Differential susceptibilities of two closely-related stored product pests, the red flour beetle (Tribolium castaneum) and the confused flour beetle (Tribolium confusum), to five selected insecticides

Journal of Stored Products Research 84 (2019) 101524

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Differential susceptibilities of two closely-related stored product pests, the red flour beetle (Tribolium castaneum) and the confused flour beetle (Tribolium confusum), to five selected insecticides Jianxiu Yao a, Chengyu Chen a, b, Hua Wu a, c, Jing Chang a, d, Kristopher Silver a, James F. Campbell e, Frank H. Arthur e, Kun Yan Zhu a, * a

Department of Entomology, Kansas State University, Manhattan, KS, 66506, USA Department of Entomology, China Agricultural University, Beijing, 100094, China College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100, China d Department of Entomology, Inner Mongolia University, Inner Mongolia, 010019, China e United States Department of Agriculture, Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS, 66502, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 April 2019 Received in revised form 22 August 2019 Accepted 30 September 2019 Available online xxx

The red flour beetle (Tribolium castaneum Herbst) and the confused flour beetle (Tribolium confusum Jacquelin du Val) are among the most commonly encountered insects infesting stored food, but their susceptibilities to different insecticides often differ significantly, which complicates efforts to manage populations in milling and processing facilities. In this study, the susceptibilities of T. castaneum and T. confusum late-stage larvae to five selected insecticides, esfenvalerate, pyrethrins, dichlorvos, methoprene and pyriproxyfen, were assessed with and without synergists using topical applications. In fourday bioassays (without flour) with dichlorvos, esfenvalerate and pyrethrins, T. castaneum larvae were less susceptible (low larval mortality) to dichlorvos and esfenvalerate than T. confusum, whereas the reverse was true for treatment with pyrethrins. Pre-treatment with one of three synergists, piperonyl butoxide (PBO, cytochrome P450 monooxygenase inhibitor), S,S,S-tributyl phosphorotrithioate (DEF, esterase inhibitor), or diethyl maleate (DEM, glutathione S-transferase inhibitor), suggested involvement of esterases in the detoxification of dichlorvos and pyrethrins, and cytochrome P450 monooxygenases in the detoxification of esfenvalerate in both species. Interestingly, pre-treatment with some synergists increased the toxicity of insecticides in only one species: DEM and DEF increased the toxicity of dichlorvos to T. castaneum, whereas only DEF increased dichlorvos toxicity to T. confusum. In 28-day bioassays with larvae treated with each of two insect growth regulators (IGRs), methoprene and pyriproxifen, T. confusum was the more tolerant species. All T. castaneum died in either the larval or pupal stages with either IGR. In contrast, only pyriproxyfen caused complete mortality in T. confusum larvae, and even the highest dose of methoprene allowed nearly 70% of T. confusum larvae to pupate, and 4.5% of adults to emerge. Our results show that although these two species are closely related, they display very different susceptibilities to different insecticides, and different metabolic detoxification mechanisms may contribute to their differential insecticide susceptibilities. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Tribolium castaneum Tribolium confusum Synergism Insecticide susceptibility

1. Introduction The red flour beetle (Tribolium castaneum Herbst) and the confused flour beetle (Tribolium confusum Jacquelin du Val) are among the most commonly encountered insects infesting stored

* Corresponding author. E-mail address: [email protected] (K.Y. Zhu). https://doi.org/10.1016/j.jspr.2019.101524 0022-474X/© 2019 Elsevier Ltd. All rights reserved.

food (Good, 1937; Campbell and Runnion, 2003; Arthur and Campbell, 2008; Campbell et al., 2015). Both larvae and adults feed on a variety of products, including cereals, flour, cake and pancake mixes, spices, chocolate, powdered milk, and dry animal food. Their infestations cause significant economic cost to wheat and rice milling industries (Good, 1937; Arthur and Campbell, 2008; Kharel et al., 2014a; Campbell et al., 2015). To manage these species in mills, a wide range of chemical insecticide treatments are used. Fumigation with methyl bromide

