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On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System
Published on: July 18, 2025
Efficient Method for the Rapid Purification of Nosema ceranae Spores
Dong-Jun Kim1, Hwi-Geon Yun1, In-Hui Kim1
1Department of Agricultural Biology, College of Agriculture, Life & Environment Science, Chungbuk National University, Cheongju 28644, Korea.
Abstract:
Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8-11 µm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.
Insights
A new method using 2 mm beads and enzymes efficiently purifies Nosema ceranae spores from honey bee midguts. This technique significantly increases spore yield for research on bee health.
Area of Science:
- Apiculture
- Mycology
- Insect Pathology
Background:
- Nosema ceranae is a fungal parasite causing honey bee mortality.
- Efficient Nosema spore purification is crucial due to non-culturability and seasonal availability.
Purpose of the Study:
- To develop a method for higher yield purification of Nosema spores from honey bee midguts.
- To optimize spore release from infected honey bee tissues.
Main Methods:
- Utilized 2 mm beads and enzymatic treatment (collagenase, trypsin) for tissue homogenization.
- Determined optimal enzyme treatment duration (1 hour) for spore yield and viability.
- Employed a single filtration step (8-11 µm pore size) to remove debris.
Main Results:
- The new method increased spore yield approximately 2.95 times compared to using 3 mm beads alone.
- Optimal enzyme treatment duration was found to be 1 hour.
- Single-step filtration effectively removed cellular debris.
Conclusions:
- This optimized method enhances Nosema ceranae spore purification efficiency.
- The technique is potentially applicable to other Nosema species.
- Improved spore yield facilitates research on honey bee diseases.
