Location: Veterinary Pest Genetics Research Unit
2024 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 initiative that began in fiscal year (FY) 2022 following a realignment as per the Program Direction and Resource Allocation Memo (PDRAM) issued in November 2021. The project has two primary objectives: Objective 1, which is newly established with no FY24 milestones, and Objective 2, which has been transferred from Project #3094-32000-042-000D, Integrated Pest Management of Cattle Fever Ticks.
Objective 1 focuses on using bioinformatic analysis of tick and fly genomes to discover new control targets and surveillance methods. ARS scientists at Kerrville, Texas, utilized Pacbio, Illumina, and Nanopore sequencing technologies to complete the whole genome sequencing and scaffolding of the U.S. strain of the Asian longhorned tick, Haemaphysalis longicornis. From this de novo genome, computationally derived vaccine targets that are predicted to immune stimulate the host immune system were identified using Scinet resources. These target antigens will be tested in an artificial feeding system with ARS partners at Pullman, Washington. Dermacentor genomes have polished genome assemblies which have now been annotated and will be used for identification of CRISPR/Cas9 target gene silencing. Additionally, in another effort, the de novo genome of the tropical bont tick, Amblyomma variegatum, has been sequenced along with the resequencing of historically preserved samples to understand genetic variation before and after outbreaks. Collaboration with the University of Houston aims to identify structural variations and sex determination pathways in domestic house flies (Musca domestica). Results of this research revealed significant chromosomal rearrangements that are crucial for regulating fly splicing loop promotor function allow for sex specific development.
Objective 2 focuses on the integrated pest management of arthropods of veterinary importance. Under Subobjective 2A, the goal is to evaluate the efficacy of new acaricides and delivery systems for tick control. ARS scientists at Kerrville, Texas, constructed and tested in vitro systems using the dual luciferase reporter system to validate gene silencing constructs targeting tick G protein-coupled receptors. Targeted gene silencing of the R. microplus leucokinin receptor was conducted, validating its impact on tick survival and reproduction. The research localized the leukokinin receptor in tick midgut surfaces using synthetic fluorescent markers.
In support of Subobjective 2B, ARS Scientists at Kerrville, Texas, with partners in Brazil have identified and formulated candidate antigens for anti-tick vaccines and tested them in cattle animal trials. Specific recombinant DNA molecules encoding tick antigens were selected as potential vaccine candidates. These candidates were formulated and tested for efficacy in FY24, demonstrating strong potential for protective immunity against R. microplus. Effective formulations are currently under review for patenting and potential for commercial use.
Subobjective 2C aims to compare the genomes of R. microplus, R. annulatus, and H. longicornis to identify sex determination genes for developing genetic control methods. ARS researchers at Edinburg, Texas, completed a comparative analysis of sex-determining genes from hard tick species, including R. microplus, R. annulatus, H. longicornis. The genome of R. microplus was significantly improved by identifying misassemblies and putative sex chromosome fragments. This improvement is vital for advancing our understanding of tick genetics and developing targeted control strategies.
Additionally, ARS scientists at Kerrville, Texas, have identified and are working on expressing a candidate target for a horn fly vaccine. Stall trials to test for immunogenicity are expected to occur towards the end of FY24. This comprehensive project aims to advance the understanding and control of veterinary pests through genomic analysis, vaccine development, and innovative pest management strategies.
Accomplishments
1. Horn fly transcriptomes of 10 populations from the southern U.S. Pesticide resistance in horn flies poses a significant threat to livestock health and productivity. Development of pesticide resistance leads to increased treatment costs and necessitates the development of alternative control strategies to manage horn fly populations. ARS scientists at Kerrville, Texas, sequenced the transcriptomes of ten horn fly populations from the southern United States with varying degrees of pesticide resistance. This comprehensive dataset provides valuable insights into biology of resistance, aiding in the development of targeted control strategies and contributing significantly to the field of insecticide resistance research. Biological processes regulating various aspects of fly biology were highly correlated with the increased susceptibility to pesticides. In addition, processes involved with chemical compound absorption and binding were highly enriched in susceptible populations, potentially identifying a specific mechanism for potential future control.
2. Development of a novel isolation protocol for microRNAs from tick ex vivo salivary gland cultures and extracellular vesicles. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in regulating gene expression with significant potential as a target for tick control. ARS scientists at Kerrville and Edinburg, Texas, developed a novel method of isolating miRNAs from tick salivary gland cultures and extracellular vesicles (EVs). This method streamlines the process of feeding and processing ticks, enabling for the enrichment of miRNAs extracted from tick tissues. This method also establishes the steps necessary for the downstream bioinformatic analysis of miRNAs and the identification of conserved and unique miRNAs found within various tick species. This new protocol also opens avenues for studying pathogen-vector interactions and identifying miRNA targets for functional studies.
Review Publications
Bendele, K.G., Guerrero, F., Lohmeyer, K.H., Foil, L., Metz, R., Johnson, C. 2023. Horn fly transcriptomes from 10 populations from the southern United States. Data in Brief. https://doi.org/10.1016/j.vetpar.2022.109699.
Leal, B., Harvey, C., Thomas, D.B., Oliva-Chavez, A. 2022. A method for the isolation of miRNAs from tick ex vivo salivary gland cultures and extracellular vesicles. The Journal of Visualized Experiments (JoVE). https://doi.org/10.3791/63618.