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STORED-PRODUCT

Ozone Toxicity and Walking Response of Populations of Sitophilus zeamais (Coleoptera: Curculionidae) A. H. SOUSA,1,2 L.R.A. FARONI,3 G. N. SILVA,3

AND

R.N.C. GUEDES1,4

J. Econ. Entomol. 105(6): 2187Ð2195 (2012); DOI: http://dx.doi.org/10.1603/EC12218

ABSTRACT Ozone is a recognized alternative to the fumigants methyl bromide and phosphine for the control of stored product insects. However, as with fumigants in general, the potential sublethal effects of ozone on targeted insect species may compromise its efÞcacy and has yet to be investigated. Here, we determined ozone toxicity of 30 Þeld-collected populations of the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae), and assessed the walking response of adult insects from these populations to sublethal ozone exposure. Time-mortality toxicity to ozone at 50 ppm concentration in a continuous 2 liter/min ßow indicated uniform susceptibility among the populations studied without any indication of ozone resistance (toxicity ratios [at LT50] ⬎ two-fold). In contrast, there was signiÞcant variation in walking activity among the maize weevil populations, which was not correlated with ozone susceptibility. This was not surprising because of the relatively uniform susceptibility to ozone among the maize weevil populations. Respiration rate affected ozone toxicity but not walking activity, whereas body mass was negatively correlated with walking activity but was not correlated with ozone toxicity. Based on our data, lower respiration rates may potentially lead to reduced ozone uptake whereas larger body mass limits walking activity. Ozone seems a promising alternative fumigant with low short-term risk of resistance development because of the high susceptibility and low variability of response to this compound. Furthermore, ozone reduces walking activity of S. zeamais that implies it likely reduces the chances of insects escaping exposure at the early stages of fumigation. KEY WORDS fumigation, ozone susceptibility, ambulatory behavior, behavioral resistance, stored product protection

Ozone (O3) is an alternative to conventional fumigants for controlling stored product insects because it is an oxidizing gas that is highly toxic to many organisms including insects, fungi, bacteria, and viruses (Khadre et al. 2001, An et al. 2007, Sousa et al. 2008, ¨ ztekin 2009). Ozone acts by damaging Isikber and O cell membranes or triggering cell death in various organisms via oxidative stress (Hollingsworth and Armstrong 2005). This is achieved because ozone is a triatomic form of oxygen with the second highest oxidation potential among chemicals, surpassed only by ßuorine (F2) (Hill and Rice 1982). The interest in ozone as an alternative fumigant for stored product pests is increasing not only because of the phasing out of methyl bromide and increased reliance on phosphine (United Nations Environment Programme [UNEP] 1995), but also because of the ever growing worldwide problems of phosphine resistance (Champ and Dyte 1976; Chaudry 2000; Col1 Departamento de Entomologia, Universidade Federal de Vic ¸ osa, Viç osa, MG 36570-000, Brazil. 2 Centro de Cie ˆ ncias Biolo´ gicas e da Natureza, Universidade Federal de Acre, Rio Branco, AC 69915-900, Brazil. 3 Departamento de Engenharia Agrõćola, Universidade Federal de Viç osa, Viç osa, MG 36570-000, Brazil. 4 Corresponding author, e-mail: [email protected].

lins et al. 2003; Pimentel et al. 2007, 2009). Ozone, which is formed when oxygen is subjected to highvoltage electric discharge, is unstable with a short half-life (20 Ð50 min) and decomposes to oxygen, a natural component of the atmosphere (Kells et al. 2001). Therefore, its environmental safety proÞle is appealing and has allowed ozone to have many uses, namely, drinking water treatment, preservation of vegetables and fruits, surface decontamination of perishable foods, and disinfection of manufacturing equipments, packing materials, and medical appliances (Graham 1997, Food and Drug Administration [FDA] 2001, Mendez et al. 2003, Zhanggui et al. 2003). Toxicity studies in target species commonly focus on lethal estimates for insecticides in general and for fumigants in particular. Much as such studies are important, the fact that studies on sublethal effects of pesticides have been neglected is hard to justify (Haynes 1988; Desneux et al. 2007; Guedes et al. 2009a,b, 2011). Insects have evolved a variety of responses to insecticides reßecting their mode of action and how these compounds may affect behavior (Moore et al. 1989, Cox et al. 1997, Bayley 2002, Guedes et al. 2008). The insect behavioral changes associated with insecticide exposure may decrease control efÞcacy and interfere with insect mobility

0022-0493/12/2187Ð2195$04.00/0 䉷 2012 Entomological Society of America

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Table 1.

