Eyes, Part Two: Fleas, Fish, and the Careful Art of Deconstruction

In my last post, I went back in time, from the well-adapted eyes we are born with, to the ancient photoreceptors used by microbes billions of years ago. Now I'm going to reverse direction, moving forward through time, from animals that had fully functioning eyes to their descendants, which today can't see a thing.

This may seem like a ridiculous mismatch to my previous post. We start out with the rise of eyes, a complex story with all sorts of twists and turns, with gene stealing, gene borrowing, gene copying; and then we turn to a simple tale of loss, of degeneration, of a few genes mutating the wrong way and--poof!--billions of years of evolution undone.

In fact, loss is never such a simple matter. I can illustrate this fact with two disparate beasts: fleas and cavefish.

Cavefish were familiar to Darwin, as were the many other blind cave dwellers, such as salamanders and insects. Darwin saw cavefish as yet another example of an animal carrying around the vestiges of its ancestors, just as we carry around the stump of a tail. As for how cavefish lost their eyes, he set natural selection aside. Darwin could not imagine how a fish in a cave would get any benefit from eyes that did a worse job than its ancestors' eyes. "I attribute their loss soley to disuse," he wrote. By disuse, Darwin may well have been thinking along the lines of his precursor, Lamarck. As fish stopped relying on their eyes in the dark, somehow their eyes degenerated, and that degeneration was passed down to the next generation of fish.

Once scientists began to decipher the molecules of heredity, such an explanation became obsolete. Instead, some scientists translated the notion of "disuse" into the language of mutations. Like any animals, a cavefish has a small but real chance of undergoing a mutation to its DNA. In some cases, these mutations can impair the fish's eyes. In a population of surface-dwelling fish, this sort of mutation would probably make it hard for a fish to find food, and might even make it an easy target for predators. The chances of the fish passing down that mutant gene to a new generation of fish would be pretty slim. But in a cave, such a mutation would have no effect on the reproductive fortunes of a fish. Over time, the population of cavefish would accumulate lots of eye mutations, until their eyes were rendered useless.

But this "neutral mutation" hypothesis isn't the only possibility. Scientists have also proposed an "energy conservation" hypothesis. Mutations that prevent cave fish from developing eyes let them save energy, boosting their odds of survival.

Scientists have tested this hypothesis in recent years by studying the fish Astyanax mexicanus. You can find perfectly normal populations of this fish in surface waters in the U.S. , but if you go into caves, you can also find some 30 populations that are blind. This transformation has happened overnight, biologically speaking: scientists estimate that it was only 10,000 years ago that populations Astyanax moved into the caves. One vivid demonstration of just how recent this move was is the fact that a cave fish and a surface fish can mate and produce healthy hybrids. The lion's share of research on Astyanax has been carried out in the laboratory of William Jeffery at the University of Maryland, and he offers an excellent summary in a paper in press in the Journal of Heredity.

Much of Jeffery's work has gone into tracking the development of the fish from eggs. The most startling thing he has found is that cavefish grow eyes for quite a long time. Just as in surface fish, the brains of cave fish embryos bulge out to the sides, stretching into stalks that end in cups. A simple retina and lens begin to form, and growing nerves begin to link the retina to the visual centers of the fish brain. After about a day, however, the cavefish eye and surface fish eye begin to take different paths. The cave fish eye fails to develop an iris or a cornea, for example. Still, many parts of the cave fish eye continue to grow as their cells multiply.

These findings alone call into question both the neutral mutation hypothesis and the energy conservation hypothesis. If mutations were building up in the cave fish genome, you wouldn't expect that the fish could advance so far in the development of their eyes. And if energy conservation was the sole advantage driving the evolution of blindness, you wouldn't expect the fish to keep producing new eye cells, even as the eye begins to deteriorate.

