JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2020-036 e2020-36 | Vol. 52 First report of molecular characterization and phylogeny of Trichuris fossor Hall, 1916 (Nematoda: Trichuridae) Malorri R. Hughes1,*, Deborah A. Duffield1, Abstract Dana K. Howe2 and Dee R. Denver2 Because species of Trichuris are morphologically similar and ranges 1Department of Biology, Portland of host preference are variable, using molecular data to evaluate spe- State University, 1719 SW 10th Ave, cies delineations is essential for properly quantifying biodiversity of SRTC Rm 246, Portland, Oregon, and relationships within Trichuridae. Trichuris fossor has been report- 97201. ed from Thomomys spp. (Rodentia: Geomyidae, ‘pocket gophers’) hosts based on morphological features alone. Partial 18S rRNA se- 2Department of Integrative Biology, quences for specimens identified as T. fossor based on morphol- Oregon State University, 3029 ogy, along with sequences from 26 additional taxa, were used for Cordley Hall, Corvallis, Oregon, a phylogenetic analysis. Evolutionary histories were constructed us- 97331. ing maximum likelihood and Bayesian inference. In both analyses, *E-mail: [email protected] the specimens fell within the Trichuris clade with 100% support and formed a distinct subclade with 100% support. These results confirm This paper was edited by that T. fossor is a distinct species and represent the first molecular Zafar Ahmad Handoo. report for it. Relatedness among species within the family were well Received for publication resolved in the BI tree. This study represents an initial effort to obtain November 7, 2019. a more comprehensive view of Trichuridae by including a new clade member, T. fossor. A better understanding of Trichuridae phylogeny could contribute to further characterization of host-associations, in- cluding species that infect livestock and humans. Keywords 18S rRNA, Western pocket gophers, Thomomys, Trichuridae, Trichuris, Trichuris fossor, Systematics, Whipworm. Nematodes of the genus Trichuris (Roederer, 1761) Understanding the diversity and phylogeny of (‘whipworms’) inhabit the ceca of a wide variety of whipworms is important; false classification limits mammalian hosts worldwide (Callejón et al., 2015; our acuity of biogeography and conceals the zo- Robles et al., 2018). Approximately 80 species are onotic potential of trichurids (Callejón et al., 2015; described based mostly on reports that have used Doležalová et al., 2015). Certain species, e.g. T. suis biometrics, morphological features, host(s) infected, (Schrank, 1788) and T. trichiura (Linnaeus, 1771), are and/or geography (Guardone et al., 2013; Doležalová problematic in developing countries and have vast et al., 2015; Callejón et al., 2016). These characteris- socioeconomic impacts via human or livestock in- tics are not reliable in all instances – many species fections (nearly 1 billion human trichuriasis infections have similar attributes with overlapping ranges of are reported globally each year) (Jex et al., 2014). measurements (Callejón et al., 2015, 2016; Vejl et al., Other species, such as T. muris (Schrank, 1788; Hall, 2017) and host preference may be more variable than 1916), have been gathering attention in biomedical previously thought (Doležalová et al., 2015). Approxi- research for potential use in immunosuppression mately 24 species been reported from rodents, many therapy (Feliu et al., 2000). A more comprehensive of which exhibit comparable morphological patterns understanding of relationships within this group (Robles et al., 2014; Doležalová et al., 2015; Eberhardt would enable predictions about how close relatives et al., 2019). interact with their host(s). © 2020 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ First molecular report of Trichuris fossor: Hughes et al. Relationships within Trichuridae have not been well Gardner and Jasmer (1983). Collected parasites were resolved using genetic approaches; results differ de- stored in 95% ethanol. Based on morphology and pre- pending on the gene(s) sequenced and the approach vious records for the hosts, nematodes found in the used for phylogenetic reconstructions (Callejón et al., ceca were tentatively identified asT. fossor (Chandler, 2015). Mitochondrial data, primarily cox1, have been 1945; Todd and Lepp, 1972; Gardner, 1985). commonly used and have allowed for high resolu- DNA extraction, amplification, and sequencing were tion of closely related lineages; however, it may be performed on individuals from each host species. Be- less credible to use with Trichuris species due to the fore beginning isolation, nematodes were transferred degree of hybridization and maternal mitochondri- to 1.5 mL microcentrifuge tubes and