Ps809.dvi

Pest Manag Sci 60:375– 380 (online: 2003)
Efficacy of insecticide mixtures against larvae
of Culex quinquefasciatus (Say) (Diptera:
Culicidae) resistant to pyrethroids and
carbamates
Vincent Corbel,1∗ Michel Raymond,2 Fabrice Chandre,1 Fr ´ed ´eric Darriet1 and
Jean-Marc Hougard1
1Institut de Recherche pour le D ´eveloppement (IRD), Laboratoire de Lutte contre les Insectes Nuisibles (LIN), 911 Avenue Agropolis, 34394
Montpellier Cedex 5, France
2Institut des Sciences de l’Evolution, Laboratoire G ´en ´etique et Environnement, CC065 Universit ´e de Montpellier II, place Eug `ene Bataillon,
34095 Montpellier, France
Abstract: The efficacy of insecticide mixtures of permethrin (pyrethroid) and propoxur (carbamate) was
tested by larval bioassays on two strains of Culex quinquefasciatus (Say), one resistant to pyrethroids
and the other resistant to carbamates. The method consisted in combining one insecticide at the
highest concentration causing no mortality (LC0) with increasing concentrations of the second one.
The concentration– mortality regression lines were determined for permethrin and propoxur alone and in
combination, and synergism ratios (SR) were calculated in order to determine the magnitude of an increase
or decrease in efficacy with use of the mixtures. With the pyrethroid-resistant strain (BK-PER), the results
showed that propoxur at LC0 significantly enhanced the insecticidal activity of permethrin (SR50 = 1.54),
especially on the upper range of the concentration– mortality regression. Conversely, when permethrin at
LC0 was tested with propoxur against the carbamate resistant strain (R-LAB), an antagonistic effect was
observed (SR50 = 0.67). With the BK-PER strain, an increased oxidative detoxification (MFO) appeared
to be the main mechanism responsible for the synergistic interaction. Nevertheless, antagonism in
the R-LAB strain is probably due to a physiological perturbation implying different target sites for
pyrethroid (ie sodium channel) and carbamate insecticides [ie acetylcholinesterase (EC 3.3.3.7) and
choline acetyltransferase (EC 2.3.1.6)].
 2003 Society of Chemical Industry
Keywords: Culex quinquefasciatus larvae; insecticide mixture; resistance mechanism; synergism; antagonism;
acetylcholinesterase; choline acetyltransferase
INTRODUCTION
recently, in two populations of A gambiae from C ˆote In 2001, resistance to insecticides concerned 540 species of arthropod, of which 198 were of medi- Given that there are few alternative insecticides in cal and veterinary importance.1 This was all the more public health coming on-stream, the main concern worrying as insecticides have, for a long time, played in resistance management strategies for vector species a major role in the control of pests and insects, consists in making a judicious use of the compounds as well as of vectors of diseases. For example in already available. The use of mixtures or recourse to West Africa, resistance to pyrethroids is widespread a strategy of rotation over time of insecticides with in Anopheles gambiae spp,2 a major malaria vector different modes of action has already made it possible in Sub-Saharan Africa and in Culex quinquefasciatus to prevent or to delay the appearance of resistance (Say),3 the main nuisance mosquito in urban envi- in the field.8 – 10 However, mixtures of appropriate ronments. Resistance to organophosphate compounds dosages of unrelated compounds may have better has developed in many species of mosquitoes of the prospects for managing resistance effectively than genera Culex4 and Anopheles.5 Resistance to carba- rotations of the types of compounds.11 – 13 This strategy mates has been noted in C quinquefasciatus6 and, more is based on the fact that, if the probability for resistance ∗ Correspondence to: Vincent Corbel, Institut de Recherche pour le D ´eveloppement (IRD), Laboratoire de Lutte contre les Insectes Nuisibles(LIN), 911 Avenue Agropolis, 34394 Montpellier Cedex 5, FranceE-mail: [email protected]/grant sponsor: Minist `ere Fran ¸cais de la Recherche programme on malaria and associated diseases (PAL+)(Received 24 February 2003; revised version received 19 June 2003; accepted 14 August 2003)Published online 21 November 2003  2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00 to one of the two insecticides is a rare and independent Organization.22 Each bioassay was repeated three event, then the probability that resistance will occur times using late third- and early fourth-instar lar- simultaneously to both insecticides of the mixture is vae of BK-PER and R-LAB C quinquefasciatus. For extremely low.14 The advantage of mixtures is that each bioassay, 20 larvae of each strain were trans- each insecticide eliminates most insects which are ferred to cups containing 99 ml of distilled water. For each bioassay, we used five cups per concentration However, the toxicological risks for humans, as well (100 larvae) and five to eight concentrations of each as the cost involved in the use of several insecticides insecticide in a range that causes 0 to 100% mortality.
