INSECTICIDEMIXTURESFOR MOSQUITO NET IMPREGNATIONAGAINST MALARIAVECTORS

CORBEL V.*, DARRIET F.*, CHANDRE F.** & HOUGARD J,M.*

Summary:

Insecticides belonging to the pyrethroid family are the only

,çpmpounds currently available for the treatment of mosquito nets.Unfortunately, some malaria vector species have developedresistance to pyrethroids and the jack of alternative chemicalcategories is a great concern. One strategy for resistancemanagement would be to treat mosquito nets with a mixtureassociating Iwo insecticides having different modes of action. Thisstudy presents the results obtained with insecticide mixturescontaining several proportions of bifenthrin (a pyrethroidinsecticide! and carbosulfan (a carbamate insecticide). Themixtures were sprayed on mosquito net samples and their efficacywere tested against a susceptible strain of Anophe/es gambiae,the major malaria vectar in Africa. A significant synergism wasobserved with a mixture containing 25 mg/m2 of bifenthrin (halfthe recommended dosage for treated nets! and 6.25 mg/m2 ofcarbosulfan (about 2 % of the recommended dosage). Theobserved mortality was significantly more thon expected in theabsence of any interaction (80 % vs 41 %) and the knock-downeffect was maintained, providing an effective barrier againstsusceptible mosquitoes.

KEYWORDS: insecticidemixture,synergy,treatedmasquitonet, Anophelesgambiae.

INTRODUCTION

nsecticide-treated mosquito nets are strongly recom-mended for malaria contrai, especiaUy in Africancountries which are severely affected by malaria

(Line, 1996). Insecticides belonging to the pyrethroidfamily are the only compounds currently available forthe treatment of mosquito nets. They ensure safe andeffective protection because they act very rapidly, withbath knockdown and lethal effects at dosages farbelow the threshold of mammalian toxicity (Zaim etal., 2000). Unfortunately, pyrethroid resistance hasalready developed in Saille populations of the major

* !RD, 911, avenue Agropolis, BP 64501, 34394 Montpellier cedex 5,France.

'* CPR, 01 BP 1500, Bouaké 01, Côte d'Ivoire.

Correspondence: Jean-Marc Hougard, LIN/!RD, 911, avenue Agro-polis, BP 64501, 34394 Montpellier Cedex 5, France.Tel.: 33 (0)4 67 04 32 23 - Fax: 33 (0)4 67 54 20 44.

Parasite, 2002, 9, 255-259

Résumé: LESMÉLANGESINSECTICIDESEN IMPRÉGNATIONDEMOUSTIQUAIRESCONTRE LES VECTEURSDU PALUDISME

Les pyréthrinoides sont actuellement les seuls insecticidesrecommandés pour l'imprégnation des moustiquaires.

Malheureusement, 10 résistance aux pyréthrinoides est déjàapparue chez plusieurs espéces de vecteurs du paludismemenaçant ainsi l'efficacité de cette méthode de lutte De plus,l'absence d'insecticide de remplacement pour l'imprégnation des

moustiquaires reste un fait préoccupant. Une stratégie de gestionde la résistance consisterait à associer sur une moustiquaire deuxinsecticides oyant des modes d'action différents. Cette étudeprésente les résultats obtenus avec des moustiquaires imprégnéesavec différentes proportions de bifenthrine (un pyréthrinoide) et decarbosulfan (un carbamate). L'efficacité de ces moustiquaires bi-imprégnées a été testée sur des moustiques adultes sensiblesd'Anopheles gambiae, principal vecteur du paludisme en Afrique.Un effet de synergie significatif a été détecté avec un mélangecomposé de 25 mg/m2 de bifenthrine (la moitié de la doseopérationnelle) et 6,25 mg/m2 de carbosulfan (2 % de la doseopérationnelle). La mortalité observée a été significativementsupérieure à celle attendue par sommation des effets (80 % contre41 %)et l'effet knock down a été maintenu, préservant ainsi uneborriére efficace contre les moustiques.

MOTSCLÉS: mélange insecticides. synergie, moustiquaire imprégnée,Anophelesgambiae.

malaria vectors, particularly Anopheles gambiae s.s. intropical Africa (Chandre et al., 1999). The lack of analternative chemical category could jeopardize theeffectiveness of treated mosquito nets against malariatransmission.

