2007 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. 4.Accomplishments 4a Maternal larvicidal gene identified. In 1992 we announced our discovery that insects can regulate their own populations by the actions of maternal larvicidal genes, but the molecular basis for this potentially useful phenomenon has remained a mystery. In cooperation with the Exelixis Pharmaceutical Co. and the Human Genome Sequencing Center, we have finally identified the molecular lesion associated with the syndrome in the red flour beetle, namely a 21.5 kb insertion of mobile DNA near the tip of chromosome 3. This segment of DNA contains extra copies of several important genes, and also includes a bacterial gene that has somehow become incorporated into the insect’s genome. Further analysis of precisely how these genes contribute to the larvicidal syndrome could spark new ways of thinking about insect population suppression. 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 A receptor for coleopteran-specific toxins produced by the insecticidal bacterium, Bacillus thurigiensis (Bt) was identified in the Bt-susceptible insect, Tenebrio molitor. The possibilities for host/range expansion of Bt toxins have not been sufficiently addressed in biopesticide development research. We found genes in the beetles Tenebrio and Tribolium that correspond to a known receptor for moth-specific Bt toxin, even though Tribolium is not susceptible to beetle-specific toxins. We detected differences in the toxin binding site in the receptor protein that could inactivate this function in Tribolium, thus rendering this insect insensitive to Bt. A peptide corresponding to the toxin binding site of the T. molitor toxin receptor was able to potentiate Bt toxicity in that species. This line of investigation could lead to increased effectiveness of next-generation Bt toxins against a wide range of pest species. 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 Tribolium genome sequence reveals large expansion in the number of digestive cysteine protease genes. The insect gut is a primary target for plant toxicants that function to deter insect herbivores, and is an excellent and underutilized target for new biopesticide design. Our analysis of the Tribolium genome sequence revealed a major expansion of a family of digestive C1 cysteine proteinase genes. We detected at least 25 such genes in Tribolium, more than double the number in Drosophila or Anopheles, and 5 times as many as in the honeybee. This unique digestive strategy in Tribolium probably reflects its nonspecialist feeding habit, its relatively acidic gut, and compensation for a reduced reliance on serine peptidases for protein digestion. As the first nonspecialist herbivore to be subjected to whole-genome sequencing, Tribolium provides the first glimpse into a potential wealth of new information on insect digestion, as well as a new set of susceptibilities for pest control intervention. 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." 4d Insect cuticle protein genes identified in the Tribolium genome sequence. Insect-specific physiological structures and processes such as chitin and molting can be targeted for design of insect-specific biopesticides. In cooperation with Kansas State University and the University of Georgia, we found approximately 100 new cuticle protein genes in the Tribolium genome sequence and used the technique of RNA interference to demonstrate that individual members of this large group of genes have specific, vital functions. This was unexpected, since the sheer number of cuticle genes suggested a high level of redundancy. This in turn suggests the possibility of many dozens of new targets for intervention in the insect cuticle alone. Examination of the formation, regulation and reutilization of the insect exoskeleton and midgut peritrophic membrane is revealing an unexpectedly 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." 4e Large-scale EST project completed for improved annotation of the Tribolium genome. Without accurate annotation and functional analysis of an insect genome sequence, the value of that sequence to science and industry is reduced. In cooperation with Kansas State University, we isolated and characterized 30,000 expressed sequences (mRNAs) from various tissues of Tribolium and compared the proteins encoded by these sequences to all of the millions of known proteins, catalogued at NCBI, as well as to all 16,400 proteins predicted from the Tribolium genome sequence. The results of this anaylsis provide a major refinement of the Tribolium genome annotation. 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." 4f Transposon-mediated germline transformation for analysis of gene function. Transposons are extremely powerful tools for genetic dissection, genome manipulation, and for revealing gene functions, but their potential to contribute to a deeper understanding of insect biology has still not been reached. We demonstrated that the piggyBac transposon can be used to illuminate the tissue specificity of insect gene expression by inserting a green fluorescent protein gene next to the section of the insect gene that regulates its transcription. Transgenic insects carrying such artificial genes displayed bright green fluorescence in only those tissues in which the test gene is normally expressed. We are now appying this technology to the functional analysis of biopesticide target genes in the insect midgut and cuticle. 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." 4g First sequence obtained for the Y chromosome of a beetle. Almost nothing is known about the molecular details of sex determination in beetles, a knowledge gap that impedes our understanding of pest insect reproduction and population biology. We examined the genome sequence of the red flour beetle, and were able to identify more than 400 kb of Y chromosomal sequence, an estimated 10-20% of the entire chromosome. This sequence contained 52 computed genes, most of which represented retrotransposons. There were no known vital genes among these 52, but there were several genes of unknown function. This is the first significant examination of the male-associated chromosome in any beetle, and sets the stage for a more detailed examination of Y chromosome structure and function. 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." 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of active CRADAs and MTAs 1 Number of patent granted 1 Number of web sites managed 3 Number of non-peer reviewed presentations and proceedings 9 Review Publications Lorenzen, M.D., Kimzey, T., Shippy, T.D., Brown, S.J., Denell, R.E., Beeman, R.W. 2007. Piggybac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Molecular Biology 16:265-275. Karumbaiah, L., Oppert, B.S., Jurat-Fuentes, J.L., Adang, M.J. 2007. Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae). Comparative Biochemistry and Physiology 146: 139-146. Vitecek, J., Petrlova, J., Adam, V., Havel, L., Kramer, K.J., Babula, P., Kizek, R., 2007. A fluorimetric sensor for detection of one living cell. Sensors 7: 222-238. Vinokurov, K.S., Elpidina, E.N., Oppert, B.S., Prabhakar, S., Zhuzhikov, D.P., Dunaevsky, Y.E., Belozersky, M.A. Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion. Comparative Biochemistry and Physiology, Part B 145: 138-146. Available: doi: 10.1016/j.cbpb.2006.05.004. Vinokurov, K.S., Elpidina, E.N., Oppert, B.S., Prabhakar, S., Zhuzhikov, D.P., Dunaevsky, Y.E., Belozersky, M.A. Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae. Comparative Biochemistry and Physiology, Part B 145: 126-137. Available: doi:10.1016/j.cbpb.2006.05.005. Prabhakar, S., Chen, M., Elpidina, E.N., Vinokurov, K.S., Smith, C.M., Marshall, J., Oppert, B.S. 2007. Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.. Insect Molecular Biology 16: 455-468. 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Insights into social insects from the genome of the honeybee Apis mellifera. Nature. 443:931-949. Miyaji, T., Kouzuma, Y., Yaguchi, J., Yatsumoto, R., Kanost, M.R., Kramer, K.J., Yonekura, M., Beeman, R.W. 2007. Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein. Journal of Insect Biochemistry and Molecular Biology 37: 960-968.