at operational dosage, are major concerns, unless One millilitre of each insecticide, at the desired con- the combined effect of the mixture is significantly centration, was added to the cups. Control treatments stronger than the sum of the single effects (synergism of 1 ml of ethanol were performed for each test. Each effect). Such a phenomenon may increase the bioassay was maintained at 27 (±1) ◦C throughout all efficacy of treatment while reducing substantially cost tests. Larval mortality was recorded after 24 h of expo- and toxicity, because of a reduction of insecticide sure, corrected by the formula of Abbott23 if necessary, amounts. Many authors have already demonstrated and data were analysed by the log-probit method of the synergistic effect on insect pests of carbamates Finney,24 using the Probit software (Prax`eme) pro- (or organophosphates) and pyrethroids.15 – 17 With grammed by Raymond et al.25 This software uses the insects of medical importance, a synergistic effect iterative method of maximum likelihood to fit a regres- between pyrethroids and carbamates was reported sion between the logarithm of concentration and the on larvae of C quinquefasciatus18 and adults of A probit of mortality. The goodness-of-fit is estimated gambiae19 susceptible to these insecticides. Given by a weighted chi-squared test. It also estimates the the development of resistance in most mosquito lethal concentrations and the slope of the regression species, we investigated the interaction between a lines with their confidence intervals (P = 0.05).
pyrethroid and a carbamate on larval stages of twoC quinquefasciatus strains, one resistant to pyrethroids Synergism study
and the other resistant to carbamates.
The effect of permethrin and propoxur in binarycombination was evaluated using late third- andearly fourth-instar larvae of BK-PER and R-LAB MATERIAL AND METHODS
C quinquefasciatus. In preliminary bioassays, the highest concentrations of permethrin and propoxur Pyrethroid- (BK-PER) and carbamate- (R-LAB) which produced no mortality (LC0) were determined resistant strains of C quinquefasciatus were used for each of the two strains of C quinquefasciatus.
for bioassays. The BK-PER strain originated from Each of the resistant strains of mosquitoes was C ˆote d’Ivoire and was maintained under con- exposed to the LC0 permethrin and propoxur stant selection pressure of permethrin. This strain individually (positive control) and in combination is homozygous for the Kdr mutation20 and also with increasing levels of propoxur and permethrin, exhibits an increased metabolic detoxification through respectively. A log-probit analysis was performed the cytochrome P450-dependant monooxygenases.3 for each insecticide individually and in combination, The R-LAB strain is resistant to carbamates and and their slopes were compared using a chi-squared organophosphates, although it remains fully suscep- parallelism test. Synergism ratios (SR) were calculated tible to pyrethroids and DDT. The R-LAB strain in order to determine the magnitude of increase or is homozygous for an insensitive acetylcholinesterase decrease of efficacy occurring with the permethrin with a genetic background identical to the susceptible and propoxur combinations. Synergism ratios were reference strain S-LAB.21 Mosquitoes were main- tained by standard methods in an insectary at 27 (±2) ◦C and 80 (±10)% relative humidity.
Synergism ratios as well as their confidence intervals Insecticides
The bioassays were carried out using technical-grade programmed by Raymond et al.25 A SR significantly permethrin (pyrethroid insecticide) and propoxur higher than 1 (ie confidence interval of SR did not (carbamate insecticide). Permethrin (cis/trans isomeric include the value 1) indicated a synergistic effect, ratio 25/75: 94.4%) and propoxur (99.6%) were whereas a SR significantly lower than 1 indicated an obtained from Agrevo (Berkhamsted, UK) and Bayer (Leverkusen, Germany), respectively. Each insecticidewas prepared in absolute ethanol and stored at 4 ◦Cthroughout the experimentation.
