben-diocarb preexposure to PBO synergist significantly increasedthe MR (Figure 3) The mean MR of deltamethrin increasedfrom 39 (95CI 294ndash492) before PBO exposure to 96(95CI 90ndash989) after PBO exposure TheMR induced bypermethrin increased from 336 before PBO exposure to100 after PBO exposure A significant difference of MR wasobserved in pyrethroid group before and after PBO exposure(1205942 = 43 df = 3 Plt 00001) Concurrently a full susceptibilitywas observed to bendiocarb after PBO exposure (MR=100)(Figure 3)

35 Biochemical Enzymatic Activities of Cytochrome P450Esterases and GST Biochemical data of enzymatic activities


0

20

40

60

80

100

Deltam

ethrin 005

Permethrin 075

Bendiocarb 01

DD

T 4

Malathion 5

Fenitrothion 1

PBO not tested

Insecticide alonePBO + Insecticide

Mor

talit

y rat

e

Figure 3 Insecticide bioassay mortality rate in wild Anophelesfunestus first generation (F1) from Tsararano Marovoay (Madagas-car) 24 hours after exposure to permethrin (075) deltamethrin(005) DDT (4) malathion (5) fenitrothion (1) and ben-diocarb (01) and effect of piperonyl butoxide (PBO) The datarepresent medians

of cytochrome P450 GST and esterases (120572 and 120573) comparedto the IPM susceptible strain are shown in Figure 4 Highlevel enzymatic activities of cytochrome P450 compared tothe susceptible IPM reference strain were observed (Mann-Whitney U= 30 Z = -651 Plt00001) (Figure 4(a)) Incontrast theGST activitywas significantly lower compared tothe IPMreference strain (Mann-WhitneyU= 1955 Z = -433Plt00001) (Figure 4(b)) suggesting the no implication ofGSTin the insecticide resistance of An funestus from Tsararano

Meanwhile a significant increased 120572-esterase activity wasobserved in the An funestus population compared to theIPM susceptible strain (Mann-WhitneyU= 1455 Z = -499Plt00001) (Figure 4(c)) In contrast no significant 120573-esteraseactivity was observed in the An funestus populations fromTsararano compared to the IPM strain (Mann-Whitney U =3925 Z = -187 P = 008) (Figure 4(d))

4 Discussion

This study conducted in Tsararano reports the levels ofinsecticide susceptibility of An funestus a main malariavector in Madagascar to the different families of insecticidesconventionally used in vector control in the country

Our study showed that An funestus is the only species ofthe funestus group present in the study area with a moderateindex circumsporozoite rate which was considerably smallerthan that obtained by Marrama et al [13] and Fontenille etal [11] in other parts of Madagascar However in many sub-Saharan Africa regions entomological inoculation rate (EIR)ofAn funestus surpasses that ofAn gambiae indicating theirmajor role in malaria transmission throughout the continent[17 33]

The study provides substantial knowledge on the resis-tance status of the wild An funestus population The resultsof insecticide bioassay trials showed a high biological level

of resistance to pyrethroid groups (deltamethrin and perme-thrin) and bendiocarb Similar resistance has been observedin southern east Africa particularly in Mozambique [34]but in Madagascar little information on An funestus isavailable in contrast to An gambiae [23 24] Furthermoreinvestigations on the biological insecticide susceptibility per-formed in sentinel sites of Madagascar in 2015 reported aresistance to pyrethroids and bendiocarb in 2037 and 824sites respectively for An gambiae Meanwhile resistanceto the pyrethroid group and bendiocarb was observed onlyin 15 and 13 sites respectively for An funestus includingin the district of Marovoay [24] However previous studiesconducted in the Central Island of Madagascar by Rako-tondraibe et al [35] and Ratovonjato et al [36] showed a fullsusceptibility to pyrethroids and DDT in wild blood feed andsemigravid An funestus population (F0) in contrast to ourstudy where F1 progeny was used according to the WHOprotocol [27] This susceptibility difference may be due byseveral factors including the intensive uses of pesticides inTsararano rice fields before 1950s [37] and also the age andphysiological status of mosquitoes (ie blood fed andorsemigravid) For insistence it has been demonstrated thatolder mosquitoes are sometimes less resistant to insecticidesespecially when resistance is conferred by the presence of adetoxifying enzyme the activity of which may decline withage [38]