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Relative toxicity of ozone to populations of Sitophilus zeamais

Population Tuna´polis (SC) Par acatu (MG) Dourados Linha Proterito (MS) Viç osa (MG) Nova Era (MG) Pedro Juan Caballero (Paraguay) Uirapuru (AM) Saõ Jose´ do Rio Pardo (SP) Amambay (Paraguay) Espõ´rito Santo do Pinhal (SP) Vicentina (MS) Abre Campo (MG) Dourados Bororo´ (MS) Votuporanga (SP) Saõ Joaõ (PE) Unaõ´ (MG) Coimbra (MG) Dourados Linha Barreirinha (MS) Guarapuava (PR) Ipora´ (GO) Piracicaba (SP) Cristalina (GO) Canarana (MT) Picos (PI) Sacramento (MG) A´ gua Boa, (MT) Ana´polis (GO) Jataõ´ (GO) Machado (MG) Guaxupe´ (MG)

No. insects

Slope (⫾ SEM)

LT50 (FL 95%) (h)

RT50 (CI 95%)

LT95 (FL 95%) (h)

RT95 (CI 95%)

␹2

P

750 900 750

4.60 ⫾ 0.26 4.57 (4.32Ð4.82) Ñ 10.40 (9.53Ð11.54) Ñ 3.52 ⫾ 0.21 5.01 (4.67Ð5.36) 1.10 (1.00Ð1.20) 14.70 (13.09Ð16.97) 1.41 (1.20Ð1.67) 5.74 ⫾ 0.46 5.03 (4.70Ð5.31) 1.10 (1.00Ð1.21) 9.73 (9.05Ð10.68) 1.07 (0.92Ð1.25)

9.27 0.16 8.81 0.18 5.57 0.35

750 750 750 750

5.35 ⫾ 0.32 5.04 (4.77Ð5.32) 1.10 (1.02Ð1.20) 10.24 (9.43Ð11.32)

1.02 (0.90Ð1.15)

5.13 0.40

6.70 ⫾ 0.42 5.31 (5.10Ð5.50) 1.16 (1.09Ð1.24) 7.11 ⫾ 0.51 5.54 (5.29Ð5.78) 1.21 (1.15Ð1.29)

1.11 (0.99Ð1.25) 10.65 0.15 1.10 (1.00Ð1.21) 6.59 0.25

750 750 900 900 750 900 900 750 900 750 900 900

6.76 ⫾ 0.39 7.42 ⫾ 0.44 5.86 ⫾ 0.38 5.51 ⫾ 0.32 8.39 ⫾ 0.56 5.29 ⫾ 0.30 4.67 ⫾ 0.34 8.24 ⫾ 0.41 5.10 ⫾ 0.32 7.96 ⫾ 0.43 5.98 ⫾ 0.34 6.17 ⫾ 0.33

5.57 (5.36Ð5.78) 5.65 (5.45Ð5.85) 5.78 (5.53Ð6.02) 5.79 (5.53Ð6.03) 5.99 (5.77Ð6.20) 6.01 (5.69Ð6.32) 6.04 (5.66Ð6.39) 6.10 (5.93Ð6.27) 6.25 (5.88Ð6.60) 6.28 (6.07Ð6.48) 6.76 (6.49Ð7.02) 6.80 (6.54Ð7.05)

1.22 (1.15Ð1.29) 1.24 (1.17Ð1.30) 1.26 (1.20Ð1.34) 1.27 (1.19Ð1.35) 1.31 (1.24Ð1.39) 1.31 (1.23Ð1.40) 1.32 (1.22Ð1.43) 1.33 (1.25Ð1.43) 1.37 (1.28Ð1.46) 1.37 (1.28Ð1.47) 1.48 (1.40Ð1.56) 1.49 (1.41Ð1.57)

9.76 (9.22Ð10.45) 9.42 (8.89Ð10.10) 11.02 (10.23Ð12.10) 11.50 (10.74Ð12.50) 9.41 (8.94Ð10.02) 12.29 (11.36Ð13.49) 13.60 (12.53Ð15.10) 9.65 (9.21Ð10.21) 13.13 (12.24Ð14.31) 10.10 (9.61Ð10.72) 12.73 (12.00Ð13.67) 12.57 (11.91Ð13.40)

