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Sampling Alfalfa Leafcutting
Bee Cells To Assess Quality

Karen Strickler, Craig Baird, and Ron Bitner


University of Idaho
College of Agriculture

Cooperative Extension System, Agricultural Experiment Station CIS 1040


  Why sample bee cells?
  Using the Parma Cocoon Testing Lab
  Laboratory services
  Advantages of using the Parma Cocoon Testing Lab
  How to collect a sample
       Loose cells
       Polystyrene boards
       Solid wood boards without a backing
  How to send a sample
  How the laboratory tests cocoons
  Analyzing loose cell samples yourself
  Interpreting conflicts with Canadian results
  Calculating how many live cells you have and how many you need
       Loose cells
       Wood boards
  Determining percent emergence after releasing bees
       Loose cells
       Solid boards
  Further readings
  The authors

Why sample bee cells?

Pollinator management is one of the largest expenses in Northwest alfalfa seed production. According to Oregon State University, a typical seed grower spends about $194 per acre for alfalfa leafcutting bee pollination. This cost varies with the price of Canadian loose cells, the number of replacement bees needed, and the management system. Successful pollination management requires accurate annual assessments of bee quality and quantity in order to adjust pollinator stocks and control costs.

Sampling bee populations allows you to:
bulletEstimate the percentage of cells containing live prepupae
bulletDetermine sources of bee mortality
bulletCalculate live cells per pound and total live bees produced in your field
bulletDetermine how many additional bees to purchase for the next season
bulletTrack changes in bee quality due to changes in management practices and weather, if you  keep records over several years
bulletConfirm that you are getting the expected live count of Canadian bees
bulletDetermine how advanced bees are in their development if temperature controls fail in cold storage facilities
bulletEvaluate loose cells after bees have emerged and been released in the field to find out if any intact cells contain bees that developed to the pupal or adult stage but were unable to chew out of their cells. This may happen if bees experience unfavorable conditions in the spring or fall.
You can sample bee cells in a variety of ways, depending on management practices. Loose cells can be cut open by hand or x-rayed by a cocoon testing facility. A small sample can be punched by hand from polystyrene boards or wood boards with a removable backing if you do not wish to have all the cells punched out of the boards. Solid wood boards without removable backing can be sampled with a larval probe.

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Using the Parma Cocoon Testing Laboratory

Note: For information on the current status of the Parma Cocoon Testing Lab, contact the Parma Research and Extension Center (208) 722-6701 x242; jbarbour @ uidaho.edu

The Parma Cocoon Testing Laboratory at the University of Idaho’s Parma Research and Extension Center opened in November 1994. Its purpose is to provide accurate estimates of the quality of bees from leafcutting bee cell samples. It can also provide an estimate of percent successful emergence and sex ratio of bee samples. Producers and pollination consultants can keep a record of their results and use this information to monitor and improve their bee populations. X-ray equipment for the Parma Cocoon Testing Laboratory was purchased jointly by the Idaho and Oregon Alfalfa Seed Commissions and the Nevada Alfalfa Seed Research and Promotion Board. There is a fee for using laboratory services.

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Laboratory services

Note: For information on the current status of the Parma Cocoon Testing Lab, contact the Parma Research and Extension Center (208) 722-6701 x242; jbarbour @ uidaho.edu

Based on x-ray examination, the laboratory will provide you with a report containing the following information about your loose cells:
bulletPercentages of cells with live larvae, chalkbrood, pollen balls, parasites, dead larvae, and machine damage
bulletA pie chart summary of the percentages
bulletMean live cells per pound
bulletA 95 percent confidence interval around the estimated mean. (There is a 95 percent chance that the live cells per pound falls somewhere between the upper and lower values of the confidence interval.)
For an additional fee and a wait of several weeks, you can get:
bulletPercent of live cells that successfully emerge
bulletSex ratio
Live cells are incubated to determine sex ratio and the percentage of live cells that successfully emerge. Bees left at warm temperatures for more than 2 weeks in the fall may appear to be alive when cut open or x-rayed but may not successfully develop into adults and emerge. An emergence and sex-ratio test can help determine if your bees have received appropriate treatment and will emerge successfully. The sex ratio information will tell you whether the expected 35 percent of your bees are females. Since females do most of the pollinating, samples with higher female counts are more valuable than samples with lower female counts.
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Advantages of using the Parma Cocoon Testing Laboratory

