Insecticide resistance, mixture potentiation and fitness in populations of the maize weevil (Sitophilus zeamais)

Crop Protection 30 (2011) 1655e1666

Contents lists available at SciVerse ScienceDirect

Crop Protection journal homepage: www.elsevier.com/locate/cropro

Insecticide resistance, mixture potentiation and fitness in populations of the maize weevil (Sitophilus zeamais) A.S. Corrêa, E.J.G. Pereira, E.M.G. Cordeiro, L.S. Braga, R.N.C. Guedes* Departamento de Entomologia, Universidade Federal de Viçosa, MG, Viçosa 36571-000, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2011 Received in revised form 23 August 2011 Accepted 27 August 2011

High levels of pyrethroid resistance and emerging organophosphate resistance in Brazilian populations of the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) led to the registration of esfenvalerate þ fenitrothion against them. Thus, a survey of physiological and behavioural resistance was carried out in 27 insect populations for two pyrethroids, esfenvalerate and permethrin, and the esfenvalerate þ fenitrothion mixture. Physiological resistance to fenitrothion was also assessed, as was the potentiation of the mixture. The potential fitness cost associated with insecticide resistance was also investigated. The resistance levels were low to fenitrothion (<14.1-fold), low to moderate to the pyrethroids (1.6e70.0-fold) and low to the pyrethroid-organophosphate mixture (<5-fold) with a high heterogeneity of response among populations. The potentiation of insecticidal activity achieved with the insecticide mixture was very high (>350-fold) reinforcing its usefulness for managing weevils. There was little variation in walking behaviour (and insecticide avoidance) among populations; there was no significant variation in fitness, body mass and respiration rate among the populations of the insect. These however, displayed variable rates of grain consumption and activity of amylase and lipase. A decrease in insecticide resistance in maize weevil populations was observed relative to previous studies, but with an initial development of resistance to the insecticide mixture. Behavioural and physiological resistance were not correlated and not associated with fitness cost. Our results support the use of esfenvalerate þ fenitrothion against the maize weevil, which is likely to have reduced the levels of pyrethroid resistance in field populations. However, resistance to this mixture seems to be evolving, justifying concerns regarding its use. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Maize weevil Pyrethroid and organophosphate resistance Walking behaviour Avoidance behaviour Fitness costs

1. Introduction The use of insecticide mixtures is a recognized strategy for managing insecticide resistant populations (Brattsten et al., 1986; Tabashnik, 1990; McKenzie, 1996). Such a strategy has been recommended against stored product insects in Brazil for a few years, but it was further reinforced recently with the commercial registration of a pyrethroid-organophosphate mixture  esfenvalerate þ fenitrothion (Cajueiro, 1988; Santos et al., 1988; Anonymous, 2010). This is the current scenario in Brazil regarding the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). This species is one of the most destructive and worldwide distributed stored grain insect pests (Rees, 1996; Danho et al., 2002).

* Corresponding author. Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG 36570 000, Brazil. Tel.: þ55 31 3899 4008; fax: þ55 31 3899 4012. E-mail addresses: [email protected], [email protected] (R.N.C. Guedes). 0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.08.022

Historically, chemical control of the maize weevil relied mainly on the use of dichlorodiphenyltrichloroethane (DDT). However, with its prohibition for agricultural purposes in the mid-1980s, its replacement by some organophosphates (malathion, pirimiphosmethyl and fenitrothion) and pyrethroids (deltamethrin, permethrin and bifenthrin) took place in Brazil (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003). Overreliance on these insecticides for maize weevil control has led to the selection of resistant populations, which was particularly acute for deltamethrin and permethrin with lower levels of resistance (<10) to organophosphates (including fenitrothion) (Fragoso et al., 2003; Ribeiro et al., 2003; Pereira et al., 2009). This situation ultimately led to the current recommendations of use of insecticide mixtures, and recent registration of esfenvalerate þ fenitrothion for use against insect-pests of stored products, including the maize weevil (Cajueiro, 1988; Santos et al., 1988; Anonymous, 2010). Although there is a long history of use of fenitrothion against stored product insects, esfenvalerate has not been long or widely used in Brazil, unlike the other pyrethroids deltamethrin, permethrin and

1656

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

bifenthrin, and even less is known about the esfenvalerate þ fenitrothion mixture (Santos et al., 1988; Guedes et al., 1995; Anonymous, 2010). Insecticide resistance may have either a physiological or behavioural basis, the latter being frequently neglected especially in stored grain insects (Georghiou, 1972; Brattsten et al., 1986; Guedes et al., 2009). Behavioural mechanisms are determined by behaviours that influence the organism’s response to selective pressures by a particular insecticide and usually increase the capacity of an insect population to escape lethal insecticide concentrations (Lockwood et al., 1984; Haynes, 1988; Hoy et al., 1998). A behavioural resistant population may retain its inherent physiological insecticide susceptibility, but its individuals exhibit behavioural responses that prevent lethal insecticide exposure. Initial studies on behavioural resistance of maize weevil to insecticides recognized its occurrence in some strains and apparent independence from physiological resistance to insecticides (Guedes et al., 2009). Selection for insecticide resistance is associated with pleiotropic effects, which may place the resistant insects at a disadvantage when insecticide use is discontinued, relaxed or altered due to associated fitness costs (Coustau et al., 2000; Arnaud and Haubruge, 2002; Foster et al., 2003). However, fitness costs associated with insecticide resistance, although frequent, are not universal (Beeman and Nanis, 1986; Arnaud and Haubruge, 2002; Fragoso et al., 2005). Allelic replacement (by a less costly one) and selection of modifier genes can mitigate the cost of insecticide resistance (Coustau et al., 2000; Raymond et al., 2001; Ribeiro et al., 2007). Curiously though, the basis of insecticide resistance costs and their mitigation is little investigated, except for recent studies with the maize weevil (Guedes et al., 2006; Araújo et al., 2008a,b; Lopes et al., 2010; Silva et al., 2010a,b). As increased respiration rate (CO2 production), along with enhanced activity of digestive enzymes (amylases) and those involved in energy metabolism (lipases), seems to be associated with mitigation of fitness costs associated with insecticide resistance in the maize weevil, their determination is useful for understanding the costs associated with insecticide resistance (Guedes et al., 2006; Oliveira et al., 2007; Araújo et al., 2008a,b; Lopes et al., 2010).

Surveys of resistance to a particular insecticide group can assist in choosing compounds efficient for controlling insect-pest populations, and the existence of fitness costs is useful information in resistance management (McKenzie and Batterham, 1994; Haubruge and Arnaud, 2001; Oliveira et al., 2007). Nonetheless, although pyrethroid resistance is well recorded in Brazilian populations of the maize weevil and even organophosphate resistance (including fenitrothion resistance) was the object of attention (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003; Pereira et al., 2009), there are no studies assessing the resistance to the recently registered pyrethroid þ organophosphate mixture esfenvalerate þ fenitrothion. However, insecticide mixtures have been the object of attention for other insect pests of stored products (Desmarchelier et al., 1981, 1987; Bengston et al., 1983; Jenson et al., 2010). In this study, we carried out a survey of resistance to fenitrothion and pyrethroids (permethrin and esfenvalerate), and to the mixture fenitrothion þ esfenvalerate in representative populations of S. zeamais from Brazil and Paraguay. In addition, we determined locomotory behavioural responses of these populations exposed to the pyrethroids and the insecticide mixture (such determinations were already carried out for fenitrothion and were not repeated here (Pereira et al., 2009; Braga et al., 2011)). We also determined the demographic performance of the populations of the weevil, and correlated these responses not only with the observed levels of physiological resistance, but also with metabolic traits potentially related to resistance-associated fitness costs. 2. Material and methods 2.1. Insects Twenty-five populations of the maize weevil from the main maize-producing states from Brazil and two from Paraguay were used in this study (Fig. 1). These populations were collected from stored maize grains in representative stored product units mostly between March and September of 2006 in the Brazilian states of Goiás (GO), Mato Grosso (MT), Mato Grosso do Sul (MS), Minas Gerais (MG), Paraná (PR), Pernambuco (PE), Santa Catarina (SC), São

Fig. 1. Sampling sites of 27 populations of the maize weevil (Sitophilus zeamais) from eight Brazilian states (Goiás (GO), Minas Gerais (MG), Mato Grosso (MT), Mato Grosso do Sul (MS), Pernambuco (PE), Paraná (PR), Santa Catarina (SC), and São Paulo (SP)) and Paraguay.

