2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Insects reduce the quality of stored grain and other stored products in the U.S. and in the world. Over 12 billion bushels of corn and wheat are grown in the U.S. each year, with a value of over 25 billion dollars. In addition, over a billion bushels of barley, oats, rice, rye, and sorghum are grown in the U.S. each year, with a value of over 3 billion dollars. It is estimated that postharvest losses to these grains due to insects are 5 to 10%, or about 1.4 to 2.8 billion dollars. Losses to processed commodities are difficult to quantify, but probably greatly exceed the losses to raw commodities. Traditional chemical controls used by the cereal foods industry are being lost due to insect resistance, reduced public acceptance, and changes in regulatory oversight. There are major gaps in our basic knowledge of the mechanisms insects use to perceive and respond to their environment, and in the systems for insect growth, development, digestion, metabolism, and survival. These knowledge gaps are hampering the development of new integrated pest management (IPM) strategies for stored-product insect pests. The need for novel systems of insect control based on knowledge of basic biology coincides with unprecedented advances in genomic analysis of pest insects, which can fuel the growth of such knowledge. The project has three major objectives:. 1)to develop new gene and protein manipulation technologies for discovery of novel biological targets in pest insects;. 2)to characterize vulnerable genetic and physiological pathways such as those involved in digestion, tissue fluid regulation, and immunity; and. 3)to develop DNA fingerprinting technologies for identifying populations and infestation sources, and for incorporation into IPM systems for stored-product insects. This research falls within five Components of National Program 304 (Crop Protection & Quarantine): Biology of Pests and Natural Enemies; Plant, Pest, and Natural Enemy Interactions; Postharvest, Pest Exclusion, and Quarantine Treatment; Pest Control Technologies; and Integrated Pest Management Systems and Areawide Suppression Programs. Because of an increased emphasis on genomic approaches to discovery of target physiology, it is also closely related to two Components of National Program 301 (Plant, Microbial, and Insect Genetic Resources, Genomics & Genetic Improvement): Genomic Characterization and Genetic Improvement; and Genome Databases and Bioinformatics. The proposed research will lead to deeper knowledge of gene families and protein pathways that regulate vital physiological processes in insect tissues. Attaining these objectives will reveal novel protein targets and could lead to safer, more selective inhibitors for insect pest control. Knowledge of Tribolium genetic content will lead to rapid advances in biological knowledge and control strategies for other similar pest species. Genetic analyses will foster improvements in molecular fingerprinting of insects, leading to a better understanding of the structure and dynamics of insect populations. Expected products include improved biocontrol agents that block molting or reproduction or that inhibit digestion, as well as novel gene and protein targets that can be inhibited by feeding on transgenic crops. Anticipated benefits include new lines of cereals that express insect control proteins to confer pest resistance. New DNA fingerprinting methods will improve the accuracy of species and biotype identification, and also facilitate monitoring of population movements and more accurate identification of sources of infestation and pest resurgence. This research will benefit farmers, producers, and academic and industrial partners involved in storage and processing of grain and grain products. Scientists will benefit from basic knowledge of insect genome organization and comparative genomic sequence, and from knowledge of gene pathways that regulate embryonic and larval growth and development, and digestive physiology. Finally, the research will benefit consumers demanding ecologically sound pest control systems founded on basic knowledge of insect biology. SCA partners are involved in whole-genome sequence analysis and annotation of Tribolium, in the evaluation of wheat protease inhibitors as biopesticides, in the search for osmoregulatory, digestive or neuroendocrine components for new biopesticide targeting, and in the RNAi-based screening of solute transporters for discovery and validation of genes involved in pesticide transport or excretion. 