Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae

Synergism between permethrin and propoxur againstCulex quinquefasciatus mosquito larvae

V. CORBEL, F . CHANDRE, F . DARRIET, F . LARDEUX and

J-M. HOUGARDLaboratoire de Lutte Contre les Insectes Nuisibles (LIN), Institut de Recherche pour le Developpement (IRD), Montpellier,

France

Abstract. To see if synergism occurs between carbamate and pyrethroid insecti-cides, we tested permethrin and propoxur as representatives of these two classes ofcompounds used for mosquito control. Larvicidal activity of both insecticides wasassessed separately and together on a susceptible strain of the mosquito Culexquinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constantratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentra-tions to plot dose/mortality regression, significant synergy occurred between them(co-toxicity coefficient¼ 2.2), not just an additive effect. For example, when themixture gave 50% mortality, the same concentrations of permethrin and propoxuralone would have given merely 2� 1% mortality. When a sublethal dose (LC0) ofpermethrin or propoxur was added to the other (range LC10–LC95), synergismoccurred up to the LC80 level. Synergistic effects were attributed to the comple-mentary modes of action by these two insecticide classes acting on differentcomponents of nerve impulse transmission. Apart from raising new possibilitiesfor Culex control, it seems appropriate to consider using such mixtures or combina-tions for insecticide-treated mosquito nets in situations with insecticide-resistantAnopheles malaria vectors.

Key words. Culex quinquefasciatus, binary mixture, co-toxicity coefficient,insecticide mixture, larval bioassays, mosquito control, permethrin, probit analysis,propoxur, resistance management, synergism

Introduction

The development of insecticide resistance in arthropods is

well documented for many pest and vector species of public

health importance (WHO, 1992; Hemingway & Ranson,

2000) and for most agricultural pests (Devonshire & Moores,

1982; Georghiou & Saito, 1983; Brown, 1986; Metcalf, 1989;

Georghiou & Lagunes-Tejada, 1991; Denholm et al., 1998).

This phenomenon is all the more alarming because there are

so few classes of available insecticides, even fewer insecticidal

modes of action, resistance and cross-resistance problems are

increasing and new products have to meet rising standards of

environmental as well as toxicological safety (Ware, 2000).

Through lack of alternatives, the management of pest popu-

lations and strategies for slowing the evolution of pesticide

resistance are based on making optimal use of existing com-

pounds (NRC, 1986; Poirie & Pasteur, 1991). For example,

to avoid selecting for any particular type of resistance, opera-

tional programmes may apply alternative classes of insec-

ticides in sequence, rotation or mosaics of compounds

acting on different target sites (Kurtak et al., 1987; Hoy,

1998; Penilla et al., 1998; Ahmad et al., 2002). Theoretical

models suggest that, under certain conditions, mixtures will

be more efficient to delay the development of resistance

compared to sequence or rotation (Tabashnik, 1989; Roush,

1993) because, if resistance to each compound is independent

and initially rare, the associated probability of resistance to

both compounds is then extremely rare (Curtis, 1985).

Correspondence: Vincent Corbel, IRD Laboratoire de Lutte

Contre les Insectes Nuisibles (LIN/IRD), 911 avenue Agropolis,

B.P. 64501, 34394 Montpellier Cedex 5, France. E-mail: corbel@

mpl.ird.fr

Medical and Veterinary Entomology (2003) 17, 158–164

158 # 2003 The Royal Entomological Society

Accordingly, in West Africa, the use of organophosphate–

pyrethroid mixtures for cotton spraying has apparently

precluded the development of pyrethroid resistance in

the cotton bollworm Helicoverpa armigera Hardwick

(Lepidoptera: Noctuidae) for more than 20 years (Martin

et al., 2000). Likewise in West African rivers, Simulium

larviciding with frequent applications of complementary

insecticides has maintained control of onchocerciasis

vectors for more than two decades without provoking

resistance problems (Hougard et al., 1997). Both these pro-

grammes have exploited negative cross-resistance between

pyrethroids and organophosphates.

