Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high‐throughput sequencing approach

Abstract

Summary Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co‐occurrence. Here, we compared the performance of a next‐generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR‐identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad‐range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

The Haptomonad Stage of Crithidia acanthocephali in Apis mellifera Hindgut

“…Crithidia acanthocephali was recently found for the first time in honey bee colonies in Spain [12] and in bumble bees [37]. It was detected in honey bee colonies throughout the year, at all seasons, and no differences were found between interior and forager bees, indicating it is a common organism in bee colonies.…”

Protocols for assessing the distribution of pathogens in individual Hymenopteran pollinators

“…The sample processing and pathogen quanti cation protocols are mostly variations on common protocols for nucleic acid extraction and analysis. An alternative to targeted screening of individual pathogens by RT-qPCR is target-free screening, using mass parallel sequencing techniques 28,34,35 . With target-free screening, the entire microbial complexity of either the RNA or the DNA phase is recovered, by mass parallel sequencing of either universal DNA barcode regions 36,37 , whole DNA metagenomes 28,38 and/or the non-ribosomal fraction of the RNA 34,39,40 .…”

“…With target-free screening, the entire microbial complexity of either the RNA or the DNA phase is recovered, by mass parallel sequencing of either universal DNA barcode regions 36,37 , whole DNA metagenomes 28,38 and/or the non-ribosomal fraction of the RNA 34,39,40 . The identity of each individual sequenced nucleic acid is determined through comparison with the large public databases, after which the composition of the sample is reconstructed from the individual identi ed reads 28,34,35 . Although this is initially much more costly than targeted screening, the advantage is that all microorganisms are investigated simultaneously with the same assay methodology.…”

An evidence-based rationale for a North American commercial bumble bee clean stock certification program

“…Newer tools may be required to confirm the presence of some parasites and pathogens in colonies. For example, next-generation sequencing technology was found to detect some pathogens that were not identified by PCR (Bartolomé et al 2021). Ion PGM sequencing detected species that were not found by classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing).…”