J. storedProd.

Res. Vol. 31, No. 2, pp. 145-150, 1995

Copyright 0 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0022-474X195 59.50+0.00

Pergamon
0022-474X(!J4)00043-3

Resistance to DDT and pyrethroids in Brazilian

populations of Sitophilus zeamais Motsch.
(Coleoptera: Curculionidae)
RAUL NARCISO C. GUEDES,* JOSk OSCAR G. LIMA, JAMILTON
and COSME D. CRUZ

P. SANTOS3

Department of Animal Biology and 2Department of General Biology, Federal University of Vigosa,
VicosalMG, 36570.000, Brazil and National Research Center of Maize and Sorghum,
CNPMSIEMBRAPA,
P.O.Box 151, Sete Lagoas, 35700.000, Brazil
(Received 26 September 1994)

Abstract-Control failures of deltamethrin used agaiust SitopAiruSzcmtis Motsch. in Brazil led

to the investigation of the possible resistance of this pest to DDT, pirimiphos-methyl and the
pyretbroids deltamethrin, cypermethrin and permetbrin, syuergized or not with piperonyl butoxide.
DDT is not now approved for use in Brazil, but was widely used in the past against this pest.
Pirhniphos-methyl and synergized deltamethrin are recommended for S. zeamais control, and
cypermethrin and permethrin are not used despite their efficiency against this pest. Bioassay tests
were carried out using the insecticide-impregnated filter paper method recommended by FAO, and
the discriminating concentrations were established based in the LC-., of each compound, syneigized
or not, for the standard susceptible population. With these tests, a cross-resistant spectrum to DDT
and pyretbroids was verified in six populations of S. zeatnais. One population showed resistance
only to DDT and deltamethrin, while three other populations showed resistance only to DDT.

Key words-DDT, pyrethroid, resistance, maize weevil, Brazil.

INTRODUCTION

The maize weevil, Sitophilus zeamais Motsch., is a very serious pest of stored maize in Brazil (Santos
et al., 1990) and chemical control is the main control method used against it (Braga et al., 1991;
Guedes, 1993). DDT was the most widely used insecticide to protect stored maize in Brazil until its
prohibition in 1985 (Mariconi, 1963; Gallo et al., 1970, 1978; Guedes, 1993), even though in 1970
DDT resistance in S. zeamais had already been detected in a field population from Capimjpolis, State
of Minas Gerais (Mello, 1970).
When organochlorines were discontinued, the use of organophosphorus compounds, especially
malathion, for control of stored grain insects greatly increased. However, malathion use decreased
after control failures in stored grain and the sudden development of resistance in several stored grain
insect pests all over the world (Champ and Dyte, 1978; Badmin, 1990; Guedes, 1990). Thus, this
compound has been replaced by pirimiphos-methyl and especially deltamethrin which, like other
pyrethroids, shows great efficacy against S. zeamais (Cajueiro, 1988; Santos er al., 1988; Braga et al.,
1991).
*Allcorrespondenceshouldbe addressedto: Dr Raul NarcisoC. Guedes,
Kansas

State University,

Manhattan,

KS 66502, U.S.A.
145

Department

of Entomology,

123 Waters

Hall,

146

Raul

NarcisoC. Guedesef al.

Despite the success of deltamethrin against stored grain insects, Santos (1988) reported control
failures with this compound when used on maize weevil populations in the States of Parana and Rio
Grande do Sul. However, these populations were successfully controlled by the organophosphorus
insecticides malathion and pirimiphos-methyl. Other pyrethroids were also tested against these
S. zeamais populations but none of them was effective. Resistance to organophosphorus insecticides
was detected in Brazilian populations of stored grain insects by Champ and Dyte (1978) and Pacheco
et al. (1990), but resistance to pyrethroids was never reported in spite of the suspicions raised by
Santos (1988).
Previous studies on pyrethroids frequently pointed to a narrow relation between pyrethroid
resistance and DDT resistance in Musca domestica L. (Busvine, 1951; Milani and Travaglino, 1957),
ticks and Pulex irritans L. (Plapp, 1976), mosquitoes (Priester and Georghiou, 1980; Malcolm and
Wood, 1982; Halliday and Georghiou, 1985), Haematobia irritans (L.) (Byford et al., 1985), Blatella
germanica (L.) (Cochran, 1987), and predaceous mites (Scott et al., 1983). The cross-resistance
between DDT and pyrethroids was reported in Spodoptera littoralis (Boisd.) (Gammon, 1980;
Riskallah et al., 1983) and in stored grain pests the phenomenon was verified in Sitophilus oryzae
(L.) by Heather (1986). This cross-resistance between DDT and pyrethroids became known as
knockdown resistance (KDR) and is due to the alteration in the site of action of these insecticides
in the resistant insects.
The question arises whether the previous selection for DDT resistance in S. zeamais is responsible
for the pyrethroid resistance in populations never exposed to pyrethroids. This phenomenon has not
been verified in this species, although there is a suspicion that this is occurring (Santos, 1988). The
cross-resistance spectrum threatens the future use of pyrethroids against S. zeamais in Brazil and
is the reason for this study.
MATERIAL

