2010 Annual Report 1a.Objectives (from AD-416) The primary goals for this project are to characterize comprehensively the structure of the genome of Tribolium castaneum (referred to hereafter as Tribolium) and the function of its constituent genes; to integrate this analysis with gene expression analysis of the gut and other tissues; to extend results from Tribolium to other beetle species; and to use the results to identify new physiological targets for pest control and to develop a deeper understanding of the population structure of stored-product pest insects. 1b.Approach (from AD-416) We propose to fully characterize the structure of the genome of the red flour beetle (Tribolium castaneum) and its constituent genes; to develop transgene technologies to reveal gene functions and vulnerable physiological pathways and to identify promising targets for insect suppression; and to develop DNA-based methods for monitoring, fingerprinting, and characterizing pest populations. We will generate and analyze expressed sequence tag (EST) data to identify genes involved with digestion, osmoregulation, immunity, metamorphosis, neuroendocrine regulation, and other vital functions. After preliminary automated genome analysis by Ensembl, we will use the Apollo interface to view, retrieve, manipulate, refine, and correct Ensembl-generated annotations in a desktop environment. Results will be integrated into InsectBase and BeetleBase. In silico analyses will be integrated with in vivo modification of the Tribolium genome using transposon vectors specifically tailored for gene disruption, discovery of gene regulatory elements, promoter analysis, and gene replacement. Identification of the digestive proteinase subgenome will be extended with genomic and proteomic studies in Tenebrio and other beetles. Microsatellites, variable repeats, hypervariable segments, single nucleotide polymorphisms, and other sequences with potential use in DNA fingerprinting will also be mined from the genome sequence and utilized for basic study of population biology and to gain insight into infestation sources and movements. 3.Progress Report This is the final report for the Project 5430-43000-026-00D terminated in April 2010. All experiments described in the Project Plan for Project 5430-43000-026-00D were completed, as elaborated below. A bridging project #5430-43000-030-00D was created until the new 5-year project #5430-43000-032-00D, currently in peer review, is implemented in early FY 2011. The outcome of this research project is an unprecendented body of new knowledge about the functional genomics of a pest insect. Achievements over the 5 years of the project include completion of the Tribolium castaneum whole-genome sequence, the first for any insect pest of agriculture; physical mapping of 95% of this sequence onto all nine T. castaneum chromosomes; and annotation of hundreds of genes involved in the biosynthesis, deposition, organization and metabolism of chitin and cuticle, as well as in digestion, osmoregulation, molting, pigmentation and other vital biological processes. We discovered the functions of more than 100 of these genes by RNAi-mediated gene knockout. We contributed tens of thousands of Tribolium EST sequences which have been incorporated into public databases and used for improved genome annotation and gene discovery. We participated in the development of the BeetleBase website and genome browser, and to the design and use of the first whole-transcriptome expression microarrays and whole-genome tiling arrays for agricultural pest species. We developed a highly efficient “jumpstarter” method for germline transformation and the first whole-genome transposon tagging technology for any non-Drosophilid arthropod, and we used these technologies to produce a library of thousands of transposon insertions that revealed new information about gene functions and expression patterns. We showed how two partially redundant classes of digestive proteinases can protect phytophagous insects from plant-derived digestive inhibitors, and demonstrated that simultaneous inhibition of both classes is a viable strategy in biopesticide design. We isolated toxin-binding-like genes in beetle guts that appear to mediate responses to ingested Bacillus thuringiensis toxins, and that could hold the key to altering the species-specificities of these toxins. We cloned and sequenced the first maternal selfish gene in any animal, and showed that the gene could be used as a natural vector to force desirable genes into populations for pest amelioration or disease suppression. We identified many microsatellites and other polymorphic DNA markers and showed how they could be used for DNA fingerprinting to differentiate local and regional pest populations. 