Efficient Sampling 101: Flowchart and Protocols for Multi-Purpose Analyses of Honey Bee Samples

By Dick Rogers, Principal Scientist/Entomologist, Bayer Bee Care Program

Dick Rogers Hive DiverI am a hive diver, and I need to be organized and efficient when I explore the depths of a hive. Let me explain:

I have been keeping and studying honey bees for just over 44 years. During this time, I found that I sometimes struggled to identify and understand what I was seeing inside honey bee hives. Many times, this was because samples were collected with a specific type of analysis in mind.

Within the first couple of years of the new millennium, I started doing research to investigate the many factors affecting honey bee health. This included checking for two species of parasitic bee mites, Nosema apis (a type of fungus), various other possible diseases and disorders, pesticide residues and Varroa resistance to varroacides. Samples of adult honey bees were needed for each test, but collecting separate samples was time consuming and a logistical nightmare. Since it was destructive sampling, taking multiple samples also had the potential to seriously reduce the adult bee population, especially of smaller or struggling colonies, and increase the risk of accidental collection of, or injury to, the queen.

To remedy the problem of having to take multiple samples, I made a list of all the key analyses that were practical and cost-effective to do based on a field-collected sample of adult honey bees. From there, I developed a flowchart/decision-tree to chart the process for achieving the greatest amount of information out of only one sample of bees. This flowchart can be found below, and I’ll describe some of the common methods I use when sampling.

Dick Rogers Bee Sampling Method Flowchart

Sampling Methods

Resistance

Collecting Samples From BeesTesting for Varroa resistance to the most commonly relied-on varroacide is a good practice to protect your hive. This is why at least once per season, preferably in the late summer, a beekeeper should perform a resistance test. I recommend collecting the bees live from a brood frame with all stages of brood directly into the test container, being careful not to collect the queen. Since a tab of the control product is already in the test chamber, the three- to six-hour exposure period starts immediately. At Bayer, we use the Rogers test method, which is a modified version of the former Central Science Lab method (now the National Bee Unit, UK). Any beekeeper who is interested in more details about this method, including test chamber specifications, tab size determination and data sheet, or who would like to participate in our annual North American varroacide efficacy survey, can contact me at dick.rogers@bayer.com.

Dick Rogers Bee Sample Method

Varroa Mite (Varroa destructor):

The final step in the resistance test requires the determination of the number of Varroa mites per 100 bees. To get this number, the sample is washed following the Currie (2013) method. Washing a sample of bees is necessary when quantitatively assessing Varroa abundance to get a number that can be compared to a provisional threshold, so the only extra work for the resistance test is to expose live bees for a timed period. After washing the bees, count the number of mites and count the number of bees washed. Record these numbers and use them to calculate mites per 100 bees.

Alcohol Washing Method for BeesVarroa Mite Picture

Honey Bee Tracheal Mite (Acarapis woodi)

Varroa MiteThe next step is to select 50-100 bees from the washed sample to process for examination for tracheal mite infestation. The number of bees selected may vary depending on how much time and resources are available, as well as the level of statistical power desired. In any case, the processing of the bees is the same: slice off the head and first section of the thorax (prothorax) of each bee and then soak the thoracic discs in a solution of KOH overnight at 37° C (99° F). This will clear the muscle tissue from the discs and make the tracheal tubes easier to see under a dissecting scope. Confirm any suspect tracheal mite detections using high power microscopic examination. Record the number of discs infested with tracheal mite and then calculate the percentage of discs infested. Instructions for the whole process for tracheal mite can be found in Shimanuki and Knox (2000).

Nosema spp (N. apis and N. ceranae)

NosemaWhen selecting bees for the tracheal mite analysis after the Varroa wash, it is also a good time to select up to 30 bees with swollen abdomens for Nosema analysis. These bees can be set aside and frozen until ready for processing, if needed. When ready to process, select a known number of these bees (10-30), separate the abdomen from the rest of the bee body, put the abdomens into a small biobag or mortar with 1 mL of water per abdomen (e.g. 30 abdomens + 30 mL water) and thoroughly crush the abdomens to expel all the gut contents. The result is a spore suspension. Thoroughly shake the suspension before removing a small amount for high power microscopic examination. More details can be found in numerous references, such as Cantwell (1970; ABJ 110:6), Rogers and Williams (2007; Bee Culture vol135) or COLOSS BEEBOOK VOL II. Using this method, followed by Nosema species identification by molecular (PCR) techniques, resulted in first detection of Nosema ceranae in Canada and the central U.S. (Williams and Rogers, 2007).

Other organisms

During microscopic examination of Nosema spore suspensions, or while using other specific slide mounts and staining techniques from bees set aside from the sample, other microscopic organisms might also be detected. Examples of other organisms include species of the genus Apicystis – a cosmopolitan neogregarine parasite of Apis mellifera and Bombus spp (Lipa and Triggiani, 1996), the bacterium Pseudomonas aeruginosa (causes septicemia in adult honey bees) and species of Spiroplasma (a group of parasitic bacteria without cell walls that cause spiroplasmosis in insects, including bees). Viruses require special collection and processing techniques and are generally outside the normal inspection capabilities of beekeepers. As such, they are not included in this flowchart.

Final Thoughts

This process is easy to follow for students, researchers, and many beekeepers.

This flowchart and sequence of analyses improve efficiency of processing samples and maximize the information that can be gained from a single sample of live collected bees at minimal cost. The techniques used are generally within the purview of many beekeepers, apiculturists, students and researchers, which makes the flowchart relevant for practical use, citizen science, training/education and research purposes.

However, this process is not suited for viruses and residues.

Although the methods outlined in this flowchart are not suitable for viruses, it is a good model for the development of a flowchart for viruses. Samples for residue analyses of crop and bee protection products also require special techniques for collection, handling and processing. Residue samples might include a number of different matrices, such as incoming nectar and pollen, stored honey and pollen, wax, brood food or adult bees. It is easy to imagine how collecting these samples, often in duplicate and triplicate, could get overwhelming and therefore requires the greatest amount of attention to special procedures and recordkeeping.

Decisions to make during the process

The flowchart contains guidance on numbers of bees needed, how to select bees for standardization, solutions required (e.g. KOH), how to examine samples, where specimens of bees go in the process, how to handle samples based on positive and negative detections, and when and what to record. Including even more details and expanding the scope to include collection and process details for virus, residue and other organisms’ samples would be useful but might be considered too complex to be of practical value to beekeepers. Therefore, the current flowchart is useful to keep handy for study and use. I hope you enjoy putting it to good use for your own research and/or monitoring purposes!