Dr. Brenda Oppert Research Molecular Biologist Biological Research Unit Grain Marketing & Production Research Center 1515 College Ave. Manhattan, KS 66502 Voice: (785) 776-2780 Fax: (785) 537-5584 brenda.oppert@ars.usda.gov www.ars.usda.gov/npa/gmprc/bru/oppert

Dr. Oppert received B.S. and M.S. degrees in biology from the University of Texas at El Paso. She received a Ph.D. in the area of protein biochemistry from Kansas State University in 1991. Since that time, Dr. Oppert has worked at GMPRC in the area of insect gut biochemistry.

Our research mission is to find better, safer ways to control insect pests found in and around food storage areas, including grain storage and processing facilities and warehouses. Often these pests are even smaller than field pests and provide challenges when trying to understand basic digestive processes and physiological responses. Therefore, we have adopted a number of proteomics and genomics techniques, including 2-D gel analysis, multiphase chromatography, protein identification by sequencing, peptide fingerprinting and mass spec, subgenome characterization, and gene expression. Specific research areas include the use of proteomics and genomics to understand insect responses to digestive proteinase inhibitors and microbial toxins. Comparisons of differential responses in insects have been particularly enlightening. This research will lead to improved control methods for stored product pests.

Discovery of more effective insect control proteins. Proteins that negatively impact insect growth and development are useful in insect control, because genes encoding insecticidal proteins can be expressed in plants to enhance host plant resistance. Proteinase inhibitors are good candidates because they disrupt insect digestion. However, insects can compensate for proteinase inhibitors by increasing the expression of digestive proteinases and/or expressing inhibitor-insensitive proteinases. Dr. Oppert was part of a team that discovered that combinations of digestive inhibitors targeting different proteinase classes provided better control of some stored product pests. Testing of other proteins by the research group led to the identification of vitamin-binding proteins that were also effective in the control of some stored product pests. Genes for these proteins can be expressed in wheat, maize, and other cereals to reduce pest damage to these products.

Biochemical techniques developed to evaluate proteinases in mixtures. Insect proteinases are being investigated to identify those that may be targeted by biopesticides. The study of stored product insect proteinases is complicated by the small size of the insect, and the collection of sufficient quantities of digestive proteinases for purification is problematic. Dr. Oppert developed two major techniques that facilitate the analysis of complex mixtures of proteinases. One technique is a fast, simple, and economical microplate assay (see Oppert et al., 1997b). The other technique provides both a qualitative and quantitative identification of proteinases in the mixture (see Oppert and Kramer, 1998). These techniques have promoted rapid and efficient identification of digestive proteinases in moth and beetle pests of stored products. Researchers from areas other than agriculture have used these assays to study proteinases prior to purification.

Elucidation of insect resistance to Bacillus thuringiensis. Proteinaceous toxins from the bacterium Bacillus thuringiensis (Bt) have been used for years in spray applications to control insect pests. Genes encoding these toxins are now being expressed in plants to control crop pests. Expression of Bt toxins in plants will increase exposure levels to insects for longer periods, thus providing increased selection pressure for the survival of resistant populations of pests. Insects can adapt to Bt toxins through an alteration in the gut receptor that binds the toxin in the early stages of toxicity. Through her work with digestive proteinases of the Indianmeal moth, a major pest of stored products, Dr. Oppert described a novel mechanism of insect resistance to Bt. Some Bt-resistant Indianmeal moths have reduced digestive proteinase activity that enables them to survive on diets containing Bt toxins. This was the first evidence of multiple adaptations in insects that result in a loss of Bt toxin efficacy. Information from this research is being used in the design of more efficient microbial toxins and in the development of effective resistance management policies.

