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Standard methods for research on Apis mellifera gut symbionts

Summary:

Gut microbes can play an important role in digestion, disease resistance, and the general health of animals, but little is known about the biology of gut symbionts in Apis mellifera . As part of the BEEBOOK series describing honey bee research methods, we provide standard protocols for studying gut symbionts. We describe non-culture-based approaches based on Next Generation Sequencing (NGS), methodology that has greatly improved our ability to identify the microbial communities associated with honey bees. We also describe Fluorescent In Situ
Hybridization (FISH) microscopy, which allows a visual examination of the microenvironments where particular microbes occur. Culturing methods are also described, as they allow the researcher to isolate particular bacteria of interest for further study or gene identification, and enable the assignment of particular functions to particular gut community members. We hope these methods will help others advance the state of knowledge regarding bee gut symbionts and the role they play in honey bee health.

    1. 2.1. Extraction, PCR, and sequencing
    2. 2.1.1. Extraction of bacterial community DNA
    3. 2.1.2. Primer choice and 16S rRNA regions
    4. 2.1.3. PCR conditions
    5. 2.1.4. Library construction
    6. 2.1.5. Sequencing
    7. 2.2. Quality filtering and data analysis
    8. 2.2.1. Quality filtering
    9. 2.2.2. Identifying operational taxonomic units (OTUs)
    10. 2.2.3. Taxonomic assignment of OTUs
    11. 2.2.4. Alpha diversity
    12. 2.2.5. Exploratory techniques: beta diversity
    13. 2.2.6. Testing for significant differences in communities
    1. 3.1. Designing probes
    2. 3.1.1. Probe sequence
    3. 3.1.2. Selecting fluorochromes
    4. 3.2. Preserving insect materials for FISH
    5. 3.3. Fixation, paraffin embedding, sectioning and mounting
    6. 3.3.1. Fixing honey bee gut samples from fresh bees
    7. 3.3.2. Fixing honey bee gut samples from preserved bees
    8. 3.3.3. Bleaching samples to reduce autofluorescence in the gut tissues
    9. 3.3.4. Dehydration, clearing, paraffin infiltration and embedding
    10. 3.3.5. Sectioning and mounting
    11. 3.4. Hybridization
    12. 3.4.1. Hybridization procedures
    13. 3.4.2. FISH controls
    14. 3.4.3. Combining probes
    15. 3.4.4. Image acquisition and adjustments
    16. 3.5. Concluding remarks about FISH
    1. 4.1. Genus Snodgrassella
    2. 4.1.1. Optimal growth conditions
    3. 4.1.2. Microbe characteristics
    4. 4.2. Genus Gilliamella
    5. 4.2.1. Optimal growth conditions
    6. 4.2.2. Gilliamella characteristics
    7. 4.3. Genus Frischella
    8. 4.3.1. Optimal growth conditions
    9. 4.3.2. Genus Frischella characteristics
    10. 4.4. Genus Lactobacillus
    11. 4.4.1. Optimal growth conditions
    12. 4.4.2. Lactobacillus characteristics
    13. 4.5. Genus Bifidobacterium
    14. 4.5.1. Optimal growth conditions
    15. 4.5.2. Bifidobacterium characteristics
    16. 4.6. Alpha-1 bacteria
    17. 4.6.1. Optimal growth conditions
    18. 4.6.2. Alpha-1 characteristics
    19. 4.7. Other bacteria
    20. 4.8. Preservation of bacterial cultures