2.List by year the currently approved milestones (indicators of research progress) 2006 Complete the physical map of the Tribolium genome. Continue manual adjustment of the genome annotation and gene discovery. Continue generating and characterizing transposon insertion lines. Initiate screening of transposon insertion libraries for enhancer patterns, stress tolerance, developmental & molting defects, pesticide resistance, or other traits. Continue sequencing and identification of gut ESTs. Continue identification of target gene families from genome annotation. Continue RNAi knockout from priority genelists. Design microarray experiments. Maintain data-stream into BeetleBase. Initiate toxin bioassays in coleopteran pests. Test additional markers with local and regional populations. 2007 Continue manual adjustment of genome annotation and gene discovery. Complete testing of promoters and transactivators & incorporation into vectors for gene tagging, enhancer trapping and misexpression. Enter data for insertional mutagenesis library into BeetleBase. Conduct Northern and qPCR analysis of proteinase gene expression. Initiate comparison of Bt toxin binding proteins in coleopteran pests. Initiate proteinase studies with Bt toxins. Initiate microarray experiments. Maintain data-stream into BeetleBase. Initiate statistical analysis of data from DNA fingerprinting study. 2008 Continue manual adjustment of genome annotation and gene discovery. Complete screening of insertional mutant libraries for physiological defects, enhancer patterns, or other traits. Design new biopesticides based on proteinase inhibitor and toxin binding studies. Conduct microarray analyses using oligo-based hybridization. Develop theories of population structure of stored-product beetles. 2009 Complete manual adjustment of genome annotation and gene discovery. Maintain data-stream into BeetleBase. Estimate the degree of sequence conservation within the coleoptera. Maintain data-stream into BeetleBase. Complete statistical analysis of the data from DNA fingerprinting study. 4a.List the single most significant research accomplishment during FY 2006. Manual Annotation Teams Organized and Implemented for the Tribolium Genome Project. Without accurate annotation and functional analysis of an insect genome sequence, the value of that sequence to science and industry is reduced. Our laboratory was selected as the official clearing house for manual annotation of all "pest biology" genes in Tribolium, and analysis has been completed on >200 such genes, including P450 (cyp) genes, cuticle protein genes, trans-membrane transporter genes, and others. The starting point for this manual effort is the putative gene list derived from the automated annotation of the Tribolium genome sequence, which was completed in May 2006 and includes 16,400 gene models. This accomplishment resulted from the joint efforts of the ARS Biological Research Unit in Manhattan, KS, Kansas State University, the Baylor College of Medicine's Human Genome Sequencing Center, the National Center for Biotechnology Information, and participants at the International Tribolium Genomics Meeting in Gottingen, Germany, in 2005. The functional analysis of genes characerized in the manual annotation will have far-reaching impact on broad knowledge of insect genome evolution, physiological adaptations in pest and beneficial beetle species, and the identification of novel targets for pest control exploitation. Research relevant to National Program 304, Crop Protection & Quarantine, Component IV-E, Postharvest, Pest Exclusion, and Quarantine Treatment-Biology and Ecology of Stored-Product Insect Pests, and specifically the goal "Improve our understanding of the genetic and physiological processes in stored product pest insects to identify new targets for pest control." 4b.List other significant research accomplishment(s), if any. Genes Identified that Regulate Chitinous Structures in Insect Cuticle and Midgut. Insect-specific physiological structures and processes such as chitin and molting can be targeted for design of insect-specific biopesticides. We found approximately 20 new cuticle/chitin genes in the Tribolium genome sequence, isolated many of the corresponding cDNAs, and described the deleterious effects on development when the functions of some of these genes are inhibited, using the technique of RNA interference. The new genes include chitinases, chitin deacetylases, syntaxin, obstructors, cuticle proteins, and peritrophins, and have functions that include cuticle synthesis, assembly, modification, and degradation. This work was done in cooperation with Kansas State University. Examination of the formation, regulation and reutilization of