Journal of Invertebrate Pathology 123 (2014) 1–5

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A coleopteran cadherin fragment synergizes toxicity of Bacillus thuringiensis toxins Cry3Aa, Cry3Bb, and Cry8Ca against lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae) Youngjin Park a, Gang Hua a, Milton D. Taylor a, Michael J. Adang a,b,⇑ a b

Department of Entomology, University of Georgia, Athens, GA 30602-2603, United States Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-2603, United States

a r t i c l e

i n f o

Article history: Received 3 June 2014 Accepted 28 August 2014 Available online 8 September 2014 Keywords: Bacillus thuringiensis Cry toxin Synergists Cadherin Lesser mealworm

a b s t r a c t The lesser mealworm, Alphitobius diaperinus, is a serious cosmopolitan pest of commercial poultry facilities because of its involvement in structural damage to poultry houses, reduction in feed conversion efficiency, and transfer of avian and human pathogens. Cry3Aa, Cry3Bb, and Cry8Ca insecticidal proteins of Bacillus thuringiensis are used to control coleopteran larvae. Cadherins localized in the midgut epithelium function as receptors for Cry toxins in lepidopteran, coleopteran, and dipteran insects. Previously, we demonstrated that the truncated cadherin (DvCad1) from Diabrotica virgifera virgifera, which consists of the C-terminal cadherin repeats (CR) 8–10 and expressed in Escherichia coli, enhanced Cry3Aa and Cry3Bb toxicity against several coleopteran species. Here we report that the DvCad1-CR8–10 enhances Cry3Aa, Cry3Bb, and Cry8Ca toxicity to lesser mealworm. Previously, by an enzyme linked immunosorbent microplate assay, we demonstrated that the DvCad1-CR8–10 binds activated-Cry3Aa (11.8 nM), Cry3Bb (1.4 nM), and now report that CR8–10 binds activated-Cry8Ca (5.7 nM) toxin. The extent of Cry toxins enhancement by DvCad1-CR8–10, which ranged from 3.30- to 5.93-fold, may have practical application for lesser mealworm control in preventing avian and human pathogen transfer in poultry facilities. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The lesser mealworm, also known as the darkling beetle, Alphitobius diaperinus (Coleoptera: Tenebrionidae), is a serious pest in the poultry house and grain storage facility (Ginden et al., 2009). The most serious problem posed by the darkling beetle is damage caused to the structure of poultry houses, where last instar larvae tunnel through soft materials looking for pupation sites (Axtell and Arends, 1990; Turner, 1986). Another problem is that they are important reservoirs of avian and human pathogens such as Salmonella typhimurium (Loeffler), Escherichia coli Lignieris, avian leukosis virus, and turkey enterovirus and rotavirus (Goodwin and Waltman, 1996; McAllister et al., 1995, 1994). In spite of high susceptibility of the beetles to several chemical insecticides, controlling A. diaperinus is very difficult owing to their Abbreviations: Bt, Bacillus thuringiensis; CR, cadherin repeat; IRM, insect resistance management. ⇑ Corresponding author at: Department of Entomology, University of Georgia, Athens, GA 30602-2603, United States. Fax: +1 706 542 2279. E-mail address: [email protected] (M.J. Adang). http://dx.doi.org/10.1016/j.jip.2014.08.008 0022-2011/Ó 2014 Elsevier Inc. All rights reserved.

hiding habit (Chernake-Leffer et al., 2007). Several registered carbaryls or pyrethroids have been used as premise treatments against A. diaperinus. This heavy reliance on insecticides for controlling A. diaperinus infestation has led to the development of resistance to these insecticides (Lambkin, 2005). Chemical pesticidal control of A. diaperinus generally provides unsatisfactory results when litter beetle populations are at outbreak levels (Geden and Steinkraus, 2003) and raises the importance of biological control (Ginden et al., 2009). The beetles have several natural enemies and pathogens including mites, protozoans, nematodes, and entomopathogenic fungi (Chernake-Leffer et al., 2007). The lesser mealworm was susceptible to the entomopathogenic nematodes Steinernematidae and Heterorhabditidae, but they showed only short-term control in field testing (Geden et al., 1987; Geden and Axtell, 1987). The entomopathogenic fungus, Beauveria bassiana, has been evaluated for control of A. diaperinus and the insect was susceptible from larval to adult stages (Geden and Steinkraus, 2003). However, B. bassiana treatments were not effective in providing long-term control because one and two treatments in one week showed only 8.1% and 57.2% mortality,

