Research ArticleInsecticides Resistance Status of An gambiae in Areas ofVarying Agrochemical Use in Cocircte DrsquoIvoire

Behi K Fodjo 12 Benjamin G Koudou123 Emmanuel Tia4

Jasmina Saric156 Prisca B Nrsquodri156 Marius G Zoh47 Christabelle S Gba12

Alida Kropf7 Nestor B Kesse1 and Mouhamadou S Chouabou1

1Centre Suisse de Recherche Scientifique en Cote drsquoIvoire (CSRS) Abidjan Cote drsquoIvoire2Universite Nangui Abrogoua Abidjan Cote drsquoIvoire3Liverpool School of Tropical Medicine Liverpool UK4Centre drsquoEntomologie Medicale et Veterinaire Universite Alassane Ouattara Bouake Cote drsquoIvoire5Swiss Tropical and Public Health Institute PO Box CH-4002 Basel Switzerland6University of Basel PO Box CH-4003 Basel Switzerland7Universite de Montpellier Montpellier France

Correspondence should be addressed to Behi K Fodjo behikouadiogmailcom

Received 15 February 2018 Revised 3 July 2018 Accepted 6 September 2018 Published 8 October 2018

Academic Editor Aparup Das

Copyright copy 2018 Behi K Fodjo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Insecticide resistance monitoring of the malaria vectors to different classes of insecticides is necessary for resistancemanagement Malaria vector control management approaches are essentially based on IRS and LLINs However insecticideresistance is caused by several sources of selection and in case the selection pressure is from agricultural practices then measuresneed to be taken to avoid a failure of the controlmethodsput in placeThe current studywasundertaken tomonitor the susceptibilityof vectors to different classes of insecticides in areas of varying agrochemical use patterns Methods A survey to determine theagricultural chemical use pattern was undertaken in ten localities across Cote drsquoIvoire In addition WHO susceptibility tests werecarried out on adults Anopheles gambiae slmosquitoes emerging from collected larvae from the sites surveyed Four insecticidesfrom each class of the four classes of insecticides were evaluated using the standard susceptibility test methods Furthermorethe target site mutations involved in resistance mechanisms were identified following the Taqman assay protocols and mosquitospecies were identified using SINE-PCR Results The mortalities of all the An gambiae sl populations were similar regardless ofthe pesticide use patternThe vectors were resistant to DDT deltamethrin and bendiocarb in all localities In contrast mosquitoesshowed high susceptibility to malathion High frequency of the Kdr-West gene allele was observed (70-100) A single Kdr-East mutation was identified in a mosquito that harboured both Ace-1 and Kdr-West genes Conclusion Cultivated marshlandsrepresenting good habitats for mosquito developmentmay deeply contribute to the selection of resistance genes given the intensiveuse of agrochemical for crop protection In view of these special attention must be given to them to mitigate mosquito resistanceto insecticides

1 Background

Malaria continues to challenge the various control strategiesthat have been deployed over several decades in endemicareas The persistence of this disease is linked to severalfactors wit the most important ones being the absence ofa vaccine resistance of the parasite to antimalarial drugsand resistance of vectors to insecticides [1ndash3] Vector control

remains one of the main pillars of malaria control and hascontributed to the reduction of the spread of malaria in theabsence of specific drug prophylaxis [4 5] Unfortunately theefficacy of long lasting insecticidal nets (LLINs) and indoorresidual spraying (IRS) which constitute the main tools ofvector control is being threatened by the resistance of the vec-tors to insecticides [5ndash10]The resistance of vectors to severalfamilies of insecticides is highly widespread and has reached

HindawiBioMed Research InternationalVolume 2018 Article ID 2874160 9 pageshttpsdoiorg10115520182874160

2 BioMed Research International

an extensive level geographically [2 11 12] The managementof those resistances is becoming a challenge for malaria con-trol programmes worldwideTheWorldHealth Organization(WHO) Global Plan for Insecticide Resistance Management(GPIRM) has proposed management approaches that areessentially based on IRS and LLINs [5] Following thoseapproaches IRS and LLNIs were supposed to be the mainsources of insecticide resistance of malaria vectors Howeverif resistance selection pressure is originated from othersources such as agriculture then the methods proposed bythe GPIRM may be much less effective Indeed the swampsused for rice and vegetable cultivation in the tropics offerideal breeding sites for mosquitoes particularly An gambiaethe main malaria vector [4 13ndash18] Some of the agriculturalpesticides used in those swamps contain the same chemicalactive ingredients like the insecticides used for public healthpurpose [19ndash22] Thus the impact of agricultural pesticideusage has been highly indexed as a source of resistance toinsecticides [22ndash27] Exposure to different pesticides maylead to varied responses and mutations on the mosquito forbetter adaptation to the environment So for the fact thatcertain agrochemicals are potent inducers of genes involvedin resistance development the use of insecticidal treated toolsfor vector control in such areas may be less effective

Several mechanisms due to mutations are involved in theresistance of malaria vectors to insecticide The knock downresistance (kdr) mutation gene caused by the substitutionof leucine to phenylalanine (L1014F) used to be specific tovectors in West Africa while the substitution of leucine toserine (L1014S) was observed mostly in East Africa [28ndash31] However this clear separation has become increasinglyblurred in recent yearswith the kdr-East gene being identifiedin Benin andCote dIvoire inWest Africa [32 33] and the kdr-West gene being found in East African vectors [34]

Increasing monitoring of insecticide resistance is neededto guide mitigation and management strategies for vectorcontrol When high level of insecticide resistance occursurgent action needs to be taken and guided by knowledgeof the mechanisms involved in resistance According toWHOrecommendations vector control interventions shouldbe based on local entomological data including the vectorcomposition and its susceptibility to insecticides [35] Alongthe same line this study is part of the insecticide resis-tance monitoring framework and aims to assess insecticidesusceptibility and resistance mechanisms of An gambiaesl collected from cultivated lowlands exposed to varyingagricultural pesticides

2 Methods

21 Study Sites The study was carried out in 10 localities (ieAbengourou Agboville Azaguie Dabou MBe San-PedroTiassale Tiebissou Toumodi and Yamoussoukro) acrossdifferent regions of Cote dIvoire The practice of agricultureis common to all settings surveyed Abengourou is locatedin the Eastern region of Cote drsquoIvoire (6∘431015840N and 3∘291015840W)Coffee cocoa rubber and palm oil are the main cash crops inAbengourou while vegetable rice and banana are the mainfood crops in the area The locality of Agboville is situated

in the forest zone in the south of Cote drsquoIvoire (5∘551015840N and4∘131015840W) with cultivation of coffee and cocoa being the mainagricultural activity of this region Food crops are dominatedby banana cassava maize yam and rice Azaguie is a citylocated in the forest zone in the south of Cote drsquoIvoire (5∘381015840Nand 4∘051015840W) and the main practiced agricultural activitiespracticed are cocoa coffee and food crops (eg cerealsrice cassava yams and bananas) The locality of Dabou issituated in the same forest area (5∘191015840N and 4∘231015840W) withcoffee cocoa rubber palm oil banana and cassava beingthe main agricultural products grown In the village Mbeat 8∘61015840N and 6∘01015840W rice cultivation is the main economicactivity of the population with 700 hectares of land devotedto rice cultivation in the area The town of San-Pedro islocated along the Atlantic Ocean at the gulf of Guinea (4∘411015840Nand 6∘391015840W) San-Pedro is located within a dense forest areawhere perennial crops are dominant Food crops includeyams potatoes cassava rice corn and smaller amounts ofvegetables (eg eggplant gumbo spice cabbage cucumberand lettuce) Tiassale is located in the forest zone in thesouth of Cote drsquoIvoire (5∘531015840N and 4∘491015840W) and relies onsimilar agricultural crops as Dabou Tiebissou and Toumodiare located in central Cote drsquoIvoire at 7∘161015840N and 5∘291015840Wand6∘551015840N and 5∘031015840W respectively Both settings have similarvegetation composed of wooded savannah and pre-forestsavannah with gallery forests The most dominant agricul-tural products in both settings are cereals tubers coffee andcashew nuts Yamoussoukro is also located in central CotedrsquoIvoire (6∘481015840N and 5∘171015840W) with the cultivation of yamscassava and rice constituting the main agricultural activities

The family of amino phosphonates aryloxyacides amideand pyrimidine constituted herbicides class commonly usedby farmers Regarding insecticides pyrethroids organophos-phates organochlorines carbamates and neonicotinoid werecommonly used

LLINs are the main vector control tools used in thesedifferent localities

22 Pesticide Use Survey A survey on the use of pesticides byfarmers was carried out in the ten localities using question-naires Between 78 and 106 farmers per locality were surveyedon their knowledge attitude and practices related to the useof pesticides in agriculture Farmers were asked about thetypes of chemicals used the doses by products the frequencyand the rules of hygiene relating to the use of pesticides

