I
nsecticide m ix t u r es fo r m o s q u it o net im p r e g n a t io nAGAINST MALARIA VECTORS
CORBEL V.*, DARRIET F.*, CHANDRE F.** & HOUGARD J.M .*
Sum mary:
Insecticides belonging to the pyrethroid family are the only compounds currently available for the treatment of mosquito nets.
Unfortunately, some malaria vector species have developed resistance to pyrethroids and the lack of alternative chemical categories is a great concern. One strategy for resistance management would be to treat mosquito nets with a mixture associating two insecticides having different modes of action. This study presents the results obtained with insecticide mixtures containing several proportions of bifenthrin (a pyrethroid insecticide) and carbosulfan (a carbamate insecticide). The mixtures were sprayed on mosquito net samples and their efficacy were tested against a susceptible strain of Anopheles gambiae, the major malaria vector in Africa. A significant synergism was observed with a mixture containing 25 m g /m 2 of bifenthrin (half the recommended dosage for treated nets) and 6 .2 5 m g /m 2 of carbosulfan (about 2 % of the recommended dosage). The observed mortality was significantly more than expected in the absence of any interaction (80 % vs 41 %) and the knock-down effect was maintained, providing an effective barrier against susceptible mosquitoes.
KEY WORDS : insecticide mixture, synergy, treated mosquito net, Anopheles gambiae.
INTRODUCTION
I
nsecticide-treated mosquito nets are strongly recom m ended for malaria control, especially in African countries w hich are severely affected by malaria (Line, 1996). Insecticides belonging to the pyrethroid family are the only com pounds currently available for the treatment o f m osquito nets. They ensure safe and effective protection because they act very rapidly, with both knockdow n and lethal effects at dosages far below the threshold o f mammalian toxicity (Zaim et al., 2000). Unfortunately, pyrethroid resistance has already developed in som e populations o f the major* IRD, 911, avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France.
** CPR, 01 BP 1500, Bouaké 01, Cote d’Ivoire.
Correspondence: Jean-Marc Hougard, LIN/IRD, 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
malaria vectors, particularly A n o p h eles g a m b i a e s.s. in tropical Africa (Chandre et al., 1999). The lack o f an alternative chem ical category could jeopardize the effectiveness o f treated m osquito nets against malaria transmission.
O ne strategy proposed for resistance m anagement is the use o f mixtures o f pesticides with different mode o f action (Tabashnik, 1989; Roush, 1993). This strategy imply that no cross resistance exist betw een the insec
ticides. For treated mosquito nets, this could be an insecticide mixture com bining a pyrethroid insecticide with a non-pyrethroid insecticide such as carbamates or organophosphates. The use o f an insecticide m ix
ture is all the more promising if there is a positive inter
action betw een the two com ponents. This has been observed in crop protection and veterinary use with mixtures o f pyrethroids with organophosphates or car
bam ates (Ali et al., 1977; Huges & Trevethan 1979;
Koziol & Witkowski, 1982; Campanhola & Plapp, 1989;
Christian et a l., 1986). A synergy has also b ee n
Mémoire
255R ésum é
: Les m élan g es in s e c tic id e s en im p rég n atio n de MOUSTIQUAIRES CONTRE LES VECTEURS DU PALUDISME Les pyréthrinoïdes sont actuellement les seuls insecticides recommandés pour l'imprégnation des moustiquaires.Malheureusement, la résistance aux pyréthrinoïdes est déjà apparue chez plusieurs espèces de vecteurs du paludisme menaçant 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 gestion de la résistance consisterait à associer sur une moustiquaire deux insecticides ayant des modes d'action différents. Cette étude présente les résultats obtenus avec des moustiquaires imprégnées avec différentes proportions de bifenthrine ¡un pyréthrinoïdel et de carbosulfan (un carbam atel L'efficacité de ces moustiquaires bi- imprégnées a été testée sur des moustiques adultes sensibles c/'Anopheles gambiae, principal vecteur du paludisme en Afrique.
Un effet de synergie significatif a été détecté avec un mélange composé de 2 5 m g /m 2 de bifenthrine lia moitié de la dose opérationnelle! et 6 ,2 5 m g /m 2 de carbosulfan [2 % de la dose opérationnelle!. La mortalité observée a été significativement supérieure à celle attendue par sommation des effets 18 0 % contre 41 %! et l'effet knock down a été maintenu, préservant ainsi une barrière efficace contre les moustiques.
