This allowed the scientists to see how much room was dedicated to the respiratory system among four species of darkling beetles ranging from 0.1 to 1.3 inches (3.2 to 33 millimeters) in length.
Insects carry oxygen to cells differently from humans. Instead of a single breathing tube, bugs have several pairs of holes known as spiracles along their bodies.
These holes connect to tubes called tracheae, which transport oxygen to cells and remove carbon dioxide.
The x-ray scans revealed that as beetles become larger, tracheae take up proportionally more room in their bodies because they need to be longer and wider to deliver enough oxygen. This, in turn, inhibits growth by crowding other organs.
The tracheae in the larger beetles took up 20 percent more room than in smaller beetles.
The area where the body and legs meet is particularly limiting, because that opening can only get so big, Harrison noted.
In the smallest beetle, tracheae take up 2 percent of the region, compared with 18 percent in the largest.
Using that information, Harrison estimated that the maximum beetle size under current oxygen levels would be about six inches (15 centimeters).
That coincides roughly with the largest known living beetle, the Titanic longhorn.
"This paper is really interesting in part because there is still a lot we don't know about how insects breathe," said Scott Kirkton, an assistant professor of biology at Union College in Schenectady, New York, who was not associated with the study.
Something in the Air
During the late Carboniferous period (354 to 290 million years ago), however, oxygen levels were much higher than they are now, partly because coal swamps that leaked the gas into the air were very common.
"Back then, there was 31 to 35 percent oxygen in the air," study lead author Kaiser said. "Now we have about 21 percent."
That meant insects needed smaller quantities of air to meet their oxygen demands, allowing the creatures to grow much larger.
"The tracheal diameter can be narrower and still deliver enough oxygen for a much larger insect," Kaiser said.
The team, though, is still trying to definitely show that this phenomenon explains why Carboniferous insects were so large.
Neither fruit flies nor beetles were around, or even had close relatives, during the Carboniferous, so the team hopes to extend its research to more ancient insects such as dragonflies, Kaiser said.
The scientists have already experimented with fruit flies in a lab at Arizona State, raising them in tanks with different levels of oxygen.
Under higher concentrations of oxygen the fruit flies definitely get bigger, Harrison said.
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