Microscopic menagerie

New view of microbes forces rethinking of what it means to be an organism

MADE OF MICROBES  The microbes dwelling in and on multicellular organisms, such as insects and humans, should be viewed as evolutionarily inseparable from their hosts, some biologists argue. 

Nicolle Rager Fuller

What is a wasp?” might seem like an overly simple question for a Ph.D. biologist to be asking. “What is a human?” Even more so.

But these are strange times in the life sciences. Seth Bordenstein of Vanderbilt University in Nashville now embraces the notion that each wasp he studies, each squirrel darting around campus — not to mention himself, every reader of science magazines and every other representative of see-it-without-a-microscope life on Earth — is really a blend of one big organism and a lot of little ones.

In recent years, research has shown that what people commonly think of as “their” bodies contain roughly 10 microbial cells for each genetically human one. The microbial mass in and on a person may amount to just a few pounds, but in terms of genetic diversity these fellow travelers overwhelm their hosts, with 400 genes for every human one. And a decent share of the metabolites sluicing through human veins originates from some microbe. By these measures, humanity is microbial.

But numbers are just the beginning.

GUT BUSTER Tsetse flies must be infected during gestation with a particular bacterium in order to develop a normal gut lining (top). Lab-raised larvae that were bacteria-free developed faulty guts (bottom) and weak immune systems that made them much more vulnerable to infection by the parasite that causes sleeping sickness. Weiss et al/PLOS Pathogens 2013
The evolutionary impact of animals’ microbial denizens can be substantial. Adult wasps of the genus Nasonia are only about 30 percent microbial, Bordenstein estimates. But those microbes keep two species apart that could otherwise interbreed.

Some researchers think of these microbes as just another part of a plant or animal’s environment, like a mountain range that keeps two related species separate. But, with a squint and a slap to the worldview, researchers like Bordenstein are exploring whether a body’s microbes are so intimate that they’re part of the organism itself. Or, if you prefer, the metaorganism.

“Ecosystem” is the word that 26 scientists used in a call for new thinking about animal-bacteria interactions that was published in February by the Proceedings of the National Academy of Sciences. The recent accumulation of knowledge about bacteria vis à vis their animal hosts “is fundamentally altering our understanding of animal biology,” the group declared.

Why would biologists get so excited about teeming microorganisms now? Even someone who missed the earliest fiddling with magnifying lenses has had 330 years to catch up on volume 14 of the Royal Society’s Philosophical Transactions, wherein merchant microscopist Antonie van Leeuwenhoek reported “to my great surprise,” that watered-down scrapings from his teeth revealed “very many small living Animals, which moved themselves very extravagantly.”

For more than three centuries after van Leeuwenhoek’s discovery, anyone interested in studying the microbial world was limited by the frustrations of “growing fuzzy things in Petri dishes,” as Corrie Moreau of the Field Museum in Chicago puts it. A fascinating microorganism might thrive in the gills of deep-ocean clams, in groundwater seeping through porous rock or in the gonads of mosquitoes. But if you couldn’t culture it in a lab dish you had no way of knowing about it. Even with clever technical advances, an estimated 99 percent of microbial life can’t be cultured, Moreau says. And what does grow may be misleading. A marginal freak may look like the dominant member of a community only because it’s the one that flourishes in the lab.

Recent genomic innovations have changed all that. In the last few years, automated systems have been developed to quickly and affordably determine the genetic signatures of thousands of individual microbes in a sample.

What a world the new technology reveals: In just 19 samples from four colonies of turtle ants, Moreau says, 445 kinds of bacteria showed up that cultures and clunkier genetic techniques had missed. Eight kinds of bacteria consistently show up in the guts of honeybees and a few other bees, but so far, nowhere else. Bedbugs need Wolbachia bacteria inside their cells to survive.

And bacteria may at last explain how the giant panda, a bamboo-eating member of the mammalian order Carnivora without a grass-grazer’s capacious fermenting gut or specialist digestive enzymes, can live on 12.5 kilograms of highly fibrous plant material a day. The bear’s puzzling digestive system turns out to gurgle with bacteria that apparently belong to groups that include competent digesters of cellulose.

Born with it

Bacteria start shaping their hosts’ lives right from the beginning. In tsetse flies, for example, inheriting genes from mom isn’t enough; larvae that don’t also inherit the right kind of bacteria don’t grow properly.