1.07 (0.97Ð1.17) 1.11 (1.01Ð1.21) 1.06 (0.95Ð1.18) 1.11 (0.99Ð1.24) 1.11 (1.01Ð1.22) 1.18 (1.06Ð1.31) 1.31 (1.15Ð1.49) 1.08 (0.97Ð1.20) 1.26 (1.15Ð1.39) 1.03 (0.94Ð1.13) 1.22 (1.12Ð1.33) 1.21 (1.10Ð1.32)

11.56 10.36 9.51 12.32 8.00 9.18 9.36 10.59 11.32 8.56 10.87 13.07

0.12 0.11 0.15 0.14 0.16 0.16 0.23 0.30 0.12 0.29 0.28 0.22

750 900 900 750 750 750 750 750 750 750 900 900

7.66 ⫾ 0.48 4.94 ⫾ 0.34 4.60 ⫾ 0.21 6.21 ⫾ 0.29 6.16 ⫾ 0.32 6.57 ⫾ 0.35 6.52 ⫾ 0.32 6.42 ⫾ 0.31 6.70 ⫾ 0.38 6.94 ⫾ 0.41 5.76 ⫾ 0.29 5.46 ⫾ 0.28

7.02 (6.70Ð7.33) 7.17 (6.63Ð7.69) 7.24 (6.90Ð7.57) 7.28 (6.99Ð7.56) 7.51 (7.17Ð7.85) 7.57 (7.24Ð7.91) 7.61 (7.26Ð7.96) 7.80 (7.47Ð8.12) 7.90 (7.56Ð8.23) 8.00 (7.64Ð8.33) 8.78 (8.36Ð9.19) 9.00 (8.53Ð9.47)

1.54 (1.45Ð1.63) 1.57 (1.44Ð1.71) 1.58 (1.45Ð1.73) 1.59 (1.50Ð1.69) 1.64 (1.55Ð1.75) 1.66 (1.55Ð1.77) 1.67 (1.56Ð1.78) 1.71 (1.60Ð1.82) 1.73 (1.63Ð1.84) 1.75 (1.65Ð1.86) 1.92 (1.80Ð2.05) 1.97 (1.83Ð2.12)

11.51 (10.83Ð12.38) 15.44 (14.15Ð17.17) 16.47 (15.38Ð17.82) 13.40 (12.68Ð14.27) 13.89 (12.95Ð15.08) 13.47 (12.62Ð14.56) 13.61 (12.87Ð14.50) 14.07 (13.32Ð15.00) 13.90 (13.04Ð14.99) 13.80 (13.03Ð14.79) 16.95 (15.94Ð18.20) 18.02 (16.80Ð19.53)

1.11 (1.01Ð1.21) 1.48 (1.32Ð1.67) 1.58 (1.40Ð1.79) 1.29 (1.17Ð1.42) 1.33 (1.21Ð1.47) 1.29 (1.16Ð1.44) 1.31 (1.19Ð1.44) 1.35 (1.24Ð1.47) 1.34 (1.22Ð1.46) 1.33 (1.21Ð1.46) 1.63 (1.48Ð1.79) 1.73 (1.56Ð1.92)

6.76 6.08 14.97 13.73 9.65 8.92 9.93 10.66 7.09 10.20 12.37 9.02

0.24 0.19 0.18 0.13 0.14 0.18 0.19 0.22 0.31 0.12 0.13 0.25

(Bayley 2002; Pereira et al. 2009; Guedes et al. 2009a, 2011; Braga et al. 2011; Correˆ a et al. 2011; Martini et al. 2012; Pimentel et al. 2012). Despite the importance of studies on sublethal effects of insecticides, studies on sublethal effects of fumigants are extremely rare, in fact, much more rare than studies on sublethal effects of conventional insecticides (Winks 1985, Winks and Waterford 1986, Nayak et al. 2003, Guedes et al. 2011, Pimentel et al. 2012). Regarding ozone fumigation against stored product insects, no sublethal (behavioral) studies have been object of attention so far, despite the high likelihood of sublethal exposure not only during the initial gas build-up and eventual exhaustion (at the beginning and Þnishing of the ozone application procedure), but throughout the ozone fumigation if the storage unit does not exhibit airtight conditions and gas escape is the consequence. In truth, lack of the airtight conditions required for high-efÞcacy fumigation is a frequent shortcoming in storage product units throughout the world (Mills 1983, Wohlgemuth and Harnisch 1986). Insect survival to ozone may take place if its behavior minimizes the gas exposure or uptake, which has been reported for conventional insecticides and particularly for phosphine more recently (Barson et al. 1992; Guedes et al. 2008, 2009a; Braga et al. 2011, Correˆ a et al. 2011; Pimentel et al. 2012). Insect behavior, particularly its walking activity, is likely to affect metabolism and consequently its respiration