On the other hand previous studies have shown that theoccurrence of theAnopheles resistance to insecticides is partlyexplained by the use of pesticides in agricultural activitiesand vector control to combat malaria [39 40] Given thatMarovoay is the second largest rice granary in MadagascarTsararano is surrounded by rice fields where insecticidesare used for pest control for an extended period of timethroughout the year Up to today no IRS has been performedin Tsararano as part of the national vector control strategyWe hypothesize that the increased resistance of An funestusto pyrethroids and bendiocarb observed in Tsararano mightbe due to the use of pesticides in agriculture Neverthelessthe causes of such widespread resistance remain unknownHowever we believe LLINs would have a limited effect inspreading resistance in this region as described elsewhere[41]

The long-term efficacy of LLINs in reducing malaria mor-bidity has recently been questioned in western Africa [17]In Madagascar 2761550 insecticide-treated nets were dis-tributed for free to the population in 2016 [42] Neverthelessa full susceptibility to organophosphates has been observed inAn funestus population therefore these insecticides shouldbe used in priority if IRS should be performed in this area

The presence or absence of kdr mutation in An funestuspopulation from Tsararano was not investigated Severalstudies have shown the absence of this mutation in Anfunestus populations [41 43] unlike in An gambiae wherethe widespread distribution of kdr-w and kdr-e has beenobserved in many African regions [5 44 45] In addition toour knowledge no kdrmutationwas observed inAn gambiaepopulations fromMadagascar [23]

Preexposure to PBO restored the full susceptibility topyrethroids and bendiocarb Similar observations have been


025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450

An arabiensis An funestus

POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein

An arabiensis An funestusPOPULATIONS

05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)

Figure 4 Detoxifying enzyme activities (enzymatic activity permg of protein) of wildAnopheles funestus first generation (F1) fromTsararanoMadagascar in comparison with Anopheles arabiensis Institut Pasteur of Madagascar susceptible strain (a) Cytochrome P450 activities(MFO) (b) Glutathione S-transferases activities (GST) (c) Alpha esterase activities (d) Beta esterase activities Red lines represent meanswith 95 confidence intervals (blue lines)

made in Mozambique [34 46] Biochemical enzymaticactivity data showed that cytochrome P450 activity wassignificantly higher in An funestus population than the IPMstrain indicating that the pyrethroid resistance in Tsararanois driven by the cytochrome P450 genes as previouslyobserved inMozambique and in theCentral AfricanRepublic[34 46] In addition we hypothesize that overexpression ofcytochrome P450s also confers a cross-resistance to carba-mates This situation is particularly likely if the pyrethroidresistance results from a change in a P450 regulatory regionrather than amutation in a single P450 structural gene whichcould produce cross-resistance to several insecticide classesincluding carbamates as previously observed inMozambique[34] Interestingly previous studies revealed that pyrethroidresistance in An funestus population is mainly driven by keycytochrome P450s genes such as CYP6P9a CYP6P9b andCYP6M7 [47 48] However the gene expression pattern ofthe detoxification linked to this cross-resistance is not wellunderstood

Furthermore a significant overexpression activity of120572-esterase was observed in An funestus from Tsararano

However no difference in120573-esterase activity was observed Incontrast GST activity was significantly lower This confirmsthe susceptibility of An funestus from Tsararano to DDT Infact the evidence of resistance to the two major classes ofinsecticides (pyrethroids and bendiocarb) in the An funestuspopulation from Tsararano may constitute a potential threatto the success of the malaria vector control programmein this region In addition given the wide distribution ofAn funestus in Madagascar further investigation in otherdistricts through the exploration of resistance profile isneeded This will provide information to elaborate adequatevector resistance management

5 Conclusions

Our study showed evidence of resistance to pyrethroidsand bendiocarb in an An funestus population Enzymaticactivities data indicated implication of cytochrome P450genes in the resistance of An funestus with a suspectedcross-resistance between pyrethroids and carbamate Thecoexistence of this cross-resistance in An funestus where


confirmed constitutes a serious concern for the future suc-cess of malaria control programme

