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Ozone toxicity to Sitophilus zeamais (Coleoptera: Curculionidae) populations under selection pressure from ozone
Journal of Stored Products Research 65 (2016) 1e5
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Ozone toxicity to Sitophilus zeamais (Coleoptera: Curculionidae) populations under selection pressure from ozone A.H. Sousa a, b, L.R.D'A. Faroni c, *, M.A.G. Pimentel d, G.N. Silva c, R.N.C. Guedes a a
Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil gicas e da Natureza, Universidade Federal do Acre, Rio Branco, AC, Brazil Departamento de Ci^ encias Biolo c Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG, Brazil d ria, Centro Nacional de Pesquisa de Milho e Sorgo, Sete Lagoas, MG, Brazil Empresa Brasileira de Pesquisa Agropecua b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2015 Received in revised form 6 November 2015 Accepted 6 November 2015 Available online xxx
Two populations of Sitophilus zeamais (Coleoptera: Curculionidae) were subjected to selection pressure with ozone (O3), and the possibility of acquiring resistance to O3 was investigated. The pattern of locomotion and the rate of respiration were evaluated following each selection cycle. Two source populations were used in the study: one was a mixture composed of 30 populations (MP), and the other was composed of the population that was the least susceptible to O3 among these 30 populations (LSP). The beetles from each source population experienced selection cycles with O3 using the lethal time for 80% (LT80) of the insect population from each generation. The O3 toxicity (50 ppm at a continuous flow rate of 2 L min1) to each generation was calculated using time-response bioassays. The locomotor pattern (distance traveled, resting period, and walking speed) and the respiratory rate (CO2 production) were also evaluated. The S. zeamais populations that were subjected to successive cycles of selection with O3 did not acquire resistance to O3, and the pattern of locomotion and the rate of respiration did not change following the selection cycles with O3. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Susceptibility Sitophilus zeamais Ozonation Alternative fumigants
1. Introduction The evolution of pest resistance to insecticides is a major obstacle in pest control programs that use chemical products (Groeters and Tabashnik, 2000). In the grain storage industry, the increased levels of resistance are primarily caused by the limited availability of different insecticides, the indiscriminate use of these insecticides for long periods, and the lack of appropriate structures for the application of fumigants (Collins et al., 2005; Sousa et al., 2009). Recent studies demonstrated that the Brazilian populations of Tribolium castaneum (Coleoptera: Tenebrionidae), Sitophilus zeamais (Coleoptera: Curculionidae), Rhyzopertha dominica (Coleoptera: Bostrichidae), and Oryzaephilus surinamensis (Coleoptera: Silvanidae) were highly resistant to phosphine, with rates of resistance that were 32.2e186.2-fold that of susceptible populations (Pimentel et al., 2007, 2009). The enrichment of the atmosphere with ozone (O3) is
* Corresponding author. Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil. E-mail address: [email protected] (L.R.D'A. Faroni). http://dx.doi.org/10.1016/j.jspr.2015.11.001 0022-474X/© 2015 Elsevier Ltd. All rights reserved.
recognized as an important alternative for the control of stored products pests (Kells et al., 2001; Sousa et al., 2008; Isikber and € Oztekin, 2009; Lu et al., 2009) because pests of stored products do not show cross-resistance between phosphine and O3. Additionally, O3 does not leave a residue in the grain because oxygen (O2) is the degradation product (Zhanggui et al., 2003; Sousa et al., 2008). Ozone is a gas that is derived from the rearrangement of oxygen atoms that occurs during electrical discharges or from exposure to high-energy electromagnetic radiation (ultraviolet light) in the atmosphere (Khadre et al., 2001; Liu et al., 2007). Ozone is an unstable molecule with a half-life of 20e50 min € (Isikber and Oztekin, 2009) that can be generated locally, which eliminates the requirements for its handling, storage, and transport. Although the toxic effects of O3 to stored products insect pests are well documented, further studies are required to assess the risk for the development of resistance in the populations that are exposed to selection pressure with O3. Based on evolutionary theory, resistance to insecticides is predicted to evolve in the populations that are maintained under selection pressure from chemicals because different traits are selected to increase the probability of survival in harsh environments (Foster et al., 2000;