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

Paulo (SP), and in the regions of Pedro Juan Caballero and Arroyo Jambure Amambay in Paraguay. They were maintained as large colonies (>200 insects per jar) for five generations without insecticide selection prior to the current bioassays to minimize eventual changes in their genetic make-up. The standard susceptible population (named “Sete Lagoas”) used was obtained from the Maize and Sorghum National Research Centre of the Brazilian Agricultural Research Corporation (EMBRAPA Milho & Sorgo, Sete Lagoas, MG, Brazil), where it has been maintained for over 20 years in the absence of insecticides. They were maintained in glass containers (1.5 l) within growth chambers (25  2  C, 70  10% RH, 12 h:12 h photoperiod (D:L)), and reared on insecticide-free whole maize grains. The bioassays were carried out under these same environmental conditions, except for the behavioural trials, which were carried out at room temperature (25  3  C). The populations of Jacarezinho (state of Paraná) and Juiz de Fora (state of Minas Gerais) were collected in the late 1980s and in the 1999, respectively; both populations exhibited high levels of DDT and pyrethroid resistance (>100 fold) based on previous studies (Guedes et al., 1994, 1995; Fragoso et al., 2003, 2007; Ribeiro et al., 2003). These populations are also maintained free of insecticide selection. 2.2. Chemicals Technical-grade permethrin (92.2% pure, Syngenta, São Paulo, SP, Brazil), esfenvalerate (96.9% pure, Iharabrás/Sumitomo, Sorocaba, SP, Brazil), and fenitrothion (96.8% pure, Iharabrás/Sumitomo, Sorocaba, SP, Brazil) were used. Insecticide concentrations were prepared using analytical-grade acetone (99.8% pure, Vetec Química Fina, Duque de Caxias, Rio de Janeiro, RJ, Brazil) as solvent. Permethrin, esfenvalerate and fenitrothion were used separately, besides the insecticide mixture of 20 parts of fenitrothion to 1 part of esfenvalerate representing the formulation recently registered and made commercially available for maize weevil control in Brazil. 2.3. Concentration-mortality bioassays Bioassays were carried out in a completely randomized experimental design with five independent replicates. Each replicate encompassed a 20 ml glass vial with its inner walls coated with dried insecticide residue (applied at the rate of 0.4 ml/vial). Twenty non-sexed adult weevils (two-weeks old) were transferred to each vial and mortality was recorded after 48-h exposure. Insects were considered dead if unable to walk when prodded with a fine hair brush (Fragoso et al., 2003; Ribeiro et al., 2003). At least six different insecticide concentrations were used to estimate each concentration-mortality curve. Control vials were treated with acetone only. 2.4. Behavioural bioassays Behavioural bioassays were carried out for all insecticides and mixture, except fenitrothion since it was already target of such studies (Pereira et al., 2009; Braga et al., 2011). Two behavioural bioassays were carried out in arenas either fully-treated or halftreated with insecticide dissolved in acetone (control treatments were treated with acetone only) (Guedes et al., 2008, 2009; Pereira et al., 2009). Filter papers (Whatman no. 1) with dried insecticide residue (applied as 1 ml solution and left to dry for 20 min, at concentration equivalent to the LC95 for the standard susceptible population) were placed on Petri dishes (9.0 cm diameter). The inner walls of each Petri dish were coated with TeflonÒ PTFE (DuPont, Wilmington, DE) to prevent the insects from escaping. Arenas with individual insects were used for each combination of

1657

sex, strain and insecticide in each behavioural bioassay (fully- and half-treated arenas), and no mortality was observed within the exposure time used for the behavioural bioassays. The movement of each insect within the arena was recorded for 10 min and digitally transferred to a computer using an automated video tracking system equipped with a CCD camera (ViewPoint Life Sciences Inc., Montreal, Canada). The video images of the arenas were maintained either undivided, for the behavioural bioassay with fully-treated arenas, or divided into two symmetrical zones: one untreated and the other treated with insecticide, for the behavioural bioassay with half-treated arenas. The parameters recorded for the fully-treated arenas were walked distance (cm), velocity (cm s1), time spent walking (s), and number of stops in the arena. For the halftreated arenas, only the time spent on the insecticide-treated and untreated zones were recorded and used to calculate the proportion of time in the untreated side of the arena. Behavioural tests were carried out between 14:00 and 18:00 h in a room with artificial, incandescent light and average temperature of 25  3  C. Adult insects (two-weeks old) were sexed based on their rostrum appearance and texture (Tolpo and Morrison, 1965) and maintained on untreated arenas for 24 h. After such acclimation, the insects were individually placed in the test arenas and their walking behaviour was recorded for 10 min. The experiments were set using a completely randomized design and following a factorial scheme (27 populations  2 sexes  3 insecticides) with 16 replicates, each one consisting of a single insect. In each trial (or replicate), the filter paper was replaced, and the side on which the insect was released in the arena was randomly chosen in each trial. 2.5. Instantaneous rate of population increase (ri), body mass, and grain consumption The experiment was carried out in glass jars (1.5 L) containing insecticide-free whole maize (250 g, 14.4% moisture content). Fifty unsexed adults of maize weevil (less than two-weeks old) were released in each jar. Three replicates were used for each population. The number of live insects, as well as grain weight and its moisture content (13.3%) were recorded after the storage period (100 days). The instantaneous rate of population increase (ri) was calculated using the formula ri ¼ [ln(Nf/Ni)]/DT, where Nf and Ni are, respectively, the final and initial number of live insects, and DT is the duration of the experiment in days (Stark and Banks, 2003). 2.6. Respirometry Carbon dioxide production was measured in a CO2 Analyzer (TR3, Sable Systems International, Las Vegas, NV, USA) using methods adapted from Guedes et al. (2006). A series of 25-ml chambers was used, each chamber containing 20 sexed insects of each population in a completely closed system. Three replicates were used for each population and CO2 production was measured in each chamber. Chambers were connected to the system for 1 h before measuring the CO2 produced by the insects. The measurements were obtained by injecting CO2-free air into the chambers for 1.5 min at a 100-ml/min flow rate. This air current directed the CO2 to an infrared reader connected to the system allowing the prompt quantification of ml CO2 produced per hour. After this measurement, insects were removed from the chambers and individually weighed on an analytical balance (Sartorius BP 210D, Göttingen, Germany) to determine their (wet) body mass. CO2 production in a control chamber without insects was also determined. The experiments were set using a completely randomized design and factorial scheme (27 populations  2 sexes) with three replicates.

1658

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

2.7. Enzymatic assays Amylase and lipase activities were respectively determined using the enzymatic kits K003 and K025 from BIOCLIN (QUIBASA e Química Básica Ltda, Belo Horizonte, Minas Gerais, Brazil), as described elsewhere (Araújo et al., 2008a; Lopes et al., 2010). For both assays, batches of five insects were homogenized in 1 ml deionized H2O and centrifuged at 1000 gmax for 15 min. Amylase activity was determined by incubating the samples with starch in the presence of iodine, a preparation that progressively loses the blue colour upon starch hydrolysis by amylase (Caraway, 1959). The absorbance is read at 660 nm. Lipase activity was determined adapting earlier described method (Cherry and Crandall, 1932), in which lipases acts over a glycerol ester releasing a chromogenic compound that is quantified at 410 nm. Protein concentration was also determined (Warburg and Christian, 1941). Activity values for amylase and lipase were expressed as amylase units (AU/dl) and international units (IU/dl), respectively. Amylase unit (AU/dl) refers to the amount of amylase that hydrolyzes 10 mg starch in 30 min at 37  C, while international unit of lipase activity (IU/dl) refers to the amount of lipase that releases 1 mmol of fatty acid per minute. The experiments were set using a completely randomized design and factorial scheme (27 populations  2 sexes) with three replicates. 2.8. Statistical analyses Concentration-mortality data were subjected to probit analysis (PROC PROBIT, SAS Institute, 2002), and 95% confidence intervals for resistance ratios were estimated following Robertson and Preisler (1992) and considered significant if not including the value 1. The overall results for fully-treated arenas were subjected to a three-way (population  sex  insecticide) multivariate analysis of variance (PROC GLM with MANOVA statement, SAS Institute, 2002), and the behavioural responses measured were subjected to Pearson correlation analyses (PROC CORR, SAS Institute, 2002). These analyses indicated that the walked distance was representative of the behavioural parameters measured, and it was further subjected to univariate analysis of variance for each insecticide (PROC GLM, SAS Institute, 2002), and subsequent Scott-Knott groupement analysis test (P < 0.001) (Scott and Knott, 1974). The results from the behavioural bioassays using half-treated arenas were subjected to univariate analysis of variance (PROC UNIVARIATE, SAS Institute, 2002), followed by Scott-Knott groupement analysis test (P < 0.001), (Scott and Knott, 1974), whenever necessary. Pearson’s correlation analysis (PROC CORR, SAS Institute, 2002) between the resistance ratio (RR at LC50), and the walked distance and proportion of time spent in the non-treated side of the arena were also carried out for each insecticide. Data on population growth and grain weight loss were subjected to univariate analyses of variance (PROC GLM, SAS Institute, 2002) while insect body mass, respiration rate, and enzyme activity were subjected to a two-way multivariate analysis of variance (population  sex) (PROC GLM with MANOVA statement, SAS Institute, 2002), and these latter response variables were subsequently subjected to univariate analyses of variance (PROC GLM, SAS Institute, 2002) and Scott-Knott’s test (Scott and Knott, 1974), when appropriate. Subsequently, (partial) canonical correlation analyses (PROC CANCORR, SAS Institute, 2002) were carried out to recognize any significant correlation of instantaneous rate of population increase with level of insecticide resistance (RR at LC50), with walked distance, and with proportion of time spent on the untreated side of the arena. Path analysis was used to identify and quantify direct and indirect interactions between the instantaneous rate of population growth (ri) and grain consumption, respiration rate, body mass, and

