Regular occurrence of atypically small spores in Apis mellifera carnica (Hymenoptera: Apidae), naturally infected with Nosema spp. (Microsporidia)

Reliable and accessible methods for differentiating the Nosema spp.microsporidia spores are still missing.However, proper identification and measuring the respective spore loads might be crucial for disease characterization, as well as for understanding the causes of emergence of natural honeybee microspopridioses and severity of their symptoms.The ultrastructure and size range of Nosema spp.spores and relationships between the spore size and spore load have been assessed in naturally infected Apis mellifera carnica.The average size of Nosema spp.spores was 5.7±2.1 × 3.05±0.75µm (n=335).We discovered that in addition to normally sized spores (3.6-7.8 × 2.3-3.8μm (n=335), atypical spores of smaller size (3.25±0.25 × 2.1±0.1 µm (n=81)), were occasionally observed.Atypical spores were separated by filtration though a 3 µm membrane filter.PCR analysis revealed that the majority of atypical spores belonged to N. apis, although N. ceranae spores were also present.With an increase in spore load, more atypical spores were observed.Ultrastructural analysis of atypical spores confirmed the PCR diagnosis and revealed the presence of a polar filament and other elements of extrusion apparatus, indicating their maturity and functionality.