Journal of Stored Products Research 45 (2009) 7174

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Journal of Stored Products Research

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Short communication

Phosphine resistance in Brazilian populations of Sitophilus zeamais Motschulsky

(Coleoptera: Curculionidae)
M.A.G. Pimentel a, L.R.DA. Faroni b, *, R.N.C. Guedes a, A.H. Sousa a, M.R. Totola c
a

Departamento de Biologia Animal, Universidade Federal de Viosa, Viosa, MG 36571-000, Brazil

Departamento de Engenharia Agrcola, Universidade Federal de Viosa, Viosa, MG 36571-000, Brazil
c
Departamento de Microbiologia Agrcola, Universidade Federal de Viosa, Viosa, MG 36571-000, Brazil
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Accepted 11 September 2008

Phosphine resistance was assessed in adults of 22 Brazilian populations of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). The concentrationmortality bioassays for the detection of phosphine resistance followed the FAO standard method. Twenty populations of S. zeamais were resistant to
phosphine and the resistance ratios at the LC50 ranged from 1.1- to 86.6-fold. This is the rst report of
phosphine resistance in populations of S. zeamais in Brazil, where previous surveys did not detect
resistance in this species. There was signicant variation in respiration rate (CO2 production) among the
populations (P < 0.05). Respiration rate was signicantly inversely correlated with phosphine resistance
for this species (P < 0.05). The populations with lower respiration rates showed higher levels of phosphine resistance, suggesting that the lower respiration rate is associated with the physiological basis of
phosphine resistance due to reduced fumigant uptake.
Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

Keywords:
Maize weevil
Fumigant resistance
Carbon dioxide production
Fumigation
Respiration rate
Brazil

1. Introduction
The maize weevil Sitophilus zeamais Motschulsky (Coleoptera:
Curculionidae) is cosmopolitan and especially abundant in warm
temperate to tropical regions such as Brazil, where it is the main
pest of stored maize (Rees, 1996). Phosphine is the primary fumigant used to protect the majority of the worlds grain and a variety
of other stored commodities from insect pests, including S. zeamais
(Chaudhry, 2000; Benhalima et al., 2004). This fumigant is playing
an increasingly important role in the protection of commodities
because of its low cost, fast diffusion in air, and lack of detectable
residue in the commodity (Chaudhry, 2000; Wang et al., 2006). The
fact that the fumigant methyl bromide is being phased out
increases the reliance on phosphine as a fumigant for storedproduct protection (UNEP, 1995). However, the lack of ideal airtight
conditions for fumigation in most storage units increases the
frequency of control failures, and thus increases the frequency of
phosphine fumigation (Pacheco et al., 1990; Chaudhry, 2000;
Benhalima et al., 2004; Lorini et al., 2007).
The 1972/1973 FAO global survey of insecticide resistance in
stored-product insects showed that about 10% of the populations
collected worldwide contained phosphine-resistant individuals,
but phosphine resistance was not detected in Brazil (Champ and

* Corresponding author. Tel.: 55 31 38991874; fax: 55 31 3899 2735.

E-mail addresses: lfaroni@ufv.br, rncguedes@gmail.com (L.R.DA. Faroni).

Dyte, 1976). However, control failures after phosphine fumigation

were reported in Brazil after this survey, and phosphine resistance
was detected in some species, but not in S. zeamais (Pacheco et al.,
1990; Sartori et al., 1990; Lorini et al., 2007; Pimentel et al., 2007).
These studies were initially based on discriminating concentrations
and neither the levels of phosphine resistance nor the mechanisms
of resistance were known. However, recent studies indicated high
levels of phosphine resistance in Brazilian populations of Oryzaephilus surinamensis (L.) (Silvanidae), Rhyzopertha dominica (F.)
(Bostrichidae), and Tribolium castaneum (Herbst) (Tenebrionidae)
(Lorini et al., 2007; Pimentel et al., 2007). An association between
phosphine resistance and reduced respiration rate was also
observed in these species, suggesting that phosphine resistance is
associated with reduced phosphine uptake (Pimentel et al., 2007).
In the present study, we report the discovery of phosphine resistance in Brazilian populations of S. zeamais and present the results
of experiments designed to test the hypothesis that phosphine
resistance is inversely correlated with respiration rate in this
species.
2. Materials and methods
2.1. Insects
Twenty-two populations of S. zeamais, collected at 19 sites in
seven Brazilian states between late 2004 and mid 2006, were used
in this study. These populations were reared in glass jars (1.5 l) in

