University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln USDA National Wildlife Research Center - Staff U.S. Department of Agriculture: Animal and Publications Plant Health Inspection Service 2020 Technical Limitations Associated With Molecular Barcoding of Arthropod Bloodmeals Taken From North American Deer Species Erin M. Borland Colorado State University - Fort Collins, [email protected] Daniel A. Hartman Colorado State University - Fort Collins, [email protected] Matthew W. Hopken USDA-APHIS NWRC, [email protected] Antoinette J. Piaggio USDA APHIS Wildlife Services, [email protected] Rebekah C. Kading Colorado State University - Fort Collins, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc Part of the Natural Resources and Conservation Commons, Natural Resources Management and Policy Commons, Other Environmental Sciences Commons, Other Veterinary Medicine Commons, Population Biology Commons, Terrestrial and Aquatic Ecology Commons, Veterinary Infectious Diseases Commons, Veterinary Microbiology and Immunobiology Commons, Veterinary Preventive Medicine, Epidemiology, and Public Health Commons, and the Zoology Commons Borland, Erin M.; Hartman, Daniel A.; Hopken, Matthew W.; Piaggio, Antoinette J.; and Kading, Rebekah C., "Technical Limitations Associated With Molecular Barcoding of Arthropod Bloodmeals Taken From North American Deer Species" (2020). USDA National Wildlife Research Center - Staff Publications. 2373. https://digitalcommons.unl.edu/icwdm_usdanwrc/2373 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA National Wildlife Research Center - Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. applyparastyle "fig//caption/p[1]" parastyle "FigCapt" applyparastyle "fig" parastyle "Figure" Journal of Medical Entomology, 57(6), 2020, 2002–2006 doi: 10.1093/jme/tjaa112 Advance Access Publication Date: 23 June 2020 Short Communication Short Communication Technical Limitations Associated With Molecular Barcoding of Arthropod Bloodmeals Taken From North American Deer Species Erin M. Borland1,3, , Daniel A. Hartman1, Matthew W. Hopken1,2, Antoinette J. Piaggio2, and Rebekah C. Kading1, 1Colorado State University, 200 W Lake Street, Fort Collins, CO 80523-1690, 2USDA/APHIS/WS National Wildlife Research Center, AADate 4101 LaPorte Ave., Fort Collins, CO 80521, and 3Corresponding author, e-mail: [email protected]. AAMonth Subject Editor: David Severson Received 12 April 2020; Editorial decision 15 May 2020 AAYear Abstract Accurate species-level identification of the source of arthropod bloodmeals is important for deciphering blood feeding patterns of field-collected specimens.Cytochrome c oxidase I (COI) mitochondrial gene sequencing has been used for this purpose; however, species resolution can be difficult to obtain from certain verte- brate genera, including Odocoileus. Sanger sequencing of mitochondrial genes was employed to identify the bloodmeal source of wild-caught mosquitoes trapped in Greeley, Colorado. Initial sequencing of the COI gene of mitochondrial DNA in bloodmeals was inadequate for species-level resolution of bloodmeals from deer in the genus Odocoileus, with current databases returning low fidelity matches to multiple genera. The use of the hypervariable D loop of the control region provided species-level identification of white-tailed deer (Order: Artiodactyla, Family: Cervidae, Odocoileus virginianus); however, taxonomic identification was successful only to genus for mule (O. hemionus hemionus) and black-tailed deer (O. hemionus columbianus). We advocate the use of multiple loci for bloodmeal analysis and the buildout of available databases to include multiple mito- chondrial reference genes for reliable host species identification. Key words: bloodmeal identification,Odocoileus , deer, Arbovirus ecology, mosquito Approximately a quarter of pathogen outbreaks in the 20th century exposed to arthropods with promiscuous feeding habits, promoting were spread by hematophagous arthropod vectors, showcasing the the spread of arthropod-borne disease. Evidence also supports the important role of vector surveillance and ecology to public health susceptibility and exposure of deer to a number of additional arbo- practice (Jones et al. 2008). Deer can act as amplification hosts for viruses around the globe (Hubalek et al. 2014). Thus, reliable tech- a number of emerging or invasive viruses, including arboviruses. niques for uncovering the deer/arthropod vector network is of critical For example, epizootic hemorrhagic disease virus (EHDV) is a sig- importance from a One Health