Buoyant density characterization of Vairimorpha apis and Vairimorpha ceranae spores

In honey bees, microsporidian infections are primarily caused by Vairimorpha (Nosema) apis and Vairimorpha (Nosema) ceranae. Mixed infections may occur where both species co-exist, making accurate species-level identification essential for epidemiological research and disease management. Although buoyant density separation is a common technique for isolating biological materials, it has been largely overlooked, and the buoyant densities of V. ceranae spores have not previously been characterized. In this study, spores isolated from infected bees were centrifuged through discrete sucrose solutions of defined concentrations and discontinuous gradient to determine their buoyant densities. Most germinated, primary, and immature spores of either species were banded atop the 57% (w/w) sucrose (1.269 g/cm³). Most V. apis environmental spores consistently banded atop the 62% (w/w) sucrose (1.298 g/cm³), whereas most V. ceranae spores banded atop the 60% (1.286 g/cm³). Interestingly, we noted that overnight storage in sucrose solutions reduced the buoyant densities of both species by approximately 2% in sucrose solutions. Such differences provide a simple centrifuge-based method for environmental spore enrichments and could serve as a non-molecular differentiation between the two species. However, both V. apis and V. ceranae exhibited distributed variation in spore densities and the distributions were overlapping for the species, indicating that buoyant density may not complete separation of Vairimorpha species in bees. These results suggest that buoyant density can still serve as an alternative diagnostic feature in laboratories without molecular tools and that spore suspensions for group inoculation assays should be in approximately 58-60% sucrose in feeders to minimize spore sedimentation.