Data Availability

The mortality rate data used to support the findings of thisstudy are included within the article and the enzymaticactivities data used to support the findings of this study areavailable from the corresponding author upon request

Ethical Approval

This study is a part of G4 project approved by the nationalethics committee in Madagascar (authorization no 122-MSANPCE-2015)

Disclosure

The funders had no role in study design data colloca-tion or analysis decision to publish or preparation of themanuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Mamadou Ousmane Ndiath conceived planned designedand supervised the study Tsiriniaina Rakotondranaivo andMihajarilala Rakotoniaina Tanjona collected identified andperformed susceptibility mosquitoes test Tsiriniaina Rako-tondranaivo Solohery Fanomezana Randriamanarivo andMihajarilala Rakotoniaina Tanjona performed laboratorywork Ines Vigan-Womas and Milijaona Randrianarivelo-josia provided technical logistical and scientific supportMamadou Ousmane Ndiath performed data analysis inter-preted the results and drafted themanuscriptMilijaonaRan-drianarivelojosia Ines Vigan-Womas and Mamadou Ous-mane Ndiath made the critical revision of the manuscript forimportant intellectual content All authors read and approvedthe final manuscript

Acknowledgments

We are grateful to Mrs Ann Allen and Dr Astrid Knoblauchwho reviewed previous versions of this manuscript We alsothank Dr Paul Howell and Dr Melissa Avery from CDC-Atlanta for CSP antibodies PCR primers and protocols Wealso thank the personnel of Unite drsquoEntomologie Medicale atInstitut Pasteur de Madagascar and the Tsararano villagersfor their cooperation throughout the study This work wassupported by theDepartment of InternationalAffairs InstitutPasteur International Network (G4 Group) awarded byOusmane NDIATH

References

[1] ldquoWorld Malaria Report 2016 Document WHOHTMGMP20162 Geneva Switzerland World Health Organizationrdquo

httpwwwwhointmalariapublicationsworld-malaria-re-port-2016en Accessed Dec 2016

[2] T Kesteman S A Rafalimanantsoa H Razafimandimby et alldquoMultiple causes of an unexpected malaria outbreak in a high-transmission area in Madagascarrdquo Malaria Journal vol 15 no1 2016

[3] T Kesteman M Randrianarivelojosia C Mattern et alldquoNationwide evaluation of malaria infections morbidity mor-tality and coverage of malaria control interventions in Mada-gascarrdquoMalaria Journal vol 13 no 1 article 465 2014

[4] ldquoRapport DLP 2016 Enquete sur les indicateurs du paludisme aMadagascarrdquo httpsdhsprogramcompubspdfMIS23MIS23pdf Accessed Dec 2016

[5] H Ranson and N Lissenden ldquoInsecticide resistance in africananopheles mosquitoes a worsening situation that needs urgentaction to maintain malaria controlrdquo Trends in Parasitology vol32 no 3 pp 187ndash196 2016

[6] D Randriantsimaniry ldquoVector control in the epidemics of theMadagascar highlandsrdquo Sante vol 5 no 6 pp 392ndash396 1995

[7] J Hamon J Mouchet G Chauvet and R Lumaret ldquoReviewof 14 years of malaria control in the french-speaking countriesof tropical africa and in madagascar considerations on thepersistence of transmission and future prospectsrdquo Bulletin de laSociete de Pathologie Exotique et de ses Filiales vol 56 pp 933ndash971 1963

[8] S Mercier and J B Razafindrakoto ldquoResults of 3 yearsrsquo cam-paigning of disinsectization of homes in Tanariverdquo Bulletin dela Societe de Pathologie Exotique et de ses Filiales vol 46 pp463ndash473 1953

[9] M Razanamparany A Bosman N Ravelomanana F Ran-driamiharisoa and L Razafimantsoa ldquoThe malaria epidemicin Antananarivo observed from Pediatric Service ldquoArdquo of theBefelatananaGeneralHospitalrdquo Parassitologia vol 31 no 1 pp89ndash99 1989

