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How leafcutter ants cultivate a fungal garden to degrade plants and provide insights into future biofuels

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How leafcutter ants cultivate a fungal garden to degrade plants and provide insights into future biofuels


Scientists have spent decades finding ways to efficiently and affordably degrade plant materials so that they can be converted into useful bioproducts that benefit everyday life.

Bio-based fuels, detergents, nutritional supplements, and even plastics are the result of this work. And while scientists have found ways to degrade plants to the extent needed to produce a range of products, certain polymers such as lignin, which is a primary ingredient in the cell wall of plants, remain incredibly difficult to affordably break down without adding pollutants back into the environment. These polymers can be left behind as waste products with no further use.

A specialized microbial community composed of fungus, leafcutter ants, and bacteria is known to naturally degrade plants, turning them into nutrients and other components that are absorbed and used by surrounding organisms and systems. But identifying all components and biochemical reactions needed for the process remained a significant challenge — until now.

As part of her Department of Energy (DOE) Early Career award, Kristin Burnum-Johnson, science group leader for Functional and Systems Biology at Pacific Northwest National Laboratory (PNNL), and a team of fellow PNNL researchers, developed an imaging method called metabolome informed proteome imaging (MIPI). This method allows scientists to peer deep down to the molecular level and view exactly what base components are part of the plant degradation process, as well as what, when, and where important biochemical reactions occur that make it possible.

Using this method, the team revealed important metabolites and enzymes that spur different biochemical reactions that are vital in the degradation process. They also revealed the purpose of resident bacteria in the system — which is to make the process even more efficient. These insights can be applied to future biofuels and bioproducts development.

The team’s research was recently published in Nature Chemical Biology.

Symbiotic relationship between leafcutter ants and fungus reveal key to success in plant degradation

For its research, the team studied a type of fungus known for its symbiotic relationship with a species of leafcutter ants — a fungus known as Leucoagaricus gongylophorus. The ants use the fungus to cultivate a fungal garden that degrades plant polymers and other material. Remnant components from this degradation process are used and consumed by a variety of organisms in the garden, allowing all to thrive.

The ants accomplish this process by cultivating fungus on fresh leaves in specialized underground structures. These structures ultimately become the fungal gardens that consume the material. Resident bacterial members help with the degradation by producing amino acids and vitamins that support the overall garden ecosystem.

“Environmental systems have evolved over millions of years to be perfect symbiotic systems,” Burnum-Johnson said. “How can we better learn from these systems than by observing how they accomplish these tasks naturally?”

But what makes this fungal community so difficult to study is its complexity. While the plants, fungus, ants, and bacteria are all active components in the plant degradation process, none of them focus on one task or reside in one location. Factor in the small-scale size of the biochemical reactions occurring at the molecular level, and an incredibly difficult puzzle presents itself. But the new MIPI imaging method developed at PNNL allows scientists to see exactly what is going on throughout the degradation process.

“We now have the tools to fully understand the intricacies of these systems and visualize them as a whole for the first time,” Burnum-Johnson said.

Revealing important components in a complex system

Using a high-powered laser, the team took scans across 12-micron-thick sections of a fungal garden — the approximate width of plastic cling film. This process helped determine locations of metabolites in the samples, which are remnant products of plant degradation. This technique also helped identify the location and abundance of plant polymers such as cellulose, xylan, and lignin, as well as other molecules in specific regions. The combined locations of these components indicated hot spots where plant material had been broken down.

From there, the team homed in on those regions to see enzymes, which are used to kick start biochemical reactions in a living system. Knowing the type and location of these enzymes allowed them to determine which microbes were a part of that process.

All of these components together helped affirm the fungus as the primary degrader of the plant material in the system. Additionally, the team determined that the bacteria present in the system transformed previously digested plant polymers into metabolites that are used as vitamins and amino acids in the system. These vitamins and amino acids benefit the entire ecosystem by accelerating fungal growth and plant degradation.

Burnum-Johnson said if scientists had used other, more traditional methods that take bulk measurements of primary components in a system, such as metabolites, enzymes, and other molecules, they would simply get an average of those materials, creating more noise and masking information.

“It dilutes the important chemical reactions of interest, often making these processes undetectable,” she said. “To analyze the complex environmental ecosystems of these fungal communities, we need to know those fine detail interactions. These conclusions can then be taken back into a lab setting and used to create biofuels and bioproducts that are important in our everyday life.”

Using knowledge of complex systems for future fungal research

Marija Velickovic, a chemist and lead author of the paper, said she initially became interested in studying the fungal garden and how it degrades lignin based on the difficulty of the project.

“Fungal gardens are the most interesting because they are one of the most complex ecosystems composed of multiple members that effectively work together,” she said. “I really wanted to map activities at the microscale level to better understand the role of each member in this complex ecosystem.”