enzyme activity (PROC REG and PROC CALIS, SAS Institute, 2002). Unlike the behavioural data, data on body mass, and activity of amylase and lipase did not satisfy the assumptions of normality and homogeneity of variances (PROC UNIVARIATE, SAS Institute, 2002) requiring data transformation (log (x þ 1) for body mass and log (x) for amylase and lipase activities). 3. Results 3.1. Concentration-mortality bioassays Control mortality was always lower than 10% and used to correct for natural mortality in the Probit analysis. Concentration-mortality data were satisfactorily described by the probit model (goodnessof-fit tests exhibiting low c2-values (<12.00) and high P-values (>0.06)), which was therefore suitable to estimate the intended toxicological parameters (Tables 1e4). The resistance ratios were estimated relative to the LC50 and LC95 for the standard susceptible population from Sete Lagoas (i.e., Sete Lagoas; Tables 1e4). The population that displayed highest LC50 for permethrin was Juiz de Fora (69.6-fold higher than LC50 for the standard susceptible population) (Table 1). This same population also exhibited the highest level of esfenvalerate resistance (66.7 at LC50) (Table 2). Resistance to fenitrothion was detected only at low levels with a maximum level of resistance at the LC50 of 14.1-fold detected for the Maracaju population (Table 3). Fenitrothion exhibited higher potency than both pyrethroids, which are used without the addition of piperonyl butoxide in their formulation unlike what takes place with deltamethrin. There was significant variation in the slopes of the regression lines among the populations, which ranged from 0.70  0.05 (Viçosa) to 1.56  0.14 (Canarana) for permethrin, from 0.56  0.06 (Espírito Santo do Pinhal) to 0.91  0.08 (Maracaju) for esfenvalerate, and from 0.70  0.09 (Maracaju and São João) to 4.01  0.21 (Guarapuava) for fenitrothion (Tables 1e3). As a result, the rank of susceptibility of the populations based on the LC50 was different from that based on the LC95 (Tables 1e3). In contrast with the isolated pyrethroid insecticides, but resembling more the levels (<15-fold) of fenitrothion resistance among the maize weevil populations surveyed, only low resistance levels to the mixture esfenvalerate þ fenitrothion were detected (Table 4). The resistance ratio (RR) based on the LC50 varied from 1.0- to 5.0-fold among the populations (Unaí). As with the isolated insecticides, there was also significant variation in the slopes of the concentration-mortality curves (between 0.64  0.15 for Votuporanga and 3.61  0.30 for Piracicaba), and the rank of susceptibility of the populations to the mixture fenitrothion þ esfenvalerate based on LC50 was different from that based on LC95 (Table 4). The potentiation ratios were estimated dividing the LC50 of the insecticides used alone by the LC50 of the insecticide mixture. The insecticide mixture led to significant potentiation of insecticidal activity when compared with fenitrothion (potentiation ratio (PR) (SEM) of 362.0 (70.3) fold) and particularly with permethrin used alone (PR (SEM) ¼ 4230.8 (1620.9) fold), although with a more variable potentiation response for this last compound. 3.2. Behavioural assays Only the pyrethroids and the insecticide mixture were used in the behavioural bioassays (with both fully-treated and half-treated arenas), because such bioassays and their results with fenitrothion have already been published elsewhere (Pereira et al., 2009; Braga et al., 2011). 3.2.1. Locomotory behaviour in fully-treated arenas Overall mobility parameters measured for S. zeamais significantly differed for insecticides (dfnum/den ¼ 15/8279.3; Wilks’

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

1659

Table 1 Relative toxicity of permethrin to populations of the maize weevil Sitophilus zeamais. Population

N

Slope (SE)

LC50 (95% FL) mg/cm2

RR50 (95%CL)

LC95 (95%FL) mg/cm2

RR95 (95%CL)

c2

P

Sete Lagoas Rio Verde Piracicaba Pedro Juan Caballero Tunapolis Vicentina Abre Campo Dourados-Bororó Guaxupé Amambay São José do Rio Pardo Jacuí Dourados-Proterito Dourados-Barreirinha Guarapuava Sacramento Canarana Nova Era Unaí Espírito Santo do Pinhal Machado Maracaju São João Viçosa Votuporanga Jacarezinho Juiz de Fora

800 800 600 600 600 600 600 800 700 800 800 700 800 700 600 600 600 800 600 800 700 600 800 800 800 780 660

1.43 0.82 1.13 1.29 1.16 0.80 0.97 1.31 0.84 0.77 0.86 0.96 1.11 0.94 1.19 1.08 1.56 1.09 1.07 1.01 0.85 0.94 0.82 0.70 1.24 1.02 1.23

1.6 1.3 1.8 2.7 2.7 2.8 2.9 3.7 4.2 4.4 4.4 4.6 5.2 5.3 5.5 5.6 5.7 6.1 7.1 8.3 9.5 9.9 13.6 24.4 25.2 88.9 110.1

1.0 (0.7e1.3) 0.8 (0.5e1.2) 1.1 (0.7e1.8) 1.7 (1.1e2.5) 1.7 (1.3e2.3) 1.8 (1.2e2.7) 1.8 (1.3e2.6) 2.3 (1.7e3.1) 2.7 (1.8e3.8) 2.8 (1.9e4.0) 2.8 (2.0e3.9) 2.9 (2.1e4.1) 3.3 (2.4e4.5) 3.3 (2.3e4.7) 3.5 (2.6e4.8) 3.6 (2.5e5.0) 3.6 (2.6e4.9) 3.9 (2.4e6.2) 4.5 (3.2e6.3) 5.2 (3.7e7.2) 6.0 (4.2e8.7) 6.2 (3.8e10.3) 8.6 (6.0e12.3) 15.4 (10.3e23.0) 15.9 (11.8e21.5) 56.2 (37.7e83.6) 69.6 (45.7e106.0)

22.3 129.9 52.3 50.2 70.1 325.0 140.5 67.0 373.5 596.3 365.6 236.6 158.7 301.2 134.2 189.8 64.3 196.2 242.2 354.4 815.5 543.4 1419.0 5384.0 529.9 3658.0 2407.0

1.0 5.9 2.4 2.3 3.2 14.7 6.3 3.0 16.9 27.0 16.5 10.7 7.1 13.6 6.0 8.6 2.9 8.8 10.9 16.0 36.9 24.5 64.2 244.1 23.8 165.0 108.3

4.5 10.4 9.4 9.1 2.7 3.3 5.9 7.2 5.9 4.8 9.5 2.1 8.9 7.2 7.2 4.1 7.1 2.0 3.9 7.1 5.2 8.7 9.4 9.8 6.0 11.9 10.7

0.62 0.11 0.05 0.06 0.61 0.50 0.21 0.30 0.31 0.57 0.15 0.84 0.18 0.21 0.13 0.39 0.13 0.92 0.42 0.31 0.40 0.07 0.15 0.13 0.42 0.06 0.06

(0.09) (0.05) (0.13) (0.16) (0.10) (0.07) (0.07) (0.10) (0.07) (0.06) (0.06) (0.08) (0.09) (0.08) (0.10) (0.11) (0.14) (0.09) (0.12) (0.10) (0.09) (0.15) (0.06) (0.05) (0.08) (0.10) (0.13)

(1.3e1.9) (0.9e1.7) (1.0e3.1) (1.4e4.2) (2.1e3.4) (2.0e3.9) (2.1e4.0) (2.9e4.5) (3.1e5.7) (3.2e6.0) (3.4e5.8) (3.4e6.1) (4.1e6.5) (3.9e7.0) (4.3e7.0) (4.3e7.5) (4.4e7.2) (4.8e7.6) (5.4e9.2) (6.3e10.6) (6.9e12.9) (4.7e20.0) (10.2e18.4) (17.5e34.9) (20.2e31.4) (57.5e138.5) (68.3e186.4)

lambda ¼ 0.9073; F ¼ 19.79; P < 0.0001), populations (dfnum/ ¼ 130/14,781, Wilks’ lambda ¼ 0.8353, F ¼ 4.23, P < 0.001), sex (dfnum/den ¼ 5/2999, Wilks’ lambda ¼ 0.9948, F ¼ 3.14, P < 0.0078), and for the interaction insecticide  population  sex (dfnum/ den ¼ 390/14,976, Wilks’ lambda ¼ 0.8491, F ¼ 1.28, P < 0.0002) when subjected to multivariate analysis of variance. Univariate analysis of variance for the individual mobility parameters of all populations varied for time in movement (from 339.9  6 to 432.4  9 s; F215,3003 ¼ 3.36, P < 0.0001), average walking velocity (0.61  0.01e0.70  0.01 cm/s; F215,3003 ¼ 3.46, den

(15.3e35.7) (78.3e244.9) (21.6e256.7) (24.3e196.2) (44.9e127.8) (163.9e836.7) (76.5e308.0) (47.5e104.0) (187.3e953.2) (301.8e1476.0) (206.1e766.3) (133.3e514.7) (99.1e299.6) (169.0e654.4) (85.9e242.3) (100.7e471.5) (44.4e106.6) (120.8e375.9) (126.4e649.2) (189.6e872.7) (365.1e2749.0) (146.6e12250.0) (710.7e3560.0) (2446.0e14961.0) (352.4e887.6) (1625.0e12653.0) (1041.0e9492.0)

(0.5e1.8) (2.9e11.9) (1.0e5.8) (1.0e4.9) (1.6e6.1) (6.0e36.1) (2.8e14.2) (1.7e5.3) (6.9e41.5) (11.1e65.4) (7.6e35.7) (4.9e23.3) (3.6e14.2) (6.2e29.8) (3.1e11.7) (3.6e20.2) (1.6e5.3) (2.4e32.2) (4.5e26.6) (6.8e37.4) (12.8e105.9) (6.3e95.2) (26.2e157.0) (91.1e653.9) (12.8e44.3) (67.8e401.5) (44.3e264.7)

