Protocols for assessing the distribution of pathogens in individual Hymenopteran pollinators

Abstract

This protocol was developed for the COST-Action “Super-B”, whose purpose was to coordinate research, outreach and policy towards sustainable pollination1-3. The protocol addresses the detection of parasites and pathogens across bee species, as one of several possible drivers of bee decline4,5. It consists of four major components:1. A sample collecting protocol, based around a dominant bee species driving pathogen distribution among other bee species2. A sample processing protocol, based on analyzing nucleic acids from individual bees3. A pathogen detection protocol, based on RT-qPCR with broad-range primers for several common pathogens4. A barcoding protocol, for accurate bee species identification The protocols have largely been adapted from existing knowledge and protocols but also include two key innovations: the use of passive reference nucleic acids and synthetic positive controls, that significantly improve the quality and robustness of the raw data, and thus the reliability of the analyses and conclusions.

“Migratory beekeeping and its influence on the prevalence and dispersal of pathogens to managed and wild bees”

“…Therefore, migratory beekeeping should implement measures to avoid the spread of honey bee pests and diseases. In that sense, molecular tools can help to perform fast, cheap and extensive testing programmes to detect the presence of honey bee pathogens and parasites ( Grangier et al, 2015 ; de Miranda et al, 2021 ). On the other hand, it is also important to keep national records of hive movements performed by beekeepers, a procedure already performed in some countries (e.g.…”

Honey bees and climate explain viral prevalence in wild bee communities on a continental scale

“…The bee sampling, identification, processing and virus detection protocols were performed as described in Miranda et al 87 , and described in short here below. Sample collection, identification, processing, and cDNA synthesis was all done by the local partner.…”

“…1 ). The field sampling was performed according to a standardized sampling protocol 87 , where each partner sampled in their respective country. At each site 30 honey bees and 30 wild bees were collected in the order they were encountered as well an additional 15 specimens of the most common wild bee.…”

“…For RNA extraction of the bees, we used the protocol described by de Miranda et al 87 . For each bee we only used the abdomen, which was dissected sterile and crushed in a TBS-buffer (50 mM TRIS.HCl PH 7.4, 150 mM NaCl).…”

Endosymbionts that threaten commercially raised and wild bumble bees (Bombus spp.)

“…For screening RNA viruses, reverse transcriptase PCR (RT-PCR) is used, which converts RNA to its complementary DNA strand (cDNA), which is then used as template in PCR (de Miranda et al 2013). Standardized protocols for PCR-based detection of a variety of common bee pathogens have recently been released (de Miranda et al 2021).…”