Microbes Gone Missing

In the early 20th century, biologists began to uncover fascinating relationships between complex organisms and their microbes: in tubeworms that had no mouth, anus or gut; in termites that fed on tough, woody plants; in cows whose grassy diet significantly lacked protein. Such observations generated excitement and prompted follow-up experiments. In those years, the absence of microbial helpers in an animal wasn’t considered particularly surprising or interesting, and it often received little more than a passing nod in the literature. Even when it was thought to merit more than that — as in a 1978 report in Science that tiny wood-eating crustaceans, unlike termites, had no stable population of gut bacteria — it ended up flying under the radar.

And so expectations quietly began to shift to a new norm, that every animal had a relationship with bacteria without which it would perish. A few voices protested this oversimplification: As early as 1953, Paul Buchner, one of the founders of symbiosis research, wrote with exasperation about the notion that obligate, fixed and functional symbioses were universal. “Again and again there have been authors who insist that endosymbiosis is an elementary principle of all organisms,” he seethed. But counterexamples drowned in the flood of studies on the importance of host-microbe symbioses, especially those that drew connections between human health and our own microbiome.

“The human microbiome has completely driven a lot of our thinking about how microbes work,” said Tobin Hammer, a postdoctoral researcher in ecology and evolutionary biology at the University of Texas, Austin. “And we often project from ourselves outwards.”

But the human example is not a good model for what’s going on in a diverse range of species, from caterpillars and butterflies to sawflies and shrimp, to some birds and bats (and perhaps even some pandas). In these animals, the microbes are sparser, more transient or unpredictable — and they don’t necessarily contribute much, if anything, to their host. “The story is more complex,” said Sarah Hird, an evolutionary biologist and microbial ecologist at the University of Connecticut, “more fuzzy.”

A transient, almost nonexistent relationship with bacteria was what Sanders saw in his tropical ants. He brought his samples back to his lab (then at Harvard University, although he is now at Cornell), where he sequenced the insects’ bacterial DNA and quantified how many microbes were present. The ant species with dense, specialized microbiomes had approximately 10,000 times more bacteria in their guts than Sanders found in the many other species he had captured. Put another way, Sanders said, if the ants were scaled to human size, some would carry a pound of microbes within them (similar to what humans harbor), others a mere coffee bean’s worth. “It’s really a profound difference.”

That difference, reported in Integrative & Comparative Biology in 2017, seemed to be associated with diet: Strictly herbivorous tree-dwelling ants were more likely to have an abundant microbiome, perhaps to make up for their protein-deficient diet; omnivorous and carnivorous ground-dwelling ants consumed more balanced meals and had negligible amounts of bacteria in their gut. Still, this pattern was inconsistent. Some of the herbivorous ants also lacked a microbiome. And the ants that did have one didn’t seem to have widespread, predictable associations with particular species of bacteria (although some sets of microbes were common to individual genera of the insects). That result marked a clear departure from mammalian microbiomes like our own, which tend to be very specific to their hosts.

The reasons why would become clearer as case studies of other organisms started to trickle in.

The Tip of the Iceberg

At around the same time that Sanders was examining ants in Peru, Hammer was in Costa Rica on an independent search for a microbiome in caterpillars. (“What better insect to have obligate relationships with bacteria than these cows of the insect world?” Sanders commented.) But try as he might, Hammer couldn’t find much bacterial DNA in the gut and fecal samples he collected. “Something really weird was going on,” he said.

When, after months of “frustrating lab work,” he realized that the animals might simply not have a stable microbiome, “it was a shift in thinking for me that was not expected at all.” He and his colleagues ultimately found that, like so many of Sanders’ ants, caterpillars had much, much lower quantities of microbes than was considered the norm. Moreover, those microbes were simply a subset of the ones found in the animals’ plant diet — “which supports the idea that they’re transiently passing through and some of them are getting digested, essentially,” Hammer said. “They’re not establishing stable populations within the gut.”

To determine whether those transient bacteria benefited the caterpillars, the researchers eliminated them with antibiotics. In other insects and animals, such a treatment tends to stunt development or kill the host outright. But it had no effect whatsoever on Hammer’s caterpillars.

Deepa Agashe, an ecologist and evolutionary biologist at the National Center for Biological Sciences in Bangalore, India, saw something similar in insects that her team collected from several locations near the greenery of their campus. The microbes they found in dragonflies and butterflies strongly correlated with the insects’ diets rather than with a particular insect species or developmental stage. The vast majority of the dragonflies’ bacterial communities seemed to have come together by chance. “Most of the bacteria were just there because they got there,” Agashe said. The insects “do not seem to be selecting for particular species of bacteria or a particular kind of bacteria.”

Repeated experiments that disrupted the butterflies’ microbial populations yielded no effect on the hosts’ growth or development. Neither did reintroducing the bacteria to their guts. “Really,” Agashe said, “they don’t seem to care about their microbes at all” — even though the butterflies feed on toxic plants and seem like perfect candidates for a full-fledged, functional microbiome that could detoxify their meals.

Like Hammer and Sanders, “initially we were scratching our heads,” Agashe said. “It was a surprising result, and actually it took us a while to wrap our heads around it.”

But maybe it shouldn’t be so surprising. As the scientists realized, when microbiomes are present, they’re often found in specific tissues — and they involve specific bacteria that influence specific traits at specific times. The bobtail squid, for example, has a symbiosis that’s limited to one species of luminous bacteria, which is sequestered in a single light-producing organ while the squid’s gut and skin remain microbe-free. Adult honeybees have important relationships with their bacteria, but the larvae don’t.

So it’s not such a leap to think there could be animals that don’t have such relationships at all, or that have relationships that play by different rules. “I think there’s now an increasing realization that there’s this whole spectrum of kinds of associations that you might find,” Agashe said.

Hammer agreed. “We’re just getting a glimpse at the tip of the iceberg,” he said.