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A.H. Sousa et al. / Journal of Stored Products Research 65 (2016) 1e5
Coustau et al., 2000; Arnaud and Haubruge, 2002; Guedes et al., 2010). Moreover, the selection of a particular trait may be associated with pleiotropy, i.e., the ability of a single gene to affect more than one trait in an individual (Boivin et al., 2001; Raymond et al., 2005; Guedes et al., 2009). Therefore, in this study, the susceptibility of populations of S. zeamais to develop resistance when subjected to selection pressure with O3 was evaluated. Additionally, we evaluated the locomotor pattern and the metabolic rate of the beetles during each selection cycle, considering that the toxicity of these insecticides has a known association with physiological and behavioral mechanisms. 2. Materials and methods 2.1. Insects Two source populations were used in this study: (1) a mixture of 30 populations that were collected in 10 Brazilian states and two Paraguayan regions (MP), and (2) the population that was the least susceptible to O3 among these 30 populations (LSP), which was in the state of Minas Gerais. The toxicity to collected in Guaxupe these populations was previously established by Sousa et al. (2012). Both populations were maintained in 1.5-L glass vials under controlled conditions (27 ± 2 C and 70 ± 5% r.h.). Previously fumigated corn grain, with a moisture content of 13% (wet basis), was used as the food substrate, and the corn was maintained at 18 C to prevent re-infestation. 2.2. Selection and O3 toxicity bioassays The adults from the MP and the LSP populations were submitted to selection cycles with O3. Initially, the lethal time for 80% (LT80) of the parental generation of each population was used. Subsequently, the corresponding LT80 for each generation was used. The insects that survived the final selection cycle were collected to obtain the progeny. Approximately 2000 beetles, aged 1e2 weeks postemergence, were used in each selection cycle. The O3 toxicity was determined with the estimations of the lethal exposure times for 50% and 95% of the populations (LT50 and LT95, respectively), using the adapted methodology from Sousa et al. (2008). The O3 concentration was set at 50 ppm (z0.11 g m3) at a continuous flow rate of 2 L min1. The O3 was administered inside plastic chambers (width, 13 cm; height, 20 cm) at 27 C ± 2 C and a relative humidity of 70% ± 5%. The insects from each population were placed in plastic cages (width, 4 cm; height, 3.5 cm) that were suspended 10 cm from the base of the fumigation chamber. The cover and bottom of the cages were composed of organza-type fabric. For the controls, O2 with a minimum purity of 99.99% was used. The experiments were performed in three replicates, each with 50 unsexed adults that were 1e2 weeks old. The mortality of the beetles was assessed after eight days of exposure to O3. ~o Jose The O3 was obtained with an O&L3.ORM (Ozone & Life, Sa ~o Paulo, Brazil), which used compressed O2 (minidos Campos, Sa mum purity of 99.99%) as the source for the O3. The O3 concentration that was indicated by the O3 generator was confirmed using a continuous O3 monitor (BMT Messetechnik GMBHeBMT 930), with accuracy of 0.001 ppmv, and the iodometric method with indirect titration (Eaton et al., 2000). 2.3. Locomotor behavior The methods used were adapted from Watson et al. (1997) and Pereira et al. (2009). The walking of individual male and female insects was observed over 10 min in acrylic arenas (3.5 cm
high 15 cm wide), with walls coated with Teflon® polytetra~o Paulo, Brazil) to avoid the influoroethylene (PTFE; DuPont, Sa sects' escape. The O3 concentration was fixed at 50 ppm (z0.11 g m3) at a continuous flow rate of 1.3 L min1. This concentration is in the sublethal range for the two populations. The gas injection and exhaustion were through two connections installed on the opposite sides of each arena. For the controls, O2 with a minimum purity of 99.99% was used. The movement of the insects within the arena was recorded by a tracking system that consists of a CCD camera that registers and digitally transfers the images to a coupled computer (ViewPoint Life Sciences Inc., Montreal, Canada). Each insect was individually placed in the center of the arena, 2 min before the beginning of the test to allow the chamber to reach ozone saturation and the insect to acclimate to the arena. The evaluated characteristics were distance (cm), resting period (s), and walking speed (mm/s). Twenty repetitions were conducted for each population. The tests were performed in a climate-controlled room (27 ± 2 C and 70 ± 5% r.h.), between 7:00 am and 