Pyrethroid resistant strain (BK-PER)
Larval bioassay procedure
Toxicity of permethrin and propoxur alone The larval bioassays were performed using a stan- The relationships between log-concentration and probit-mortality with permethrin (χ 2 = 5.85, df = 5) Pest Manag Sci 60:375– 380 (online: 2003)
Efficacy of insecticide mixtures against Culex quinquefasciatus larvae and propoxur (χ 2 = 4.41, df = 5) were statistically to appear at LC30 and increased with increasing fitted by straight lines (P > 0.05) and mortality never permethrin concentrations (Table 1).
exceeded 5% in the control. The slope of the regression The LC0 value for permethrin was 0.01 mg litre−1.
line with permethrin [1.29 (±0.14)] confirmed the When this LC0 was combined with increasing con- polyfactorial nature of resistance in the BK-PER strain.
centrations of propoxur, the slope for the mix- ture [3.51 (±0.19)] was not changed from that 50 and LC95 values of permethrin, 0.40 and 7.53 mg litre−1, respectively (Table 1), also confirmed for propoxur alone [4.15 (±0.37)] (χ2 parallelism its high resistance level to pyrethroids. There was a test = 9.62, df = 9, P = 0.38). The LC50 of the mix- resistance factor (RF) of >300 compared with the ture (0.35 mg litre−1) was not significantly different susceptible reference strain of C quinquefasciatus.18 from that of propoxur (0.38 mg litre−1), indicating The slope of the regression line with propoxur [4.15 that there was no synergism with this combina- (±0.37)] was steeper than that for permethrin. The LC50 and LC95 values were 0.38 and 0.94 mg litre−1,respectively (RF = 4).
Carbamate resistant strain (R-LAB)
Toxicity of permethrin and propoxur alone Toxicity of permethrin and propoxur in The relationships between log-concentration and probit-mortality with permethrin (χ 2 = 8.82, df = 6) For propoxur, the LC0 value was 0.1 mg litre−1 with and propoxur (χ 2 = 7.07, df = 6) were well fitted the BK-PER strain. When this LC0 was combined by straight lines (P > 0.05) and mortality never with increasing concentrations of permethrin, the exceeded 5% in the control batches. The slopes of slope for the mixture [1.82 (±0.15)] had increased the regression lines of propoxur [9.8 (±0.96)] and significantly compared with that for permethrin permethrin [8.4 (±0.90)] were steep, indicating a alone [1.29 (±0.14)] (χ2 parallelism test = 22.3, strong homogeneity of the mosquitoes with respect df = 11, P = 0.02). These results indicated that the to the toxic effect of the two insecticides.
heterogeneity of larval response to the mixture was The high LC50 and LC95 values of propoxur slightly lower than to permethrin alone. The LC50 (180 and 266 mg litre−1, respectively) confirmed of the mixture (0.26 mg litre−1) was approximately two fold less than that for permethrin (LC50 = 0.40 mg litre−1). Significant synergism ratios started susceptible reference strain of C quinquefasciatus (S-LAB).18 Conversely, the R-LAB strain displayed a great Table 1. Efficacy of permethrin with and without propoxur at LC0
against a pyrethroid-resistant strain of Culex quinquefasciatus values (1.2 × 10−3 and 1.9 × 10−3 mg litre−1, respec-tively) were comparable with those for the susceptible reference strain S-LAB; LC50 and LC95 values forthe latter, were 1.5 × 10−3 and 2.5 × 10−3 mg litre−1, Toxicity of permethrin and propoxur in The LC0 value of permethrin was 4 × 10−4 mg litre−1.
A significant antagonistic effect appeared when this LC0 was combined with increasing concentrations of propoxur. The LC50 of propoxur increased from 180 mg litre−1, when used alone, to 269 mg litre−1, when used in combination with permethrin at LC50 (Table 2). Because both regression lines were parallel (χ 2 parallelism test = 13.2, df = 8, P = 0.10), the synergism ratios were statistically the same for all the lethal concentrations (SR = 0.67).
The LC0 value of propoxur was 80 mg litre−1. When this LC0 was combined with increasing concentrations of permethrin, the slope for the mixture [8.4 (±0.90)] did not significantly change compared with that for permethrin alone [9.2 (±0.99)] (χ2 parallelism test = 19.0, df = 11, P = 0.06). In addition, there was no significant difference between the LC50 values a Not significantly different from 1 (confidence interval of SR includes of permethrin alone and permethrin mixed with propoxur (1.2 × 10−3 mg litre−1 for each).
