Supported by: www.ricolafoundation.org www.evacranetrust.org www.veto-pharma.com www.vetsuisse.unibe.ch www.ibrabee.org.uk
Generic selectors
Exact matches only
Search in title
Search in content
Search in posts
Search in pages
Filter by Categories
Articles
BEEBOOK Volume I
BEEBOOK Volume II
BEEBOOK Volume III
Events
Jobs
News
Register Login

Standard methods for molecular research in Apis mellifera

Summary:

From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al . (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.

    1. 1.1. Introduction
    2. 1.2. Sample collection
    3. 1.2.1. Adult bees
    4. 1.2.1.1. Nurse bees
    5. 1.2.1.2. Foraging bees
    6. 1.2.2. Pupae
    7. 1.2.3. Larvae
    8. 1.2.4. Eggs
    9. 1.2.5. Extracted guts
    10. 1.2.6. Drone endophalus and semen
    11. 1.2.7. Faeces
    12. 1.2.8. Dead bee samples
    13. 1.3. Sample transport
    14. 1.3.1. Freezing with dry ice
    15. 1.3.2. Freezing with “wet??? ice
    16. 1.3.3. Live transport
    17. 1.3.4. Chemical stabilizers
    18. 1.3.5. Sample collection cards
    19. 1.4. Long-term sample storage
    20. 1.4.1. Freezing
    21. 1.4.2. Drying
    22. 1.4.3. Chemical stabilizers
    1. 2.1. Introduction
    2. 2.2. Sample homogenization
    3. 2.2.1. Bead-mill homogenizers
    4. 2.2.2. Blender
    5. 2.2.3. Paint shaker
    6. 2.2.4. Mortar and pestle
    7. 2.2.5. Mesh bags
    8. 2.2.6. Micropestle
    9. 2.2.7. Robotic extraction
    1. 3.1. Introduction
    2. 3.2. Genomic DNA extraction from adult bees
    3. 3.2.1. DNA extraction using CTAB
    4. 3.2.2. DNA extraction using Qiagen Blood and Tissue DNA kits
    5. 3.2.3. DNA extraction using Chelex
    6. 3.3. DNA detection using southern blots with DIG labelling
    7. 3.3.1. Restriction enzyme digestion and agarose gel electrophoresis
    8. 3.3.2. Assembly of the transfer setup and transfer of DNA from gel to membrane
    9. 3.3.3. Synthesis of DIG-labelled DNA probe
    10. 3.3.4. Hybridizing the DIG-labelled DNA Probe to DNA on the Blot
    1. 4.1. Introduction
    2. 4.2. Affinity column purification
    3. 4.3. Acid Phenol RNA extraction from adult bees
    4. 4.3.1. TRIzol extraction
    5. 4.3.2. Bulk Extraction of RNA from 50-100 whole bees using the acid-phenol method
    6. 4.3.3. RNA lysis/stabilization buffer
    7. 4.4. RNA quality assessment
    8. 4.5. cDNA synthesis from total RNA
    9. 4.5.1. Reverse Transcription of RNA
    10. 4.6. Qualitative RT-PCR for honey bee and pathogen targets
    11. 4.7. Quantitative RT-PCR for honey bee and pathogen targets
    12. 4.7.1. One-Step versus Two-Step RT-PCR
    13. 4.7.2. One-Step RT-qPCR
    14. 4.7.3. Two-Step RT-qPCR
    15. 4.7.4. Two-step Quantitative PCR for high-throughput assays
    16. 4.7.5. Multiplex RT-(q)PCR
    17. 4.8. Primer and probe design
    18. 4.8.1. Primer length, melting temperature and composition
    19. 4.8.2. Annealing temperature
    20. 4.8.3. Cycling parameters
    21. 4.9. Assay optimization
    22. 4.9.1. Primer-dimers and other PCR artefacts
    23. 4.9.2. Primer concentration
    24. 4.9.3. Magnesium concentration
    25. 4.10. Quantitation controls
    26. 4.10.1. External reference standards
    27. 4.10.2. Internal reference standards
    28. 4.10.2.1. Exogenous internal reference standards
    29. 4.10.2.2. Internal reference standards
    30. 4.10.2.3. External standard for viral target quantification
    31. 4.11. Microarrays
    32. 4.12. Northern blots using DIG labelling
    33. 4.12.1. Agarose / Formaldehyde gel electrophoresis