[10] M S Razanamparany F A Randriamiharisoa N J Razanam-parany and V Ramialimanana ldquoThe malaria epidemic inAntananarivo from 1983 to 1994 as seen through the PediatricService A in the Befelatanana General Hospitalrdquo Sante vol 5no 6 pp 382ndash385 1995

[11] D Fontenille and I Rakotoarivony ldquoReappearance of anophelesfunestus as a malaria vector in the antananarivo region Mada-gascarrdquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 82 no 4 pp 644-645 1988

[12] J P Lepers D Fontenille M D Andriamangatiana-Rason PDeloron and P Coulanges ldquoEcological factors in the renewedoutbreak of malaria in Madagascarrdquo Bulletin de Societe dePathologie Exotique vol 83 pp 330ndash341 1990

[13] L Marrama E Rajaonarivelo S Laventure and P RabarisonldquoAnopheles funestus and rice culture on the Plateau of Mada-gascarrdquo CahiersSante vol 5 no 6 pp 415ndash419 1995

[14] D Fontenille and F Simard ldquoUnravelling complexities inhuman malaria transmission dynamics in Africa through acomprehensive knowledge of vector populationsrdquo ComparativeImmunology Microbiology amp Infectious Diseases vol 27 no 5pp 357ndash375 2004

[15] B Greenwood and K Koram ldquoMalaria control in AfricaProgress but still much to dordquo The Lancet vol 383 no 9930pp 1703-1704 2014

[16] S Sougoufara S M Diedhiou S Doucoure et al ldquoBiting byAnopheles funestus in broad daylight after use of long-lastinginsecticidal nets A new challenge to malaria eliminationrdquoMalaria Journal vol 13 no 1 article 125 2014


[17] J-F Trape A Tall N Diagne et al ldquoMalaria morbidity andpyrethroid resistance after the introduction of insecticide-treated bednets and artemisinin-based combination therapiesa longitudinal studyrdquo The Lancet Infectious Diseases vol 11 no12 pp 925ndash932 2011

[18] M Zaim A Aitio and N Nakashima ldquoSafety of pyrethroid-treatedmosquito netsrdquoMedical andVeterinary Entomology vol14 no 1 pp 1ndash5 2000

[19] J Hemingway H Ranson A Magill et al ldquoAverting a malariadisaster Will insecticide resistance derail malaria controlrdquoTheLancet vol 387 no 10029 pp 1785ndash1788 2016

[20] H Ranson R Nrsquoguessan J Lines N Moiroux Z Nkuniand V Corbel ldquoPyrethroid resistance in African anophelinemosquitoes what are the implications for malaria controlrdquoTrends in Parasitology vol 27 no 2 pp 91ndash98 2011

[21] J RatovonjatoM Randrianarivelojosia M E Rakotondrainibeet al ldquoEntomological and parasitological impacts of indoorresidual spraying with DDT alphacypermethrin anddeltamethrin in the western foothill area of MadagascarrdquoMalaria Journal vol 13 no 1 2014

[22] ldquoRapport DLP 2017 Plan strategique national de lutte contrele paludisme a Madagascar 2018-2022rdquo httpswwwpmigovdocsdefault-sourcedefault-document-librarymalaria-opera-tional-plansfy17fy-2017-madagascar-malaria-operational-planpdfsfvrsn=6f Accessed Nov 2017

[23] J-D Rakotoson C M Fornadel A Belemvire et al ldquoInsec-ticide resistance status of three malaria vectors Anophelesgambiae (sl) An funestus and An mascarensis from thesouth central and east coasts of Madagascarrdquo Parasites ampVectors vol 10 no 1 2017

[24] ldquoPMI Report 2016 PresidentrsquosMalaria Initiative Country Insec-ticide Susceptibility Summariesrdquo August 2016 httpswwwpmigovdocsdefault-sourcedefault-document-librarytools-curriculapmi-insecticide-susceptibility-summary-august-2016pdfsfvrsn=8 Accessed Nov 2016

[25] M T Gillies and D De Meillon ldquoThe Anophelinae of AfricaSouth of the Saharardquo Publications of the South African Institutefor Medical Research vol 54 p 343 1968

