Dr. Brenda Oppert Research Molecular Biologist Stored Product Insect Research Unit Center for Grain & Animal Health Research 1515 College Ave. Manhattan, KS 66502 Voice: (785) 776-2780 Fax: (785) 537-5584 brenda.oppert@ars.usda.gov www.ars.usda.gov/npa/cgahr/spiru/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 CGAHR 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.

Functional Genomics Applied To Stored Product Pest Problems
New insect control products for storage pests are urgently needed to replace those that are undergoing deregulation or to circumvent resistant pest populations. With the sequencing of the red flour beetle genome, we now have new tools to develop control products based on vulnerabilities in the biology of the beetle. We developed microarrays that contain representative sequences from each gene in the red flour beetle genome to identify sequences that are highly expressed in a particular tissue. Initial studies have identified gut transcripts as potential new targets for biopesticides. Beetle genome microarrays are also being used to probe the beetle's response to dietary inhibitors, toxins, and current control products, such as phosphine. We've found that beetle larvae fed dietary inhibitors mount a complex genetic response to survive. This information has provided us with details about genes that are transcribed by the beetle to survive inhibitor damage, and also genes that encode enzymes critical to digestion of protein in the diet. Because a closely related beetle, the yellow mealworm, has no sequenced genome, we are using high throughput sequencing to identify highly expressed genes in the gut of mealworm larvae. By comparing genetic transcripts in mealworm larvae exposed or not to a bacterial toxin, we are beginning to understand the complex response of mealworm larvae to toxins. All functional genomics applications are highly dependent on sophisticated bioinformatics algorithms, which we are developing to analyze insect sequences and data. As we identify potential new targets for biopesticides, we are using high throughput bioassays to evaluate purified proteins, peptides, or a process of interfering in the synthesis of genetic transcripts, called RNA interference. Biopesticides have applications in the integrated pest management of storage pests in sprays, formulations, or transgenic cereals. (Cooperators: Drs. Jeff Lord, Jeff Fabrick, Juan Luis Jurat-Fuentes, Elena Elpidina, John Tomich)

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.

Stored-Product Insect Images Database *

Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts

Some insecticides are based on toxins produced by bacteria or other microbes, but these insecticides often don’t work well for beetle pests. In order to make these microbial toxins more effective for controlling beetles, we need to understand how they kill the insect. Most of the microbial toxins used for insect control are produced by the bacterium Bacillus thuringiensis, and we used one of these toxins in our study. We know that there are enzymes in the beetle gut that are involved in activating the toxin and making it soluble so that it can bind to the lining of the insect gut. Binding of the toxin starts a chain of events that we still don’t fully understand, but leads to killing the insect. To better understand this process, we determined how many enzymes are in the gut of mealworm larvae, and what changes are induced in these enzymes when mealworm larvae are fed toxin. Our results suggest that that mealworm larvae exposed to toxin are attempting to decrease toxicity of the toxin while maintaining efficient digestion. Knowledge of how the gut enzymes in mealworm larvae are affected by microbial toxins may help us to improve these important biopesticides for beetle pest control.
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Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata, Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment

Bacillus thuringiensis (Bt) is a bacterium that produces toxins used in the control of insect pests. Bt toxins generally are not effective against many beetle pests, limiting the use of Bt toxins in integrated pest management. We evaluated a novel peptide from a toxin receptor in the beetle Tenebrio molitor to determine the potential to enhance the activity of Bt toxins against the beetles Crioceris quatuordecimpunctata, Phaedon brassicae, and Colaphellus bowringi, serious pests of vegetables in China. The activity of Bt toxin Cry3Aa was increased as much as 15.3-fold in these pests when the peptide was added, compared to the activity of Cry3Aa alone. The data demonstrate that the peptide has potential as an additive in Bt sprays or incorporated into transgenic Bt crops to protect against beetle pests.
     

Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests

To be successful, stored product pests need to have enzymes capable of efficiently digesting their main dietary proteins, which have many glutamine and proline amino acids. We describe for the first time one enzyme, a glutamine-specific peptidase, in the gut of Tenebrio molitor larvae that are capable of digesting proteins by hydrolyzing at glutamine residues. Our characterization of these peptidases indicates that they are previously studied enzymes belonging to the class of cysteine peptidases, hypothesized to have evolved in storage pests to protect the insect from serine protease inhibitors found in cereals. We now propose that storage pests also have retained cysteine peptidases to efficiently digest cereal proteins. These results may be exploited to develop new control products for storage pests.
     