P < 0.0001) and the number of stops in the arena (752  28e992  24; F215,3003 ¼ 3.82, P < 0.0001). Walked distance was positively correlated with walking time (r ¼ 0.84, P < 0.0001) and with the average walking velocity (r ¼ 0.66, P < 0.000), whereas it was negatively correlated with the number of stops (r ¼ 0.43, P < 0.0001). Thus, only the walked distance was considered in subsequent analyses as its trends and differences are representative of the other parameters assessed. When considered separately, such behavioural trait also showed significant contribution for differences among maize weevil populations and

Table 2 Relative toxicity of esfenvalerate to populations of the maize weevil Sitophilus zeamais. Population

N

Slope (SE)

LC50 (95% FL) mg/cm2

RR50 (95%CL)

LC95 (95%FL) mg/cm2

RR95 (95%CL)

c2

P

Sete Lagoas Piracicaba Unaí Canarana Dourados-Bororó Maracaju Guaxupé Vicentina Pedro Juan Caballero Abre Campo Dourados-Proterito Rio Verde Tunapolis Amabay Jacuí Sacramento Guarapuava Machado Viçosa São José do Rio Pardo Dourados-Barreirinha Votuporanga São João Nova Era Espírito Santo do Pinhal Jacarezinho Juiz de Fora

700 700 760 800 700 800 800 800 800 700 780 800 700 800 780 800 800 800 580 800 800 800 800 700 800 800 700

0.80 (0.06) 0.64 (0.07) 0.85 (0.05) 0.75 (0.05) 0.81 (0.06) 0.91 (0.08) 0.70 (0.06) 0.75 (0.06) 0.70 (0.05) 0.67 (0.06) 0.84 (0.07) 0.84 (0.07) 0.71 (0.07) 0.76 (0.07) 0.86 (0.08) 0.83 (0.06) 0.70 (0.07) 0.66 (0.06) 0.80 (0.10) 0.78 (0.08) 0.90 (0.10) 0.70 (0.07) 0.70 (0.05) 0.58 (0.07) 0.56 (0.06) 0.65 (0.07) 0.80 (0.08)

6.4 5.9 4.0 7.4 7.6 8.7 9.8 10.5 10.6 11.9 12.8 13.0 13.6 14.7 18.6 19.9 20.6 24.5 24.6 25.7 32.6 35.5 38.7 41.6 87.7 162.5 427.7

1.0 (0.6e1.6) 0.9 (0.4e0.9) 0.6 (0.5e1.5) 1.2 (0.7e1.8) 1.2 (0.7e1.9) 1.4 (0.8e2.1) 1.5 (1.0e2.5) 1.6 (1.0e2.6) 1.7(1.0e2.6) 1.8 (1.1e3.0) 2.0 (1.3e3.1) 2.0 (1.3e3.1) 2.1 (1.3e3.4) 2.3 (1.4e3.7) 2.9 (1.8e4.6) 3.1 (2.0e4.9) 3.2 (1.9e5.3) 3.8 (2.3e6.4) 3.8 (2.0e7.3) 4.0 (2.5e6.5) 5.1 (3.2e8.0) 5.5 (3.2e9.6) 6.0 (3.8e9.6) 6.5 (3.5e12.9) 13.7 (6.9e26.9) 25.3 (14.2e45.2) 66.6 (41.4e107.3)

711.7 (326.9e2001.0) 2273.0 (989.5e6831.0) 347.5 (187.2e802.0) 1160.0 (593.2e2751.0) 827.7 (405.5e2140.0) 570.0 (308.2e1330.0) 2148.0 (961.2e6349.0) 1617.0 (747.2e4658.0) 2340.0 (1081.0e6437.0) 3420.0 (1326.0e13160.0) 1160.0 (582.3e2951.0) 1169.0 (597.6e2873.0) 2836.0 (1113.0e11076.0) 2128.0 (934.0e6745.0) 1522.0 (692.9e4752.0) 1937.0 (944.2e5048.0) 4746.0 (1697.0e21765.0) 7674.0 (2587.0e37301.0) 2747.0 (784.6e31060.0) 3290.0 (1278.0e13421.0) 2188.0 (926.6e8068.0) 7803.0 (2376.0e50855.0) 8364.0 (3800.0e23923.0) 27621.0 (6465.0e269280.0) 74752.0 (15543.0e821625.0) 54146.0 (14179.0e470125.0) 48843.0 (19825.0e185096.0)

1.0 3.2 0.5 1.6 1.2 0.8 3.0 2.3 3.3 4.8 1.6 1.6 3.9 3.0 2.1 2.7 6.7 10.8 3.9 4.6 3.1 10.9 11.7 38.8 105.0 76.1 68.6

4.4 3.7 2.7 1.0 4.0 3.8 4.4 5.2 5.2 2.7 8.1 1.9 3.1 5.4 6.7 4.0 1.2 7.8 8.8 5.5 5.2 11.1 5.9 8.1 2.0 11.6 7.0

0.49 0.71 0.75 0.98 0.55 0.70 0.62 0.52 0.52 0.74 0.23 0.93 0.69 0.49 0.34 0.67 0.98 0.25 0.07 0.48 0.52 0.08 0.44 0.15 0.92 0.07 0.22

(4.6e9.2) (4.1e8.6) (3.0e5.3) (5.5e10.1) (5.6e10.5) (6.4e11.6) (7.1e13.8) (7.5e14.5) (7.7e14.8) (8.4e17.0) (9.5e17.2) (9.7e17.3) (9.5e19.4) (10.6e20.7) (13.7e25.7) (14.8e27.2) (14.4e30.4) (16.8e37.6) (9.7e51.9) (18.3e37.4) (24.4e45.4) (20.5e63.0) (27.8e54.2) (25.5e75.2) (52.2e176.6) (90.3e306.5) (307.0e611.9)

(0.3e3.5) (0.2e1.5) (0.9e11.8) (0.5e5.2) (0.3e3.9) (0.3e2.5) (0.8e10.9) (0.6e8.0) (0.9e11.5) (1.2e20.0) (0.5e5.4) (0.5e5.3) (0.9e16.5) (0.8e11.1) (0.6e7.7) (0.8e9.1) (1.5e30.5) (2.3e51.8) (0.9e16.6) (1.1e19.5) (0.8e12.0) (2.5e47.5) (3.3e41.9) (5.4e284.0) (13.0e861.0) (15.5e375.9) (17.0e277.1)

1660

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

Table 3 Relative toxicity of fenitrothion to populations of the maize weevil Sitophilus zeamais. Population

N

Slope (SE)

LC50 (95% FL) mg/cm2

RR50 (95%CL)a

LC95 (95%FL) mg/cm2

RR95 (95%CL)

c2

P

Sete Lagoas Nova Era Jacuí Dourados-Barreirinha Dourados-Proterito Unaí Amambay Rio Verde Sacramento Piracicaba Guaxupé Guarapuava São José do Rio Pardo Dourados-Bororó São João Juiz de Fora Viçosa Tunapolis Jacarezinho Abre Campo Canarana Espírito Santo do Pinhal Votuporanga Vicentina Pedro Juan Caballero Machado Maracaju

600 680 620 700 720 480 780 600 780 580 820 700 560 540 680 640 740 560 660 560 700 660 700 460 600 640 720

1.00 0.84 1.05 0.80 0.76 1.00 0.58 1.10 2.01 1.81 2.12 4.01 1.94 0.98 0.70 1.06 0.72 2.06 1.76 0.73 1.64 0.71 0.74 1.29 1.07 0.65 0.70

0.8 0.7 0.7 0.8 0.8 0.9 0.9 1.2 1.5 2.0 2.0 1.8 2.1 2.2 2.8 2.9 3.0 3.1 2.4 3.4 3.6 4.5 4.6 6.3 5.7 7.5 11.6

1.0 0.9 0.9 0.9 1.0 1.1 1.1 1.5 1.8 2.4 2.5 2.5 2.6 2.7 3.5 3.5 3.6 3.7 3.9 4.1 4.3 5.5 5.6 7.7 7.0 9.2 14.1

3.2 2.6 3.2 9.6 11.8 4.7 56.6 4.2 2.9 4.2 3.8 2.5 4.2 8.9 58.1 10.2 53.1 5.9 0.6 21.8 8.1 112.9 85.6 17.9 20.4 242.6 229.4

1.0 0.8 1.0 3.1 3.7 1.5 17.7 1.3 0.9 1.3 1.2 0.8 1.3 2.8 18.1 3.2 16.6 1.8 3.0 6.8 2.5 35.3 26.7 5.6 6.4 75.8 71.7

6.7 6.4 6.2 9.3 7.6 8.7 6.2 3.1 5.2 4.5 11.7 8.5 8.3 5.1 8.0 7.9 6.1 7.1 9.1 6.8 6.1 8.0 10.9 8.2 7.4 5.3 7.0

0.15 0.27 0.28 0.16 0.18 0.07 0.40 0.70 0.64 0.35 0.11 0.20 0.08 0.41 0.16 0.16 0.41 0.13 0.11 0.23 0.41 0.15 0.10 0.10 0.11 0.39 0.20

(0.09) (0.07) (0.10) (0.08) (0.07) (0.13) (0.06) (0.17) (0.26) (0.10) (0.21) (0.28) (0.33) (0.07) (0.07) (0.11) (0.08) (0.57) (0.08) (0.06) (0.32) (0.07) (0.10) (0.30) (0.16) (0.08) (0.09)

(0.7e1.0) (0.7e0.9) (0.5e1.3) (0.5e1.1) (0.6e1.1) (0.2e1.3) (0.5e1.6) (0.9e1.5) (1.3e1.7) (1.8e2.1) (1.8e2.2) (1.7e1.9) (1.7e2.5) (1.9e2.6) (1.7e3.5) (2.4e3.3) (2.1e2.8) (2.8e3.3) (2.3e2.6) (2.9e3.9) (3.2e3.9) (2.9e6.9) (2.3e8.7) (4.3e8.9) (5.2e6.4) (3.8e15.5) (5.1e17.6)

insecticide treatment (F26,3003 ¼ 7.48, P < 0.0001; and F2,3003 ¼ 17.80, P < 0.01, respectively). The interaction insecticide  population  sex was also significant (F78,3003 ¼ 1.35, P ¼ 0.02) for this trait, but the sex differences were negligible (when the means where compared using Scott-Knott’s test at P < 0.05) and therefore not presented in Fig. 2.