23 Susceptibility Test The susceptibility of mosquitoes wasassessed through the WHO cylinder test [36] and themosquitoes used for the test were An gambiae sl wildstrain species Larvae were collected from the different sitecultivated swamps

After collection the larvae of different areas were trans-ferred and reared to adult stage in the insectary of the CentreSuisse de Recherches Scientifiques en Cote drsquoIvoire (CSRS) at27 plusmn 2∘C and a relative humidity of 70 plusmn 10 After emer-gence the adults were fed with cotton balls soaked in honeysolution diluted to 10 The susceptibility test performedaccording to the WHO protocol [36] involved exposureof 3ndash5-day old nonblood-fed female adults to a diagnostic

BioMed Research International 3

dosage of the following insecticides DDT (4) deltamethrin(005) bendiocarb (01) and malathion (5) Anophelesgambiae Kisumu strain was used as the reference susceptiblestrain and was tested simultaneously with the field pop-ulations The mortality of the different tests achieved wasinterpreted according to the criteria proposed by WHO [36]as follows mortality between 98 and 100 implies that thevectors are susceptible mortality between 90 and 97 indi-cates the presence of resistance genes in the vector populationwhich must be confirmed and mortality less than 90 con-firms the existence of resistance gene in the test population

The resistance ratio (RR) of vectors to the various insec-ticides was determined from reports of the knock down timeof 50 of the population (Kdt

50) of wild mosquitoes and

those of the susceptible Kisumu strain This ratio expressesthe level of resistance of the field strain compared with thesusceptible Kisumu strain based on the knock down effectThe time at which 50 of the test population were knockeddown (KDT

50) was determined using PoloPlus software via

log-probit analysis

24 Molecular Analysis of the Mosquito Vector

241 DNAExtraction Genomic DNAof the mosquitoes wasextracted according to the method of Collins et al [37] Inbrief whole mosquitoes were soaked in 2 cetyl trimethylammonium bromide (CTAB) The mosquitoes were crushedindividually in 200 120583l of CTAB and incubated at 65∘C for5 min A total of 200 120583l of chloroform were added andthe resulting mixture was centrifuged for 5 min at 12000rpm The supernatant was pipetted into a new 15 ml tubeto which 200 120583l isopropanol was added the mixture wascentrifuged for 15 min at 12000 rpm to precipitate the DNAThe supernatant was discarded subsequently and the DNApellet formed at the bottom of tubes was purified with 70ethanol After a further centrifugation step at 12000 rpm for5 min the ethanol was removed and the pellet dried on thebench over the night The extracted DNA was reconstitutedin 20 120583l DNase-free water (Sigma-Aldrich United Kingdom)prior to storage at -20∘C

25 Identification of Anopheles gambiae ss The differentspecies of An gambiae (An gambiae ss and An coluzzii)were determined according to the SINE-PCR method pre-viously described [38] The primer F61A of sequence51015840-TCGCCTTAGACCTTGCGTTA-31015840 was used to deter-mine An coluzzii and the primer R61B of sequence 51015840-CGCTTCAAGAATTCGAGATAC-31015840 for An gambiae ssThe incubation took place in a thermocycler of LongGenetype (A200 Gradient Thermal cycler LongGene ScientificInstruments Co Ltd Hangzhou PR China) according to thefollowing programme 94∘C for 5min 94∘C for 25 s and 54∘Cfor 30 s 72∘C for 1 min repeated 35 times and a final stepat 72∘C for 10 min to terminate the reaction An agarose gelwas prepared at 15 in TBE (TrisborateEDTA) containingethidium bromide at 10 mgml The PCR product was loadedon gel and allowed to migrate under a voltage of 140 V for 1h The result was visualized with a UV illuminator (BioDoc-It Imaging System Upland CA USA) The profile of the

0

20

40

60

80

Percentages

Insecticides

Abamectin Acetamiprid Deltamethrin AtrazineLambdacyhalothrin Carbofuran Chlorpyriphos-ethyl BifenthrinCypermethrin Diazinon Fipronil ImidaclopridIndoxacarb Alphamethrin Malathion Permethrin

Pyrethroids

Neonicotin

oids

Organop

hosphates

Carbam

ates

Triazines

Phenylpyrazoles

Avermectin

s

Figure 1 Proportion of different insecticide families used in the tenlocalities

expected bands by species was 479 bp forAn gambiae coluzziiand 249 bp for An gambiae ss

26 Identification of Resistance Genes The real time PCRwas used to investigate the presence of insecticide resistancegenes including kdr-East and West and Ace-1 [39] Thereaction was carried out in an Agilent Stratagene MX3005qPCR thermocycler (Agilent Technologies Santa Clara CAUSA) for each gene in a final volume of 10 120583l containingSensiMix and the specific probe containing FAM and HEXfluorochromes FAM was used to detect the mutant allelewhile HEX detected the wild-type susceptible allele Theamplification conditions used were 10 min at 95∘C 40 cyclesof 10 s at 95∘C and 45 s at 60∘C Genotypes were scoredfrom dual colour scatter plots produced by the device afterincubation

3 Results

31 Frequency of Pesticide Usage The results of pesticideusage surveys showed that 975of the farmers surveyed usedat least one pesticide Three groups of pesticides were foundin these ten localities Among these pesticides herbicidesaccounted for 619 insecticides were 335 and fungicides46 The proportion of herbicides used in all localities washigher than that of insecticides except San-Pedro which is acocoa growing areawhere insecticides accounted for 647ofthe pesticides used Among the insecticides pyrethroids arethe most commonly used (76 ) (Figure 1)

Pesticides were used at different frequencies dependingon the crop areas The low proportion of pesticides used havebeen observed in the area of culture of cashew yams andcassava in the localities of Toumodi and Tiebissou while highproportion was observed in the rice fields and vegetable plotsof Agboville Tiassale and Dabou The average number ofpesticides treatment by farmer per year varied from 22 to 76according the locality

32 Susceptibility of Vectors to Insecticides Impregnatedpapers used for susceptibility testing resulted in 100 mor-tality of the susceptible Kisumu strain indicating the good

4 BioMed Research International

Tieacutebiss

ou

Toum

odi

Mbeacute

Yamou

ssok

ro

Abengourou

San-Peacutedro

Agboville

Tiassaleacute

Dabou

Azaguieacute

Localities

120

100

80

60

40

20

0

Percentages

DDT Deltamethrin Bendiocarb Malathion

Figure 2 Level of resistance of Anopheles gambiae according toinsecticide families in 10 localities in Cote dIvoire according tothe intensity of agricultural pesticide use The mortality generatedby malathion remains higher than that of other insecticides in alllocalities followed by bendiocarb deltamethrin and DDT

quality of treated papers Furthermore control tube mor-talities were less than 5 implying any Abbotrsquos correctionAccording to the susceptibility tests conducted on wild Angambiaemosquitoes the mortalities generated by each insec-ticide showed variations between the localities (Figure 2)The resistance to DDT and deltamethrin was very high atthe various sites assessed Thus the highest mortality againstDDT was 22 and was observed in the locality of Dabouwhile the highest mortality against deltamethrin was 4 asobserved in San-Pedro For bendiocarb all mortality ratesrecorded were below 40 In contrast malathion recorded100 mortality in Abengourou Mbe and Tiebissou 99 inSan-Pedro and 98 in Tiassale In Toumodi and Yamous-soukro the mortality caused by malathion was 967 and923 respectively while in Dabou the vectors developedresistance to malathion with a mortality of 793

33 Resistance Ratio and Knock Down The RR50

variedamong localities (Table 1) The lowest RR

50of deltamethrin

was 35 as observed in Dabou and Yamoussoukro Howeverthe RR

50for deltamethrin could not be determined in Tiebis-

sou because nomosquito had been knocked down during theexposure timeThe highest rate of mosquitoes knocked downby deltamethrin (26) was observed in Dabou from the 18thminute The few mosquitoes knocked down by DDT (12)were after 30 min and were observed at Azaguie The RR

50

of bendiocarb varied between 25 and 39 The highest rateof mosquitoes knocked down by bendiocarb was observedat Toumodi (53) Concerning malathion the RR

50varied

between 15 and 24 with 100 of the mosquitoes of thelocalities Abengourou and San-Pedro being knocked downbefore the end of the exposure time and after the 23rd minuteMalathion recorded 100 knock down rate after 18 minutesat Tiebissou and represented the fastest knock down effect

34 Molecular Species Among the 436 An gambiae speci-mens characterized An coluzzii was the most represented

species with a proportion of 822 followed by An gam-biae ss with 161 Nine mosquitoes were characterizedas hybrids (21) In the locality of Agboville all mosquitospecimens analyzed were An coluzzii (Figure 3)

