Standard methods for research on Apis mellifera gut symbionts.

Authors: Philipp Engel, Rosalind James, Ryuichi Koga, Waldan K Kwong, Quinn McFrederick, Nancy A Moran.

Table of contents

1. Introduction
2. Bacterial community analysis using next-generation sequencing (NGS)
   2.1. Extraction, PCR, and sequencing

      2.1.1. Extraction of bacterial community DNA
      2.1.2. Primer choice and 16S rRNA regions.
      2.1.3. PCR conditions
      2.1.4. Library construction
      2.1.5. Sequencing
   2.2. Quality filtering and data analysis
      2.2.1. Quality filtering
      2.2.2. Identifying operational taxonomic units (OTUs)
      2.2.3. Taxonomic assignment of OTUs
      2.2.4. Alpha diversity
      2.2.5. Exploratory techniques: beta diversity
      2.2.6. Testing for significant differences in communities
3. Fluorescent in situ hybridization (FISH) for image analysis of specific microorganisms in the digestive tract
   3.1. Designing Probes

      3.1.1. Probe sequence
      3.1.2. Selecting fluorochromes
   3.2. Preserving insect materials for FISH
   3.3. Fixation, paraffin embedding, sectioning and mounting

      3.3.1. Fixing honey bee gut samples from fresh bees
      3.3.2. Fixing honey bee gut samples from preserved bees
      3.3.3. Bleaching samples to reduce autofluorescence in the gut tissues
      3.3.4. Dehydration, clearing, paraffin infiltration and embedding
      3.3.5. Sectioning and mounting
   3.4. Hybridization
      3.4.1. Hybridization procedures
      3.4.2. FISH controls
      3.4.3. Combining probes
      3.4.4. Image acquisition and adjustments
   3.5. Concluding remarks about FISH
4. Culture conditions for the dominant members of the bee gut microbiota
   4.1. Genus Snodgrassella

      4.1.1. Optimal growth conditions
      4.1.2. Microbe characteristics
   4.2. Genus Gilliamella
      4.2.1. Optimal growth conditions
      4.2.2. Gilliamella characteristics
   4.3. Gamma-2
      4.3.1. Optimal growth conditions
      4.3.2. Gamma-2 characteristics
   4.4. Genus Lactobacillus
      4.4.1. Optimal growth conditions
      4.4.2. Lactobacillus characteristics
   4.5. Genus Bifidobacterium
      4.5.1. Optimal growth conditions
      4.5.2. Bifidobacterium characteristics
   4.6. Alpha-1 bacteria
      4.6.1. Optimal growth conditions
      4.6.2. Alpha-1 characteristics
   4.7. Other bacteria
   4.8. Preservation of bacterial cultures
5. Conclusions and outlook

6. Acknowledgements
7. References