2005 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? What does it matter? Ticks can cause direct economic impact because of a general loss of host condition due to irritation, anemia, secondary infection, paralysis, and toxicoses. However, their major impact upon animal production is their ability to vector a wide spectrum of pathogenic microorganisms, including protozoa, rickettsiae, spirochetes, and viruses. Disease transmitted by ticks is a major factor in limiting animal production in many subtropical and tropical regions of the world. In North America, the tick vectors of Babesia bovis and B. bigemina, the protozoan agents of Cattle Fever, are the southern cattle tick, Boophilus microplus, and the cattle tick, Boophilus annulatus. These tick species were declared eradicated from the U.S. in 1943. A quarantine zone was created on the Texas-Mexico border, and an import-dipping program has been maintained to prevent reentry of the tick vector into the U.S. Reentry of the tick and babesiosis into susceptible U.S. cattle herds would present serious economic consequences for U.S. cattle producers. Effective control of ticks is paramount to the control of animal and human tick-borne diseases. Ticks have been controlled primarily with pesticides. Development of pesticide resistance and a lack of new classes of pesticides in development have stimulated interest in novel means of tick control that are both environmentally safe and sustainable in the U.S. and in other regions of the world. Genomic and proteomic information for Boophilus sp. is quite limited. Information obtained from this project will allow for the elucidation of the complex interactions between the tick-host-pathogen. This information should aid in the definition of novel non-chemical control alternatives. The long-term goal of this research is to develop the biological and molecular databases that will provide for the rational design of effective control technologies for the important animal disease vector, Boophilus microplus. The specific objectives for the 5 years of this project are: 1. Develop genomic resources that would facilitate the initiation of a Boophilus microplus genome project. 2. Develop techniques of gene silencing with B. microplus as a tool for use in gene function studies. 3. Investigate components of the B. microplus proteome that are involved in the various events associated with successful tick feeding and the facilitation of pathogen transmission. This would specifically include expressed proteins of the salivary gland and midgut, and lifestage-related differences in protein expression. It is anticipated that this integrated long-term approach to the problem of tick control based upon the development of molecular genomic and proteomic databases will provide important physiological targets for the rational design of control technologies for the cattle fever tick, B. microplus. The research to be undertaken falls under NP 104 - Veterinary, Medical and Urban Entomology and addresses goal: 3.1 Genomics and Host-Pathogen Interaction. Goal 3.1.1 specifies the use of ultrastructural methods, as well as expressed sequence tag (EST) and bacterial artificial chromosome (BAC) libraries, to investigate the basis of tick digestion, salivation, egg production, and susceptibility to protozoan parasites of livestock. This goal includes identifying and cloning genes crucial to these processes and designing agents that will inhibit or disrupt their actions. Goal 3.1.2 specifies the organization of and participation in an international consortium to sequence the entire genome of the tick B. microplus and manage the bioinformatics to allow comparison of homologous genes in different genera. Achieving these objectives will provide benefits to producers, regulators of animal diseases, and other scientists. The public will benefit from a continued flow of safe and affordable food products. The producers will realize the economic benefits of avoiding costly quarantine and control efforts. In addition to the importance of a safe and nutritious food supply, is the importance of developing environmentally safe and sustainable control technologies for ticks and the diseases they transmit. Knowledge of the tick genome and gene products will provide the database from which to develop those new control technologies. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (2005) Cot Analysis BAC Library Synthesis BAC Library End Sequencing BAC Clone Screening EST Sequencing EST Annotation and Gene Index Develop a List of Tick Researchers With Genomic Interest Draft a White Paper Proposing a Boophilus microplus Genome Project Submit a White Paper to Funding Agencies Construction of dsRNAs Year 2 (2006) BAC Clone Sequencing EST Annotation and Gene Index Introduction of dsRNAs in vivo RNAi silencing of additional targets Proteome Analysis: Eggs Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Production of stage-specific Antisera Preparation of stage-specific antigens Identification of antigens; larval Identification of antigens; nymphal Purification of anticoagulants adult female Isolation/Characterization of anti-thrombin gene Immunogenicity of anti-thrombin in the bovine Inhibition of anti-thrombin by specific antibody elicited by natural exposure Year 3 (2007) EST Annotation and Gene Index Quantitation of target mRNAs and Proteins RNAi silencing of additional targets Proteome Analysis: Unfed Larvae Proteome Analysis: Unfed Nymphs Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Salivary Gland Protein Polymorphisms Identification of antigens; adult male Purification of anticoagulants nymphal Evaluation of anti-thrombin gene polymorphisms and tissue expression Inhibition of anti-thrombin by specific antibody elicited by vaccination Year 4 (2008) EST Annotation and Gene Index RNAi silencing of additional targets Proteome Analysis: Unfed Adult Male and Female Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Identification of antigens; adult female Purification of anticoagulants larval Evaluation of anti-thrombin gene polymorphisms and tissue expression Year 5 (2009) EST Annotation and Gene Index RNAi silencing of additional targets Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Purification of anticoagulants adult male Vaccination with anti-thrombin and Challenge 4a.What was the single most significant accomplishment this past year? Submission of a Genomic White Paper ARS scientists at the Knipling-Bushland U. S. Livestock Insects Research Laboratory (KBUSLIRL) and the Cattle Tick Fever Research Laboratory (CFTRL) in collaboration with scientists at The Institute for Genomic Research (TIGR) and the Center for Disease Control (CDC) were able to complete genomic objectives 1 through 9 of the project plan, FY2005 milestones. This newly acquired knowledge and compilation of genomic resources has led to the completion of a white paper entitled “Proposal for the sequencing of a new target genome: the southern cattle tick, Boophilus microplus”, F. D. Guerrero, V. M. Nene, J. E. George, S. C. Barker, and P. Willadsen, describing the resources, rationale, needs and scientific support for a B. microplus genome sequencing project. The completed white paper was submitted to funding agencies (NHGRI and NIAID-NIH) in FY 2005. Submission of the white paper represents a major accomplishment in the first year of this research project, and it represents the initial and essential event in securing adequate funding for the conduct of a sequencing project. 