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was widely used to treat mills, but its use has been phased out because it is an ozone depleting substance. Other fumigants such as sulfuryl fluoride or cylinderized phosphine are used as alternatives to methyl bromide but in general structural fumigations are applied less regularly than in the past (Subramanyam and Hagstrum, 2012). Instead, a range of other insecticides applied as aerosols or residual treatments are becoming favored alternatives, with increasing utilization of reduced risk materials, such as pyrethrins, pyrethroids, and insect growth regulators (IGRs) (Arthur and Fontenot, 2012; Arthur, 2015). Currently, five insecticides can be used as aerosol insecticides in the United States to control insect pests in milling and processing facilities: dichlorvos, esfenvalerate, pyrethrins, and the IGRs methoprene and pyriproxyfen. In some applications, the insecticides are mixed with one or more synergists that inhibit detoxification enzymes of insects (cytochrome P450 monooxygenases, esterases, and glutathione S-transferases) (Bodnaryk et al., 1984; Kharel et al., 2014a). For example, a commercial aerosol, Entech Fog 10, containing 1.0% pyrethrins, 2.0% piperonyl butoxide (PBO), 3.33% N-octyl bicycloheptane dicarboximide, and 93.7% other ingredients, has been used to control all life stages of both T. castaneum and T. confusum (Kharel et al., 2014a). T. castaneum and T. confusum are closely related species, having very similar morphological and biological characteristics (Baldwin and Fasulo, 2003). Previous studies have shown that despite their similarities, the two species have significantly different susceptibilities to organophosphates, pyrethriods, chlorfenapyr, diatomaceous earth and neonicotinoids (without synergist) (Arthur, 2000, 2008; Arthur and Fontenot, 2012; Athanassiou et al., 2013). For example, T. castaneum is more tolerant to organophosphates than T. confusum (Zettler, 1991), but the mechanisms responsible for these differences are still poorly understood. Both species were found together in all flour samples in mill (Hawkin et al., 2011). The significant differences in the susceptibility of these two species to aerosols can result in poor control efficacy in one species accompanied by high efficacy in the other species using the same insecticide (Arthur and Fontenot, 2012). As a result, this variability could pose big challenges for consistent control of the insect pests in stored grains. This study evaluated the differences in insecticide susceptibility between T. castaneum and T. confusum to the five aerosol insecticides, including an organophosphate (dichlorvos), a pyrethrin I and II mixture (pyrethrins), a pyrethroid (esfenvalerate), and two IGRs (methoprene and pyriproxyfen), that are commercially available for controlling insect pests in milling and processing facilities in the United States. In addition, three common synergists, PBO (cytochrome P450 monooxygenase inhibitor), S,S,S-tributyl phosphorotrithioate (DEF, esterase inhibitor), and diethyl maleate (DEM, glutathione S-transferase inhibitor), were also used to assess whether the differences in susceptibility to these insecticides between T. castaneum and T. confusum could be attributed to the activity of the common metabolic enzymes involved in insecticide detoxification. 2. Materials and methods 2.1. Insects T. castaneum and T. confusum colonies were obtained from the Center for Grain and Animal Health Research, USDA-ARS (Manhattan, KS, USA). All insects were reared on whole-wheat flour containing 5% (by weight) brewers' yeast at 28
C and 65% RH in growth chambers at Kansas State University (Manhattan, KS, USA). The adults were allowed to lay eggs in fresh flour for 24 h. After 24 h, all adults were removed and eggs were carefully reared with fresh flour provided weekly.