35 Resistance Genes The kdr-East kdr-West and Ace-1 genes were all identified in the vectors Among theseresistance genes kdr-West was the most frequently expressedwithin the population of vectors with an allelic frequencyranged between 07 (Tiebissou) and 1 (Abengourou) All thesamples tested had at least one resistant allele to the kdr-West geneThe proportion of homozygous individuals of thismutant allele was 719 in general while that of heterozygousindividuals was 281 As for the kdr-East gene 985 of thegenes expressed were of the susceptible genotype and only asingle sample of the locality of Tiebissouwas heterozygous forthis gene This same mosquito harboured the kdr-West geneand the Ace-1 gene at the heterozygous stage Concerningthe Ace-1 mutation the allelic frequency of the mutant generanged between 007 and 05 with 0014 for homozygotes and054 for heterozygotes (Table 2)

4 Discussion

The susceptibility tests carried out on An gambiae popula-tions sampled in 10 different localities across Cote drsquoIvoirehighlight the extent of resistance of this vector to thefour classes of insecticides commonly used for malariavector control The level of resistance developed againstdeltamethrin DDT and bendiocarb was found to be veryhigh and in line with the trends reported by previous studies[11 12 40] The strong resistance observed with high kdrallelic frequencies suggested a fixation of this resistance genewithin the population of An gambiae in Cote drsquoIvoire Themortalities generated among the An gambiae populationwere similar regardless of the pesticide use patterns as highlevel of resistance where found in low moderate and highpesticide used area A similar level of insecticide resistanceacross areas of different pesticide exposure-intensity may beexplained by vector control activities in each of the areasassessed such as the distribution of LLINs by the nationalmalaria control program (NMCP) since decade A specificinfluence of agrochemicals to development of resistance ofAn gambiae populations could not be dissociated in thecurrent set upHowever we were unable to locate agriculturalareas with complete absence of pesticide-use therefore therole that agrochemicals may play in fostering insecticideresistance should not be dismissed Both the presence ofhybrids and themigration of vectors can contribute to geneticshuffling and favour the homogenization of populations ofresistant vectors Large-scale aerial spraying of agriculturalpesticides in plantations by agroindustrial firms that arewidely established in Cote drsquoIvoire should also be consideredin future studies

Although the use of DDT is prohibited in agriculture aswell as for public health in Cote drsquoIvoire the current studyrevealed a high resistance of mosquitoes to this productThishigh DDT resistance observed in the mosquito populations

BioMed Research International 5

Table 1 Resistance ratio (RR50) of mosquito populations (An gambiae) to deltamethrin malathion bendiocarb and DDT CI

50 confidence

interval at 50 KdT50 knockdowntime of 50 of the population KdT50 of the wild strain divided by KdT

50of the Kisumu reference strain

RR50 resistance ratio at 50 cannot be determined (no knockdown)

Insecticides by localities Kdt50 (CI50) kisimu (min) Kdt50 (CI50) wild strain (min) RR50

AzaguieDeltamethrin (005) 319 (258ndash385) 1143 (903ndash1911) 36Malathion (5) 296 (284ndash309) 513 (487ndash546) 17Bendiocarb (01) 316 (305ndash326) 795 (725ndash933) 25DDT (4) 223 (217ndash229) TiebissouDeltamethrin (005) 319 (258ndash385) Malathion (5) 316 (305ndash326) 460 (450ndash471) 15Bendiocarb (01) 187 (181ndash193) 565 (543ndash592) 3DDT (4) 223 (217ndash229) AbengourouDeltamethrin (005) 217 (207ndash226) 802 (729ndash922) 37Malathion (5) 314 (299ndash329) 337 (329ndash344) 11Bendiocarb (01) 282 (269ndash295) 376 (337ndash452) 29DDT (4) 223 (217ndash229) AgbovilleDeltamethrin (005) 217 (207ndash226) 9932 (855ndash1228) 46Malathion (5) 282 (269ndash295) 619 (578ndash678) 2Bendiocarb (01) 282 (269ndash295) 4193 (2247ndash14923) 149DDT (4) 223 (217ndash229) San-PedroDeltamethrin (005) 217 (207ndash226) 8345 (751ndash978) 39Malathion (5) 316 (305ndash326) 3368 (329ndash344) 11Bendiocarb (5) 255 (231ndash277) 376 (337ndash451) 15DDT (4) 223 (217ndash229) YamoussoukroDeltamthrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 724 (679ndash780) 23Bendiocarb (01) 187 (181ndash193) 646 (614ndash687) 35DDT (4) 223 (217ndash229) DabouDeltamethrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 66 (622ndash710) 21Bendiocarb (01) 187 (181ndash193) 724 (656ndash824) 39DDT (4) 223 (217ndash229) MBeDeltamethrin (005) 297 (298ndash305) Malathion (5) 316 (305ndash326) 729 (688ndash791) 23Bendiocarb (01) 187 (181ndash193) 703 (667ndash756) 38DDT (4) 223 (217ndash229) TiassaleDeltamethrin (005) 217 (207ndash226) 2204 (1516ndash4317) 102Malathion (5) 314 (299ndash329) 634 (605ndash669) 2Bendiocarb (01) 282 (269ndash295) 1235 (948ndash2274) 44DDT (4) 223 (217ndash229) ToumodiDeltamethrin (005) 319 (258ndash385) 2992 (1556ndash25214) 94Malathion (5) 316 (305ndash326) 554 (541ndash670) 18Bendiocarb (01) 187 (181ndash193) 507 (488ndash629) 27DDT (4) 223 (217ndash229)

6 BioMed Research International

0 25 50 100 150 200

Kilometers

An coluzziiAn gambiaeHybrid

N

Figure 3 Proportion of different species by locality

Table 2 Allelic frequency of different mutational genes in malariavectors in 10 localities in Cote drsquoIvoire The allelic frequency is therate of presence of the resistance allele within the vector populations

Localities West-KdrFreq(R)

East-KdrFreq(R)

Ace-1Freq(R)

Azaguie 092 0 033Tiebissou 07 003 013Toumodi 08 0 033Mbe 077 0 013Yamoussoukro 096 0 032Abengourou 1 0 020San-Pedro 093 0 007Agboville 079 0 05Tiassale 07 0 04Dabou 09 0 043

across the country may be attributed to both the strongresidues of this product in the nature [41] having beenused in the past and the cross-resistance between DDT andpyrethroids which were shown to account for more than 90of chemical used by farmers [12]

InTiebissou a singleAn gambiae ssmosquitowas foundto possess the kdr-West (L1014F) kdr-East (L1014S) and Ace-1(G119S)mutations simultaneouslyThe co-occurrence of boththe kdr mutations (L1014FS) has been reported from WestCentral and also East Africa [42ndash44]However this is the firsttime these three alleles associated with insecticide resistancewere found in a single specimen in Cote drsquoIvoire

The presence of hybrids is a very serious threat to vectorcontrol as this can favour the spread of these traits Henceour research highlights the urgent need for a compound witha novel mode of action in addition to those already existingto eliminate such individuals within the vector population

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

BioMed Research International 3

dosage of the following insecticides DDT (4) deltamethrin(005) bendiocarb (01) and malathion (5) Anophelesgambiae Kisumu strain was used as the reference susceptiblestrain and was tested simultaneously with the field pop-ulations The mortality of the different tests achieved wasinterpreted according to the criteria proposed by WHO [36]as follows mortality between 98 and 100 implies that thevectors are susceptible mortality between 90 and 97 indi-cates the presence of resistance genes in the vector populationwhich must be confirmed and mortality less than 90 con-firms the existence of resistance gene in the test population

The resistance ratio (RR) of vectors to the various insec-ticides was determined from reports of the knock down timeof 50 of the population (Kdt

50) of wild mosquitoes and

those of the susceptible Kisumu strain This ratio expressesthe level of resistance of the field strain compared with thesusceptible Kisumu strain based on the knock down effectThe time at which 50 of the test population were knockeddown (KDT

50) was determined using PoloPlus software via

log-probit analysis

24 Molecular Analysis of the Mosquito Vector

241 DNAExtraction Genomic DNAof the mosquitoes wasextracted according to the method of Collins et al [37] Inbrief whole mosquitoes were soaked in 2 cetyl trimethylammonium bromide (CTAB) The mosquitoes were crushedindividually in 200 120583l of CTAB and incubated at 65∘C for5 min A total of 200 120583l of chloroform were added andthe resulting mixture was centrifuged for 5 min at 12000rpm The supernatant was pipetted into a new 15 ml tubeto which 200 120583l isopropanol was added the mixture wascentrifuged for 15 min at 12000 rpm to precipitate the DNAThe supernatant was discarded subsequently and the DNApellet formed at the bottom of tubes was purified with 70ethanol After a further centrifugation step at 12000 rpm for5 min the ethanol was removed and the pellet dried on thebench over the night The extracted DNA was reconstitutedin 20 120583l DNase-free water (Sigma-Aldrich United Kingdom)prior to storage at -20∘C