[26] T N Nepomichene L Andrianaivolambo S Boyer and CBourgouin ldquoEfficient method for establishing F1 progeny fromwild populations of Anopheles mosquitoesrdquo Malaria Journalvol 16 no 1 article 21 2017

[27] ldquoTest procedures for insecticide resistance monitoring inmalaria vector mosquitoesrdquo WHO CDSCPCMALD edi-tor 2016 2nd edGeneva Switzerland World Health Organi-zation httpappswhointirishandle10665250677 AccessedNov 2016

[28] S Randriamaherijaona H J Velonirina and S Boyer ldquoSus-ceptibility status of Anopheles arabiensis (Diptera Culicidae)commonly used as biological materials for evaluations ofmalaria vector control tools in Madagascarrdquo Malaria Journalvol 15 no 1 article no 338 2016

[29] WG Brogdon J CMcAllister and J Vulule ldquoHeme peroxidaseactivity measured in single mosquitoes identifies individualsexpressing an elevated oxidase for insecticide resistancerdquo Jour-nal of the American Mosquito Control Association vol 13 no 3pp 233ndash237 1997

[30] J Hemingway andWG Brogdon ldquoTechniques toDetect Insec-ticide Resistance Mechanisms (Field and Laboratory Manual)rdquo1998

[31] M L O Sangba T Deketramete S P Wango M Kazanji MAkogbeto andMONdiath ldquoInsecticide resistance status of the

Anopheles funestus population in Central African Republic Achallenge in the warrdquo Parasites amp Vectors vol 9 no 1 2016

[32] M LDustin ldquoVisualizing Immune SystemComplexityrdquo ScienceSignaling vol 2 no 66 pp mr4ndashmr4 2009

[33] A Cohuet F Simard J-C Toto P Kengne M Coetzee andD Fontenille ldquoSpecies identification within the Anophelesfunestus group of malaria vectors in Cameroon and evidencefor a new speciesrdquo The American Journal of Tropical Medicineand Hygiene vol 69 no 2 pp 200ndash205 2003

[34] N Cuamba J CMorganH Irving A Steven andC SWondjildquoHigh level of pyrethroid resistance in an Anopheles funestuspopulation of the chokwe district in mozambiquerdquo PLoS ONEvol 5 no 6 2010

[35] M E Rakotondraibe G Le Goff E Rajaonarivelo et al ldquoInsec-ticide sensitivity in malaria vectors in high altitude Madagascarafter five years of vector controlrdquoArchives de lrsquoInstitut Pasteur deMadagascar vol 66 no 1-2 pp 32ndash35 2000

[36] J Ratovonjato G Le Goff E Rajaonarivelo E M Rako-tondraibe and V Robert ldquoRecent observations on the sen-sitivity to pyrethroids and DDT of Anopheles arabiensis andAnopheles funestus in the central Highlands of Madagascarpreliminary results on the absence of the kdr mutation in Anarabiensisrdquo Archives de lrsquoInstitut Pasteur de Madagascar vol 69no 1-2 pp 63ndash69 2003

[37] ldquoLes irrigations en 1925 dans la plaine de Marovoayrdquo in Extraitdu bulletin economique de Madagascar 23e Annee No 1 Servicede lrsquohydraulique et des Ameliorations Agricoles 1926

[38] J D Lines and N S Nassor ldquoDDT resistance in Anophelesgambiae declines with mosquito agerdquo Medical and VeterinaryEntomology vol 5 no 3 pp 261ndash265 1991

[39] C Czeher R Labbo I Arzika and J-B Duchemin ldquoEvi-dence of increasing Leu-Phe knockdown resistance mutationin Anopheles gambiae from Niger following a nationwidelong-lasting insecticide-treated nets implementationrdquo MalariaJournal vol 7 article no 189 2008

[40] J M Vulule R F Beach F K Atieli J M Roberts DL Mount and R W Mwangi ldquoReduced susceptibility ofAnopheles gambiae to permethrin associated with the useof permethrin-impregnated bednets and curtains in KenyardquoMedical andVeterinary Entomology vol 8 no 1 pp 71ndash75 1994