Pest Manag Sci 60:375– 380 (online: 2003)
Table 2. Efficacy of propoxur with and without permethrin at LC0
organophosphate insecticides may be competitive sub- against a carbamate resistant strain of Culex quinquefasciatus strates for the same oxidase, thus increasing the toxicity of the mixture. In addition, Gunning et al28demonstrated that synergism between fenvalerate and organophosphate insecticides in the cotton pest tion by organophosphates of esterases involved in In our study, it is likely that a similar phe- nomenon occurred with the pyrethroid-resistant strain BK-PER which exhibited an increased metabolic detoxification by the cytochrome P450-dependant monooxygenases.3 The monooxygenase action on propoxur would prevent (or delay) the degradation of permethrin, hence providing a level of synergism by competitive substrate inhibition. The non-specific esterases (NSE) were probably not involved in syner- gism since Chandre et al3 demonstrated that efficacy of permethrin was unchanged in BK-PER after addi- tion of DEF (S,S,S-tributyl phosphorotrithioate), an Conversely, the mechanism by which permethrin antagonized the propoxur in the carbamate-resistant strain R-LAB appeared more complex. It is obvious that an inhibition of metabolic detoxification (esterase or oxidase activities) by one of the two compounds of the mixture cannot explain such interaction.
a Synergism ratios were identical for all lethal concentrations since In a previous study, we have shown that similar the propoxur regression lines with and without LC0 permethrin were combinations of propoxur with permethrin displayed parallels (P > 0.05).
synergistic interactions against susceptible larvae of Cquinquefasciatus (S-LAB).18 Moreover, this synergism DISCUSSION
was maintained even when oxidase activity was In this study, bioassays were carried out to evaluate inhibited by piperonyl butoxide, an oxidase inhibitor the insecticidal activities of permethrin and propoxur, (V Corbel, unpublished data). Thus, it is surprising alone and in combination, against pyrethroid- and to note that synergism occurred in the susceptible carbamate-resistant larvae of C quinquefasciatus. The reference strain S-LAB whereas antagonism appeared results indicated three types of relationship between in the carbamate resistant strain R-LAB, both having the insecticides, depending on the resistance mecha- an identical enzymatic background, only differing by an insensitive acetylcholinesterase (AChE).
Bourguet et al29 reported that in the resistant strain With the pyrethroid – resistant strain (BK-PER), the R-LAB, AChE (EC 3.3.3.7) (the primary target of pyrethroid toxicity was significantly increased when carbamates) was unaffected by propoxur, even at adding a sub-lethal concentration of the carbamate concentrations giving 100% mortality, because of its insecticide. This increase, which was more acute complete insensitivity to carbamates. These authors at LC95, resulted from a synergistic interaction showed that, when AChE was highly insensitive to carbamates, another enzyme responsible for the With the carbamate-resistant strain (R-LAB), the synthesis of acetylcholine (choline acetyl transferase opposite situation was observed. The carbamate or ChAT EC 2.3.1.6) became the second target of efficacy decreased when combined with a sub-lethal propoxur, and insect death occurred through a lack concentration of pyrethroid (antagonism).
of acetylcholine in the synapses. We thought that Finally, neither synergistic nor antagonistic interac- inhibition of either AChE (for S-LAB) or ChAT tions occurred with either strain when mosquitoes (for R-LAB) by propoxur would explain the opposite were resistant to the insecticide used at non-toxic interactions observed with the insecticide mixtures in A general model has been developed to explain syn- Salgado et al30 demonstrated that the repetitive ergism between insecticides.26 The model indicated firing of nerves induced by pyrethroids stimulated that ‘one toxicant interferes with the metabolic detox- an acetylcholine release within the synaptic gap, ification of the second toxicant, thereby potentiating even when low concentrations of permethrin were the toxicity of the latter compound’. Indeed, Kulkrani used. Consequently, with the R-LAB strain, the and Hodgson27 demonstrated that pyrethroid and acetylcholine released by permethrin at LC0 may Pest Manag Sci 60:375– 380 (online: 2003)
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