2.2. Insecticides and synergists The technical grade insecticides, esfenvalerate (purity 99.5%), pyrethrins (total pyrethrin I and II, 48.7%), methoprene (99.0%), pyriproxyfen (99.5%), and dichlorvos (99.5%), were purchased from Chem Service (West Chester, PA). Additional pyrethrins (total pyrethrin I and II, 79.85%) were also purchased from Sigma-Aldrich (St. Louis, MO). All the insecticides were dissolved in acetone at appropriate concentrations for our bioassays. The technical grade insecticide synergist DEF (97.7%) was purchased from Chem Service, whereas the other two synergists PBO (90%) and DEM (97%) were purchased from Sigma-Aldrich. All the synergists were prepared as 2% solutions in acetone for topical application. 2.3. Bioassay of conventional insecticides The larval susceptibility of each insect species to each of three conventional insecticides was determined by topical application. Serial dilutions of dichlorvos (0, 625, 1250, 2500, 5000, and 10000 mg/L), pyrethrins (0, 200, 400, 800, 1600, and 3200 mg/L), or esfenvalerate (0, 50, 100, 200, 400, and 800 mg/L) were made in acetone, and 0.5 ml of each insecticide solution was applied topically on the dorsal mesothorax of each larva by using a micro-applicator (Burkard Manufacturing Co., Ltd., Hertfordshire, UK) (SanadaMorimuraMatsumura, 2011). At least 60 larvae (16e18 day old) were used per dilution per insecticide. In total, more than 360 larvae of each species were used for each insecticide bioassay. The application of 0.5 ml acetone/per larva was used as a control. Mortality was recorded daily until four days after application. No food was provided. 2.4. Synergistic interaction with insecticides Three detoxification enzyme inhibitors, PBO, DEF, and DEM, were used alone and in combination with three insecticides (esfenvalerate, pyrethrins and dichlorvos) on both T. castaneum and T. confusum larvae. Sublethal concentrations (LC20-LC25) of esfenvalerate (50 mg/ml), pyrethrins (700 mg/ml), and dichlorvos (700 mg/ ml) were used in these experiments with or without 2% PBO, 2% DEM, or 2% DEF in acetone. Slightly different numbers (20e30) of the larvae (16e18 day old) were used in each of three replicates. The larvae were treated topically (dorsal mesothorax) with 0.5 ml 2% PBO, 2% DEM, 2% DEF or acetone only for 2 h, respectively. Subsequently, 0.5 ml of a sublethal concentration of each insecticide was topically applied to each larva. Beetle mortality was recorded daily until four days after treatment. 2.5. Bioassay of insect growth regulators (IGRs) Stock solutions of methoprene and pyriproxyfen (1000 mg/L) were prepared in acetone. In total, more than 180 larvae of each species were used in the IGR experiments and at least 60 larvae (17day old) were treated topically with 0.5 ml acetone containing 100 mg/ml or 1000 mg/ml of methoprene or pyriproxyfen. The larvae treated with acetone only were used as negative controls. After application, at least 20 insects were reared in one petri dish (90 mm diameter) containing 10 g whole wheat flour and 9 petri dishes were used in each IGR assay (at least 20 individuals/per dish and three dishes per treatment). Larval mortality, pupation rate, pupal mortality, and adult emergence rate were recorded weekly for four weeks. 2.6. Statistical analysis Probit analysis was used to calculate the median lethal dose

