Presence of Nasonia wasps and the bacterium Arsenophonus nasoniae in calliphorid fly pupae collected from great tit nests in Coimbra, Portugal.

During the breeding season of 2021, we sampled pupae from great tit nests at Mata Nacional do Choupal (40º13’N, 8º27’W), a mixed woodland on the periphery of the city of Coimbra, Portugal (more details of the study area in (Norte et al., 2009). Old nest material from all nest boxes had been previously removed at the end of the previous breeding season to avoid the accumulation of overwintering <em>Protocalliphora</em> spp. adults (Gold and Dahlsten, 1983). Once all fledglings had left their nests in May/June, the nests were collected in plastic bags and transported to the laboratory. Blowfly puparia were sorted manually from the nest material and were stored in 1.5 mL tubes with absolute ethanol for further genetic analysis. <br> To determine the presence of <em>N. vitripennis </em>and its endosymbiont <em>A. nasoniae</em>, up to five pupae collected from each nest were surface sterilised with 70% ethanol, added to a 2 ml Eppendorf tube, crushed and homogenised using a sterile pestle. Fly pupal shells were removed with the aid of a sterile forceps and DNA was extracted from the homogenised sample using a Zymoresearch Quick DNA TM Miniprep plus kit (Zymoresearch, USA). These were then subject to diagnostic QC PCR of the COI gene with HCO/LCO primers described above (this amplifies fly DNA in the absence of other parties, and establishes template quality). Then, PCR assays for wasp and microbe were performed. Amplicons for the <em>A. nasoniae</em> PCR assay were sequenced using the Sanger method to confirm identity. <br> Column A: Nest ID Column B: Collection date for fly pupae Column C: Number of fly pupae combined for testing. Column D: Presence of Nasonia (- = not present += present) Column E: Presence of A. nasoniae (- = not present += present)