Targeted ITS2 rDNA Sequencing to Analyze Morphological Strain Variants in Two Geographically Separated Populations of the Digenean Trematode Proterometra macrostoma

Noah Kelner Earlham College, Maleeka Shrestha Earlham College
Faculty Sponsor(s): Peter Blair Earlham College
Trematodes are a globally diverse and highly successful class of flatworm. Cercariae are a larval stage of Digenean trematodes that emerge from aquatic gastropods, an intermediate host, eventually infecting a vertebrate, the definitive host, where the parasite matures into the adult form. Proterometra macrostoma, a fairly common trematode found in the Midwest, was originally described to have eight morphological variants from specimens collected from North Elkhorn Creek, Scott County, Kentucky (Riley and Uglem 1995). In 2014, we identified a population of P. macrostoma cercariae emerging from the freshwater gastropod, Pleurocera semicarinata livescens in Clear Creek, Wayne County, Indiana. Our prior research identified complete large subunit rDNA sequence identity (~1400 bp) between both phenotypic variants and geographically separated populations. The goal of this study was to utilize direct sequencing of the more variable internal transcribed spacer-2 (ITS2) region from individual cercariae to validate our prior results. Two independent PCR primer sets were used to amplify the majority of the ITS2 region (>400 bp). Independent recombinant clones of the resulting amplicons were produced and verified by restriction digest. Each strand of the recombinants was independently sequenced and analyzed using BioEdit software. The results of our direct ITS2 rDNA sequencing validates our prior findings by confirming a lack of allopatric speciation between the two geographical areas and phenotypic variants. Future studies would include sequencing of other target genes from unique chromosomal regions to ultimately validate our findings.
Oral Presentation

When & Where

02:00 PM
Jordan Hall 172