Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR

• Nosema microsporidia are endoprevalent parasites of managed and wild bees. • Current molecular methods do not enable accurate Nosema quantification. • We developed qPCR methods to quantify N. apis , N. ceranae , and N. bombi. • Accuracy of parasite loads was validated on honey bee, bumble bee, and mason bee. • qPCR methods open perspectives in nosemosis diagnosis and in bee epidemiology. Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees ( Apis spp.), while Nosema bombi has been described in bumble bees ( Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 ( RPB1 ) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee ( Apis mellifera ) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log 10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log 10 copies/honey bee. Method accuracy was also verified in bumble bee ( Bombus terrestris ) and mason bee ( Osmia bicornis ) homogenates in the range of 5 to 10 log 10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite’s eco-dynamics in managed and wild bees.