Pollen DNA metabarcoding approach to profile the nectar source for urban beekeeping in Tokyo Bay Area, Koto-ku

Apis mellifera are a honey bee that introduced around the world as industrial beekeeping species. Recently, urban beekeeping has attracted attention for the purpose of ecosystem protection and urban greening. This study investigated to understand nectar sources by pollen DNA metabarcoding though urban beekeeping in Koto-ku, Tokyo. The Musashino university beekeeping using honey bee, A. mellifera, was carried out on the roof of the second building at Musashino University Ariake Campus in the Ariake district of Koto-ku on the Tokyo Bay coast. The honey containing pollen was sampled from a relatively new hive that divided into three periods of early, middle and late months between mid-March, 2018 to mid-October, 2018. After adding water to reduce viscosity of honey, whole liquid phase was grinded with beads. Genomic DNA was extracted using an MPure Bacterial DNA Extraction Kit. The extracted DNA solution was mixed with a final concentration of 5% PVPP and purified by collecting the supernatant after centrifugation. An amplicon library targeting a part of rbcL region in chloroplast genome was constructed by 2-step tailed PCR approach. A mixture of multiple libraries was sequenced by 2 x 300 bp paired-end reads using a MiSeq platform. A total of 32 spots could be sampled except early July. In early-May, early-, mid-, and late-June, mid-July, early- and mid-August, mid-September, and early-October, samples were taken from multiple hives. Sequence data of 6,994-95,832 paired-end reads were output for each sample.

西方蜜蜂（Apis mellifera）是作为工业养蜂物种被引种至全球的蜂种。近年来，以生态保护与城市绿化为目标的城市养蜂受到广泛关注。本研究针对东京都江东区的城市养蜂项目，通过花粉DNA宏条形码（pollen DNA metabarcoding）技术探究其蜜源植物。武藏野大学的养蜂实验使用西方蜜蜂（A. mellifera），实验点位设于东京湾沿岸江东区有明地区的武藏野大学有明校区第二教学楼楼顶。研究于2018年3月中旬至2018年10月中旬期间开展，从一个相对较新的蜂箱中采集含花粉的蜂蜜样本，并按月份分为早、中、晚三个时段进行采样，除7月初外共计完成32组有效采样。其中，在5月初、6月初、6月中旬、6月下旬、7月中旬、8月初、8月中旬、9月中旬以及10月初，均从多个蜂箱采集样本。实验流程如下：先向蜂蜜中加水以降低其粘度，随后将全部液相进行珠磨均质处理；使用MPure细菌DNA提取试剂盒提取基因组DNA；向提取得到的DNA溶液中添加终浓度为5%的聚乙烯吡咯烷酮（PVPP），经离心后收集上清液完成DNA纯化；采用两步尾引物PCR法构建靶向叶绿体基因组rbcL区域的扩增子文库；将多个文库混合后，使用MiSeq平台以2×300 bp双端reads进行测序。每个样本产出的双端reads数量介于6994至95832之间。