Leggy beetles show how insects lost limbs.

A beetle larva with a chorus line of legs is spotlighting the evolutionary steps that insects took to get from multilimbed centipedes to the six-legged specimens common today.

Evolving from millipede to beetle is a genetic two-step, say biologist Randy L. Bennett of Brigham Young University in Provo, Utah, and his colleagues at the University of Wisconsin-Madison. The researchers discovered that suppressing a couple of genes in grubs of the red flour beetle, Tribolium castaneum, could make the larvae grow extra legs.

"It's almost as if you're going back in evolution," says geneticist Mario R. Capecchi of the University of Utah in Salt Lake City. Early insects had many identical segments, each with its own set of legs, he says. Somewhere along the line, the segments evolved separate identities. The abdominal segments lost their limbs, leaving only the six thorax legs.

Bennett and his colleagues knew that in fruit flies, two genes called Ultrabithorax (Ubx) and abdominal-A (abd-A) control the number of legs and wings that the insects sport. The genes work together to keep fruit fly abdomens limbless. The scientists wanted to know if the same two genes gave beetles leg-free abdomens as well, Bennett says.

The researchers injected beetle eggs with double-stranded RNA copies of the beetle versions of Ubx and abd-A. Researchers use the technique, known as RNA interference or RNAi, to shut down specific genes (SN: 1/15/2000, p. 36). Originally developed to make mutant worms, RNAi has become a popular way to inactivate genes in many animals, including mice, fruit flies, and, recently, beetles.

When the scientists took out abd-A alone, the beetle larvae grew nublike appendages called pleuropodia on all segments but didn't make legs. Knocking out Ubx alone gave "something on the first abdominal segment that looks like it's trying to form a leg," Bennett says. When the researchers removed both genes, "we got legs everywhere," he says. The larvae died before maturing.

These experiments, reported in the April 25 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, give clues to the molecular process that led to the six-legged body plan, says Nipam Patel, a developmental biologist at the University of Chicago. "They were able to take something that had been hinted at and actually show it experimentally," Patel says.

In insects like fruit flies, both Ubx and abd-A turn off the leg-development program, Patel says. In crustaceans and centipedes, however, both proteins encoded by the genes can be found even in segments that have legs. Patel speculates that these proteins were present in the common ancestor of all insects but didn't suppress legs. The beetles represent an intermediate stage of evolution, he says, in which neither abd-A nor Ubx alone can completely suppress leg growth but each modifies limb formation.

These results confirm that evolution is often a process of rearranging genetic networks to make different developmental programs, says Bennett. "You don't have to invent new genes to evolve. You can just play with what you have," he says.

Although he praised the study as "a nice contribution from a different system," Capecchi warns that scientists should exercise caution when interpreting the results of RNAi experiments. "We still don't really know how RNAi works," he says.