Method I. Small Replicates
By Dr. Jay Evans, USDA-ARS Bee Research Laboratory, Beltsville, MD USA.
- 16 oz. Solo cups with lids. Poke about 50 holes around the cup walls using a flame-heated fork, avoid fumes
- 12 x 12 x 12 or so styrofoam cooler, hole in lid with surgical/tygon tube slipped through
- CO2 supply pressurized in a canister. (Dry ice can be used. In that case a chunk of dry ice would be placed in a separate chamber and somehow the sublimating fumes but not the cold can be directed into the chamber with bees. In a pinch bees can be anesthetized by cold, in small groups in a regular freezer, but timing is hard to regulate since they cluster, and the stress is hard on them. We have lost more bees that way)
- Pasteur pipettes, clipped just at bulb.
- Available bees.
- Stressor, parasite, etc.
- Sucrose solution 1:1 by weight/ final volume.
- 5 ' surgical tubing, maybe 1/4" diam? It doesn't matter much, just whatever is attached to the canister.
- Incubator set at 33 degrees C (91.4 degrees F) with pan of water below racks for humidity.
One possible protocol
- Smoke colony.
- Brush bees from an outer frame into a holding cup (can be at higher density, 200-250 bees/cup, just don't let them overheat).
- Place cup into styrofoam chamber and run CO2 from canister for 45 secs or so, checking to see when bees are KO'd.
- Pour bees into individual cups, 40 bees/cup. Put the lids on tight on and set on a cookie tray.
- Something to challenge bees (e.g., 10x solution with a chemical, with a spore suspension, with a hemolymph dilution from sick bees, etc.)
- Place sucrose syrup to which you have added challenges or controls into pipettes. (We use a sterile plastic dish, pour about 10 mls solution in and draw into the bulbs.)
- Set up at least three replicates/treatments as 40-bee cups and place into incubator.
- Collect bes at 72 hours, freeze the samples until dead.
- Dissect bee midguts from the bodies. If you haven't done so there are many protocols on the web, or I can film it, just involves holding the thorax with one pair of forceps while tugging the 4th or so abdominal plate away to the distal (butt) side, the abdomen should separate and the midgut looks cloudy and is between the crop and the rectum, which are both more 'bulblike'.
- As you suggest a simple bacterial culture (MR+S plates?) might do the trick here on out, or there are genetic screens for bacterial loads.
- A parallel set of bees can be maintained in the incubator, checking each day and counting the dead bees on the bottom.
Method II. 1,000 to 2,000 bees per treatment group
By Dr. Judy Wu-Smart, University of Nebraska, Lincoln
For each package of bees (containing 2 -3lbs of bees or 7-10,000 bees) split into 5 treatment groups (containing 1 to 2,000 bees):
- Control (split into three separate cages): one cage should have control + probiotics1
- Glyphosate (low dose)
- Glyphosate (high dose)
- Glyphosate (low dose) + probiotics1
- Glyphosate (high dose) + probiotics1
Continuously monitor and record mortality through out the experiment. Take a subset of X bees (ex: 100 bees) from each group to examine the gut fauna (or whatever measures) pre-treatment. Treat each treatment group chronically for X days (ex: one week). After chronic exposure is complete:
- take another subset of X bees (ex: 100 bees) from each group to examine the gut fauna (or whatever measures) to examine differences among treatments after chronic exposure.
- supply remaining bees with new untreated syrup and water.
After chronic treatment, give each treatment group (and leave one of the control cages alone) probiotics2. That gives you two controls with and one without probiotics to see if probiotics affect untreated bees. Take another subset of X bees (ex:100 bees) from each group to examine the gut fauna again.
This last part is optional but if probiotics can help mitigate effects from herbicide exposure then I think it may help answer an interesting question of whether the probiotics act as a preventive benefit (probiotic1=if present during exposure) or if it'll have an affect given after the exposure (probiotic2). Probiotic1 and probiotic2 are the same thing just different in the sense of when it was present in exposed bees. This design tests whether probiotics given concurrently (probiotics1+probiotics2) or after herbicide treatment (probiotics2 only) will affect bees exposed to glyphosate.
Finally, leave the rest of the bees in cages to monitor longevity of bees and continue to record mortality each or every other day.
Please keep in mind this would be essentially one rep and it would be wise to do at least 3 reps (using different packages).
Also, note pollen is an important factor I did not mention because I'm not sure if the probiotics will contain any pollen/protein source but that will affect longevity severely (which is why there are a number of control groups).
You can either feed pollen supplement to all cages or none just note that the lack of pollen will make bees die faster in cages.
Also note that the number of bees in the controls will be smaller because the controls have been split into three groups but you generally do not need to dissect or sample the same number of bees then the treatment bees particularly if you find little variation and mortality among control bees (which is usually the case)(ex: sample 50 instead of 100 bees). Take measures as proportions so that they may be compared across treatments more easily.
To summarize treatment groups in the design above:
|Treatment groups||After herbicide treatment (add probiotics2)|
|1) Control (split into three separate cages):
|1) Control (split into three separate cages):
|2) Glyphosate (low dose)||2) Glyphosate (low dose) + probiotics2|
|3) Glyphosate (high dose)||3) Glyphosate (high dose) + probiotics2|
|4) Glyphosate (low) + probiotics1||4) Glyphosate (low) + probiotics1+ probiotics2|
|5) Glyphosate (high) + probiotics1||5) Glyphosate (high) + probiotics1+ probiotics2|
Other issues worth mentioning:
- Bees from queenless packages will vary in age and may senesce more quickly in the absence of brood and queen pheromone.
- Getting bees into cages may also be a challenge. Chilling a package will cause bees to cluster consider the use of CO2 but be careful as too much will kill them too.