(0.7e1.4) (0.6e1.3) (0.6e1.3) (0.6e1.4) (0.7e1.6) (0.7e1.7) (0.7e1.8) (0.9e1.8) (1.6e2.1) (1.9e2.7) (2.0e2.8) (2.4e2.8) (2.2e2.9) (2.4e3.1) (2.1e5.8) (3.0e4.0) (2.4e5.4) (3.1e4.2) (2.9e5.3) (3.5e4.6) (3.9e4.7) (3.6e8.4) (3.3e9.6) (6.4e9.3) (6.5e7.6) (5.4e16.0) (7.3e18.1)

(2.6e4.0) (1.4e3.4) (2.8e4.0) (6.2e12.2) (8.2e14.7) (1.2e6.9) (32.3e81.1) (3.3e6.0) (2.6e3.5) (3.7e4.8) (3.4e4.5) (2.3e2.8) (3.4e6.1) (7.5e11.0) (24.7e94.6) (8.4e13.5) (35.5e81.1) (5.1e7.3) (0.1e18.4) (16.3e32.3) (7.1e9.5) (10.2e1180.5) (9.0e1216.0) (11.2e212.3) (16.4e27.6) (31.8e978.3) (28.3e1229.8)

(0.5e2.0) (0.3e1.9) (0.6e1.9) (1.0e9.0) (1.2e11.4) (0.5e12.2) (3.8e81.4) (0.6e2.0) (0.6e1.5) (0.6e1.8) (0.7e2.6) (0.5e1.6) (0.9e1.8) (2.1e3.2) (4.6e70.2) (2.3e4.1) (4.5e61.5) (1.4e2.3) (2.8e3.1) (4.7e8.7) (1.9e3.0) (12.8e112.3) (3.5e380.5) (2.1e8.6) (5.0e34.5) (12.2e450.1) (10.9e473.5)

behaviour), the effect of sex was negligible (and without a general trend), and there was significant variation among populations and insecticides (P < 0.0001). Some maize weevil populations exhibited avoidance to esfenvalerate and permethrin, but not to the esfenvalerate þ fenitrothion mixture (Fig. 3). 3.3. Correlations between physiological and behavioural resistance

3.2.2. Walking behaviour in partially-treated arenas Although the interaction insecticide  population  sex was also significant (F52, 2237 ¼ 1.41; P ¼ 0.03) for the proportion of time spent on the non-treated half of the arena (i.e. avoidance

The levels of permethrin resistance among the maize weevil populations were highly significant and positively correlated with the levels of esfenvalerate resistance (r ¼ 0.91, P < 0.0001), but not

Table 4 Relative toxicity of esfenvalerate þ fenitrothion (1:20) to populations of the maize weevil Sitophilus zeamais. Population

N

Slope (SE)

LC50 (95% FL) mg/cm2

RR50 (95%CL)a

LC95 (95%FL) mg/cm2

RR95 (95%CL)

c2

P

Sete Lagoas Piracicaba Amambay Rio Verde Sacramento Dourados-Bororó Canarana Maracaju Guaxupé Viçosa São José do Rio Pardo São João Nova Era Espírito Santo do Pinhal Jacuí Vicentina Machado Abre Campo Juiz de Fora Tunapolis Dourados-Barreirinha Guarapuava Pedro Juan Caballero Votuporanga Dourados-Proterito Jacarezinho Unaí

600 780 780 700 700 540 680 740 600 640 600 760 600 580 600 700 700 700 600 660 580 700 600 600 780 560 760

2.2 3.6 1.9 2.2 2.2 1.4 1.9 2.2 2.0 1.8 1.6 2.5 1.6 1.1 0.7 1.7 1.1 1.6 2.6 1.4 1.0 0.9 1.2 0.6 1.4 1.2 1.4

0.005 0.005 0.005 0.006 0.006 0.006 0.006 0.006 0.006 0.007 0.007 0.007 0.008 0.008 0.008 0.008 0.008 0.008 0.010 0.012 0.013 0.017 0.018 0.018 0.020 0.021 0.026

1.0 1.0 1.0 1.1 1.1 1.1 1.2 1.2 1.2 1.3 1.4 1.4 1.5 1.5 1.5 1.5 1.6 1.6 1.8 2.3 2.4 3.4 3.4 3.5 3.9 4.1 5.0

0.029 0.014 0.038 0.032 0.033 0.089 0.044 0.035 0.042 0.056 0.073 0.033 0.078 0.204 1.165 0.067 0.291 0.096 0.041 0.195 0.468 1.020 0.407 6.751 0.331 0.519 0.395

1.0 0.5 1.3 2.2 1.1 3.1 1.5 1.2 1.5 1.9 2.5 1.1 2.7 7.0 40.2 2.3 10.1 3.3 1.4 6.7 16.1 35.2 14.5 232.8 11.4 17.9 13.6

7.8 9.0 10.4 3.2 8.9 8.5 10.9 8.7 5.1 9.8 9.1 4.5 8.9 1.2 6.7 9.2 9.1 8.2 5.4 8.7 5.3 2.7 5.1 6.0 2.6 9.4 6.5

0.10 0.17 0.06 0.67 0.11 0.08 0.05 0.19 0.27 0.08 0.06 0.61 0.06 0.88 0.15 0.10 0.11 0.15 0.25 0.12 0.26 0.75 0.27 0.20 0.86 0.05 0.37

(0.4) (0.3) (0.3) (0.2) (0.2) (0.4) (0.3) (0.2) (0.2) (0.2) (0.3) (0.2) (0.3) (0.2) (0.1) (0.2) (0.1) (0.2) (0.2) (0.1) (0.2) (0.1) (0.2) (0.1) (0.1) (0.2) (0.1)

(0.003e0.007) (0.005e0.005) (0.004e0.007) (0.005e0.006) (0.005e0.006) (0.002e0.009) (0.004e0.008) (0.005e0.007) (0.006e0.007) (0.005e0.009) (0.004e0.010) (0.007e0.008) (0.005e0.011) (0.006e0.009) (0.005e0.010) (0.007e0.009) (0.007e0.010) (0.007e0.010) (0.009e0.011) (0.010e0.015) (0.010e0.016) (0.014e0.024) (0.015e0.023) (0.013e0.032) (0.017e0.026) (0.013e0.039) (0.021e0.034)

(0.7e1.3) (0.8e1.2) (0.8e1.4) (0.9e1.4) (0.9e1.4) (0.8e1.7) (0.9e1.6) (0.9e1.5) (1.0e1.6) (1.0e1.8) (1.0e1.9) (1.1e1.8) (1.0e2.0) (1.1e2.0) (1.0e2.2) (1.2e1.9) (1.2e2.2) (1.3e2.1) (1.5e2.3) (1.8e3.0) (1.8e3.3) (2.4e4.7) (2.5e4.6) (2.3e5.6) (2.9e5.3) (2.7e6.0) (3.7e6.8)

(0.018e0.105) (0.012e0.017) (0.022e0.136) (0.025e0.045) (0.025e0.049) (0.032e40.46) (0.026e0.131) (0.027e0.048) (0.029e0.072) (0.035e0.123) (0.036e0.390) (0.026e0.044) (0.040e0.395) (0.108e0.590) (0.309e25.45) (0.044e0.131) (0.142e0.978) (0.064e0.173) (0.033e0.055) (0.112e0.463) (0.201e2.183) (0.364e6.506) (0.197e1.367) (0.923e1402.00) (0.187e0.753) (0.155e18.36) (0.221e0.913)

(0.5e2.0) (0.3e0.8) (0.6e2.8) (0.6e2.0) (0.6e2.0) (0.8e11.6) (0.7e3.1) (0.7e2.1) (0.8e2.8) (1.0e3.7) (1.1e5.8) (0.7e1.9) (1.2e6.2) (2.8e17.9) (6.0e277.1) (1.1e4.7) (3.6e28.1) (1.7e6.6) (0.8e2.5) (3.0e15.6) (4.9e53.6) (8.7e145.6) (5.1e39.5) (12.5e4474.6) (5.0e26.2) (4.7e69.7) (5.9e31.6)

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

1661

Fig. 2. Distance walked (mean  standard error) during 10 min by individuals from populations of Sitophilus zeamais in surfaces fully treated with insecticides (in addition to a control on untreated surface). Populations with mean walked distances grouped by the same bar do not differ by the Scott-Knott’s test (P > 0.05).