25 Identification of Anopheles gambiae ss The differentspecies of An gambiae (An gambiae ss and An coluzzii)were determined according to the SINE-PCR method pre-viously described [38] The primer F61A of sequence51015840-TCGCCTTAGACCTTGCGTTA-31015840 was used to deter-mine An coluzzii and the primer R61B of sequence 51015840-CGCTTCAAGAATTCGAGATAC-31015840 for An gambiae ssThe incubation took place in a thermocycler of LongGenetype (A200 Gradient Thermal cycler LongGene ScientificInstruments Co Ltd Hangzhou PR China) according to thefollowing programme 94∘C for 5min 94∘C for 25 s and 54∘Cfor 30 s 72∘C for 1 min repeated 35 times and a final stepat 72∘C for 10 min to terminate the reaction An agarose gelwas prepared at 15 in TBE (TrisborateEDTA) containingethidium bromide at 10 mgml The PCR product was loadedon gel and allowed to migrate under a voltage of 140 V for 1h The result was visualized with a UV illuminator (BioDoc-It Imaging System Upland CA USA) The profile of the

0

20

40

60

80

Percentages

Insecticides

Abamectin Acetamiprid Deltamethrin AtrazineLambdacyhalothrin Carbofuran Chlorpyriphos-ethyl BifenthrinCypermethrin Diazinon Fipronil ImidaclopridIndoxacarb Alphamethrin Malathion Permethrin

Pyrethroids

Neonicotin

oids

Organop

hosphates

Carbam

ates

Triazines

Phenylpyrazoles

Avermectin

s

Figure 1 Proportion of different insecticide families used in the tenlocalities

expected bands by species was 479 bp forAn gambiae coluzziiand 249 bp for An gambiae ss

26 Identification of Resistance Genes The real time PCRwas used to investigate the presence of insecticide resistancegenes including kdr-East and West and Ace-1 [39] Thereaction was carried out in an Agilent Stratagene MX3005qPCR thermocycler (Agilent Technologies Santa Clara CAUSA) for each gene in a final volume of 10 120583l containingSensiMix and the specific probe containing FAM and HEXfluorochromes FAM was used to detect the mutant allelewhile HEX detected the wild-type susceptible allele Theamplification conditions used were 10 min at 95∘C 40 cyclesof 10 s at 95∘C and 45 s at 60∘C Genotypes were scoredfrom dual colour scatter plots produced by the device afterincubation

3 Results

31 Frequency of Pesticide Usage The results of pesticideusage surveys showed that 975of the farmers surveyed usedat least one pesticide Three groups of pesticides were foundin these ten localities Among these pesticides herbicidesaccounted for 619 insecticides were 335 and fungicides46 The proportion of herbicides used in all localities washigher than that of insecticides except San-Pedro which is acocoa growing areawhere insecticides accounted for 647ofthe pesticides used Among the insecticides pyrethroids arethe most commonly used (76 ) (Figure 1)

Pesticides were used at different frequencies dependingon the crop areas The low proportion of pesticides used havebeen observed in the area of culture of cashew yams andcassava in the localities of Toumodi and Tiebissou while highproportion was observed in the rice fields and vegetable plotsof Agboville Tiassale and Dabou The average number ofpesticides treatment by farmer per year varied from 22 to 76according the locality

32 Susceptibility of Vectors to Insecticides Impregnatedpapers used for susceptibility testing resulted in 100 mor-tality of the susceptible Kisumu strain indicating the good

4 BioMed Research International

Tieacutebiss

ou

Toum

odi

Mbeacute

Yamou

ssok

ro

Abengourou

San-Peacutedro

Agboville

Tiassaleacute

Dabou

Azaguieacute

Localities

120

100

80

60

40

20

0

Percentages

DDT Deltamethrin Bendiocarb Malathion

Figure 2 Level of resistance of Anopheles gambiae according toinsecticide families in 10 localities in Cote dIvoire according tothe intensity of agricultural pesticide use The mortality generatedby malathion remains higher than that of other insecticides in alllocalities followed by bendiocarb deltamethrin and DDT

quality of treated papers Furthermore control tube mor-talities were less than 5 implying any Abbotrsquos correctionAccording to the susceptibility tests conducted on wild Angambiaemosquitoes the mortalities generated by each insec-ticide showed variations between the localities (Figure 2)The resistance to DDT and deltamethrin was very high atthe various sites assessed Thus the highest mortality againstDDT was 22 and was observed in the locality of Dabouwhile the highest mortality against deltamethrin was 4 asobserved in San-Pedro For bendiocarb all mortality ratesrecorded were below 40 In contrast malathion recorded100 mortality in Abengourou Mbe and Tiebissou 99 inSan-Pedro and 98 in Tiassale In Toumodi and Yamous-soukro the mortality caused by malathion was 967 and923 respectively while in Dabou the vectors developedresistance to malathion with a mortality of 793

33 Resistance Ratio and Knock Down The RR50

variedamong localities (Table 1) The lowest RR

50of deltamethrin

was 35 as observed in Dabou and Yamoussoukro Howeverthe RR

50for deltamethrin could not be determined in Tiebis-

sou because nomosquito had been knocked down during theexposure timeThe highest rate of mosquitoes knocked downby deltamethrin (26) was observed in Dabou from the 18thminute The few mosquitoes knocked down by DDT (12)were after 30 min and were observed at Azaguie The RR

50

of bendiocarb varied between 25 and 39 The highest rateof mosquitoes knocked down by bendiocarb was observedat Toumodi (53) Concerning malathion the RR

50varied

between 15 and 24 with 100 of the mosquitoes of thelocalities Abengourou and San-Pedro being knocked downbefore the end of the exposure time and after the 23rd minuteMalathion recorded 100 knock down rate after 18 minutesat Tiebissou and represented the fastest knock down effect

34 Molecular Species Among the 436 An gambiae speci-mens characterized An coluzzii was the most represented

species with a proportion of 822 followed by An gam-biae ss with 161 Nine mosquitoes were characterizedas hybrids (21) In the locality of Agboville all mosquitospecimens analyzed were An coluzzii (Figure 3)

35 Resistance Genes The kdr-East kdr-West and Ace-1 genes were all identified in the vectors Among theseresistance genes kdr-West was the most frequently expressedwithin the population of vectors with an allelic frequencyranged between 07 (Tiebissou) and 1 (Abengourou) All thesamples tested had at least one resistant allele to the kdr-West geneThe proportion of homozygous individuals of thismutant allele was 719 in general while that of heterozygousindividuals was 281 As for the kdr-East gene 985 of thegenes expressed were of the susceptible genotype and only asingle sample of the locality of Tiebissouwas heterozygous forthis gene This same mosquito harboured the kdr-West geneand the Ace-1 gene at the heterozygous stage Concerningthe Ace-1 mutation the allelic frequency of the mutant generanged between 007 and 05 with 0014 for homozygotes and054 for heterozygotes (Table 2)

4 Discussion

The susceptibility tests carried out on An gambiae popula-tions sampled in 10 different localities across Cote drsquoIvoirehighlight the extent of resistance of this vector to thefour classes of insecticides commonly used for malariavector control The level of resistance developed againstdeltamethrin DDT and bendiocarb was found to be veryhigh and in line with the trends reported by previous studies[11 12 40] The strong resistance observed with high kdrallelic frequencies suggested a fixation of this resistance genewithin the population of An gambiae in Cote drsquoIvoire Themortalities generated among the An gambiae populationwere similar regardless of the pesticide use patterns as highlevel of resistance where found in low moderate and highpesticide used area A similar level of insecticide resistanceacross areas of different pesticide exposure-intensity may beexplained by vector control activities in each of the areasassessed such as the distribution of LLINs by the nationalmalaria control program (NMCP) since decade A specificinfluence of agrochemicals to development of resistance ofAn gambiae populations could not be dissociated in thecurrent set upHowever we were unable to locate agriculturalareas with complete absence of pesticide-use therefore therole that agrochemicals may play in fostering insecticideresistance should not be dismissed Both the presence ofhybrids and themigration of vectors can contribute to geneticshuffling and favour the homogenization of populations ofresistant vectors Large-scale aerial spraying of agriculturalpesticides in plantations by agroindustrial firms that arewidely established in Cote drsquoIvoire should also be consideredin future studies

Although the use of DDT is prohibited in agriculture aswell as for public health in Cote drsquoIvoire the current studyrevealed a high resistance of mosquitoes to this productThishigh DDT resistance observed in the mosquito populations

BioMed Research International 5

Table 1 Resistance ratio (RR50) of mosquito populations (An gambiae) to deltamethrin malathion bendiocarb and DDT CI

50 confidence

interval at 50 KdT50 knockdowntime of 50 of the population KdT50 of the wild strain divided by KdT

50of the Kisumu reference strain

RR50 resistance ratio at 50 cannot be determined (no knockdown)

Insecticides by localities Kdt50 (CI50) kisimu (min) Kdt50 (CI50) wild strain (min) RR50

