KaleidaGraph helps researchers discover a key pheromone in honeybees – essential for U.S. agricultural health

A recent discovery unveils the chemical secret that gives old bees the authority to keep young bees home babysitting instead of going out on the town. A hard-to-detect pheromone explains a phenomenon that somehow older forager bees exert influence over the younger nurse bees in a hive, keeping them grounded until they are more mature, and thus more ready to handle the demands of their critical responsibilities. A research team that spanned from the United States, to France and Canada explained how the bees kept an exquisitely consistent balance between the ones that go out to collect nectar and pollen and defend the hive, and those that stay home and nurture the larvae. This balance is controlled by the elder bees, those that typically spend the final one to three weeks of their five-week lifespan out in the field. Experiments showed that if a significant number of forager bees didn’t come home, the young nurse bees would mature ahead of schedule and head out to become foragers themselves. If the older bees were kept inside more than usual – as in an extended rain shower – fewer young bees would mature, but instead stick to brood care.

But the question was always, why? Pheromones are a chemical signal emitted by animals, insects and humans. Some, called releaser pheromones, are like a quick
conversation that changes behavior, such as those that inspire sexual attraction. Since releasers change behaviors immediately, they historically have been easier to identify. Hundreds of releaser pheromones have been chemically identified, whereas only four (including this new one) have been identified as primer pheromones. Primer pheromones are more difficult to work with because they impart behavioral changes in a much longer time scale, taking days or sometimes weeks to see an effect.

Researchers spent years futilely searching for a primer pheromone. After many dead ends, the group came upon a crucial difference between forager bees and nurse bees: Forager bees carry a mother load of a chemical called ethyl oleate in the abdominal reservoir in which they store nectar. That led them to identify ethyl oleate as another kind of pheromone – called primer pheromone.

How the primer pheromone was discovered
Discovering the primer pheromone was no easy task. Juvenile hormone (JH) measurement played a key role in finding the primer pheromone because the primer pheromone regulates the nurse bee to forager bee transition, and high juvenile hormone (JH) is an excellent indicator of foraging status. JH titers had to be measured (using a radioimmunoassay) in many experiments leading to the final discovery of the pheromone.

Samples containing blood (in .5 ml acetonitrile) are extracted for juvenile hormone (JH). JH is incubated with anitbody and radiolabeled JH. After 2 hours of incubation, the free JH is stripped by dextran coated charcoal. The supernatant contains the radiolabeled JH.

Scintillation cocktail (about 3.5 ml) is then added. The cocktail converts the energy of radioactive decay (beta particles from tritium in this case) into photons. The samples are then loaded into a Scintillation Counter which can measure the radioactivity. A liquid scintillation counter can “see” and calculate the amount of radioactivity in each vial. The Counter prints averages of each set of duplicates. A program opens the file generated by the Counter and writes the calculated averages into a file, which can be opened by Excel. “I used to use SAS for nonlinear regressing to fit a model,” states Zachary Huang, Michigan State University entomologist and 10 year KaleidaGraph user. “When I use a 5 parameter model, often the SAS program would not “converge” and I have to try many different initial values. I had to go back to the two parameter model to suit SAS.”

“I now use KaleidaGraph exclusively for the 5-parameter nonlinear regression. It never ‘crashes’ and works extremely well with any initial values. I found the “autolink” feature very useful, as I do not need to train all students how to do it. Simply use autolink, extract the data, and copy the new data, and update the plot to get the 5 parameters.”

For more information on Zachary Huang and his research with honeybees, visit www.cyberbee.net. The first page of this document contains an excerpt from an article written by Sue Nichols, MSU.

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