9.34 (8.80Ð10.06) 9.45 (8.89Ð10.20)

rate, which is a potential determinant of ozone uptake, as observed for phosphine (Pimentel et al. 2007, 2009). These responses may vary with insect population and ozone exposure may further increase such variation. Although some studies have investigated ozone toxicity among populations of stored-product insect species, none have been conducted on the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), which is a key pest of stored products in the Afrotropical and Neotropical regions. The objectives of the current study were to assess ozone toxicity using 30 Þeld populations of S. zeamais and investigate their walking activity when exposed to sublethal concentrations of ozone. We were not expecting signiÞcant variation in ozone toxicity among populations based on early Þndings for other stored product species (Sousa et al. 2008), but signiÞcant variation in walking activity may occur among populations potentially affecting ozone efÞcacy. Materials and Methods Insects. In total, 30 populations of S. zeamais were collected between 2006 and 2008 from storage units in the Brazilian states of Amazonas (AM), Goia´s (GO), Mato Grosso (MT), Mato Grosso do Sul (MS), Minas Gerais (MG), Parana´ (PR), Pernambuco (PE), Piauõ´ (PI), Santa Catarina (SC), and Saõ Paulo (SP), and the Paraguayan locations of Pedro Juan Caballero and Arroyo Jambure Amambay (Table 1); some of these

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SOUSA ET AL.: OZONE TOXICITY AND WALKING RESPONSE IN S. zeamais

populations are resistant to phosphine (Pimentel et al. 2009). The insects were reared in 1.5 liter glass jars maintained in rearing chambers under constant conditions of 27 ⫾ 2⬚C, 70 ⫾ 5% relative humidity (RH), and 24 h scotophase. Insecticide-free maize (13% moisture content) that had previously been fumigated to prevent cross-infestation was used as a food substrate. Ozone Toxicity. The toxicity of ozone in each of the 30 populations of S. zeamais was determined by timemortality bioassays using Þve to six independent exposure times (varying from 4 to 19 h of exposure) and ozone concentration set at 50 ppm (⬇0.11 g/m) with a continuous ßow of 2 liter/min. Ozone application was performed inside cylindrical plastic chambers (13 cm diameter ⫻ 20 cm high) at 27 ⫾ 2⬚C and relative humidity of 70 ⫾ 5%. Ozone injection and exhaustion was accomplished by a connection installed at 6 cm from the base of each chamber and one at the top (cover), respectively. The same system was used providing only oxygen (99.99% pure) to allow estimation of natural mortality for use as a control treatment. Pure oxygen was preferred to the natural air because of the high humidity of the later, which prevented the ozone generator from functioning compromising the maintenance of the ozone concentration. Insects in each population were placed in plastic cages (4 cm wide ⫻ 3.5 cm high) suspended 10 cm from the base of the chambers. The cages had their top and bottom made of an organzalike fabric (i.e., a thin, plain weave, sheer fabric), allowing free passage of ozone and oxygen. Three replicates of 50 unsexed 1- to 4-wk-old insects were used for each exposure time, for each insect population. After each exposure time, the insects were removed from the fumigation chamber. Insect mortality was assessed after 8 d of each period of exposure to ozone. Ozone Generation and System Operation. Ozone was produced with an O&L3.ORM ozone generator (Ozone & Life, Saõ Jose´ dos Campos, SP, Brazil), which uses compressed 99.99% pure oxygen as input. At the exit of the oxygen cylinder, a device with two air exits was installed with ßow rate of 6 liter/min, one passing through an ozone generator and the other going to the system using only oxygen (control). Ozone and oxygen were uniformly distributed to the chambers containing insects. Residual ozone and oxygen in the exhaust were connected to a PVC tube (4.5 cm wide ⫻ 56 cm high) containing an 8-cm water column before being released into the atmosphere, minimizing the contribution to ionization by the electrons released from the chamber walls (“wall effect”). In the generation of ozone, oxygen gas ßows through a refrigerated reactor, where an electric current is discharged through a dielectric barrier. This type of discharge is produced by applying a high voltage between two parallel electrodes with a sandwiched dielectric glass and one free space through which oxygen ßows. In this space a discharge is produced in the form of Þlaments, where electrons are generated with sufÞcient energy to cause the breakdown of oxygen molecules and forming ozone (O3). The ozone concentration indicated by the ozone generator was conÞrmed by a continuous ozone monitor

