Location: Livestock Arthropod Pests Research
2017 Annual Report
Objectives
Objective 1: Sequencing and annotation of the genome of the horn fly and cattle fever tick.
Subobjective 1A: Assembly and annotation of the cattle tick genome sequence.
Subobjective 1B: Sequencing, assembly, and annotation of the genome sequence of the horn fly.
Objective 2: Investigate molecular-based control and surveillance technologies.
Subobjective 2A: Identify candidate antigens for anti-biting fly and anti-tick vaccines and formulate as vaccines for animal trials.
Subobjective 2B: Identification of gene-based mechanisms of pesticide resistance and develop gene-based surveillance assays to monitor gene flow.
Objective 3: Increase sequence information and genetic annotation of livestock pests, focusing on biological aspects likely to be affected by climate change.
Approach
Utilize advanced bioinformatic assembly and annotation protocols to attain a draft annotated R. microplus genome sequence of sufficient quality for publication in international peer-reviewed journals. The assembled and annotated sequence will be made available for the scientific community at CattleTickBase (http://ccg.murdoch.edu.au/cattletickbase) and GenBank.
Sequence the horn fly genomic DNA with similar protocols and caveats utilized to sequence the tick genome. The horn fly genomic DNA to be sequenced will be obtained from a laboratory colony maintained since 1961 at our laboratory, reared in cages and feeding upon cotton pads saturated with citrated bovine blood. The assembled sequence will be available for the scientific community by submission of the data to GenBank.
Identify candidate vaccine antigens through reverse vaccinology from datasets obtained from prior project or as part of objective 1 of this project. Utilize real-time PCR to study metabolic-based pesticide resistance and quantify gene expression of specific horn fly cytochrome P450s in populations of horn flies with known metabolic resistance-based mechanisms.
Use a transcriptomic approach to sequence nuclear and mitochondrial genes from tick collections in Indian and Philippine collections to use for phylogenetic comparisons to the Texas outbreak R. microplus Deutsch population that was used in prior R. microplus transcriptome studies.
Progress Report
Objective 1. The genome of the cattle fever tick has been assembled and annotated, and a manuscript reporting this achievement has been accepted for publication by International Journal for Parasitology. The sequences from this assembled genome have been released to the scientific community through Murdoch University's CattleTickBase, National Center for Biotechnology Information's Whole Genome Shotgun Database, NIAID's VectorBase, and USDA's AgData Commons. Sequences from this genome and transcriptome have been mined in our new Cooperative Research and Development Agreement (CRADA) research project to identify and produce novel anti-tick vaccine antigens. The horn fly genome sequence has been annotated and a manuscript reporting this accomplishment has been submitted to an international journal for peer review. A new cooperative research and development agreement partnership is exploring the cattle tick and horn fly genome and transcriptome sequences using a reverse vaccinology approach to develop vaccine-based control technologies effective against ticks and biting flies.
Objective 2: The activities of several horn fly genes were surveyed in pyrethroid resistant horn fly populations sampled from various ranches in the U.S. The genes selected for analysis function in metabolism of the fly's ingested blood meal and are involved in pesticide degradation in other fly species. A straightforward relationship between the activity level of these genes in the sampled horn flies and pesticide resistance level was not evident. A more comprehensive set of horn fly genes have been discovered from the newly available horn fly genome sequence and this larger gene set is being evaluated for effects on pesticide resistance. A new CRADA was initiated to identify and evaluate horn fly proteins that could serve as the active ingredient in an anti-biting fly vaccine for cattle. The experimental approach will use a genome-based technique called reverse vaccinology. This approach uses computational predictions of effectiveness in a vaccine and we have identified 10 proteins that are in the production phase for evaluations. Four anti-cattle tick vaccines have been prepared for evaluations to be conducted by CRADA partner in cattle trials.