Bacillus thuringiensis Cry3Aa toxin increases the susceptibility of Crioceris quatuordecimpunctata to Beauveria bassiana infection

The spotted asparagus beetle is a major pest of asparagus world-wide. There is a need for viable alternatives to chemical insecticides for their control that are compatible with integrated pest management. The most apparent candidate for such an alternative is a commonly used bacterial insecticide, but it is not effective for this beetle. We demonstrated that non-lethal exposure of the beetle to the bacterial insecticide made it more susceptible to a commercial insecticidal fungus. This research will help to provide growers with safe, environmentally friendly alternatives to chemical insecticides.
     

Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin

Responses of coleopteran storage pests to microbial toxins has not been well-studied. We used high throughput sequencing to determine the effect of Bacillus thuringiensis Cry3Aa protoxin on transcript expression in the gut of Tenebrio molitor larvae. Transcripts associated with mitochondrial respiration, membrane restructuring, immunity, and signaling were increased, while transcripts associated with the metabolism of food and storage proteins were repressed in Cry3Aa-intoxicated larvae. Knowledge of the intricate responses to toxin in intoxicated larvae can provide information critical to the improvement of microbial toxins for coleopteran pest control.
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Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors

Previously, our studies determined that flour beetle larvae respond to dietary inhibitors by shifting from one class of proteases to another. This response is problematic if cereal inhibitors are to be incorporated into IPM strategies for storage pest control. To study this at the gene level, whole-genome microarrays were used to evaluate the inhibitor response. The data demonstrated that the response by beetle larvae to dietary inhibitors involves a complicated adjustment of gene expression. However, the study also provided clues as to how to better utilize inhibitors for beetle pest control. Therefore, effective control of beetles may soon be possible with a strategy that can anticipate beetle responses to inhibitors.
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Magazine Articles