MOTS CLES
: mélange insecticides, synergie, moustiquaire imprégnée, Anopheles gambiae.Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2002093255
CORBEL V., DARRIET F., CHANDRE F. & HOUGARD J.M .
observed recently betw een a pyrethroid and a carba
mate on susceptible and resistant larvae o f C u lex q u in - q u efa scia tu s, a mosquito o f medical importance (Cor
bel et al., 2002).
The present study is aimed to mosquito nets samples impregnated with insecticide mixtures com bining low doses o f pyrethroid and carbam ate formulations ( “bi
treated” mosquito nets). Their efficacy w ere com pared to “m ono-treated” net samples on a susceptible strain of A n o p h eles g a m b i a e s. s. Carbosulfan (2,3-dihydro-2,2- dim etyl-7-benzofuranyl [(dibutylam ino)thio] methyl- carbam ate) has b een selected among carbamates. In contrast to pyrethroids and DDT, this insecticide family does not induce any knock-dow n effect. Carbosulfan is currently used for public health despite its relative toxicity to mammals because it has show n a good effi
cacy in net im pregnation at 300 mg/m2, particularly against pyrethroid-resistant populations o f A n o p h eles and C u lex mosquitoes (Kolaczinski et al., 2000). Bifen- thrin (2 methyl t l , l ’-biphenyl]-3-yl)methyl 3-(2-chloro- 3 ,3 ,3-trifluoro-1-propenyl)-2,2-dim ethyl-cyclopropane- carboxylate) has b een selected am ong pyrethroids, as it is potentially a good candidate insecticide for malaria prevention. It is used world wide against a wide range o f agricultural pests and has already b een tested suc
cessfully under laboratory conditions against malaria vectors (Hougard et al., 2002). Preliminary field tests have confirm ed its efficacy against pyrethroid-resistant populations o f A. g a m b i a e and C. q u in q u e fa s c ia tu s at 50 mg/m2 (Guillet et al., 2001).
MATERIAL A N D M ETHODS
T
ests w ere conducted using Kisumu, a fully susceptible reference strain o f A. g a m b i a e origi
nated from Kenya and initially maintained under standard laboratory conditions (27 ± 2° C and 80 % relative humidity).
The net samples w ere impregnated with com m ercial formulations. Bifenthrin was formulated as a suspen
sion concentrate (SC) containing 8 % active ingredient (Talstar® 80 SC, FMC, Philadelphia, USA). Carbosulfan was formulated as a m icrocapsule suspension contai
ning 2.5 % active ingredient (Marshall® 25 CS, FMC, Phi
ladelphia, USA).
The efficacy o f insecticides, alone or in association, was assessed according to the cone test m ethod (Anony
mous WHO, 1998). Five unfed females aged from two to five days w ere placed in a plastic co n e that was positioned over treated net sample. After three minutes the m osquitoes w ere rem oved from the net sample, placed in 150 ml plastic cups and supplied with a 10 % honey solution as a food source. The mosquitoes were held for 24 hours in an environmental cham ber at 27° C
± 2 ° C and 80 % ± 10 % RH. Each replicate consisted
o f three-four cones and each test sample was replicated three times. The percentage o f knocked down m os
quitoes was noted after a three minutes exposure on the treated net, at fixed time intervals, varying betw een two to 10 minutes according to the intensity o f the k nock down effect. T he p ercentage mortality was recorded after 24 hours.
Data w ere analyzed using a log-probit program based on Finney (1971). The statistical analysis calculated the rates at w hich 50 % and 95 % o f m osquitoes would be knock down (respectively: KDt50 and KDt95). The difference betw een two KDt50 or two KDt95 values was considered as statistically significant only if there is no overlap o f their confidence limits. Each test was car
ried out with an untreated control m osquito net. If the control mortality was above 5 %, the “observed” mor
tality is adjusted by Abbott’s formula (Abbott, 1925).
P ercen t m ortalities w ere com p ared using the Chi square test at a 95 % Confidence Interval.
The netting material was a multifilament polyester 100 denier (weight in grams o f 9.000 m yarn), m esh size 156 (25 holes/cm2), from Siamdutch (Bangkok, Thai
land). For treatment, pieces o f netting w ere folded in three equal parts and put in a disposable petri dish.