Velickovic performed all the hands-on experiments in the lab, collecting material for the slides, scanning the samples to view and identify metabolites in each of the sections, and identifying hot spots of lignocellulose degradation.

Both Velickovic and Burnum-Johnson said they are ecstatic about their team’s success.

“We actually accomplished what we set out for,” Burnum-Johnson said. “Especially in science, that isn’t guaranteed.”

The team plans to use its findings for further research, with specific plans to study how fungal communities respond and protect themselves amid disturbances and other perturbations.

“We now have an understanding of how these natural systems degrade plant material very well,” Burnum-Johnson said. “By looking at complex environmental systems at this level, we can understand how they are performing that activity and capitalize on it to make biofuels and bioproducts.”

The work was funded by DOE’s Office of Science. Additionally, researchers accessed mass spectrometry imaging and computing and proteomics resources at the Environmental Molecular Sciences Laboratory, an Office of Science user facility located at PNNL.



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‘Ice bucket challenge’ reveals that bacteria can anticipate the seasons

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How leafcutter ants cultivate a fungal garden to degrade plants and provide insights into future biofuels


Bacteria use their internal 24-hour clocks to anticipate the arrival of new seasons, according to research carried out with the assistance of an ‘ice bucket challenge.’ 

This discovery may have profound implications for understanding the role that circadian rhythms – a molecular version of a clock – play in adapting species to climate change, from migrating animals to flowering plants.  

The team behind the findings gave populations of blue-green algae (cyanobacteria) different artificial day lengths at a constant warm temperature. Samples on plates received either short days, equinox days (equal light and dark), or long days, for eight days.  

After this treatment, the blue-green algae were plunged into ice for two hours and survival rates monitored.   

Samples that had been exposed to a succession of short days (eight hours light and 16 hours dark) in preparation for the icy challenge achieved survival rates of 75%, up to three times higher than colonies that had not been primed in this way. 

One short day was not enough to increase the bacteria’s resistance to cold. Only after several short days, and optimally six to eight days, did the bacteria’s life chances significantly improve. 

In cyanobacteria which had genes that make up their biological clock removed, survival rates were the same regardless of day lengths. This indicates that photoperiodism (the ability to measure the day-night cycle and change one’s physiology in anticipation of the upcoming season) is critical in preparing bacteria for longer-term environmental changes such as a new season or shifts in climate. 

“The findings indicate that bacteria in nature use their internal clocks to measure day length and when the number of short days reaches a certain point, as they do in autumn/fall, they ‘switch’ to a different physiology in anticipation of the wintry challenges that lie ahead,” explained first author of the study, Dr Luísa Jabbur, who was a researcher at Vanderbilt University, Tennessee, in the laboratory of Prof. Carl Johnson when this study took place, and is now a BBSRC Discovery Fellow at the John Innes Centre.  

The Johnson lab has a long history of studying the circadian clock of cyanobacteria, both from a mechanistic and an ecological perspective. 

Previous studies have shown that bacteria have a version of a biological clock, which could allow them to measure differences in day-night length, offering an evolutionary advantage. 

This study, which appears in Science, is the first time that anyone has shown that photoperiodism in bacteria has evolved to anticipate seasonal cues.  

Based on these findings a whole new horizon of scientific exploration awaits. A key question is: how does an organism with a lifespan of between six and 24 hours evolve a mechanism that enables it not merely to react to, but to anticipate, future conditions? 

“It’s like they are signalling to their daughter cells and their granddaughter cells, passing information that the days are getting short, you need to do something,” said Dr Jabbur. 

Dr Jabbur and colleagues at the John Innes Centre will, as part of her BBSRC Discovery Fellowship, use cyanobacteria as a fast-reproducing model species to understand how photoperiodic responses might evolve in other species during climate change, with hopeful applications to major crops.  

A key part of this work will be to understand more about the molecular memory systems by which information is passed from generation to generation in species. Research will investigate the possibility that an accumulation of compounds during the night on short days acts as a molecular switch that triggers change to a different physiology or phenotype.  

For Dr Jabbur the findings amount to an early-career scientific breakthrough in the face of initial scepticism from her scientific mentor and the corresponding author of the paper, Professor Carl Johnson. 

“As well as being a fascinating person and an inspiration, Carl sings in the Nashville Symphony Chorus, and he has an operatic laugh! It echoed round the department when I first outlined my idea for the icy challenge, to see if photoperiod was a cue for cyanobacteria in their natural element,” said Dr Jabbur. 

“To be fair he told me to go away and try it, and as I went, he showed me a sign on his door with the Frank Westheimer quote: ‘Progress is made by young scientists who carry out experiments that old scientists say would not work.’ 