	Insect Proteinase Database *
	Stored-Product Insect Images Database *

Proteolytic Enzyme Activity of Kentucky and Kansas Bacillus thuringiensis Susceptible and Resistant Indianmeal Moths Reared on Transgenic Bt Corn

Bacillus thuringiensis (Bt) transformed plants are effective for controlling many insect pests, but insect resistance threatens the long term effectiveness of these toxins. Proteinase-mediated mechanisms may be involved in resistance to Bt and are investigated in this experiment.
     Poster

Magazine Articles

Avidin: An Egg-Citing Insecticidal Protein in Corn

News, Miscellaneous

Missing Enzyme Linked to Bt-Resistance in Insects

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PDF	Goiptar, I.A., I.Y. Filippova, E.N. Lysogorskaya, E.S. Oksenoit, K.S. Vinokurov, D.P. Zhuzhikov, N.V. Bulushova, I.A. Zalunin, Y.E. Dunaevsky, M.A. Belozersky, B. Oppert, and E.N. Elpidina. 2008. Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut. Biochimie 90: 508-514.
PDF	Janarthanan, S., P. Suresh, G. Radke, T.D. Morgan, and B. Oppert. 2008. Arcelins from an Indian wild pulse, Lablab purpureus, and insecticidal activity in storage pests. J. Agric. Food Chem. 56: 1676-1682.
PDF	Park, Y., J. Aikins, L.J. Wang, R.W. Beeman, B. Oppert, J.C. Lord, S.J. Brown, M.D. Lorenzen, S. Richards, G.M. Weinstock, and R.A. Gibbs. 2008. Analysis of transcriptome data in the red flour beetle, Tribolium castaneum. Insect Biochem. Mol. Biol. 38: 380-386.
PDF	Tamez-Guerra, P., J.A. Valadez-Lira, J.M. Alcocer-Gonzalez, B. Oppert, R. Gomez-Flores, R. Tamez-Guerra, and C. Rodriguez-Padilla. 2008. Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hubner) exposed to Bacillus thuringiensis. J. Invertebr. Pathol. 98: 218-227.
PDF	Tribolium Sequencing Consortium (includes R.W. Beeman, M.D. Lorenzen, B. Oppert, J. Lord, K. Kramer, Y. Arakane). 2008. The genome of the model beetle and pest Tribolium castaneum. Nature 452: 949-955.
PDF	Karumbaiah, L., B. Oppert, J.L. Jurat-Fuentes, and M.J. Adang. 2007. Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae). Comp. Biochem. Physiol. Part B 146: 139-146.
PDF	Li, H., L.L. Buschman, F. Huang, K.Y. Zhu, B. Bonning, and B. Oppert. 2007. DiPel-selected Ostrinia nubilalis larvae are not resistant to transgenic corn expressing Bacillus thuringiensis Cry1Ab. J. Econ. Entomol. 100: 1862-1870.
	Li, H., L.L. Buschman, K.Y. Zhu, F. Huang, and B. Oppert. 2007. Resistance to Bacillus thuringiensis endotoxins in the European corn borer, Ostrinia nubilalis. Biopestic. Int. 3: 96-107.
PDF	Prabhakar, S., M.S. Chen, E.N. Elpidina, K.S. Vinokurov, C.M. Smith, J. Marshall, and B. Oppert. 2007. Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L. Insect Mol. Biol. 16: 455-468.
PDF	Oppert, B. 2006. Two-dimensional analysis of proteinase activity. J. Biochem. Biophys. Methods 67: 173-179.
PDF	Oppert, B., P. Walters, and M. Zuercher. 2006. Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae). Bull. Entomol. Res. 96: 167-172.
PDF	Tamez-Guerra, P.G. Damas, M.M. Iracheta, B. Oppert, R. Gomez-Flores, and C. Rodriguez-Padilla. 2006. Differences in susceptibility and physiological fitness of Mexican field Trichoplusia ni strains exposed to Bacillus thuringiensis. J. Econ. Entomol. 99: 937-945.
PDF	Vinokurov, K.S., E.N. Elpidina, B. Oppert, S. Prabhakar, D.P. Zhuzhikov, Y.E. Dunaevsky, and M.A. Belozersky. 2006. Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion. Comp. Biochem. Physiol. Part B 145: 138-146.
