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Standard methods for physiology and biochemistry research in Apis mellifera

Summary:

Despite their tremendous economic importance, and apart from certain topics in the field of neurophysiology such as vision, olfaction, learning and memory, honey bees are not a typical model system for studying general questions of insect physiology. The reason is their social lifestyle, which sets them apart from a “typical insect??? and, during social evolution, has resulted in the restructuring of certain physiological pathways and biochemical characteristics in this insect. Not surprisingly, the questions that have attracted most attention by researchers working on honey bee physiology and biochemistry in general are core topics specifically related to social organization, such as caste development, reproductive division of labour and polyethism within the worker caste. With certain proteins playing key roles in these processes, such as the major royal jelly proteins (MRJPs), including royalactin and hexamerins in caste development, and vitellogenin in reproductive division of labour and age polyethism, a major section herein will present and discuss basic laboratory protocols for protein analyses established and standardized to address such questions in bees. A second major topic concerns endocrine mechanisms underlying processes of queen and worker development, as well as reproduction and polyethism, especially the roles of juvenile hormone and ecdysteroids. Sensitive techniques for the quantification of juvenile hormone levels circulating in haemolymph, as well as its synthesis by the corpora allata are described. Although these require certain instrumentation and a considerable degree of sophistication in the analysis procedures, we considered that presenting these techniques would be of interest to laboratories planning to specialize in such analyses. Since biogenic amines are both neurotransmitters and regulators of endocrine glands, we also present a standard method for the detection and analysis of certain biogenic amines of interest. Further questions that cross borders between individual and social physiology are related to energy metabolism and thermoregulation. Thus a further three sections are dedicated to protocols on carbohydrate quantification in body fluid, body temperature measurement and respirometry.