AND METHODS

Insect samples were collected in six different Brazilian States. Three of the cultures were from
insect rearing laboratories and the others from field and grain storage facilities (Table 1). The
populations were reared and the bioassays were carried out under constant conditions of 25C and
70% r.h.
Adult insects were tested by exposure to insecticide-impregnated filter paper according to the
standard technique recommended by FAO (Anonymous, 1974), but acetone was used to dissolve the
technical grade insecticides. The exposure time was 6 h for all insecticides except DDT, for which
the exposure time was 24 h due to its slower effect on the insects. The insecticides used were DDT,
pirimiphos-methyl and the pyrethroids deltamethrin, cypermethrin and permethrin. These were used
alone or in mixture with piperonyl butoxide (PBO), an inhibitory synergist that acts upon mixed
function oxidases (Brindley and Selim, 1984; Collins, 1990; B.-Bernard and Philogene, 1993), and
were mixed in the proportions commercially used in the Brazilian deltamethrin formulation for

Table 1. Code number and origin of Brazilian populations of S. reumais

Origin
Population
code number
1
2
:+
5
6
7
8
9
10
II
12

County

State

Place

Piracicaba
Vicosa
Sate Lagoas
Sete Lagoas
Patos de Minas
Jacarezinho
Jacarezinho
Ponta Grossa
Santa Helena
Inhumas
Cachoeiro do Itapemirim
Santa Cruz do Sul

SLO Paul0
Minas Gerais
Minas Gerais
Minas Gerais
Minas Gerais
ParanO
Paran
Param
GoiPs
Goils
Espirito Santo
Rio Grande do Sul

Laboratory
Field crop
Field crop
Laboratory
Storage facility
Field crop
Storage facility
Field crop
Storage facility
Storage facility
Field crop
Storage facility

*Standard susceptible population.

tHybrid population developed from survivors of pyrethroid selections of insects from populations
7. 10 and 12.

Pyrethroid-resistant

147

S. ream&

Table 2. Toxicity of indicated insecticides to the standard laboratory susceptible population of S. :eamais
Insecticide

EPuation*

Deltamethrin
Cypermethrin
Permethrin
Deltametrin+PBOt
Cypermethrin + PBOt
Permethrin + PBOt
DDT
Pirimiphos-methyl

Y=7.390+1.996X
Y=6.201+2.012X
Y=5.751+2.664X
Y= 12.443+3.684X
Y= 10.766+3.702X
Y=8.616+ 1.662X
Y=5.304+1.107X
Y=7.865+3.391X

*Y =probit; X= log concentration

tPB0, piperonyl butoxide.

L&I (95% CL) (mail)

0.063
0.253
0.523
0.009
0.028
0.007
0.531
0.143

(0.051~.074)
(0.203-0.300)
(0.468-0.596)
(0.00&0.011)
(0.02SO.031)
(0.005-0.008)
(0.4030.707)
(0.129-0.157)

LCPP(95% CL) (mail)

0.932
3.639
3.915
0.041
0.118
0.168
67.673
0.696

(0,587-l ,938)
(2.475-6.331)
(2.394-9.830)
(0.032-0.569)
(0.0934.166)
(0.098-0.387)
(25.470-307.596)
(0.56Ho.916)

Y2

Prob.

