Lab Experiment I. Effect of a common agricultural herbicide on honey bee gut bacteria (Small Replicates)

From DataCenter
Jump to: navigation, search

Summary

Bees in Solo cups
Bee gut bacteria cultures.

The effect of field realistic dosages of a common agricultural herbicide on honey bee gut bacteria was measured. Three replicates of six groups consisting of 30 to 50 bees in each group were fed sucrose solution with Roundup™ added at the following concentrations:


Group Field Realistic Dose Glyphosate
1 none (control) 0.0 mg/l
2 half minimum 0.7 mg/l
3 minimum 1.4 mg/l
4 mid range 4.5 mg/l
5 maximum 7.6mg/l
6 twice maximum 15.2 mg/l


Small replicates kept in Solo cups were used to learn lab skills, develop procedures and to test equipment. Periodically throughout the experiment, the dead bees in each Solo cup were counted. The live bees were knocked out, sampled, killed and their gut bacteria cultured.

This experiment was followed by Lab Experiment II. Effect of a common agricultural herbicide on honey bee gut bacteria (Medium Replicates)

Background

Spraying Tomatoes on Rabun Gap-Nacoochee School campus, Rabun Gap, GA, USA

The agricultural use of fungicides, herbicides and insecticides is increasing. Concerns have been raised that individually, or through synergistic effects, these compounds are causing Colony Collapse Disorder or otherwise contributing to colony loss. Concerns have been raised that glyphosate, contained in the popular herbicide Roundup®, is affecting animal and human health.

Difficulties with obtaining bees in north Georgia in the winter required starting with small replicates. Methods used were suggested by Dr. Jay Evans: Lab Procedures Method I. Small Replicates. Purpose is to develop necessary skills.

Skills

  1. Experimental Design.
  2. Use analytical balances, micro pipettes, autoclave.
  3. Mix agar, pour plates.
  4. Dilutions.
  5. Remove the gut from bees and prepare samples.
  6. Sterile Techniques.
  7. Organization.
  8. Time Management.

Design Experiment

Questions

  1. What are you capable of doing?
  2. How many cultures can you prepare, how much time do you have to count CFUs?
  3. How much error will you tolerate? How to reduce error?
  4. How will you handle the bees? Knock them out? Cold or CO2?
  5. Sample the gut from live bees or ones that have died?
  6. How to feed them, how to kill them?
  7. What data? Mass, mortality?
  8. Feed pure glyphosate or common agricultural herbicides that contain glyphosate such as Roundup™
  9. How much glyphosate in feed?

Field Realistic Dose

Field realistic dosages are from Goldsborough and Brown, 1988; Feng et al., 1990; Giesy et al., 2000. These amounts were chosen to represent relevant exposure levels to GLYP that the bees would experience.

Experimental Procedure

  • 18 replicates of 30-50 (50 preferred) bees per replicate in 16 oz solo cuts
  • Mass, food intake, mortality and morbidity will be recorded daily (Monday thru Saturday)
  • Feed sucrose solution 1:1 w/w with sterile water. Amounts of Glyphosate (GLYP), in the form of Roundup™, added to the sucrose solution, represent one half the minimum field realistic dosage, minimum field realistic dosage, mid range dosage, maximum field realistic dosage and 2x the maximum. Roundup™ was chosen after an informal survey of feed stores revealed 90% of local farmers used Roundup™ brand herbicide.
  1. One group of 3 replicates fed with no GLYP added to sucrose (control)
  2. One group of 3 replicates fed with 0.7mg/l GLYP added to sucrose
  3. One group of 3 replicates fed with 1.4mg/l GLYP added to sucrose
  4. One group of 3 replicates fed with 4.5 mg/l GLYP added to sucrose
  5. One group of 3 replicates fed with 7.6 mg/l GLYP added to sucrose
  6. One group of 3 replicates fed with 15.2 mg/l GLYP added to sucrose
  • Bees will be kept in an incubator at approx 85 oF and 55% RH
  • Bees will be knocked down either with CO2 or refrigerated at 4 oC to be sampled
  • Bees will be dissected and gut removed and placed in nutrient broth, then diluted to plate onto MR+S agar plates
  • Plates are incubated for 24-48 hours then counted.
  • Solo cup replicates will be sampled on Monday, Wednesday and Friday weekly for a maximum of two weeks after feeding begins or until sample size is to small to be useful.
  • A sample of bees will be taken at the beginning of the lab before any feeding to assay the amount of bacteria present at the beginning of the project
  • At the end of the experiment, the remaining bees will be flash frozen and sent to UGA to measure the amount of GLYP in each group of bees.