7:00 pm. 2.4. Respiratory rate The production of carbon dioxide (CO2) was measured using a TR3C CO2 analyzer (Sable Systems International, Las Vegas, USA). For this purpose, 25-mL respiratory chambers that contained 20 adults of each population were used, with each connected to a completely closed system. Four replicates were used for each population, and the CO2 production was measured in each chamber at a controlled temperature of 27 C ± 2 C after a 15-h acclimation period. CO2-free air was injected into the chambers for 2 min at a flow rate of 100 mL min1. An infrared sensor reader was connected to the system output for the quantification of the CO2 (mL CO2 h1 per insect). The controls were empty respiratory chambers, and the values were used to normalize the respiratory rate data for each population. 2.5. Statistical analyses The data of the time-response bioassays were subjected to probit analysis (PROC PROBIT; SAS Institute, 2011). The confidence intervals for the toxicity ratios (TRs) were calculated according to Robertson and Preisler (1992), and the lethal time (LT) values were considered significantly different (P < 0.05) when the confidence intervals of the TRs did not include the value 1. For the locomotor parameters, multivariate covariance analyses were performed (population generation treatments, with and without O3) (PROC GLM with MANOVA procedure; SAS Institute, 2011). The CO2 data were subjected to an analysis of covariance (population generation) (PROC GLM; SAS Institute, 2011). 3. Results 3.1. O3 toxicity For the two populations, the variation in the toxicity of O3 between generations was markedly low (<2-fold; Table 1), which indicated the lack of variation in the populations in the susceptibility to O3. Because no resistance to O3 was observed, the tests were terminated in the F2 generation. The TRs of the LT50 values varied between 1.00- and 1.09-fold for the mixed population (MP) and between 1.00- and 1.03-fold for the population least susceptible to O3 (LSP). The slopes of the timeemortality curves were similar between the populations, which indicated homogeneity in the responses to O3 in the selection cycles.
A.H. Sousa et al. / Journal of Stored Products Research 65 (2016) 1e5
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Table 1 Relative O3 toxicity to adults in the population least susceptible to O3 (LSP) and in the mixed population (MP) of Sitophilus zeamais after two selection cycles using the LT80 and O3 at a concentration of 50 ppm. Population
Generation
Slope (±SEM)
LT50 (95% FL) (h)
TR50 (95% CL)
LT95 (95% FL) (h)
TR95 (95% CL)
c2
P
LSP
Parental F1 F2 Parental F1 F2
5.46 5.81 6.98 4.71 5.27 5.20
9.00 9.77 9.07 6.58 6.69 6.80
e 1.09 1.01 e 1.02 1.03
18.02 18.75 15.60 14.71 13.72 14.09
e 1.04 1.16 e 1.07 1.04
9.02 14.80 13.69 11.69 10.17 9.30
0.25 0.25 0.25 0.11 0.12 0.16
MP
(±0.28) (±0.27) (±0.35) (±0.25) (±0.28) (±0.34)
(8.53e9.47) (9.41e10.13) (8.72e9.40) (6.25e6.91) (6.30e7.06) (6.30e7.26)
3.2. Locomotor behavior The distance traveled varied significantly between the populations (F1,168 ¼ 7.12; P ¼ 0.0084) and between the treatments with and without O3 (F1,168 ¼ 16.75; P ¼ 0.0001). However, no significant variations in these parameters between the generations subjected to selection pressure with O3 (F2,168 ¼ 1.70; P ¼ 0.1854) were observed. A significant interaction was observed between populations the treatments with and without O3 (F1,168 ¼ 18.57; P ¼ 0.0001). However, a significant interaction did not occur for the populations the generations (F2,168 ¼ 2.75; P ¼ 0.1059), generations the treatments with and without O3 (F2,168 ¼ 1.90; P ¼ 0.1525), or a three-way interaction (F2,168 ¼ 1.89; P ¼ 0.1541). With respect to the resting period, there was a significant variation between the populations (F1,168 ¼ 11.03; P ¼ 0.0011) and between the treatments with and without O3 (F1,168 ¼ 9.23; P ¼ 0.0028). However, these parameters did not vary significantly between the generations subjected to selection pressure with O3 (F2,168 ¼ 1.64; P ¼ 0.1964). A significant interaction was observed between populations the treatments with and without O3
(1.02e1.16) (0.95e1.06) (0.94e1.10) (0.94e1.13)
(16.80e19.53) (17.72e20.02) (14.89e16.45) (13.57e16.20) (12.78e14.89) (13.20e15.24)
(0.94e1.15) (1.07e1.25) (0.95e1.21) (0.94e1.16)