4.13
1.57
3.36
1.86
3.22
7.98
1.63
3.61

0.123
0.539
0.184
0.602
0.197
0.054
0.554
0.306

(mg a.i./ml).

stored grain insect control (1 part pyrethroid : 10 parts PBO). Synergized deltamethrin and
pirimiphos-methyl are recommended for stored grain pests in Brazil, while cypermethrin and
permethrin are not recommended.
Discriminating concentrations (DC) were based on the LC99.9estimated for the standard susceptible
population from CENA, where it has been reared since the foundation of the institution without
insecticide selection pressure. LC99.9were obtained by probit analysis of the bioassay results (Finney,
1971) in which at least four concentrations for each insecticide were used together with a control
where only acetone was used. For each concentration three replicates of 40 insects from the standard
susceptible laboratory populations were used. Three samples of 40 adult insects for each population
were exposed to the DC of the different insecticides with or without synergist. The mortality results
obtained were compared with the mortality in the standard laboratory susceptible population by the
one-sided Z test at 95% confidence level with correction for continuity (Roush and Miller, 1986).
RESULTS

The equations for the standard laboratory susceptible population of S. zeamais obtained by probit
analysis are presented in Table 2. The DCs based on the estimated LG.9 (Table 3) and submitted
to each insect population provided the results presented in Tables 4 and 5. Three different
resistant patterns were observed: (1) six populations (4,5,7,9, 10 and 12) showed resistance to DDT
and synergized and non-synergized pyrethroids; (2) one population (11) showed resistance to
DDT and non-synergized deltamethrin; and (3) three populations (1, 3 and 8) showed resistance
only to DDT. All populations were susceptible to the organophosphorus insecticide pirimiphosmethyl.
The mortalities caused by DDT and pyrethroids in the populations resistant to them
simultaneously were extremely low (ranging from 0.0% to 29.2%) except in Population 12. In
Population 11 of S. zeamais, which is resistant to DDT and synergized pyrethroids, the mortality
caused by these insecticides were 52.5 and 80.8% respectively. Populations 2, 3 and 8, which were
resistant only to DDT, had mortalities of 43.4, 85.0 and 72.5% respectively. The highest mortality
for all insecticides was reached in the standard susceptible population of maize weevil (1).

Table 3. Insecticide discriminating concentrations and their respectwe doses based on the LCwv
determined for the standard laboratory susceptible population used to screen the S. xmais
populations for insecticide resistance
Insecticide
Deltamethrin
Cypermethrin
Permethrin
Deltamethrin + PBO*
Cypermethrin + PBO
Pennethrin + PBO*
DDT
Pirimiphos-methyl
PBO, piperonyl butoxide.

LCW9(mg/l)
2.24
8.69
5.54
0.07
0.19
0.48
329.02
1.17

Discriminating
concentration (mg/l)
2.50
8.70
5.60
0.07
0.20
0.50
330.00
I .20

Discriminating
dose (mg/cm)
0.0325
0.1130
0.0728
o.ooo9
0.0026
0.0065
4.2874
0.0156

148

Raul Narciso C. Guedes et al.

Table 4. Mortality of Brazilian populations of S. zeamais from discriminating
concentrations of DDT and pyrethroids
Mortalitv (%P at discriminatina concentrations
code no.

Deltamethrin
100.0
98.5
100.0
o.o*
9.4*
99.2
2.5%
99.2
0.0.
0.8
80.8
45.0*

2
3
4
5
6
7
8
9
10
11
12

Cypermethrin
99.2
98.3
99.2
o.o*
15.8*
100.0
3.3
100.0
2.5+
o.o*
99.2
45.0

Permethrin
100.0
91.4
100.0
o.o*
29.2
100.0
1.I
loo.0
0.0;
1.7
100.0
91.8*

DDT
94.3
43.4
85.0
o.o*
7,s
94.2
0.8*
12.5,
2.5
0.8
52.5
18.3

Mortalities followed by asterisks are significantly different from the mortality in

the standard population by the one-sided 2 test at 95% confidence level with
correction for continuity.

