Location: Livestock Arthropod Pests Research
2016 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: Sequencing and annotation of the genome of the horn fly and cattle fever tick. The assembly and annotation of the horn fly genome and 18 tissue and/or life stage-specific transcriptomes has been completed. The analysis of the horn fly genome is on-going, specifically directed toward genes involved in pesticide resistance and metabolism. The cattle fever tick genome has been assembled and annotated and a manuscript reporting this accomplishment and the genome analysis has been submitted to international journal peer-review. The cattle tick genome assembly is serving as a model for evaluating novel bioinformatics algorithms for their usefulness in assembling large and complex genomes seen in other tick species.
Objective 2: Investigate molecular-based control and surveillance technologies. Horn fly genes that encode proteins involved in metabolism or sequestration of pesticides have been identified in the newly assembled and annotated transcriptome datasets and their expression levels in pyrethroid resistant populations of horn flies determined. A manuscript reporting this information is being drafted in conjunction with bioinformaticians at Texas A&M University. Two cattle stall test trials of ARS-developed anti-cattle tick vaccine Antigen 2 were conducted with CRADA partner. Production of ARS-patented anti-cattle tick vaccine Antigen 1 was inconsistent and new production methods have been used to produce new yeast strains that secrete the recombinant Antigen 1 into the growth media, facilitating purification. Forty strains have been screened for secreted production, however a high yielding strain has not been found. A chimeric protein-coding region has been designed for production and secretion of the immuno-reactive segments of Antigen 1 and this chimera is being screened for Antigen 1.
Objective 3: Increase sequence information and genetic annotation of livestock pests, focusing on biological aspects likely to be affected by climate change. A collaborative project was initiated with French government researchers at their laboratory in the Caribbean island of Guadeloupe. Tissues from the tropical bont tick, Amblyomma variegatum, were dissected to obtain ovary, gut, salivary gland, and synganglia tissues for transcriptome sequencing. Genomic DNA was purified to serve as source material for a genome sequencing project. A genome sequencing and reverse vaccinology project for the cattle tick, Rhipicephalus annulatus, was initiated with Texas A&M University AgriLife researchers. Specific tick tissues and life stages have been collected for genome and transcriptome sequencing by a Pacific Biosciences Sequel platform recently implemented for large complex genomes such as found in ticks.
Accomplishments
1. Assembly and annotation of the cattle tick genome. The genome of an organism is like a master template that guides the organism's development, metabolism, and responses to environmental perturbations within its ecosystem. ARS scientists at Kerrville, Texas, in collaboration with researchers at Murdoch University's Centre for Comparative Genomics, Murdoch, Australia have assembled and analyzed the genome of the cattle tick, identifying its full complement of genes and their sequences. Knowing these sequences will facilitate the development of novel tick control technologies aimed at specific gene products targeted by new pesticides or anti-tick vaccines.
A collaboration between ARS scientists at Kerrville, Texas, and the University of Texas-El Paso Bioinformatics Program has entered its final year of funding from a successful grant application to USDA/NIFA Hispanic Serving Institutions Education Grants Program. The grant was provided to support a graduate level student internships to conduct bioinformatic analysis of research data generated at the ARS lab in Kerrville. The overall objective for this collaboration is the identification of G-Protein Coupled Receptors (GPCRs), proteins that would be attractive targets for development of novel pesticides. One Master's student participated with an internship at the ARS-Kerrville lab in FY 2016, and two other Master's students participated while located in El Paso. As direct outcome, one paper was published in an international peer-reviewed parasitology journal and one additional manuscript has been drafted and is in journal review.
Review Publications
Temeyer, K.B., Tuckow, A.P. 2016. Tick salivary acetylcholinesterase: A probable immunomodulator of host-parasite interactions. Journal of Medical Entomology. 53:500-504.
Coates, B.S., Poelchau, M.F., Childers, C., Evans, J.D., Handler, A.M., Guerrero, F., Skoda, S.R., Hopper, K.R., Wintermantel, W.M., Ling, K., Hunter, W.B., Oppert, B.S., Perez De Leon, A.A., Hackett, K.J., Shoemaker, D.D. 2015. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Current impacts and future prospects. Trends in Entomology. 11(1):1-27.
Hussein, H.E., Scoles, G.A., Ueti, M.W., Suarez, C.E., Adham, F.K., Guerrero, F., Bastos, R.G. 2015. Targeted silencing of the aquaporin 2 gene of rhipicephalus (Boophilus) microplus reduces tick fitness. Parasites & Vectors. doi: 10.1186/s13071-015-1226-2.
Guerrero, F., Kellogg, A., Ogrey, A., Heekin, A.M., Barrero, R., Bellgard, M., Dowd, S., Leung, M. 2016. Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus. Ticks and Tick Borne Diseases. 7(5):670-677.