repeatedly rinsed al heredity seen in this genus (Callejón et al., 2015; with DI water (5 rinses of 1 mL dH2O) to remove all trac- Doležalová et al., 2015). Nuclear data have provided es of ethanol. Nematodes were then transferred into higher support for relationships than mitochondrial fresh PCR tubes and mechanically homogenized be- data (Doležalová et al., 2015). The nuclear ITS1-ITS2 fore extracting with the DNeasy Blood and Tissue Kit genes offer markers that allow closely related species (Qiagen) following the manufacturer’s protocols. Over- to be detected (Eberhardt et al., 2019) and ITS1-5.8S- lapping fragments of 18S rRNA were amplified using the ITS2 has been used to show relations among rumi- primers G18S4F (5′-GCTTGTCTCAAAGATTAAGCC-3′), nant- and rodent-infecting species (Doležalová et al., 136R (5′-TGATCCTTCTCGCAGGTTCACCTAC-3′), 652F 2015). However, the number of variants of RNA genes (5′-GCAGCCGCGGTAATTCCAGCTC-3′), and 647R (5′-CAT (including the ITS2 region) makes their utility in dis- TCTTGGCAAATGCTTTCGC-3′) (Callejón et al., 2013). entangling the phylogeny of Trichuris less opportune, After PCR products were visualized on a 1.5% agarose particularly given that the amount of ploidy is un- gel, they were SPRI-purified (Elkin et al., 2001) and pre- known (Doležalová et al., 2015). The 18S rRNA gene pared for direct end sequencing. Sequencing reactions has been used to infer the placement of trichurids were processed by the Center for Genome Research within Nematoda as well as to elucidate relationships and Biocomputing (CGRB; Oregon State University, within Trichuridae and is less prone to result in unclear Corvallis, OR). multiple alignments (Callejón et al., 2013, 2015; Guar- Sequences were examined using MEGA v. 7.0.26 done et al., 2013; Doležalová et al., 2015). To date, (Kumar et al., 2016). Forward and reverse sequenc- both nuclear and mitochondrial data have suggested es for individual segments were combined by align- that Trichuris may be a polyphyletic genus; species or ment using MUSCLE, followed by combining the groups within the genus, e.g. T. trichiura and T. suis, two overlapping segments. Low-quality ends were may also be polyphyletic (Doležalová et al., 2015). trimmed and a BLAST search against the NCBI nr Trichuris fossor (Hall, 1916) has been reported only database was performed. Sequence information from from hosts belonging to the genus Thomomys (Wied- the BLAST match for 26 related taxa was incorporat- Neuwied 1839) (Rodentia: Geomyidae) (Todd and ed into a phylogenetic comparison. These additional Lepp, 1972; Gardner, 1985; but see Falcón-Ordaz, Trichuris spp. included in the analyses infect dogs, 1993). Descriptions have been based on morphology humans, pigs, sheep, and other rodents (murids, cri- and host preference and Trichuris from geomyid cetids, and arvicolids). Sequences were aligned using hosts have never been sequenced (Eberhardt et al., MUSCLE, ends were trimmed, and 1,644 base pairs 2019). The aim of this study was to serve as the first remained. The newly generated sequences were molecular report for T. fossor. The 18S rRNA gene was submitted to the GenBank database under acces- sequenced for four specimens identified putatively as sion numbers MT071351, MT071352, MT071353, and T. fossor collected from separate Thomomys (western MT071354. pocket gophers) species hosts. Phylogenetic analyses were performed in MEGA v. 7.0.26 (Kumar et al., 2016) and in BEAST2 v. 2.6.1 Materials and methods (Bouckaert et al., 2019). MEGA determined that Ki- mura 2-parameter with invariant sites and a gamma Thomomys bulbivorus (Richardson, 1829; Brandt, distribution was the best fit substitution model for this 1855) were salvaged from trappers working in Yam- data based on Bayesian information criterion. An evo- hill County, Oregon, in April 2018. Thomomys bottae lutionary history was inferred based on this model us- (Eydoux and Gervais, 1836), T. mazama (Merriam, ing the maximum likelihood (ML) method and a con- 1897), and T. talpoides (Richardson, 1828) were col- sensus tree was generated using 1,000 bootstrapping lected from Curry, Jackson, and Grant County, re- replicates in MEGA. The Bayesian inference (BI) anal- spectively, Oregon in August 2019. Complete intestinal ysis was prepared in BEAUti v. 2.6.0 (Bouckaert et al., tracts were examined following procedures outlined by 2019) and performed in BEAST2 v. 2.6.0 (Bouckaert 2 JOURNAL OF NEMATOLOGY et al., 2019). The analysis used the HKY substitution sister taxa with 100% support in both analyses. model with equal frequencies (K2P + I + G is not availa- These findings are consistent with previous studies ble in BEAST2) and ran for 1×107 generations. Tracer v (Feldman and Ramirez, 2014; Borba