The citrus mealybug looks like a walking dandruff flake, or perhaps a woodlouse that’s been rolled in flour. It’s also the insect version of a Russian nesting doll. If you look inside its cells, you’ll find a bacterium called Tremblaya princeps. And if you look inside Tremblaya, you’ll find yet another bacterium called Moranella endobia.

The two bacteria aren’t just passing hitchhikers. They’re symbionts— constant fixtures of the mealybug’s cells, and necessary for its survival. The trio cooperates to manufacture essential nutrients, such as amino acids. This involves a chain of chemical reactions, and it takes enzymes from all three partners to complete every step. Imagine a single production line with machines from three different manufacturers. Raw ingredients enter; amino acids come out.

As if this wasn’t complicated enough, some of these machines are built using genetic instructions that are loaned from three other groups of bacteria. These microbes probably lived inside the mealybug’s ancestors and transferred some genes into the insect’s genome. So, six different branches on the tree of life have come together to allow this three-way partnership to make the nutrients they need! “It’s almost too fantastic,” says John McCutcheon from the University of Montana, who has studied this hierarchy.

“This is really the kind of finding that would have blown away Charles Darwin or early geneticists,” says Nancy Moran from Yale University, who studies insect symbionts.

Three-in-one

The mealybug’s hitchhikers were first seen under the microscope in the 1950s, but only Moranella was recognised as a bacterium. Tremblaya was thought to be a special package created by the insect. It was Carol von Dohlen from Utah State University who recognised this middle partner for what it was, and described the bug-in-a-bug-in-a-bug arrangement.

In 2011, von Dohlen and McCutcheon sequenced the genomes of Tremblaya and Moranella and found that the latter was almost four times bigger, even though it sits inside the former. In fact, Tremblaya has the smallest genome of any known bacterium. Many species shrink their genomes through extreme organisation—packing their genes into ever-tighter spaces. But Tremblaya’s genome, though tiny, was also flabby. It had many wasted spaces and dead genes.

It’s also missing crucial genes, including an entire group that’s essential for building proteins. These genes are a few billion years old, and were present in the last common ancestor of all living thingslast common ancestor of all living things. They’re as indispensable for life as genes can get. There should be 20 of them. Some symbionts have lost a few. Tremblaya doesn’t have any. How does it survive?

McCutcheon suspected that Moranella probably takes up the slack. His team, led by Czech graduate student Filip Husnik, have now confirmed as much. They looked at the oat mealybug, whose cells also contain Tremblaya but not Moranella. The genome of the oat Tremblaya is also very small, but it’s still bigger and more tightly organised than that of the citrus Tremblaya. It also gives the oat mealybug all the enzymes that the citrus mealybug gets from both its partners combined.

So, at some point in the history, the mealybugs became colonised by Tremblaya and the two struck up a lasting partnership. Tremblaya lost many of the genes for a free-living existence, and its genome shrank considerably. Later, Moranella entered their alliance. By relying on the genes of this new partner, Tremblaya could lose even more of its own genes and become superlatively small.

View Images Mealybug cells, showing Tremblaya (red), Moranella (green) and mealybug nuclei (blue). Credit: Ryuichi Koga, National Institute of Advanced Industrial Science and Technology, Japan

No, wait, six-in-one

In some cases, symbionts have relocated their genes to the genomes of their hosts, so the missing Tremblaya genes may have ended up in the mealybug’s DNA. Indeed, the team found that the bug’s genome contains at least 22 genes of bacterial origin. But in a surprising twist, none of these came from Tremblaya or Moranella!

Instead, they hailed from three separate lineages of bacteria. All three groups contain members that regularly colonise the cells of insects, but none of them are found in the mealybug today. Maybe they colonised the insect’s ancestors, donated their genes, and have since disappeared. “These genes are like the ghosts of symbionts-past,” explains Molly Hunter from the University of Arizona. There are many examples of bacteria donating their genes to animals but “this degree is impressive, especially since its from so many different sources,” Hunter adds.

These borrowed genes aren’t sitting idly by. They are also involved in making amino acids, plugging holes in the production line that neither Tremblaya nor Moranella can fill. The citrus mealybug is effectively a mash-up of six different species, three of which aren’t even there!

So, what do you call these things?

The history of life is full of bacteria that have become permanent residents in other cells. Your own cells, and those of every animal, plant and fungus, contain small structures called mitochondria, which used to be free-living bacteria. Now, they’re organelles—compartments within larger cells that perform specialised tasks. The mitochondria, for example, are batteries that provide us with energy.

So, is Tremblaya a symbiont or has it already become more of an organelle? “The distinction is a matter of debate and definition,” says Martin Kaltenpoth from the Max Planck Institute for Chemical Ecology. Tremblaya permanently lives inside other cells, helps its host to survive, and has lost many genes. These are all ticks for the organelle column.

But there are two big differences. As free-roaming bacteria transformed into mitochondria, they shrunk by shunting many of their genes into their hosts. Tremblaya hasn’t done that. It became small by relying on Moranella. Also, organelles tend to be permanent and irreplaceable. If all your mitochondria suddenly vanished, you’d die very quickly. But Tremblaya can be replaced. In some mealybugs, a different bacterium has usurped it. “I don’t want to call it an organelle. I really don’t,” says McCutcheon.

Rather than quibbling over labels, it’s more important to him to work out the details of this partnership, and there are still many mysteries left to solve. For example, how exactly does Moranella share its enzymes with Tremblaya? After all, Moranella doesn’t make any of the transporter proteins that would normally export molecules. In 2011, the team suggested that Moranella might just burst apart, releasing its contents inside Tremblaya. “That was wild speculation. We just couldn’t figure anything else out,” says McCutcheon.

But they may have been right. Moranella’s cell wall—the layer that keeps its insides inside—is made from molecules called peptidoglycans, which the bacterium can’t make on its own. Instead, it relies on genes from the mealybug, including those that were loaned from the other bacterial groups! By switching off these genes, the bug could potentially destabilise Moranella, causing it to burst and release its contents to Tremblaya. Maybe it controls the relationship between its current symbionts using genes borrowed from its old symbionts.

Wouldn’t that destroy Tremblaya as well? No, because it has lost so many essential genes that it can’t make its own cell wall or membrane. McCutcheon suspects that it somehow gets those barriers from the mealybug. If that’s true, it would be amazing. It would mean that Tremblaya relies on the mealybug to define its own boundaries. Without its host, it would be a bunch of molecules floating away in liquid!

“This is still wild speculation but at least we can now do experiments,” says McCutcheon. He could, for example, switch off the mealybug’s peptidoglycan-making genes and see what happens to Moranella numbers.

McCutcheon’s peers are already very impressed. “John is well-known for his outstanding contributions to understanding the evolution of intracellular mutualisms in insects, and this is yet another excellent piece of work from his group,” says Kaltenpoth. “I thought this paper was a masterwork,” adds Hunter.

Reference: Husnik, Nikoh, Koga, Ross, Duncan, Fujie, Tanaka, Satoh, Bachtrog, Wilson, von Dohlen, Fukatsu & McCutcheon. 2013. Horizontal Gene Transfer from Diverse Bacteria to an Insect Genome Enables a Tripartite Nested Mealybug Symbiosis. Cell http://dx.doi.org/10.1016/j.cell.2013.05.040