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Y. Park et al. / Journal of Invertebrate Pathology 123 (2014) 1–5

respectively, on larval stages of A. diaperinus (Geden and Steinkraus, 2003). The successful usage of spore-forming Bacillus thuringiensis (Bt) strains and their parasporal Cry proteins for pest insect control is well-documented (Bravo et al., 2011), prompting evaluation of Bt isolates with known coleopteran activities for toxicity to A. diaperinus larvae (Adang, 2011). The outcome from the study was the identification of Bt var. tenebrionis and Bt var. japonensis (Buibui strain) as having toxicity to A. diaperinus larvae. The activity of the Bt japonsensis Buibui strain and its Cry8Ca toxin against A. diaperinus was unexpected as the strain and toxin’s host range has been reported for coleopteran larvae from the family Scarabaeidae including chafer (Anomala cuprea) and Japanese beetle (Popillia japonica) (Hori et al., 1994; Suzuki et al., 1992). Cadherins have been identified as receptors of Cry toxins in insects (Pigott and Ellar, 2007) and peptide fragments of cadherins developed as synergists of Cry toxicity (Chen et al., 2007). Evidence suggests that the relationship between receptor and synergist function is the ability of cadherin peptides to induce Cry toxin oligomerization forming a pre-pore structure (Fabrick et al., 2009; Soberon et al., 2007). Cadherins are known receptors of Cry3Aa toxin in Tenebrio molitor (Fabrick et al., 2009) and Cry3Bb toxin in A. diaperinus (Hua et al., 2014). Critical evidence of in vivo receptor function in both studies was that silencing of cadherin expression through RNA interference (RNAi) resulted in highly reduced susceptibility to Cry3Aa or Cry3Bb. With respect to cadherin peptide synergy of coleopteran-active toxins, a fragment of DvCad1 cadherin from western corn rootworm, Diabrotica virgifera virgifera (Park et al., 2009) enhanced Cry3Aa and Cry3Bb toxicity to larvae of D. virgifera virgifera, the southern corn rootworm, Diabrotica undecimpunctata howardi, western corn rootworm, and Colorado potato beetle, Leptinotarsa decemlineata, from the family Chrysomelidae (Park et al., 2009). Similarly, a fragment from cadherin TmCad1 increased toxicity of Cry3Aa to larvae of three chrysomelid beetles that are important vegetable pests (Gao et al., 2011). Recently, Hua et al. (2014) showed that a fragment of cadherin AdCad1 from A. diaperinus larvae could overcome reduced Cry3Bb toxicity caused by suppression of AdCad1 mediated by RNAi . The goal of this study was to quantify the toxicities of Cry3Aa, Cry3Bb, and Cry8Ca toxins to A. diaperinus larvae and test the abilities of a cadherin repeat (CR) 8–10 fragment from D. virgifera virgifera cadherin (DvCad1) containing Cry3 toxin binding regions to enhance Cry3Aa, Cry3Bb, and Cry8Ca toxicity to A. diaperinus larvae. The toxicities of the tested Cry toxins and their toxicity enhancement by a DvCad1 cadherin fragment suggests the potential for A. diaperinus control with Bt Cry proteins will be possible.

2. Materials and methods 2.1. Preparation of Bt Cry proteins and DvCad1 CR-8–10 peptide B. thuringiensis var. tenebrionis producing Cry3Aa (Sekar et al., 1987) and B. thuringiensis var. japonensis Buibui producing Cry8Ca protein (Sato et al., 1994) were cultured and crystals purified as described (Park et al., 2009). Construction and expression of Cry3Bb (Donovan et al., 1992) in E. coli BL21 (DE3)/pRIL (Stratagene, La Jolla, CA) was described previously (Park et al., 2009). Truncated DvCad1-CR8–10 peptide (called CR8–10) was overexpressed and purified from inclusion bodies on a HiTrap Ni2+-chelating HP column (GE Healthcare, Piscataway, NJ) according to (Chen et al., 2007). The final Cry protein quantity was determined based on band density of Coomassie brilliant blue-stained 15% SDS–PAGE gels by gel image analysis (Alpha Innotech, San Leandro, CA) using bovine serum albumin (BSA) as a standard.

