Frequency of A. nasoniae in calliphorid fly pupae collected from European bird's nests

<em>Nasonia vitripennis </em>commonly oviposits in calliphorid ‘filth fly’ pupae found in birds’ nests. Nests of blue tit (<em>Cyanistes caeruleus</em>), great tit (<em>Parus major</em>) and other passerine birds were collected shortly following fledging from the nest and placed in bags. Collections were made in June/July 2012 from the area surrounding Harjavalta Town, Finland; from Lausanne, Switzerland; from Kraslava, Latvia; and from Marbury, Cheshire in the UK. Each nest was given an ID number, was manually inspected for calliphorid/protocalliphorid pupae, and these pupae removed individually to a tube for any parasitising wasps to emerge. Where more than one pupa was collected from the same nest this was noted, as pupae from the same nest may be parasitised by the same individual wasp. The pupae, which were monitored daily, were placed at 25 °C for 14 days to allow for <em>N. vitripennis</em> wasps to emerge. <br> Arsenophonus nasoniae presence in <em>N. vitripennis</em> was then assayed. DNA was prepared using the Chelex method. Template quality was assessed based on COI amplification using primers HCO/LCO (Folmer et al, (1994), with 30 PCR cycles of 93°C for 15s, 47°C for 1m, 72°C for 1m and an expected amplicon size of. c. 710bp. Templates passing this quality control were then tested for <em>A. nasoniae</em> using primers amplifying the metalloprotease gene (M1f GGGTCACATACCTATTTT, M1r GTAGTCGCCTGGGTGGG), with 30 cycles of 93°C for 15s, 55°C for 45s, 72°C for 30s and an expected amplicon size of 594bp . <br> Sheet 1: Pupa ID: Unique identifier for fly pupa collected from birds nest. Note, the initial number represents the nest ID (column B) and a suffix letter indicates independent pupae from that nest. Nest ID: Unique identified for the birds nest the pupa was collected from. Arse +/-: Presence of Arsenophonus (+) in Nasonia emerging from that wasp as ascertained through PCR assay. Null value means Arsenophonus not detected. Location: Place the nest/pupa was located. <br> Sheet 2: Summary data. <br>