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with a 0.001-ppm precision (BMT 930, BMT Messtechnik GMBH, Berlin, Germany) and the iodometric method using indirect titration (Eaton et al. 2000). Walking Bioassay. The methods used were adapted from Guedes et al. (2009a) and Pimentel et al. (2012). Walking activity of unsexed (individual) insects was recorded for 10 min in acrylic chambers (15 cm diameter ⫻ 3.5 cm high), whose walls were coated with Teßon PTFE (DuPont, Saõ Paulo, Brazil) to avoid insect escape. Ozone concentration was set at 50 ppm (0.11 g/m) in continuous ßow of 1.3 liter/min. The injection and exhaustion of the gas were carried out through two opposite connections installed in each chamber. The same bioassays were run with only oxygen introduced into the chamber. The walking activity of each insect in the chamber was recorded by a tracking system consisting of a CCD camera that digitally records and transfers the images to an attached computer (ViewPoint Life Sciences Inc., Montreal, Canada). Each insect was released in the center of the chamber, 2 min before initiating the tests. In each case, the chamber was Þrst saturated with ozone to allow the insect to acclimate to the conditions. This acclimation period was determined based on preliminary tests and correspond to the time necessary for the insects to establish a constant walking pattern of activity. The characteristics evaluated were the distance walked (cm), walking velocity (mm/s), and resting time (s). In total, 20 replicates were used for each population. Each arena was replaced by another one previously opened, cleaned (with water and neutral detergent) and fully aerated after use. The bioassays were carried out in a room with controlled temperature (27.0 ⫾ 2.0⬚C) between 7 a.m. and 7 p.m. Respiration Rate and Body Mass. The production of carbon dioxide (CO2), expressed as ␮l CO2/insect/h, was measured using a TR3C respirometer equipped with a CO2 Analyzer (Sable System International, Las Vegas, NV) (Guedes et al. 2006, Sousa et al. 2008). Respirometric chambers with volumes of 25 ml, each containing 20 unsexed adult insects, were connected to a completely closed system. Production of CO2 was measured 15 h after acclimatization of insects in the chambers. The chambers were connected to the system for 1 h before measuring CO2 produced by insects. The measurements were obtained by injecting CO2free air into the chambers for 2.0 min at a 100-ml/min ßow rate. This air current directed the CO2 to an infrared reader connected to the system thereby allowing prompt quantiÞcation of CO2. Levels of CO2 were also quantiÞed in the control chamber. After measuring CO2 production, the insects were removed from the chambers and weighed using an analytical balance (Sartorius BP 210D, Go¨ ttingen, Germany). Respiration rate values were not normalized by body mass because this procedure masks the effect of individual insects (Packard and Boardman 1999, Haynes 2001). Four replicates were conducted for each population. Statistical Analyses. The time-mortality data were subjected to probit analysis (PROC PROBIT; SAS Institute 2008). ConÞdence intervals for the toxicity ratios (TRs) were calculated based on Robertson and

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Fig. 1. Distance walked (mean ⫾ SE) by insects from different populations of S. zeamais in the presence or absence of ozone. A sublethal ozone concentration of 50 ppm for 10 min was used. Histogram bars grouped by the same letter are not signiÞcantly different based on the ScottÐKnottÕs test (P ⬍ 0.05).