2.2. Binding affinity of Cry toxins to cadherin peptides The affinity of Cry8Ca binding to CR8–10 was measured using Cry-coated microtiter plates and an enzyme-linked immunosorbent assay (ELISA) as described previously for determining the affinity of Cry3Aa and Cry3Bb binding to CR8–10 (Park et al., 2009). Purified cadherin peptide was biotinylated, dialyzed against 200 mM NaCl, 20 mM Na2CO3 (pH 8.0) and stored in aliquots at 4 °C until needed for the binding assays. The microtiter plates (high-binding, 96-well Immulon 2HB plates; Thermo Fisher Scientific, Inc., Waltham, MA) were coated with chyomotrypsinactivated Cry8Ca at 1.0 lg/well in coating buffer (100 mM Na2CO3, pH 9.6) with or without a 1000-fold molar excess of unlabeled CR8–10. After washing and blocking, plates were incubated with horseradish peroxidase-conjugated streptavidin (SA-HRP; Pierce), and then incubated with HRP chromogenic substrate (1-Step Ultra TMB-ELISA, Thermo Fisher Scientific, Inc.). Color development was stopped by adding 3 M sulfuric acid, and absorbance was measured at 450 nm using a microplate reader (MDS Analytical Technologies, Sunnyvale, CA). The data was analyzed using Sigma Plot software Version 11.0 (SPSS Science, Chicago, IL). 2.3. Insects and Cry toxin diet-overlay bioassay An A. diaperinus colony was kept in containers and maintained at room temperature on pine shavings and chicken feed (Layena, Purina Mills) with apple slices to increase humidity. Details of A. diaperinus maintenance and diet surface bioassays conducted on a semi-solid chicken feed diet are reported (Hua et al., 2014). Cry crystals or crystals plus CR8–10 inclusions were serially diluted with sterile deionized water and then overlaid onto the diet surface and air-dried. One newly hatched larva was transferred into each well; the trays were sealed with perforated lids (C-D International, Pitman, NJ) and then covered with brown paper to provide a dark environment. Each bioassay was conducted with 16 larvae per replicate and two replicates per concentration. The trays were incubated at 28 °C for 3 days before larval mortality was scored. The concentrations of Cry3Aa and Cry8Ca crystals used for determining concentration response by A. diaperinus were 0.1, 1, 5, 10, 50, and 100 lg/cm2; for Cry3Bb the concentrations used were 0.1, 1, 5, 10, 50, 100, and 500 lg/cm2. The optimal ratio of Cry toxin to CR8–10 was determined by performing bioassays with a fixed amount of Cry toxins and an increasing amount of CR8–10 (i.e., 1:0, 1:1, 1:10, and 1:100 mass ratios of Cry toxin-cadherin peptide). We determined the effect of a constant 1:10 Cry:Cadherin peptide mass ratio on the LC50 values of each toxin using Cry3Aa crystals at 0.01, 0.1, 1, 5, 10, 50, and 100 lg/cm2 and Cry3Bb and Cry8Ca crystals at 0.01, 0.1, 1, 5, 10, and 100 lg/cm2. The LC50s of each experiment were calculated using the EPA Probit Analysis Program, version 1.5 (U.S. Environmental Protection Agency, Cincinnati, OH). The differences in the LC50s were considered significant if the 95% confidence limits did not overlap. Pairwise chisquare analysis was performed to analyze the effect of the synergist in the dose–response bioassays. Mortality data from the effect of the synergist in the dose–response and mass ratio bioassays were normalized using arcsine-square root (v) transformation and were analyzed using analysis of variance (ANOVA) with the significance level set at an a value of 0.05. When significant F values were detected, the means were separated using a Fisher protected least significant difference (LSD) test to compare the treatment means with the control and with each other. All calculations were performed using PROC GLM and PROC UNIVARIATE of the Statistical Analysis System (SAS 2002–2003, version 9.1; SAS Institute, Cary, NC). All data are presented in the original scale and asterisk or different letters above the error bars indicate a significant difference between means.

