Unbiased RNA Shotgun Metagenomics in Social and Solitary Wild Bees Detects Associations with Eukaryote Parasites and New Viruses

<div><p>The diversity of eukaryote organisms and viruses associated with wild bees remains poorly characterized in contrast to the well-documented pathosphere of the western honey bee, <i>Apis mellifera</i>. Using a deliberate RNA shotgun metagenomic sequencing strategy in combination with a dedicated bioinformatics workflow, we identified the (micro-)organisms and viruses associated with two bumble bee hosts, <i>Bombus terrestris</i> and <i>Bombus pascuorum</i>, and two solitary bee hosts, <i>Osmia cornuta</i> and <i>Andrena vaga</i>. Ion Torrent semiconductor sequencing generated approximately 3.8 million high quality reads. The most significant eukaryote associations were two protozoan, <i>Apicystis bombi</i> and <i>Crithidia bombi</i>, and one nematode parasite <i>Sphaerularia bombi</i> in bumble bees. The trypanosome protozoan <i>C</i>. <i>bombi</i> was also found in the solitary bee <i>O</i>. <i>cornuta</i>. Next to the identification of three honey bee viruses Black queen cell virus, Sacbrood virus and Varroa destructor virus-1 and four plant viruses, we describe two novel RNA viruses Scaldis River bee virus (SRBV) and Ganda bee virus (GABV) based on their partial genomic sequences. The novel viruses belong to the class of negative-sense RNA viruses, SRBV is related to the order Mononegavirales whereas GABV is related to the family <i>Bunyaviridae</i>. The potential biological role of both viruses in bees is discussed in the context of recent advances in the field of arthropod viruses. Further, fragmentary sequence evidence for other undescribed viruses is presented, among which a nudivirus in <i>O</i>. <i>cornuta</i> and an unclassified virus related to Chronic bee paralysis virus in <i>B</i>. <i>terrestris</i>. Our findings extend the current knowledge of wild bee parasites in general and addsto the growing evidence of unexplored arthropod viruses in valuable insects.</p>