The way tsetse flies start their lives “is eerily similar to what happens in mammals,” says Brian L. Weiss of Yale University. In most insects, “the female will just lay a bunch of eggs and fly away.” Tsetse females, however, gestate one fertilized egg at a time inside what amounts to a uterus. Glands inside the uterus produce a white milklike liquid rich in fats and proteins. After suckling for its first three larval stages, the youngster weighs about as much as its mother. Then she gives birth.

Gorging on mother’s milk doses the infant with a Wigglesworthia bacterium, which Weiss describes as looking like a hot dog. Wigglesworthia can live only inside a tsetse fly, and flies deprived of it don’t give birth.

Weiss was able to deduce what Wigglesworthia does in development by dosing moms with B vitamins to artificially keep their bacteria-free larvae alive. The larvae grew up but never formed a decent immune system. Flies deprived of bacteria as larvae also failed to form a real gut lining, Weiss and his colleagues reported in April in PLOS Pathogens.

A faulty gut lining in a tsetse fly is a serious problem, and not just for the flies. Even though they’re famous for spreading the trypanosome parasite that causes sleeping sickness, only 1 to 5 percent of normal tsetse flies become carriers when feeding on infected blood. With faulty guts, though, more than 50 percent of bacterially starved, skimpy-gut flies turn into carriers.

Other studies have turned up similar examples of microbial power in animal development. Females of the parasitic wasp Asobara tabida need a Wolbachia bacterial strain in order to form wasp eggs. Developing mice can’t form normal capillaries in their guts without a standard set of microbes being present. And young lab mice may even need their gut bacteria for proper brain development, a research team in Sweden reported in 2011. Mice raised without normal gut microbes were unusually active and bold in tests, as if their brains weren’t wired the same way as those of regular shadow-loving, skittish mice. Returning gut bacteria to germfree mice re-created normal caution in their offspring. But it failed in adults with brains that were already mature.

Moms of a variety of species appear to microbially prep their young, says Bordenstein. Vesicomyid clams that need microbial help to survive at deep-sea vents, some sponges and cockroaches release eggs already loaded with bacteria. When stinkbugs lay eggs, the capsules get smeared with mom’s bacteria-rich excrement. When the youngsters hatch, they gobble the egg case, smear and all.

Reports of mother-to-child bacterial transmission appear to be so widespread among animals, Bordenstein argues, that it’s time to consider them the norm. He and Vanderbilt colleague Lisa Funkhouser published a manifesto in August in PLOS Biology calling for an end to “the sterile-womb paradigm.”

Bacterial billboards

Other paradigms are drawing strength from microbiologists’ recently developed ability to genetically probe bacterial communities. Since the mid-1970s, biologists have suspected that in many mammals a microbial community ferments various sweats, oozes and excretions into distinctive scents that reveal age, health and much more to knowing noses in a select social circle.

The notion sounds plausible, but attempts to test it have stalled for years. Culturing bacteria from various mammal scent glands has generally yielded only one or two, or sometimes five, kinds. This paltry haul seemed too limited to convey all the information that biologists think is wafting around.

With modern genetic tools to identify bacteria, Kevin Theis of Michigan State University in East Lansing and his colleagues are revisiting the classic hypothesis of messaging by fermentation. His scent-marking research subjects are spotted and striped hyenas.

Say it with scent Striped and spotted hyenas (shown, top) both use a smelly paste exuded from pouches under their tails to mark territory and communicate with one another. A two-dimensional statistical plot showing variation among bacterial communities hosted by individual hyenas of the two species (bottom left) looks very much like one showing variation in chemical components of their scents (bottom right). Sam DCruz/ShutterStock; K. Theis et al/PNAS 2013

“Pretty robust,” is how Theis rates the funk wafting off hyena scent marks. Both species evert a pouch just under the tail and dab a pungent paste produced by sebaceous glands onto a grass stem or other convenient landmark. The paste smells to Theis like pine mulch fermenting after a rain. It could encode territorial information as well as olfactory gossip such as who’s growing eager for a mate, already pregnant or perhaps ill.

Hyenas have a lot to smear and sniff about. Spotted hyenas live in hierarchical clans of dozens of animals. “It’s like watching a soap opera,” says Theis. Striped hyenas spend more time alone and form smaller groups, but still need to keep up with their kind while they forage, rest and travel.

So far, Theis says, he’s found more bacterial genera just in the scent paste of adult female spotted hyenas than researchers had discovered in 15 earlier studies of any mammalian scent gland.

