Standard methods for physiology and biochemistry research in Apis mellifera

Authors: Klaus Hartfelder, Márcia M G Bitondi, Colin S Brent, Karina R Guidugli-Lazzarini, Zilá L P Simões, Anton Stabentheiner, Érica D Tanaka, and Ying Wang.

Table of contents



1. Protein analysis for honey bee samples
   1.1 Introduction
   1.2. Quantification of total protein content in samples

      1.2.1. The Bradford assay
      1.2.2. The bicinchoninic acid (BCA) assay
   1.3. One-dimensional SDS gel electrophoresis of proteins
      1.3.1. Preparing samples
      1.3.2. Preparing and running vertical slab gels
      1.3.3. Staining gels with Coomassie Brilliant Blue
      1.3.4. Staining gels with silver salts
   1.4. Western blotting and immunodetection of proteins separated by SDS-PAGE
   1.5. Rocket immunoelectrophoresis
   1.6. Immunofluorescence detection of proteins in tissue: tubulin localization in ovariole whole mounts as an example of a working protocol

      1.6.1. Buffers
      1.6.2. Dissection and fixation of ovarioles
2. Measurement of glucose and trehalose in honey bee haemolymph
   2.1 Sample Collection
   2.2. Preparation of the reagents

      2.2.1. Benzoic acid solution
      2.2.2. Glucose standard stock solution (1 mg/ml)
      2.2.3. Enzyme mix solution
   2.3. Glucose assay
   2.4. Trehalose assay

3. Analysis of juvenile hormone and ecdysteroid levels in honey bees
   3.1. General instructions on haemolymph sample collection and glassware preparation for juvenile hormone assays

      3.1.1. Haemolymph collection
      3.1.2. Glassware preparation For GC-MS analysis For JH-RIA
   3.2. Juvenile hormone extraction, purification and quantification by GC-MS
      3.2.1. JH sample purification and quantification by GC-MSD
   3.3. Juvenile hormone quantification by radioimmunoassay
      3.3.1. JH extraction
      3.3.2. Preparation of RIA solutions JH-III standard Phosphate buffer Saturated ammonium sulphate Solution of radioactive JH (RIA tracer solution) Antibody solution
      3.3.3. Running a JH RIA
      3.3.4. Data analysis
      3.3.5. User safety
   3.4. Quantification of ecdysteroids by radioimmunoassay
      3.4.1. Sample preparation for haemolymph for tissue in case of lipid-rich samples (e.g. whole larva)
      3.4.2. Preparation of RIA solutions 20-hydroxyecdysone (20E) standard Phosphate buffer Saturated ammonium sulphate Solution of radioactive ecdysone (RIA tracer solution) Antibody solution (RIA serum)
         3.4.3. Running an ecdysteroid RIA
         3.4.4. Data analysis
         3.4.5. User Safety
   3.5. Measuring the rate of juvenile hormone biosynthesis by the paired corpora allata
      3.5.1. Purifying and preparing radioactive methionine for use in assay
      3.5.2. Measuring the rate of JH biosynthesis
      3.5.3. Data analysis
      3.5.4. User Safety
4. Biogenic amine extraction and quantification by HPLC-ECD
   4. 1. Introduction
   4.2. Dissection of the brain from the head capsule
   4.3. Pre-analysis preparation of the HPLC
   4.4. Preparation of internal and external standards

      4.4.1 Stock dilutions (1 x 10^6 pg/μl)
      4.4.2. External and Internal standard Dilution ES-A Dilution IS-A (5000 pg/μl) Dilution IS-B (500/250 pg/μl) Standard Curve
   4.5. HPLC separation of standards
   4.6. Sample preparation
   4.7. Separation and quantification of biogenic amines by HPLC

5. Temperature, radiation and humidity measurement in honey bees
   5.1. Contact thermosensors

      5.1.1. Thermocouples and thermoneedles Thermocouple types Calibration Use inside colonies
      5.1.2. Thermoresistors (thermistors, Pt100)
   5.2. Non-contact temperature measurement
      5.2.1. Infrared spot thermometers
      5.2.2. Infrared thermography Main thermographic camera types Honey bee cuticle infrared emissivity Thermography camera calibration with reference radiator Attenuation of infrared transmissive films
   5.3. Operative temperature
   5.4. Radiation sensors

      5.4.1. Star pyranometers (according to Dirmhirn)
      5.4.2. Photoelectric pyranometers
      5.4.3. Measurement of the short-wave radiation balance
5.5. Humidity measurement
6. Respiration and energetics measurement in honey bees

   6.1. Flow-through respirometry

      6.1.1. Measurement arrangements, measurement chambers and accessories General setup Air drying, CO2 scrubbing and tubing Serial measurement arrangement Parallel measurement arrangement Measurement chambers
      6.1.2. O2 consumption Fuel cell devices Paramagnetic devices Calibration Indirect calorimetry: calculation of energy turnover
      6.1.3. CO2 production Measurement range selection DIRGA Calibration Respiratory quotient (RQ): calculation of energy turnover from CO2 measurements
      6.1.4. H2O balance
      6.1.5. Impact of flow control and measurement chamber size on sensitivity and temporal resolution
      6.1.6. Controlling relative humidity
      6.1.7. Closed chamber method (CO2 accumulation)
      6.1.8. Thermolimit respirometry
   6.2. Chemical-optical oxygen sensors
   6.3. Manometric and volumetric respirometry
   6.4. Titration methods
   6.5. Isotopic tracer techniques
   6.6. Calorimetry
   6.7. Energetics derived from measurement of sugar consumption
   6.8. Activity monitoring

      6.8.1. Video and thermography
      6.8.2. Optical activity detectors