Objective 3: Genomic DNA of the cattle tick, Rhipicephalus annulatus, has been purified for a genome sequencing project conducted with Texas A&M AgriLife collaborators. Additionally, under this collaboration, the genome assembly of the cattle tick, Rhipicephalus microplus, will be supplemented by new long-read technology sequences (50X coverage addition) and the entire genome sequence database reassembled for comparative genome studies between the two cattle tick species. The testes transcriptome from the New World screwworm has been assembled and annotated and published in the international peer-reviewed literature. As an emergency response assistance to USDA-APHIS dealing with an outbreak of New World screwworm on the U.S. mainland, we sequenced a mitochondrial DNA gene from individual outbreak larval samples for phylogenetic studies aimed at determining the geographic source of the outbreak. A new collaborative project with New Zealand government researchers has begun to sequence the genome of the New Zealand cattle tick, Haemaphysalis longicornis, with the aim of developing an anti-tick vaccine efficacious against this species. The effects of Arundo donax canopy protection on cattle tick larval survival in the Quarantine Zone are being studied at the molecular level. An experiment was completed that exposed R. microplus larvae to environmental conditions in native buffel grass vs. under a canopy provided by a mature stand of A. donax.
Accomplishments
1. Publication and release of the cattle tick genome sequence. The genome of an organism contains the information necessary to allow that organism to develop and survive over its lifetime. The genome of the cattle tick, Rhipicephalus microplus, is huge and complex to sequence, containing over twice the amount of DNA as the human genome. ARS scientists at Kerrville, Texas, in collaboration with researchers at Murdoch University's Centre for Comparative Genomics, Murdoch, Australia have released and published the genome sequence for this cattle tick. This dataset contains sequences from genes involved in evasion of bovine host immune response, pesticide resistance, maintenance of pathogens, and feeding, among others. This new comprehensive sequence information is facilitating tick vaccine research and pesticide resistance monitoring.
Review Publications
Bendele, K.G., Guerrero, F., Cameron, C., Perez De Leon, A.A. 2016. The testes transcriptome of the New World Screwworm, Cochliomyia hominivorax. Data in Brief. 9:1141-1146.
Gatto Brito, L., Da Silva Barbieri, F., Barros Rocha, R., De Sena Oliveira, M.C., Guerrero, F., Foil, L.D. 2015. Horn fly population dynamics as prediction tool for the fixation of pesticide resistance. Technical Report. 75:1677-8618.
Viol, M., Guerrero, F., Cesar Miranda, B., Conceicao Costa, M., Hudson Loiola, S., Dias De Melo, G., Helena De Souza, A., Takami Kanamura, C., Valerio Garcia, M., Andreotti, R., Marcal Felix De Lima, V., Denise Saraiva, K. 2016. Identification of Leishmania spp. promastigotes in the intestines, ovaries and salivary glands of Rhipicephalus sanguineus actively infesting dogs. Parasitology Research. 115:3479-3484.
Munoz, S., Guerrero, F., Kellogg, A., Heekin, A.M., Leung, M. 2017. Bioinformatic prediction of G protein-coupled receptor encoding sequences from the transcriptome of the foreleg, including the Haller’s organ, of the cattle tick, Rhipicephalus australis. Chemical Senses. 12(2):e0172326.
Martinez Evora, P., Sanches, G.S., Guerrero, F., Perez De Leon, A.A., Bechara, G.H. 2017. Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs. Revista Brasileira de Parasitologia Veterinaria. 26(1):60-66.
Goolsby, J., Guerrero, F., Gaskin, J.F., Bendele, K.G., Azhahianambi, P., Amalin, D., Flores-Cruz, M., Kashefi, J., Smith, L., Saini, R.K., Racelis, A., Perez De Leon, A.A. 2016. Molecular comparison of cattle fever ticks from native and introduced ranges with insights into optimal search areas for classical biological control agents. Southwestern Entomologist. 41(3):595-604.
Concha, C., Palavesam, A., Guerrero, F., Sagel, A., Li, F., Pardo, T., Hernandez, Y., Quintero, G., Vasquez, M., Phillips, P.L., McMillan, W., Skoda, S.R., Scott, M.J. 2016. A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. Proceedings of the Royal Society of London B. 14:72.
Ziska, L., A. Crimmins, A. Auclair, S. DeGrasse, J.F. Garofalo, A.S. Khan, I. Loladze, A.A. Pérez de León, A. Showler, J. Thurston, and I. Walls, 2016: Ch. 7: Food Safety, Nutrition, and Distribution. In: Basu, R., English, P. et al., editors. The Impacts of Climate Change on Human Health in the United States: A Scientific Assessment. Washington, D.C.: Global Change Research Program. 189–216. doi:10.7930/J0ZP4417