“It did work, first time. Then I repeated the experiments. There is something very precious about looking at a set of plates with bacteria on them and realizing that in that moment you know something that nobody else knows.” 

Bacteria can anticipate the seasons: Photoperiodism in cyanobacteria appears in Science.  



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New filtration material could remove long-lasting chemicals from water

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How leafcutter ants cultivate a fungal garden to degrade plants and provide insights into future biofuels


Water contamination by the chemicals used in today’s technology is a rapidly growing problem globally. A recent studyby the U.S. Centers for Disease Control found that 98 percent of people tested had detectable levels of PFAS, a family of particularly long-lasting compounds, also known as forever chemicals, in their bloodstream.

A new filtration material developed by researchers at MIT might provide a nature-based solution to this stubborn contamination issue. The material, based on natural silk and cellulose, can remove a wide variety of these persistent chemicals as well as heavy metals. And, its antimicrobial properties can help keep the filters from fouling.

The findings are described in the journal ACS Nano, in a paper by MIT postdoc Yilin Zhang, professor of civil and environmental engineering Benedetto Marelli, and four others from MIT.

PFAS chemicals are present in a wide range of products, including cosmetics, food packaging, water-resistant clothing, firefighting foams, and antistick coating for cookware. A recent study identified 57,000 sites contaminated by these chemicals in the U.S. alone. The U.S. Environmental Protection Agency has estimated that PFAS remediation will cost $1.5 billion per year, in order to meet new regulations that call for limiting the compound to less than 7 parts per trillion in drinking water.

Contamination by PFAS and similar compounds “is actually a very big deal, and current solutions may only partially resolve this problem very efficiently or economically,” Zhang says. “That’s why we came up with this protein and cellulose-based, fully natural solution,” he says.

“We came to the project by chance,” Marelli notes. The initial technology that made the filtration material possible was developed by his group for a completely unrelated purpose — as a way to make a labelling system to counter the spread of counterfeit seeds, which are often of inferior quality. His team devised a way of processing silk proteins into uniform nanoscale crystals, or “nanofibrils,” through an environmentally benign, water-based drop-casting method at room temperature.

Zhang suggested that their new nanofibrillar material might be effective at filtering contaminants, but initial attempts with the silk nanofibrils alone didn’t work. The team decided to try adding another material: cellulose, which is abundantly available and can be obtained from agricultural wood pulp waste. The researchers used a self-assembly method in which the silk fibroin protein is suspended in water and then templated into nanofibrils by inserting “seeds” of cellulose nanocrystals. This causes the previously disordered silk molecules to line up together along the seeds, forming the basis of a hybrid material with distinct new properties.

By integrating cellulose into the silk-based fibrils that could be formed into a thin membrane, and then tuning the electrical charge of the cellulose, the researchers produced a material that was highly effective at removing contaminants in lab tests.

The electrical charge of the cellulose, they found, also gave it strong antimicrobial properties. This is a significant advantage, since one of the primary causes of failure in filtration membranes is fouling by bacteria and fungi. The antimicrobial properties of this material should greatly reduce that fouling issue, the researchers say.

“These materials can really compete with the current standard materials in water filtration when it comes to extracting metal ions and these emerging contaminants, and they can also outperform some of them currently,” Marelli says. In lab tests, the materials were able to extract orders of magnitude more of the contaminants from water than the currently used standard materials, activated carbon or granular activated carbon.

While the new work serves as a proof of principle, Marelli says, the team plans to continue working on improving the material, especially in terms of durability and availability of source materials. While the silk proteins used can be available as a byproduct of the silk textile industry, if this material were to be scaled up to address the global needs for water filtration, the supply might be insufficient. Also, alternative protein materials may turn out to perform the same function at lower cost.

Initially, the material would likely be used as a point-of-use filter, something that could be attached to a kitchen faucet, Zhang says. Eventually, it could be scaled up to provide filtration for municipal water supplies, but only after testing demonstrates that this would not pose any risk of introducing any contamination into the water supply. But one big advantage of the material, he says, is that both the silk and the cellulose constituents are considered food-grade substances, so any contamination is unlikely.

“Most of the normal materials available today are focusing on one class of contaminants or solving single problems,” Zhang says. “I think we are among the first to address all of these simultaneously.”

The research team included MIT postdocs Hui Sun and Meng Li, graduate student Maxwell Kalinowski, and recent graduate Yunteng Cao PhD ’22, now a postdoc at Yale. The work was supported by the Office of Naval Research, the National Science Foundation, and the Singapore-MIT Alliance for Research and Technology.



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‘Some pterosaurs would flap, others would soar’ — new study further confirms the flight capability of these giants of the skies

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Some species of pterosaurs flew by flapping their wings while others soared like vultures, demonstrates a new study published in the peer-reviewed Journal of Vertebrate Paleontology.