with those to the mixture esfenvalerate þ fenitrothion (r ¼ 0.25, P > 0.05). Fenitrothion resistance was not correlated with any of the pyrethroids and the insecticide mixture (P > 0.05). Importantly, no significant correlation (P > 0.05) was detected between the resistance ratios to the insecticides and behavioural traits assessed, which is also true for fenitrothion. However, on arenas with esfenvalerate, the insects that walked longer distances preferred the untreated side of the arena (r ¼ 0.43, P < 0.05), although this was not observed for the other insecticides. 3.4. Population growth, respiration rate, and enzymatic assays There was no significant differences in the instantaneous rate of population growth (ri) among maize weevil populations (0.0213  0.0004/day) (F26,54 ¼ 0.73, P ¼ 0.80). Nevertheless, significant differences in grain consumption were detected among populations (F54,26 ¼ 2.11, Pp ¼ 0.01) (Fig. 4). Significant differences among insect populations were also evident for respiration rate, body mass, amylase and lipase activities (dfnum/den ¼ 104/419.1, Wilks’ lambda ¼ 0.0335, F ¼ 5.46, P < 0.001), and for the interaction population  sex (dfnum/ den ¼ 104/419.1, Wilks’ lambda ¼ 0.1425, F ¼ 2.56, P < 0.0001), and thus further univariate analysis of variance was carried out for each of these traits. Differences in respiration rate (1.75  0.03 mL de CO2 h1/insect) (F53, 108 ¼ 0.99, P > 0.50) and body mass (2.96  0.018 mg/insect) (F53, 108 ¼ 0.94, P > 0.60) were not significant. In contrast, the interaction population  sex was significant for amylase (F54, 26 ¼ 3.46, P < 0.0001) and lipase activity (F54, 26 ¼ 3.46, P < 0.0001), and these were then subjected to ScottKnott’s test (Fig. 5).

Mean amylase activity in females ranged from 521  76 to 10,957  498 AU/dl/mg protein (Guaxupé and Pedro Juan Caballero, respectively), while in males the magnitude of the variation was 822  266 (Dourados-Bororó) and 8893  1266 AU/dl/mg protein (Pedro Juan Cabalhero). Males of Canarana, Guarapuava, Maracaju, Abre Campo, and Jacuí had higher amylase activity relative to females in these populations, and conversely females of Amambay and Dourados-Bororó had higher amylase activity than males in these populations (Fig. 5a). Regarding lipase activity, the Juiz de Fora population exhibited highest specific activity among the females (1113  69 IU/dl/mg protein), while Tunápolis exhibited the lowest activity (46  9 IU/ dl/mg protein). For males, Juiz de Fora exhibited the highest activity (637  47 IU/dl/mg protein), while Unaí exhibited the lowest lipase activity (56  10 IU/dl/mg protein). The females of Dourados-Bororó, Viçosa and Guarapuava had higher lipase activity relative the males in these populations, while the opposite occurred in populations of Dourados-Barreirinha, Canarana, Maracaju, and Machado, with males exhibiting increased activity relative to females (Fig. 5b).

3.5. Overall correlations and path analysis There was no significant (partial) canonical correlation between instantaneous rate of population growth (ri) and levels of insecticide resistance (at LC50) (r ¼ 0.26, P ¼ 0.80), distance walked (r ¼ 0.25, P ¼ 0.79) and proportion of time spent in the non-treated half of arena (r ¼ 0.36, P ¼ 0.53) determined for each insecticide (including fenitrothion).

1662

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

Fig. 3. Proportion of time spent (mean  standard error) by individuals from populations of Sitophilus zeamais in the untreated half of the arena (in half-treated arenas) during 10 min. Populations grouped by the same bar line do not differ by the Scott-Knott’s test (P > 0.05).

Fig. 4. Grain consumption (mean  standard error) by populations of Sitophilus zeamais. Populations grouped by the same bar line are not significantly different by Scott-Knott’s test (P > 0.05).

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

1663

Fig. 5. Specific activity of (a) amylase and (b) lipase in males and females of populations of the maize weevil Sitophilus zeamais. Populations grouped by the same bar line do not differ by the Scott-Knott’s test (P > 0.05). Asterisks indicate significant differences between the sexes for a given population (Fisher’s F at P < 0.05).

Direct, indirect, and total effects of grain consumption, respiration rate, body mass, amylase and lipase activity on the ri determined for each of the maize weevil populations were assessed by path analysis as shown by the path diagram in Fig. 6. The sex effect was not considered in the model because of its negligible effect. No significant departures from expected covariance matrices were observed (c2 ¼ 2.90, df ¼ 3, P ¼ 0.41) indicating that the path model used is valid. Amylase and lipase activity were correlated although none of them had significant effect in grain consumption by maize weevil populations (Fig. 6, Table 5), but both exhibited significant effect (direct and indirect) on body mass (Fig. 6, Table 5). The direct effect was represented by an arrow linking both variables, while in the indirect effect such influence is mediated by an intermediary variable (e.g., amylase indirectly affects rate of increase by means of body mass) (Fig. 6); the total effect of a variable is the result of its direct and indirect effects (Table 5). The respiration rate correlated with grain consumption, but not with insect body mass (Fig. 6). The instantaneous rate of growth (ri) was significantly affected by grain consumption, but not by body mass. However, the overall direct, indirect, and total effects of the variables tested on the ri were not significant (R2 ¼ 0.05, P ¼ 0.56) (Table 5).

et al., 2008a), resulting in reduced levels of resistance to pyrethroids relative to those originally found in the field populations when collected. Such high levels of pyrethroid resistance detected in Brazilian populations of maize weevil in the late 1980s resulted from cross-resistance to DDT (Guedes et al., 1994, 1995; Fragoso et al., 2003; Ribeiro et al., 2003). A recent study with the DDT- and pyrethroid-resistant populations of the maize weevil from Brazil confirmed target site alteration as the primary mechanism of resistance to these compounds (mutation T929I in the sodium channel, following the housefly numbering; Araújo et al., 2011). Esfenvalerate was recently registered for control of stored grain insects in Brazil and its use is recommended only in combination

4. Discussion Concentration-mortality bioassays for esfenvalerate and permethrin clearly showed a reduction in pyrethroid resistance in Brazilian populations of the maize weevil relative to earlier studies (Guedes et al., 1995, 2009; Ribeiro et al., 2003), in which resistance levels exceeded 1000-fold. The populations that exhibited the highest resistance levels to esfenvalerate and permethrin were from Juiz de Fora and Jacarezinho, respectively. These two populations have been intensively studied and maintained in the laboratory in the absence of insecticides for over 15 years (Guedes et al., 1994, 1995, 2006, 2009; Fragoso et al., 2003; Ribeiro et al., 2003; Araújo

Fig. 6. Path analysis diagram for the influence of body mass, respiration rate, grain consumption, and amylase and lipase activity on the instantaneous rate of population growth (ri) of populations of the maize weevil Sitophilus zeamais. The result of c2 goodness-of-fit for the path model is indicated. One-headed arrows indicate causal relationship (regression) while doubled-headed arrows indicate correlation between the variables. Significance levels are represented by asterisks (*P < 0.05. **P < 0.01), and the thickness of each line is proportional to the strength of the relationship. Direct, indirect and total values for path coefficients are fully presented in Table 5.

1664

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

Table 5 Direct (DE), indirect (IE) and total (TE) effects in the path diagram of Fig. 6 for the model on the influence of amylase and lipase activity, grain consumption and body mass on the population growth rate of populations of the maize weevil Sitophilus zeamais. Variable

Amylase activity (U amylase/dl/mg) Lipase activity (U lipase/dl/mg) Grain consumption (g) Insect body mass (mg) R2 P

Grain consumption (g)

Instantaneous growth rate (ri)

Body mass (mg/insect)

