Development of PCR Blocking Primers Enabling DNA Metabarcoding Analysis of Dietary Composition in Hematophagous Sea Lamprey

Since the establishment of the invasive sea lamprey (Petromyzon marinus) in the Great Lakes, extensive management efforts have attempted to reduce negative impacts on native fishes. Despite a significant reduction in sea lamprey population abundance following application of several control methods, uncertainties remain concerning damage caused by sea lamprey predation on Great Lakes fish populations. While conventional dietary assessments are hindered by the hematophagous nature of sea lamprey, DNA metabarcoding offers a promising alternative by identifying prey species DNA from sea lamprey digestive tract samples. DNA metabarcoding has been used for dietary analyses in numerous species, including lampreys; however, initial assessments using polymerase chain reaction (PCR) primers designed to amplify the 12S rRNA gene in vertebrate taxa indicated a high presence of sea lamprey amplification product per sample. To minimize sea lamprey DNA co-amplification, we designed and tested eight blocking primers that suppress the amplification of sea lamprey 12S sequences while allowing amplification of host species DNA. This approach allowed for the use of a single marker to amplify a taxonomically diverse suite of host species, in contrast to previous studies that used multiple taxon-specific primer pairs (e.g. Salmonidae, Cyprinidae, and Catostomidae). Candidate blocking primers evaluated in this study differed in base pair length, end sequence modification, and purification method. Samples with different sea lamprey-to-host DNA ratios were subjected to multiple detection methods including gel electrophoresis, quantitative PCR, and DNA metabarcoding to assess the ability of each blocking primer to selectively suppress amplification of the sea lamprey 12S gene region. All blocking primers tested performed well and demonstrated high effectiveness. Results show that the blocking primers evaluated can facilitate molecular diet analysis in sea lamprey, allowing the amplification of a taxonomically diverse range of host fish species with universal primers.