0022-474X/$ see front matter Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jspr.2008.09.001

72

M.A.G. Pimentel et al. / Journal of Stored Products Research 45 (2009) 7174

Table 1
Relative toxicity of phosphine to 22 populations of Sitophilus zeamais. The insect populations are identied by the Brazilian County of origin and its respective State (Goias (GO),
Mato Grosso (MT), Minas Gerais (MG), Parana (PR), Piau (PI), Santa Catarina (SC), Sao Paulo (SP)).
Populations
d

Guaxupe I (MG)
Coimbra (MG)
Jacu (MG)
Piracicaba (SP)
Tunapolis (SC)
Sacramento (MG)
Rio Verde (GO)
Nova Era (MG)
Abre Campo II (MG)
Ipora (GO)
Canarana (MT)
Picos (PI)
Viosa (MG)
Abre Campo I (MG)
Jata (GO)
Guaxupe II (MG)
Cristalina (GO)
Guarapuava (PR)
Machado (MG)
Paracatu (MG)
Una I (MG)
Una II (MG)
a
b
c
d

No.a

Slope  SEMb

LC50 (95% FL), mg l1

RR50c (95% CL)

LC95 (95% FL), mg l1

RR95 (95% CL)

c2

600
400
600
700
700
600
800
900
600
600
500
500
500
400
500
600
900
600
600
800
900
500

3.29  0.24
6.48  0.61
4.01  0.30
7.13  0.49
4.61  0.31
4.95  0.54
5.79  0.37
3.58  0.21
5.15  0.45
3.38  0.24
8.29  0.67
5.63  0.49
3.32  0.29
2.32  0.36
2.48  0.23
2.17  0.19
1.68  0.15
1.76  0.18
2.29  0.20
1.64  0.12
2.77  0.19
3.48  0.26

2.58
2.84
4.04
4.08
4.27
4.40
4.60
4.89
6.13
6.14
6.55
6.48
7.17
7.28
7.74
8.99
10.12
34.65
63.40
137.27
157.59
223.78

8.19 (7.099.81)
5.09 (4.635.80)
10.39 (9.0212.47)
6.94 (6.517.53)
9.70 (8.7311.09)
9.46 (8.2611.60)
8.86 (8.269.66)
14.08 (12.5216.25)
12.78 (11.4114.92)
18.81 (16.1122.88)
10.35 (9.6611.33)
12.69 (11.3714.73)
22.41 (18.7828.55)
37.30 (26.4570.10)
35.73 (29.1947.14)
51.56 (41.1369.94)
97.01 (73.29143.15)
298.43 (208.17504.36)
332.58 (266.94446.96)
1,373.59 (1,004.762,062.67)
618.54 (542.10728.93)
664.49 (567.18816.03)

7.64
1.62
4.96
7.22
6.49
4.10
3.53
7.48
8.34
2.47
3.64
2.63
7.44
3.15
6.72
4.85
6.28
6.55
8.67
8.20
9.13
7.92

0.18
0.65
0.29
0.21
0.26
0.54
0.74
0.38
0.14
0.78
0.30
0.45
0.11
0.21
0.24
0.30
0.39
0.16
0.12
0.22
0.17
0.16

(2.312.85)
(2.673.00)
(3.714.40)
(3.924.24)
(4.024.53)
(4.134.66)
(4.404.80)
(4.575.21)
(5.766.51)
(5.556.76)
(6.276.84)
(6.096.88)
(6.527.84)
(6.078.29)
(6.568.87)
(7.5910.36)
(8.5511.60)
(29.9239.81)
(54.5172.21)
(120.33156.79)
(140.83173.48)
(204.56244.35)

1.10
1.56
1.58
1.65
1.70
1.78
1.89
2.37
2.37
2.54
2.51
2.78
2.82
3.00
3.48
3.92
13.41
24.53
53.11
60.97
86.58

(0.971.24)
(1.361.79)
(1.411.77)
(1.461.87)
(1.511.92)
(1.592.00)
(1.672.14)
(2.102.68)
(2.052.74)
(2.262.84)
(2.222.83)
(2.413.19)
(2.343.38)
(2.493.60)
(2.884.20)
(3.254.71)
(11.2216.01)
(20.5729.26)
(44.7962.97)
(52.5370.77)
(75.3899.44)

