Standard methods for varroa research

Authors: Vincent Dietemann, Francesco Nazzi, Stephen J Martin, Denis L Anderson, Barbara Locke, Keith S Delaplane, Quentin Wauquiez, Cindy Tannahill, Eva Frey; Bettina Ziegelmann, Peter Rosenkranz, James D Ellis.

Table of contents

Authors
Summary
1. Introduction
2. Taxonomy and systematics

   2.1. Taxonomy
   2.2. Collection of mites for identification

      2.2.1. Mite appearance
      2.2.2. Where to find mites
      2.2.3. Sampling techniques
      2.2.4. Storage of mite samples
         2.2.4.1. Storage medium and conditions
         2.2.4.2. Storage and collection container
      2.2.5. Sample shipping
   2.3. Morphological methods for identifying Varroa
      2.3.1. Sample preparation
      2.3.1.1. Recipe for Nesbitt’s Solution
      2.3.1.2. Recipe for Hoyer’s medium
      2.3.2. Sample identification
   2.4. Molecular methods and systematics
      2.4.1. DNA extraction
      2.4.2. DNA amplification
      2.4.3. DNA sequencing
      2.4.4. Species identification
      2.4.5. Haplogroup and haplotype identification
      2.4.6. Kinship determination with microsatellites
   2.5. Perspectives on the taxonomy of Varroa spp.
3. Laboratory techniques
   3.1. Collecting mites

      3.1.1. Manual collection
      3.1.2. Icing sugar
      3.1.3. Washing with water
      3.1.4. Collection from brood
         3.1.4.1. Collection from L5 larvae
         3.1.4.2. Collection from capped cells
         3.1.4.2.1. Opening each cell
            3.1.4.2.2. Opening large number of cells and washing the brood
   3.2. Rearing mites in the laboratory
      3.2.1. Maintaining mites in the laboratory
         3.2.1.1. Maintaining mites on adult honey bees
         3.2.1.2. Maintaining mites on honey bee brood
         3.2.1.3. Artificial diet
      3.2.2. Breeding mites in the laboratory
         3.2.2.1. Natural infestation
         3.2.2.2. Artificial infestation
   3.3. Assessing reproduction in the laboratory
      3.3.1. Assessing fertility
      3.3.2. Assessing oogenesis
   3.4. Marking techniques
      3.4.1. Oogenesis
      3.4.2. Feeding site
      3.4.3. Marking mites
   3.5. Infecting varroa with secondary diseases
      3.5.1. Microinjection
      3.5.2. Dipping
   3.6. Bioassays
      3.6.1. Experimental conditions
         3.6.1.1. Environment
         3.6.1.2. Dosage of chemicals
         3.6.1.3. Mites to be used in the tests
      3.6.2. Bioassays in Varroa chemical ecology
         3.6.2.1. Cell invasion
            3.6.2.1.1. Mites to be used
            3.6.2.1.2. Experimental setup
            3.6.2.1.3. Data analysis
         3.6.2.2. Oogenesis
           3.6.2.2.1. Mites used in the bioassay
           3.6.2.2.2. Experimental setup to test the activation of oogenesis
              3.6.2.2.2.1. In the field
              3.6.2.2.2.2. In the laboratory
           3.6.2.2.3. Experimental setup to test oviposition
         3.6.2.3. Orientation inside the sealed cell
            3.6.2.3.1. Mites to be used
            3.6.2.3.2. Experimental setup
            3.6.2.3.3. Data analysis
         3.6.2.4. Phoretic phase
            3.6.2.4.1. Mites to be used
            3.6.2.4.2. Experimental setup
            3.6.2.4.3. Data analysis
         3.6.2.5. Mating bioassays
            3.6.2.5.1. Mites used in the bioassay
            3.6.2.5.2. Experimental setup
      3.6.3. Bioassays to quantify the susceptibility of the Varroa mite to acaricides
         3.6.3.1. Mites used in susceptibility bioassays
         3.6.3.2. Bioassays for contact substances
         3.6.3.3. Bioassays for volatile substances
         3.6.3.4. Data analysis
4. Field methods
   4.1. Diagnostic techniques
      4.1.1. Debris examination
      4.1.2. Brood examination
      4.1.3. Bee examination
   4.2. Measuring colony infestation rate
      4.2.1. Acaricide treatment
      4.2.2. Whole colony estimate
      4.2.3. Measuring the infestation rate of brood and adult bees
         4.2.3.1. Infestation rates of adult bees
            4.2.3.1.1. Sampling
            4.2.3.1.2. Dislodging mites from bees
               4.2.3.1.2.1. Powdered sugar
               4.2.3.1.2.2. Ether wash
               4.2.3.1.2.3. Warm/soapy water or ethanol (75%)
               4.2.3.1.2.4. Assessing the efficiency of dislodging method
         4.2.3.2. Infestation rates of brood
         4.2.3.3. Evaluation of total mite population size in the colony
      4.2.4. Natural mite fall
      4.2.5. Sub-sampling mites to count on a bottom board
   4.3. Estimating reproduction parameters
      4.3.1. Assessing reproductive success
      4.3.2. When to measure reproductive success?
      4.3.3. How to measure reproductive success?
      4.3.4. Assessing oogenesis
   4.4. Estimating  damage thresholds
      4.4.1. How to estimate damage thresholds
         4.4.1.1 Colony establishment
         4.4.1.2 Experimental treatments, sample size, and colony arrangements
         4.4.1.3 Dependent variables and sampling protocols
         4.4.1.4 Analyses, interpretation, and pitfalls
      4.4.2. Regional variations in reported damage thresholds
   4.5. Standardising field trials
      4.5.1. Starting conditions
         4.5.1.1. Obtaining mite free colonies
         4.5.1.2. Obtaining residue free hives
      4.5.2. Artificial mite infestations
         4.5.2.1. How many mites to introduce?
        
4.5.2.2. How to introduce varroa mites in colonies?
         4.5.2.3. How to introduce varroa mites in cells?
            4.5.2.3.1. Manual infestation
            4.5.2.3.2.  Natural infestation
      4.5.3. Field bioassays of semiochemicals
         4.5.3.1. Cell invasion
         4.5.3.2. Mite reproduction
      4.5.4. Testing varroacides in the field
         4.5.4.1. Preliminary tests
         4.5.4.2. Efficacy tests
            4.5.4.2.1. Statistical analysis
            4.5.4.2.2. Hives
           
4.5.4.2.3. Colonies
            4.5.4.2.4. Location
            4.5.4.2.5. Treatment
            4.5.4.2.6. Observations and parameters
               4.5.4.2.6.1. Assessment of efficacy
               4.5.4.2.6.2. Assessment of safety of product for honeybees
         4.5.4.3. Resistance pattern
  4.6. Breeding mites in colonies
 
4.7. Brood attractiveness
5. Acknowledgements
6. References