    34. 4.12.2. Assembly of the transfer setup and transfer of RNA from gel to membrane
    35. 4.12.3. Preparation of DIG labelling (non-radioactive) probe
    36. 4.12.4. Hybridization analysis
    37. 4.13. In situ hybridization
    38. 4.13.1. Tissue fixation
    39. 4.13.2. Preparation of DIG labelling (non-radioactive) probe
    40. 4.13.3. Hybridization analysis
    1. 5.1. Introduction
    1. 6.1. Introduction
    2. 6.2. Mitochondrial DNA analysis
    3. 6.3. Nuclear DNA analysis
    4. 6.3.1. Microsatellites
    5. 6.3.1.1. Microsatellite reaction mix
    6. 6.3.1.2. Primers for multiplexed honey bee microsatellite loci
    7. 6.3.1.3. Thermal cycling conditions for multiplex PCR
    8. 6.3.1.4. Size estimation of PCR products
    9. 6.3.2. Single-nucleotide polymorphisms (SNPs)
    1. 7.1. Introduction
    2. 7.2. Obtaining and formatting sequences of interest for phylogenetics
    3. 7.3. Sequence data in FASTA format
    4. 7.4. Alignment of sequence data
    5. 7.4.1. Clustal
    6. 7.4.2. MUSCLE
    7. 7.5. Trimming aligned sequence data to equal length
    8. 7.6. Performing phylogenetic analyses
    9. 7.6.1. Using MEGA
    10. 7.6.1.1. Converting data to MEGA format
    11. 7.6.1.2. Constructing and testing phylogenetic trees
    12. 7.6.2. Using SATé
    13. 7.6.2.1. External tools
    14. 7.6.2.2. Sequence import and tree building
    15. 7.6.2.3. Job Settings
    16. 7.6.2.4. SATé Settings
    17. 7.6.3. Building trees using distance and character based methods
    1. 8.1. Introduction
    2. 8.2. Honey bee genome project
    3. 8.3. Honey bee parasite and pathogen genomes
    4. 8.4. Comparative genomics
    5. 8.5. Second-generation sequencing
    6. 8.6. Genomic sequence assembly
    7. 8.7. Transcriptomic analyses (“RNASeq???)
    8. 8.8. Metagenomics
    9. 8.8.1. RNA versus DNA sampling
    10. 8.8.2. Sample preparation
    11. 8.8.3. Amplicon-based or shotgun sequencing
    12. 8.8.4. Assembly of shotgun sequences vs. read mapping
    13. 8.8.5. Databases for metagenomics
    14. 8.8.6. Post-assignment statistics
    1. 9.1. Introduction
    2. 9.2. Tissue fixation and tissue sectioning exemplified with gut tissue
    3. 9.3. Fixation of cultured cells grown in suspension
    4. 9.4. FISH-analysis of tissue sections and fixed insect cells
    1. 10.1. Introduction
    2. 10.2. Production of RNA interfering molecules
    3. 10.2.1. siRNA design and synthesis
    4. 10.2.2. Production of dsRNA
    5. 10.3. RNAi Applications
    6. 10.3.1. RNAi in adult honey bees via feeding
    7. 10.3.2. RNAi in honey bee larvae via feeding
    8. 10.3.3. Gene knock-down by abdominal injection of targetspecific dsRNA/siRNA
    9. 10.4. Concluding remarks
    1. 11.1. Introduction
    2. 11.2. DNA methylation in honey bees
    3. 11.3. DNA extraction from various tissues for methylation analysis
    4. 11.4. High-throughput sequencing of targeted regions
    5. 11.4.1. Fragmentation of DNA
    6. 11.4.2. End-repair of sheared DNA
    7. 11.4.3. Adaptor ligation
    8. 11.4.4. Size selection of adapter-ligated fragments
    9. 11.4.5. Bisulfite conversion and amplification of the final library
    10. 11.4.6. Validation of the libraries
    11. 11.4.7. Sequencing and data analysis
    12. 11.5. Mapping and methylation assessment
    13. 11.6. Methylation dynamics and expression of individual genes
    14. 11.6.1. Amplicon sequence selection
    15. 11.6.2. Bisulfite DNA conversion
    16. 11.6.3. Bisulfite PCR
    17. 11.7. RNA extraction
    18. 11.8. cDNA synthesis and template quantification