The blends of stinky volatile compounds that striped and spotted hyenas use to communicate are distinct enough that biologists can distinguish the two species by their scent marks. And, as would be predicted if microbes were making the scents, the two species likewise have distinctive microbial communities that align with those scent differences, Theis and his colleagues report November 11 in the Proceedings of the National Academy of Sciences. The link between odor difference and community difference supports a main pillar of the hypothesis that the microbes are the message.

The researchers also detected some patterns within species suggesting that the communities shifted with events such as pregnancy. This paper marks the closest anyone has come to demonstrating the whole fermented-message idea, Theis says.

Choice microbes

Microbial residents do more than broadcast scented status updates. Bacteria also appear to steer their hosts away from some mates.

One startling example, described in 2010, grew out of a peculiar side effect of rearing fruit flies on different diets. In earlier experiments, researchers had noticed that lineages of fruit flies fed for 25 generations on different diets became less likely to mate with each other.

Follow-up tests at Tel Aviv University found that Drosophila melanogaster flies rejected opposite-diet flies as potential mates after just one generation of eating molasses rather than starch. At Tel Aviv, Eugene Rosenberg and Ilana Zilber­Rosenberg had been formulating ideas on the importance of what they called the holo­genome, the sum of genetic information in a host species and its microbial residents. To test this comprehensive view of the fruit fly, researchers fed the flies antibiotics to kill the insects’ microbial communities. Without microbial influence, the lineages took to mating with each other again.

Inoculating reconciled fly lineages with different microbial communities resurrected the mating barrier. What made the difference, researchers proposed, were diet-based shifts in gut microbes that in turn influenced sex pheromones.

A breed apart When two related species of Nasonia wasp mate (left), their hybrid male offspring (g/v and v/g) usually don’t survive. With a dose of antibiotics, however, hybrid males are almost as viable as nonhybrids (right). R. Brucker and S. Bordenstein/Science 2013

Observing a microbial effect on mate choice makes it sensible to ask a very big question: Could these teeming microscopic masses control the evolutionary fate of whole species?

In jewel wasps, for example, a genetic barrier that keeps two species apart turns out to have a previously overlooked microbial aspect (SN: 8/10/13, p. 13), Bordenstein and Vanderbilt colleague Robert Brucker reported in the Aug. 9 Science.

Two kinds of jewel wasp, Nasonia giraulti and Nasonia vitripennis, split off from a common ancestor about a million years ago. If the two species happen to mate now, the second-generation male larvae develop a dark splotch and die. Geneticists have traced this lethal incompatibility in detail, finding genetic differences between the species that appear to influence hybrid survival.

To test for a possible missing microbial something, Brucker dosed doomed hybrids with an antibiotic. Their resident microbes died, but many of the hybrid wasps lived. A mismatch between their parents’ differing microbes and their genes seemed to be killing hybrids.

As a further test, Brucker gave the unexpectedly alive germfree hybrids some of the gut bacteria that hybrids normally have. No longer germ-free, the hybrids died.

The experiment supports Bordenstein’s view that evolutionary forces act not just on an animal’s DNA but on the sum of its own genome and those of its microbial residents.

Microbial incompatibility appears to render hybrid male wasp larvae of two Nasonia species much less viable than purebreds. R. Brucker and S. Bordenstein/Science 2013

Of course microbes matter, says Tadashi Fukami of Stanford University. But he isn’t ready to declare them and their hosts a single evolutionary entity. He studies the microbial communities living in flowers’ nectar, and applauds increased attention to microbial influences. Yet he says that he would expect the hologenome theory of evolution to apply only in specialized, albeit interesting, cases. The discussion reminds him of debates over what’s called group selection. The idea that evolution acts on groups of organisms caused excitement and controversy when first proposed. But now Fukami and a fair number of other evolutionary biologists don’t find many cases in which it applies.

Still, appreciating microbes’ evolutionary significance could upend some fundamental ideas taught in introductory biology, says developmental biologist Scott Gilbert of Swarthmore College in Pennsylvania. Learning that a normal set of mouse genes isn’t sufficient to grow a healthy mouse body “set off all kinds of gongs and whistles,” he remembers. “All this stuff about ‘you are who you are depending on your nuclear genes’ was demonstrably not true,” he says, if microbes living symbiotically in the body amount to a second mode of inheritance.

He’s embracing the idea of animals as composite beings. On occasion he finishes scientific presentations with a closing PowerPoint slide that credits the talk not to him alone, but to “Team Scott Gilbert.”

Susan Milius is the life sciences writer, covering organismal biology and evolution, and has a special passion for plants, fungi and invertebrates. She studied biology and English literature.

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