0.62 (0.510.76)
1.27 (1.011.60)
0.85 (0.711.01)
1.18 (0.971.45)
1.15 (0.911.46)
1.08 (0.901.30)
1.72 (1.392.12)
1.56 (1.261.93)
2.30 (1.812.92)
1.26 (1.051.51)
1.55 (1.261.91)
2.74 (2.103.57)
4.56 (2.827.39)
4.37 (3.275.83)
6.31 (4.628.61)
11.89 (8.2117.23)
36.57 (22.9658.27)
40.70 (30.0155.18)
168.42 (113.32250.31)
75.62 (60.6294.33)
81.16 (63.55103.66)

No. number of insects tested.

SEM standard error of mean.
RR resistance ratio.
Standard susceptible population.

the laboratory (28  1  C, 70  5% relative humidity (r.h.)). Whole

maize grains free of insecticide residues (13% moisture content)
were used as the food source for S. zeamais. Each collected population was subjected to the concentrationresponse bioassays
after maintenance for at least three generations in the laboratory.
2.2. Concentrationresponse curves
Fumigation of adult insects was based on the FAO method (FAO,
1975), and took place at 25  C and 70% r.h. Phosphine (ca 86% pure)
was produced using aluminium phosphide tablets (0.6 g) (Casa
Bernardo, Sao Paulo, Brazil) in acidied water (5% sulfuric acid). The
range of four to eight phosphine concentrations used to establish
the concentrationmortality curves was determined after preliminary tests including the FAO-recommended discriminating
concentration of phosphine (0.05 mg l1) (FAO, 1975). Non-sexed
adult beetles (13 weeks old) were conned in ventilated plastic
containers inside gas-tight desiccators. There were 50 adults per
container and two containers per desiccator. Two replicates with 50
insects each were used for each of the four to eight concentrations
tested against each population. Phosphine was injected with gastight syringes through a septum in the lid of each desiccator to
produce the required concentration. After fumigation, the
containers were removed from the desiccators and kept for 14 days
(25  C and 70% r.h.), after which mortality was assessed. The
mortality data were corrected for the mortality in control treatments where only air was injected.
2.3. Respirometry assays
The production of carbon dioxide was measured in a carbon
dioxide analyzer (TR 2 Sable System International, Las Vegas, NV,
USA) using methods adapted from earlier work (Guedes et al.,
2006; Pimentel et al., 2007). A series of 25 ml asks was used,
each ask containing 20 insects of each population in a completely
closed system. Three replicates were used for each population
and carbon dioxide production was measured in each ask at
a controlled temperature (25  C) after a period of 5 h

acclimatization. Carbon dioxide-free air was injected into the

asks for 2 min at 600 ml min1. An infrared reader was connected
to the outlet of the system to quantify carbon dioxide (mmol).
Respiration rate was not normalized by weight since the procedure was likely to mask the physiological basis of insecticide
resistance and its mitigation (Haynes, 2001; Guedes et al., 2006;
Pimentel et al., 2007).
2.4. Statistical analysis
The concentrationmortality data were submitted to probit
analysis (PROC PROBIT; SAS Institute, 2002). The resistance ratios
(RRs) and their respective condence intervals (95%) were calculated as described by Robertson and Preisler (1992). Carbon dioxide
production (mmol h1 insect1) was subjected to analysis of variance (P < 0.05, PROC GLM) (SAS Institute, 2002), and subsequent
Tukeys HSD test, if there was signicant variation among populations. The carbon dioxide production was also subjected to
correlation analysis against the resistance ratio (at LC50) (PROC
CORR; SAS Institute, 2002). Experiments were conducted in
a completely randomized design.
3. Results
3.1. Resistance level
Mortality values predicted by the probit model did not differ
signicantly from the bioassay observed values (P > 0.05), and it
was therefore appropriate to use the probit model for the
concentrationmortality analyses (Table 1). There was signicant
variation in susceptibility among the populations of S. zeamais. The
concentrationmortality curves indicated that ve populations of S.
zeamais (Guarapuava, Machado, Paracatu, Una I, and Una II) were
moderately resistant to phosphine (RR50 between 10- and 100fold), while low levels of phosphine resistance (RR50 < 10-fold)
prevailed in a further 15 of the 22 populations assessed. The
majority of the slopes of the concentrationmortality curves were
similar among the more phosphine-resistant populations of