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(LD50) in bioassay of each conventional insecticide, and Chi-square tests were applied to ensure the goodness-of-fit of the model using SAS v9.2 (SAS Institute, Cary, NC). After the percentage data of mortality, pupation and emergence rate were arcsine square roottransformed, the data were examined to see whether they are normally distributed using Minitab software. Statistical analyses were performed for mean separation (at p  0.05) among different synergists with each conventional insecticide. The different susceptibilities among different IGR concentrations treatments were also analyzed using Tukey post-hoc multiple comparison test at p < 0.05 (Sgolastra et al., 2017). 3. Results and discussion 3.1. Differences in susceptibility between T. castaneum and T. confusum to three conventional insecticides The LD50 values of T. castaneum and T. confusum differed for pyrethrins and esfenvalerate, but not for dichlorvos based on the criterion whether their 95% confidence intervals (CIs) of the same insecticide between the two insect species overlap or not (Table 1). The LD50s for dichlorvos on T. castaneum and T. confusum larvae were 1849 and 1261 mg/g, respectively. Although T. confusum appeared slightly more susceptible to dichlorvos than T. castaneum, their 95% CIs of the LD50 values overlapped. Larvae of both Tribolium species were not highly susceptible to dichlorvos through topical application, and the LD50 was over 1000 mg/g. Even though dichlorvos is known for its broad spectrum activity and high efficacy in storage insect pest control, more than 50% field populations or strains of T. castaneum in the southern United States were resistant to dichlorvos (Halliday et al., 1988). Zettler (1991) also found 24% of T. confusum and 64% of T. castaneum strains were resistant to dichlorvos after screened 17 T. confusum and 28 T. castaneum populations in the United States. The low toxicities of dichlorvos to both species in the laboratory observed in this study might be interpreted as suggesting treatment with dichlorvos will be ineffective at providing effective control of these species in commercial environments. However, this conclusion is not supported by evidence from field settings where application of dichlorvos as an aerosol results in highly efficient control of both species in air-proof storage facilities. Interestingly, in these field studies, dichlorvos does not provide any good residual control (Subramanyam et al., 2014), which is consistent with our data and suggests that T. castaneum and T. confusum may be much more susceptible to aerosol forms of dichlorvos than liquid. Laboratory studies evaluating efficacy of insecticides should include similar treatment protocols to those used in the field. The LD50 values for T. castaneum and T. confusum larvae treated with pyrethrins were 142.6 and 852.5 mg/g, respectively, indicating T. castaneum larvae were approximately 6-fold more susceptible than T. confusum to pyrethrins. Zettler (1991) observed no resistance in these two Tribolium species to pyrethrins or resmethrin after screening 17 T. confusum and 28 T. castaneum field

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populations. However, pyrethrin-resistant strains of T. castaneum were reported in Australia in 1990 and this resistance ultimately resulted in failures to control T. castaneum (Collins, 1990). In addition, recovery from knockdown after pyrethroid application is a common phenomenon of both T. confusum and T. castaneum larvae (Arthur et al., 2014; Tucker et al., 2014), which was also noticeable in topical application of both pyrethrins and esfenvalerate in this study. At 2-weeks after pyrethrins application, recovery from knockdown increased with the presence of flour and the survival rate reached more than 70%. Between pyrethrins and esfenvalerate, the latter was more toxic to the two Tribolium species than the former (Table 1). The LD50 values for esfenvalerate on T. castaneum and T. confusum were 82.5 and 11.9 mg/g, respectively, indicating that T. confusum is approximately 7-fold more susceptible than T. castaneum to esfenvalerate. These results are similar to previous publications on the control efficacies of pyrethrins containing 2% PBO and esfenvalerate (trade name: Conquer®), against T. castaneum in grains storage facilities (Toews et al., 2010). In this study, mortality in esfenvalerate-treated dishes averaged greater than 90%, whereas the mortality in dishes treated with pyrethrins at the same rate averaged around 60%. 3.2. Role of detoxification enzymes in the differential susceptibilities of T. castaneum and T. confusum to insecticides Treatment with a sublethal dose of dichlorvos (700 mg/ml) resulted in 14.5% and 26.7% mortalities in T. castaneum and T. confusum larvae, respectively, after 96 h (Fig. 1A). Exposures of either T. castaneum or T. confusum larvae to 2% PBO for 2 h prior to insecticide exposures had no effect on the susceptibility of these insects to dichlorvos (Fig. 1B and C). In contrast, application of 2% DEF resulted in significantly increased mortality up to 75.97 ± 4.67% and 76.52 ± 5.64% in T. castaneum and T. confusum, respectively (Fig. 1B and C). These results suggest that esterases might be major detoxification enzymes in the larvae of both Tribolium species. However, pretreatment of larvae with 2% DEM increased mortality in T. castaneum, but not in T. confusum (Fig. 1B and C), suggesting that the higher tolerance of T. castaneum to dichlorvos might be due to the involvement of both esterases and glutathione S-transferases. Thus, whereas esterases are important detoxification enzymes in both species for dichlorvos, the action of glutathione Stransferases is important in the detoxification of dichlorvos only in T. castaneum. Pretreatment of larvae with each of the three synergists followed by treatment with pyrethrins or esfenvalerate also revealed apparent differences in the role of detoxification enzymes in insecticides toxicities between T. castaneum and T. confusum. Treatment of larvae with a sublethal dose (700 mg/ml) of pyrethrins resulted in 3.5-fold greater mortality in T. castaneum than T. confusum (Fig. 2A). When combined with DEF, mortality due to treatment with pyrethrins increased in both T. castaneum and T. confusum, whereas DEM had no effect on larval mortality (Fig. 2B and C). The combination of treatment with pyrethrins and PBO,

