The study of diet is central to wildlife ecology, management and conservation. Metabarcoding increased the capability to identify species contributing to wildlife diet and blocking primers can maximize the detection of prey. Relative read abundance (RRA) of different prey species has been used as semi-quantitative approach, assuming that RRA reflects species contribution to diet. However, this approach has been contested and it is unclear how blocking primers might affect the result. We tested accuracy of RRA to estimate diet by feeding captive wolves six different diets. We analyzed samples without and with four blocking primer concentrations (5, 10, 15 and 20x) to provide insight into the validity of the semi-quantitative metabarcoding approach and the influence of blocking primer in results. RRA produced a highly accurate representation of actual contribution to diet, that was best without blocking primer (0.775 ± 0.033; P <0.001; R2=0.815) with no difference in the number of diet items detected when compared with analyses with blocking primer. Adding blocking primer resulted in higher proportions of reads of diet items, as opposed to wolf sequences, but did not increase the probability of detecting diet components, increased detections of items not fed to wolves, and produced slightly less accurate estimates of diet composition. Finally, resampling suggested that sample sizes beyond 30 scats reduced the variation in results. While our results are promising and support the use of metabarcoding to determine volumetric contribution of items to diet, caution and further research are needed before safe extrapolation to filed studies.

Agriculture expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from faecal eDNA. To help understand the impact of agricultural expansion, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the faeces of farmland birds. The capacity to identify diet and parasites of two existing and three developed mini-barcodes was tested “in silico” in relation to the behaviour of a reference eukaryotic primer set. Of the mini-barcodes tested, MiniB18S_81, one of our designs, showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa sequences. This primer pair was tested on faeces samples from five different steppe bird species. According to our data, this barcode shows good taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes and Apicomplexa, at the order level. The mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasitology of birds, essential for conservation and management.