DE

IE

TE

DE

IE

TE

DE

IE

TE

0.08 0.15 e e

e e e e 0.05 0.56

0.08 0.15 e e

0.36 0.12 0.40 e

0.03 0.06 e e 0.43 0.004**

0.39 0.18 0.40 e

e e 0.67 0.01

0.00 0.08 0.06 e 0.03 0.45

0.00 0.08 0.61 0.01

with fenitrothion. However, the levels of resistance to this insecticide in the insect populations studied are comparable to those observed for permethrin, a compound with a longer history of use for stored grains in the country (Fragoso et al., 2003; Ribeiro et al., 2003, 2007; Guedes et al., 2006). Thus, esfenvalerate resistance in Brazilian populations of the maize weevil is likely a consequence of cross-resistance to other pyrethroids (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003). This hypothesis is reinforced by the positive and significant correlation between resistance to permethrin and esfenvalerate here observed. Resistance to malathion, and more recently to pirimiphos-methyl and fenitrothion, has also been reported in Brazil, but only lower levels of resistance (<10) were reported (Fragoso et al., 2003; Ribeiro et al., 2003; Pereira et al., 2009; Braga et al., 2011), as also observed in our study. The potency of fenitrothion was higher than that of the pyrethroids for the maize weevil. This is the likely consequence of the lack of use of the insecticide synergist (i.e., compounds which, in sub-lethal concentrations, increases the toxicity of the other compound in the mixture) piperonyl butoxide (PBO) in the commercial formulations of pyrethroids registered for stored product protection in Brazil, with the exception of the deltamethrin formulations which contains 10 parts of PBO to one of the insecticide (Cajueiro, 1988; Santos et al., 1988; Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003). This is so because PBO greatly enhances pyrethroid toxicity to stored product insects, particularly to the maize weevil, by inhibiting cytochrome P450-dependent monooxygenases (Guedes et al., 1995, 1997; Ribeiro et al., 2003). The popular recommendations of use of organophosphate-pyrethroid mixture initiated in the 1990’s in Brazil led to the recent registration of esfenvalerate þ fenitrothion for use against insect-pests of stored products, including the maize weevil (Cajueiro, 1988; Santos et al.,1988; Anonymous, 2010). The great potentiation of insecticidal activity in the esfenvalerate þ fenitrothion mixture lays credence to its use as potential alternative for the insecticides used alone. Resistance bioassays with the mixture esfenvalerate þ fenitrothion suggest existence of multiple resistance mechanisms to pyrethroids and organophosphates, not only because of the lack of correlation between resistance to pyrethroids, fenitrothion and the insecticide mixture, but also because the main pyrethroid resistance mechanism recognized in Brazilian populations of the maize weevil is the kdr-like mechanism, which is exclusive of pyrethroids and DDT (Fragoso et al., 2003, 2007; Ribeiro et al., 2003; Pereira et al., 2009; Araújo et al., 2011). However, we believe that the low levels of resistance obtained (even for pyrethroids, which were high in the recent past) are directly related to the recent approval and broad use of this formulated mixture against insect-pests of stored grains, which is probably allowing efficient control of maize weevil populations resistant to either pyrethroids, or organophosphate insecticides. The low slopes obtained in the mortality curves reinforces this hypothesis and highlights the wide variability of responses to the insecticides, indicating high capacity for resistance evolution in the maize weevil even to insecticide mixture. Increased activity of detoxification enzymes, a secondary mechanism of resistance to organophosphates and pyrethroids already reported in

Brazilian maize weevil populations, enables cross-resistance to these insecticides in various insects-pests (Brattsten et al., 1986; Fragoso et al., 2003, 2007). In Spodoptera litura for instance, cross-resistance between pyrethroids and organophosphates or carbamates is due to increased activity of esterases and cytochrome P450-dependent monooxygenases (Huang and Han, 2007). The behavioural patterns of insect locomotion in both arenas used varied with population. The different patterns of locomotory behaviour observed among S. zeamais populations is possibly related to differences in metabolism of the insects, which can influence insect behaviour (Guedes et al., 2009), although these differences were not clearly related to the activity of carbohydrateand lipid-metabolizing enzymes in the populations of the maize weevil studied. If the variation observed is underlined by inheritable differences in insect sensory processes, this could lead to the development of behavioural resistance to insecticides in populations of the maize weevil (Chareonviriyaphap et al., 1997; Hoy et al., 1998; Desneux et al., 2007; Guedes et al., 2009; Braga et al., 2011). In the present study, we did not observe any positive or negative correlation between behavioural and physiological resistance. The negative correlation between behavioural and physiological resistance to insecticides was earlier hypothesized based on the fact that behavioural resistance may lead to reduced insecticide exposure, consequently minimizing selection pressure for evolution of physiological mechanisms of insecticide resistance (Georghiou, 1972). Nevertheless, an alternative view was suggested indicating the possibility for evolution of both behavioural and physiological resistance based on two main relationships (Lockwood et al., 1984). Firstly, all behaviour has a physiological basis, being a physical expression of an organism’s internal physiology; and secondly, that co-existence of behavioural and physiological resistance may take place if a single physiological or behavioural mechanism does not provide sufficient level of resistance to a specific insecticide. A third possibility, of independence between physiological and behavioural resistance, was also recognized by Lockwood et al. (1984), and reported among mosquitoes and weevils (Chareonviriyaphap et al., 1997; Guedes et al., 2009). Here we also obtained support for this last possibility due to the lack of correlation between resistance ratios and behavioural traits measured, probably resulting from the distinct genetic background of the populations studied and their distinct selection pressure by these insecticides. The demographic performance assessed through the instantaneous rate of population growth (ri) showed no significant differences among the populations, indicating absence of measurable fitness cost in the insect populations studied. This comes as no surprise given the low levels of insecticide resistance detected, which did not seem to cause any measurable negative effects on the potential for population growth (Pereira et al., 2009). Additionally, the lack of correlation between ri and physiological and behavioural patterns of insecticide resistance observed reinforce the low influence of the resistance levels detected on the demographic performance of the maize weevil populations.

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

The path diagram revealed a major role of amylase and lipase activity as well as grain consumption on weevil body mass. Increased body mass, which is directly related to energy acquisition by digestive enzymes (Araújo et al., 2008a,b), has been identified as a trait of influence on ri in maize weevil populations (Fragoso et al., 2005; Guedes et al., 2006), and consequently, with a strong relationship with the mitigation of fitness costs in some insecticideresistant populations (Guedes et al., 2006; Oliveira et al., 2007; Araújo et al., 2008a). However, these studies were carried out with a limited number of maize weevil populations and it is important to recognize how general is this relationship using a larger and more representative group of insect populations. This was carried out in the present study and we were unable to detect such relationship between body mass (which was related to amylase activity) and ri probably because of the low variation in these traits among the populations studied and the low to moderate levels of insecticide resistance observed, which did not seem to incur in fitness costs. In conclusion, the use of insecticide mixture remains a viable approach for maize weevil control and its widespread use seems to have decreased the levels of pyrethroid resistance observed in field-collected populations. However, resistance to the esfenvalerate þ fenitrothion mixture seems to be evolving and there is enough genotypic variation in this trait to justify concerns regarding its use. Alternative approaches are necessary to extend the field use of such mixtures. Acknowledgements We thank Drs. J.P. Santos, L.R.D’A. Faroni, M.A.G. Pimentel, and L.B. Silva for providing some of the insect populations used in this study. We also would like to thank Syngenta, Iharabras and Sumitomo for providing us with the technical grade insecticides. Financial support provided by the National Council of Scientific and Technological Development (CNPq), CAPES Foundation (Brazilian Ministry of Education) and the Minas Gerais State Foundation for Research Aid (FAPEMIG) was greatly appreciated. The comments and suggestions provided by Dr. J.V. Cross and the anonymous referees were also greatly appreciated. References Anonymous, 2010. Agrofit: Sistema de Agrotóxicos Fitossanitários. Ministério da Agricultura, Pecuária, e Abastecimento. Brasília, Brazil. http://extranet. agricultura.gov.br/agrofit_cons/principal_agrofit_cons (accessed June 5, 2010). Araújo, R.A., Guedes, R.N.C., Oliveira, M.G.A., Ferreira, G.H., 2008a. Enhanced proteolytic and cellulolytic activity in insecticide-resistant strains of the maize weevil, Sitophilus zeamais. J. Stored Prod. Res. 44, 354e359. Araújo, R.A., Guedes, R.N.C., Oliveira, M.G.A., Ferreira, G.H., 2008b. Enhanced activity of carbohydrate- and lipid-metabolizing enzymes in insecticide-resistant populations of the maize weevil, Sitophilus zeamais. Bull. Entomol. Res. 98, 417e424. Araújo, R.A., Williamson, M.S., Bass, C., Field, L.M., Duce, I.R., 2011. Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel. Insect Mol. Biol. 20, 437e445. Arnaud, L., Haubruge, E., 2002. Insecticide resistance enhances reproductive success in a beetle. Evolution 56, 2435e2444. Beeman, R.W., Nanis, S.M., 1986. Malathion resistance alleles and their fitness in the red flour beetle (Coleoptera: Tenebrionidae). J. Econ. Entomol. 79, 580e587. Bengston, M., Davies, R.A.H., Desmarchelier, J.M., Henning, R., Murray, W., Simpson, B.W., Snelson, J.T., Sticka, R., Wallbank, B.E., 1983. Organosphosphothioates and synergised synthetic pyrethroids as grain protectants on bulk wheat. Pestic. Sci. 12, 365e374. Braga, L.S., Corrêa, A.S., Pereira, E.J.G., Guedes, R.N.C., 2011. Face or flee? Fenitrothion resistance and behavioral response in populations of the maize weevil, Sitophilus zeamais. J. Stored Prod. Res. 47, 161e167. Brattsten, L.B., Holyoke Jr., C.W., Leeper, J.R., Raffa, K.F., 1986. Insecticide resistance: challenge to pest management and basic research. Science 231, 1255e1260. Cajueiro, IVM, 1988. Controle Químico de Sitophilus zeamais Motschulsky, 1855 (Coleoptera: Curculionidae), em grãos de sorgo, Sorghum bicolor (L.) Moench, em Laboratório. MS thesis, University of São Paulo, Piracicaba, SP, Brazil. Caraway, W.T., 1959. A stable starch substrate for the determination of amylase in serum and other body fluids. Am. J. Clin. Pathol. 32, 97e99.