Avidin: An Egg-Citing Insecticidal Protein in Corn

News, Miscellaneous

Missing Enzyme Linked to Bt-Resistance in Insects

PDF	Oppert, B., and T.D. Morgan. 2013. Improved high-throughput bioassay for Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). J. Stored Prod. Res. 52: 68-73.
PDF	Gao, Y., B.S. Oppert, J.C. Lord, C. Liu, and Z. Lei. 2012. Bacillus thuringiensis Cry3Aa toxin increases the susceptibility of Crioceris quatuordecimpunctata to Beauveria bassiana infection. J. Invertebr. Pathol. 109: 260-263.
PDF	Goptar, I.A., T.A. Semashko, S.A. Danilenko, E.N. Lysogorskaya, E.S. Oksenoit, D.P. Zhuzhikov, M.A. Belozersky, Y.E. Dunaevsky, B. Oppert, I.Y. Filippova, and E.N. Elpidina. 2012. Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests. Comp. Biochem. Physiol. Part B 161: 148-154.
PDF	Oppert, B., A.G. Martynov, and E.N. Elpidina. 2012. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts. Comp. Biochem. Physiol. Part D 7: 233-243.
PDF	Oppert, B.S., S.E. Dowd, P. Bouffard, L. Li, A. Conesa, M.D. Lorenzen, M. Toutges, J. Marshall, D.L. Huestis, J.A. Fabrick, C. Oppert, and J.L. Jurat-Fuentes. 2012. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin. PLoS ONE 7(e34624), 12 pp.
PDF	Valadez-Lira, J.A., J.M. Alcocer-Gonzalez, G. Damas, G. Nuñez-Mejiá, B. Oppert, C. Rodriguez-Padilla, and P. Tamez-Guerra. 2012. Comparative evaluation of phenoloxidase activity in different larval stages of four lepidopteran pests after exposure to Bacillus thuringiensis. J. Insect Sci. 12(80),11 pp.
PDF	Yao, J., L.L. Buschman, B. Oppert, C. Khajuria, and K.Y. Zhu. 2012. Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae. PloS ONE 7(8): e44090.
PDF	Gao, Y., J. L. Jurat-Fuentes, B. Oppert, J.A. Fabrick, C. Liu, J. Gao, and Z. Lei. 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.
PDF	Oppert, B., T.D. Morgan, and K.J. Kramer. 2011. Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests. Pest Manag. Sci. 67: 568-573.
PDF	Toutges, M.J., K.L. Hartzer, J.C. Lord, and B.S. Oppert. 2011. Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum. J. Agric. Food Chem. 58: 8948-8951.
PDF	Willis, J.D., B.S. Oppert, C. Oppert, W.E. Klingeman, and J.L. Jurat-Fuentes. 2011. Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae). J. Insect Physiol. 57: 300-306.
PDF	Dubovenko, A.G., Y.E. Dunaevsky, M.A. Belozersky, B. Oppert, J.C. Lord, and E.N. Elpidina. 2010. Trypsin-like proteins of the fungi as possible markers of pathogenicity. Fungal Biol. 114: 151-159.
PDF	Gao, Y., Y. Hu, Q. Fu, J. Zhang, B.S. Oppert, F. Lai, and Z. Zhang. 2010. Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis. J. Invertebr. Pathol. 105: 11-15.
PDF	Liu, C., Y. Gao, C. Ning, K. Wu, B.S. Oppert, and Y. Gao. 2010. Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera. J. Insect Physiol. 56: 718-724.
PDF	Lord, J.C., K. Hartzer, M. Toutges, and B. Oppert. 2010. Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge. J. Microbiol. Meth. 80: 219-221.
PDF	Oppert B.S., E.N. Elpidina, M. Toutges, and S. Mazumdar-Leighton. 2010. Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors. Comp. Biochem. Physiol. Part D 5: 280-287.
PDF	Oppert, B.S. 2010. Rapid bioassay to screen potential biopesticides in Tenebrio molitor larvae. Biopestic. Int. 6: 67-73.
PDF	Oppert, B.S., R.T. Ellis, and J. Babcock. 2010. Effects of Cry1F and Cry34Ab1/35Ab1 on storage pests. J. Stored Prod. Res. 46: 143-148.
PDF	Oppert, C., W.E. Klingeman, J.D. Willis, B. Oppert, and J.L. Jurat-Fuentes. 2010. Prospecting for cellulolytic activity in insect digestive fluids. Comp. Biochem. Physiol. Part B 155: 145-154.
PDF	Willis, J.D., W.E. Klingeman, C. Oppert, B.S. Oppert, and J.L. Jurat-Fuentes. 2010. Characterization of cellulolytic activity from digestive fluids of Dissosteira carolina (Orthoptera: Acrididae). Comp. Biochem. Physiol. Part B 157: 267-272.
PDF	Crook, D.J., S. Prabhakar, and B. Oppert. 2009. Protein digestion in larvae of the red oak borer Enaphalodes rufulus. Physiol. Entomol. 34: 152-157.
PDF	Fabrick, J., C. Oppert, M.D. Lorenzen, K. Morris, B. Oppert, and J.L. Jurat-Fuentes. 2009. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J. Biol. Chem. 284: 18401-18410.
PDF	Huestis, D.L., B. Oppert, and J.L. Marshall. 2009. Geographic distributions of Idh-1 alleles in a cricket are linked to differential enzyme kinetic performance across thermal environments. BMC Evol. Biol. 9(113), 12 pp.
PDF	Liu, C., K. Wu, Y. Wu, Y. Gao, C. Ning, and B. Oppert. 2009. Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment. J. Insect Physiol. 55: 686-693.
PDF	Marshall, J.L., D.L. Huestis, Y. Hiromasa, S. Wheeler, C. Oppert, S.A. Marshall, J.M. Tomich, and B. Oppert. 2009. Identification, RNAi knockdown, and functional analysis of an ejaculate protein that mediates a postmating, prezygotic phenotype in a cricket. PloS ONE 4(e7537), 10 pp.
PDF	Morris, K., M.D. Lorenzen, Y. Hiromasa, J.M. Tomich, C. Oppert, E.N. Elpidina, K.Vinokurov, J.L. Jurat-Fuentes, J. Fabrick, and B. Oppert. 2009. Tribolium castaneum larval gut transcriptome and proteome: a resource for the study of the coleopteran gut. J. Proteome Res. 8: 3889-3898.
PDF	Vinokurov, K.S., E.N. Elpidina, D.P. Zhuzhikov, B. Oppert, D. Kodrik, and F. Sehnal. 2009. Digestive proteolysis organization in two closely related Tenebrionid beetles: Red flour beetle (Tribolium castaneum)and confused flour beetle (Tribolium confusum). Arch. Insect Biochem. Physiol. 70: 254-279.
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.
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