Y. Park et al. / Journal of Invertebrate Pathology 123 (2014) 1–5

3. Results 3.1. Toxicity of Cry toxins to A. diaperinus larvae The toxicity of Cry3Aa, Cry3Bb, or Cry8Ca crystals against A. diaperinus larvae was tested in bioassays and the dose-mortality curves are shown in Fig. 1 (Panels A, B and C). The LC50s values calculated by Probit analysis were 9.58 lg Cry3Aa/cm2 (4.54–18.17), 26.52 lg Cry3Bb/cm2 (15.79–44.80), and 7.71 lg Cry8Ca/cm2 (4.39–12.73) (Table 1). There is a significant difference between

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Cry8Ca and Cry3Bb, but not between Cry3Bb and Cry3Aa LC50s to A. diaperinus. Larval mortality in controls ranged between 0% and 3.12%. 3.2. DvCad-CR8–10 enhances toxicity of Cry toxins to A. diaperinus larvae Inclusions of CR8–10 produced in E. coli were composed of the expected 45-kDa peptide (data not shown). Purified inclusion bodies were tested for their ability to enhance toxicity of Cry3Aa,

Fig. 1. The DvCad1-CR8–10 fragment enhances Cry3Aa, Cry3Bb, and Cry8Ca toxicity to newly hatched A. diaperinus larvae. Larvae were exposed to diet treated with Cry3Aa (A and D), Cry3Bb (B and E), or Cry8Ca (C and F) crystals alone or Cry plus CR8–10 inclusions at 1:10 (A–C) or various toxin/peptide ratios (D–F). In panel D, the Cry3Aa concentration was 1.0 lg/cm2. In panel E, the Cry3Bb concentration was 5.0 lg/cm2. In panel F, the Cry8Ca concentration was 1.0 lg/cm2. Mortality was scored on day 3. Each data point represents the mean ± standard error of the results from a bioassay with 32 larvae per concentration. In panels A–C, an asterisk denotes a significant difference (chi-square analysis, P < 0.05) between larval mortality with Cry treatment and that with Cry plus CR8-10 treatment at the same toxin dose. In panels D–F, different letters above the error bars indicate significant differences between means (ANOVA, P = 0.0001).

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Table 1 Effect of CR8–10 peptide on the toxicity of Cry3Aa, Cry3Bb, and Cry8Ca to lesser mealworm, A. diaperinus. Treatment b

Cry3Aa Cry3Aa + CR8–10e Cry3Bbc Cry3Bb + CR8–10 Cry8Cad Cry8Ca + CR8–10

No. of larvae

LC50 (95% CL)a

Slope ± SE

v2

224 256 224 224 224 224

9.58 (4.54 ± 18.17) 1.81 (0.92 ± 3.13) 26.52 (15.79 ± 44.80) 8.82 (3.72 ± 20.98) 7.71 (4.39 ± 12.73) 1.30 (0.53 ± 2.53)

0.84 ± 0.14 1.01 ± 0.12 1.18 ± 0.16 0.73 ± 0.14 1.12 ± 0.18 0.89 ± 0.14

1.70 2.83 1.71 0.46 1.15 1.65

RTf 5.29 3.00 5.93

a

LC50s (with 95% confidence limits) expressed as micrograms of Cry toxin per square centimeter of diet surface for the bioassay. The LC50s for each experiment were calculated using the EPA Probit Analysis Program version 1.5. SE indicates standard error. b Cry3Aa crystals were purified from a sporulated B. thuringiensis subsp. tenebrionis culture. c Cry3Bb crystal inclusions were prepared from recombinant E. coli. d Cry8Ca crystals were purified from a sporulated B. thuringiensis subsp. japonensis Buibui culture. e CR8–10 crystal inclusions were isolated from E. coli expressing the CR8–10 region of cadherin. f RT means relative toxicity and was determined by dividing the LC50 of a Cry toxin alone by the LC50 of the Cry toxin plus CR8–10.

Fig. 2. SDS–PAGE of Cry8Ca and binding affinity of Cry8Ca for CR8–10 peptide. (Panel A) Solubilized Cry8Ca from crystals and after chymotrypsin treatment and purification (designated with XT). (Panel B) The binding of biotinylated CR8–10 to Cry8Ca was determined with an ELISA binding assay. Microtiter plates coated with chymotrypsin treated Cry8Ca were incubated with increasing concentrations of biotinylated-CR8–10 alone, or with 1000-fold excess peptide to determine specific binding. Each data point is the mean of the results from two experiments done in duplicate. Error bars depict standard deviations. Binding affinities (Kd) were calculated based on specifically bound biotinylated CR8–10 using a one-site saturation binding equation.