One strategy proposed for resistance management isthe use of mixtures of pesticides with different modeof action (Tabashnik, 1989; Roush, 1993). This strategyimply that no cross resistance exist between the insec-ticides. For treated mosquito nets, this could be aninsecticide mixture combining a pyrethroid insecticidewith a non-pyrethroid insecticide such as carbamatesor organophosphates. The use of an insecticide mix-ture is aU the more promising if there is a positive inter-action between the two components. This has beenobserved in crop protection and veterinary use withmixtures of pyrethroids with organophosphates or car-bamates (Ali et al., 1977; Huges & Trevethan 1979;Koziol & Witkowski, 1982; Campanhola & Plapp, 1989;Christian et al., 1986). A synergy has also been

255

observed recently between a pyrethraid and a carba-mate on susceptible and resistant larvae of Culex quin-quefasciatus, a mosquito of medical importance (Cor-bel et al., 2002).

The present study is aimed to mosquito nets samplesimpregnated with insecticide mixtures combining lowdoses of pyrethraid and carbamate formulations ("bi-treated" mosquito nets). Their efficacy were comparedto "mono-treated" net samples on a susceptible strainof Anopheles gambiae s. s. Carbosulfan (2,3-dihydra-2,2-dimetyl- 7-benzofuranyl [(dibutylamino)thio] methyl-carbamate) has been selected among carbamates. lncontrast to pyrethraids and DDT, this insecticide familydoes not induce any knock-down effect. Carbosulfanis currently used for public health despite its relativetoxicity to mammals because it has shawn a good effi-cacy in net impregnation at 300 mg/m2, particularlyagainst pyrethraid-resistant populations of Anophelesand Culex mosquitoes (Kolaczinski et al., 2000). Bifen-thrin (2 methyl [1,1'-bipheny!]-3-yl)methyl 3-(2-chlora-3,3,3-trifluora-1-prapenyl)-2,2-dimethyl-cycloprapane-carboxylate) has been selected among pyrethraids, asit is potentially a good candidate insecticide for malariaprevention. It is used world wide against a wide rangeof agricultural pests and has already been tested suc-cessfully under laboratory conditions against malariavectors (Hougard et al., 2002). Preliminary field testshave confirmed its efficacy against pyrethraid-resistantpopulations of A. gambiae and C. quinquefasciatus at50 mg/m2 (Guillet et al., 2001).

MATERIALAND METHODS

ests were conducted using Kisumu, a fully sus-ceptible reference strain of A. gambiae origi-nated fram Kenya and initially maintained under

standard laboratory conditions (27 :t 2° C and 80 %relative humidity).The net samples were impregnated with commercialformulations. Bifenthrin was formulated as a suspen-sion concentrate (SC) containing 8 % active ingredient(Talstar@ 80 SC, FMC, Philadelphia, USA). Carbosulfanwas formulated as a micracapsule suspension contai-ning 2.5 % active ingredient (Marshall@ 25 CS, FMC, Phi-ladelphia, USA).The efficacy of insecticides, alone or in association, wasassessed according to the cane test method (Anony-mous WHO, 1998). Five unfed females aged fram twoto five days were placed in a plastic cane that waspositioned over treated net sample. After three minutesthe mosquitoes were removed fram the net sample,placed in 150 ml plastic clipS and supplied with a 10 %honey solution as a food source. The mosquitoes wereheld for 24 hours in an enviranmental chamber at 27° C

:t 2° C and 80 % :t 10 % RH. Each replicate consisted

of three-four canes and each test sample was replicatedthree times. The percentage of knocked clown mos-quitoes was noted after a three minutes exposure onthe treated net, at fixed time intervals, varying betweentwo to 10 minutes according to the intensity of theknock clown effect. The percentage mortality wasrecorded after 24 hours.

Data were analyzed using a log-prabit pragram basedon Finney (1971). The statistical analysis calculated therates at which 50 % and 95 % of mosquitoes wouldbe knock clown (respectively: KDtso and KDt9S)' Thedifference between two KDtso or two KDt9Svalues wasconsidered as statistically significant only if there is nooverlap of their confidence limits. Each test was car-ried out with an untreated contrai mosquito net. If thecontrai mortality was above 5 %, the "observed" mor-tality is adjusted by Abbott's formula (Abbott, 1925).Percent mortalities were compared using the Chisquare test at a 95 % Confidence Interval.The netting material was a multifilament polyester 100denier (weight in grams of 9.000 m yarn), mesh sile156 (25 holes/cm2), fram Siamdutch (Bangkok, Thai-land). For treatment, pieces of netting were folded inthree equal parts and put in a disposable petri dish.A volume of formulation suspension corresponding tospecifie absorbency of the net and prepared imme-diately before treatment was drapped regularly ontothe surface. Net were then carefully soaked with fin-gers (fitted with plastic gloves) in order to ensure aregular distribution of the solution. Once impregnated,nets were kept to dry in the box. Tests were made fivedays after treatment in order to avoid testing depositsof markedly different ages, which might have differentimpact on mosquito behaviour and affect the results.

