Setting the Clock
To demonstrate that monarchs have an internal clock and that the clock is set by daylight, the researchers examined the time of day when adult monarchs emerge from their hard-shelled pupa, called the chrysalis.
The time of emergence from the chrysalis is a reliable marker of circadian function in other insects, according to the researchers. Monarch butterflies normally emerge in the morning hours.
To test if this timing is controlled by an internal clock, the researchers exposed the chrysalids to total darkness.
"If you put an animal in an environment where there are no time cues, which is a default for circadian clocks, circadian rhythms continue to tick and keep time," said Reppert.
When the butterflies were placed in total darkness, they maintained their emergence cycle. But when exposed to constant light the butterflies emerged throughout the day, an indication that the circadian cycle was disrupted. "It gummed up the circadian clock so it did not work properly," said Reppert.
To measure the circadian clock after the butterflies had emerged from the chrysalis, the researchers cloned the monarch's so-called period gene, known as per, which is known to set the internal clock in fruit flies.
They found that the gene's activity was high during dark hours and low during light hours, but when exposed to constant daylight for several days the rhythm was blunted.
"Constant light thus disrupts the underlying clockwork mechanism, leading to the disruption of output rhythms," Reppert and colleagues wrote in Science.
Next, the researchers studied how the manipulation of the daily light and dark cycles affects the orientation and navigation ability of the butterflies.
After being exposed to a light-dark cycle in the lab that resembled the outdoor lighting of the northeastern United States during autumn, the researchers placed the butterflies outdoors in a 40-gallon (176-liter) flight simulator with a directional recording device.
The simulator was developed in 2001 by biologists Henrik Mouritsen at the University of Oldenburg in Germany and Barrie Frost at Queen's University in Ontario, Canada and is considered the breakthrough that allowed this monarch migration research.
"I predicted that the machine would open a floodgate of research questions that could potentially be answered," said Brower. "This study is an example of that."
The butterflies are tethered to the recording device with a metal wire. They have complete freedom of movement on the horizontal axis, but cannot move up and down, explained Reppert. Air blown up from the floor simulates a lofting breeze.
When placed in the flight simulator after being housed in the autumn-like, light-dark cycle, the butterflies oriented themselves to the southwest, as if they were flying towards their Mexican wintering grounds.
Butterflies housed in a 1 a.m.-to-1 p.m., light-dark cycle flew towards the southeast, as expected. "By shifting the clock, you get a predicted shift in direction in where the animals orient," said Reppert.
However, when exposed to constant light before being placed in the flight simulator, the butterflies flew directly towards the sun, an indication that the direct light had disrupted the circadian rhythm, thus making it impossible for them get a sense of direction.
The team also found that while ultraviolet light is required for sun-compass navigation, some other wavelength of light was required for setting the butterflies' internal clocks. The difference may provide a means for untangling the two biological processes, they report.
"What the research is doing is allowing us to get into how navigation occurs from the circadian clock vantage point," said Reppert.
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