Note: For information on the current status of the Parma Cocoon Testing Lab, contact the Parma Research and Extension Center (208) 722-6701 x242; jbarbour @ uidaho.edu

An x-ray report provides more detailed information about loose cells than most growers get if they open cells themselves. The Parma Cocoon Testing Laboratory sample includes about 500 cells, more than most growers have time to open. Submitting samples to the Parma Cocoon Testing Laboratory may save you time. Usually results are ready within 2 to 3 work days. The report also provides an objective written confirmation that you are getting the live count of Canadian bees that you expect.
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How to collect a sample

(See also Wayne Mennie's sampling advice)  
Cell moisture content 
may change after cells 
are punched out. This 
can alter estimates of 
live larva per pound. 
See  “Interpreting Conflicts with Canadian Results.”
Loose cells—Be sure that loose cells are well tumbled, because the weight of leaf debris is  not taken into consideration in calculating live larvae per pound for loose cells. The accuracy of your sample depends on how representative it is of the entire population of bees. Take a handful 
of cells from two or three different depths in each container to be tested. Mix these cells well in a separate container, and measure out 100 grams (3.5 ounces or about 2 cups) per x-ray to send to the laboratory. Please do not mix Canadian bee samples with local bee samples because bee quality differs greatly between sources. 

Polystyrene boards—Growers who buy solid polystyrene boards can punch out one or two nest tunnels per board with an Allen wrench or other straight tool. Tell the lab that your sample was hand-punched from boards rather than from tumbled loose cells so that the entire sample will be weighed to take into account leaf remains when live larvae per pound is calculated. If you know the average weight of empty boards and filled boards, you should be able to calculate the live count per board from the information the lab provides. 


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Sampling solid wood boards without a backing

Wood boards are sampled with a larval probe, which can be purchased from some suppliers of wood boards. A probe is a corkscrew wire the diameter and depth of the holes in solid  wood boards. The corkscrew is glued inside a pen case.

Larval probes work best when most cells contain live prepupae that have spun their cocoons. If many cells contain pollen balls, it may be difficult to separate individual cells. The probe itself may also stab some live larvae, making it difficult to know if the larva was live or dead before sampling. Despite these problems, a larval probe is the best method we currently have to estimate bee quality in solid wood boards.

The bee board is probed most easily if it is standing upright. Insert the probe into a hole by pushing gently and turning counterclockwise until it can go no deeper. Then pull straight out to remove the cells. Remove the cells from the probe by holding them gently and turning the probe clockwise. If the probe has not cut through the leaves and cocoon of a cell so that the larva is visible inside, you may need to use the point of the probe or a knife to cut open the cocoon.

Count the number of live larvae each time that you probe a hole. Try to sample one hole in each of at least 50 boards to get a representative sample of your bees. You also should estimate the percentage of filled holes per board.
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How to send a sample

Place bee cells in a paper bag (so air can reach the cells) with your name, address, and phone number and a fax number if you wish to have results faxed to you. Include all phone numbers where you can be reached. To maintain confidentiality, results cannot be phoned or faxed to numbers not received with the sample.

If you send more than one sample, clearly label each sample so that they will not be confused. Place the paper bag in a container to prevent crushing during shipping. A clean margarine container, coffee can, or similar container will do. Insulate with packing material such as plastic peanuts or crumpled newspapers.

Ship your package via U.S. mail, UPS, or Federal Express, or deliver it by hand. Try to avoid shipping by surface mail if temperatures are expected to be especially hot or cold. Clearly mark the outside of the package “LIVE MATERIAL. AVOID EXTREME HEAT OR COLD.”

Address samples to:

Parma Cocoon Testing Laboratory
Parma Research and Extension Center
29603 U of I Lane
Parma, ID 83660

Contact the Parma Cocoon Testing Laboratory for current fees and include payment with your sample. Call 208-722-6701 weekdays, 8 a.m.-12 p.m. and 1 p.m.-5 p.m. mountain time.