4.Accomplishments 1. Using functional genomics to solve storage pest problems. Studies of the complex response of storage pests to biopesticides have been advanced significantly by new genetic tools, such as high throughput sequencing and microarray analysis. To delineate the mode of action of bacterial toxins in coleopteran pests, ARS scientists in Manhattan, KS, have used high throughput sequencing to provide a snapshot of intoxication of the yellow mealworm by microbial toxins and to develop microarrays for analyzing global patterns of gene expression in the gut of yellow mealworm larvae exposed to biopesticides. Further, these advanced technologies were used to develop whole genome microarrays for the red flour beetle that have been used to monitor changes in gene expression in response to dietary inhibitors. These data could lead to improved pest management strategies for beetle storage pests through the development of more targeted and environmentally friendly biopesticides. 2. Insect “yellow” genes characterized. The chemistry of insect exoskeleton is still only poorly understood, but represents a sensitive aspect of insect physiology that could be exploited by appropriately-targeted biopesticides. ARS scientists in Manhattan, KS, identified fourteen new genes in the red flour beetle that seem to have roles in the maturation (hardening and darkening) of the insect cuticle. Inhibiting the expression of some of these genes resulted in failure of the exoskeleton to either darken or ripen normally. In some cases such gene knockout prevented the affected insects from shedding the old skin, resulting in the death of the insect. A deeper understanding of insect exoskeleton could reveal new weaknesses that may lead to new methods of insect control. 3. Insect Uridine diphosphate N-acetylglucosamine pyrophosphorylase genes characterized. Chitinous structures on insects such as the exoskeleton and digestive sac are vital for insect survival and could be exploited by appropriately-targeted biopesticides. ARS scientists in Manhattan, KS, identified two new “UAP” genes in the red flour beetle that are required to generate the basic building blocks not only of chitin, but also other vital insect sugar polymers and sugar-proteins. All other invertebrates examined have only one UAP gene. One of the two genes is unique to red flour beetles and functions in nutrition and growth. If this unique gene is eliminated, the insects die, apparently of starvation. Our ongoing gene discovery efforts in this pest insect continue to reveal new weaknesses that may lead to new methods of insect control. 4. Microarrays (Genomic tiling arrays) and next-generation sequencing (Seq-Cap and Illumina) for polymorphism detection. Microarrays and new, high-throughput sequencing technologies are revolutionizing the analysis of genomes and transcriptomes. ARS scientists in Manhattan, KS, developed several novel applications of these new technologies for biotyping pest insects. These developments could greatly facilitate DNA fingerprinting or related approaches to strain/biotype differentiation in pest insects. Review Publications Marshall, J.L., Huestis, D.L., Hiromasa, Y., Oppert, C., Marshall, S.A., Tomich, J., Oppert, B.S. 2009. Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket. PLoS One 4(10): e7537-e7546. Trauner, J., Schinko, J.B., Lorenzen, M.D., Shippy, T.D., Wimmer, E.A., Beeman, R.W., Klingler, M., Bucher, G., Brown, S.J. 2009. Large-Scale Insertional Mutagenesis of the Coleopteran Stored Grain Pest, the Red Flour Beetle Tribolium castaneum, Identifies Embryonic Lethal Mutations and Enhancer Traps. Biomed Central (BMC) Genomics. 7:73. Begum, K., Li, B., Beeman, R.W., Park, Y. 2010. Functions of Ion Transport Peptide and Ion Transport Peptide-Like in the Red Flour Beetle Tribolium castaneum. Insect Biochemistry and Molecular Biology. 39: 717-725. Kim, H., Murphy, T., Xia, J., Caragea, D., Park, Y., Beeman, R.W., Lorenzen, M.D., Manak, J., Butcher, S., Brown, S.J. 2010. BeetleBase in 2010: Revisions to Provide Comprehensive Genomic Information for Tribolium castaneum. Nucleic Acids Research. 38: D437-D442. Broehan, G., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S., Merzendorfer, H. 2010. Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference. Insect Biochemistry and Molecular Biology. 40(3):274-283. doi:10.1016/j.ibmb.2009.10.009. Arakane, Y., Dittmer, N.T., Kramer, K.J., Muthukrishnan, S., Beeman, R.W., Kanost, M.R. 2010. Identification, mRNA expression and functional analysis of several yellow family genes in Tribolium castaneum. Insect Biochemistry and Molecular Biology. 40(3):259-266. doi:10.1016/j.ibmb.2010.01.012. Dubovenko, A.G., Dunaevsky, Y.Y., Belozersky, M.A., Oppert, B.S., Lord, J.C., Elpidina, E.N. 2010. Trypsin-like Proteins of the Fungi as Possible Markers of Phytopathogenicity. Fungal Biology. 114: 151-159. doi:10.1016/j.funbio.2009.11.004 Jasrapuria, S., Arakane, Y., Osman, G., Kramer, K.J., Beeman, R.W., Muthukrishnan, S. 2010. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function. Insect Biochemistry and Molecular Biology. 40(3):214-227. doi:doi:10.1016/j.ibmb.2010.01.011. Oppert, C., Klingeman, W., Willis, J.D., Oppert, B.S., Jurat-Fuentes, J.L. 2010. Prospecting for Cellulolytic Activity in Insect Digestive Fluids. Comparative Biochemistry and Physiology. 155: 145-154. doi:10.1016/j.cbpb.2009.10.014.