Apis mellifera used for the research are sourced from the bee farm of National Chung Hsing University. Apart from the bees used for experiments, the bees are kept in an open space on the rooftop of a building. Each bee colony consists of a healthy queen and 8 brood frames containing larvae, pupae, honey, and pollen. During seasons with a lack of pollen and nectar, the bees are provided with "pollen patty" as a substitute. The pollen patty is made by mixing pollen and sucrose in a 1:5 ratio with appropriate water content. Additionally, the bees are fed with a 50% sucrose solution.
We collected active foragers captured upon their return to the nest and maintained them in an insect growth chamber at 32-34°C with a relative humidity of 60%±10%, keeping the environment dark.We housed groups of 30 foragers bees in cages made of acrylic plastic (3 mm thick) with dimensions of 10 × 10 × 10 cm and a volume of 1000 mL (改自Wu et al., 2021). Inside the cages, the honey bees were provided with food in the form of a 35mm diameter dish containing 2 grams of Brassica napus pollen patty. The pollen patty was made by 2 : 1 (w/v) dry pollen mix with 50% (w/v) sucrose solution.Additionally, we supplied the bees with a small bird feeder (Wu et al., 2021) containing 15 mL of 50% sucrose solution.
First, bees were provided a sugar water diet containing either PQQ or a solvent control under laboratory conditions to evaluate the effects of the PQQ substance alone on the bees (control versus PQQ).
Dissolve 10mg PQQ in distilled water to prepare a 2500 ppm solution as a stock solution and store it at 4℃. Add an appropriate amount to a 50% sucrose solution to prepare a sucrose solution with a PQQ concentration of 200nM.
Each group of 30 bees into a breeding box and put them into the nest foundation. The control group was given 50% sucrose solution and pollen patty, and the treatment group was given 50% sucrose solution with 200nM PQQ and pollen patty. pollen patty and sucrose solution were changed and recorded every day.Then the dead bees will be taken out.
Experimental purpose: Testing the learning ability of bees.
1- Hexanol
50% sucrose solution
1.5mL eppendorf
tampon
15 mm diameter round filter paper
25mL syringe
Microcentrifuge tube rack
method
We collected frames of sealed brood from multiple bee colonies and maintained them in an insect growth chamber at 32-34°C with a relative humidity of 60%±10%, keeping the environment dark. After 12 hours, we gathered the emerged worker bees. We housed groups of 30 worker bees in cages made of acrylic plastic (3 mm thick) with dimensions of 10 × 10 × 10 cm and a volume of 1000 mL (改自Wu et al., 2021). Inside the cages, the honey bees were provided with food in the form of a 35mm diameter dish containing 3 grams of Brassica napus pollen patty. The pollen patty was made by mixing 50 grams of pollen with 15 mL of a 50% sucrose solution. Additionally, we supplied the bees with a small bird feeder (Wu et al., 2021) containing 10 mL of 30% sucrose solution.
1. Raise with 30% sucrose and rape pollen bee food in an an insect growth chamber at 32-34℃ and relative humidity of 60±10% for 5 days. The experiment started on the 6th day of age.
2. After taking out the pollen patty and sucrose solution, place the entire Cage in a 4°C refrigerator and check it every 10 minutes until the bees faint.
3. Cut off the tip of the 1.5mL eppendorf, and put the bee in so that the bee's head can be extended and the head and mouthparts can be moved. A strip of cotton is stuffed at the bottom to prevent it from escaping.
4. Wait for 5 hours to allow the bees to recover and have enough time to be hungry. Use 3% sucrose solution to test whether the bees have normal proboscis reflex. Select the bees with normal responses and put them back into the growth box for 30 minutes before starting the test.
5. Drop 2μL of 1-hexanol on the round filter paper and put it into the syringe.
6. Then deliver the odor with a syringe containing 1-hexanol odor within 4 seconds, followed by 1 μL of 50% sucrose solution (US) 2 seconds later, with an interval of 10 minutes. Each bee was primed six times with 10-min intertrial intervals, which facilitates olfactory learning in bees ( Menzel, 2001 ).