A simple molecular assay differentiates two burrowing mayfly species, Hexagenia limbata and H. rigida (Ephemeroptera: Ephemeridae), in western Lake Erie

Burrowing mayflies (Hexagenia spp.) are important indicators of mesotrophic water conditions, and have been studied for several decades in conjunction with the changing trophic status of western Lake Erie. However, most studies on the biology of burrowing mayflies have failed to differentiate between immature H. limbata and H. rigida, the life stages that are most commonly collected. We developed a simple molecular assay to facilitate species-level identification of the two mayflies as an easier alternative to sequencing the COI gene. We analyzed a total of 152 cytochrome c oxidase, subunit I barcoding sequences from the Barcode of Life Data System (BOLD) for these mayfly species and developed a diagnostic primer set. We tested our primers on 46 random burrowing Lake Erie mayfly nymphs using both conventional PCR and qPCR. Results of the two methods were consistent and unambiguous. Sequencing of the COI barcoding region in the mayflies confirmed that the PCR assays correctly identified mayflies in all cases. Our assay is simple and effective, and will facilitate species-level identification of burrowing mayflies in future studies on their biology where the two species co-occur.