Saturday, 18 May 2013

3-D Scans Reveal Caterpillars Turning Into Butterflies....

Left: Chrysalis of a painted lady butterfly, showing breathing tubes (blue) and guts (red), at day 1 (left), day 13 (centre) and day 16 (right).
The transformation from caterpillar to butterfly is one of the most exquisite in the natural world. Within the chrysalis, an inching, cylindrical eating machine remakes itself into a beautiful flying creature that drinks through a straw.

This strategy—known as holometaboly, or complete metamorphosis—partitions youngsters and adults into completely different worlds, so that neither competes with the other. It’s such a successful way of life that it’s used by the majority of insects (and therefore, the majority of all animals).

Butterflies, ants, beetles and flies all radically remodel their bodies within a pupa as they develop from larvae to adults.

But what goes on inside a pupa? We know that a larva releases enzymes that break down many of its tissues into their constituent proteins. Textbooks will commonly talk about the insect dissolving into a kind of “soup”, but that’s not entirely accurate. Some organs stay intact. Others, like muscles, break down into clumps of cells that can be re-used, like a Lego sculpture decomposing into bricks. And some cells create imaginal discs—structures that produce adult body parts. There’s a pair for the antennae, a pair for the eyes, one for each leg and wing, and so on. So if the pupa contains a soup, it’s an organised broth full of chunky bits.

We know this because scientists have dissected lots of pupae, although they’ve mostly trained their scalpels on fruit flies and blowflies. By its nature, such work always destroys the insect that’s being observed. It also only provides a snapshot in time. If you want to work out what happens as metamorphosis progresses, you need to cut open many pupae that you think are at different stages of development.

But now, two teams of scientists have started to captured intimate series of images showing the same caterpillars metamorphosing inside their pupae. Both teams used a technique called micro-CT, in which X-rays capture cross-sections of an object that can be combined into a three-dimensional virtual model.

By dissecting these models rather than the actual insects, the teams could see the structures of specific organs, like the guts or breathing tubes. They could also watch the organs change over time by repeatedly scanning the same chrysalis over many days. And since insects tolerate high doses of radiation, this procedure doesn’t seem to harm them, much less kill them.

One team analysed the caterpillar of the stunning blue morpho just before it started metamorphosis and a week into the process. They analysed the structure of the tracheae—the network of breathing tubes that carry oxygen throughout the insect’s body. Their work was done with the BBC as part of a documentary on metamorphosis—it was publicised in March but hasn’t been published yet.

The second project had its origins in crime-fighting. Thomas Simonsen from London’s Natural History Museum started using micro-CT to look at the pupae for blowflies. These insects lay their eggs on fresh corpses, whether it’s “someone who has been murdered or a deer in a forest”. They appear so predictably that you can estimate a body’s time of death based on where its blowflies are in their life cycle. This gets trickier once the flies turn into pupae, since those all look the same from the outside. But by scanning their insides using micro-CT, Simonsen hoped to get better estimates for how old they are.

From flies, he turned his attention to his favourite subjects—butterflies and moths. He worked with Tristan Rowe and Russell Garwood from the University of Manchester, who regularly scanned the cocoons of painted lady butterflies, some every day.

Painted lady butterfly chrysalis during metamorphosis. Breathing tubes in blue; guts in red.
Painted lady butterfly chrysalis during metamorphosis. Breathing tubes in blue; guts in red.

The scans showed that the caterpillar’s guts quickly change shape, becoming narrower, shorter and more convoluted. Meanwhile, the tracheal tubes become bigger, although their arrangement barely changes. The common wisdom is that “almost everything is massively reorganised in the pupa,” says Simonsen. That’s largely true, but not for the tracheal system. From its first day as a chrysalis, the painted lady already has the breathing tubes of an adult butterfly. “If there is remodelling, it happens very quickly in the first hours of pupation,” says Garwood. Alternatively, it happens when the butterfly is still a caterpillar.

This doesn’t drastically change what we knew about metamorphosis. There are some small insights—it seems that midway through the transformation, the big breathing tube that delivers oxygen to the flight muscles reattaches itself to a different set of openings on the insect’s torso. But the big picture stays the same. “I think it will provide instructive images for textbooks, but I don’t think it provided surprising new insights,” says David Champlin at the University of Southern Maine, who studies metamorphosis.

There are other limitations. The technique’s resolution is rather low. You cannot stain individual tissues or proteins with coloured molecules, while still keeping the animal alive. And the scanners can only pick up a limited number of organs. Brains and nerves, for example, are invisible to them, although Garwood hopes that new technological advances will overcome that hurdle.

Micro-CT scans may not revolutionise what we know about metamorphosis but Garwood hopes that their advantages will give scientists new options for their experiments. For example, the scans use up fewer individuals, since you can scan the same ones over time. This could free up insect specialists to move beyond the usual suspects like fruit flies, and study the development of rare or valuable species without harming them. They could look at how pesticides affect the development of bees, or how mutations in different genes change the process of metamorphosis. Champlin agrees. “It would be great to compare the normal animal with a variety of mutant strains defective for specific genes,” he says.

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