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.
# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 158–164
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).
Insecticide synergism against mosquito larvae 161
# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 158–164
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.
# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 158–164
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.
# 2003 The Royal Entomological Society, Medical and Veterinary Entomology, 17, 158–164