Developing a Fecal DNA Method With Blocking Primers to Determine Red Knots' (Calidris canutus) Diets Noninvasively

Understanding the diets of migratory shorebirds such as the Red Knot (Calidris canutus) is essential for evaluating habitat quality, informing conservation policies, and monitoring ecosystem health. Fecal DNA can be complementary to traditional approaches to understanding shorebirds' diets, as it offers a noninvasive, efficient, and high-resolution alternative for dietary analyses. However, one significant challenge with fecal DNA is the presence of host DNA, which can dominate polymerase chain reaction (PCR) amplification, hindering the detection of ingested prey DNA. In this study, we designed a blocking primer that reduced DNA amplification in Red Knots (Calidris canutus) by 68.3% in the absence of the blocking primer to 34.6% when the primer was used (paired t = 4.11, df = 10, p = 0.002; Wilcoxon signed-rank p = 0.003). The method was validated on field samples (n = 52) from Cape Cod, MA, and multiple prey taxa were identified, including horseshoe crab eggs, a major prey item in this area. The success of this study confirms the utility of blocking primers in dietary assessment involving host-dominated eDNA samples.