AzaguieDeltamethrin (005) 319 (258ndash385) 1143 (903ndash1911) 36Malathion (5) 296 (284ndash309) 513 (487ndash546) 17Bendiocarb (01) 316 (305ndash326) 795 (725ndash933) 25DDT (4) 223 (217ndash229) TiebissouDeltamethrin (005) 319 (258ndash385) Malathion (5) 316 (305ndash326) 460 (450ndash471) 15Bendiocarb (01) 187 (181ndash193) 565 (543ndash592) 3DDT (4) 223 (217ndash229) AbengourouDeltamethrin (005) 217 (207ndash226) 802 (729ndash922) 37Malathion (5) 314 (299ndash329) 337 (329ndash344) 11Bendiocarb (01) 282 (269ndash295) 376 (337ndash452) 29DDT (4) 223 (217ndash229) AgbovilleDeltamethrin (005) 217 (207ndash226) 9932 (855ndash1228) 46Malathion (5) 282 (269ndash295) 619 (578ndash678) 2Bendiocarb (01) 282 (269ndash295) 4193 (2247ndash14923) 149DDT (4) 223 (217ndash229) San-PedroDeltamethrin (005) 217 (207ndash226) 8345 (751ndash978) 39Malathion (5) 316 (305ndash326) 3368 (329ndash344) 11Bendiocarb (5) 255 (231ndash277) 376 (337ndash451) 15DDT (4) 223 (217ndash229) YamoussoukroDeltamthrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 724 (679ndash780) 23Bendiocarb (01) 187 (181ndash193) 646 (614ndash687) 35DDT (4) 223 (217ndash229) DabouDeltamethrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 66 (622ndash710) 21Bendiocarb (01) 187 (181ndash193) 724 (656ndash824) 39DDT (4) 223 (217ndash229) MBeDeltamethrin (005) 297 (298ndash305) Malathion (5) 316 (305ndash326) 729 (688ndash791) 23Bendiocarb (01) 187 (181ndash193) 703 (667ndash756) 38DDT (4) 223 (217ndash229) TiassaleDeltamethrin (005) 217 (207ndash226) 2204 (1516ndash4317) 102Malathion (5) 314 (299ndash329) 634 (605ndash669) 2Bendiocarb (01) 282 (269ndash295) 1235 (948ndash2274) 44DDT (4) 223 (217ndash229) ToumodiDeltamethrin (005) 319 (258ndash385) 2992 (1556ndash25214) 94Malathion (5) 316 (305ndash326) 554 (541ndash670) 18Bendiocarb (01) 187 (181ndash193) 507 (488ndash629) 27DDT (4) 223 (217ndash229)

6 BioMed Research International

0 25 50 100 150 200

Kilometers

An coluzziiAn gambiaeHybrid

N

Figure 3 Proportion of different species by locality

Table 2 Allelic frequency of different mutational genes in malariavectors in 10 localities in Cote drsquoIvoire The allelic frequency is therate of presence of the resistance allele within the vector populations

Localities West-KdrFreq(R)

East-KdrFreq(R)

Ace-1Freq(R)

Azaguie 092 0 033Tiebissou 07 003 013Toumodi 08 0 033Mbe 077 0 013Yamoussoukro 096 0 032Abengourou 1 0 020San-Pedro 093 0 007Agboville 079 0 05Tiassale 07 0 04Dabou 09 0 043

across the country may be attributed to both the strongresidues of this product in the nature [41] having beenused in the past and the cross-resistance between DDT andpyrethroids which were shown to account for more than 90of chemical used by farmers [12]

InTiebissou a singleAn gambiae ssmosquitowas foundto possess the kdr-West (L1014F) kdr-East (L1014S) and Ace-1(G119S)mutations simultaneouslyThe co-occurrence of boththe kdr mutations (L1014FS) has been reported from WestCentral and also East Africa [42ndash44]However this is the firsttime these three alleles associated with insecticide resistancewere found in a single specimen in Cote drsquoIvoire

The presence of hybrids is a very serious threat to vectorcontrol as this can favour the spread of these traits Henceour research highlights the urgent need for a compound witha novel mode of action in addition to those already existingto eliminate such individuals within the vector population

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

4 BioMed Research International

Tieacutebiss

ou

Toum

odi

Mbeacute

Yamou

ssok

ro

Abengourou

San-Peacutedro

Agboville

Tiassaleacute

Dabou

Azaguieacute

Localities

120

100

80

60

40

20

0

Percentages

DDT Deltamethrin Bendiocarb Malathion

Figure 2 Level of resistance of Anopheles gambiae according toinsecticide families in 10 localities in Cote dIvoire according tothe intensity of agricultural pesticide use The mortality generatedby malathion remains higher than that of other insecticides in alllocalities followed by bendiocarb deltamethrin and DDT

quality of treated papers Furthermore control tube mor-talities were less than 5 implying any Abbotrsquos correctionAccording to the susceptibility tests conducted on wild Angambiaemosquitoes the mortalities generated by each insec-ticide showed variations between the localities (Figure 2)The resistance to DDT and deltamethrin was very high atthe various sites assessed Thus the highest mortality againstDDT was 22 and was observed in the locality of Dabouwhile the highest mortality against deltamethrin was 4 asobserved in San-Pedro For bendiocarb all mortality ratesrecorded were below 40 In contrast malathion recorded100 mortality in Abengourou Mbe and Tiebissou 99 inSan-Pedro and 98 in Tiassale In Toumodi and Yamous-soukro the mortality caused by malathion was 967 and923 respectively while in Dabou the vectors developedresistance to malathion with a mortality of 793

33 Resistance Ratio and Knock Down The RR50

variedamong localities (Table 1) The lowest RR

50of deltamethrin

was 35 as observed in Dabou and Yamoussoukro Howeverthe RR

50for deltamethrin could not be determined in Tiebis-

sou because nomosquito had been knocked down during theexposure timeThe highest rate of mosquitoes knocked downby deltamethrin (26) was observed in Dabou from the 18thminute The few mosquitoes knocked down by DDT (12)were after 30 min and were observed at Azaguie The RR

50

of bendiocarb varied between 25 and 39 The highest rateof mosquitoes knocked down by bendiocarb was observedat Toumodi (53) Concerning malathion the RR

50varied

between 15 and 24 with 100 of the mosquitoes of thelocalities Abengourou and San-Pedro being knocked downbefore the end of the exposure time and after the 23rd minuteMalathion recorded 100 knock down rate after 18 minutesat Tiebissou and represented the fastest knock down effect

34 Molecular Species Among the 436 An gambiae speci-mens characterized An coluzzii was the most represented

species with a proportion of 822 followed by An gam-biae ss with 161 Nine mosquitoes were characterizedas hybrids (21) In the locality of Agboville all mosquitospecimens analyzed were An coluzzii (Figure 3)

35 Resistance Genes The kdr-East kdr-West and Ace-1 genes were all identified in the vectors Among theseresistance genes kdr-West was the most frequently expressedwithin the population of vectors with an allelic frequencyranged between 07 (Tiebissou) and 1 (Abengourou) All thesamples tested had at least one resistant allele to the kdr-West geneThe proportion of homozygous individuals of thismutant allele was 719 in general while that of heterozygousindividuals was 281 As for the kdr-East gene 985 of thegenes expressed were of the susceptible genotype and only asingle sample of the locality of Tiebissouwas heterozygous forthis gene This same mosquito harboured the kdr-West geneand the Ace-1 gene at the heterozygous stage Concerningthe Ace-1 mutation the allelic frequency of the mutant generanged between 007 and 05 with 0014 for homozygotes and054 for heterozygotes (Table 2)

4 Discussion

The susceptibility tests carried out on An gambiae popula-tions sampled in 10 different localities across Cote drsquoIvoirehighlight the extent of resistance of this vector to thefour classes of insecticides commonly used for malariavector control The level of resistance developed againstdeltamethrin DDT and bendiocarb was found to be veryhigh and in line with the trends reported by previous studies[11 12 40] The strong resistance observed with high kdrallelic frequencies suggested a fixation of this resistance genewithin the population of An gambiae in Cote drsquoIvoire Themortalities generated among the An gambiae populationwere similar regardless of the pesticide use patterns as highlevel of resistance where found in low moderate and highpesticide used area A similar level of insecticide resistanceacross areas of different pesticide exposure-intensity may beexplained by vector control activities in each of the areasassessed such as the distribution of LLINs by the nationalmalaria control program (NMCP) since decade A specificinfluence of agrochemicals to development of resistance ofAn gambiae populations could not be dissociated in thecurrent set upHowever we were unable to locate agriculturalareas with complete absence of pesticide-use therefore therole that agrochemicals may play in fostering insecticideresistance should not be dismissed Both the presence ofhybrids and themigration of vectors can contribute to geneticshuffling and favour the homogenization of populations ofresistant vectors Large-scale aerial spraying of agriculturalpesticides in plantations by agroindustrial firms that arewidely established in Cote drsquoIvoire should also be consideredin future studies