DISCUSSION

Resistance to four different insecticides with the same mode of action suggests a case of
cross-resistance to DDT and pyrethroids in 6 of the 11 S. zeumais populations studied. This
possibility was previously suggested by Santos (1988) in his study of the effect of pyrethroids in the
S. zeumais populations from Parana and Rio Grande do Sul and is reinforced by the history of the
widespread use of DDT in the past against Brazilian stored product insects. More than half of the
insect populations studied, from four different Brazilian States, showed resistance to deltamethrin,
cypermethrin and permethrin.
Cypermethrin and permethrin were never used against stored grain pests in Brazil and deltamethrin
showed control failures when first used against some of the S. zeumuis populations studied by Santos
(1988). Some reports about this phenomenon have already been verified in some agricultural pests
like S. littoralis (Boisd.) (Gamon, 1980) and S. oryzue (Heather, 1986), and this resistance pattern
is generally due to the knockdown resistance (KDR) mechanism (i.e. alteration of the site of action
of DDT and pyrethroids). The detection of resistance bioassays using pyrethroids synergized with
piperonyl butoxide and pirimiphos-methyl (Table 4) provided further evidence that KDR is the
resistance mechanism involved. The pyrethroid cross-resistance is probably due to the alteration in
the site of action of these insecticides because even with piperonyl butoxide, pyrethroids had little
effect over the cross-resistant maize weevil populations. This pattern fits with the KDR mechanism.
Pirimiphos-methyl, on the other hand, was highly effective against all of the insect populations, and
none of them exhibited resistance to this organophosphate insecticide.
Population 11, which was resistant to DDT and deltamethrin, did not show resistance to synergized
deltamethrin and pirimiphos-methyl. Thus, the deltamethrin resistance may be due to the activity
of mixed function oxidase and the DDT resistance due to activity of this enzymatic system or of the
Table 5. Mortality of Brazilian populations of S. reamois from discriminating concentrations of synergized pyrethroids
and oirimiuhos-methvl
Mortality (%) at discriminating concentrations
Population
code no.
I

2
3
4
5
6
7
8
9
IO
11
12

Deltamethrin + PBOb

Cvuermethrin + PBO

Permethrin + PBOh

100.0
100.0
100.0
0.0

100.0

100.0

100.0

99.2
100.0

100.0

100.0

6.3.
100.0
6.1*
100.0
o.o*
0.0
96.0
45.0

53.7
100.0
3.3*
100.0
3.3
7.58
100.0
30.8

I .-I

99.2
0.0
25.5

100.0
4.0*
100.0
2.5*
I.14
100.0

58.9*

PirimiDhos-methvl

100.0
100.0
100.0

100.0
100.0
100.0

100.0

99.2
100.0
100.0

Mortalities followed by asterisks are significantly different from the mortality in the standard laboratory population by
the one-sided 2 test at 95% confidence level with correction for continuity.
PBO. piperonyl butoxide.

Pyrethroid-resistant

S. zeamais

149

enzyme DDT dehydrochlorinase (DDTase). Populations 2, 3 and 8 did not show resistance to
pyrethroids and pirimiphos-methyl. Thus, their resistance to DDT seems to be specific, probably
involving DDT dehydrochlorination to DDE by the action of DDTase, a very common resistance
mechanism to this insecticide (Plapp, 1976; Champ and Dyte, 1978).
Some insect populations from the same geographical origin, such as 3 and 4, and 6 and 7, presented
extreme differences in the resistance detection tests, requiring additional explanations. Population
6 did not show resistance to any insecticide and Population 3 was resistant only to DDT. In contrast,
populations 4 and 7 did show resistance to DDT and pyrethroids even when the latter were synergized
with piperonyl butoxide.
Populations 3 and 4 were reared in the Stored Grain Insects Laboratory of CNPMS/EMBRAPA
and the distinct patterns observed are easily explained. Population 3 is the insect population generally
used as standard susceptible population for insecticide tests in this Research Center and was collected
in a maize field in Sete Lagoas and reared in the absence of insecticides. The second insect population
from Sete Lagoas (4) is a hybrid population developed with insects from Santa Cruz do Sul,
Jacarezinho and Inhumas submitted to pyrethroid selection pressure in laboratory, showing a
resistance spectrum similar to these populations.
The insect Populations 6 and 7, both from the same vicinity, showed different resistance patterns
due to reasons distinct from those of populations 3 and 4. Population 6 was collected in a maize field,
while Population 7 was collected from stored maize after a S. zeamais control failure with synergized
deltamethrin. The use of this pesticide had just been initiated. This reinforces the hypothesis that
DDT was responsible for the fast deltamethrin resistance development in places where this insecticide
had never been used before.
Pyrethroid use has been widely expanded in Brazil. These insecticides are used in a great range
of conditions against several insect-pests, including stored grain pests. However, the future utilization
of these compounds has been threatened by the fast evolution of resistance to pyrethroids in
conditions in which they have never been used, like the populations of S. zeamais reported here. In
this context KDR may be playing an important role since it is responsible for a typical spectrum of
high resistance levels to DDT and pyrethroids, through the reduced sensitivity of the active site of
these compounds.
Acknowledgements-The
technical assistance and suggestions of N. M. P. Guedes, F. Teixeira and F. Zolnerkevic were greatly
appreciated. We thank the Minas Gerais State Sponsor Agency (FAPEMIG) for the financial support provided and the
organizations Quimio/Roussel Uclaf, Imperial Chemical Industries and Shell International Chemical Company Ltd for
providing us with the technical grade insecticides. We also would like to thank B. A. Dover, E. G. Jay and an anonymous
referee for reviewing this manuscript.

