While our blood cells bustle around carrying red iron-containing hemoglobin to deliver oxygen to our tissues, crabs and lobsters use a blue copper-containing protein called hemocyanin to transport their oxygen. Cryptocyanin, another protein found lurking in crabs' bloodstreams, is closely related to hemocyanin but can't transport oxygen. So why do crabs bother to produce it, Nora Terwilliger wondered(p. 2467).

Terwilliger's first clue surfaced when the late crustacean biologist Dorothy Skinner suggested that differences in hemocyanins between juvenile and adult crabs might be related to crabs' molt cycles; crabs regularly have to shed, or molt, their hard outer shell because it limits their growth. To find out if Skinner was right, Terwilliger tracked crabs' blood protein levels during the molt cycle. She caught some crab larvae in an Oregon bay, took them back to the lab and sampled their blood as they grew. But to her surprise cryptocyanin, not hemocyanin, changed throughout the cycle; cryptocyanin peaked during premolt and plummeted just before crabs crawled out of their old shells.

Intrigued by this finding, Terwilliger reasoned that hemocyanin and cryptocyanin are regulated by different mechanisms. To understand how the proteins are regulated, Terwilliger and Margaret Ryan teamed up with David Towle to examine the mRNA expression of both proteins in different crab tissues during the molt cycle. They found that both proteins are expressed in the hepatopancreas, a digestive organ. They saw that hemocyanin mRNA levels are relatively constant throughout the molt cycle, but cryptocyanin mRNA levels follow the same pattern as the presence of cryptocyanin in the bloodstream during the molt cycle. The fact that hemocyanin and cryptocyanin are present in different amounts and at different times during the molt cycle suggests that the two proteins have different control mechanisms,' Terwilliger says.

Suspecting that cryptocyanin might be under hormonal control, Terwilliger and Ryan snipped off crabs' eyestalks, which contain a molt-inhibiting hormone. Monitoring the crabs' bloodstreams during subsequent molt cycles,they saw that hemocyanin levels were unaffected, but cryptocyanin disappeared.Cryptocyanin is regulated by molting hormones,' Terwilliger concludes.

Since they had found such different mRNA expression patterns and control mechanisms for the two proteins, Terwilliger and Ryan figured that the proteins must be synthesized in different cells. To discover which types of hepatopancreas cell synthesize the proteins, they made labelled probes to locate the mRNA in the cells. They were astonished to find that hemocyanin and cryptocyanin are in fact synthesized in the same cell type.

Despite being produced by the same cells, the two proteins have very different jobs. Terwilliger and Ryan uncovered a final clue concerning cryptocyanin's function when they discovered that the protein is present in crabs' newly secreted exoskeletons. They conclude that when cryptocyanin lost its oxygen-transport abilities, it was assigned a new job by evolution: to help growing crabs build roomier shells.

Terwilliger, N. B., Ryan, M. C. and Towle, D.(
2005
). Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister.
J. Exp. Biol.
208
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