Another potential reason for combining insecticides

could be to gain a synergistic effect, whereby the effective

concentration of each active ingredient might be reduced for

operational use – although the cost of treatment with a

mixture might be higher than either product alone. Insecti-

cidal synergism between organophosphate and pyrethroid

compounds has been widely investigated and such mixtures

have been employed against agricultural pests (Hughes &

Trevethan, 1979; Koziol & Witkowski, 1982; Ascher et al.,

1986; Wu, 1995; Bynum et al., 1997). In contrast, mixtures

of two insecticide classes have been neglected for controlling

pests and vectors of public health importance, despite the

massive commercialization of synergized pyrethrins and

pyrethroids as consumer pesticides for personal and domestic

use (Jones, 1998). Moreover, it is possible for vector popula-

tions to show negative cross-resistance between pyrethroid

and carbamate or organophosphate insecticides, as reported

for Culex quinquefasciatus (Georghiou et al., 1983; Peiris &

Hemingway, 1990) and for the blackfly Simulium sanctipauli

V. & D. (Diptera: Simuliidae) (Kurtak et al., 1987). Thus it is

worth exploring new combinations of insecticides to optimize

their dose-efficacy. For evaluation of complementary

mixtures against mosquitoes, we used a standard larval

bioassay procedure to assess whether larvicidal treatment

with a mixture of carbamate plus pyrethroid yields additive,

antagonistic or synergistic effects.

Materials and methods

Mosquitoes

The standard susceptible ‘S-Lab’ strain of Culex

quinquefasciatus, originally fromCalifornia (Georghiou et al.,

1966), was employed for larval bioassays. This tropical

pest mosquito is the urban vector of Bancroftian filariasis

and the S-Lab strain is free of any detectable insecticide

resistance mechanism.

Insecticides

Two technical grade compounds were used, representing

carbamate and pyrethroid classes of insecticide, viz. propoxur

99.4% (Bayer AG, Leverkusen, Germany) and permethrin

94.4% cis : trans 25 : 75 (Agrevo plc, Berkhamsted, U.K.).

Stock solutions were prepared in absolute ethanol and stored

at 4�C for no more than 2months.

Larval bioassay procedure

Bioassays followed the standard W.H.O. (1970) protocol

with 20 larvae (late third and early fourth instar) in 100mL

of insecticide solution at the required concentration, freshly

prepared by adding 1mL of stock ethanol concentrate to

99mL of distilled water in a plastic cup. Each set of bio-

assays used five to eight serial concentrations providing a

range of mortality from nil to 100%, triplicated for five lots

of 20 larvae per concentration. The untreated control had

1mL of ethanol without insecticide added to the water and

the temperature was maintained at 27�C throughout all tests.

Larval mortality was recorded after 24-h exposure, corrected

by the formula of Abbott (1925) if necessary, and results

were analysed by the log-Probit method of Finney (1971),

programmed by Raymond et al. (1997). Mortality rates were

compared by w2 test at 0.05% level of significance.

Evaluation of mixture

When treatment with a mixture results in more or less

than the expected additive effect (summation), it may be

deduced that synergism or antagonism occurs between the

two insecticides. To reveal which of these three possibilities

(antagonism, summation, synergism) results from a mixture

of carbamate (propoxur) and pyrethroid (permethrin)

applied as larvicide, we tried two evaluation methods.

Binary mixture. Binary mixture of both insecticides,

at the LC50 ratio, was bioassayed at serial concentrations.

If the dose/response regression lines for these insecticides

are parallel, their mixture ratio is constant. Any synergistic/

antagostic effect was assessed by calculating the co-toxicity

coefficient (CC), which gives a quantitative measure of any

interaction between the two compounds. On the assump-

tion that the modes of action of the two insecticides are

independent, a theoretical LC50 for the mixture was calcu-

lated by the formula of Bliss (1939) as: (LC50 of insecticide

A alone� percentage of A in the mixture)þ (LC50 of insec-

ticide B alone� percentage of B in the mixture). The CC

was then quantified by dividing the theoretical LC50 by the

observed LC50 of the mixture. Values of the CC signifi-

cantly >1 indicate degrees of synergism, values �1 indicate

summation, and values <1 indicate degrees of antagonism

(Sun & Johnson, 1960). Results were also assessed by com-

paring the observed percentage mortality – for a given

concentration of the mixture – to the theoretical mortality

expected, calculated by adding the observed mortality rates

due to that concentration of each insecticide, taking into

account that they act on those surviving exposure to each

other.