REFERENCES
Anonymous (1974) Recommended methods for the detection and measurement of resistance of agricultural pests to
insecticides: tentative method for adults of some major beetle pests of stored cereals with malathion or lindane-FAO
Method no. 15. FAO PI. Pror. Bull. 22, 127-131.
B.-Bernard C. and Philogene B. J. R. (1993) Insecticide synergists: role, importance and perspectives. J. Toxicol. environ.
Health 38, 199-233.

Badmin J. S. (1990) IRAC survey of resistance of stored grain pests: results and progress. In Proc. 5th Int. Work. Conf.
Srored-Prod. Prof. (Edited by Fleurat-Lessard F. and Ducom P.), pp. 973-981. Bordeaux, France.
Braga G. C., Guedes R. N. C., Silva F. A. P. and Castro L. H. (1991) Avaliacao da eficiencia de inseticidas, isolados e em
misturas, no controle de Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) em milbo armazenado. R. Ceres
38, 522-528.
Brindley W. A. and Selim A. A. (1984) Synergism and antagonism in the analysis of insecticide resistance. Erviron. Em. 13,
348-353.
Busvine J. R. (1951) Mechanism of resistance to insecticides in houseflies. Narure 168, 193-195.
Byford R. L., Quisenberry S. S., Sparks T. C. and Lockwood J. A. (1985) Spectrum of insecticide cross-resistance in
pyrethroids-resistant populations of Haematobia irritans (Diptera: Muscidae). J. econ. Em. 78, 768-773.
Cajueiro I. V. M. (1988) Controle quimico de Sitophilus zeamais Motschulsky, 1855 (Coleoptera: Curculionidae), em graos
de sorgo Sorghum bicolor (L.) Moench. em laboratorio. M.S. thesis, Escola Superior de Agricultura Luiz de Queiroz, Brazil,
Champ B. R. and Dyte C. E. (1978) Informe de la Prospeccion Mundial de la FAO Sobre Suscepribilidad a 10s Insecricidas
de las Plagas de Granos Almacenados. FAO/UN, Rome.
Cochran D. G. (1987) Selection for pyrethroid resistance in German cockroach (Dictyoptera: Blatellidae). J. econ. Enr. 80,
1117-1121.
Collins P. J. (1990) A new resistance to pyrethroids in Tribolium castaneum (Herbst.). Pestic. SC;. 28, 101-I 15.
Finney D. J. (1971) Probir Analysis. University Press, Cambridge, UK.
Gallo D., Nakano O., Wiendl F. M., Silveira Neto S. and Carvalho R. P. L. (1970) Manual de Entomologia. Agronomica
Ceres, Slo Paulo, Brazil.

150

Raul Narciso C. Guedes et al.

