Mealybugs only eat plant sap but sap doesn't contain all the essential amino acids the insects need to survive - so mealybugs have developed a symbiotic relationship with two species of bacteria, one living inside the other in a situation unique to known biology, to manufacture the nutrients sap doesn't provide.

The net result: The bacteria get a comfy mealybug home, and the bugs get the nutrition they need to live.

University of Montana microbiologist John McCutcheon describes such mutually beneficial relationships used to solve life's little problems as "almost hilariously complicated. But animal-bacterial relationships are extremely common in nature, and it's my goal in life to help people understand that it's normal."

McCutcheon and his research partners recently delved deeper into the genes involved in the "tripartite nested mealybug symbiosis," published in Cell. The researchers discovered the already complex three-way symbiosis actually depends on genes from six different organisms – three more than the number of species that currently exist in the symbiosis.

Tremblaya princeps is the larger of the two bacteria species living within special organs inside mealybugs. Tremblaya houses the smaller bacterial species, Moranella endobia, within its cytoplasm. But what makes Tremblaya truly odd is the size of its genome, or genetic code. With only 120 genes, its genome is the smallest known and smaller than many scientists consider necessary for life. By comparison, common E. coli bacteria have about 4,200 genes and humans have about 21,000.

"We wanted to discover how this genome got so small," McCutcheon said. "We suspected Tremblaya's genome may have gotten smaller by transferring genes to the host animal, which is called horizontal transfer."

The researchers looked for genes in the mealybug genome that resemble bacteria genes. However, after extensive analysis they only found one weak possibility for horizontal transfer from Tremblaya.

"Our hypothesis that Tremblaya was transferring genes to the host was dead wrong," said McCutcheon. They did, however, find 22 other bacterial genes mixed in with the mealybug code – genes that seem to support activities missing in Tremblaya, Moranella and the mealybug.

How can this be?

"The genes are probably from historical bacterial infections," McCutcheon said. "These bacteria are no longer present in the mealybugs we work with, but their horizontally transferred genes are, and these genes allow the symbiosis to work."

The research team also examined a strain of Tremblaya that doesn't have Moranella living inside it. This variety employs about 50 more genes than the one containing Moranella, which strongly suggests Moranella plays a key role in allowing the insect-dwelling Tremblaya to operate with such a tiny genome.

McCutcheon said Tremblaya, with its shrinking genome, in many ways resembles organelles called mitochondria – tiny structures found within all plant and animal cells that scientists believe started out as symbiotic bacteria in the early history of life. The mealybug/bacteria relationship he studies may illustrate one pathway bacteria take in becoming essential and highly integrated components of other cells.

"So this research really touches on some fundamental questions of the origin of life," he said. "It's exciting to see if we can get some insight into the origin of organelles."

McCutcheon said this study involved an international cast of 12 collaborators. Filip Husnik, the study's lead author, is a Czech doctoral student from the University of South Bohemia who worked in McCutcheon's UM lab. Other team members were from Japan, England, California, Utah and Florida.

"Our work illustrates how an animal's interactions with bacteria can drive hidden organismal complexity," McCutcheon said. "A tree is more than a tree, and an animal is more than an animal. They are really mosaics of plants and animals and bacteria all working together."