(F1,168 ¼ 8.40; P ¼ 0.0042). However, a significant interaction did not occur for the populations the generations (F2,168 ¼ 1.28; P ¼ 0.2798), generations the treatments with and without O3 (F2,168 ¼ 0.68; P ¼ 0.5051), or a three-way interaction (F2,168 ¼ 0.43; P ¼ 0.6493). The locomotor patterns are represented by the walking trail in Fig. 1. In the control, the distance traveled was longer in the LSP population than in the MP population. We also observed that, in the control, the resting period was smaller in the LSP than in the MP population. However, no significant differences were observed between the populations following exposure to O3. With respect to the walking speed, no significant variations were observed between the populations (F1,168 ¼ 0.037; P ¼ 0.8474), the treatments with and without O3 (F1,168 ¼ 0.68; P ¼ 0.4083), and generations (F2,168 ¼ 0.069; P ¼ 0.9336). 3.3. Respiratory rate The respiratory rate (mL CO2/insect/h) did not vary significantly between generations following selection cycles with O3
Fig. 1. Distance walked (A and B) and resting period (C and D) (±standard error of the mean) of individuals in the mixed population (MP) and in the population least susceptible to O3 (LSP) of Sitophilus zeamais subjected to O3 treatment (50 ppm) and O2 treatment (control) for 10 min. The histograms with continuous bars did not differ between the populations, according to the F-test (P < 0.05), and averages with asterisks indicate significant differences, according to the F-test (P < 0.05), between populations exposed or not to O3.
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In conclusion, no evidence for the development of O3 resistance was found in the two S. zeamais populations exposed to selection pressure with O3; similarly, no changes were observed in the locomotor patterns or in the respiratory rates in these populations. Notably, the susceptibility to O3 can be maintained with the practical use of insecticides as long as rational application strategies are established, including insect pest monitoring and the association of O3 with other control strategies. Acknowledgments The financial support provided by the CNPq (Grant number: 475182/2004-9) and FAPEMIG (Grant number: CAG 266/06) was greatly appreciated, as was the technical assistance provided by Dr. W. I. Urruchi. Fig. 2. Respiratory rates (±standard error of the mean) of Sitophilus zeamais in the mixed population (MP) and in the population least susceptible (LSP) to O3. Histogram bars with the identical letters did not differ significantly, according to the F-test (P < 0.05).
(F2,18 ¼ 0.025; P ¼ 0.9754). However, the production of CO2 in the MP population was significantly greater than that in the LSP population (F1,18 ¼ 33.18; P ¼ 0.0001) (Fig. 2), with a variation of 21.81%. The interaction of population generation was not significant (F2,18 ¼ 0.032; P ¼ 0.9689). 4. Discussion The assessment of the risk of resistance predicts the likelihood that insect pests will develop resistance to insecticides (Tabashnik, 1992; McKenzie and Batterham, 1998). For O3, no significant changes in the susceptibility were observed in the populations subjected to selection pressure with O3. Therefore, for these populations, genetic variability in the response to the toxicity of O3 was absent. Additionally, these results reinforced observations of low variability in the susceptibility to O3 in populations of stored products insect pests, as documented in Brazil, China, and Australia (Zhanggui et al., 2003; Sousa et al., 2008). Although no significant differences were observed in the locomotor activity of the populations subjected to selection pressure with O3, the lowest innate locomotor activity of the population or a decrease in the activity in the presence of O3 did not result in the development of resistance to O3 in these insect populations. This result is important because a decrease in locomotor activity of insects with insecticides decreases the exposure to the deleterious effects of the compounds. Similarly, a decrease in the respiratory rate decreases insect exposure to fumigants because the respiratory system is the primary entry route for gases in these animals (Chaudhry, 1997; Pimentel et al., 2009). In the present study, the innate variation in the production of CO2 in the two source populations did not result in a decrease in the toxicity of the O3 following the selection cycles. The low genetic variability in the S. zeamais populations for susceptibility to O3 indicates that the risk of development of resistance to this fumigant is low in the short-term. However, the lack of resistance to O3 in laboratory conditions does not guarantee the identical results in the field, particularly when laboratory populations often have limited genetic variability compared with field populations (McKenzie and Batterham, 1998). The extrapolation of these results to field conditions might also be hindered by the lack of adequate structures for the application of fumigants in storage units, which results in the exposure of insects to sublethal doses of fumigants (Pimentel et al., 2007, 2009).