Although the use of DDT is prohibited in agriculture aswell as for public health in Cote drsquoIvoire the current studyrevealed a high resistance of mosquitoes to this productThishigh DDT resistance observed in the mosquito populations

BioMed Research International 5

Table 1 Resistance ratio (RR50) of mosquito populations (An gambiae) to deltamethrin malathion bendiocarb and DDT CI

50 confidence

interval at 50 KdT50 knockdowntime of 50 of the population KdT50 of the wild strain divided by KdT

50of the Kisumu reference strain

RR50 resistance ratio at 50 cannot be determined (no knockdown)

Insecticides by localities Kdt50 (CI50) kisimu (min) Kdt50 (CI50) wild strain (min) RR50

AzaguieDeltamethrin (005) 319 (258ndash385) 1143 (903ndash1911) 36Malathion (5) 296 (284ndash309) 513 (487ndash546) 17Bendiocarb (01) 316 (305ndash326) 795 (725ndash933) 25DDT (4) 223 (217ndash229) TiebissouDeltamethrin (005) 319 (258ndash385) Malathion (5) 316 (305ndash326) 460 (450ndash471) 15Bendiocarb (01) 187 (181ndash193) 565 (543ndash592) 3DDT (4) 223 (217ndash229) AbengourouDeltamethrin (005) 217 (207ndash226) 802 (729ndash922) 37Malathion (5) 314 (299ndash329) 337 (329ndash344) 11Bendiocarb (01) 282 (269ndash295) 376 (337ndash452) 29DDT (4) 223 (217ndash229) AgbovilleDeltamethrin (005) 217 (207ndash226) 9932 (855ndash1228) 46Malathion (5) 282 (269ndash295) 619 (578ndash678) 2Bendiocarb (01) 282 (269ndash295) 4193 (2247ndash14923) 149DDT (4) 223 (217ndash229) San-PedroDeltamethrin (005) 217 (207ndash226) 8345 (751ndash978) 39Malathion (5) 316 (305ndash326) 3368 (329ndash344) 11Bendiocarb (5) 255 (231ndash277) 376 (337ndash451) 15DDT (4) 223 (217ndash229) YamoussoukroDeltamthrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 724 (679ndash780) 23Bendiocarb (01) 187 (181ndash193) 646 (614ndash687) 35DDT (4) 223 (217ndash229) DabouDeltamethrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 66 (622ndash710) 21Bendiocarb (01) 187 (181ndash193) 724 (656ndash824) 39DDT (4) 223 (217ndash229) MBeDeltamethrin (005) 297 (298ndash305) Malathion (5) 316 (305ndash326) 729 (688ndash791) 23Bendiocarb (01) 187 (181ndash193) 703 (667ndash756) 38DDT (4) 223 (217ndash229) TiassaleDeltamethrin (005) 217 (207ndash226) 2204 (1516ndash4317) 102Malathion (5) 314 (299ndash329) 634 (605ndash669) 2Bendiocarb (01) 282 (269ndash295) 1235 (948ndash2274) 44DDT (4) 223 (217ndash229) ToumodiDeltamethrin (005) 319 (258ndash385) 2992 (1556ndash25214) 94Malathion (5) 316 (305ndash326) 554 (541ndash670) 18Bendiocarb (01) 187 (181ndash193) 507 (488ndash629) 27DDT (4) 223 (217ndash229)

6 BioMed Research International

0 25 50 100 150 200

Kilometers

An coluzziiAn gambiaeHybrid

N

Figure 3 Proportion of different species by locality

Table 2 Allelic frequency of different mutational genes in malariavectors in 10 localities in Cote drsquoIvoire The allelic frequency is therate of presence of the resistance allele within the vector populations

Localities West-KdrFreq(R)

East-KdrFreq(R)

Ace-1Freq(R)

Azaguie 092 0 033Tiebissou 07 003 013Toumodi 08 0 033Mbe 077 0 013Yamoussoukro 096 0 032Abengourou 1 0 020San-Pedro 093 0 007Agboville 079 0 05Tiassale 07 0 04Dabou 09 0 043

across the country may be attributed to both the strongresidues of this product in the nature [41] having beenused in the past and the cross-resistance between DDT andpyrethroids which were shown to account for more than 90of chemical used by farmers [12]

InTiebissou a singleAn gambiae ssmosquitowas foundto possess the kdr-West (L1014F) kdr-East (L1014S) and Ace-1(G119S)mutations simultaneouslyThe co-occurrence of boththe kdr mutations (L1014FS) has been reported from WestCentral and also East Africa [42ndash44]However this is the firsttime these three alleles associated with insecticide resistancewere found in a single specimen in Cote drsquoIvoire

The presence of hybrids is a very serious threat to vectorcontrol as this can favour the spread of these traits Henceour research highlights the urgent need for a compound witha novel mode of action in addition to those already existingto eliminate such individuals within the vector population

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

BioMed Research International 5

Table 1 Resistance ratio (RR50) of mosquito populations (An gambiae) to deltamethrin malathion bendiocarb and DDT CI

50 confidence

interval at 50 KdT50 knockdowntime of 50 of the population KdT50 of the wild strain divided by KdT

50of the Kisumu reference strain

RR50 resistance ratio at 50 cannot be determined (no knockdown)

Insecticides by localities Kdt50 (CI50) kisimu (min) Kdt50 (CI50) wild strain (min) RR50

AzaguieDeltamethrin (005) 319 (258ndash385) 1143 (903ndash1911) 36Malathion (5) 296 (284ndash309) 513 (487ndash546) 17Bendiocarb (01) 316 (305ndash326) 795 (725ndash933) 25DDT (4) 223 (217ndash229) TiebissouDeltamethrin (005) 319 (258ndash385) Malathion (5) 316 (305ndash326) 460 (450ndash471) 15Bendiocarb (01) 187 (181ndash193) 565 (543ndash592) 3DDT (4) 223 (217ndash229) AbengourouDeltamethrin (005) 217 (207ndash226) 802 (729ndash922) 37Malathion (5) 314 (299ndash329) 337 (329ndash344) 11Bendiocarb (01) 282 (269ndash295) 376 (337ndash452) 29DDT (4) 223 (217ndash229) AgbovilleDeltamethrin (005) 217 (207ndash226) 9932 (855ndash1228) 46Malathion (5) 282 (269ndash295) 619 (578ndash678) 2Bendiocarb (01) 282 (269ndash295) 4193 (2247ndash14923) 149DDT (4) 223 (217ndash229) San-PedroDeltamethrin (005) 217 (207ndash226) 8345 (751ndash978) 39Malathion (5) 316 (305ndash326) 3368 (329ndash344) 11Bendiocarb (5) 255 (231ndash277) 376 (337ndash451) 15DDT (4) 223 (217ndash229) YamoussoukroDeltamthrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 724 (679ndash780) 23Bendiocarb (01) 187 (181ndash193) 646 (614ndash687) 35DDT (4) 223 (217ndash229) DabouDeltamethrin (005) 319 (258ndash385) 1119 (886ndash1603) 35Malathion (5) 316 (305ndash326) 66 (622ndash710) 21Bendiocarb (01) 187 (181ndash193) 724 (656ndash824) 39DDT (4) 223 (217ndash229) MBeDeltamethrin (005) 297 (298ndash305) Malathion (5) 316 (305ndash326) 729 (688ndash791) 23Bendiocarb (01) 187 (181ndash193) 703 (667ndash756) 38DDT (4) 223 (217ndash229) TiassaleDeltamethrin (005) 217 (207ndash226) 2204 (1516ndash4317) 102Malathion (5) 314 (299ndash329) 634 (605ndash669) 2Bendiocarb (01) 282 (269ndash295) 1235 (948ndash2274) 44DDT (4) 223 (217ndash229) ToumodiDeltamethrin (005) 319 (258ndash385) 2992 (1556ndash25214) 94Malathion (5) 316 (305ndash326) 554 (541ndash670) 18Bendiocarb (01) 187 (181ndash193) 507 (488ndash629) 27DDT (4) 223 (217ndash229)

6 BioMed Research International

0 25 50 100 150 200

Kilometers

An coluzziiAn gambiaeHybrid

N

Figure 3 Proportion of different species by locality

Table 2 Allelic frequency of different mutational genes in malariavectors in 10 localities in Cote drsquoIvoire The allelic frequency is therate of presence of the resistance allele within the vector populations

Localities West-KdrFreq(R)

East-KdrFreq(R)

Ace-1Freq(R)

Azaguie 092 0 033Tiebissou 07 003 013Toumodi 08 0 033Mbe 077 0 013Yamoussoukro 096 0 032Abengourou 1 0 020San-Pedro 093 0 007Agboville 079 0 05Tiassale 07 0 04Dabou 09 0 043

across the country may be attributed to both the strongresidues of this product in the nature [41] having beenused in the past and the cross-resistance between DDT andpyrethroids which were shown to account for more than 90of chemical used by farmers [12]