Even the degeneration of the eye challenges both of these hypotheses. The eye doesn't collapse into a stew of chaos; it is dismantled in a stately choreography. The cells in the lens release some signal that instructs other eye cells to begin to commit suicide. In surface fish, the lens sends signals that do just the opposite, allowing the eye to develop fully. Jeffery and his colleagues found that if they transplanted just the lens of a surface fish into the eye of a cave fish, the cave fish grew a completely normal eye. What's more, the transplant triggered new nerve fibers to project from the retina to the brain, and the part of the cave fish's brain that handles vision even grew. It's possible that a transplanted lens allows a cave fish to see. Despite being blind, the cavefish still retains its original circuit of eye-building genes.

Jeffery and his colleagues have also tracked the degeneration of the eye at the level of genes. The neutral mutation hypothesis would lead you to expect that cave fish would express fewer genes in the eye than surface fish, because many of them would have been destroyed by mutations. But this is not the case, Jeffery and his colleagues have found. Instead, they're starting to identify some genes that make more of their proteins in the eyes of cave fish than in those of surface fish, and even some genes that aren't active in the eyes of surface fish at all.

One particularly important protein in the developpment of cavefish eyes is known as Hedgehog. In all vertebrates, Hedgehog plays a vital role in the development of the eye, starting at its earliest stage. Initially, the cells that will give rise to the eyes form a single cluster. Cells in the midline of the embryo start producing Hedgehog, which somehow signal the cells in the middle of this eye cluster to stop developing. As a result, only the cells on the far sides continue to develop, thus producing two separate eyes. Mutations that interfere with the production of Hedgehog can cause a gruesome birth defect in humans called cyclopia, in which a single cyclops-like eye develops.

Cave fish have evolved in the opposite direction: they produce more Hedgehog, rather than less. The extra protein stops the development of a wider expanse of the original eye-cell cluster, leaving few cells to progress. Jeffery and his colleagues confirmed this by boosting the production of Hedgehog in surface Astyanax. Not only do they develop smaller eyes, but they suffer the same lens-directed degeneration seen in cavefish. This means that the degeneration of cavefish eyes requires cells beyond the eyes to help coordinate the process.

What's most remarkable about this choreography is that it has evolved again and again. Studies on Astyanax DNA suggest that populations of surface fish have repeatedly invaded caves, and each time they have gone blind. Jeffery and his colleagues have started comparing the development of embryos from different populations, and they find the cavefish have evolved blindness through the same patterns of gene activity.

This parallel evolution is hardly what you'd expect from a random blast of neutral mutations. Nor does Jeffery believe that energy conservation can explain it. Males and females show no difference in the development of their eyes, despite the fact that females need a lot more energy to make their eggs. Likewise, some populations of cave fish get lots of energy because they live under colonies of bats that can drop food and guano into the water. Despite this luxurious conditions, these fish are no different than their leaner cousins.

Jeffery thinks that Hedgehog may be the key to understanding what's really driving the evolution of cavefish. Like many genes involved in development, Hedgehog has many different jobs. It is known to be essential for the development of tastebuds, for example, as well as teeth and the bones that make up the head. And in cave fish, all of these features are significantly different from surface fish. It's possible that these changes are adaptations that help the cave fish feed more efficiently. These changes were only made possible by cranking up the production of Hedgehog. A side effect of this increase was the destruction of the cave fish eyes. But because eyes aren't essential in the dark, this wasn't such a big price to pay. If Jeffery is right, Darwin's real mistake with cave fish wasn't falling back on a Lamarckian explanation. It was not recognizing how powerful natural selection could be.

Jeffery and his colleagues have managed learned so much about the evolution of cavefish eyes because they figured out how to turn Astyanax into a laboratory organism, which can be studied as carefully as a fruit fly or a lab rat. This sort of transformation takes many years, and only a few species have what it takes. Many other animals have lost their eyes, but in most cases, scientists can only glean less direct clues. Still, the stories they have to tell can be just as interesting. Most interesting of all is the fact that different evolutionary forces seem to have been at work.

Case in point: fleas.