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Ozone toxicity to Sitophilus zeamais (Coleoptera: Curculionidae) populations under selection pressure from ozone A.H. Sousa a, b, L.R.D'A. Faroni c, *, M.A.G. Pimentel d, G.N. Silva c, R.N.C. Guedes a a
Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil gicas e da Natureza, Universidade Federal do Acre, Rio Branco, AC, Brazil Departamento de Ci^ encias Biolo c Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG, Brazil d ria, Centro Nacional de Pesquisa de Milho e Sorgo, Sete Lagoas, MG, Brazil Empresa Brasileira de Pesquisa Agropecua b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2015 Received in revised form 6 November 2015 Accepted 6 November 2015 Available online xxx
Two populations of Sitophilus zeamais (Coleoptera: Curculionidae) were subjected to selection pressure with ozone (O3), and the possibility of acquiring resistance to O3 was investigated. The pattern of locomotion and the rate of respiration were evaluated following each selection cycle. Two source populations were used in the study: one was a mixture composed of 30 populations (MP), and the other was composed of the population that was the least susceptible to O3 among these 30 populations (LSP). The beetles from each source population experienced selection cycles with O3 using the lethal time for 80% (LT80) of the insect population from each generation. The O3 toxicity (50 ppm at a continuous flow rate of 2 L min1) to each generation was calculated using time-response bioassays. The locomotor pattern (distance traveled, resting period, and walking speed) and the respiratory rate (CO2 production) were also evaluated. The S. zeamais populations that were subjected to successive cycles of selection with O3 did not acquire resistance to O3, and the pattern of locomotion and the rate of respiration did not change following the selection cycles with O3. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Susceptibility Sitophilus zeamais Ozonation Alternative fumigants
1. Introduction The evolution of pest resistance to insecticides is a major obstacle in pest control programs that use chemical products (Groeters and Tabashnik, 2000). In the grain storage industry, the increased levels of resistance are primarily caused by the limited availability of different insecticides, the indiscriminate use of these insecticides for long periods, and the lack of appropriate structures for the application of fumigants (Collins et al., 2005; Sousa et al., 2009). Recent studies demonstrated that the Brazilian populations of Tribolium castaneum (Coleoptera: Tenebrionidae), Sitophilus zeamais (Coleoptera: Curculionidae), Rhyzopertha dominica (Coleoptera: Bostrichidae), and Oryzaephilus surinamensis (Coleoptera: Silvanidae) were highly resistant to phosphine, with rates of resistance that were 32.2e186.2-fold that of susceptible populations (Pimentel et al., 2007, 2009). The enrichment of the atmosphere with ozone (O3) is
* Corresponding author. Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil. E-mail address: [email protected] (L.R.D'A. Faroni). http://dx.doi.org/10.1016/j.jspr.2015.11.001 0022-474X/© 2015 Elsevier Ltd. All rights reserved.
recognized as an important alternative for the control of stored products pests (Kells et al., 2001; Sousa et al., 2008; Isikber and € Oztekin, 2009; Lu et al., 2009) because pests of stored products do not show cross-resistance between phosphine and O3. Additionally, O3 does not leave a residue in the grain because oxygen (O2) is the degradation product (Zhanggui et al., 2003; Sousa et al., 2008). Ozone is a gas that is derived from the rearrangement of oxygen atoms that occurs during electrical discharges or from exposure to high-energy electromagnetic radiation (ultraviolet light) in the atmosphere (Khadre et al., 2001; Liu et al., 2007). Ozone is an unstable molecule with a half-life of 20e50 min € (Isikber and Oztekin, 2009) that can be generated locally, which eliminates the requirements for its handling, storage, and transport. Although the toxic effects of O3 to stored products insect pests are well documented, further studies are required to assess the risk for the development of resistance in the populations that are exposed to selection pressure with O3. Based on evolutionary theory, resistance to insecticides is predicted to evolve in the populations that are maintained under selection pressure from chemicals because different traits are selected to increase the probability of survival in harsh environments (Foster et al., 2000;