[41] C Mulamba J M Riveron S S Ibrahim et al ldquoWidespreadpyrethroid and DDT resistance in the major malaria vectoranopheles funestus in East Africa is driven by metabolicresistance mechanismsrdquo PLoS ONE vol 9 no 10 2014

[42] ldquoNet Mapping Projet Raport 2017rdquo httpnetmappingprojectallianceformalariapreventioncomdatatitle selective=Allampre-gion selective=Abiaampyear selective=Allampfield non endemicvalue=0 Accessed Dec 2016

[43] R Djouaka H Irving Z Tukur and C S Wondji ldquoExploringmechanisms of multiple insecticide resistance in a populationof the malaria vector Anopheles funestus in Beninrdquo PLoS ONEvol 6 no 11 2011

[44] MO Ndiath A Cailleau E Orlandi-Pradines et al ldquoEmergingknock-down resistance in Anopheles arabiensis populations ofDakar Senegal First evidence of a high prevalence of kdr-emutation in West African urban areardquoMalaria Journal vol 14no 1 2015

[45] M O Ndiath K Eiglmeier M L Ole Sangba I Holm MKazanji and K D Vernick ldquoComposition and genetics ofmalaria vector populations in the Central African RepublicrdquoMalaria Journal vol 15 no 1 article 387 2016


[46] R G Kloke E Nhamahanga R H Hunt and M CoetzeeldquoVectorial status and insecticide resistance of Anopheles funes-tus from a sugar estate in southern Mozambiquerdquo Parasites ampVectors vol 4 no 1 article 16 2011

[47] J M Riveron S S Ibrahim E Chanda et al ldquoThe highlypolymorphicCYP6M7cytochromeP450 gene partnerswith thedirectionally selected CYP6P9a and CYP6P9b genes to expandthe pyrethroid resistance front in the malaria vector Anophelesfunestus in Africardquo BMC Genomics vol 15 no 1 article 8172014

[48] J M Riveron H Irving M Ndula et al ldquoDirectionally selectedcytochrome P450 alleles are driving the spread of pyrethroidresistance in the major malaria vector Anopheles funestusrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 110 no 1 pp 252ndash257 2013

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


the prosperity and economic development of the country asthe economic cost of malaria is estimated at more than 50millionUSDper year [4] Consequently inMadagascar thesemalaria control measures must be increased to achieve theglobal malaria action plan (GMAP) [5]

Madagascar deployed traditional vector control methodsas early as 1949 [6 7] The global malaria eradicationcampaign in the 1950s and 1960 which was based on thelarge-scale use of dichlorodiphenyltrichloroethane (DDT)was successful in Madagascar and there was a significantdrop in national malaria transmission from 1959 until theearly 1970s [8] Unfortunately these effortswere not sustainedand recurring malaria outbreaks were observed in the centralhighlands in the 1980s [9 10]The upsurge of the disease wasdue to several factors such as the abandonment of indoorresidual spraying (IRS) insufficient funding for malariacontrol the erosion of the public health system and the poorknowledge of the malaria vector biology (including that ofAnopheles funestus the main vector involved in the malariaepidemics in the central highlands) as well as the Anophelesresistance to insecticides [11ndash13]

The diversity of Madagascarrsquos ecosystems with 5 epi-demiological zones several vectors 2 major parasites (Pfalciparum and P vivax) and the changes they experienceas a result of human activities (eg the use of insecticidesfor agriculture and vector control) among others rendersthe malaria vector system extremely complex and constantlyevolving Today it is widely accepted that vector controlstrategies will only be effective in the long term if there is athorough knowledge of vector biology and particularly thestatus of insecticide resistance [14 15] The failure to takeinto account insecticide susceptibility of the potential vectorsinvolved in malaria transmission has contributed to failuresin malaria control throughout Africa [5]

The most effective way to prevent malaria transmissionis to avoid human-vector contact hence the importance ofvector control [5] However in many countries where vectorcontrol strategies including IRS and long lasting insecticidetreated bed nets (LLINs) have been implemented on alarge scale profound vector changes have been observedThese range from behavioral changes to insecticide resis-tance which greatly undermine the success of current vectorcontrol [16 17] This situation is all the more compellingwhen considering that Anopheles exhibits a strong resistanceto pyrethroids [5] the only insecticide recommended andapproved by the WHO for bed net impregnation [18] Inseveral countries resistance to pyrethroids has been reportedin major malaria vectors including An gambiae sl andAn funestus [19 20] This has become an almost universalproblem and may seriously impair the progress noticed infighting malaria [5]