Note: For information on the current status of the Parma Cocoon Testing Lab, contact the Parma Research and Extension Center (208) 722-6701 x242; jbarbour @ uidaho.edu

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How the laboratory tests cocoons

When a sample arrives, all information about the sample including date of arrival is recorded. If the sample cannot be processed immediately, it is put into cold storage (5°-10°C = 40°-50°F) until it can be processed. Samples are processed on a first-come, first-served basis.

Loose cells—When ready to be processed, the sample is divided into five subsamples of 10 grams each, laid on contact paper, and x-rayed. X-rays are evaluated, and cells whose contents are unclear are opened for confirmation. Results are then recorded, tabulated, and graphed. A copy of the results is sent to you along with the x-ray.

Boards—For samples hand punched from solid polystyrene boards, the entire lot is weighed. Loose leaf debris is separated from good cells and weighed. The sample is divided into 10-gram subsamples, and five of these subsamples are chosen at random for x-ray analysis. This avoids a potential bias for leaving heavier cells (e.g., pollen balls) in the bottom of the pile. Crushed cells containing dried prepupae with no mold are counted as live. When calculating live larvae per pound, average live larvae per subsample is divided by 10 grams plus a fraction of the weight of leaf debris, which depends on the number of 10-gram subsamples. This is multiplied by 454 to convert grams to pounds.

Emergence and sex ratio—Samples are accepted for emergence/sex ratio testing between January and April. The cells are first x-rayed as described above. Cells that appear live in the x-ray are removed from the contact paper and placed in small plastic trays with individual wells for each cell. The trays are labeled with the grower’s name and the date the test is begun. Trays are stored at 85° to 88°F until all live bees have cut through their cells.

Although bees stored at 41°F for 6 months should complete emergence in about 30 days, bees stored for only 3 or 4 months may take several weeks longer to complete emergence. When emergence is complete, the sex of emerged bees is recorded. Cells that have not emerged are opened to determine at what stage the bees died. A report is mailed as soon as the data are compiled.
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Analyzing loose cell samples yourself

Many growers prefer to analyze their own bee cell samples. The advantage is that results are obtained on the spot without any cost other than the time to sample. Opening cells yourself gives you direct visual, tactile, (and often olfactory!) information about the quality of the bees.

If you analyze your own bee samples, select a representative sample in the manner described above. Weigh out 0.35 ounces (10 grams) of cells instead of 3.5 ounces. This should be about 100 cells. Alternatively, count out 100 cells and then weigh them. It is a good idea to take several such samples and calculate average live cells per ounce.

Use a small, sharp utility knife, razor blade, or pen knife to cut the cap off each cell or to carefully slice across the length of the cell just below the surface of the leaves. Cut through the leaves and cocoon but not into the larva itself.

Live larvae will be full and white and will spring back when pressed with a pencil point. If you are not sure whether the larva is alive, lay the larva on the table and briefly press on the head area until it is nearly flat. If it springs back after you release the pressure, then the larva was healthy. If the head area stays depressed, the larva was dead or dying.

Count the live larvae in your sample.

Percent live larvae = number of live larvae x 100.
                                       number of cells opened
If you wish to calculate live larvae per pound, use one of these formulae, depending on your measurement units:
Live larvae per pound = number of live larvae x 16
                                              ounces cells opened
= number of live larvae x 454.
         grams cells opened

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Interpreting conflicts with Canadian results

The number of live larvae per pound that you calculate or that is calculated by the Parma Cocoon Testing Laboratory may differ from that calculated by the Canadian Cocoon Testing Lab in Brooks, Alberta. Differences result from changes in the percentage of live larvae, changes in the number of cells per gram, and/or from natural variability in the sample.

If the percentages of live larvae for the two samples are close, the quality of bees has not changed since the Brooks Lab sample. If the percentage of live larvae has decreased, check to see if the percentage of dead larvae has increased. Mortality may have occurred from unfavorable conditions in your storage facilities or from damage incurred during sampling. Consider these possibilities before you blame your supplier.