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Coustau et al., 2000; Arnaud and Haubruge, 2002; Guedes et al., 2010). Moreover, the selection of a particular trait may be associated with pleiotropy, i.e., the ability of a single gene to affect more than one trait in an individual (Boivin et al., 2001; Raymond et al., 2005; Guedes et al., 2009). Therefore, in this study, the susceptibility of populations of S. zeamais to develop resistance when subjected to selection pressure with O3 was evaluated. Additionally, we evaluated the locomotor pattern and the metabolic rate of the beetles during each selection cycle, considering that the toxicity of these insecticides has a known association with physiological and behavioral mechanisms. 2. Materials and methods 2.1. Insects Two source populations were used in this study: (1) a mixture of 30 populations that were collected in 10 Brazilian states and two Paraguayan regions (MP), and (2) the population that was the least susceptible to O3 among these 30 populations (LSP), which was in the state of Minas Gerais. The toxicity to collected in Guaxupe these populations was previously established by Sousa et al. (2012). Both populations were maintained in 1.5-L glass vials under controlled conditions (27 ± 2 C and 70 ± 5% r.h.). Previously fumigated corn grain, with a moisture content of 13% (wet basis), was used as the food substrate, and the corn was maintained at 18 C to prevent re-infestation. 2.2. Selection and O3 toxicity bioassays The adults from the MP and the LSP populations were submitted to selection cycles with O3. Initially, the lethal time for 80% (LT80) of the parental generation of each population was used. Subsequently, the corresponding LT80 for each generation was used. The insects that survived the final selection cycle were collected to obtain the progeny. Approximately 2000 beetles, aged 1e2 weeks postemergence, were used in each selection cycle. The O3 toxicity was determined with the estimations of the lethal exposure times for 50% and 95% of the populations (LT50 and LT95, respectively), using the adapted methodology from Sousa et al. (2008). The O3 concentration was set at 50 ppm (z0.11 g m3) at a continuous flow rate of 2 L min1. The O3 was administered inside plastic chambers (width, 13 cm; height, 20 cm) at 27 C ± 2 C and a relative humidity of 70% ± 5%. The insects from each population were placed in plastic cages (width, 4 cm; height, 3.5 cm) that were suspended 10 cm from the base of the fumigation chamber. The cover and bottom of the cages were composed of organza-type fabric. For the controls, O2 with a minimum purity of 99.99% was used. The experiments were performed in three replicates, each with 50 unsexed adults that were 1e2 weeks old. The mortality of the beetles was assessed after eight days of exposure to O3. ~o Jose The O3 was obtained with an O&L3.ORM (Ozone & Life, Sa ~o Paulo, Brazil), which used compressed O2 (minidos Campos, Sa mum purity of 99.99%) as the source for the O3. The O3 concentration that was indicated by the O3 generator was confirmed using a continuous O3 monitor (BMT Messetechnik GMBHeBMT 930), with accuracy of 0.001 ppmv, and the iodometric method with indirect titration (Eaton et al., 2000). 2.3. Locomotor behavior The methods used were adapted from Watson et al. (1997) and Pereira et al. (2009). The walking of individual male and female insects was observed over 10 min in acrylic arenas (3.5 cm
high 15 cm wide), with walls coated with Teflon® polytetra~o Paulo, Brazil) to avoid the influoroethylene (PTFE; DuPont, Sa sects' escape. The O3 concentration was fixed at 50 ppm (z0.11 g m3) at a continuous flow rate of 1.3 L min1. This concentration is in the sublethal range for the two populations. The gas injection and exhaustion were through two connections installed on the opposite sides of each arena. For the controls, O2 with a minimum purity of 99.99% was used. The movement of the insects within the arena was recorded by a tracking system that consists of a CCD camera that registers and digitally transfers the images to a coupled computer (ViewPoint Life Sciences Inc., Montreal, Canada). Each insect was individually placed in the center of the arena, 2 min before the beginning of the test to allow the chamber to reach ozone saturation and the insect to acclimate to the arena. The evaluated characteristics were distance (cm), resting period (s), and walking speed (mm/s). Twenty repetitions were conducted for each population. The tests were performed in a climate-controlled room (27 ± 2 C and 70 ± 5% r.h.), between 7:00 am and 7:00 pm. 2.4. Respiratory rate The production of carbon dioxide (CO2) was measured using a TR3C CO2 analyzer (Sable Systems International, Las Vegas, USA). For this purpose, 25-mL respiratory chambers that contained 20 adults of each population were used, with each connected to a completely closed system. Four replicates were used for each population, and the CO2 production was measured in each chamber at a controlled temperature of 27 C ± 2 C after a 15-h acclimation period. CO2-free air was injected into the chambers for 2 min at a flow rate of 100 mL min1. An infrared sensor reader was connected to the system output for the quantification of the CO2 (mL CO2 h1 per insect). The controls were empty respiratory chambers, and the values were used to normalize the respiratory rate data for each population. 