Cry3Bb, and Cry8Ca to newly hatched A. diperinus larvae. We applied 1 lg Cry3Aa/cm2, 5 lg Cry3Bb/cm2 and 1 lg Cry8Ca/cm2 to diet surface alone or with Cry:CR8–10 mass ratios of 1:1: 1:10 and 1:100. Maximal enhancement by CR8–10 occurred at a mass ratio between 1:10 and 1:100 for each Cry protein (Fig. 1D–F). The enhancement effect on LC50 values was determined using increasing amounts of each Cry while maintaining a Cry:Cadherin peptide mass ratio of 1:10. The LC50s of each toxin with CR8–10 were 1.81 (0.92–3.13) lg Cry3Aa/cm2, 8.82 (3.72–20.98) lg Cry3Bb/cm2, and 1.30 (0.53–2.53) lg Cry8Ca/cm2 (Table 1). Relative toxicity is calculated by dividing the Cry toxin plus CR8–10 LC50 by Cry toxin alone LC50. There was a 5.2-fold enhancement of Cry3Aa toxicity, 3.0-fold enhancement for Cry3Bb, and 5.9-fold for Cry8Ca (Table 1). 3.3. Activated Cry3Aa, Cry3Bb and Cry8Ca toxins bind the DvCad1CR8–10 peptide The affinity of DvCad-CR8–10 binding to Cry3Aa, Cry3Bb, and Cry8Ca were analyzed by an ELISA binding assay (Park et al., 2009). Chymotrypsinized Cry3Aa and C3Bb toxins used in binding assays were 55-kDa as shown previously (Park et al., 2009). The 130-kDa Cry8Ca protoxin was digested to 53-kDa by chymotrypsin

(Fig 2A). The values for CR8–10 binding to Cry3Aa and Cry3Bb reported in our previous study were Kd = 11.8 ± 0.4 nM for Cry3Aa and 1.4 ± 0.2 nM for Cry3Bb (Park et al., 2009). In this study, using a one-site saturation fit model, the calculated Kd CR8–10 peptide binding to Cry8Ca was 5.7 ± 1.1 (R2 = 0.98). The binding assay has shown that CR8–10 peptide binds to Cry8Ca with high affinity similar to how the cadherin peptide binds Cry3 toxins. 4. Discussion The quantitative toxicity of Bt Cry3Aa, Cry3Bb, and Cry8Ca to lesser mealworm, A. diaperinus, was established in this study. The LC50 values for Cry3Aa and Cry8Ca were similar at 9.58 lg/cm2 and 7.71 lg/cm2, respectively. Cry3Bb was slightly less toxic at a LC50 value of 26.52 lg/cm2. These values are comparable to the reported toxicity of Cry3Aa to another tenebrionid, T. molitor (LC50 = 21 lg/cm2) (Belfiore et al., 1994). While the toxicity of each Cry protein against A. diaperinus show the possibility of practical application of these toxins to control this pest in the poultry house, the LC50 values are relatively low compared to that of a highlyactive Cry protein against lepidopteran larvae. Possibly, the modes of action of Cry3Aa and Cry8Ca differ sufficiently that they may be deployed together in a single biopesticide.