We tested on Kisumu strain different impregnated netswith the insecticides alone and in mixtures with the

objectives to find the better insecticide combination in

term of KdTso/9s and mortality. Five samples of mos-Quito net were used as reference samples: two withbifenthrin alone at 25 and 12.5 mg/m2, three with car-bosulfan alone at 100, 25 and 6.25 mg/m2, and onewith no insecticide (contrai sample). Six other sampleswere impregnated with different bifenthrin 1 carbo-sultan mixtures using the five doses enumerated above.These dosages have been selected as they inducedseparately a low range of mortality (5 % - 40 %), thusallowing a better detection of any synergism effect(Table O. By comparing the results observed witheach mixture with the results theoretically expected inthe absence of any interaction (un-correlated jointaction), it was possible to determine the effect of suchinsecticide mixtures on adult mosquitoes The expectedmortality was calculated by multiplying the percentagesurvival on each insecticide tested separately and sub-tracting the result fram 100 %. ln the same way, theKDt theoretically expected for a given insecticide mix-

ture, in the absence of any interaction, is equal ta thesum of the KDt of each insecticide. There is said ta

be synergy when the observed results are significantlyhigher than the expected results (positive interaction).There is said ta be antagonism when the observedresults are significantly lower than the expected results(negative interaction).

RESULTS

allies shawn in Table 1 were not adjusted Silicethe contraI mortality was very low (no KDeffect was observed witt the untreated

samples). The result obtained witt carbosulfan aloneconfirmed the absence of KD effect that characterise

the carbamate family, whatever the dosages tested.Results witt bifenthrin alone confirmed the existence

a KD effect, but there was no apparent dose/effect cor-relation Silice an overlap of the confidence intervals

was observed for KDtso as well as for KD~s' Witt theselow concentrations, mortality levels recorded for eachinsecticide used separately were low (maximum 38 %for bifenthrin and 15 % for carbosulfan).

Witt insecticide mixtures, a reduction of the KD effectwas noted when the dosage of carbosulfan increasedand the dosage of bifenthrin decreased. This antago-nism was more evident at the KDt9Slevels, particularlywitt mixtures containing the lower dosage of bifen-thrin 02.5 mg/m2) and the higher dosage of carbo-sulfan 000 mg/m2): the speed of the KD effect wasalmost halved (KDtso: 12.2 min ; KD~s: 50.6 min) com-pared witt that observed witt bifenthrin alone at12.5 mg/m2 (KDtso: 6.8 min; KDt9S: 26.0 min). Conver-sely, a very significant synergy (P < 0.0001) was

observed witt the insecticide mixture containing thelower dosage of carbosulfan (6.25 mg/m2) and thehigher dosage of bifenthrin (25 mg/m2): the percen-rage mortality observed witt the mixture (80% ) wastwice the expected one in the absence of any inter-action (41.1 %). Except for 12.5 mg/m2 bifenthrin and100 mg/m2 carbosulfan, where a slight synergy wasobserved, no significant difference was noted witt theother insecticide mixtures between the observed and

expected mortalities.

DISCUSSION

The lack of knock clown effect at low dosages of car-bosulfan was confirmed. Results observed witt the dif-

ferent mixtures showed that carbosulfan, at a relativelyhigh dosage 000 mg/m2) , could act as an inhibitor ofthe KD effect of pyrethraids. The irritant praperties ofcarbosulfan against Anopheles and Mansonia specieswas demonstrated by Klein et Darriet 0989) by housespraying of experimental huts. At a relatively highdosage, the irritant effect of carbosulfan could decreasethe duration of tarsal contact witt the substrate, thusreducing the KD effect of bifenthrin.Regarding mortality, a very significant synergy (P <0.0001) was noted at the highest dosage of bifenthrin(25 mg/m2) and the lowest of carbosulfan (6.25 mg/m2).Although these insecticides do not have the sametarget sites, it is likely that their combination resultedin a complex molecular interaction. Pyrethraids act onthe nervous system by modifying the gating kineticsof voltage-sensitive sodium channels (Lund & Nara-hashi, 1983). As witt the other pyrethraids, bifenthrinkeeps the sodium channels open temporarily, thus cau-

N, number tested. CI, 95 % confidence interval. Percent mortalities were compared using a Chi square test at 95 % confidence interval.