Table 1 Comparative susceptibilities of T. castaneum and T. confusum larvae to three conventional insecticides based on a four-day bioassay using topical application. Insecticide

Insect species

LD50 (95% CI) (mg/g)

Slope ± SE

c2 (P)

Body weight (mg/larva)

Dichlorvos

T. T. T. T. T. T.

1849.0 (1313.0e3132.0) 1261.0 (915.0e1943.0) 82.5 (69.0e99.6) 11.9 (9.2e17.1) 142.6 (94.7e191.8) 852.5 (650.7e1257.2)

0.44 ± 0.07 0.39 ± 0.068 0.81 ± 0.08 0.67 ± 0.08 0.48 ± 0.07 0.70 ± 0.09

5.24 19.0 7.98 11.5 6.63 4.88

3.31 ± 0.044 3.35 ± 0.015 3.17 ± 0.046 3.30 ± 0.049 3.13 ± 0.061 3.41 ± 0.022

Esfenvalerate Pyrethrins

castaneum confusum castaneum confusum castaneum confusum

(0.97) (0.77) (0.85) (0.98) (0.92) (0.98)

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Fig. 1. Comparative susceptibilities of T. castaneum and T. confusum larvae to dichlorvos (i.e., DDVP) synergized with each of three synergists (PBO, cytochrome P450 monooxygenase inhibitor; DEM, glutathione S-transferase inhibitor; and DEF, esterase inhibitor). (A) Mortality caused by topical application of 700 mg/ml dichlorvos in T. castaneum and T. confusum larvae after 96 h. (B) Mortality in T. castaneum larvae treated with each of three synergists followed by dichlorvos. (C) Mortality in T. confusum larvae treated with each of three synergists followed by dichlorvos. The results are the mean and standard errors from three experimental replicates, each with a group of 20e25 larvae.

Fig. 2. Comparative susceptibilities of T. castaneum and T. confusum larvae to pyrethrins synergized with each of three synergists (PBO, cytochrome P450 monooxygenase inhibitor; DEM, glutathione S-transferase inhibitor; and DEF, esterase inhibitor). (A) Mortality caused by topical application of 700 mg/mL pyrethrins in T. castaneum and T. confusum larvae after 96 h. (B) Mortality in T. castaneum larvae treated with each of three synergists followed by pyrethrins. (C) Mortality in T. confusum larvae treated with each of three synergists followed by pyrethrins. The results are the mean and standard errors from three experimental replicates, each with a group of 20e25 larvae.