Complementary treatment. Complementary treatments

were evaluated by varying the concentration of one insecticide

against a fixed concentration of the other. The procedure

Insecticide synergism against mosquito larvae 159

# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 158–164

involved adding a constant and very low concentration of one

compound (actually LC0, the highest concentration which

caused no mortality) to various doses of the other insecticide.

The LC0 of each insecticide was determined by preliminary

bioassays. In addition to the untreated (negative) controls,

there were ‘positive controls’ (four cups per replicate) of the

LC0 to check that the mortality rate was not higher than the

negative control. For each insecticide, regression lines were

established with and without ‘synergist’ and a test of parallel-

ism was computed. For a given LC of one insecticide, the

Synergist Ratio (SR) was determined by calculating the ratio

between LCs with and without the other insecticide LC0 as a

potential synergist/antagonist. The value SR¼ 1 indicates no

synergism of one insecticide by LC0 of the other; SR< 1

shows anatagonism whereas SR> 1 shows the degree of

synergism.

Results of larval bioassays

Binary mixture

The log dose/probit mortality relationships for each

insecticide tested separately (Fig. 1) were well fitted by

straight lines (P> 0.05): their LC50 values were determined

as 1.5� 10�3mg/L for permethrin and 9.4� 10�2mg/L for

propoxur. As both regression lines were parallel (parallel-

ism test: P> 0.05), the mixture of permethrin and propoxur

was prepared with their LC50 ratio, i.e. 1 : 60. The dose/

mortality relationship for the mixture also fitted a straight

line (P> 0.05), but with a slope (3.9) significantly weaker

than those observed with permethrin (7.1) and propoxur

(7.4). The co-toxicity coefficient calculated for the mixture

was 2.2, showing that the observed LC50 of the mixture was

less than half (2.2-fold lower) than expected for summation

of effects of the two insecticides. This value, significantly

above 1, indicated a synergistic effect. For each concentra-

tion of the mixture, the expected mortality was significantly

lower (P< 0.05) than observed (Table 1). This synergism

effect was more pronounced at the lower range of concen-

trations, as indicated by the weaker slope of the regression

line for the mixture compared to each insecticide alone. The

mixture LC50 corresponded to concentrations of permethrin

and propoxur giving only 1% mortality separately (mid-line

of Table 1).

Complementary treatment

The log dose/probit mortality regression lines using one

insecticide as a synergist for the other are shown in Figs 2

and 3. The synergisitic LC0 concentrations were determined

as 4.10�4 mg/L for permethrin and 1.10�2 mg/L for pro-

poxur. For ‘positive controls’ of both insecticides the mor-

tality (2%) never exceeded that of the ‘negative controls’.

Positive effects were detected using low concentrations of

propoxur or permethrin as a synergist of the other. With

propoxur as the synergist (Fig. 2), the slope of dose/

response for permethrin was significantly lower than for

permethrin alone (4.1 vs. 7.4, respectively). With permethrin

as the synergist (Fig. 3), the slope of dose/response for

propoxur was less than half that for propoxur alone (2.8

vs. 7.1). Synergist ratios recorded from LC10 (SR10) to LC80

(SR80) differed significantly from 1 and were significantly

higher when permethrin synergized propoxur, up to the

LC80 level (Table 2). At high concentrations (>LC80), the

synergist ratios were not statistically different from 1, indi-

cating summation instead of synergism.

