Location: Livestock Arthropod Pests Research
2015 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: Sequence and annotate the genome of the horn fly and cattle fever tick. We have completed the sequencing of the horn fly genome using next- and 3rd-generation sequencing technologies. We have also sequenced transcriptomes from 18 different tissues, organs, and life stages of the horn fly, which enables assembly of the horn fly genome. The genome of the cattle tick, Rhipicephalus microplus, has been assembled to a Version 2.0. This assembly is a draft quality assembly utilizing Illumina-based protocols that were focused upon the gene-rich region of the genome. A manuscript to report cattle tick genome assembly has been started. The assembly of the complex repetitive fraction of the genome, that was sequenced using Pacific Biosciences 3rd-generation technology, is ongoing and approximately 50% completed.
Objective 2: Investigate molecular-based control and surveillance technologies. We have used horn fly transcriptome datasets to identify several transcripts encoding membrane proteins with critical roles in the horn fly life cycle. Two of these transcripts have been selected for testing as anti-fly vaccine candidate antigens and are being expressed in Pichia pastoris. Evaluation of anti-tick vaccines has progressed with CRADA partners. A CRADA-partnered cattle trial to evaluate two ARS-patented anti-cattle tick vaccine antigens, RmAPQ1 (R. microplus aquaporin 1) and RmGMP (R. microplus gut membrane protein), for efficacy against R. microplus has begun at the ARS-Cattle Fever Tick Research Laboratory. ARS Antigen 7 has been produced in sufficient quantities for cattle trials conducted by a Brazilian CRADA partner to test for efficacy against R. microplus. Three other ARS antigens are in the production stages for similar evaluations by the same CRADA partner. Cattle tick and horn fly transcriptome datasets acquired from Illumina-based deep sequencing technology have been analyzed to obtain a dataset of transcripts encoding enzymes involved in metabolic resistance to pesticides. Several insecticide resistant populations of horn flies and cattle ticks have been tested with PCR-based assays to quantify expression of some of these metabolic resistance-associated transcripts. We chose to identify and characterize a horn fly cDNA encoding a protein previously identified in mosquitoes as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) delta-endotoxin. This target was chosen because we demonstrated that Bti is insecticidal to horn flies, and mosquitoes have not developed resistance to Bti despite many years of mosquitocidal use. We identified a horn fly cDNA encoding the Bti receptor and are working to identify portions of the protein that may be suitable subunit immunogens to develop a vaccine to protect cattle from horn fly infestations.
Objective 3: Increase sequence information and genetic annotation of livestock pests, focusing on biological aspects likely to be affected by climate change. The mitochondrial gene, Cox1, is often used to determine phylogenetic relationships between populations and species. We have sequenced a diagnostic fragment of Cox1 and determined the phylogeny of putative R. microplus samples collected by ARS scientists and collaborators from India and the Philippines in relation to a laboratory R. microplus Texas outbreak strain known as Deutsch.
Accomplishments
1. Sequencing the genome of the horn fly. The genome of an organism is like a master template that guides the individual's development, metabolism, and responses to environmental perturbations, determining the organism's success within its ecosystem. ARS scientists at Kerrville, Texas, in collaboration with researchers at National Center for Genome Resources, Santa Fe, New Mexico, have completed the sequencing of the horn fly genome. The genome sequence will be computationally assembled and annotated to identify the full complement of genes in the horn fly. Knowing the gene sequences of the horn fly will facilitate the development of new fly control technologies by allowing the identification of specific gene products that can be targets for new insect-specific pesticides and anti-fly vaccines.
Review Publications
Xu, Q., Guerrero, F., Palavesam, A., Perez De Leon, A.A. 2015. Use of electroporation as an option to transform the Horn Fly, Haematobia irritans: a species recalcitrant to microinjection. Insect Science. 00:1-9.
Meganathan, P.R., Singh, B., Wells, J.D., Guerrero, F., Ray, D.A. 2014. A 454 sequencing approach to dipteran mitochondrial genome research. Genomics. 105:53-60.
Mccooke, J., Guerrero, F., Barrero, R., Black, M., Hunter, A., Bell, C., Faye, S., Miller, R., Bellgard, M. 2015. The mitochondrial genome of a Texas outbreak strain of the cattle tick, Rhipicephalus (Boophilus) microplus, derived from whole genome sequencing Pacific Biosciences and Illumina reads. Elsevier. Gene. 571(1):135-141.
Bendele, K.G., Guerrero, F., Miller, R., Li, A.Y., Barrero, R., Moolhuijzen, P., Black, M., Mccooke, J., Meyer, J., Hill, C., Bellgard, M. 2015. Acetylcholinesterase 1 in populations of organophosphate-resistant North American strains of the cattle tick, Rhipicephalus microplus (Acari: Ixodidae). Parasitology Research. 114(8):3027-3040.
Rodriguez-Vivas, R.I., Perez-Cogollo, L.C., Rosado-Aguilar, J.A., Ojeda-Chi, M.M., Trinidad-Martinez, I., Miller, R., Li, A.Y., Perez De Leon, A.A., Guerrero, F., Klafke, G.M. 2014. Rhipicephalus (Boophilus) microplus resistant to acaricides and ivermectin in cattle farms of Mexico. Brazilian Journal of Veterinary Parasitology. 23(2):113-122.
Tuckow, A.P., Temeyer, K.B. 2015. Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in Rhipicephalus (Boophilus) microplus cell lines. Insect Molecular Biology. 24(4):454-456.
Guerrero, F., Perez De Leon, A.A., Rodriguez-Vivas, R.I., Jonsson, N., Miller, R., Andreotti, R. 2014. Acaricide research and development, resistance and resistance monitoring. In: Sonenshine, D.E., Roe, M.R., editors. Biology of Ticks. New York, NY: Oxford University Press. p. 353-381.
Guerrero, F., Andreotti, R., Bendele, K.G., Cunha, R.C., Miller, R., Yeater, K.M., Perez De Leon, A.A. 2014. Rhipicephalus (Boophilus) microplus aquaporin as an effective vaccine antigen to protect against cattle tick infestations. Parasites & Vectors. 7:475.
Barrero, R., Guerrero, F., Moolhuijzen, P., Goolsby, J., Bellgard, S., Tidwell, J.P., Bellgard, M. 2015. Shoot transcriptome of the Giant Reed, Arundo donax. Genomics Data. 3:1-6.
Bellgard, M.I., Sleeman, M.W., Guerrero, F., Fletcher, S., Baynam, G., Goldblatt, J., Rubinstein, Y., Bell, C., Groft, S., Barrero, R., Bittles, A.H., Wilton, S.D., Mason, C.E., Weeramanthri, T. 2014. Rare Disease Roadmap: Navigating the challenges and barriers to deliver improved outcomes for patients living with a Rare Disease. Health Policy and Technology. 3(4):325-335.