Live larvae per pound may change over time as cell moisture content changes, without a change in the number of live bees. If the number of cells per 10-gram sample has increased between samples, cells may have dried since the last test. Cells per pound will have increased, but total pounds of cells purchased will have decreased.

Conversely, if the number of cells per 10-gram sample has decreased between samples, cells may have picked up moisture since the last test. Cells per pound will have decreased, but total pounds purchased will have increased. In either case, if the percentage of live larvae is similar between the two samples, the total number of live bees purchased (or gallons of live bees purchased) has not changed. The total pounds of cells and the number of live bees in your purchase should be determined at the same time as live larvae per pound.

Also remember that there is natural variability between samples because the cells themselves vary. The Parma Cocoon Testing Laboratory calculates a confidence interval (an upper and lower value) around the estimate of live cells per pound that takes into account variability between 10-gram samples. If another estimate falls within this interval, the two estimates are not significantly

The more variable the subsamples, the greater is the difference between the upper and lower values of the 95 percent confidence interval. The true variability may be even greater, because one 50-gram sample of cells may not be representative of a very large lot. Also, different people reading the same x-ray may disagree about the contents of a few cells. All of these factors can explain differences in the results from different laboratories.
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Calculating how many live cells you have and how many you need

Current recommendations are to allow at least 20,000 live bees per acre. Many growers report that they get faster pollination using 30,000 to 50,000 live bees per acre. It is not clear that this number is necessary in normal years. Excess bees may drift if nest holes or untripped flowers are scarce. However, extra bees may be needed in cool, cloudy weather when bee flight is restricted. Bees are usually sold by the gallon, which is defined as 10,000 live bees, so most growers try to use 3 to 5 gallons of live bees per acre.

Loose cells

Method 1—Multiply your estimate of live larvae per pound of loose cells by the total weight of loose cells that you have purchased or punched out to calculate the total number of live larvae that you expect to emerge in the spring. You should weigh your total cells at the same time that you estimate live larvae per pound so both weights are consistent with the moisture content of the cells. Divide by 10,000 to get the number of gallons of live larvae. 
1 “gallon” of bees = 10,000 live larvae or 
3,500 live female bees. 
Suppose you have 4,194 live larvae per pound and 124 pounds of loose cells: 
     4,194 larvae per pound x 124 pounds = 520,000 live larvae. 
     520,000 live larvae ??10,0000 live larvae per gallon = 52 gallons of live larvae. 
If you aim to have 4 gallons of bees per acre, this would be adequate for 13 acres.

Method 2—Estimate the total volume of cells that you have by counting how many 5-gallon buckets or other containers of known volume they will fill. Multiply this total volume by the percentage of live larvae divided by 100 to get total volume of live cells. A gallon of live larvae has approximately 10,000 cells. (You can get a more accurate estimate of cells per gallon by counting the number of cells in a 1-cup measuring cup and multiplying by 16, the number of cups in a gallon. Expect about 625 cells per cup.) 

Suppose you have 25 1/3 5-gallon drums filled with loose cells: 
     25.33 drums x 5 gallons per drum = 126.65 total gallons of loose cells. 
Suppose your loose cells are 41% live larvae: 
     126.65 gallons loose cells x 0.41 = 52 gallons of live larvae. 
Method 3—If you have had emergence/sex ratio testing, you can better estimate the number of live bees. First adjust the number of gallons of live larvae to reflect the percentage that actually emerge: Multiply the number of gallons of total live cells by the percent emergence divided by 100. 

To determine how many gallons of female bees you have, multiply the number of live emerged bees by the percentage of females divided by 100. Aim to release 7,000 to 17,500 females per acre (the equivalent of 2 to 5 gallons or 20,000 to 50,000 live bees. This assumes an average of 35 percent females in the population.)

Suppose the results of an emergence test indicate that 92 percent of live larvae will emerge 
from your sample and suppose that you have 52 gallons of loose cells:
52 gallons x 0.92 = 47.8 gallons live adults. 
47.8 gallons x 10,000 live bees per gallon = 478,000 live adults. 
Suppose 44% of the emerging bees are females:
478,000 live adults x 0.44 = 210,000 females. 
If you are aiming for the equivalent of 4 gallons of live bees per acre (40,000 bees), you 
want to use 14,000 females per acre (40,000 bees x 0.35). Thus, 210,000 females should 
be adequate to pollinate 15 acres: 
210,000 females ??14,000 females per acre = 15 acres. 
Note that by considering the percentage of emerged females, which is greater than the 
average of 35 percent, we determined that these bees should adequately pollinate more 
acres than was indicated by the number of total live larvae.