A volume o f formulation suspension corresponding to specific absorbency o f the net and prepared im me
diately before treatment was dropped regularly onto the surface. Net w ere then carefully soaked with fin
gers (fitted with plastic gloves) in order to ensure a regular distribution o f the solution. O nce impregnated, nets were kept to dry in the box. Tests w ere made five days after treatment in order to avoid testing deposits o f markedly different ages, w hich might have different impact on mosquito behaviour and affect the results.
W e tested on Kisumu strain different impregnated nets with the insecticides alone and in mixtures with the objectives to find the better insecticide com bination in term o f KdT50/95 and mortality. Five samples o f m os
quito net w ere used as reference samples: two with bifenthrin alone at 25 and 12.5 mg/m2, three with car
bosulfan alone at 100, 25 and 6.25 mg/m2, and one with no insecticide (control sample). Six other samples w ere im pregnated with different bifenthrin / carbo
sulfan mixtures using the five doses enumerated above.
These dosages have b een selected as they induced separately a low range o f mortality (5 % - 4 0 %), thus allowing a better detection o f any synergism effect (Table I). By com paring the results observed with each mixture with the results theoretically expected in the absence o f any interaction (un-correlated joint action), it was possible to determ ine the effect o f such insecticide mixtures on adult mosquitoes The expected mortality was calculated by multiplying the percentage survival on each insecticide tested separately and sub
tracting the result from 100 %. In the same way, the KDt theoretically expected for a given insecticide m ix
In se c t ic id e m ix t u r es f o r m o s q u it o nets
ture, in the absence o f any interaction, is equal to the sum o f the KDt o f each insecticide. There is said to be synergy w hen the observed results are significantly higher than the expected results (positive interaction).
There is said to be antagonism w hen the observed results are significantly low er than the expected results (negative interaction).
RESULTS
V
alues show n in Table I w ere not adjusted since the control mortality was very low (no KD e f f e c t w as o b s e r v e d w ith th e u n tre a te d sam ples). The result obtained with carbosulfan alone confirm ed the absence o f KD effect that characterise the carbam ate family, w hatever the dosages tested.Results with bifenthrin alone confirm ed the existence a KD effect, but there was no apparent dose/effect cor
relation since an overlap o f the confidence intervals was observed for KDt50 as well as for KDt95. With these low concentrations, mortality levels recorded for each insecticide used separately w ere low (maximum 38 % for bifenthrin and 15 % for carbosulfan).
With insecticide mixtures, a reduction o f the KD effect was noted w hen the dosage o f carbosulfan increased and the dosage o f bifenthrin decreased. This antago
nism was more evident at the KDt95 levels, particularly with mixtures containing the low er dosage o f bifen
thrin (12.5 mg/m2) and the higher dosage o f carbo
sulfan (100 mg/m2): the speed o f the KD effect was almost halved (KDt50: 12.2 min ; KDt95: 50.6 min) com pared with that observed with bifenthrin alone at 12.5 mg/m2 (KDt50: 6.8 min; KDt95: 26.0 min). Conver
sely, a very significant synergy ( P < 0.0 0 0 1 ) w as
observed with the insecticide mixture containing the low er dosage o f carbosulfan (6.25 mg/m2) and the higher dosage o f bifenthrin (25 mg/m2): the percen
tage mortality observed with the mixture ( 8 0 % ) was twice the expected one in the absence o f any inter
action (41.1 %). Except for 12.5 mg/m2 bifenthrin and 100 mg/m2 carbosulfan, w here a slight synergy was observed, no significant difference was noted with the other insecticide mixtures betw een the observed and expected mortalities.
DISCU SSIO N
The lack o f knock down effect at low dosages o f car
bosulfan was confirmed. Results observed with the dif
ferent mixtures show ed that carbosulfan, at a relatively high dosage (100 mg/m2), could act as an inhibitor o f the KD effect o f pyrethroids. The irritant properties o f carbosulfan against A n o p h eles and M a n s o n ia species was demonstrated by Klein et Darriet (1989) by house spraying o f experim ental huts. At a relatively high dosage, the irritant effect o f carbosulfan could decrease the duration o f tarsal contact with the substrate, thus reducing the KD effect o f bifenthrin.
Regarding mortality, a very significant synergy (P <
0.0001) was noted at the highest dosage o f bifenthrin (25 mg/m2) and the lowest o f carbosulfan (6.25 mg/m2).