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however, resulted in increased mortality in T. confusum, but not T. castaneum. These results suggest a role for esterases in pyrethrins detoxification in both species, as well as for cytochrome P450s in T. confusum only. Kharel et al. (2014b) reported that the larvae and adults of both T. castanuem and T. confusum were more tolerant to pyrethrins aerosol than eggs and pupae, and there was no significant difference in larval susceptibilities between two species after an aerosol application. However, we found that T. castaneum larvae are more susceptible to pyrethrins than T. confusum through topical application (Table 1 and Fig. 2). In addition, the synergism bioassays showed that esterases were involved in detoxification of pyrethrins in both species, but only P450s were involved in T. confusum. These results differ from those of Collins (1990) who reported that pyrethroid resistance in Tribolium species was almost completely suppressed by PBO. Our data suggest, however, that the higher tolerance of T. confusum than T. castaneum larvae to pyrethrins may be due to the involvement of both cytochrome P450s and esterases in the detoxification of pyrethrins in this species. A sublethal concentration (50 mg/ml) of esfenvalerate caused 2.7-fold higher mortality in T. confusum than T. castaneum (Fig. 3A), which was consistent with our initial experiments showing higher susceptibility of T. confusum adults (Table 1). Pretreatment with PBO significantly increased the toxicity of esfenvalerate to both T. castaneum and T. confusum, whereas pre-treatment with DEM had no effect (Fig. 3B and C). In contrast, pretreatment with DEF significantly increased the toxicity of esfenvalerate to T. confusum, but not T. castaneum. These findings suggest that whereas cytochrome P450s are important in the detoxification of esfenvalerate in both species, esterases are only active in the metabolism of esfenvalerate in T. confusum. Arthur and Gillenwater (1990) reported that PBO significantly enhanced the efficacy of esfenvalerate (0.25% esfenvalerate with 1.25% PBO) and almost killed 100% of T. castaneum after two rounds of applications. Here, when the larvae were exposed to esfenvalerate, T. castaneum larvae were more tolerant than T. confusum larvae and the addition of PBO increased T. castaneum larval mortality from 14.5 ± 1.6% to 29.7 ± 3.9%. However, both esterases and cytochrome P450s were involved in detoxification of esfenvalerate in T. confusum larvae. The mortality increased from 39.7 ± 2.4% under esfenvalerate alone to 66.3 ± 5.15 and 76.2 ± 4.2% with addition of PBO and DEF, respectively (Fig. 3C). 3.3. Comparison of the differences in susceptibility between larvae of T. castaneum and T. confusum to methoprene and pyriproxyfen

Fig. 3. Comparative susceptibilities of T. castaneum and T. confusum larvae to esfenvalerate (Esf) synergized with each of three synergists (PBO, cytochrome P450 monooxygenase inhibitor; DEM, glutathione S-transferase inhibitor; and DEF, esterase inhibitor). (A) Mortality caused by topical application of 50 mg/mL esfenvalerate in T. castaneum and T. confusum larvae after 96 h. (B) Mortality in T. castaneum larvae treated with each of three synergists followed by esfenvalerate. (C) Mortality in

Methoprene, hydroprene and pyriproxyfen are three IGRs that are regarded as reduced risk insecticides, and tested in laboratory and storage facilities. Hydroprene is the most volatile chemical among other IGR candidates, with a lack of residual persistence on surfaces, which compromises the control efficacy. The presence of the flour residual in storage facility apparently further compromised residual control (Arthur and Hoernemann, 2004; Arthur et al., 2009). Thus, hydroprene is no longer considered a good residual control aerosol. Pyriproxyfen showed much higher residual activity than hydroprene and greater control efficacy than hydroprene and methoprene against both Tribolium species (Arthur et al., 2009). In this study, T. castaneum and T. confusum were tested for their susceptibilities to methoprene and pyriproxyfen. Specifically, T. castaneum and T. confusum larvae were treated with 100 or T. confusum larvae treated with each of three synergists followed by esfenvalerate. The results are the mean and standard errors from three experimental replicates, each with a group of 20e25 larvae.