4b.List other significant accomplishments, if any. Publication of Gene Index 1 ARS scientists at the KBUSLIRL in collaboration with scientists at TIGR, in an effort to develop genomic resources that would facilitate the initiation of a B. microplus genome project, have assembled a genomic database entitled Gene Index version 1 that has been published and posted on TIGR website. The database includes 45,000 EST sequences that have been assembled into a gene index of approximately 13,000 coding regions covering an estimated 35-60% of B. microplus genes. This database is available to investigators worldwide as a resource for their research projects. Salivary Gland Specific EST Sequences In complement with Gene Index 1, and to elucidate gene expression patterns that provide for successful tick feeding, ARS scientists at the KBUSLIL and CFTRL have developed tissue-specific cDNA libraries that were constructed using salivary glands of adult female B. microplus ticks that had fed on a bovine host for 1-2 days (early-feeding) and for 4-5 days (late-feeding). At present, 403 and 255 unique sequences have been annotated from the 1-2 day and the 4-5 day libraries, respectively. These data are currently being utilized to isolate and characterize full-length novel sequences from B. microplus that may facilitate successful feeding of the tick. 4c.List any significant activities that support special target populations. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Submission of a Genomic White Paper - ARS scientists at the Knipling-Bushland U. S. Livestock Insects Research Laboratory (KBUSLIRL) and the Cattle Tick Fever Research Laboratory (CFTRL) in collaboration with scientists at The Institute for Genomic Research (TIGR) and the Center for Disease Control (CDC) were able to complete genomic objectives 1 through 9 of the project plan, FY 2005 milestones. This newly acquired knowledge and compilation of genomic resources has led to the completion of a white paper entitled “Proposal for the sequencing of a new target genome: the southern cattle tick, Boophilus microplus”, F. D. Guerrero, V. M. Nene, J. E. George, S. C. Barker, and P. Willadsen, describing the resources, rationale, needs and scientific support for a B. microplus genome sequencing project. The completed white paper was submitted to funding agencies (NHGRI and NIAID-NIH) in FY 2005. Submission of the white paper represents a major accomplishment in the first year of this research project, and it represents the initial and essential event in securing adequate funding for the conduct of a sequencing project. The ever increasing genomic and proteomic databases that are originating from this research project, and are being made available to investigators worldwide, should substantially impact the rational design of future strategies for the control of ticks and the pathogens they transmit. This accomplishment addresses genomic milestones 1 through 9 of this project plan for FY 2005. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of the National Program 104 Action Plan. In addition, the accomplishment addresses Performance Measure 3.2.1 of the ARS Strategic Plan in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health. Publication of Gene Index 1 – ARS scientists at the KBUSLIRL in collaboration with scientists at TIGR, in an effort to develop genomic resources that would facilitate the initiation of a B. microplus genome project, have assembled a genomic database entitled Gene Index version 1 that has been published and posted on TIGR website. The database includes 45,000 EST sequences that have been assembled into a gene index of approximately 13,000 coding regions covering an estimated 35-60% of B. microplus genes. This database is available to investigators worldwide as a resource for their research projects. This accomplishment addresses genomic milestones 5, EST sequencing, and 6, EST annotation and gene index, of this project plan for FY 2005. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of the National Program 104 Action Plan. In addition, the accomplishment addresses Performance Measure 3.2.1 of the ARS Strategic Plan in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health. 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? Publication of Gene Index 1 and posting on the TIGR website of 45,000 EST sequences, assembled into a gene index of approximately 13,000 coding regions covering an estimated 35-60% of B. microplus genes represents a significant advance in B. microplus genomics that is available to investigators worldwide. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None Review Publications Guerrero, F., Lysyk, T.J., Kalischuk-Tymensen, L. 2004. Expressed sequence tags and new gene coding regions from the horn fly. Southwestern Entomologist. 29(3):193-208. Poels, J., Nachman, R.J., Akerman, K.E., Oonk, H.B., Guerrero, F., De Loof, A., Janecka, A.E., Torfs, H., Vanden Broeck, J. 2005. Pharmacology of stomoxytachykinin receptor depends on second messenger system. Peptides. 26:109-114. Ullmann, A.J., Lima, C.M.R., Guerrero, F.D., Piesman, J., Black IV, W.C. 2005. Genome size and organization in the blacklegged tick, Ixodes scapularis and the southern cattle tick, Boophilis microplus. Insect Molecular Biology. 14(2):217-222. Untalan, P.M., Guerrero, F.D., Haines, L.R., Pearson, T.W. 2005. Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus. Insect Biochemistry and Molecular Biology. 35:141-151. Guerrero, F.D., Miller, R.J., Rousseau, M.-E, Sunkara, S., Quackenbush, J., Lee, Y., Nene, V. 2005. BmiGI: A database of cDNAs expressed in Boophilus microplus, the tropical/southern cattle tick. Insect Biochemistry and Molecular Biology. 35:585-595.