the insect exoskeleton and midgut peritrophic membrane is revealing a great variety of candidate genes for biopesticide-mediated disruption. Research relevant to National Program 304, Crop Protection & Quarantine, Component IV-E, Postharvest, Pest Exclusion, and Quarantine Treatment-Biology and Ecology of Stored-Product Insect Pests, and specifically the goal "Improve our understanding of the genetic and physiological processes in stored product pest insects to identify new targets for pest control." Biochemical Analysis of Beetle Digestion Yields Valuable Insights. A comprehensive biochemical and genetic study of digestive proteases in Tenebrio molitor has yielded valuable insights into the complexity of digestion in this beetle storage pest. Multiple forms of cysteine- and serine-type proteases were compartmentalized, presumably for maximal activity and ensuring an efficient as well as tightly-regulated control of food digestion. These enzymes operate in a concerted fashion to digest oat proteins. Enzymes that initiate digestion were speculated to be primary targets for inhibitors, such as plant protease inhibitors, that can be developed as new control products for beetle storage pests. This information also will be used to study the regulation of digestive enzymes in the genome of the closely related beetle, Tribolium castaneum. Research relevant to National Program 304, Crop Protection & Quarantine, Component IV-E, Postharvest, Pest Exclusion, and Quarantine Treatment-Biology and Ecology of Stored-Product Insect Pests, and specifically the goal "Improve our understanding of the genetic and physiological processes in stored product pest insects to identify new targets for pest control." 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. We demonstrated that flour beetle larvae challenged with proteinase inhibitors compensated for the loss of digestive activity by over-production of other classes of proteinases. This flexibility in the response of an insect to targeting by digestive inhibitors will be an important consideration in the design of new, binary or multiple-component biopesticides. We have examined a group of maternally-acting larvicidal "selfish genes" that are unique to the animal kingdom, and have made substantial progress towards unravelling the mechanism of this unusual system of insect reproductive suppression. We have shown that insects utilize two different chitin synthase enzymes for production of the major structural polysaccharide comprising the exoskeleton and gut peritrophic membrane, and we have characterized their genes from several insect species. We conclusively identified the elusive insect tanning enzyme. We identified many other genes not previously known to be involved in peritrophic membrane and cuticle formation and reutilization, including chitinases, chitin deacetylases, obstructors, cuticle proteins, and peritrophins. These fundamental discoveries contributed to the development and refinement of the first model system for insect cuticle assembly, which includes a cuticular protein, a cross-linking agent precursor, and a cross-linking enzyme. This system will be employed to screen for compounds that inhibit cuticle formation, which have potential for development as insect growth regulators. Our progress in characterizing the genome of a pest beetle has led to high density chromosome maps and to the first completed genome sequence for an agronomic pest species. We obtained many cDNA sequences from Tribolium and Tenebrio which have been made available to the scientific community. We developed CRADA agreements with biotechnology and agri-business firms for validation of biopesticide target genes. We developed the first facile system for gene transfer and genetic manipulation in a pest beetle, based on mobile DNA vectors. We have initiated expression microarray experiments which will enable whole-transcriptome screening of genes involved in various biological processes. We co-organized the whole-genome annotation effort, initiated in 2006. Research relevant to National Program 304, Crop Protection & Quarantine, Component IV-E, Postharvest, Pest Exclusion, and Quarantine Treatment-Biology and Ecology of Stored-Product Insect Pests, and specifically the goal "Improve our understanding of the genetic and physiological processes in stored product pest insects to identify new targets for pest control." 