PDF	Vinokuroz, K.S., E.N. Elpidina, B. Oppert, S. Prabhakar, D.P. Zhuzhikov, Y.E. Dunaevsky, and M.A. Belozersky. 2006. Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae. Comp. Biochem. Physiol. Part B 145: 126-137.
PDF	Elpidina, E.N., T.A. Tsybina, Y.E. Dunaevsky, M.A. Belozersky, D.P. Zhuzhikov, and B. Oppert. 2005. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie 87: 771-779.
PDF	Li, H., B. Oppert, R.A. Higgins, F. Huang, L.L. Buschman, and K.Y. Zhu. 2005. Susceptibility of Dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins. J. Econ. Entomol. 98: 1333-1340.
PDF	Li, H., B. Oppert, R.A. Higgins, F. Huang, L.L. Buschman, J. Gao, and K.Y. Zhu. 2005. Characterization of cDNAs encoding three trypsin-like proteinases and mRNA quantitative analysis in Bt-resistant and -susceptible strains of Ostrinia nubilalis. Insect Biochem. Mol. Biol. 35: 847-860.
PDF	Oppert, B., T.D. Morgan, K. Hartzer, and K.J. Kramer. 2005. Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. Part C 140: 53-58.
PDF	Tsybina, T.A., Y.E. Dunaevsky, M.A. Belozersky, D.P. Zhuzhikov, B. Oppert, and E.N. Elpidina. 2005. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70: 300-305.
PDF	Vinokurov, K.S., B. Oppert, and E.N. Elpidina. 2005. An overlay technique for postelectrophoretic analysis of proteinase spectra in complex mixtures using p-nitroanilide substrates. Analyt. Biochem. 337: 164-166.
PDF	Zhu, Y.C., X. Liu, A.S. Maddur, B. Oppert, and M.S Chen. 2005. Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (say)]. Insect Biochem. Mol. Biol. 35: 23-32.
PDF	Li, H., B. Oppert, R.A. Higgins, F. Huang, K.Y. Zhu, and L.L. Buschman. 2004. Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae). Insect Biochem. Mol. Biol. 34: 753-762.
PDF	Li, H., J. González-Cabrera, B. Oppert, J. Ferré, R.A. Higgins, L.L. Buschman, G.A. Radke, K.Y. Zhu, and F. Huang. 2004. Binding analyses of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis. Biochem. Biophys. Res. Comm. 323: 52-57.
PDF	Candas, M., O. Loseva, B. Oppert, P. Kosaraju, and L.A. Bulla, Jr. 2003. Insect resistance to Bacillus thuringiensis: Alterations in the Indianmeal moth larval gut proteome. Mol. Cell. Proteomics 2: 19-28.
Available at NAL DDR	Li, H., B. Oppert, K.Y. Zhu, R.A. Higgins, F. Huang, and L.L. Buschman. 2003. Transgenic plants expressing Bacillus thuringiensis delta-endotoxins. Entomologia Sinica 10: 155-166.
PDF	Oppert, B., T.D. Morgan, K. Hartzer, B. Lenarcic, K. Galesa, J. Brzin, V. Turk, K. Yoza, K. Ohtsubo, and K.J. Kramer. 2003. Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. Part C 134: 481-490.
PDF	Zhu, Y.C., F. Zeng, and B. Oppert. 2003. Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae). Insect Biochem. Mol. Biol. 33: 889-899.
PDF	Oppert, B., K. Hartzer, and M. Zuercher. 2002. Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae). Bull. Entomol. Res. 92: 331-336.
PDF	Herrero, S., B. Oppert, and J. Ferre. 2001. Different mechanisms of resistance to Bacillus thuringiensis toxins in the Indianmeal moth. Appl. Environ. Microbiol. 67: 1085-1089.
PDF	Oppert, B., K. Hartzer, and C.M. Smith. 2000. Digestive proteinases of alfalfa weevil, Hypera postica, (Gyllenhal) (Coleoptera: Curculionidae). Trans. Kansas Acad. Sci. 103: 99-110.
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