| Summary: | Birds face many challenges during a breeding attempt including predation, food availability, pathogens and parasite load. Cyanistes caeruleus (blue tit) prefer human placed nest boxes, however the stable microclimate presented within the nest box exacerbates the challenges posed by ectoparasites and potentially pathogenic bacteria, with reductions in breeding success reported. The addition of green plant material to help control these deleterious effects has been reported within Mediterranean climes but comparable studies have not been undertaken in temperate regions. This study introduces novel molecular approaches including next generation sequencing methods to assess the nest microbiome. This approach avoids the culturing bias of previous work in this area. Terminal-Restriction Fragment length Polymorphism (T-RFLP) analysis was used to assess bacterial richness progression through the breeding attempt with more traditional methods used to assess bacterial load. DNA barcoding was performed to identify bacteria, ectoparasites and vascular plant fragments present within the nest with vegetation surveys conducted around a subset of nests to assess if any active selection of plant material was occurring. Bacterial species richness and load were relatively stable between nest build and clutch completion with a significant increase in both post fledging, following the introduction of nestling faeces in the nest and reduced time for nest sanitation. DNA barcoding provided marked increases in the taxonomic knowledge of nest dwelling biota with 169 bacterial taxa, thirteen species of ectoparasite and 154 vascular plant taxa identified. Although ectoparasites and pathogenic bacteria were detected within the nest no effect was seen upon hatching or fledging success. It is more likely that a reduction in fitness would be observed post fledging. A high proportion of plant material containing volatile compounds was recorded within the nest, however active selection could not be confirmed.
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