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? DNA sequences were deposited in GenBank, making them available to the global research community free of charge. We have filled numerous requests from researchers and instructors in academia, industry, and from other federal laboratories for genetic strains, plasmids, information, or advice. We and our collaborators founded the Tribolium insertional mutant stock center, which will serve as a source for researchers worldwide. We are co-organizers of the whole-genome manual annotation project, the results of which will be available to the public through the HGSC Tribolium genome browser and BeetleBase. Our research has had an inordinately large impact on redirecting the research efforts of other labs from government, industry, and academia in new, productive directions. Review Publications Oppert, B.S., Walters, P., Zuercher, M.C. 2006. Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Leconte) (Coleoptera: Tenebrionidae). Bulletin of Entomological Research 96: 167-172. Lewis, S.M., Kobel, A., Fedina, T., Beeman, R.W. 2005. Sperm stratification and paternity success in red flour beetles. Physiological Entomology 30: 303-307. Arakane, Y., Muthukrishnan, S., Kramer, K.J., Specht, C.A., Tomoyasu, Y., Lorenzen, M.D., Kanost, M., Beeman, R.W. 2005. The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix. Insect Molecular Biology 14: 453-463. Bolognesi, R., Arakane, Y., Muthukrishnan, S., Kramer, K.J., Terra, W.R., Ferreira, C., Beeman, R.W. 2005. Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda. Insect Biochemistry and Molecular Biology 35: 1249-1259. Elpidina, E.N., Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B.S. 2005. Purification and characterization of a chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochemie 87: 771-779. Oppert, B.S. 2006. Two-dimensional analysis of proteinase activity. Journal of Biochemical and Biophysical Methods 67: 173-179. Li, H., Oppert, B.S., Higgins, R.A., Huang, F., Buschman, L.L., Gao, J., Zhu, K.Y. 2005. Characterization of cDNAs encoding three trypsin-like proteinases and quantitative analysis of mRNA in Bt-resistant and -susceptible strains of Ostrinia Nubilalis. Insect Biochemistry and Molecular Biology 35: 847-860. Petrek, J., Vitecek, J., Vlasinova, H., Kizek, R., Kramer, K.J., Adam, V., Klejdus, B., Havel, L., Beeman, R.W. 2005. Application of computer imaging, stripping voltammetry and mass spectrometry to study the effect of lead (Pb-EDTA) on the growth and viability of early somatic embryos of Norway spruce (Picea abies/L.Karst.). Analytical Bioanalytical Chemistry 383: 576-586. Matsumiya, M., Arakane, Y., Haga, A., Muthukrishnan, S., Kramer, K.J., Beeman, R.W. 2006. Substrate specificity of chitinases from two species of fish, greenling, hexagrammos otakii, and common mackerel, scomber japonicus, and the insect, tobacco hornworm, manduca sexta. Insect Biochemistry and Molecular Biology 70: 971-979. Suderman, R.J., Dittmer, N.T., Kanost, M.R., Kramer, K.J., Beeman, R.W. 2006. Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols. Insect Biochemistry and Molecular Biology36: 353-365. Tamez-Guerra, P., Damas, G., Iracheta, M.M., Oppert, B.S., Gomez-Flores, R., Rodriguez-Padilla, C. Diferences in susceptibility and physiological fitness of Mexican field Trichoplusia ni (Hubner) strains exposed to Bacillus thuringiensis. Journal of Economic Entomology 99: 937-945. Hogenkamp, D.G., Arakane, Y., Zimoch, L., Merzendorfer, H., Kramer, K.J., Beeman, R.W., Kanost, M.R., Specht, C.A., Muthukrishnan, S. 2005. Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development. Journal of Insect Biochemistry and Molecular Biology 35: 529-540. Beeman, R.W., Lorenzen, M.D., Brown, S.J., Stuart, J.J. 2005. The selfish gene medea in tribolium castaneum: an update [Abstract]. Tribolium Transgenics Meeting and International Tribolium Genetics Meeting, Gottingen, Germany, August 1-5, 2005. Beeman, R.W., Lorenzen, M.D., Brown, S.J., Gibbs, R., Weinstock, G., Richards, S., Liu, Y. 2005. Integration of the recombination and physical maps with the genome sequence of tribolium castaneum [Abstract]. Tribolium Transgenic Meeting and International Tribolium Genetics Meeting, Gottingen, Germany, August 1-5, 2005. Lorenzen, M.D., Wang, L., Brown, S.J., Beeman, R.W. 2005. Annotation of the Tribolium castaneum genome [Abstract]. Presentation at the International Tribolium Genetics Conference, Gottingen, Germany, August 1-5, 2005. Beeman, R.W., Lorenzen, M.D., Brown, S.J. 2005. Unveiling the first beetle genome [Abstract]. National Entomological Society of America Annual Meeting, Fort Lauderdale, FL, November 6-9, 2005. Lorenzen, M.D., Beeman, R.W. 2006. The tribolium genome project and its impact on pest management strategies [Abstract]. Presentation, 2006 International Plant Resistance to Insects Workshop, West Lafayette, IN, April 9-12, 2006.