Table I. - Compared activity of bifenthrin and carbosulfan formulations, alone and in association, against susceptible Anopheles gambiae.

KDtso (min) KDs (min) Mortality (%)

Insecticide N Observed CI Expected Observed CI Expected Observed Expected X2 P

Bifenthrin

25 mg/m2 61 6.0 5.2-6.8 26.8 21.2-37.8 38012.5 mg/m2 58 6.8 6.0-7.6 26.0 21.5-33.7 26.0

Carbosulfan

100 mg/m2 51 no efect no effect 15025 mg/m2 52 no effect no effect 4.0

6.25 mg/m2 50 no effcet no effcet 50

Bifenthrin 25 mg/m2+ Carbo. 100 mg/m2 59 8.0 69-90 6.0 42.8 34.1-58.6 26.8 370 473 1.2 > 0.05+ Carbo. 25 mg/m2 62 5.7 4.8-6.6 6.0 30.2 23.9-42.3 26.8 530 40.5 2.1 > 0.05+ Carbo. 6.25 mg/m2 60 54 4.6-6.1 6.0 20.7 17.6-25.6 26.8 80.0 41.1 18.5 < 0.05

Bifenthrin 12.5 mg/m2+ Carbo. 100 mg/m2 64 12.2 11.1-134 6.8 50.6 41.1-68.1 26.0 56.0 37.1 4.5 < 0.0001+ Carbo. 25 mg/m2 63 7.7 6.8-8.4 6.8 26.2 22.5-321 26.0 35.0 29.0 0.6 > 0.05+ Carbo. 6.25 mg/m2 63 6.1 5.3-7.0 6.8 29.3 24.8-424 26.0 46.0 29.7 3.4 > 0.05

sing repeated dis charges of action potentials whichincrease the amount of acetylcholine (ACh) in thesynapses (Salgado, 1983). Carbosulfan, as with theother carbamates, is an inhibitor of acetylcholineterase(AChE). Such inhibition also contributes to increased

ACh concentration at level which cou Id explain thesynergy observed. The absence of synergy at higherconcentrations of carbosulfan could be due either to

the shorter contact between mosquitoes and impre-gnated nets, as observed by reduced KD effect, or toa secondary target site inhibition as Choline Acetyl-transferase (ChAT). ChAT is a protein implicated in syn-thesis of ACh and is a secondary target for carbamates(Bourguet et al., 1997). When adding a higher dosageof carbosulfan (100 mg/m2), ChAT could become an

insecticide target, leading to a decrease of ACh pro-duction which could balance the accumulation of AChdue to the other mechanisms.

Because carbamates are patent inhibitors of ACh andbecause carbosulfan degrades to a more toxic com-pound (carbofuran) (2,3-dihydro-2,2-dimethyl-n- 7-ben-zofuranyl methylcarbamate) by the loss of the (dibu-tylamino)thio group, the toxicological risk of their useon mosquito nets has to be carefully considered in rela-tion to human safety (Anonymous WHO, 1986). It isencouraging that the optimum interaction obtained inthe susceptible Kisumu strain was with the lower doseof carbosulfan (about 50 rimes less than the operationaldose). However, an insecticide mixture must be consi-

dered as a new compound that needs a long series oftoxicological studies before it would be cleared for use

on mosquito nets (Zaim et al., 2000). Another impor-tant advantage with this ratio was the absence of anyantagonism of the KD effect that allows the mixture

to keep its fast acting properties on nets and continueto provide an effective barrier against susceptible mos-quitoes (Miller & Gibson, 1994).

Preliminary field studies in experimental huts havebeen carried out in Côte d'Ivoire by using a "two inone" combination of bifenthrin and carbosulfan appliedto different parts of the saille bednet (Guillet et al.,2001). Promising results have been obtained against thelocal pyrethroid resistant populations of A. gambiae s. s.and C. quinquefasciatus. This study will be com-pleted in the saille area by using mosquito nets treatedwith a mixture of bifenthrin and carbosulfan at an

optimum ratio giving mortality LInder laboratory condi-tions.

ACKNOWLEDGEMENTS

e thank the Ministère français de la recher-che on malaria and associated communi-

cable diseases for developing countries

(PAt+ program) for funding this work and the FMC Cor-poration for providing the formulations of bifenthrinand carbosulfan.

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Reçu le 15 avril 2002Accepté le 13 mai 2002