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>丽蝇蛹集金小蜂（Nasonia vitripennis）通常将卵产于鸟巢内发现的丽蝇科腐生蝇蛹中。研究人员于雏鸟离巢后不久采集青山雀（Cyanistes caeruleus）、大山雀（Parus major）及其他雀形目鸟类的巢穴并装入密封袋中。样本采集于2012年6至7月，采集地点覆盖芬兰哈尔亚瓦尔塔镇周边区域、瑞士洛桑、拉脱维亚克拉斯洛瓦，以及英国柴郡马伯里。每个鸟巢均被赋予唯一编号，研究人员人工检视巢内的丽蝇科/原丽蝇属（Protocalliphora）蛹，并将单头蛹单独转移至试管中以待寄生蜂羽化。若同一鸟巢采集到多头蛹，则予以记录——因同一巢内的蛹可能被同一只雌蜂寄生。所有蛹均置于25℃恒温环境下培养14天以促进丽蝇蛹集金小蜂羽化，期间每日进行观测记录。 随后通过分子实验检测丽蝇蛹集金小蜂样本中是否存在纳森菌（Arsenophonus nasoniae）。采用Chelex法提取基因组DNA。以引物HCO/LCO（Folmer等，1994）扩增COI基因以评估模板质量，聚合酶链式反应（PCR）设置30个循环：93℃变性15秒、47℃退火1分钟、72℃延伸1分钟，预期扩增片段大小约为710bp。通过质量检测的DNA模板进一步使用扩增金属蛋白酶基因的引物对（M1f：GGGTCACATACCTATTTT，M1r：GTAGTCGCCTGGGTGGG）进行纳森菌检测，PCR反应设置30个循环：93℃变性15秒、55℃退火45秒、72℃延伸30秒，预期扩增片段大小为594bp。 工作表1： - 蛹编号（Pupa ID）：从鸟巢采集的蝇蛹唯一标识符。注：编号前缀代表对应鸟巢编号（B列），后缀字母表示该巢内的独立采集蛹样本。 - 鸟巢编号（Nest ID）：采集蛹所在鸟巢的唯一标识符。 - Arse +/-：通过PCR检测确认，从该蛹羽化的丽蝇蛹集金小蜂体内是否存在纳森菌（"+"表示存在）。空值表示未检测到纳森菌。 - 采集地点（Location）：鸟巢/蛹的实际采集地点。 工作表2：汇总统计数据。