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Fig. 4. Comparative susceptibilities of T. castaneum and T. confusum larvae treated with two concentrations (100 mg/ml and 1000 mg/ml) of each of two IGRs (methoprene and pyriproxyfen) by topical application. (A) Mortality in T. castaneum larvae treated with methoprene; (B) Mortality in T. confusum larvae treated with methoprene; (C) Mortality in T. castaneum larvae treated with pyriproxyfen; and (D) Mortality in T. confusum larvae treated with pyriproxyfen. Each data point represents the mean ± standard error of four experimental replicates, each with 15e20 larvae. Larval mortality was monitored at day 3 and then weekly up to 28 days post-application. The asterisk “*” indicates significant difference at different time points.

1000 mg/ml methoprene or pyriproxyfen, and the insects were observed for larval mortality, pupation rate, pupal mortality, and adult emergence for 28 days (Figs. 4e7). Methoprene had no effect on larval mortality in either species for up to 28 days following treatment (Fig. 4A and B). Pyriproxyfen increased larval mortality in T. castaneum at 1000 mg/ml and in T. confusum at100 mg/ml, but not at 1000 mg/ml (Fig. 4C and D). The average higher mortalities of T. confusum larvae under pyriproxyfen (100 mg/ml) might be caused by experimental variations (e.g., handling of the larvae during insecticide bioassay) because the mortality reached close to 15% at 3-day post application which was zero or less than 5% under any other treatments. In contrast to our results with larval mortality, treatment with 100 mg/ml methoprene significantly decreased the pupation rate in T. castaneum, but not T. confusum, at day 7 (Fig. 5A and B; levels were similar to those of controls by day 14), 1000 mg/ml methoprene significantly reduced the rate of pupation in T. confusum and halted larval development in T. castaneum and completely preventing pupation. Pyriproxyfen at 100 mg/ml also slowed pupation in both species at day 7, though this effect was also temporary and beetles were pupating at levels similar to controls by day 14. At 1000 mg/ml, however, pyriproxyfen completely and nearly

completely (only 4% of larvae) eliminated pupation in T. castaneum and T. confusum, respectively (Fig. 5C and D). Pupal mortality was also evaluated with methoprene and pyriproxyfen in both species following treatment of larvae. Only 100 mg/ml methoprene or pyriproxyfen were examined in T. castaneum in these studies because the higher (1000 mg/ml) treatment level prevented pupation (Fig. 5A and C). Treatment of T. castaneum with methoprene or pyriproxyfen resulted in 100% mortality by day 28 (Fig. 6A and C). In contrast, 100 mg/ml methoprene had no effect on T. confusum (Figs. 5D & 6B). However, 100 and 1000 mg/ml pyriproxyfen and 1000 mg/ml methoprene both caused 100% pupal mortality in T. confusum (Fig. 6B and D). Furthermore, no adult emergence was observed in T. confusum with 1000 mg/ml methoprene or 100 mg/ml pyriproxyfen (Fig. 7A and B). One hundred percent of T. confusum pupae that survived treatment with 100 mg/ml methoprene, however, emerged as adults (Fig. 7A). No T. castaneum pupae survived treatment with either methoprene or pyriproxyfen, so adult emergence was not examined. Sutton et al. (2011) also reported that application of methoprene formulation caused more than 90% of larvae of both species permanently arrested at either larval or deformed pupal stage, with less than 2% of deformed emergence. Arthur (2010) found that T. castaneum was

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Fig. 5. Comparisons of the pupation rates after T. castaneum and T. confusum larvae were treated with two concentrations (100 mg/ml and 1000 mg/ml) of each of two IGRs (methoprene and pyriproxyfen). (A) T. castaneum larvae treated with methoprene; (B) T. confusum larvae treated with methoprene; (C) T. castaneum larvae treated with pyriproxyfen; and (D) T. confusum larvae treated with pyriproxyfen. Each data point represents the mean ± standard error of four experimental replicates, each with 15e20 larvae. Pupation rates were monitored day 3 and then weekly up to 28 days post-application. The asterisk “*” indicates significant difference at different time points.