98

95

90

80

7060504030

20

10

5

2

% M

OR

TALI

TY

10–4 10–3 10–2 10–11

3

4

5

6

7

DOSAGE (mg/L)

Permethrin

MixturePropoxur

Fig. 1. Dose/mortality regression line for permethrinþpropoxur mixture (ratio 1 : 60) activity on Culex quinquefasciatus larvae, compared

with permethrin and propoxur alone.

160 Corbel et al.


Discussion

By both methods of evaluation, we found generally positive

synergism between permethrin and propoxur against

Cx. quinquefasciatus larvae. When the binary mixture was

applied, the LC50 co-toxicity coefficient of 2.2 indicated

synergism between the two insecticides. Comparison of the

observed and expected mortalities confirmed this positive

interaction across a wide range of concentrations, the effect

being inversely proportional to mixture concentration.

Complementary treatments, when sublethal dose of each

insecticide was used to synergize the other, also resulted in

significantly greater mortality than expected up to the LC80.

This synergistic effect is presumably due to this combination

of carbamate and pyrethroid insecticides together over-

whelming the mosquito’s detoxification defense mechanisms

(Hemingway & Ranson, 2000), by reinforcing their com-

bined impact though different biochemical mode(s) of action,

but could reflect variable tolerance (polymorphism) of the

mosquito population. Heterogeneity of the detoxifying

enzyme systems (e.g. oxidases or esterases) that counteract

these insecticides could be involved, but is unlikely for such a

susceptible strain inbred for >30 years under laboratory con-ditions. Chandre et al. (1998) synergized the same insecticides

with two enzyme inhibitors (piperonyl butoxide as oxidase

inhibitor and tribufos as esterase inhibitor) against the same

mosquito strain: regression lines of each insecticide with or

without synergist were parallel, indicating little heterogeneity

of these enzymes in the S-Lab strain.

Presumably the observed synergism comes from com-

bined impact of these two complementary insecticides

simultaneously acting in different ways, magnifying their

efficacy. The main physiological targets of pyrethroids

and carbamates (Ware, 2000) are, respectively, voltage-

dependent sodium channel and acetylcholinesterase, both

involved with nerve function of the insect. By inhibiting

acetylcholinesterase, propoxur causes accumulation of

acetylcholine involved in neurotransmission at the synapse

(Champ, 1985). At the same time, permethrin keeps the

sodium channels open (Lund and Narahashi, 1983), inducing

repetitive impulses which result in synaptic acetylcholine

release (Salgado et al., 1983). Repeated discharge of action

potential induced by a sublethal dose of permethrin, together

with inhibition of acetylcholinesterase by propoxur, leads to

98

95

90

80

7060504030

20

10

5

2

% M

OR

TALI

TY

10–4 10–3 10–2

3

4

5

6

7

DOSAGE (mg/L)

PermethrinPermethrin + LC Propoxur0

Fig. 2. Dose/mortality regression line for permethrin activity on Culex quinquefasciatus larvae, compared with permethrin synergised by

propoxur LC0.

Table 1. Observed and expected activity of the binary mixture of permethrinþpropoxur (ratio 1 : 60) against Culex quinquefasciatus larvae,

compared with observed activity of permethrin or propoxur alone

Concentration

(10�3mg/L)

Binary mixture

mortality (%)

Observed Expected

Permethrin

Concentration

(10�3mg/L)

Mortality

(%)

Propoxur

Concentration

(10�3mg/L)

Mortality

(%)

20.2 10 0* 0.33 0 19.9 0

31.5 30 0* 0.52 0 31.0 0

42.8 50 2* 0.70 1 42.1 1

58.1 70 14.5* 0.95 10 57.2 5

90.6 90 70* 1.48 50 89.1 40

*Significantly lower than observed mortality (P< 0.05 by w2 test).



acetylcholine excess at the synapses, causing increased mor-

tality at low doses. The reciprocal interaction would have

occurred when a sublethal dose of propoxur synergized

permethrin. Many questions remain, however, particularly

to explain the decrease of synergism at higher concentra-

tions.