InTiebissou a singleAn gambiae ssmosquitowas foundto possess the kdr-West (L1014F) kdr-East (L1014S) and Ace-1(G119S)mutations simultaneouslyThe co-occurrence of boththe kdr mutations (L1014FS) has been reported from WestCentral and also East Africa [42ndash44]However this is the firsttime these three alleles associated with insecticide resistancewere found in a single specimen in Cote drsquoIvoire

The presence of hybrids is a very serious threat to vectorcontrol as this can favour the spread of these traits Henceour research highlights the urgent need for a compound witha novel mode of action in addition to those already existingto eliminate such individuals within the vector population

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

6 BioMed Research International

0 25 50 100 150 200

Kilometers

An coluzziiAn gambiaeHybrid

N

Figure 3 Proportion of different species by locality

Table 2 Allelic frequency of different mutational genes in malariavectors in 10 localities in Cote drsquoIvoire The allelic frequency is therate of presence of the resistance allele within the vector populations

Localities West-KdrFreq(R)

East-KdrFreq(R)

Ace-1Freq(R)

Azaguie 092 0 033Tiebissou 07 003 013Toumodi 08 0 033Mbe 077 0 013Yamoussoukro 096 0 032Abengourou 1 0 020San-Pedro 093 0 007Agboville 079 0 05Tiassale 07 0 04Dabou 09 0 043

across the country may be attributed to both the strongresidues of this product in the nature [41] having beenused in the past and the cross-resistance between DDT andpyrethroids which were shown to account for more than 90of chemical used by farmers [12]

InTiebissou a singleAn gambiae ssmosquitowas foundto possess the kdr-West (L1014F) kdr-East (L1014S) and Ace-1(G119S)mutations simultaneouslyThe co-occurrence of boththe kdr mutations (L1014FS) has been reported from WestCentral and also East Africa [42ndash44]However this is the firsttime these three alleles associated with insecticide resistancewere found in a single specimen in Cote drsquoIvoire

The presence of hybrids is a very serious threat to vectorcontrol as this can favour the spread of these traits Henceour research highlights the urgent need for a compound witha novel mode of action in addition to those already existingto eliminate such individuals within the vector population

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

BioMed Research International 7

Although Ace-1 mutation can lead to resistance to bothcarbamates and organophosphates the high resistance levelto carbamates in one hand and low resistance to organophos-phates in another hand found in the current study suggeststhat the resistance mechanism to these compounds involvedother pathways and requires further investigations

The overall situation of resistance as observed in the cur-rent study no longer allows dissociating the impact of publichealth insecticides to that of the agricultural pesticide usagesYet this knowledge is key to evaluate the actual contributionof each domain to the selection of resistant individuals owingto the strong focus on vector control in current malariacontrol programmes In addition until specific insecticidesdedicated solely to vector control are available combinedmanagement policies need to be established to deal withresistance of vectors to these same set of compounds used inagricultural and for public health purpose

All the actors involved in vector control have unani-mously agreed on the need of an adequate resistance man-agement practices yet most programme or project activitiesare limited to suggestionswithout practical fieldmanagementactionsThe proof of the extent of the resistance phenomenonmust be conducive to raise awareness and training of farmersand users of public health insecticides for better managementof insecticide resistance To this end lsquofield schoolrsquo projects totrain farmers on the proper way to use the pesticides shouldbe initiated in areas of intense cultivation

In a context of resistance generalization a debate onthe usefulness of continued implementation of LLINs arisesAlthough LLINs provide personal protection the strong lev-els of resistance observed with pyrethroids used for impreg-nating bed nets may put communities at risk owing to theincrement of resistance selection By eliminating susceptibleindividuals the propagation of resistance genes may beintensified by the multiplication of resistant individuals Areintroduction of no insecticide impregnated bed nets maybe therefore preferable in such setting providing personalprotection in the same way as impregnated LLINs but withthe advantage of not selecting for mutant genes The use oforganophosphates in the current context in IRS may be agood alternative considering the susceptibility of the vectorsto malathion There is a real danger that resistance if notmonitored closely may lead to a loss of all the gains madein recent decades in malaria control notably the fall of theglobal burden of malaria by nearly 29 [45] It is therefore ofutmost importance to take appropriate and context-specificmeasures

5 Conclusions

This study revealed strong resistance of malaria vectors todeltamethrin DDT and bendiocarb in 10 varying agrochem-icals use areas in Cote drsquoIvoire Malathion induced highmortality in most localities but vector resistance against thisinsecticide observed in Dabou and Agboville may be thebeginning of widespread resistance in the near future if noappropriate management measures are taken

In view of the abundance of cultivated marshlands andtheir intense use of pesticides special attention must be givento them to prevent the emergence of resistant mosquitoes

The strong resistance to pyrethroids observed and thehigh frequency of resistance genes may hinder the effective-ness of mosquito nets However organophosphates appearsto be a good alternative for vector control The identificationof individual mosquitoes possessing all three mutation-typeresistance genes appears to be an additional threat to currentvector control tools if this trait were to spread more widelyand urges close surveillance

Abbreviations

Ace-1 Insensitive acetylcholinesteraseCTAB Cetyl trimethyl ammonium bromideDDT DichlorodiphenyltrichloroethaneDNA Deoxyribonucleic acidGPIRM Global Plan for Insecticide Resistance

ManagementIRS Indoor residual sprayingKdr Knockdown resistanceKdT50 Knockdown time of 50LLINs Long lasting insecticidal netsPCR Polymerase chain reactionRR Resistance ratioWHO World Health Organization

Data Availability

Data generated or analyzed during this study are included inthis published article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Behi K Fodjo carried out the field sampling and labo-ratory analysis and drafted the manuscript Benjamin GKoudou supervised laboratory analysis and the whole studyEmmanuel Tia supervised field sampling Jasmina Saricrevised the manuscript and offered editorial support NestorB Kesse and Prisca B Nrsquodri helped with the field samplingand Alida Kropf and Christabelle S Gba and Marius GZoh helped with the laboratory analyses Mouhamadou SChouaıbou conceived and initiated the study and helped draftthe manuscript All authors have seen and approved that lastdraft All authors read and approved the final version of themanuscript

Acknowledgments

The authors thank Dr Yapi Richard CSRS researcher forhis support in the design of the map This study was fundedthrough the Wellcome Trust Project Ref 103995

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

8 BioMed Research International

Supplementary Materials

Thefirst 40 tables are themosquito susceptibly test data sheetsfrom Abengourou Abidjan Agboville Azaguie DabouMrsquobe San-Pedro Soubre Tiassale Tiebissou Toumodiand Yamoussoukro localities to deltamethrin bendiocarbmalathion and DDT Table 41 is the mutation data sheetsAnd the last table is the proportion of different insecticidefamilies used in the ten localities (Supplementary Materials)

References

[1] H Yassine and M A Osta ldquoAnopheles gambiae innate immu-nityrdquo Cellular Microbiology vol 12 no 1 pp 1ndash9 2010

[2] WHO Insecticides resistance World Health OrganizationGeneva 2015

[3] P L Alonso G Brown M Arevalo-Herrera et al ldquoA ResearchAgenda to Underpin Malaria Eradicationrdquo PLoS Medicine vol8 no 1 p e1000406 2011

[4] K G Konan A B Kone Y L Konan et al ldquoResistancedrsquoAnopheles gambiae sl aux pyrethrinoıdes et au DDT aTiassalekro village de riziculture irriguee en zone sud forestierede Cote-drsquoIvoirerdquo Bulletin de la Societe de Pathologie Exotiquevol 104 no 4 pp 303ndash306 2011

[5] WHOGlobal plan for insecticide resistance management WorldHealth Organization Geneva 2012

[6] WHO World malaria report World Health OrganizationGeneva 2011

[7] M C Akogbeto and H Djouaka Noukpo ldquoUtilisation desinsecticides agricoles au Beninrdquo Entomologie Medicale vol 98no 5 pp 400ndash405 2005

[8] A Philbert S L Lyantagaye and G Nkwengulila ldquoA Review ofAgricultural Pesticides Use and the Selection for Resistance toInsecticides in Malaria Vectorsrdquo Advances in Entomology vol02 no 03 pp 120ndash128 2014

[9] M B M Cisse C Keita A Dicko et al ldquoCharacterizing theinsecticide resistance of Anopheles gambiae in Malirdquo MalariaJournal vol 14 no 1 2015

[10] MC Reid andF EMcKenzie ldquoThe contribution of agriculturalinsecticide use to increasing insecticide resistance in Africanmalaria vectorsrdquoMalaria Journal vol 15 no 1 2016

[11] C V A Edi B G Koudou C M Jones D Weetman and HRanson ldquoMultiple-insecticide resistance in Anopheles gambiaemosquitoes Southern Cote drsquoIvoirerdquo Emerging Infectious Dis-eases vol 18 no 9 pp 1508ndash1511 2012