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The potential and properties of cadherin peptides as Cry synergists has been expanded since their first description (Chen et al., 2007). High affinity binding of cadherin peptide to Cry toxin is correlated with the ability to synergize toxins (Chen et al., 2007; Fabrick et al., 2009; Hua et al., 2008; Park et al., 2009; Peng et al., 2010). With respect to cadherin fragments that synergize Cry toxicity to coleopteran larvae, we showed that DvCad1-CR8– 10 cadherin fragment binds Cry3Aa (Kd = 11.8 nM) and Cry3Bb (Kd = 1.4 nM). Here we show that CR8–10 cadherin fragment also binds Cry8Ca (Kd = 7.1 nM) with high affinity. In lepidopteran and coleopteran insects, it has been demonstrated the synergistic cadherin fragments induce oligomerization and pre-pore formation of Cry toxins (Fabrick et al., 2009; Soberon et al., 2007), a common step in the action of Cry toxins. Protection of Cry toxins from degradation by larval gut proteases has also been correlated with increased synergism of toxicity (Rahman et al., 2012). Recently, we (Hua et al., 2014) reported the ability of a cadherin fragment from AdCad1 to not only synergize Cry3Bb toxicity to lesser mealworm, but also overcome resistance mediated by dsRNA inhibition of AdCad1 expression in vivo. Truncated cadherin DvCad1 CR8–10 increased the Cry3Aa, Cry3Bb, or Cry8Ca toxicity against lesser mealworm 3.00- to 5.93-fold. This increase in larval toxicity is comparable to the 6.4-, 8.4-, and 13.1-fold enhancement of Cry3Bb toxicity to L. decemlineata, D. virgifera virgifera, and D. undecimpunctata howardi, respectively, reported by (Park et al., 2009), but slightly less that the maximal 15.3-fold increase in Cry3Aa toxicity to chrysomelid pests of vegetables (Gao et al., 2011). The range of Cry3 and Cry8 toxicity enhancement is considerably less than the 112-fold enhancement of Cry1Ac toxicity to Helicoverpa zea by the CR7–12 fragment of MsCad1 cadherin (Abdullah et al., 2009). Insect resistance management is a critical component for pest control by various Bt toxins including Bt crops. The conventional strategy for delaying pest resistance to Bt crops increases survival of susceptible insects with a refuge of host plants that do not produce Bt toxins and by combining Cry toxins that have distinct modes of action in Bt crops. Although factors such as feeding behavior and environmental conditions in poultry house can impact A. diaperinus survival, a peptide synergist of several active Cry toxins (Cry3Aa, Cry3Bb, and Cry8Ca) is encouraging for the potential of Bt to provide answers to the challenges of A. diaperinus management.

Acknowledgment This study was supported by U.S. NIFA award number 201065105-20590 to M.J. Adang (University of Georgia).

References Abdullah, M.A., Moussa, S., Taylor, M.D., Adang, M.J., 2009. Manduca sexta (Lepidoptera: Sphingidae) cadherin fragments function as synergists for Cry1A and Cry1C Bacillus thuringiensis toxins against noctuid moths Helicoverpa zea, Agrotis ipsilon and Spodoptera exigua. Pest Manag. Sci. 65, 1097–1103. Adang, M.J., 2011. Enhancement of Bacillus thuringiensis Cry toxicities to lesser mealworm Alphitobius diaperinus. U.S. Application No 13/056,380. Axtell, R.C., Arends, J.J., 1990. Ecology and management of arthropod pests of poultry. Annu. Rev. Entomol. 35, 101–126. Belfiore, C.J., Vadlamudi, R.K., Osman, Y.A., Bulla Jr., L.A., 1994. A specific binding protein from Tenebrio molitor for the insecticidal toxin of Bacillus thuringiensis subsp. tenebrionis. Biochem. Biophys. Res. Commun. 200, 359–364. Bravo, A., Likitvivatanavong, S., Gill, S.S., Soberon, M., 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 41, 423–431.

