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                    U.S. DEPARTMENT OF AGRICULTURE
 Veterinary Pest Genetics Research Unit: Kerrville, TX
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ARS Home Plains Area Kerrville, Texas Knipling-Bushland U.S. Livestock Insects Research Laboratory Veterinary Pest Genetics Research Unit Research





- Saelao, Perot
- 2023
- 2022
- Publications
- Food Animal Production (101)
- Animal Health (103)
- Veterinary, Medical and Urban Entomology (104)

Research Project: Genetics of Veterinary Pests **Location:
                            Veterinary Pest Genetics Research Unit** 2023 Annual Report **Objectives**
                                

Objective 1: Perform bioinformatic analysis of tick and fly genomes to find new targets of control and methods of surveillance.
Objective 2: Develop genetic methods to control ticks and dipteran pests of livestock and wildlife, to include new vaccines through reverse vaccinology, gene editing/silencing, gene drives, and other genetic approaches to mitigate acaricide resistance. **Approach**
                                

Genetic studies on arthropods of medical and veterinary importance helps identify and elucidate gene activities in target organisms. Genomic techniques provide information on population genetics and support identifying invasive insect source populations and migration pathways, the development of gene targets for disruption or RNAi gene suppression, identifying pest species that are difficult to identify morphologically, reverse vaccinology for the discovery of new antigen candidates, and more rapid discovery of natural enemies using next generation sequencing and metagenomics. Research on fly and tick metagenomes (the entire genomic complement found in the environment) and microbiomes (the universe of microbes living in association with each pest) generates information about pathogens and commensal and symbiotic microorganisms that can be used to solve agricultural problems associated with these organisms. Scientists have worked with U.S. university partners and international institutions to generate ‘omic resources such as transcriptomes, microbiomes, and genomes for a number of livestock and human pests, including biting midges, mosquitoes, and house flies. Mosquito mitochondrial genomes sequenced using nanopore technology support the development of databases for rapid identification of field samples. Sequencing of pooled RNA-Seq is valuable for gene expression analysis of pathogens that cause disease in humans, livestock, and other animals. Understanding of genetics and bionomics are critical in the developing sustainable integrated pest management (IPM) programs. Defining the problem and selecting appropriate control strategies requires obtaining background information on pest identification (systematics and taxonomy), distribution (spatial and temporal), and behavior (particularly behaviors that cause or have the potential to cause damage). Fundamental research on pest genetics generates information that can be used to identify weaknesses of the pest; findings can also be used to help develop models that assess entomological and/or epidemiological risk to host populations. Genetic studies of pests are rapidly generating a wealth of information that can be used to develop new and adaptive pest control measures using CRISPR CAS9 and Gene Drive technologies. In practice, each genetically based control method is applied individually and locally, so it is especially challenging to project if or how laboratory results will be replicated in integrated field studies. Developing new genetic and chemical pest control measures that result in commercial IPM products often requires coordinating stakeholder efforts, funding, and other resources. **Progress Report**
                                

Project #3094-22320-001-000D, Genetics of Veterinary Pests, is a new project beginning in fiscal year (FY) 2022 as the result of a realignment Program Direction and Resource Allocation Memo (PDRAM)(Nov. 2021). Objective 1 is newly created (no FY22 milestones) and Objective 2 was transferred from Project #3094-32000-042-000D, Integrated Pest Management of Cattle Fever Ticks.
In support of Objective 1, perform bioinformatic analysis of tick and fly genomes to find new targets of control and methods of surveillance, ARS scientists at Kerrville, Texas, utilized recently acquired Pacbio and Illumina sequencers to complete the whole genome sequencing and scaffolding of the U.S. strain of the Asian longhorned tick, Haemaphysalis longicornis. Candidate targets for reverse vaccinology have been identified for the development of target antigen. For sequencing of two Dermacentor and two Amblyomma tick genomes. 
In support of Subobjective 2A, evaluate the efficacy of novel acaricides and delivery systems against ticks on livestock, and through the targeted treatment of infested pastures and protected and sensitive habitats, ARS scientists at Kerrville, Texas, constructed and tested in vitro (cell culture) using the dual luciferase reporter system to validate and select constructs effective for gene silencing of tick G protein coupled receptors (GPCR). Constructs that were shown to be effective silencing constructs in vitro were injected into unfed adult female ticks that were placed onto cattle hosts for further development.  Silencing efficiencies in vivo were determined at 3- and 5-days post injection by dissection and quantitative polymerase chain reaction (PCR) assessment of target gene expression in silenced and control tick samples. Effects of tick gene silencing was assessed morphologically during tick dissections, and by evaluation of tick survival and reproduction in silenced ticks compared to controls. GPCR targets evaluated included the pyrokinin receptor and periviscerokinin receptor of Rhipicephalus microplus. Results of these studies demonstrating effects on tick viability and reproduction were published. Continuing research is underway to assess effects of synthetic effector (agonists/antagonists) molecules targeting the periviscerokinin receptor, utilizing gene silencing to validate target specificity of the small molecules.
ARS researchers at Kerrville, Texas, selected specific recombinant DNA molecules encoding specific tick antigens as potential anti-tick vaccine antigen candidates in support of Subobjective 2B, identify candidate antigens for anti-tick vaccines and formulate as vaccines for animal trials. Computational methods and reverse vaccinology algorithms were used to screen and select candidates for their anti-tick vaccine potential. Of the selected protein antigen candidates that were chosen for recombinant protein analysis in P. pastoris, two candidates were successfully purified in quantities sufficient to formulate vaccine doses. These two candidate vaccines were utilized to conduct and complete cattle stall tests to evaluate vaccine efficacy against R. microplus. 
In support of Subobjective 2C, compare the genomes of R. microplus, R. annulatus, and H. longicornis to identify sex determination genes for the development of genetic control methods, ARS researchers at Edinburg, Texas, completed a comparative analysis of sex determining genes from 17 species of hard ticks including R. microplus, R. annulatus, H. longicornis, D. andersoni, A. maculatum, R. sanguineus, and I. scapularis. Two Ornithodoros soft tick species and four species of mites were also included in the analysis. This analysis was used to identify sex determination pathway gene orthologues for comparison to R. microplus. Additionally, ARS researchers in collaboration with Texas A&M University demonstrated that the R. icroplus reference genome was in need of an update and identified potential target genes for development of genetic control methods in the R. microplus. **Accomplishments** **Review Publications**
                            *Wulff, J.P., Temeyer, K.B., Tidwell, J.P., Schlechte, K.G., Lohmeyer, K.H., Peitroantonio, P.V. 2022. Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight, and reproductive output. Parasites & Vectors. https://doi.org/10.1186/s13071-022-05457-7.*
                                *Temeyer, K.B., Schlechte, K.G., Gross, A.D., Lohmeyer, K.H. 2023. Identification, baculoviral expression and biochemical characterization of a novel cholinesterase of Amblyomma americanum (Acari: Ixodidae). International Journal of Molecular Sciences. https://doi.org/10.3390/ijms24097681.*
                                *Dos Santos, E.G., Dos Santos Bezerra, W.A., Temeyer, K.B., Perez De Leon, A.A., Costa-Juior, L.M., Dos Santos Soares, A.M. 2021. Effect of essential oils on native and recombinant acetylcholinesterase of Rhipicephalus microplus. Brazilian Journal of Veterinary Parasitology. 30. Article e002221. https://doi.org/10.1590/S1984-29612021024.*


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