2.5. Statistical analyses The data of the time-response bioassays were subjected to probit analysis (PROC PROBIT; SAS Institute, 2011). The confidence intervals for the toxicity ratios (TRs) were calculated according to Robertson and Preisler (1992), and the lethal time (LT) values were considered significantly different (P < 0.05) when the confidence intervals of the TRs did not include the value 1. For the locomotor parameters, multivariate covariance analyses were performed (population generation treatments, with and without O3) (PROC GLM with MANOVA procedure; SAS Institute, 2011). The CO2 data were subjected to an analysis of covariance (population generation) (PROC GLM; SAS Institute, 2011). 3. Results 3.1. O3 toxicity For the two populations, the variation in the toxicity of O3 between generations was markedly low (<2-fold; Table 1), which indicated the lack of variation in the populations in the susceptibility to O3. Because no resistance to O3 was observed, the tests were terminated in the F2 generation. The TRs of the LT50 values varied between 1.00- and 1.09-fold for the mixed population (MP) and between 1.00- and 1.03-fold for the population least susceptible to O3 (LSP). The slopes of the timeemortality curves were similar between the populations, which indicated homogeneity in the responses to O3 in the selection cycles.
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Table 1 Relative O3 toxicity to adults in the population least susceptible to O3 (LSP) and in the mixed population (MP) of Sitophilus zeamais after two selection cycles using the LT80 and O3 at a concentration of 50 ppm. Population
Generation
Slope (±SEM)
LT50 (95% FL) (h)
TR50 (95% CL)
LT95 (95% FL) (h)
TR95 (95% CL)
c2
P
LSP
Parental F1 F2 Parental F1 F2
5.46 5.81 6.98 4.71 5.27 5.20
9.00 9.77 9.07 6.58 6.69 6.80
e 1.09 1.01 e 1.02 1.03
18.02 18.75 15.60 14.71 13.72 14.09
e 1.04 1.16 e 1.07 1.04
9.02 14.80 13.69 11.69 10.17 9.30
0.25 0.25 0.25 0.11 0.12 0.16
MP
(±0.28) (±0.27) (±0.35) (±0.25) (±0.28) (±0.34)
(8.53e9.47) (9.41e10.13) (8.72e9.40) (6.25e6.91) (6.30e7.06) (6.30e7.26)
3.2. Locomotor behavior The distance traveled varied significantly between the populations (F1,168 ¼ 7.12; P ¼ 0.0084) and between the treatments with and without O3 (F1,168 ¼ 16.75; P ¼ 0.0001). However, no significant variations in these parameters between the generations subjected to selection pressure with O3 (F2,168 ¼ 1.70; P ¼ 0.1854) were observed. A significant interaction was observed between populations the treatments with and without O3 (F1,168 ¼ 18.57; P ¼ 0.0001). However, a significant interaction did not occur for the populations the generations (F2,168 ¼ 2.75; P ¼ 0.1059), generations the treatments with and without O3 (F2,168 ¼ 1.90; P ¼ 0.1525), or a three-way interaction (F2,168 ¼ 1.89; P ¼ 0.1541). With respect to the resting period, there was a significant variation between the populations (F1,168 ¼ 11.03; P ¼ 0.0011) and between the treatments with and without O3 (F1,168 ¼ 9.23; P ¼ 0.0028). However, these parameters did not vary significantly between the generations subjected to selection pressure with O3 (F2,168 ¼ 1.64; P ¼ 0.1964). A significant interaction was observed between populations the treatments with and without O3
(1.02e1.16) (0.95e1.06) (0.94e1.10) (0.94e1.13)
(16.80e19.53) (17.72e20.02) (14.89e16.45) (13.57e16.20) (12.78e14.89) (13.20e15.24)
(0.94e1.15) (1.07e1.25) (0.95e1.21) (0.94e1.16)
(F1,168 ¼ 8.40; P ¼ 0.0042). However, a significant interaction did not occur for the populations the generations (F2,168 ¼ 1.28; P ¼ 0.2798), generations the treatments with and without O3 (F2,168 ¼ 0.68; P ¼ 0.5051), or a three-way interaction (F2,168 ¼ 0.43; P ¼ 0.6493). The locomotor patterns are represented by the walking trail in Fig. 1. In the control, the distance traveled was longer in the LSP population than in the MP population. We also observed that, in the control, the resting period was smaller in the LSP than in the MP population. However, no significant differences were observed between the populations following exposure to O3. With respect to the walking speed, no significant variations were observed between the populations (F1,168 ¼ 0.037; P ¼ 0.8474), the treatments with and without O3 (F1,168 ¼ 0.68; P ¼ 0.4083), and generations (F2,168 ¼ 0.069; P ¼ 0.9336). 3.3. Respiratory rate The respiratory rate (mL CO2/insect/h) did not vary significantly between generations following selection cycles with O3
