Location: Livestock Arthropod Pests Research2018 Annual Report
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.
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.
Objective 1. The genome of the horn fly has been sequenced, assembled, annotated, and published. The sequences from this genome have been released to the scientific community through National Center for Biotechnology Information's Whole Genome Shotgun Database. Predicted gene sequences from this work have been analyzed to identify novel targets for fly control technology. An additional level of long-read sequencing has been completed on the cattle fever tick, Rhipicephalus microplus, to improve the assembly and gene prediction accuracy and bring that tick's genome sequence assembly to the reference quality level. Objective 2. A comprehensive set of over 150 horn fly genes that encode metabolic enzymes has been derived from the genome assembled and published by ARS-Kerrville, Texas scientists. The expression of these genes has been analyzed to produce a set of genes that are over-expressed in pesticide resistant horn fly adults. This gene set is being tested to find individual genes which can be used to predict insecticide resistance in populations of flies on cattle. Through an agreement initiated in FY17, the collaborator has produced, as anti-tick vaccine antigen, four cattle tick proteins as recombinant proteins in yeast protein production systems for evaluation. The production of 10 horn fly proteins in a recombinant protein yeast expression system has been initiated. These proteins were identified through reverse vaccinology analysis at ARS-Kerrville, Texas as part of the agreement and will be evaluated for effectiveness as active ingredients in anti-fly vaccine. A capillary feeding protocol for cattle ticks has been implemented as a method to pre-screen anti-tick vaccine antigen candidates prior to full-scale pen testing on cattle. Antigens showing efficacy in the capillary feeding protocol are prioritized for testing in the more resource intensive cattle stall tests. Objective 3. The genome of the cattle fever tick, Rhipicephalus annulatus, has been sequenced and assembled in a collaboration with Texas A&M AgriLife Genomics and Bioinformatics Service, College Station, Texas. Under this collaboration, the genome of the cattle tick, Rhipicephalus microplus, has been resequenced using the new Pac Bio Sequel long-read technology with the aim of bringing the genome assembly of this globally important tick to the reference quality gold standard level. As an emergency response to the outbreak and establishment of the longhorned tick, Haemaphysalis longicornis, in the U.S., the genome of this tick has been sequenced and the assembly and annotation has been prioritized to facilitate the initiation of a reverse vaccinology approach to identify anti-tick vaccine candidates effective against this species. The genome of a strain of the tropical bont tick, Amblyomma variegatum, from the Caribbean island of Guadeloupe was sequenced in a collaboration between ARS scientists at Kerrville, Texas and external researchers at the National Center for Genome Resources, Santa Fe, New Mexico and the French Agricultural Research Centre for International Development (CIRAD).
1. Publication and release of the assembled horn fly genome sequence. The genome of the horn fly contains the biological blueprint necessary to allow the fly to develop and survive over its lifetime, including how the fly responds to external stimuli and threats such as pesticides. ARS scientists at Kerrville, Texas collaborated with researchers at National Center for Genome Resources, Santa Fe, New Mexico and Texas A&M University, College Station, Texas to sequence, assemble, and annotate this fly's genome. Genes involved in pesticide resistance and metabolism, sex determination, and blood feeding were identified and described. This new comprehensive dataset is being used to identify the various mechanisms used by the fly to resist pesticides and develop novel fly control technology that will be relevant to agriculture.
2. Sequencing the genome of the longhorned tick. The longhorned tick, Haemaphysalis longicornis, is a known serious pest of livestock in Asia where it occurs. It is an aggressive biter with a diverse host range and a vector of several viral, bacterial, and protozoan agents of livestock and human diseases. This tick has recently established populations in at least 3 U.S. states and as an emergency response assistance to USDA-APHIS, ARS scientists at Kerrville, Texas collaborated with researchers at Texas A&M University AgriLife in College Station, Texas to sequence the genome of the tick. This completed genome opens new avenues of research on tick control methodology, including vaccine development and detection of pesticide resistance-associated genes.
Rodriguez Valle, M., Guerrero, F. 2018. Anti-tick vaccines in the omics era. Frontiers in Bioscience. 10:122-136.
Swale, D., Li, Z., Guerrero, F., Perez De Leon, A.A., Foil, L. 2017. Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster. Pesticide Biochemistry and Physiology. 141:41-49.
Barrero, R., Guerrero, F., Black, M., McCooke, J., Schilkey, F., Perez De Leon, A.A., Miller, R., Bruns, S., Dobry, J., Mikhaylenko, G., Stormo, K., Bell, C., Tao, Q., Bogden, R., Moolhuijzen, P., Hunter, A., Bellgard, M. 2017. Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: Assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome. International Journal for Parasitology. 47(9):569-583.
Klafke, G.M., Miller, R., Tidwell, J.P., Barreto, R., Guerrero, F., Kaufman, P., Perez De Leon, A.A. 2017. Mutation in the sodium channel gene corresponds with phenotypic resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to pyrethroids. Journal of Medical Entomology. 54(6):1639-1642.
Konganti, K., Guerrero, F., Schilkey, F., Ngam, P., Jacobi, J., Umale, P., Perez De Leon, A.A., Threadgill, D. 2018. A whole genome assembly of the horn fly, Haematobia irritans, and prediction of genes with roles in metabolism and sex determination. G3, Genes/Genomes/Genetics. 8:1675-1686.