Preisler (1992). Values of lethal times (LTs) were considered signiÞcantly different if their 95% CIs do not encompass the value 1. Walking activity data were subjected to two-way analysis of variance (ANOVA) to determine the effects of ozone exposure and population on walking activity (PROC GLM; SAS Institute 2008), followed by ScottÐKnott groupment analysis when necessary (P ⬍ 0.05) (Scott and Knott 1974, Sistema de Ana´lises Estatõ´sticas e Gene´ ticas [SAEG] 2005). CO2 production and body mass data were also subjected to ANOVA to determine the effects of population, followed by ScottÐKnot test (P ⬍ 0.05) when appropriate. Data on CO2 production and body mass were also

subjected to ANOVA, followed by ScottÐKnott test when appropriate (P ⬍ 0.05) (Scott and Knott 1974, SAEG 2005). Correlation analyses were conducted to recognize the relationship between ozone toxicity, walking activity, or respiration rate and body mass (PROC CORR; SAS Institute 2008). Results Ozone Toxicity. Ozone toxicity to S. zeamais was determined using time-mortality curves (Table 1). There was little variation in ozone toxicity among insect populations. Toxicity ratios ranged from 1.00 to 1.97 at LT50 and 1.00 Ð1.73 at LT95. The slopes of the

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Fig. 2. Walking velocity (mean ⫾ SE) by insects from different populations of S. zeamais in the presence or absence of ozone. A sublethal ozone concentration of 50 ppm for 10 min was used. Histogram bars grouped by the same letter are not signiÞcantly different based on the ScottÐKnottÕs test (P ⬍ 0.05).

time-mortality curves were also similar among insect populations with relative homogeneity of response among and within populations. Walking Behavior. The walking behavior varied signiÞcantly among populations and there was signiÞcant interference of ozone exposure in such behavior for each S. zeamais population (i.e., populations-ozone interaction was signiÞcant for distance walked [F29,1140 ⫽ 2.67; P ⬍ 0.001], walking velocity [F29,1140 ⫽ 2.31; P ⬍ 0.001], and resting time [F29,1140 ⫽ 2.19; P ⬍ 0.001]). The overall effect of ozone exposure was reducing the distance walked (Fig. 1) and walking velocity (Fig. 2). However, ozone exposure resulted in increased resting time (Fig. 3). The effects of ozone exposure on the afore-

mentioned three parameters varied with population hence the signiÞcant interactions between ozone exposure and population reported. Distance walked, walking velocity, and resting time in the presence of ozone were positively correlated when in absence of ozone (n ⫽ 30, r ⫽ 0.63, P ⫽ 0.0002; n ⫽ 30, r ⫽ 0.65, P ⫽ 0.0001; and n ⫽ 30, r ⫽ 0.61, P ⫽ 0.0004, respectively). Respiration Rate and Body Mass. Respiration rate varied in 41.24% among populations of the maize weevil (F29,90 ⫽ 7.01; dferror ⫽ 90; P ⬍ 0.001) (Fig. 4A). Individual insect body mass also varied (in 70.77%) among populations of the maize weevil (F29,90 ⫽ 13.37; dferror ⫽ 90; P ⬍ 0.001) (Fig. 4B). Respiration rate was positively correlated with body mass (n ⫽ 90; r ⫽ 0.60; P ⬍ 0.001).

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Fig. 3. Resting time (mean ⫾ SE) by insects from different populations of S. zeamais in the presence or absence of ozone. A sublethal ozone concentration of 50 ppm for 10 min was used. Histogram bars grouped by the same letter are not signiÞcantly different based on the ScottÐKnottÕs test (P ⬍ 0.05).

Relationship Between Ozone Toxicity, Walking Activity, and Metabolism. Ozone toxicity (LT50) was not correlated with walking activity (P ⬎ 0.05), or body mass (P ⬎ 0.05), but was positively affected by respiration rate (n ⫽ 30; r ⫽ 0.41; P ⫽ 0.02). This means that ozone toxicity is greater at higher respiration rates. Respiration rate did not affect walking activity (P ⬎ 0.05), whereas insect body mass was negatively correlated with walking (n ⫽ 30; r ⬎ ⫺0.47; P ⬍ 0.01). Discussion Ozone toxicity has been assessed in populations of red ßour beetle (Tribolium castaneum Herbst), of

lesser grain borer (Rhyzopertha dominica F.), and of sawtoothed grain beetle (Oryzaephilus surinamensis L.) (Sousa et al. 2008). This is the Þrst study on the effects of ozone on S. zeamais. Our results are similar to those for the aforementioned species where little variation in ozone toxicity was found among populations studied (Sousa et al. 2008). All the S. zeamais populations we tested were susceptible to ozone and their response was relatively homogeneous indicating present lack of variation in ozone resistance development and therefore low risk of short-term development of ozone resistance. Similar Þndings were also reported for the red ßour beetle, the lesser grain borer, and the sawtoothed grain beetle (Sousa et al. 2008).

