The overall goal of this experiment is to evaluate how pesticide residues in the environment affect individual honeybees in the natural condition of beehive colonies. The main advantage of this method is that it surveys the effects of pesticides or other contaminants of interest on both individual honeybees and the whole colonies. Visual demonstration of this method is critical as the manipulation of pupae is difficult because they are fragile.
Demonstrating the procedure will be Ko Chong-Yu a PHD student from my laboratory. Prior to the experiment, feed each colony a liter of 50%sugar syrup. Replenish the food reservoir weekly, a healthy colony will have nine frames of bee combs and one queen laying eggs.
To begin, locate the queen in the colony and replace at least one frame on her side of the colony with an empty frame. Then insert the queen excluders to isolate the queen to a four frame section containing at least one empty frame. The next day, check the frames for freshly laid eggs and put the eggs apart from the queen for 72 hours.
Then remove these frames, brush off the worker bees, and prepare to treat the larvae in the brood cells. For each treatment cover 50 randomly selected one day old larvae in the frame with transparent slides. Secure the slides with thumbtacks, then make identifying information on the slides with permanent marker.
Now using BLD warmed to 35 degrees celsius, make different concentrations of PPN treatment ranging from 0.1 to 100 parts per million in 100 mL volumes. For the next three days, treat the brood cell groups. To treat the larvae, uncover them and add 10 microliters of the PPN BLD solution to each brood cell.
Repeat the treatment the next day using 10 microliters of treatment solution, and repeat the treatment on the third day using 20 microliters of treatment solution. After the third treatment, let them develop in the colony In the experimental design, replicate each treatment group four times using different colonies. On day seven, inspect the PPN treated larvae, record the mortality and capping rates in the treatment groups.
On day 13, record the eclosion rate of each treatment group. First, remove the bee wax from the capped brood cells. Then remove the pupae using tipped tweezers to grip them very lightly, and gently tug them out.
When removing the pupae, use grip between head and thorax and take pupae out very gently. It will be necessary to practice many times to obtain a survival rate of at least 90%prior to running experiment. Transfer the pupae into 24 welled tissue culture plates lined with two layers of laboratory tissue.
Be sure to note any damage in mortality that occurs during the transfer. Keep the culture plates at 34 degrees celsius with 70%relative humidity until emergence. Over the next eight days, record the eclosion rate.
Set up as before with a healthy nine frame colony where the queen is limited to a four frame section containing at least one empty frame. Call the four frame section part A and the five frame section part B.Be sure the queen excluders divide the colony completely so the queen cannot move between sections. This division is vital to collecting groups of one day old larvae.
The next day, label a group of 100 brood cells with freshly laid eggs as before. This is group one, return it to part A for three days. Two days later, transfer the queen to part B to lay eggs.
The next day, label 100 freshly laid eggs in part B as group two and return the section to part B in the colony. After two more days, move the queen back to part A.Now repeat the process to make another group and continue exchanging the queen between part A to part B every three days until nine groups of one day old eggs have been assigned on three day intervals. During the first five days of development, score the hatching rate of each labeled group of brood cells.
After 11 days of development, start recording the number of capped brood cells in each group. On day 13 of the experiment, treat the colony with 50%sugar syrup containing PPN at 10 or 100 parts per million. Load a feed box with one liter of treatment and connect it to the colony.
Group one is an untreated control as those cells should now be capped. Starting on day 17 record the eclosion rate of each group. To do this, transfer the larvae as previously demonstrated to a 24 well plate, then incubate the plate and later record the eclosion rate.
After 49 days, the eclosion data from all nine groups should be known. Individual brood cells were treated as described with PPN BLD. The negative effect of the pesticide on the larval stage honeybees were easily observed and were dosage dependent.
These effects included melanization and wing deformation. To simulate honey bee colonies suffering from environmental PPN residue, entire colonies were fed PPN syrup. Group one is a control as these bees developed to adults prior to treatment.
Group two and three were initially effected in the larva stage, and groups four through nine were initially effected as eggs. Changes in hatching, capping, eclosion, and deformities occurred with PPN treatments. 100 parts per million of PPN was more effective than 10 parts per million, except on the hatching rate.
Moreover, after the treatments, many pupae died with black cuticles or by failing to emerge. In the 100 ppm PPN treatment, capped cells were destroyed and the injured pupae were removed from the colony. Once mastered, the colony treatment procedure can be done in about one month, and the daily manipulation of pupa can be finished in about two hours.
While attempting this procedure, it's important to remember to regularly check the condition of your colony and become well-practiced at handling pupae before you perform an experiment. Don't forget that working with honeybees can be hazardous due to bee venom allergies. Precautions such as wearing protecting clothing should always be taken while performing this procedure.
