Knowledge of feeding ecology of declining species, such as farmland birds, is essential to address their conservation requirements, especially when their habitats are suffering important reductions of trophic resources. In this study, we apply a metabarcoding approach to describe the diet composition of six of the most significant farmland birds inhabiting European cereal pseudo-steppes: little bustard, great bustard, pin-tailed sandgrouse, black-bellied sandgrouse, red-legged partridge, and common quail. We further studied seasonal diet variations (autumn to spring) in all species but the common quail, whose diet was studied during spring and summer. We show that study species´ diets mostly consisted of plants, although in the case of little bustard and great bustard arthropods are also highly relevant. Among arthropods, we found high proportions of thrips, arachnids, and springtails, which were previously unreported in their diet, and some taxa that could be used as antiparasitic food. Moreover, we report that little bustard’s diet is the least rich of that of all studied species, and that diet of all these species is less diverse in winter than in autumn and spring. Diet composition of these declining species supports the importance of natural and semi-natural vegetation and landscape mosaics that can provide a wide variety of arthropods, plants, and seeds all year-round.

Agricultural 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 the 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 fecal environmental DNA. To help to understand the impact of agricultural expansion on biodiversity, this study sought to design and identify mini‐barcodes that might simultaneously assess diet and intestinal parasites from the feces of farmland birds. The capacity to identify diet and parasites of 2 existing and 3 newly developed mini‐barcodes was tested “in silico” in relation to the behavior of a reference eukaryotic barcode. Among the newly designed mini‐barcodes, MiniB18S_81 showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa. Moreover, when it was tested on fecal samples from 5 different steppe bird species, MiniB18S_81 showed high taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes, and Apicomplexa at the order level. Thus, the mini‐barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasites of birds, essential for conservation and management.

Many farmland bird populations are declining, and their negative trends are often associated with changes in land-use or farming practices, including the use of agrochemicals. The red-legged partridge (RLP) is a Mediterranean farmland game species of high socio-economic importance whose populations are thought to have declined sharply since the mid-20th century associated with farmland changes. However, no large-scale studies have tested whether abundance or trends of RLP are related to farmland composition or management. We used hierarchical distance sampling models to estimate RLP abundance in 2010 in central Spain (Castilla-La Mancha), a main European population stronghold of this species. We studied associations between RLP density and land-uses (including variation in management: irrigated crops or organic farming). We also assessed regional abundance variation over seven years (2010-2017) and its relationship with changes in land-use. Our results show that RLP abundance increased with the availability of natural vegetation and traditional rain-fed vineyards, but decreased with increasing proportions of tree crops and irrigated vineyards; the latter association was less pronounced in areas sensitive to nitrate contamination in water, where the amount of fertilizers applied in farmland and use of certain farming practices is more strictly regulated. These results support the idea that increases in intensive vineyards are detrimental to the RLP. We also report a strong population decline of RLP in the region, with a 51% abundance reduction in seven years. This decline was steeper in areas where more natural vegetation had been lost and where ecological tree crops had increased. Overall, our results indicate that changes in land-use (type of crop, or the destruction of natural vegetation in farmland) and farming practices (e.g. use of irrigation in certain crops, use of nitrates) have important impacts on this farmland bird, affecting both spatial distribution and population dynamics.

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.