In Madagascar IRS using DDT has been implementedsince the 1950s whereas LLINs were widely distributed inthe last decade [9 21] Malaria control still relies on the useof LLINs throughout the country and on IRS in the centralhighland area and in a small area on the eastern coast [22]

In malaria endemic regions mosquito resistance canincrease rapidly following the implementation of vectorcontrol [17] Therefore it is crucial to monitor the resistance

Figure 1 Map of Marovoay district (Madagascar) showing studyTsararano area

Figure 2 Photograph showing the vast rice fields at TsararanoMarovoay constituting potential larval breeding site for Anophelesfunestus

to insecticides particularly in countries where the policyof universal bed net coverage is applied Recent studiesdemonstrated the occurrence of the resistance to pyrethroidsand DDT in An arabiensis population in several districtsof Madagascar [23] However little information is availableon the status and insecticide resistance mechanisms in Anfunestus one of the main malaria vectors [24] In this paperwe report the insecticide susceptibility in wild An funestuspopulation fromTsararano and the involved resistancemech-anisms

2 Methods

21 Study Site This study was carried out in the village ofTsararano (S 16∘10101584042410158401015840 E 046∘40101584013610158401015840) located in the dis-trict ofMarovoay in the Boeny region approximately 540 kmnorthwest of Antananarivo the capital city of Madagascar(Figure 1) The village has about 1200 inhabitants It has atropical climate and rainfall occurs from October to AprilThe average annual temperature is 29∘C and the averagerainfall is 1500mm Tsararano is situated on themarshy bankof a permanent stream river the Betsiboka In this secondlargest rice granary region of Madagascar agriculture is themain activity (Figure 2) Anopheles larval sites are presentall year round and malaria is endemic No IRS has beenimplemented in the district since the 1950s however LLINswere distributed for the fifth round in December 2015








50and KDT

95















3 Results







50time lt289min (95CI 257-322)



50was





0

20

40

60

80

100

Deltam

ethrin 005

Permethrin 075

Bendiocarb 01

DD

T 4

Malathion 5

Fenitrothion 1

PBO not tested


Mor

talit

y rat

e




4 Discussion










025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450


POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein


05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)





5 Conclusions




Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























50and KDT

95















3 Results







50time lt289min (95CI 257-322)



50was





0

20

40

60

80

100

Deltam

ethrin 005

Permethrin 075

Bendiocarb 01

DD

T 4

Malathion 5

Fenitrothion 1

PBO not tested


Mor

talit

y rat

e




4 Discussion










025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450


POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein


05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)





5 Conclusions




Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
































3 Results







50time lt289min (95CI 257-322)



50was





0

20

40

60

80

100

Deltam

ethrin 005

Permethrin 075

Bendiocarb 01

DD

T 4

Malathion 5

Fenitrothion 1

PBO not tested


Mor

talit

y rat

e




4 Discussion










025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450


POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein


05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)





5 Conclusions




Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

20

40

60

80

100

Deltam

ethrin 005

Permethrin 075

Bendiocarb 01

DD

T 4

Malathion 5

Fenitrothion 1

PBO not tested


Mor

talit

y rat

e




4 Discussion










025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450


POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein


05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)





5 Conclusions




Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















025

020

015

010

005

000

nmol

GSH

conj

m

in

mg

prot

ein

Cytochrome P450


POPULATIONS(a)

nmol

GSH

conj

m

in

mg

prot

ein


05

04

03

02

01

00

GST

(b)


03

02

01

00

m

ol

Na

min

mg

prot

ein

Esterase Alpha

(c)


020

015

010

005

000

m

ol

Na

min

mg

prot

ein

Esterase Beta

(d)





5 Conclusions




Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Data Availability


Ethical Approval


Disclosure






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















evidence of insecticide resistance to pyrethroids and bendiocarb...

Documents