New Developments Concerning the Circadian Rhythm Experiments

 

 

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You get up every day at the crack of dawn (or noon, hey, it’s college), combat the rumbles in your tummy with some cereal, wash it down with OJ, and start your work day.  At midday, you scarf a sandwich and some chips to satisfy the scathing growling in your stomach.  In the evening, a salad and soup combo pleases the stomach god.  The point is, there’s a time when your stomach has the rumbles that only food can satisfy, and these rumbles often occur on some kind of general schedule.  From sleeping, to eating, to defecating, our bodies run on a natural, internal cycle called a circadian rhythm.  The same is true of plants, fungi, and other animals.

Circadian rhythms can be tweaked or even reset by things like temperature and sunlight.  If you’ve ever read about light therapy with individuals who often travel across time zones, you’ve read something about circadian rhythms.  But it gets more complicated than that.  Though they are endogenously generated, circadian rhythms are not self-contained.  They can interact in ways that are crucial for survival.

When herbivorous insects get the rumblies and start looking for something green to munch on, they interact with their food not only in the context of their own circadian rhythm, but also with that of their food’s.  Plants defend themselves from herbivory using a variety of chemical compounds, including secondary metabolites such as alkaloids, terpenoids, and phenolics, but keeping a strong, chemical defense running at all times is not always cost-effective.  Especially for plants in areas where the diversity and concentration of herbivorous insects is low, the need for constant, constitutive defense is minimal.  Instead, many plants have paid attention to the feeding schedules of those pesky insects, and have adjusted their circadian rhythms accordingly so that when the insects are hungriest, the plants are the most well-defended.

Last summer, during High Performance Liquid Chromatography (HPLC) trials, my lab buddy, Jake, and Professor Puzey found that the chemical signatures of milkweed defenses varied with the time of day during which they were collected.  This sparked the idea: what if we could design and perform an experiment to test the night and day cycles of milkweed defense using a growth chamber in which we had control of the lights?

Professor Puzey found a paper in which a similar trial had been performed on Arabidopsis (http://www.pnas.org/content/109/12/4674.full).  We decided to model our trial based on their experimental setup.  The concept is simple.  First, because A. syriaca (the common milkweed) is clonal (sends out stems from a lateral, running root), we will separate a single plant into many clones and then split these clones into two groups.  In separate growth chambers, we would adapt each group to a 12-hour night/day cycle opposite of that of the other group’s cycle.  We would simultaneously adapt two groups of a single species of insect which feeds upon milkweed to corresponding 12-hour night/day cycles.  During the actual trial, insects and plants would be matched either with corresponding 12-hour day/night cycles, or with opposite day/night cycles.  The effectiveness and strength of the plants’ circadian production of secondary metabolites would be measured in terms of herbivory.  If our hypothesis is correct that A. syriaca produces its cardenolide and phenolic defenses based on a circadian cycle, we predict herbivory scores should be much less for plants adapted to the same circadian rhythm as that of the insects than for those following an opposite cycle.

Professor Puzey and I met yesterday to discuss our summer plans.  Over the last few weeks, Jake and I have created 73 clones or replicates of milkweed plants in the Millington Greenhouse and have mapped the position of every plant in both Millington and at the greenhouse at Sullivan.  Professor Puzey mentioned a master list which includes a chemical profile of every plant or genotype that we have collected.  This information will be useful in both informing us on which plant we should use for our circadian experiment as well as which plants I should use for my diversity experiment.

We want to start getting the plants used to our circadian cycle as soon as possible, and since ISC 3 might not be available as early as we thought, Professor Puzey suggested using an interesting temperature-controlled lighting chamber that he inherited in the ISC.  It looks a bit old and clunky, but also sort of sci-fi, which is exciting.  As long as we make our replicates small, they should fit no problem.

The plan right now is to do both weighing and circadian experiments over the summer.  The circadian project will require a little help as we have to run the lighting chamber in 12-hour intervals.  Since sleep is also important, Professor Puzey suggested we need to ask someone to help us take turns turning on and off the light.

Planning is going well and I feel like we have a good base on which to build.  It looks like Jake and I will have our hands full with the replicates.  Weighing the caterpillars over the summer will also be a full job, but a fun one.  I’m excited to see how things come together.