The use of mixtures of complementary insecticides,

whether or not they have different modes of action, to

control insect pests and vectors of public health importance

could improve their joint dose-efficacy through synergism,

thus reducing the amounts applied (saving cost and improv-

ing safety) and opposing resistance. Efficacy of some insec-

ticides is enhanced by mixing with enzyme inhibitors as

synergists, for example with tribufos against esterases and

piperonyl butoxide against oxidases; the latter is crucial for

the potency of pyrethroid-based aerosols and sustains the

domestic pest control industry by overcoming incipient

pyrethroid resistance problems (Sawicki, 1985; Jones,

1998). As pyrethroid-treated nets are increasingly used to

provide personal and collective protection against malaria

transmission by Anopheles mosquitoes (Lengeler et al.,

1996), we are considering ways to limit the rise and spread

of pyrethroid resistance in anophelines (Chandre et al.,

1999; Hargreaves et al., 2000), perhaps by involving other

classes of insecticides, notably organophosphates and

carbamates (Miller et al., 1991; Curtis et al., 1998; Fanello

et al., 1999; Kolaczinski et al., 2000). However, these

alternatives lack the fast knockdown activity of pyrethroids

and could be more hazardous toxicologically (IPCS,

2002). Fortunately, we found considerable synergism of

carbamateþ pyrethroid mixture against Anopheles gambiae

females, allowing reduction of the carbamate dose to a level

presenting little or no risk for human safety (Corbel et al.,

2002).

98

95

90

80

7060504030

20

10

5

2

% M

OR

TALI

TY

10–2 10–1 1

3

4

5

6

7

DOSAGE (mg/L)

PropoxurPropoxur Permethrin+ LC0

Fig. 3. Dose/mortality regression line for propoxur activity on Culex quinquefasciatus larvae, compared with propoxur synergised by

permethrin LC0.

Table 2. Synergism of permethrin and propoxur by each other against Culex quinquefasciatus larvae at LC10–LC95 levels. Synergist Ratio of 1

indicates no synergy; ratios >1 indicate degrees of synergism (range shows 95% confidence limits)

Permethrin Propoxur

Mortality %(LC level)

Without

propoxur

(mg/L)

With

propoxur

LC0(mg/L) Synergist Ratio

Without

permethrin

(mg/L)

With

permethrin

LC0 (mg/L) Synergist Ratio

10 0.0010 0.0005 2.00 (1.64–2.25) 0.063 0.016 3.93 (3.30–4.62)

20 0.0011 0.0006 1.83 (1.54–1.96) 0.072 0.023 3.13 (2.74–3.60)

30 0.0012 0.0008 1.50 (1.46–1.77) 0.080 0.030 2.66 (2.39–2.97)

40 0.0013 0.0009 1.44 (1.40–1.63) 0.087 0.037 2.35 (2.12–2.54)

50 0.0015 0.0010 1.50 (1.33–1.52) 0.094 0.046 2.04 (1.88–2.20)

60 0.0016 0.0012 1.33 (1.25–1.43) 0.102 0.057 1.79 (1.67–1.92)

70 0.0018 0.0014 1.29 (1.16–1.35) 0.111 0.071 1.56 (1.46–1.68)

80 0.0020 0.0017 1.18 (1.06–1.28) 0.123 0.091 1.35 (1.22–1.45)

90 0.0023 0.0021 *1.10 (0.92–1.20) 0.141 0.132 *1.07 (0.95–1.19)

95 0.0025 0.0026 *0.96 (0.81–1.13) 0.158 0.177 *0.89 (0.77–1.01)

*Not significantly different from 1 (P< 0.05 by w2 test).

162 Corbel et al.


Acknowledgements

We are grateful to M. Raymond, P. Carnevale and

T. Martin for helpful comments and discussion. We also

thank the Ministere Francais de la Recherche, programme

on malaria and associated communicable diseases for

developing countries (PALþ program), for funding this

work. Bayer (Leverkusen, Germany) and Agrevo

(Berkhamsted, U.K.) donated the samples of propoxur

and permethrin.

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Accepted 8 February 2002

164 Corbel et al.


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