M.A.G. Pimentel et al. / Journal of Stored Products Research 45 (2009) 7174

Fig. 1. Respiration rate (SEM) of Brazilian population of Sitophilus zeamais collected

in the states of Goias (GO), Mato Grosso (MT), Minas Gerais (MG), Parana (PR), Santa
Catarina (SC), Sao Paulo (SP) and Piau (PI). Histogram bars (SEM) indicate the mean
of three independent replicates and the vertical lines connect undistinguishable
populations based on Tukeys HSD test (P < 0.05).

S. zeamais, unlike the more susceptible populations, which showed

higher slopes indicating more variability of response to phosphine
(Robertson and Preisler, 1992).
3.2. Respiration rate
The production of carbon dioxide varied signicantly among
populations of S. zeamais (F21,47 4.16, P < 0.0004), ranging from
0.21  0.10 to 0.54  0.04 mmol h1 insect1 (Machado (MG) and
Sacramento (MG), respectively) (Fig. 1). Correlation analysis
between average CO2 production and resistance ratio for LC50 (i.e.,
RR50) of each population was negative and signicant (r 0.44;
P < 0.04; n 22). The populations with the highest carbon dioxide
production had the smallest RRs.
4. Discussion
The results of the present study show the occurrence of phosphine resistance in 20 populations of S. zeamais from the states of
Goias, Mato Grosso, Minas Gerais, Parana and Piau. The levels of
phosphine resistance are low (15 populations) to moderate (ve
populations) suggestive of an emerging problem. This is the rst
report of phosphine resistance in populations of S. zeamais in Brazil,
where the recommended phosphine application rate (2.0 mg l1)
should still be effective against the Brazilian populations of S. zeamais, except for the few instances where moderate to high levels of
phosphine resistance (at LC95) are present (e.g., Paracatu (MG)).

73

This is in contrast to previous surveys in Brazil (Champ and Dyte,

1976; Pacheco et al., 1990; Sartori et al., 1990).
A number of factors may contribute to the observed increase in
phosphine resistance in Brazilian populations of S. zeamais. Some of
these factors are fumigation in unsealed silos, lack of monitoring of
phosphine concentration during fumigation, extensive use of only
one fumigant, favorable weather conditions for the insect pest and
little or no insect management practiced in farms and others
storage facilities (Pacheco et al., 1990; Lorini et al., 2007). The
common procedure to cope with these pest management shortcomings in Brazilian stored product units is to increase the
frequency of phosphine fumigation, which further increases the
selection for phosphine resistance in insect pests of stored products, including S. zeamais. In addition, the movement of insects as
a result of the commodity trade is a likely contributor for the spread
of phosphine resistance in stored-product insects.
The main reported mechanism of phosphine resistance is
associated with the active exclusion of the gas (Price, 1981, 1984;
Chaudhry and Price, 1990). This mechanism results in a lower
phosphine uptake by resistant insects compared with their
susceptible counterparts, as reported in different species of storedproduct insects (Price, 1981, 1984; Chaudhry and Price, 1990;
Chaudhry, 1997, 2000). Our results with populations of S. zeamais
support such claims, and further suggest that the lower phosphine
uptake might be caused by the reduced respiration rate of the
resistant populations, as also observed in recent work with O.
surinamensis, R. dominica, and T. castaneum (Pimentel et al., 2007).
There is an urgent need for a thorough review of fumigation and
storage practices in Brazil to identify and implement improvements
in currently used fumigation methods. Future demographic studies
will allow the assessment of potential tness disadvantages associated with phosphine resistance in populations of S. zeamais. Such
studies will provide further insights for designing suitable
management programs for phosphine-resistant populations of S.
zeamais. In addition, the association between high respiration rate
and phosphine susceptibility suggests the possibility of modifying
the storage environment to increase respiration rate as a potential
way to manage phosphine resistance in stored-product insects.
Acknowledgements
The authors would like to thank F.H. da Silva and M.D. Batista for
technical assistance, and the editor (Americas) and reviewers for
their comments and suggestions. Financial support was provided
by the CNPq and FAPEMIG was also greatly appreciated.
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