1665

Chareonviriyaphap, T., Roberts, D.R., Andre, R.G., Harlan, H., Bangs, M.J., 1997. Pesticide avoidance behavior in Anopheles albimanus Wiedmann. J. Am. Mosq. Cont. Assoc. 13, 171e183. Cherry, I.S., Crandall, L.A., 1932. The specificity of pancreatic lipase: its appearance in the blood after pancreatic injury. Am. J. Physiol. 100, 266e270. Coustau, C., Chevillon, C., ffrench-Constant, R., 2000. Resistance to xenobiotics and parasites: can we count the cost? Trends Ecol. Evol. 15, 378e383. Danho, M., Gaspar, C., Haubruge, E., 2002. The impact of grain quality on the biology of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae): oviposition, distribution of eggs, adult emergence, body weight and sex ratio. J. Stored Prod. Res. 38, 259e266. Desmarchelier, J., Bengston, M., Connell, M., Henning, R., Ridley, E., Ripp, E., Sierakowski, C., Sticka, R., Snelson, J., Wilson, A., 1981. Extensive pilot use of the grain protectant combinations, fenitrothion plus bioresmethrin and pirmiphosmethyl plus bioresmethrin. Pestic. Sci. 12, 365e374. Desmarchelier, J., Bengston, M., Davies, R., Elder, B., Hart, R., Henning, R., Murray, W., Ridley, E., Ripp, E., Sierakowski, C., Sticka, R., Snelson, J., Wallbank, B., Wilson, A., 1987. Assessment of the grain protectants chlorpyrifos-methyl plus bioresmethrin, fenitrothion plus (1R)-phenothrin, methacrifos and pirmiphosmethyl plus carbaryl under practical conditions in Australia. Pestic. Sci. 20, 271e288. Desneux, N., Decourtye, A., Delpuech, J.M., 2007. The sublethal effects of pesticides on beneficial arthropods. Annu. Rev. Entomol. 52, 181e206. Foster, S.P., Young, S., Williamson, M.S., Duce, I., Denholm, I., Devine, G.J., 2003. Analogous pleiotropic effects of insecticide resistance genotypes in peachpotato aphids and houseflies. Heredity 91, 98e106. Fragoso, D.B., Guedes, R.N.C., Rezende, S.T., 2003. Glutathione S-transferase detoxification as a potential pyrethroid resistance mechanism in the weevil, Sithophilus zeamais. Entomol. Exp. Appl. 109, 21e29. Fragoso, D.B., Guedes, R.N.C., Peternelli, L.A., 2005. Developmental rates and population growth of insecticide-resistant and susceptible populations of Sitophilus zeamais. J. Stored Prod. Res. 41, 271e281. Fragoso, D.B., Guedes, R.N.C., Oliveira, M.G.A., 2007. Partial characterization of glutathione S-transferases in pyrethroid-resistance and e susceptible populations of the maize weevil, Sitophilus zeamais. J. Stored Prod. Res. 43, 167e170. Georghiou, G.P., 1972. The evolution of resistance to pesticides. Annu. Rev. Ecol. Syst. 3, 133e168. Guedes, R.N.C., Lima, J.O.G., Santos, J.P., Cruz, C.D., 1994. Inheritance of deltamethrin resistance in a Brazilian strain of maize weevil (Sitophilus zeamais Motsch.) (Coleoptera: Curculionidae). Int. J. Pest Manag. 40, 103e106. Guedes, R.N.C., Lima, J.O.G., Santos, J.P., Cruz, C.D., 1995. Resistance to DDT and pyrethroids in Brazilian populations of Sitophilus zeamais Motsch. (Coleoptera: Curculionidae). J. Stored Prod. Res. 31, 145e150. Guedes, R.N.C., Kambhampati, S., Zhu, B.A., Dover, B.A., 1997. Biochemical mechanisms of organophosphate resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae) populations from the United states and Brazil. Bull. Entomol. Res. 87, 581e586. Guedes, R.N.C., Oliveira, E.E., Guedes, N.M.P., Ribeiro, B.M., Serrão, J.E., 2006. Cost and mitigation of insecticide resistance in the maize weevil, Sitophilus zeamais. Physiol. Entomol. 31, 30e38. Guedes, R.N.C., Campbell, J.F., Arthur, F.H., Opit, G.P., Zhu, K.Y., Throne, J.E., 2008. Acute lethal and behavioral sublethal responses of two stored-product psocids to surface insecticides. Pest Manag. Sci. 64, 1314e1322. Guedes, N.M.P., Guedes, R.N.C., Ferreira, G.H., Silva, L.B., 2009. Flight take-off and walking behavior of insecticide-susceptible and eresistant strains of Sitophilus zeamais exposed to deltamethrin. Bull. Entomol. Res. 99, 393e400. Haubruge, E., Arnaud, L., 2001. Fitness consequences of malathion-specific resistance in the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera, Tenebrionidae), and selection for resistance in the absence of insecticide. J. Econ. Entomol. 94, 552e557. Haynes, K.F., 1988. Sublethal effects of neurotoxic insecticides on insect behavior. Annu. Rev. Entomol. 33, 149e168. Hoy, C.W., Head, G.P., Hall, F.R., 1998. Spatial heterogeneity and insect adaptation to toxins. Annu. Rev. Entomol. 43, 571e594. Huang, S., Han, Z., 2007. Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China. Pestic. Biochem. Physiol. 87, 14e22. Jenson, E.A., Arthur, F.H., Nechols, J.R., 2010. Methoprene and synerrgized pyrethrins as aerosol treatments to control Plodia interpunctella (Hübner), the Indian meal moth (Lepidoptera: Pyralidae). J. Stored Prod. Res. 46, 103e110. Lockwood, J.A., Sparks, T.C., Story, R.N., 1984. Evolution of insect resistance to insecticides: a reevaluation of the roles of physiology and behavior. Bull. Entomol. Soc. Am. 30, 41e51. Lopes, K.V.G., Silva, L.B., Reis, A.P., Oliveira, M.G.A., Guedes, R.N.C., 2010. Modified aamylase activity among insecticide-resistant and esusceptible strains of the maize weevil, Sitophilus zeamais. J. Insect Physiol. 56, 1050e1057. McKenzie, J.A., Batterham, P., 1994. The genetic, molecular and phenotypic consequences of selection for an insecticide resistance. Trends Ecol. Evol. 9, 166e169. McKenzie, J.A., 1996. Applying the theory: the better management of resistance and pests. In: McKenzie, J.A. (Ed.), Ecological and Evolutionary Aspects of Insecticide Resistance. Academic, The Austin, pp. 149e173. Oliveira, E.E., Guedes, R.N.C., Tótola, M.R., de Marco, P., 2007. Competition between insecticide-susceptible and resistant populations of the maize weevil, Sitophilus zeamais. Chemosphere 69, 17e24.

1666

A.S. Corrêa et al. / Crop Protection 30 (2011) 1655e1666

Pereira, C.J., Pereira, E.J.G., Cordeiro, E.M.G., Della Lucia, T.M.C., Tótola, M.R., Guedes, R.N.C., 2009. Organophosphate resistance in the maize weevil Sitophilus zeamais: magnitude, costs and behavior. Crop Prot. 28, 168e173. Raymond, M., Berticat, C., Weill, M., Pasteur, N., Chevillon, C., 2001. Insecticide resistance in mosquitoes Culex pipiens: what have we learned about adaptation? Genetica 112/113, 287e296. Rees, D.J., 1996. Coleoptera. In: Subramayam, Bh., Hagstrum, D.W. (Eds.), Integrated Management of Insects in Stored Products. Marcel Dekker, New York, pp. 1e39. Ribeiro, B.M., Guedes, R.N.C., Oliveira, E.E., Santos, J.P., 2003. Insecticide resistance and synergism in Brazilian populations of Sitophilus zeamais (Coleoptera: Curculionidae). J. Stored Prod. Res. 39, 21e31. Ribeiro, B., Guedes, R.N.C., Corrêa, A.S., Santos, C.T., 2007. Fluctuating asymmetry in insecticide-resistant and insecticide-susceptible strains of the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae). Arch. Environ. Cont. Toxicol. 53, 77e83. Robertson, J.L., Preisler, H.K., 1992. Pesticide Bioassays with Arthropods. CRC, Boca Raton. Santos, J.P., Bitran, E., Nakano, O., 1988. Avaliação residual de diversos inseticidas para proteção de sementes de milho contra insetos durante o armazenamento. In: Anais do 16 Congresso Brasileiro de Milho. EMBRAPA, Brasília, Brazil, pp. 268e275.

SAS Institute, 2002. SAS User’s Manual, Version 9.1. SAS Institute, Cary, NC, USA. Scott, A.J., Knott, M., 1974. Cluster-analysis method for grouping means in analysis of variance. Biometrics 30, 507e512. Silva, L.B., Reis, A.P., Pereira, E.J.G., Oliveira, M.G.A., Guedes, R.N.C., 2010a. Partial purification and characterization of trypsin-like proteinases from insecticideresistant and susceptible strains of the maize weevil, Sitophilus zeamais. Comp. Biochem. Physiol. B. 155, 15e19. Silva, L.B., Reis, A.P., Pereira, E.J.G., Oliveira, M.G.A., Guedes, R.N.C., 2010b. Altered cysteine proteinase activity in insecticide-resistant strains of the maize weevil: purification and characterization. Comp. Biochem. Physiol. B. 157, 80e87. Stark, J.D., Banks, J.E., 2003. Population-level effects of pesticides and other toxicants on arthropods. Annu. Rev. Entomol. 48, 505e519. Tabashnik, B.E., 1990. Modeling and evaluation of resistance management tactics, in Pesticide Resistance in Arthropods. In: Roush, R.T., Tabashnik, B.E. (Eds.), Pesticide Resistance in Arthropods. Chapman & Hall, New York, pp. 153e182. Tolpo, N.C., Morrison, E.O., 1965. Sex determination by snout characteristics of Sitophilus zeamais Motschulsky. Tex. J. Sci. 7, 122e124. Warburg, O., Christian, W., 1941. Isolierung und kristallisation des garungs ferments enolase. Biochemische Zeitsch 310, 384e421.