Scientists know very little about the vision of fleas. As insects, fleas have inherited the standard insect eye, which consists of slender columns tightly packed together. But this standard insect eye has undergone drastic changes in fleas. Some fleas have what look like simple eyespots. Others seem to lack any eye at all. To learn about this transformation, a team of biologists from Brigham Young University have compared fleas to their relatives, which still have eyes.

This wouldn't have been possible even a few years ago, because scientists have only recently worked out the "flea tree." Fleas evolved from a group of insects with particularLY sharp vision. Their cousins include scorpionflies, which rely on their image-forming eyes to help them scavenge dead insects. Their closest relatives are "snow fleas" (Boreidae). These wingless insects live in mountains, where they feed on moss. They have small eyes, but can see well enough to jump away if you try to catch them. So it appears that fleas are the product of a long-term evolution towards simpler eyes.

The scientists used this tree to track the evolution of some of the molecules that are essential for vision. Known as opsins, they respond to light by triggering a chemical reaction that sends a signal from the eye to the brain. Opsins can be sensitive to different colors, depending on their shape, which depends in turn on the DNA sequence in their genes. The scientists isolated the gene for green opsins from 11 species of scorpionflies, snow fleas, and true fleas.

The scientists then compared the DNA sequences for signs of change. A mutation to an opsin gene may have no effect on the opsin molecule itself, or it may alter its structure dramatically. The difference depends on where in the DNA sequence that mutation strikes. The scientists found that most changes that occurred during the evolution of fleas had no effect on the actual opsins. They confirmed this by using the DNA sequence of the opsin genes to create computer models of the opsin molecules themselves. Even in fleas, the green opsin molecule has basically the same structure as in scorpionflies--despite their radically different eyes.

Just because a gene hasn't changed for millions of years doesn't mean that it hasn't been experiencing natural selection. The scientists found evidence that the opsin gene has been experience a special kind of natural selection in fleas and their relatives, known as purifying selection. Purifying selection occurs if even the slightest change to the structure of a molecule puts a serious dent in the reproductive success of an animal. The fact that fleas have experience purifying selection on their opsin gene means that it remains essential to their survival. (The details of their work appear in a paper in press at the journal Molecular Biology and Evolution.)

So what on Earth are the fleas doing with their opsins? The scientists doubt that the fleas are using them in their eyes. They point out that flea eyes are covered over in a tough layer of chitin, and they lack the lenses and other structures that would let them see. But in many animals, ranging from pigeons to salmon to butterflies, opsins have also been found outside the eye. In some animals, they grow inside the brain, while in others they grow on the abdomen or other parts of the body. Recent studies suggest that these opsins set the pace for biological clocks by registering the change of light from day to night.

This brings us back around to the very origin of eyes, which I described in my first post. Long before full-fledged eyes evolved, light-sensitive molecules may have existed in microbes, allowing them to change their movements during night and day. These molecules may have been incorporated into early eyes, making it possible for animals to see. But this transition didn't mean that photoreceptors could no longer serve their original function. Early insects may have used opsins both within their eyes to see and outside of their eyes as biological clocks. Later, some lineages of insects lost their eyes. Some may have lost them in dark caves. Fleas, on the other hand, lost their eyes as they became parasites. Instead of navigating through a complex landscape in search of a particular prey, they just hopped from one host to the next. But they still relied on opsins to run their biological clocks. The authors point out that scientists have also found opsins in other animals that have lost their eyes. The animals? None other than Astyanax.

What's particularly remarkable about the new study is how strongly the flea opsin resisted any evolutionary change--even after it was no longer being used in the flea eye. The molecule need the same functional structure for both jobs. As I mentioned at the beginning of my previous post, Charles Darwin recognized that the complexity of the eye might appear to pose a major challenge to his theory. To some people, it still does; they argue that the components of the eye cannot function on their own, and so they could never have existed on their own. By this reasoning, it would be impossible for one of these components--an opsin, for example--to do anything useful if it wasn't inside an eye.

The flea apparently sees things differently.