[12] L Djogbenou ldquoVector control methods against malaria andvector resistance to insecticides in Africardquo Medecine TropicaleRevue du Corps de Sante Colonial vol 69 no 2 pp 160ndash1642009

[13] J Dossou-Yovo J M C Doannio S Diarrassouba andG Chauvancy ldquoDiarrassouba S Chauvancy G Impactdrsquoamenagements de rizieres sur la transmission du paludismedans la ville de Bouake Cote drsquoIvoirerdquo Bulletin de la Societe dePathologie Exotique vol 91 no 4 pp 327ndash333 1998

[14] A M Kouassi ldquoCaracterisation drsquoune modification eventuellede la relation pluie-debit en Afrique de lrsquoOuest cas du bassinversant du Nrsquozi (Bandama) en Cote drsquoIvoireThese de DoctoratUniversite de Cocody-Abidjan Cote drsquoIvoire 210 prdquo Physio-Geo no Volume 4 pp 3-4 2010

[15] A Nrsquogo A Kouadi J Deguy A Hien A Goula and I SavaneldquoInfluence de la dynamique de lrsquooccupation du sol sur la quantite

de perte de sol au sud du bassin versant du sassandra (cotedrsquoivoire)rdquo International Journal of Advanced Research vol 6 no4 pp 830ndash838 2018

[16] B Matthys E K NrsquoGoran M Kone et al ldquoUrban agriculturalland use and characterization of mosquito larval habitats in amedium-sized town of Cote drsquoIvoirerdquo Journal of Vector Ecologyvol 31 no 2 pp 319ndash333 2006

[17] D A Lalithanjalie and G I S W Weerakkodi ldquoDensity anddiversity of mosquito larvae associated with rice field andmarshland habitats in two climatically different areas in SriLanka Intrdquo Journal of the Entomological Research Society vol02 no 02 pp 59ndash71 2014

[18] J Chabi P K Baidoo A K Datsomor et al ldquoInsecticidesusceptibility of natural populations of Anopheles coluzzii andAnopheles gambiae (sensu stricto) from Okyereko irrigationsite Ghana West Africardquo Parasites amp Vectors vol 9 no 1 2016

[19] A W M Yadouleton A Asidi R F Djouaka J Brama CD Agossou and M C Akogbeto ldquoDevelopment of vegetablefarming A cause of the emergence of insecticide resistance inpopulations of Anopheles gambiae in urban areas of BeninrdquoMalaria Journal vol 8 no 1 article no 103 2009

[20] A Yadouleton T Martin G Padonou et al ldquoCotton pest man-agement practices and the selection of pyrethroid resistance inAnopheles gambiae population in Northern Beninrdquo Parasites ampVectors vol 4 no 1 2011

[21] T E Nkya I AkhouayriW Kisinza and J-P David ldquoImpact ofenvironment on mosquito response to pyrethroid insecticidesfacts evidences and prospectsrdquo Insect Biochemistry and Molec-ular Biology vol 43 no 4 pp 407ndash416 2013

[22] M S Chouaıbou B K Fodjo G Fokou et al ldquoInfluenceof the agrochemicals used for rice and vegetable cultivationon insecticide resistance in malaria vectors in southern CotedrsquoIvoirerdquoMalaria Journal vol 15 no 1 article no 426 2016

[23] S Dongus D Nyika K Kannady et al ldquoUrban agricultureand Anopheles habitats in Dar es Salaam Tanzaniardquo GeospatialHealth vol 3 no 2 pp 189ndash210 2009

[24] M J Vanek B Shoo D Mtasiwa et al ldquoCommunity-basedsurveillance of malaria vector larval habitats A baseline studyin urban Dar es Salaam Tanzaniardquo BMC Public Health vol 62006

[25] S M Muriu E J Muturi J I Shililu et al ldquoHost choice andmultiple blood feeding behaviour of malaria vectors and otheranophelines in Mwea rice scheme KenyardquoMalaria Journal vol7 article no 43 2008

[26] D S Luc A Benoit D Laurette and M Michel ldquoIndirectevidence that agricultural pesticides select for insecticide resis-tance in the malaria vector Anopheles gambiaerdquo Journal ofVector Ecology vol 41 no 1 pp 34ndash40 2016

[27] T ENkya R Poupardin F Laporte et al ldquoImpact of agricultureon the selection of insecticide resistance in the malaria vectorAnopheles gambiae A multigenerational study in controlledconditionsrdquoParasitesampVectors vol 7 no 1 article no 480 2014

[28] F Chandre F Darrier L Manga et al ldquoStatus of pyrethroidresistance in Anopheles gambiae sensu latordquo Bulletin of theWorld Health Organization vol 77 no 3 pp 230ndash234 1999

[29] T S Awolola I O Oyewole C N Amajoh et al ldquoDistributionof the molecular forms of Anopheles gambiae and pyrethroidknock down resistance gene in Nigeriardquo Acta Tropica vol 95no 3 pp 204ndash209 2005

[30] J Etang E Fondjo F Chandre et al ldquoShort report First reportof knockdown mutations in the malaria vector Anopheles

BioMed Research International 9

gambiae from Cameroonrdquo The American Journal of TropicalMedicine and Hygiene vol 74 no 5 pp 795ndash797 2006

[31] J Pinto A Lynd N Elissa et al ldquoCo-occurrence of East andWest African kdr mutation suggests high levels of pyrethroidinsecticides in Anopheles gambiae from Libreville GabonrdquoMedical and Veterinary Entomology vol 20 no 1 pp 27ndash322006

[32] I Djegbe O Boussari A Sidick et al ldquoDynamics of insecticideresistance in malaria vectors in Benin First evidence of thepresence of L1014S kdr mutation in Anopheles gambiae fromWest AfricardquoMalaria Journal vol 10 2011

[33] M Chouaıbou F B Kouadio E Tia and L Djogbenou ldquoFirstreport of the East African kdr mutation in an Anophelesgambiae mosquito in Cote drsquoIvoirerdquo Wellcome Open Researchvol 2 2017

[34] V Katrijn V B Wim R Patricia B Thierry and C MarcldquoDetection of the East and West African kdr mutation inAnopheles gambiae and Anopheles arabiensis from Ugandausing a new assay based on FRETMelt Curve analysisrdquo inMalaria Journal vol 5 p 16 2006

[35] WHO ldquoGlobal technical strategy for malaria 2016-2030rdquo TechRep World Health Organization Geneva 2015

[36] WHO Test procedures for insecticide resistance monitoring inmalaria vectormosquitoesWorldHealthOrganizationGeneva2013

[37] F H Collins M A Mendez M O Rasmussen P C MehaffeyN J Besansky and V Finnerty ldquoA ribosomal RNA geneprobe differentiates member species of the Anopheles gambiaecomplexrdquo The American Journal of Tropical Medicine andHygiene vol 37 no 1 pp 37ndash41 1987

[38] F Santolamazza EMancini F Simard YQi Z Tu andADellaTorre ldquoInsertion polymorphisms of SINE200 retrotransposonswithin speciation islands of Anopheles gambiae molecularformsrdquo Malaria Journal vol 7 article no 163 2008

[39] Chris Bass Dimitra Nikou John Vontas M J Donnelly M SWilliamson and L M Field ldquoThe Vector Population Monitor-ing Tool (VPMT) High-Throughput DNA-Based Diagnosticsfor the Monitoring of Mosquito Vector Populationsrdquo MalariaResearch and Treatment vol 2010 Article ID 190434 8 pages2010

[40] M S Chouaibou J Chabi G V Bingham et al ldquoIncrease insusceptibility to insecticides with aging of wild Anopheles gam-biaemosquitoes from Cote drsquoIvoirerdquo BMC Infectious Diseasesvol 12 no 1 2012

[41] P Menchai L Van Zwieten S Kimber N Ahmad P S CRao and G Hose ldquoBioavailable DDT residues in sedimentsLaboratory assessment of ageing effects using semi-permeablemembrane devicesrdquo Environmental Pollution vol 153 no 1 pp110ndash118 2008

[42] RKDabireMNamountougouADiabate et al ldquoDistributionand frequency of kdr mutations within Anopheles gambiaesl populations and first report of the Ace1G119S mutation inAnopheles arabiensis from Burkina Faso (West Africa)rdquo PLoSONE vol 9 no 7 2014

[43] J Pinto A Lynd N Elissa et al ldquoCo-occurrence of East andWest African kdr mutations suggests high levels of resistance topyrethroid insecticides in Anopheles gambiae from LibrevilleGabonrdquo Medical and Veterinary Entomology vol 20 no 1 pp27ndash32 2006

[44] K Verhaeghen W Van Bortel P Roelants T Backeljau andM Coosemans ldquoDetection of the East and West African kdr

Mutation in Anopheles gambiae and Anopheles arabiensisfrom Uganda Using a New Assay Based onFRETMelt CurveAnalysisrdquoMalaria Journal vol 5 article no 16 2006

[45] WHO World Malaria Report World Health OrganizationGeneva 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom