Aerosol insecticides are being used in flour mill pest management programs, but there is limited information on their efficacy on different insect life stages. In this study, we evaluated the efficacy of synergized pyrethrin applied as an aerosol against eggs, larvae, pupae, and adults of the red flour beetle, Tribolium castaneum (Herbst), and the confused flour beetle, Tribolium confusum Jacquelin du Val. Effects of direct and indirect exposure were evaluated by exposing each life stage to the aerosol and then transferring to untreated flour, transferring untreated insects to treated flour, or exposing both the insects and the flour to the aerosol. The aerosol produced >88% mortality of both species and all life stages when insects were directly treated and transferred to either treated or untreated flour. Mortality was significantly reduced when insects were either treated together with flour or untreated insects were transferred to treated flour (indirect exposure to the aerosol). Larvae and adults of both species were more tolerant compared with eggs and pupae. Recovery of moribund adults in the indirect exposure treatments was greater compared with recovery of moribund insects in the direct exposure treatments. Good sanitation before aerosol application could facilitate direct exposure of insects and thus increase aerosol efficacy inside flour mills.
Flour milling facilities typically consist of different components such as grain receiving and storage, precleaners or cleaners, milling and processing arenas, and packaging and shipping warehouses (Williams and Rosentrater 2007). The constant availability of whole grains and milled products provides excellent feeding and oviposition sites for many stored-product insects. The red flour beetle, Tribolium castaneum (Herbst), and the confused flour beetle, Tribolium confusum Jacquelin du Val, are two of the most important pests in the United States associated with flour mills (Good 1937, Campbell and Runnion 2003, Arthur and Campbell 2008, Toews et al. 2009). These species can successfully exploit food patches of varying size and quality (Campbell and Hagstrum 2002, Campbell et al. 2010, Ming and Cheng 2012) that are generated during various milling operations, such as grinding, milling, sifting, and moving of processed food products. In a flour mill, T. castaneum and T. confusum can cause economic impact by feeding and through contamination and damage of finished products, which may result in return and rejection of goods, loss of customer goodwill, and failure to meet regulatory requirements for adulterated food (Campbell et al. 2002).
In the United States, flour milling facilities use whole structure treatments such as heat treatment and fumigation with sulfuryl fluoride or cylinderized phosphine, or more targeted treatments such as aerosols and surface treatments with residual insecticides to control insect pests. Heat treatments may not be suitable in older facilities and can be cost-intensive, whereas use of sulfuryl fluoride and cylinderized phosphine require facility shutdown and regulatory requirements of developing and complying with a fumigation management plan. Phosphine is corrosive to metals, and therefore is not a fumigant of choice for use in flour mills (Bond et al. 1984). Hence, there is increasing interest in targeted treatments in general and aerosols in particular (Campbell et al. 2010).
Aerosol application is a method of applying a liquid insecticide in the form of fog or mist consisting of droplet sizes ranging from 5 to 50 μm (Peckman and Arthur 2006), which are deposited on mill surfaces. The disadvantage associated with the aerosols is that they may not distribute into areas that are obstructed by equipment or into sites that harbor hidden infestations in spillage and accumulated food dust. Nonetheless, the exposed food materials may have some residual effects and insects could be indirectly exposed through treated foods (Arthur 2010). However, there are few published data regarding efficacy of aerosols against insects using these different exposure methods.
Currently, insecticides, such as dichlorvos, synergized pyrethrin, pyrethroids (Arthur and Campbell 2008), and the insect growth regulators methoprene (Sutton et al. 2011) and pyriproxyfen (Arthur et al. 2010), are being used as aerosol insecticides. Pyrethrin is of special interest because of its low mammalian toxicity and efficacy against a broad range of stored-product insects. Commercial formulations used in milling and processing facilities often contain the synergist piperonyl butoxide, which enhances the biological activity of pyrethrins (Brooke 1958).
Most published studies on the susceptibility of flour beetles to pyrethrin have been conducted against adults, but in field conditions, adults may comprise only ≈5–10% of the total population (Toews et al. 2005; Campbell et al. 2010a,b; Arthur et al. 2013). Several studies examined direct and indirect effects of aerosols on life stages of flour beetles in food patches in small-scale studies (Toews et al. 2010), but there are no studies evaluating the susceptibility of different individual life stages of T. castaneum and T. confusum to pyrethrin. Therefore, the objectives of this study were to determine: 1) efficacy of synergized pyrethrin aerosol at different direct and indirect exposure scenarios that could be encountered in flour mills, 2) differences in susceptibility between T. castaneum and T. confusum, and 3) differences in susceptibility among different life stages of T. castaneum and T. confusum.
Materials and Methods
The colonies of T. castaneum and T. confusum were obtained from the U.S. Department of Agriculture–Agricultural Research Service (USDA–ARS) Center for Grain and Animal Health Research (CGAHR), Manhattan, KS, and reared on a diet of wheat flour (with 5% by weight of brewer's yeast) in a laboratory incubator set at 27°C, 70% relative humidity (RH), and in constant darkness. These colonies have been maintained at CGAHR for >30 yr and are considered to be pesticide-susceptible. At these environmental conditions described above, egg-to-adult development takes ≈6 wk. The life stages for each of the two species used for the study were 2-d-old eggs, late-stage larvae (4 wk old), pupae (5 wk old), and 1-wk-old adults. The aerosol study was conducted in two experimental sheds (6 m by 2.9 m by 2.6 m; 45.24 m3) in Manhattan, KS. Environmental conditions inside the shed were maintained at 27°C and 70% RH, and monitored with HOBO data loggers (Onset Computer Corporation, Bourne, MA).
The insecticide used in the test, Entech Fog 10 (Environmental Protection Agency Reg. No. 40391-10), manufactured by Entech Systems Corporation, Kenner, LA, comprises 1.0% pyrethrin, 2.0% piperonyl butoxide (technical), 3.33% N-octyl bicycloheptane dicarboximide, and 93.67% other ingredients. One of the sheds was used for exposing insects to the insecticide and other was used as the untreated control.
An exposure arena consisted of 9-cm-diameter glass petri dishes with a surface area of ≈63 cm2. Twenty individuals of a particular life stage for each species were prepared in a petri dish with or without 5 g of wheat flour. Petri dishes containing only flour but no insects were also prepared. Altogether, there were 40 dishes per shed: 16 dishes containing only a life stage for T. castaneum or T. confusum, 16 dishes containing only flour, and 8 dishes with a life stage of an insect and flour together. All 40 dishes were placed randomly in a 10 by 4 grid marked inside each shed (Fig. 1).
The insecticide was delivered using a handheld aerosol applicator, Fogmaster Jr 5330 (The Fogmaster Corporation, Deerfield Beach, FL). The output of insecticide for this sprayer at the setting on the dial was calibrated to be ≈21 ml/min. The label rate for the insecticide is 29.56 ml of the formulation/28 m3; hence, the amount of insecticide needed for the sheds was 47 ml. The time required to deliver the insecticide was calculated as 2 min and 16 s. The aerosol was applied by standing at a distance of 2.8 m away from the grid and by pivoting the Fogmaster Jr 5330 slowly from side to side at ≈3 m above the floor. The insects and flour dishes were held in both treated and control sheds for 2 h after the aerosol application. Control sheds did not receive any aerosol application, but the treatments and dishes were handled similar to that described for aerosol-treated sheds.
After the 2 h of exposure, the dishes were removed from the sheds. To mimic different direct and indirect ways that insects could be exposed to aerosol insecticides within a flour mill, the following treatments were created for each life stage: insect treated with aerosol and transferred to treated flour (TI + TF), treated insect transferred to untreated flour (TI + UF), insect untreated and transferred to aerosol treated flour (UI + TF), untreated insect transferred to untreated flour (UI + UF; control treatment), insects and flour combined and both treated together (TIF), and insects and flour combined and both untreated (UIF; control treatment). After these transfers were completed, all treatment arenas were placed in an incubator set at 27°C and 70% RH. This entire process was repeated (replicated) six times at weekly intervals.
For each replicate, adults of each species were assessed at 2, 5, 8, and 15 d after exposure to the aerosols, and classified as follows: live (morphologically normal adults), moribund adults (which were on their backs and capable of reflex movement), and dead (unable to move when prodded with a probe). Untreated controls were classified in the same manner. Exposed eggs, larvae, and pupae were held in the incubator for 40, 28, and 21 d, respectively, to assess the number of dead individuals out of the total exposed. To observe the effects of different treatments and to characterize differences between the two Tribolium species, total affected individuals were calculated for all developmental stages in each treatment. For adults, affected individuals comprised the total dead and moribund individuals, whereas for immature stages total affected comprised of all the dead, moribund, and developmentally arrested individuals that failed to complete development normally to the adult stage. The percentage of insects dead in the immature stage during the holding time was corrected for control mortality using Abbott's (1925) formula to compare susceptibility of stages to the aerosol.
Statistical analyses were conducted with the General Linear Models procedure of the Statistical Analysis System (SAS Institute 2002–2008, SAS Institute, Cary, NC). The number of affected insects (dead, moribund, and arrested) in each treatment was converted to percentage values, which were transformed to angular values (Zar 2010) before subjecting data to statistical analysis. However, the data presented in figures and texts are the untransformed values. The total affected data by species and exposure methods were subjected to a two-way analysis of variance (ANOVA) to determine differences in affected individuals produced for each life stage. After two-way ANOVA, one-way ANOVAs were performed to determine differences among exposure method and species. Means were separated using the Waller–Duncan k-ratio t-test (SAS Institute 2002–2008). Similarly, for immature stages, the mortality data by exposure methods and stages were analyzed by two-way ANOVA and subsequently by one-way ANOVA to determine the susceptibility among the stages. Differences among means were considered significant at α = 0.05 level.
The two-way ANOVA showed affected individuals were significantly different among exposure methods for adults, pupae, larvae, and eggs (F = 179.4, F = 137.5, F = 112.9, F = 149.1, respectively; df = 5, 60; P < 0.01 for all). The two species were not different in any of the developmental stages: adults, pupae, larvae, and eggs (F = 0.8, P = 0.38; F = 0.01, P = 1.0; F = 0.41, P = 0.5; F = 1.1, P = 0.29, respectively; df = 1, 60 for all). There was no interaction between exposure method and species for the total affected individuals produced for developmental stage: adult stage at 15 d, pupae at 21 d, larvae at 28 d, and eggs at 35 d postexposure to aerosol (F = 1.0, P = 0.40; F = 0.3, P = 0.9; F = 0.25, P = 0.93; F = 0.6, P = 0.72, respectively; df = 5, 60 for all). The subsequent one-way ANOVA showed significant differences among exposure methods for both T. castaneum adults, pupae, larvae, and eggs (F = 61.7, F = 87.2, F = 59.3, F = 73.0, respectively), and T. confusum adults, pupae, larvae, and eggs (F = 101.1, F = 103.0, F = 73.1, F = 127.0, respectively, df = 5, 30 and P < 0.01 for all).
Exposing insects directly to the aerosol resulted in a significantly higher number of affected individuals, whether or not they were transferred to treated or untreated flour, compared with exposing insects with flour; transferring untreated insects to petri dishes containing treated flour reduced the total affected individuals in both the species (Table 1). However, for adult stages, total higher number of affected individuals was observed in both species when insects and flour were exposed together. This may be because of a higher number of moribund individuals in those treatments (Fig. 2 and Fig. 3).
When adults were exposed without food and transferred to treated or untreated flour postexposure, no normal live beetles were found at 2, 5, 8, and 15 d, in contrast to the indirect exposure treatments (insects exposed to aerosol with flour or untreated insects transferred to treated flour). In the indirect exposures, many adults were moribund after the initial exposures but were able to recover with time (Fig. 2 and Fig. 3). Recovery was ≈15% in T. castaneum and 8% in T. confusum exposed with flour. Recovery of adults that were untreated but placed on the flour that had been exposed to the aerosol was ≈43% for T. castaneum and 25% for T. confusum (Fig. 2 and Fig. 3).
In addition, the susceptibility of the immature stages was compared across the exposure methods. For T. castaneum, the mean ± SE mortality for controls UI + UF (untreated insect transferred to untreated flour) and UIF (insects and flour combined and both untreated) were 5.0 ± 1.8% and 2.5 ± 1.28%, respectively, for larvae; 4.2 ± 2.4% and 5.8 ± 2.4%, respectively, for pupae; and 28.3 ± 2.5% and 16.7 ± 1.0%, respectively, for eggs. Similarly, for T. confusum, the mean ± SE mortality for controls UI + UF and UFI were 1.7 ± 1.0% for both controls in larvae; 2.5 ± 1.7% and 4.2 ± 0.8%, respectively, for pupae; and 20.8 ± 4.2 and 19.2 ± 3.5%, respectively, for eggs. Therefore, the mortality data were corrected for control mortality (Abbott 1925).
For T. castaneum, the two-way ANOVA showed no interaction between stages and exposure methods for the mortality produced among the immature stages (F = 1.2; df = 6, 60; P = 0.29), whereas the stages and exposure methods both were significant (F = 6.3; df = 2, 60; P < 0.01; and F = 16.6; df = 3, 60; P < 0.01; respectively). The one-way ANOVA for stages showed no differences among the stages when treated insects were transferred to treated flour, treated insects were transferred to untreated flour, insects were treated together with flour, or insects were transferred to treated flour (F = 1.0, P = 0.39; F = 2.9, P = 0.1; F = 0.2, P = 0.83; F = 2.9, P = 0.1, respectively, df = 2, 15 for all) (Table 2).
For T. confusum, the two-way ANOVA by stages and exposure methods showed significant interaction between the stages and exposure methods in terms of mortality produced among the immature stages (F = 2.8, df = 6, 60, P = 0.02). Also, the stages and exposure methods were significant (F = 21.2, df = 2, 60, P < 0.01; F = 38.9, df = 3, 60, P < 0.01, respectively). The one way ANOVA for stages showed no differences among the stages when treated insects were transferred to treated flour and treated insects were transferred to untreated flour (F = 2.2, P = 0.15; F = 1.7, P = 0.22, respectively; df = 2, 15 for both). However, when insects and flour were exposed together and when untreated insects were transferred to treated flour, larvae were the least susceptible stage followed by pupae (F = 6.7 and F = 10.0, respectively; df = 2, 15; P < 0.01 for both), but eggs were the most susceptible of all the developmental stages (Table 2).
Our results indicated that direct exposure to pyrethrin aerosol was effective on all life stages of T. castaneum and T. confusum. Similar results have been reported by Toews et al. (2010), who showed mortality of generally >80% on all developmental stages of T. castaneum exposed to pyrethrin aerosol. Also, our study showed reduced efficacy when various developmental stages were either treated together with flour or untreated insects were transferred to treated flour (indirect exposure to aerosol). In addition, moribund adults initially observed in indirect exposure treatments were able to recover as healthy adults over time, which is consistent with other reports showing greatly reduced efficacy of pyrethrin when adult flour beetles were provided with a food source after they were exposed (Arthur and Campbell 2008).
These results emphasize the importance of hygienic and sanitary measures in milling facilities in conjunction with insecticide applications, as the presence of food materials limits the exposure to insecticides, enables insects to remove insecticide particles from body surfaces, and provides nutritional support for recovery (Arthur 2000). In addition, cleaning procedures followed by the mill sanitarians may influence insect movement through increased searching for food patches, which potentially increases their chances of contacting the applied aerosols (Roesli et al. 2003). However, milling facilities have considerable structural complexity because of different types of processing equipment and are continuously generating spillage and food residues. Hence, populations could develop in hidden areas where food residues accumulate and would have reduced exposure to the aerosol. Our study showed a sharp increase in recovery of moribund adults after 5 d when these adults were indirectly exposed to pyrethrin, which may be because of little residual activity of synergized pyrethrin (Arthur 2010). This indicates that the moribund insects should be removed from the facilities shortly after aerosol treatment.
Furthermore, we observed no differences between the two exposed Tribolium species to pyrethrin aerosol, which is different from previous published studies using the same laboratory colonies and exposure method. Previous results reported greater susceptibility of T. castaneum compared with T. confusum for synergized pyrethrin (Arthur 2008) and the combination of pyrethrin and methoprene (Sutton et al. 2011). The discrepancy in the species susceptibility observed in those studies may be due to several reasons. Arthur (2008) held the insects overnight in the facility after they were exposed to pyrethrin, and removed them the next day. In the current test, we removed the insects after 2 h. The longer holding time in the treated facility may have contributed for the increased susceptibility of T. castaneum. Similarly, Sutton et al. (2011) conducted tests by exposing larvae of both species on concrete dishes containing wheat flour that had been previously exposed to a combination of 1% pyrethrin + methoprene or 3% pyrethrin + methoprene. For 0–16 wk postexposure, larvae were added to the treated flour using different dishes each time. They showed generally higher number of adult emergence of T. confusum larvae than that of T. castaneum. However, adult emergence of T. confusum larvae was reduced at the 3% pyrethrin rate compared with the 1% rate. Residual control is generally assumed to be due to the growth regulator methoprene added to pyrethrin. Nevertheless, the results of that study indicated some residual effect of pyrethrin or an additive effect between the two components that may have affected the relative susceptibility of the two species. Our study was conducted with the formulation containing only 1% pyrethrin.
Our study showed that larvae were more tolerant than the immobile pupae and eggs stages in T. confusum. Even though the susceptibility of the stages was not significant in T. castaneum, there was a clear trend that the eggs were the most susceptible stage followed by the pupae. Furthermore, fewer affected individuals were produced when the adult stage was exposed to aerosol and adults exhibited high recovery, which also suggests that adults were less susceptible to some extent. This result is different from previous studies evaluating fumigant efficacy because they cited eggs and pupae as the most tolerant stages of stored-product insects to phosphine: phosphine (Rajendran 1992, Pike 1994, Bell 2000), carbonyl sulfide (Zettler et al. 1997, Zettler and Arthur 2000), sulfuryl fluoride (Bell and Savvidou 1999, Hartzer et al. 2010, Tsai et al. 2011), and propylene oxide (Isikber et al. 2004). Greater tolerance of eggs and pupae to insecticides has often been attributed to their lower metabolic activity. In our study, larvae were more tolerant than other immature stages, especially when they were indirectly exposed to the aerosol (when treated with flour and when untreated insects were transferred to treated flour). It is plausible that during aerosol treatment the larvae burrowed into the flour (5 g flour available per dish), thereby reducing their exposure to the aerosol. In contrast, fumigants would penetrate through the flour and come into contact with the larvae. We assumed the pyrethrin would have limited penetration into the flour, and therefore reduced contact with the larvae. In conclusion, this study showed that synergized pyrethrin aerosol was only effective when all life stages of T. castaneum and T. confusum were directly exposed. The indirect exposure was much less effective, suggesting that the presence of food material may compromise aerosol efficacy by providing harborage sites where infestations can persist and insects can escape the exposure (Campbell and Hagstrum 2002, Toews et al. 2010). Integrating sanitation with aerosols could facilitate more direct exposure to aerosols by reducing the available food material, and therefore increase the effectiveness of the aerosol treatment in the flour mills.
We would like to express our sincere thanks to Brian Barnett, Rich Hammel, and Kris Hartzer (USDA–ARS Center for Grain and Animal Health Research, Manhattan, KS) for their technical assistance. We appreciate Entech Corporation providing the insecticide used in this study. This research was partially supported by the U.S. Department of Agriculture, National Institute of Food and Agriculture (NIFA) Methyl Bromide Transitions program (grant 2010-51102-21660). Insect voucher specimens were deposited at the Kansas State University Museum of Entomological and Prairie Arthropod Research under the voucher no. 226. This paper is contribution number 14-029-J of the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS.