BRIEF RESEARCH REPORT published: 15 February 2021 doi: 10.3389/fvets.2021.639400 Host Bloodmeal Identification in Cave-Dwelling Ornithodoros turicata Dugès (Ixodida: Argasidae), Texas, USA Rachel E. Busselman 1, Mark F. Olson 2, Viridiana Martinez 3, Edward Davila 1, Cierra Briggs 2,4, Devon S. Eldridge 2,5, Bailee Higgins 2, Brittany Bass 2, Thomas L. Cropper 6, Theresa M. Casey 6, Theresa Edwards 7, Pete D. Teel 2, Sarah A. Hamer 1 and Gabriel L. Hamer 2* 1 Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, United States, 2 Department of Entomology, Texas A&M AgriLife Research, College Station, TX, United States, 3 Department of Ecology and Conservation Biology, Texas A&M University, College Station, TX, United States, 4 Department of Entomology, Cornell University, Ithaca, NY, United States, 5 Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, Knoxville, TN, United States, 6 59th Medical Wing, Joint Base San Antonio, Lackland, San Antonio, TX, United States, 7 Texas Parks and Wildlife Department, Government Canyon State Natural Area, San Antonio, TX, United States Edited by: Tick-host bloodmeal associations are important factors when characterizing risks of Sebastián Muñoz-Leal, associated pathogen transmission and applying appropriate management strategies. University of Concepcion, Chile Despite their biological importance, comparatively little is known about soft tick Reviewed by: (Argasidae) host associations in the United States compared to hard ticks (Ixodidae). In Markéta Nováková, Masaryk University, Czechia this study, we evaluated a PCR and direct Sanger sequencing method for identifying the Deon Bakkes, bloodmeal hosts of soft ticks. We collected 381 cave-associated Ornithodoros turicata Agricultural Research Council of South Africa, South Africa near San Antonio, Texas, USA, and also utilized eight colony-reared specimens fed *Correspondence: artificially on known host blood sources over 1.5 years ago. We correctly identified the Gabriel L. Hamer vertebrate host bloodmeals of two colony-reared ticks (chicken and pig) up to 1,105 [email protected] days post-feeding, and identified bloodmeal hosts from 19 out of 168 field-collected soft Specialty section: ticks, including raccoon (78.9%), black vulture (10.5%), Texas black rattlesnake (5.3%), This article was submitted to and human (5.3%). Our results confirm the retention of vertebrate blood DNA in soft ticks Parasitology, and advance the knowledge of argasid host associations in cave-dwelling O. turicata. a section of the journal Frontiers in Veterinary Science Keywords: soft ticks, Argasidae, Ornithodoros turicata, blood meal, host identification Received: 08 December 2020 Accepted: 20 January 2021 Published: 15 February 2021 INTRODUCTION Citation: Busselman RE, Olson MF, Martinez V, The identification of arthropod host-feeding patterns through bloodmeal analysis can provide key Davila E, Briggs C, Eldridge DS, information for vertebrate host contact and pathogen transmission networks (1–4). Bloodmeal Higgins B, Bass B, Cropper TL, analysis methods based on the detection of vertebrate DNA left in the residual bloodmeal are widely Casey TM, Edwards T, Teel PD, used across diverse arthropod taxa. For example, reservoir hosts of Leishmania were identified by Hamer SA and Hamer GL (2021) Host studying previous bloodmeals of sand flies (5), and host-feeding patterns in mosquitoes allowed Bloodmeal Identification in Cave-Dwelling Ornithodoros turicata for an enhanced understanding of the reservoirs of West Nile virus (6, 7). Molecular analysis of Dugès (Ixodida: Argasidae), Texas, bloodmeals has also been used to identify a broad host community for Culicoides, vectors of avian USA. Front. Vet. Sci. 8:639400. Haemosporida infections (8), and of triatomines, vectors of Trypanosoma cruzi, agent of Chagas doi: 10.3389/fvets.2021.639400 disease (9). Frontiers in Veterinary Science | www.frontiersin.org 1 February 2021 | Volume 8 | Article 639400 Busselman et al. Bloodmeal Analysis of Soft Ticks Bloodmeal analysis, applied to ticks, has repeatedly been caves over 2 days for 18 and 21.5h, respectively (Figure 2). associated with limited success (10, 11), likely owing to DNA These caves are closed to park visitors and were selected based degradation during the molt and many months since prior on a previous study which demonstrated robust O. turicata bloodmeal acquisition. Given their importance as vectors of populations in these caves (31). Traps consisted of 1.9L coolers human pathogens, several studies have conducted bloodmeal filled with dry ice (cooler spout open) bolted through the bottom ◦ analysis of hard ticks (Ixodidae), identifying vertebrate hosts to a 0.41 m2 untreated 3-ply pine plywood with edges cut at 45 in 20–93% of