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Chen, J., Hua, G., Jurat-Fuentes, J.L., Abdullah, M.A., Adang, M.J., 2007. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin. Proc. Natl. Acad. Sci. USA 104, 13901–13906. Chernake-Leffer, A.M., Almeida, L.M., Sosa-Gómez, D.R., Anjos, A., Vogado, K.M., 2007. Populational fluctuation and spatial distribution of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) in a poultry house, Cascavel, Parana state. Brazil. Braz. J. Biol. 67, 209–213. Donovan, W.P., Rupar, M.J., Slaney, A.C., Malvar, T., Gawron-Burke, C., Johnson, T.B., 1992. Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species. Appl. Environ. Microbiol. 58, 3921– 3927. Fabrick, J., Oppert, C., Lorenzen, M.D., Morris, K., Oppert, B., Jurat-Fuentes, J.L., 2009. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J. Biol. Chem. 284, 18401–18410. Gao, Y., Jurat-Fuentes, J.L., Oppert, B., Fabrick, J.A., Liu, C., Gao, J., Lei, Z., 2011. Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment. Pest. Manag. Sci. 67, 1076–1081. Geden, C.J., Arends, J.J., Axtell, R.C., 1987. Field trials of Steinernema feltiae (Nematoda: Steinernematidae) for control of Alphitobius diaperinus (Coleoptera: Tenebrionidae) in commercial broiler and turkey houses. J. Econ. Entomol. 80, 136–141. Geden, C.J., Axtell, R.C., 1987. Factors affecting climbing and tunneling behavior of the lesser mealworm (Coleoptera: Tenebrionidae). J. Econ. Entomol. 80, 1197– 1204. Geden, C.J., Steinkraus, D.C., 2003. Evaluation of three formulations of Beauveria bassiana for control of lesser mealworm and hide beetle in Georgia poultry houses. J. Econ. Entomol. 96, 1602–1607. Ginden, G., Glazer, I., Mishoutchenko, A., Samish, M., 2009. Entomopathogenic fungi as a potential control agent against the lesser mealworm, Alphitobius diaperinus in broiler houses. BioControl 54, 549–558. Goodwin, M.A., Waltman, W.D., 1996. Transmission of Eimeria, viruses, and bacteria to chicks: Darkling beetles (Alphitobius diaperinus) as vectors of pathogens. J. Appl. Poult. Res. 5, 51–55. Hori, H., Suzuki, N., Ogiwara, K., Himejima, M., Indrasith, L.S., Minami, M., Asano, S., Sato, R., Ohba, M., Iwahana, H., 1994. Characterization of larvicidal toxin protein from Bacillus thuringiensis serovar japonensis strain Buibui specific for scarabaeid beetles. J. Appl. Bacteriol. 76, 307–313. Hua, G., Park, Y., Adang, M.J., 2014. Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis. Insect Biochem. Mol. Biol. 45, 11–17. Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101–5110. Lambkin, T.A., 2005. Baseline responses of adult Alphitobius diaperinus (Coleoptera: Tenebrionidae) to fenitrothion and susceptibility status of populations in Queensland and New South Wales. Australia. J. Econ. Entomol. 98, 938–942. McAllister, J.C., Steelman, C.D., Newberry, L.A., Skeeles, J.K., 1995. Isolation of infectious bursal disease virus from the lesser mealworm, Alphitobius diaperinus (Panzer). Poult. Sci. 74, 45–49. McAllister, J.C., Steelman, C.D., Skeeles, J.K., 1994. Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae). J. Med. Entomol. 31, 369–372. Park, Y., Abdullah, M.A., Taylor, M.D., Rahman, K., Adang, M.J., 2009. Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to coleopteran larvae by a toxin-binding fragment of an insect cadherin. Appl. Environ. Microbiol. 75, 3086–3092. Peng, D., Xu, X., Ruan, L., Yu, Z., Sun, M., 2010. Enhancing Cry1Ac toxicity by expression of the Helicoverpa armigera cadherin fragment in Bacillus thuringiensis. Res. Microbiol. 161, 383–389. Pigott, C.R., Ellar, D.J., 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Molec. Biol. Rev. 71, 255–281. Rahman, K., Abdullah, M.A., Ambati, S., Taylor, M.D., Adang, M.J., 2012. Differential protection of Cry1Fa toxin by cadherin orthologs against Spodoptera frugiperda larval gut proteases correlates with increased synergism. Appl. Environ. Microbiol. 78, 354–362. Sato, R., Takeuchi, K., Ogiwara, K., Minami, M., Kaji, Y., Suzuki, N., Hori, H., Asano, S., Ohba, M., Iwahana, H., 1994. Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects. Curr. Microbiol. 28, 15–19. Sekar, V., Thompson, D.V., Maroney, M.J., Bookland, R.G., Adang, M.J., 1987. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc. Natl. Acad. Sci. U.S.A. 84, 7036– 7040. Soberon, M., Pardo-Lopez, L., Lopez, I., Gomez, I., Tabashnik, B.E., Bravo, A., 2007. Engineering modified Bt toxins to counter insect resistance. Science 318, 1640– 1642. Suzuki, N., Hori, H., Ogiwara, K., Asano, S., Sato, r., Sato, r., Ohba, M., Iwahana, H., 1992. Insecticidal spectrum of a novel isolate of Bacillus thuringiensis Serovar japonensis. Biol. Control 2, 138–142. Turner, E.C., 1986. Structural and litter pests. Poult. Sci. 65, 644–648.