Although these insecticides do not have the same target sites, it is likely that their com bination resulted in a com plex m olecular interaction. Pyrethroids act on the nervous system by modifying the gating kinetics o f voltage-sensitive sodium channels (Lund & Nara- hashi, 1983). As with the other pyrethroids, bifenthrin keeps the sodium channels open temporarily, thus cau
Insecticide Bifenthrin
25 mg/m2
N
61
KDt Observed
6.0
:50 (m in)
Cl 1
5.2-6.8
Expected
K Observed
26.8
Dt95 (m in) Mortality (% )
Cl Expected Observed Expected x2 P
21.2-37.8 - 38.0
12.5 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 15.0
25 mg/m2 52 no effect - - no effect 4.0
6.25 mg/m2 50 no effcet - - no effcet 5.0
Bifenthrin 25 mg/m2
+ Carbo. 100 mg/m2 59 8.0 6.9-9.0 6.0 42.8 34.1-58.6 26.8 37.0 47.3 1.2 > 0.05
+ Carbo. 25 mg/m2 62 5.7 4.8-Ö.6 6.0 30.2 23.9-42.3 26.8 53.0 40.5 2.1 > 0.05
+ Carbo. 6.25 mg/m2 60 5.4 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 11.1-13.4 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 22.5-32.1 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 24.8-42.4 26.0 46.0 29.7 3.4 > 0.05
N, number tested. Cl, 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 gam biae.
Parasite, 2002, 9, 255-259
Mémoire
2 5 7CORBEL V.. DARRIET F., CHANDRE F. & HOUGARD J.M .
sing repeated discharges o f action potentials w hich increase the amount o f acetylcholine (ACh) in the synapses (Salgado, 1983). Carbosulfan, as with the other carbamates, is an inhibitor o f acetylcholineterase (AChE). Such inhibition also contributes to increased ACh concentration at level w hich could explain the synergy observed. The absence o f synergy at higher concentrations o f carbosulfan could be due either to the shorter contact betw een m osquitoes and im pre
gnated nets, as observed by reduced KD effect, or to a secondary target site inhibition as Choline Acetyl- transferase (ChAT). ChAT is a protein implicated in syn
thesis o f ACh and is a secondary target for carbamates (Bourguet et al., 1997). W hen adding a higher dosage o f carbosulfan (100 mg/m2), ChAT could b ecom e an insecticide target, leading to a decrease o f ACh pro
duction w hich could balance the accum ulation o f ACh due to the other mechanisms.
B ecause carbam ates are potent inhibitors o f ACh and because carbosulfan degrades to a more toxic com pound (carbofuran) (2,3-dihydro-2,2-dimethyl-n-7-ben- zofuranyl m ethylcarbam ate) by the loss o f the (dibu- tylam ino)thio group, the toxicological risk o f their use on mosquito nets has to be carefully considered in rela
tion to hum an safety (Anonymous WHO, 1986). It is encouraging that the optimum interaction obtained in the susceptible Kisumu strain was with the low er dose o f carbosulfan (about 50 times less than the operational dose). However, an insecticide mixture must be consi
dered as a new com pound that needs a long series o f toxicological studies before it would be cleared for use on m osquito nets (Zaim et al., 2000). Another impor
tant advantage with this ratio was the absence o f any antagonism o f the KD effect that allows the mixture to keep its fast acting properties on nets and continue to provide an effective barrier against susceptible m os
quitoes (Miller & Gibson, 1994).
Preliminary field studies in experim ental huts have been carried out in Cote d’Ivoire by using a “two in on e” combination o f bifenthrin and carbosulfan applied to different parts o f the sam e bednet (Guillet et al., 2001). Promising results have been obtained against the local pyrethroid resistant populations o f A. g a m b ia e s . s.
and C. q u in q u e fa s c ia tu s . This study will be co m pleted in the same area by using mosquito nets treated with a mixture o f bifenthrin and carbosulfan at an optimum ratio giving mortality under laboratory condi
tions.
ACKNOWLEDGEMENTS
W
e thank the Ministère français de la recherche on malaria and associated com m uni
ca b le d iseases for d evelo p in g cou n tries
(PAL+ program) for funding this work and the FMC Cor
poration for providing the formulations o f bifenthrin and carbosulfan.
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Reçu le 15 avril 2002 Accepté le 13 mai 2002
Parasite, 2002, 9, 255-259