Fig. 1. Distance walked (A and B) and resting period (C and D) (±standard error of the mean) of individuals in the mixed population (MP) and in the population least susceptible to O3 (LSP) of Sitophilus zeamais subjected to O3 treatment (50 ppm) and O2 treatment (control) for 10 min. The histograms with continuous bars did not differ between the populations, according to the F-test (P < 0.05), and averages with asterisks indicate significant differences, according to the F-test (P < 0.05), between populations exposed or not to O3.
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In conclusion, no evidence for the development of O3 resistance was found in the two S. zeamais populations exposed to selection pressure with O3; similarly, no changes were observed in the locomotor patterns or in the respiratory rates in these populations. Notably, the susceptibility to O3 can be maintained with the practical use of insecticides as long as rational application strategies are established, including insect pest monitoring and the association of O3 with other control strategies. Acknowledgments The financial support provided by the CNPq (Grant number: 475182/2004-9) and FAPEMIG (Grant number: CAG 266/06) was greatly appreciated, as was the technical assistance provided by Dr. W. I. Urruchi. Fig. 2. Respiratory rates (±standard error of the mean) of Sitophilus zeamais in the mixed population (MP) and in the population least susceptible (LSP) to O3. Histogram bars with the identical letters did not differ significantly, according to the F-test (P < 0.05).
(F2,18 ¼ 0.025; P ¼ 0.9754). However, the production of CO2 in the MP population was significantly greater than that in the LSP population (F1,18 ¼ 33.18; P ¼ 0.0001) (Fig. 2), with a variation of 21.81%. The interaction of population generation was not significant (F2,18 ¼ 0.032; P ¼ 0.9689). 4. Discussion The assessment of the risk of resistance predicts the likelihood that insect pests will develop resistance to insecticides (Tabashnik, 1992; McKenzie and Batterham, 1998). For O3, no significant changes in the susceptibility were observed in the populations subjected to selection pressure with O3. Therefore, for these populations, genetic variability in the response to the toxicity of O3 was absent. Additionally, these results reinforced observations of low variability in the susceptibility to O3 in populations of stored products insect pests, as documented in Brazil, China, and Australia (Zhanggui et al., 2003; Sousa et al., 2008). Although no significant differences were observed in the locomotor activity of the populations subjected to selection pressure with O3, the lowest innate locomotor activity of the population or a decrease in the activity in the presence of O3 did not result in the development of resistance to O3 in these insect populations. This result is important because a decrease in locomotor activity of insects with insecticides decreases the exposure to the deleterious effects of the compounds. Similarly, a decrease in the respiratory rate decreases insect exposure to fumigants because the respiratory system is the primary entry route for gases in these animals (Chaudhry, 1997; Pimentel et al., 2009). In the present study, the innate variation in the production of CO2 in the two source populations did not result in a decrease in the toxicity of the O3 following the selection cycles. The low genetic variability in the S. zeamais populations for susceptibility to O3 indicates that the risk of development of resistance to this fumigant is low in the short-term. However, the lack of resistance to O3 in laboratory conditions does not guarantee the identical results in the field, particularly when laboratory populations often have limited genetic variability compared with field populations (McKenzie and Batterham, 1998). The extrapolation of these results to field conditions might also be hindered by the lack of adequate structures for the application of fumigants in storage units, which results in the exposure of insects to sublethal doses of fumigants (Pimentel et al., 2007, 2009).
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