Location: Tick and Biting Fly Research
2005 Annual Report
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.
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
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.
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.
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.