Equipment and Supplies

Analytical Balance Ohaus Model AP110S
Micro Pipette Eppendorf
Autoclave Tuttnauer 2540M
Vortex Mixer Baxter Scientific Products SIP Vortex Mixer Catalog S8223-1
Hotplate with Magnetic Stirrer Flinn Scientific Model AP1087
Incubator for cultures Blue M Electric Co. Model 100A
Incubator for bees
Petri dishes diSPo Petri Dish D1906 100x15 mm
Micro Pipette Tips
MRS Agar
Lactobacillus Nutrient

Setup and check equipment

Calibration

  1. Checking the micro pipettes using the analytical balances. The reference sample was DI water at 4C. Calibration was volume/mass.

Prepare incubator

Prepare trial batch of plates

Bee Anatomy

Dry Run

Procedure

Prepare Solo cups

Melt holes in 18 solo cups (3 replicates of 6 samples each). Cut stems from plastic pipettes. Stems need to be as short as possible such that the pipette bulb is within reach of the bee proboscus.

Obtain bees

Removing bees from hives.

Approximately 1/2 lbs of bees were shaken from outsides frames of 3 hives in Athens, Georgia into a .5 lb cage.

Prepare Feed

Roundup Plus,™ (18% Glyphosate, 0.73% Diquat) was added to the sucrose solution at the following concentrations:

Group Field Realistic Dose Glyphosate
1 none (control) 0.0 mg/l
2 half minimum 0.7 mg/l
3 minimum 1.4 mg/l
4 mid range 4.5 mg/l
5 maximum 7.6mg/l
6 twice maximum 15.2 mg/l


Calculate Herbicide Dilutions

Prepare Herbicide Dilutions

  1. Placed 10 mL of Roundup™ in 1 L graduated cylinder.
  2. Filled with water.


Transfer bees to Solo Cups

Bees added to Solo cups and feeding begun-Loading bees into cups presented several problems. The regulator on the carbon dioxide tank froze up and the bees were still active during the loading procedure. We were not able to get even counts into the cups and are depending on initial masses to quantify.

There is difficulty with the incubator maintaining temperature and humidity levels. We have two heaters and humidifiers running 24 hours per day. Data on temp and humidity recorded in hive tool as RGNS Incubator.


Feed

Gas with CO2

The regulator on the carbon dioxide tank froze up and the bees were still active during the loading procedure. We were not able to get even counts into the cups and are depending on initial masses to quantify.


Prepare sample tube

Prepare nutrient Using the Eppendorf Pipettes, put 500 uL in each tube.


Sample bacteria from bee's gut

Removing bee gut.
Bee gut samples.

Prepare 18 samples (3 from each cage) from the gut of 3 bees. The mid and hind gut were sampled from three bees per cup, then preped and plated. Student challenges were organization, time management, and sterile technique. By the end of the 75 minute period, the class was able to quickly prepare and plate bacteria samples.


  1. Autoclave adiquats.
  2. Prepare 18 adiquats by numbering them and filling with 500 micro L of nutrient.
  3. Gas bees to knock them out.
  4. Remove 9 bees from the cage. (There will be three replicates of 3 bees each.)
  5. Using forceps and a dissecting tool, pull abdomen from thorax.
  6. Remove the gut from 3 bees and place in adiquat.
  7. Smash the gut against the side of the adiquat.
  8. Close lid on the adiquat.
  9. Record the number of the cage and adiquat.
  10. Mix for 30 seconds on Vortex Mixer.

Prepare Plates

Ratio is: 100 Ml of Distilled H2O 7 grams of Lactobacilli MRS Agar Alpha Bioscience


  1. Put 200 ml of H2O in 300 Ml beaker.
  2. Add stirrer to beaker
  3. Set heat and stir to 10
  4. Add 14 g Agar
  5. After 3 to 5 minutes it should boil.
  6. Lower stir and heat to 1-2 for 1 minute.
  7. Put on gloves.
  8. Pour plates.

Prepare Dilutions

Inoculate Plates

Incubate

Count Culture forming units

Bee gut bacteria cultures.

Record data

Repeat steps


Observations

Initially, there was a large percentage die off in several of the solo cups, but no pattern was obvious. Reason for the die off is unknown, possible initial divisions, incubator problems, other unknown factors. It was later determined that the die off may have been due to troubles with the pipette feeders.

Color and texture of gut.


Data

The data from the solo cup lab was meaningless, but the students learned procedure for handling bees and culturing bacteria.

Problems

  1. Solo cups
    1. If pipette feeders cut too long, won't work and bees starve.
    2. Can't get the dead bees out of the Solo cups. Difficult to count. When knocked out, the live bees all fall to the bottom with the dead bees. After 2 weeks there were decaying bees in bottom of the Solo cups.
  2. Couldn't get the incubator to 85 F and 55% RH.
  3. The Y-Section 3 Compartment Disposable Plastic Petri Dishes were difficult to use.
  4. Regulator on the CO2 tank freezes up.

Conclusions

  1. Need better way to feed.
  2. Need way to remove dead bees if experiment runs more than 48 hours.
  3. Need better incubator.
  4. Use a different type of C02 tank and regulator.