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Fig. 6. Comparisons of pupal mortalities after T. castaneum and T. confusum larvae were treated with two concentrations (100 mg/ml and 1000 mg/ml) of each of two IGRs (methoprene and pyriproxyfen). (A) T. castaneum larvae treated with methoprene; (B) T. confusum larvae treated with methoprene; (C) T. castaneum larvae treated with pyriproxyfen; and (D) T. confusum larvae treated with pyriproxyfen. Each data point represents the mean ± standard error of four experimental replicates, each with 15e20 larvae. Pupal mortality was monitored at day 3 and then weekly up to 28 days post-application. The asterisk “*” indicates significant difference at different time points.

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4. Conclusion In this study, we found significant differences in susceptibility to the same insecticide between the two closely related Tribolium species. The different susceptibilities appeared to be at least in part due to the involvement of different detoxification enzymes between the two species. For instance, T. confusum pretreated with either PBO or DEF followed by pyrethrins significantly increased insect mortality as compared with those treated with pyrethrins only, whereas in T. castaneum, only PBO increased larval susceptibility to pyrethrins. These results indicated that both esterases and cytochrome P450s were involved in the detoxification of pyrethrins in T. confusum, but only cytochrome P450s were involved in T. castaneum. Furthermore, T. confusum was more tolerant to methoprene or pyriproxyfen than T. castaneum. Because these beetle species typically occur in the same commodities, field applications of insecticides would need to take these differences into consideration in order to maximize the control efficacy for the both species. Acknowledgement This research was supported by the U.S. Department of Agriculture (USDA) Agricultural Research Service Specific Cooperative Agreement (agreement number 58-3020-5-029) and the USDA National Institute of Food and Agriculture (NIFA) Methyl Bromide Transition program (grant number 2016-51102-25816). This manuscript is contribution No. 20-048-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA or by Kansas State University. USDA and Kansas State University are equal opportunity employers. Fig. 7. Comparisons of adult emergence after T. confusum larvae were treated with two concentrations (100 mg/ml and 1000 mg/ml) of each of two IGRs (methoprene and pyriproxyfen). (A) T. confusum treated with methoprene; (B) T. confusum treated with pyriproxyfen. Each data point represents the mean ± standard error of four experimental replicates, each with 15e20 larvae. The adult emergence of each treatment was recorded at day 3 and then weekly up to 28 days post-application. The asterisk “*” indicates significant difference at different time points.

more susceptible to both methoprene and pyriproxyfen than T. confusum. The topical application also indicated that the larvae of T. confusum were more tolerant to both methoprene and priproxyfen than T. castaneum because some T. confusum adults emerged after the application of each of the two IGRs (Fig. 7), whereas all T. castaneum were arrested or killed at larval stage. The applications of IGRs alone do not typically reach a desirable efficacy for managing stored product insect pests because of IGRs’ slow actions and differential susceptibilities among different pest species. Thus, binary combinations of insecticides with different modes of action have been widely tested in storage facilities (Arthur, 2010; Kharel et al., 2014a and b; Campbell et al., 2015; Tucker et al., 2015). For example, insects treated using 3% pyrethrins þ33.6% methoprene significantly decreased adult emergence rate (Sutton et al., 2011). The combination of pyrethrins þ pyriproxyfen (containing 0.7% pyrethrins, 5% PBO and 0.3% pyriproxyfen) exhibited higher control efficacy on T. confusum than a combination of pyrethrins þ methoprene (containing 1% pyrethrins, 5.0% PBO and 33.6% methoprene) (Scheff et al., 2018). These studies further showed that pyriproxyfen has higher toxicities to both species than methoprene and the binary (or triple) mixture elicits higher control efficacies than either one of the compounds alone.

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