analyzed ticks (12, 13). Given the challenges of angles to improve tick access to the surface of the board. Double PCR-Sanger sequencing-based bloodmeal analysis of hard ticks, sided carpet tape (Roberts, Boca Raton, FL) was applied to the alternative strategies have been evaluated to identify bloodmeal surface of the board, and insect glue boards (Bell Laboratories, hosts, including analysis of the variation in stable isotopes in fed Madison, WI) were cut into strips and applied to the carpet tape ticks (10, 14, 15), reverse line blot (16, 17), and proteomics (18). and under the corners of the plywood board. The ticks were In comparison, relatively few studies have attempted to identify removed from the sticky tape on-site and placed into ethanol- the bloodmeal hosts of argasid ticks (soft ticks). filled 1.5ml tubes; each vial contained 3–10 ticks depending Ornithodoros turicata, found in the southwestern on the number of ticks caught at each trap each day. Samples ◦ United States and Florida (19), is a vector of human and were transported to the laboratory and stored at 4 C until animal pathogens. O. turicata is a known vector of tick-borne DNA extraction. relapsing fever caused by Borrelia turicatae (20), and is also a putative vector for transmission of African swine fever virus, an emerging disease in Africa, Europe, and most recently Asia Tick Processing and DNA Extraction (21, 22). This DNA virus is transmitted by soft ticks of the O. turicata specimens were measured, identified to species and Ornithodoros genus and is highly pathogenic to domestic swine life stage by morphological features, and sexed if adults (32). A (23). While African swine fever has yet to be detected in the U.S., subset of 124 ticks were photographed to serve as a reference recent studies have identified O. turicata as a most likely vector for confirming life stage and sex (Supplemental Data Sheet 1). should the virus reach the US (24, 25). Photos were taken early in the identification process and Ornithodoros turicata is found in caves or burrows occupied throughout tick cataloging as needed when life stage or sex was or visited by diverse vertebrate hosts (19). Larvae, nymphs difficult to determine. We attempted to record the size of the and adults attach, blood-feed, and drop from a host quickly bloodmeal based on the shape of the abdomen and presence (typically 15–20min); thus, they are seldom collected from hosts of blood; however, the storage in ethanol produced a dark during blood feeding events, complicating knowledge of tick- red color in all specimens and visual bloodmeal scoring was host associations (26). Further, they can survive for years between not reliable. bloodmeals as nymphs and adults (27). The first 20 ticks collected at each site, followed by equal Identification of the host community that supports O. turicata numbers of the largest ticks from each cave (based on length), populations could be useful in providing an ecological basis were processed further using the following methods until a for vector control and disease management. We previously threshold of 40% of the total number of collected ticks had conducted a bloodmeal analysis study using quantitative PCR for been processed. To minimize exogenous DNA on the exterior the identification of vertebrates in experimentally fed O. turicata surface of ticks, ticks were washed in ethanol for 5s, then a 10% (28). The results demonstrated vertebrate DNA could be detected bleach solution for 15 s, and finally two consecutive 15 s DNA- 330 days post-feeding and through multiple molts, suggesting free water rinses immediately after (10, 33, 34). On a sterile longer retention of bloodmeal DNA in soft ticks compared microscope slide over ice, the legs were then removed, placed ◦ to hard ticks. The current study builds on these prior results in ethanol, and stored at −40 C to decrease the amount of by conducting a PCR-Sanger sequencing bloodmeal analysis tick DNA processed and to preserve samples of each tick for protocol on O. turicata fed experimentally on known hosts as well future use. The tick bodies were placed in clean tubes, flash- as field-collected specimens from cave environments in Texas frozen in liquid nitrogen, and crushed with a sterile pestle (Wards near the location of recent outbreaks of TBRF in humans (29, 30). Sciences, Rochester, NY), which was discarded after each use. The crushed tissue was then lysed, and DNA was extracted using MATERIALS AND METHODS the MagMAX CORE Nucleic Acid Purification Kit (Applied Biosystems ThermoFisher Scientific, Waltham, Massachusetts) Tick Collections according to the manufacturer’s protocol. Negative and positive We obtained eight adult O. turicata specimens (five male, three controls, including