    1. 1.1. Introduction
    2. 1.2. Quantification of total protein content in samples
    3. 1.2.1. The Bradford assay
    4. 1.2.2. The bicinchoninic acid (BCA) assay
    5. 1.3. One-dimensional SDS gel electrophoresis of proteins
    6. 1.3.1. Preparing samples
    7. 1.3.2. Preparing and running vertical slab gels
    8. 1.3.3. Staining gels with Coomassie Brilliant Blue
    9. 1.3.4. Staining gels with silver salts
    10. 1.4. Western blotting and immunodetection of proteins separated by SDS-PAGE
    11. 1.5. Rocket immunoelectrophoresis
    12. 1.6. Immunofluorescence detection of proteins in tissue: Tubulin localization in ovariole whole mounts as an example of a working protocol
    13. 1.6.1. Buffers
    14. 1.6.2. Dissection and fixation of ovarioles
    1. 2.1. Sample Collection
    2. 2.2. Preparation of the reagents
    3. 2.2.1. Benzoic acid solution
    4. 2.2.2. Glucose standard stock solution (1 mg/ml)
    5. 2.2.3. Enzyme mix solution
    6. 2.3. Glucose assay
    7. 2.4. Trehalose assay
    1. 3.1. General instructions on haemolymph sample collection and glassware preparation for juvenile hormone assays
    2. 3.1.1. Haemolymph collection
    3. 3.1.2. Glassware preparation
    4. 3.1.2.1. For GC-MS analysis
    5. 3.1.2.2. For JH-RIA
    6. 3.2. Juvenile hormone extraction, purification and quantification by GC-MS
    7. 3.2.1. JH sample purification and quantification by GC-MSD
    8. 3.3. Juvenile hormone quantification by radioimmunoassay
    9. 3.3.1. JH extraction
    10. 3.3.2. Preparation of RIA solutions
    11. 3.3.2.1. JH-III standard
    12. 3.3.2.2. Phosphate buffer
    13. 3.3.2.3. Saturated ammonium sulphate
    14. 3.3.2.4. Solution of radioactive JH (RIA tracer solution)
    15. 3.3.2.5. Antibody solution
    16. 3.3.3. Running a JH RIA
    17. 3.3.4. Data analysis
    18. 3.3.5. User safety
    19. 3.4. Quantification of ecdysteroids by radioimmunoassay
    20. 3.4.1. Sample preparation
    21. 3.4.1.1. for haemolymph
    22. 3.4.1.2. for tissue
    23. 3.4.1.3. in case of lipid-rich samples (e.g. whole larva)
    24. 3.4.2. Preparation of RIA solutions
    25. 3.4.2.1. 20-hydroxyecdysone (20E) standard
    26. 3.4.2.2. Phosphate buffer
    27. 3.4.2.3. Saturated ammonium sulphate
    28. 3.4.2.4. Solution of radioactive ecdysone (RIA tracer solution)
    29. 3.4.2.5. Antibody solution (RIA serum)
    30. 3.4.3. Running an ecdysteroid RIA
    31. 3.4.4. Data analysis
    32. 3.4.5. User Safety
    33. 3.5. Measuring the rate of juvenile hormone biosynthesis by the paired corpora allata
    34. 3.5.1. Purifying and preparing radioactive methionine for use in assay
    35. 3.5.2. Measuring the rate of JH biosynthesis
    36. 3.5.3. Data analysis
    37. 3.5.4. User Safety
    1. 4.1. Introduction
    2. 4.2. Dissection of the brain from the head capsule
    3. 4.3. Pre-analysis preparation of the HPLC
    4. 4.4. Preparation of internal and external standards
    5. 4.4.1. Stock dilutions (1 x 106 pg/μl)
    6. 4.4.2. External and Internal standard
    7. 4.4.2.1. Dilution ES-A
    8. 4.4.2.2. Dilution IS-A (5000 pg/μl)
    9. 4.4.2.3. Dilution IS-B (500/250 pg/μl)
    10. 4.4.2.4. Standard Curve
    11. 4.5. HPLC separation of standards
    12. 4.6. Sample preparation
    13. 4.7. Separation and quantification of biogenic amines by HPLC
    1. 5.1. Contact thermosensors
    2. 5.1.1. Thermocouples and thermoneedles
    3. 5.1.1.1. Thermocouple types
    4. 5.1.1.2. Calibration
    5. 5.1.1.3. Use inside colonies
    6. 5.1.2. Thermoresistors (thermistors, Pt100)
    7. 5.2. Non-contact temperature measurement
    8. 5.2.1. Infrared spot thermometers
    9. 5.2.2. Infrared thermography
    10. 5.2.2.1. Main thermographic camera types
    11. 5.2.2.2. Honey bee cuticle infrared emissivity
    12. 5.2.2.3. Thermography camera calibration with reference radiator
    13. 5.2.2.4. Attenuation of infrared transmissive films
    14. 5.3. Operative temperature
    15. 5.4. Radiation sensors
    16. 5.4.1. Star pyranometers (according to Dirmhirn)
    17. 5.4.2. Photoelectric pyranometers
    18. 5.4.3. Measurement of the short-wave radiation balance
    19. 5.5. Humidity measurement
    1. 6.1. Flow-through respirometry
    2. 6.1.1. Measurement arrangements, measurement chambers and accessories
    3. 6.1.1.1. General setup
    4. 6.1.1.2. Air drying, CO2 scrubbing and tubing
    5. 6.1.1.3. Serial measurement arrangement
    6. 6.1.1.4. Parallel measurement arrangement
    7. 6.1.1.5. Measurement chambers
    8. 6.1.2. O2 consumption
    9. 6.1.2.1. Fuel cell devices
    10. 6.1.2.2. Paramagnetic devices
    11. 6.1.2.3. Calibration
    12. 6.1.2.4. Indirect calorimetry: calculation of energy turnover
    13. 6.1.3. CO2 production
    14. 6.1.3.1. Measurement range selection
    15. 6.1.3.2. DIRGA Calibration
    16. 6.1.3.3. Respiratory quotient (RQ): calculation of energy turnover from CO2 measurements
    17. 6.1.4. H2O balance
    18. 6.1.5. Impact of flow control and measurement chamber size on sensitivity and temporal resolution
    19. 6.1.6. Controlling relative humidity
    20. 6.1.7. Closed chamber method (CO2 accumulation)
    21. 6.1.8. Thermolimit respirometry
    22. 6.2. Chemical-optical oxygen sensors
    23. 6.3. Manometric and volumetric respirometry
    24. 6.4. Titration methods
    25. 6.5. Isotopic tracer techniques
    26. 6.6. Calorimetry
    27. 6.7. Energetics derived from measurement of sugar consumption
    28. 6.8. Activity monitoring
    29. 6.8.1. Video and thermography
    30. 6.8.2. Optical activity detectors