Gallo D., Nakano O., Silveira Neto S., Carvalho R. P. L., Bat&a G. C., Berti Filho E., Parra J. R. P., Zucchi R. A. and
Alves S. B. (1978) Manual de Emomoloaia Aaricola. Astronomica Ceres. f&o Paula. Brazil.
Gammon D. W: (1980) Pyrethroid resistan& in astrain of Spodoptera Iirror& is correlated with decrease sensitivity of the
CNS in vitro. Pestic. Biochem. Physiol. 13, 53-62.
Guedes R. N. C. (1990) Resistincia a inseticidas: Desafio para o controle de pragas de griios armaxenados. Seiua SO,24-29.
Guedes R. N. C. (1993) Detec&o e heranca da resist&cia ao DDT e a piretroides em Sitophilus zeamais Motschulsky
(Coleoptera: Curcuhonidae). MS. thesis, Universidade Federal de Vigosa, Brazil.
Halliday W. R. and Georghiou G. P. (1985) Inheritance of resistance to permethrin and DDT in the Southern house mosquito
(Diptera: Culicidae). J. econ. Em. 78, 762-767.
Heather N. W. (1986) Sex-linked resistance to pyrethroids in Sirophilusoryzae (L.) (Coleoptera: Curculionidae). J. stored Prod.
Res. 22, 15-20.

Malcolm C. A. and Wood R. J. (1982) Location of a gene confering resistance to knockdown by permethrin and bioresmethrin
in adults of the BKPM3 strain of Aedes aegypti. Genetica 59,233-237.
Mariwni F. A. M. (1963) Inseticidas e seu emprego no combate a pragas. Agronomica Ceres, SHo Paulo, Brazil.
Mello E. J. R. (1970) Constata9Ho de resist&tcia ao DDT e lindane em Sitophilus oryzae (L.) em milho armaxenado na
localidade de Capinopohs, Minas Gerais. In Reunicio Bras. Milho, pp. 131-134. Porto Alegre, Brazil.
Milani R. and Travaglino A. (1957) Ricerche sulla resistenxa al DDT in Musca domestica concatenaxionse del gene kdr
(knockdown resistance) con due mutanti morfologi. Riu. Paras. 18, 199.
Pacheco I. A., Sartori M. R. and Bolonhexi S. (1990) Resistance to malathion, pirimiphos-methyl and fenitrothion in
Coleoptera from stored grains. In Proc. 5th Znt. Work. Co& Stored-Prod. Prot. (Edited by Fleurrat-Lessard F. and
Ducom P.), pp. 1029-1037. Bordeaux, France.
Plapp F. W. (1976) Biochemical genetics of insecticide resistance. Ann. Rev. Em. 21, 179-197.
Priester T. M. and Georghiou G. P. (1980) Cross-resistance spectrum in pyrethroid-resistant Culex quinquefasciatus. Pestic.
Sci. 11, 617-624.
Riskallah M. R., Abd-Elghafar S. F., Abo-Elghar M. R. and Nassar M. E. (1983) Development of resistance and
cross-resistance in fenvalerate and deltamethrin selected strains of Spodoptera littoralis (Boisd.). Pesric. Sci. 14,508-512.
Roush R. T. and Miller G. L. (1986) Considerations for design of insecticide resistance monitoring programs. J. econ .Ent.
79,293-298.

Santos J. P. (1988) ComparacHo entre populacbes de Sitophilus zeamais quanto a resistencia a inseticidas piretroides e
fosforados. In iesumos 15 Gong. Nat. MiZho e Sorgo, i. 71. Piracicaba, SzIo Paulo, Brazil.
Santos J. P.. Bitran E. and Nakano 0. (1988) Avaliacgo residual de diversos inseticidas nara a mote&o de sementes de miiho
contra ihsetos durante o armaxenamento. In A&s 16~ Cong. Not. MiZho, pp. 268-275. Brasilia, Brazil.
Santos J. P., Maia J. D. G. and Crux I. (1990) Efeito da infesta@ pelo gorgulho (Sifophilus zeamais) e traca (Sirotroga
cerealella) sobre a germinaflo de sementes de milho. Pesq. Agrop. bras. 25, 1687-1692.
Scott J. G., Croft B. A. and Wagner S. W. (1983) Studies on the mechanism of permethrin resistance in Amblysius fallacis
(Acarina: Phystoseiidae) relative to previous insecticide use on apple. J. econ. Em. 76, 6-10.