ABOVE: Stinkbug (Plautia stali) hatchlings acquiring symbiotic bacteria from their eggshells MINORU MORIYAMA

Insects are an incredibly successful class of animals: They’ve conquered every continent and spread throughout virtually every terrestrial ecosystem. But they didn’t do it alone. Many species of insects owe at least some of their success to nutrient-producing symbiotic bacteria, which allow the insects to survive on diets that would be impossible for other animals or without the bacteria’s assistance. 

While the partnerships between insects and bacteria have been studied for decades, how they form has remained something of a mystery. Now, in a study published August 4 in Nature Microbiology, researchers directed bacterial evolution in the lab to forge a new insect-bacteria partnership, providing insights into how these symbioses may evolve in nature.

“This study is very novel and exciting because it shows the surprising ease with which microbes can evolve to become symbionts,” Alison Ravenscraft, an insect microbiome researcher at the University of Texas at Arlington who was not involved in this study, tells The Scientist over email. 

In this study, researchers at the National Institute of Advanced Industrial Science and Technology in Japan (AIST) used the stinkbug (Plautia stali) as a model. This species has specialized crypts in its gut where it houses a bacterial partner (Pantoea sp.) that is crucial to the bug’s survival. While researchers aren’t 100 percent sure what makes the bacteria so essential to P. stali, study coauthor and AIST symbiotic evolution researcher Takema Fukatsu tells The Scientist that other species of stinkbugs rely on symbiotic bacteria to produce B vitamins and essential amino acids, so it’s likely P. stali’s symbiotic bacteria perform a similar function. 

When researchers replaced the stinkbug’s Pantoea with a hypermutating strain of E. coli, only five to ten percent of the bugs survived to adulthood, and those that did were stunted and brown in color—a sharp contrast from the vibrant green of a healthy stinkbug.

a close-up of a bright green stinkbug facing the camera
Adult stinkbug (Plautia stali)

In 19 groups of stinkbugs with the hypermutating E. coli, scientists took the healthiest bugs from each generation (determined either by growth or by the healthiest coloring) and passed their bacteria on to the next, repeating the process for 12 generations. Over time, the E. coli strains in two of the groups of bugs seemed to become beneficial for the stinkbugs: Bugs inoculated with these strains had improved coloring and were generally more likely to survive to adulthood compared to prior generations.

By analyzing the genomes of these two strains before and after they became beneficial, scientists determined that both strains had mutations that disrupted the carbon catabolite repression (CCR) pathway. The CCR pathway helps bacteria to survive when glucose (their preferred carbon source) is scarce by allowing them to consume other carbon sources for energy, and it involves the up- or down-regulation of hundreds of other genes.

This study is very novel and exciting because it shows the surprising ease with which microbes can evolve to become symbionts.

—Alison Ravenscraft, University of Texas at Arlington 

Researchers aren’t yet sure exactly why these mutations are beneficial to E. coli, but one hypothesis is that they may ease the bacteria’s adjustment to the gut crypt environment. 

Inside the stinkbug’s crypts, the bacteria may not have access to sufficient glucose, says Fukatsu. Normally, E. coli might try to turn on the CCR pathway, but inside the stinkbug there aren’t other carbon sources for them to exploit. 

“Hence,” says Fukatsu, “the bacteria may repeatedly switch their metabolism in vain, which must be a heavy metabolic cost for the bacteria. Eliminating such metabolic cost by inactivating the CCR pathway is a possible source of benefit for the bacteria, and ultimately also for the host.”

Fukatsu says that avoiding this metabolic cost may result in more efficient bacterial production of nutrients essential to the host, or may simply allow more bacteria to survive inside the host, ultimately resulting in greater amounts of the nutrients required by the stinkbug.

While E. coli’s shift towards an insect mutualist appeared to be mediated by the CCR pathway in this study, Fukatsu thinks that a CCR mutation is just one of the many paths leading to mutualism. He says that experiments currently in progress in the lab have created many strains of E. coli whose beneficial effects on the stinkbugs do not seem to be related to the CCR pathway. 

“There are so many genes that contribute to the establishment and maintenance of symbiosis in the system,” he says. “We are now looking for these other symbiosis genes. . . . Elucidating all the pathways will give us a more complete, more comprehensive understanding of the mechanisms of symbiosis.”

This is highly important field of study, says Ravenscraft. “We are coming to realize that so much of animals’ and plants’ biology depends on microbial partners. . . . Studying how mutualisms evolve will help us understand how bacteria provide crucial functions to both us, and the animals and plants we depend on.”