July, 2021 — Be it bird, bug, or bacterium—no organism on Earth exists in isolation. We all live and interact with other living things, and these relationships between species shape the world around us.

This natural harmony is easy to picture on a global scale: exotic creatures eating fruit in a lush and colorful rainforest, tall cacti thriving under the brutal desert sun, or bumblebees spreading pollen in a field of wildflowers. 

But what about the many “invisible” ecosystems that make you you?

Like Earth, the human body is host to an eclectic array of communities, each containing many interacting microbes. These tiny ecosystems, called microbiomes, influence our physiology in myriad ways. In particular, the interplay between the microbiota in the human gut—many trillions of bacteria, viruses, and fungi—plays an outsized role in shaping our health.

Despite its importance, researchers lack a mechanistic understanding of how the gut microbiome as a whole is greater than the sum of its parts. In other words, to what extent do individual microbes influence us, and to what degree are these impacts determined by the interconnected and overlapping interactions between unique species? Studying the underlying biology that governs these relationships is absolutely critical to advancing our understanding of human health. However, the sheer number of different microbes in our gut presents a challenge to cataloging and understanding the effects of their synergy.

Using the natural simplicity of the fruit fly gut microbiome as a model system, the Ludington Lab at the Carnegie Institution for Science’s Department of Embryology is starting to untangle this complex web of interactions. 


Electron microscopy of a cross section of the fruit fly gut. The bacteria are seen as dark, round shapes. They are separated from the fly cells by a light gray band of an unknown extracellular substance. // Ren Dodge & Mike Sepanski.

 

Dr. William Ludington, who joined Embryology as a Staff Member in August 2018, deploys advanced techniques in bioinformatics, microbiology, state-of-the-art imaging, mathematical modeling, and molecular biology to comprehensively map the submicroscopic network of the fly gut. His work is revealing fundamental biological principles that can be used to understand the ecosystems inside our own bodies. 
 
“The classic way we think about bacteria is in a black-and-white context as agents of disease—either you have it, or you don’t,” explains Ludington. “This isn’t the case for the gut microbiome. Our research shows that the effects of a particular species depend on the context of which other microbial species are also present in the community.”
 

Live imaging of a fruit fly gut. The Bellymount System, developed by a collaboration between Dr. Lucy O’Brien and Dr. KC Huang of Stanford University and Dr. William Ludington of Carnegie, allows researchers to peer into the live tissue of the fruit fly gut in real-time. // Courtesy of Leslie Koyama and Lucy O’Brien.

 

To accomplish the level of precision necessary to analyze this complex and unmapped ecosystem, Ludington raises germ-free flies which serve as living ‘Petri dishes’. The lab can populate the gut of these flies with any bacterium (or combination of bacteria) they choose, then monitor the effects at single-cell resolution and in real-time—a revolutionary technique in biology. 
 
“When people have done other kinds of microbiome experiments, they are asking ‘what’s there?’ But we know exactly what’s there,” explains Ludington. “We’re not just being descriptive. We’re saying, ‘we have this specific system, and when we put this in or take that away, we can see the effect come and go.’ So, you can really establish causation of the specific bacteria.”
 

Handling live flies is a delicate task. Lab manager Daniel Martinez uses a CO2 pad and custom “grabbing” device to prepare fruit flies for an experiment under sterile conditions.

 

Before an experiment, a lab member must carefully place living flies in individual chambers of a high-throughput assay dish, called a 96-well plate. To collect the flies, they are first put on a sterile, porous pad that emits CO2—quickly knocking them out. The four-pronged apparatus seen above uses filtered air and gentle suction to “grab” four flies simultaneously, all while keeping them intact and free of germs. Former Ludington Lab researcher Maria Jaime developed this custom device with help from building maintenance specialist Ted Cooper.
 
High throughput screening produces a readout that allows the lab to rapidly quantify the genetic differences from fly to fly. “It's all about controlling variation,” says Ludington. “Everything we're doing is about controlling variation so that we know a precise perturbation causes a precise physiological reaction in the fly.”
 

Johns Hopkins University rotation student Gemechu Mekonnen prepares flies for the “Smurf assay.”

 

Above, rotation student Gemechu Mekonnenon collects fruit flies to analyze their gut permeability. Called the “Smurf assay,” he will feed the flies blue-dyed food. If the entire fly turns blue to the naked eye, Mekonnenon knows bacteria have weakened the integrity of its gut.

 

With other kinds of microbiome experiments, the researchers are asking ‘what’s there?’ But we know exactly what’s there. We’re saying, ‘we have this specific system, and when we put this in or take that away, we can see the effect come and go.” 

Drosophila melanogaster (the fruit fly) shares 60 percent of its DNA with humans. // Sanjay Acharya

 

The spark that led to Ludington’s innovative work came shortly after joining the Department of Embryology, where he began to collaborate with Director Emeritus (and current Staff Scientist) Dr. Allan Spradling—one of the founders of modern fly genetics. In the 1980s, Spradling pioneered research and techniques that led to a much greater understanding of fruit fly biology, including manipulating its genome. In no small part because of Spradling, the fruit fly is now the most ubiquitous and powerful model organism used to understand human biology; directly responsible for six Nobel Prizes in Physiology and Medicine. 

