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

Abstract

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, Apis mellifera. 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, Bombus terrestris and Bombus pascuorum, and two solitary bee hosts, Osmia cornuta and Andrena vaga. Ion Torrent semiconductor sequencing generated approximately 3.8 million high quality reads. The most significant eukaryote associations were two protozoan, Apicystis bombi and Crithidia bombi, and one nematode parasite Sphaerularia bombi in bumble bees. The trypanosome protozoan C. bombi was also found in the solitary bee O. cornuta. 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 Bunyaviridae. 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 O. cornuta and an unclassified virus related to Chronic bee paralysis virus in B. terrestris. Our findings extend the current knowledge of wild bee parasites in general and addsto the growing evidence of unexplored arthropod viruses in valuable insects.

Investigating the viral ecology of global bee communities with high-throughput metagenomics

“…This approach allowed us to search for novel viruses, begin to determine the distribution of these viruses (and viral families) across the world, and begin to evaluate the extent to which A. mellifera and wild bee populations share these viruses. As in other studies [28,29], we identified several viruses that share sequence homology to plant viruses and thus may be the result of contamination, but given that these were found in multiple populations and locations, it raises the question of whether or not these viruses may use bees as hosts.…”

“…Positive sense RNA ((+)ssRNA) viruses are the most common type of virus that have been identified in bees thus far, with a vast majority of the viruses possessing this genome architecture. Most of these viruses belong to the viral order Picornavirales (including DWV, BQCV, IAPV, and SBV), but recently additionally studies have added to the list of viral orders that infect bees, including Tymovirales [6,29]. Here we have identified novel virus sequences that share sequence homology with three (+)ssRNA virus families, Dicistroviridae, Secoviridae, and Tymoviridae.…”

“…Only recently have negative sense RNA ((-)ssRNA) viruses been identified in bees [6,29]. One of these, Apis mellifera Rhabdovirus 1 (ARV-1), from the family Rhabdoviridae [6], was detected in A. mellifera populations in South Africa, Tonga, and the Netherlands.…”

“…Similarly, recent metagenomics studies of bees have identified many viruses that were originally described in plants [28,29]. It has been assumed that these are simply contaminants from the pollen and nectar in the bees' diets, but these viruses may in fact be using bees as hosts.…”

“…Using this approach, several recent studies have identified novel viruses in populations of bees (A. mellifera, Bombus terrestris, Bombus pascuorum, Osmia cornuta, and Andrena vaga) collected from sites in the Netherlands, South Africa, Tonga, Israel and Belgium [6,29,34]. However, while these studies have provided valuable information about viral communities within particular species or regions, they are limited in scope, in that they focus on a single bee species in multiple locations (A. mellifera populations in Israel [34] or the Netherlands, South Africa, and Tonga [6]) or a selection of wild bees in a single region (Belgium [29]). Furthermore, two of these studies [6,29] did not purify viral particles prior to sequencing, and thus had samples with a high percentage of reads corresponding to the host bee species, which can limit detection of rare viral species.…”