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Fig. 4. Respiration rate (mean ⫾ SE) (A) and body mass (mean ⫾ SE) (B) of insects from different populations of S. zeamais. Means followed by the same bars are not signiÞcantly different by the ScottÐKnottÕs test (P ⬍ 0.05).

The overall susceptibility and lack of variability in ozone toxicity among populations of different stored product insect species is a stark contrast with what has been reported for phosphine resistance. Resistance to phosphine has existed from the time of the FAO global survey of the phenomenon and is still increasing (Champ and Dyte 1976; Chaudry 2000; Collins et al. 2003; Pimentel et al. 2007, 2009). We found ozone toxicity to be positively correlated with respiration rate in populations of S. zeamais, and

this is despite the low variation in the former. Such correlation was not detected in populations of red ßour beetle, lesser grain borer, and sawtoothed grain beetle (Sousa et al. 2008). This is probably because of the small variation in ozone toxicity observed among populations of each species tested and the modest number of populations surveyed for each species (Sousa et al. 2008). Increased respiration increases fumigant uptake, which has been recognized as a phosphine resistance mechanism in populations of

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stored product insects (Pimentel et al. 2007, 2009, 2012; Nath et al. 2011). Therefore, the association between high respiration rate and high ozone toxicity was expected and detected in our study despite the relatively small variation in the later. Walking activity greatly varied among populations of S. zeamais but this activity was not correlated with ozone toxicity. Exposure of S. zeamais to sublethal ozone levels reduces walking activity. Variation in walking activity among populations was expected and has been reported in different species of stored product insects, including the maize weevil (Guedes et al. 2009a, Pereira et al. 2009, Braga et al. 2011, Correˆ a et al. 2011, Pimentel et al. 2012). Such variation may affect ozone efÞcacy by affecting respiration rate for instance (Pimentel et al. 2012). Based on our data, walking activity did not affect respiration rate but was affected by body mass. High body mass was associated with reduced walking activity, which is likely to reduce the chance of escaping ozone exposure. Ozone exposure resulted in reduced walking of S. zeamais minimizing the chance of escape from exposure. The magnitude of this effect varied with population, but reduced walking activity will reduce the chances of escaping from lethal exposure during gas build-up and eventual exhaustion during the early and late stages of ozone fumigation, respectively, in stored product units. In contrast, the lack of acceptable standards of gas-tightness in stored product units, which is fairly common in several parts of the world (Mills 1983, Wohlgemuth and Harnisch 1986, Bell 2000), is likely to prevent reaching effective ozone concentrations in the storage unit impairing its efÞcacy. However, this problem with ozone fumigation is probably not as serious as with phosphine fumigation (Pimentel et al. 2012), because of the lower insect mobility observed under ozone exposure. In summary, we found negligible variability in ozone toxicity among S. zeamais populations and all populations were susceptible to ozone. In contrast, there was signiÞcant variation in walking activity among populations and walking activity was generally reduced by ozone exposure. Ozone toxicity and walking activity were uncorrelated, but insect body mass affected walking activity. The fact that ozone toxicity is correlated with respiration rate means that reduced ozone uptake could become a mechanism for resistance. Ozone seems a promising alternative fumigant with low short-term risk of resistance development because of the high susceptibility and low variability of response to this compound not only among populations of S. zeamais, but also of other species of stored product insects (e.g., Sousa et al., 2008). The ability of ozone to reduce insect walking activity means it will probably be effective against stored-product insect pests because it limits their ability to escape exposure. However, the potential use of ozone varies with different types of storages because this gas may cause extensive corrosion in metal silos for instance (Bonjur et al. 2011). As the types of storages may also affect both ozone use and insect behavior, Þeld studies under different types of storages are necessary to better

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assess the potential of ozone as an alternative fumigant for storage product protection.

Acknowledgments We are grateful for the funding and fellowships provided by the following Brazilian agencies: CAPES Foundation (Brazilian Ministry of Education), National Council for ScientiÞc and Technological Development (CNPq), and Minas Gerais State Foundation for Research Aid (FAPEMIG).

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