perspective (Sinclair 2019). If we un- nificant arboviral pathogen of white-tailed deer and cattle, trans- derstand more about these transmission dynamics, we can better pro- mitted by biting midges Culicoides spp. Latreille 1809 (Diptera: tect human populations from spillover of disease. Ceratopogonidae). Multiple serotypes of EHDV currently circulate in Molecular identification of vector bloodmeal source to the the United States, presenting a health threat to domestic and wild ru- species level has become an increasingly important tool to under- minants (Shope et al. 1960, Ruder et al. 2016). Current data indicate stand the transmission dynamics of arthropod-vectored pathogens that white-tailed deer Odocoileus virginianus Zimmermann 1780 (Kent 2009). Nucleotide-based approaches are widely used and a (Artiodactyla: Cervidae) are also highly susceptible to Rift Valley multitude of published PCR assays targeting mitochondrial DNA fever virus (RVFV) infection and could serve as an amplification host (mtDNA) are available (Kent 2009, Brinkmann et al. 2016, Logue if this virus is introduced to the United States (Kakani et al. 2010; et al. 2016). Concerns about the use of mtDNA sequences for spe- Hartley et al. 2011; Golnar et al. 2014, 2018; Wilson et al. 2018). cies delimitation have been raised because ancestral polymorphisms Modeling further suggests that deer could enhance spillover of some can persist long after species divergence due to incomplete lin- pathogens into human populations because of their close proximity eage sorting, as well as introgression between species dating back to high-density urban areas (Kakani et al. 2010); deer tend to inter- millions of years (Mallet and Willmott 2003). North American face with humans frequently in these areas and could potentially be deer of the family Cervidae includes the genus Odocoileus, with © The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. 2002 All rights reserved. For permissions, please e-mail: [email protected]. Journal of Medical Entomology, 2020, Vol. 57, No. 6 2003 two species O. virginianus (white-tailed deer) and Odocoileus volumes (Table 1). The COI primer cocktails were prepared using a hemionus Rafinesque 1817 (Artiodactyla: Cervidae) (mule deer), 1:1:2 ratio as described previously (Ivanova et al. 2006). The primer each with multiple subspecies (Wilson and Reeder 2005). Genetic H16501 paired most closely with available reference sequences and studies including both species have identified instances of limited was utilized (Table 1) (Purdue et al. 2000). Amplification products divergence and shared haplotypes which has been interpreted in were verified by electrophoresis on 1.2% agarose gels (Invitrogen, two ways: historic introgression (Carr et al. 1986), and incom- Carlsbad, CA), and were purified using the QIAquick PCR plete lineage sorting (Cronin et al. 1988). Reports of introgression Purification Kit (Qiagen, Venlo, The Netherlands). between these two species has found O. virginianus mtDNA in Sanger sequencing was performed by Quintara Biosciences O. hemionus individuals (Carr et al. 1986), and later studies sug- (Berkeley, CA). COI sequencing was performed using primers gested that interspecies gene flow is not unidirectional (Ballinger targeting M13-tail motifs present on the amplification primers et al. 1992). The complexity of shared genetic history and po- (Table 1). Raw sequences were trimmed and analyzed for call tential for recent hybrids can make it challenging to separate the quality and nucleotide polymorphisms using Geneious 11.1 software species with small fragments of mtDNA as used for barcoding of (Biomatters Ltd., Auckland, New Zealand). Sequences were aligned bloodmeals from arthropods. by Pairwise/Multiple alignment using the Geneious Alignment algo- As part of a larger study on mosquito community composition rithm, global alignment with free end gaps, and a 93% similarity and blood feeding behavior, we identified blood meals from a di- cost matrix. Sequences greater than 200 nucleotides in length were versity of mosquito species and locations in northern Colorado submitted to National Center for Biotechnology Information (NCBI) (Hartman et al. 2019). Using existing and widely-used protocols, we GenBank (Table 2). identified some blood meals to the family Cervidae, but were unable CR sequences were generated for a single control sample to obtain greater resolution to genus or species. Here, the utility of (WG091117-016; Odocoileus hemionus hemionus) and compared two mtDNA loci, COI and the d-loop control region (CR),