 
“One of the main reasons I came to Embryology was with the hopes of working with Allan,” says Ludington. “Having worked in the field for so long, he has deep knowledge and a real openness to working on new areas—such as trying to understand the genes evolved to promote organismal interactions.”
 
 
Dr. Allan Spradling at work in his lab at the Department of Embryology. Circa 1980.

 

Now, Ludington and Spradling are teaming up to study a poorly understood region of the fly gut. Their preliminary results suggest it cultivates symbiotic bacteria. Because this discovery happened in the small and well-understood fruit fly, their genetic tools should quickly allow a deep mechanistic understanding of this symbiotic niche. 
 
“This commensal niche in flies had never been seen before,'' explains Ludington. “And because we found it in this workhorse of animal genetics that Allan helped establish, we can now go to the public stock centers and simply request the genetic mutants that are already available. It saves a ton of time having all the mutants already made.”
 

Since starting operations in mid-2018, the Ludington Lab has produced results at an explosive rate, honing in on individual gut bacteria interactions and their effects on targeted biological processes—like the immune response. 


Each vial in the Ludington Lab’s incubation chambers holds a different genome of fruit fly.

 

In the image above, Ludington reaches for a vial from one of his incubation chambers. Each vial contains a different genetic stock of fruit fly—some are wild-type, with no known mutations, and others have lab-induced mutations in a specific immune gene. By comparing the distinct effects of the same bacterium in both types of fly, Ludington and his team can study how it affects the immune system. 

But it’s not enough to only understand and manipulate the genetics of the fruit fly. To have complete control over the equation, the lab must also turn “on” and “off” specific genes and proteins in the bacteria themselves—and know where to find them. 

Below, postdoctoral fellow Karina Gutierrez Garcia uses advanced bioinformatics to analyze the genetic and evolutionary history of Lactobacillus plantarum—the most common bacteria found in wild fruit flies


Karina Gutierrez-Garcia studies the genetics of Lactobacillus plantarum. Each purple and gray line to her left represents a separate genome of the bacteria. Her right screen displays a graphical analysis of its evolutionary tree. 

 

“If we take any random fly that we see, it's likely to have Lactobacillus,” explains Ludington. “It’s also found in the human digestive system and in fermented foods worldwide. We suspect this may be because fruit flies are spreading it.”
 
Gutierrez-Garcia is sifting through the data to find a workable set of “target genes of interest.” For example, she might find a gene that helps the bacteria grab hold of a host molecule that allows it to live inside the fly. If the gene is turned off, the bacteria will no longer recognize the fly and establish the symbiotic relationship, resulting in its expulsion. 
 
“We are working to characterize the fly genetics of colonization—and also the bacterial genetics of colonization.”  William Ludington

Predoctoral associate Kevin Aumiller uses a custom fluorescent lightbox to count bacterial colonies from a single fly. Measuring the number of bacteria in the host is one of the most common aspects of the lab’s experiments.

 

In the first of the two images below, postdoctoral associate Robert Scheffler displays colonies of fluorescent-labeled Lactobacillus plantarum. By expressing the mCherry protein, the lab can turn the bacteria a pinkish-red color—allowing for easy visualization under a microscope. In the second image, Johns Hopkins Ph.D. student Haolong Zhu analyzes advanced microscopy data from a fly gut colonized with mCherry-expressed Lactobacillus.


By tagging bacteria with a fluorescent protein, researchers in the Ludington Lab can easily locate it in the gut. Top: Robert Scheffler. Bottom: Haolong Zhu.

 

Below, senior research associate Ren Dodge dissects a fly to prepare a cross-section for imaging at Embryology’s microscopy suite. There, an advanced electron microscope will produce high-magnification images that detail important properties of the fly’s gut bacteria. 


Ren Dodge carefully dissects a fly to prepare its gut for electron microscopy.

 

“We do a lot of experiments where we rip the fly apart,” says Ludington. “But that destroys its gut morphology. We knew that we also needed non-disruptive techniques that let us precisely quantify the geometry of the gut.”

So, Ludington has started to collaborate with the University of California’s Lawrence Berkeley National Lab, which uses a powerful X-ray machine called a synchrotron to produce detailed micro-CT scans of un-damaged flies.  


Ludington Lab manager Daniel Martinez studies a micro-CT scan of a fly gut. The images to his right were generated at the Lawrence Berkeley National Lab’s Synchrotron facility. 

 

“It's all about controlling variation. Everything we're doing is about controlling variation so that we know a precise perturbation causes a precise physiological reaction in the fly.”

 

Just two-and-a-half years after starting his lab at the Department of Embryology (the blink of an eye in biological science), Ludington has opened doors to many new possibilities for microbiome research and beyond—including the potential to help develop improved and novel treatments for human disease. He has already gained the attention and financial support of major scientific organizations, including the National Institution for Health (NIH), The National Science Foundation (NSF), and most recently, the Research Corporation For Science Advancement (RCSA). Stay tuned for updates! 

 

Want to learn more? Visit The Ludington Lab