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Wood boards

If you are using a probe to sample filled boards, divide the number of live larvae that you encounter by the number of holes that you sample to get the average number of live larvae per hole. Then multiply average live larvae per hole by the number of filled holes in a block. For solid wood blocks that are completely filled, this is about 2,096 holes. For partially filled boards, this is 2,096 multiplied by the fraction of holes that are full (for example, 3/4, 1/2, or 1/4).
Suppose you average 1.65 larvae per hole. A full board would have about 
1.65 larvae per hole x 2,096 holes per board= 3,458 live larvae per board. 
Since 1 gallon is defined as 10,000 live cells, you have the equivalent of
0.35 gallons of bees per board
(3,458 live larvae per board ??10,000 live larvae per gallon). 
To have 20,000 live bees per acre you will need
20,000 live bees per acre ??3,458 live larvae per board = 5.8 boards per acre. 

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Determining percent emergence after releasing bees

Fall treatment of bees may affect spring emergence. Bees that experience warm temperatures for 20 days or more may look alive when cut open or x-rayed during the winter, but they may not have enough energy reserves to emerge. Sampling cells after emergence can tell you whether emergence was as high as the percent live larvae value suggested. If it was not, you may want to consider whether your fall management techniques or those of your supplier are adequate. Bees should be moved to 60°F (15°C) as soon as possible, then gradually cooled to between 40° and 50°F (5° to 10°C) and not allowed to warm up until spring.

Loose cells

There is no easy way to determine percent emergence from the weight of empty cells. However, percent emergence measured after bee release can be compared with the percentage of cells with live larvae measured during the winter.

To sample emerged loose cells, wait until no new bees emerge from your trays. Take a handful of cells from each shelter, or from a selection of trays in your incubator, and mix them together in a container. Randomly select 100 cells from the mixture, and check to see if there is an emergence hole.

If you check 100 cells, the number of cells with holes is an estimate of percent emergence and should be close to the percentage of live larvae calculated during the winter. If more cells than expected are still sealed, you may want to open some to see if dead pupae or adults are inside. The Parma Cocoon Testing Laboratory will provide an x-ray and count of emerged cells for growers who would like a written assessment.
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Solid boards

To check emergence in solid boards, put two or three representative boards in a phase-out shelter. If the boards are supposed to have a high live count, most nests should have a hole at the nest entrance when emergence is complete. If more holes than expected are still capped, use a larval probe for wood boards or hand punch a few nest tunnels from polystyrene boards to determine how many dead pupae and adults are in the tunnels.


Further readings

Alfalfa Seed Production and Integrated Pest Management, WREP 12, $5.00
Order from Bulletin Office, Cooper Publications Building, Washington State University, Pullman, WA 99164-5912,
(509) 335-2857.
How and Where Were They Raised? Critical Management Considerations in Megachile. Proceedings of the 26th Northwest Alfalfa Seed Growers Winter Seed School. pp. 27-35.

The authors—Karen Strickler, Pollination Ecologist, Department of Plant, Soil, and Entomological Sciences, UI Parma Research and Extension Center; Craig Baird, Extension Entomology Specialist, Department of Plant, Soil, and Entomological Sciences, UI Parma
Research and Extension Center; Ron Bitner, International Pollination Systems, Caldwell, Idaho.

Issued in furtherance of cooperative Extension work in agriculture and home economics, Acts of May 8 and June 30, 1914, in cooperation with the U.S. Department of Agriculture, LeRoy D. Luft, Director of Cooperative Extension System, University of Idaho, Moscow, Idaho 83844. The University of Idaho provides equal opportunity in education and employment on the basis of race, color, religion, national origin, gender, age, disability, or status as a Vietnam-era veteran, as required by state and federal laws.

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August 4, 2000, August 8, 2009
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