It has long been debated whether the largest pterosaurs could fly at all.

However, “remarkable” and “rare” three-dimensional fossils of two different large-bodied azhdarchoid pterosaur species — including one new-to-science — have enabled scientists to hypothesize that not only could the largest pterosaurs take to the air, but their flight styles could differ too.

The new findings are led by experts from the University of Michigan, in the US, the Natural Resources Authority and Yarmouk University, in Jordan, and the Saudi Geological Survey, in Saudi Arabia.

Their paper details how these fossils — which date back to the latest Cretaceous period (approximately 72 to 66 million years ago) — were remarkably three-dimensionally preserved within the two different sites that preserve a nearshore environment on the margin of Afro-Arabia, an ancient landmass that included both Africa and the Arabian Peninsula. The research team used high-resolution computed tomography (CT) scans to then analyze the internal structure of the wing bones.

“The dig team was extremely surprised to find three-dimensionally preserved pterosaur bones, this is a very rare occurrence,” explains lead author Dr Kierstin Rosenbach, from the Department of Earth and Environmental Sciences of the University of Michigan.

“Since pterosaur bones are hollow, they are very fragile and are more likely to be found flattened like a pancake, if they are preserved at all.

“With 3D preservation being so rare, we do not have a lot of information about what pterosaur bones look like on the inside, so I wanted to CT scan them.

“It was entirely possible that nothing was preserved inside, or that CT scanners were not sensitive enough to differentiate fossil bone tissue from the surrounding matrix.”

Luckily, though, what the team uncovered was “remarkable,” via “exciting internal structures not only preserved, but visible in the CT scanner.”

CT scans reveal one soars; one flaps!

Newly collected specimens of the already-known giant pterosaur, Arambourgiania philadelphiae, confirm its 10-meter wingspan and provide the first details of its bone structure. CT images revealed that the interior of its humerus, which is hollow, contains a series of ridges that spiral up and down the bone.

This resembles structures in the interior of wing bones of vultures. The spiral ridges are hypothesized to resist the torsional loadings associated with soaring (sustained powered flight that requires launch and maintenance flapping).

The other specimen analyzed was the new-to-science Inabtanin alarabia, which had a five-meter wingspan. The team named it after the place where it was excavated — near a large grape-colored hill, called Tal Inab. The generic name combines the Arabic words “inab,” for grape, and “tanin” for dragon. ‘Alarabia’ refers to the Arabian Peninsula.

Inabtanin is one of the most complete pterosaurs ever recovered from Afro-Arabia, and the CT scans revealed the structure of its flight bones was completely different from that of Arambourgiania.

The interior of the flight bones were crisscrossed by arrangement with struts that match those found in the wing bones of modern flapping birds.

This indicates it was adapted to resist bending loads associated with flapping flight, and so it is likely that Inabtanin flew this way — although this does not preclude occasional use of other flight styles too.

“The struts found in Inabtanin were cool to see, though not unusual,” says Dr Rosenbach.

“The ridges in Arambourgiania were completely unexpected, we weren’t sure what we were seeing at first!

“Being able to see the full 3D model of Arambourgiania’s humerus lined with helical ridges was just so exciting.”

What explains this difference?

The discovery of diverse flight styles in differently-sized pterosaurs is “exciting,” the experts state, because it opens a window into how these animals lived. It also poses interesting questions, like to what extent flight style is correlated with body size and which flight style is more common among pterosaurs.

“There is such limited information on the internal bone structure of pterosaurs across time, it is difficult to say with certainty which flight style came first,” Dr Rosenbach adds.

“If we look to other flying vertebrate groups, birds and bats, we can see that flapping is by far the most common flight behavior.

“Even birds that soar or glide require some flapping to get in the air and maintain flight.

“This leads me to believe that flapping flight is the default condition, and that the behavior of soaring would perhaps evolve later if it were advantageous for the pterosaur population in a specific environment; in this case the open ocean.”

Co-author Professor Jeff Wilson Mantilla, Curator at Michigan’s Museum of Paleontology, and Dr Iyad Zalmout, from the Saudi Geological Survey, found these specimens in 2007 at sites in the north and south of Jordan.

Professor Jeff Wilson Mantilla says the “variations likely reflect responses to mechanical forces applied on the pterosaurs’ wings during flight.”

Enabling further study of vertebrate flight

Concluding, Dr Rosenbach states: “Pterosaurs were the earliest and largest vertebrates to evolve powered flight, but they are the only major volant group that has gone extinct.

“Attempts to-date to understand their flight mechanics have relied on aerodynamic principles and analogy with extant birds and bats.

“This study provides a framework for further investigation of the correlation between internal bone structure and flight capacity and behavior, and will hopefully lead to broader sampling of flight bone structure in pterosaur specimens.”



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