Island Fox Microbiome at the Smithsonian!

FIF's 2020 Research Grant recipient Dr. Alexandra DeCandia recently spoke about her work investigating the island fox microbiome. 

Watch the video of the program

Here's a behind the scenes look at her process extracting the DNA of different microbes from the island fox swab samples.

Dr. Allie reports: 

I inventoried over 800 swabs and over 300 blood samples collected from Channel Island foxes and island spotted skunks in 2020. I then extracted DNA and prepared samples for microbiome sequencing (a process called "library preparation"). The last step of the library preparation is cleaning the library–in essence, removing all of the DNA fragments that I don't want to ultimately sequence.

Although it may seem like a straightforward process, this took a bit of troubleshooting. After a few failed attempts, I worked with the lab manager at the Smithsonian National Zoo's Center for Conservation Genomics to learn how to run an "eGel". In this approach, you put your library in an agarose gel (which looks like a think rectangle of gelatin), run an electrical current through the gel to separate DNA fragments based on size...

(under UV light, the fragments look like glowing bands of the gel), and then pull out the band you want to sequence (in my case, fragments roughly 400 base pairs in length).

Then the DNA had to be treated to remove "gel particles and other potential contaminants."

Overall, this was a nerve-wracking process, but thankfully it worked!

Dr. Allie has submitted all seven sequencing libraries to the Princeton University Genomics Core Facility for sequencing. She reported on some preliminary findings during her "Date with a Fox" presentation. This is Fox Science at work! 

Your donations support this cutting-edge science that will provide important information regarding island fox health.  

Swab collection continues.

What is a microbiome? and Why is this important for island foxes?

Fox Foto Friday - Island Fox Science in Progress

What are these? Look closely. These are swabs from island fox ears and backsides being processed in the lab at the Smithsonian Conservation Biology Institute in Washington D.C.

This is island fox science in progress! FIF 2020 Research Grant recipient Dr. Allie DeCandia updated us on her work investigating the microbiome of island foxes.

"Between July and December 2020, collaborators across all six Channel Islands collected ear canal swabs, anal swabs, and blood samples from 50–60 foxes per subspecies. After collection in the field, these samples were shipped to Princeton University for temporary storage until all samples arrived on the east coast." In March, DeCandia moved the samples to her lab at the Smithsonian. She spent April and May inventorying samples.

"My collaborators did a fantastic job sampling foxes!" DeCandia says. "In total, I inventoried 851 swabs over the course of 23 hyper-focused laboratory hours (which were thankfully not consecutive)."

Processing the samples must be meticulous because she is investigating DNA.

"I sterilized scissors (bleach dunk / water dunk / superheat in a Bacinerator), snipped off the swab tip, placed the swab tip in a sterile microcentrifuge tube, recorded metadata written on the original swab container, and repeated the process for each swab collected."

The coordinating 300+ blood samples are now being inventoried and the individual island fox DNA extracted. DeCandia will spend the summer extracting microbial DNA from the swabs and preparing samples for "microbiome sequencing." Over the fall/winter, she'll be deep in analyzing the data.

"I am ecstatic to be working with so many amazing researchers in the field and in the lab," DeCandia says, "and can't wait to analyze this impressive dataset in the months ahead!"

FIF grant funds are processing the DNA extraction. 

Your donations support this cutting edge research.

If an island fox is an environment for microbes, what was lost when island fox populations declined to just a few individuals? How is an entire species' health impacted if they recover from near extinction, but have lost some of the microbial biodiversity that protected them from bacteria infection or supported healthy digestion? 

Island fox microbiome investigation will help us understand island fox health and may have implications for other endangered species.

Your donations supporting research are an investment in the island fox's long-term survival.

Friends of the Island Fox is taking applications

through August 31 for our 

2021 Research Grant  (see application)

Fox Foto Friday - Island Foxes in Washington D.C.?

What smells like an island fox? Came from an island fox? But is contained on the tip of a cotton swab? A sample of island fox microbiome.

Island fox being swab with cotton swab

Friends of the Island Fox recently heard from FIF 2020 Research Grant recipient, Alexandria DeCandia, Ph.D.

DeCandia wrote "I wanted to let you know that microbiome samples from all six islands have arrived on the east coast :)" Swabs taken from island foxes during health checks have been transferred to a lab in Washington D.C. She added, "I'm very much looking forward to the next steps of this research!"

FIF's Research Grant is funding the DNA analysis of the swabs to get a picture of the biodiversity of microbes living on and in island foxes.

What Is She Looking For?

DeCandia's research is cutting edge science. Her early analysis of Catalina Island foxes led to some important findings on the possible relationships between bacteria in island fox ear canals and a tendency toward extreme immune system response to ear mites. This next stage of research builds on that work.

Maintaining healthy island foxes across the islands requires greater in depth understanding of their health, diet, position in the ecosystem, and interactions with other species, including humans. 

Friends of the Island Fox invests in research to invest in the island fox's future. Applications for the FIF 2021 Research Grant will be available April 15, 2021.

FIF Research Grant to Investigate Diversity of Island Fox Microbiome

Friends of the Island Fox is happy to announce Alexandra DeCandia, Ph.D. is the recipient of FIF's 2020 Research Grant.

You may remember DeCandia's article from April 2020 regarding her doctoral work at Princeton University: Mites, Microbes, and Cancer in Santa Catalina Island Foxes. Microbes can be found on the skin, in the digestive system, and in connection with the body's openings.

A healthy animal has a diversity of microbes. In this way, a single island fox is like an island. If something should happen to the biodiversity of microbes on an individual, the ecosystem on that individual might become out of balance. Some microbes might thrive, while others perish. An imbalance of microbes can impact an animal's overall health.

When island foxes on a specific island go through a near-extinction population bottleneck, there is a potential for a loss of microbe diversity that can be passed on to surviving island foxes. 

DeCandia investigated if there was a connection between the diversity of microbes on Catalina Island foxes and an unusual prevalence of cancer in this subspecies.

Looking into island fox ear canal

What she found was "evidence of disrupted microbial communities in mite-infected ear canals that may contribute to sustained inflammation." Inflammation can play a role in cancer and this microbial imbalance may be connected to why Santa Catalina Island foxes are the only island foxes known to develop cancerous tumors in their ear canals.

DeCandia's work was published in Molecular Ecology and when she presented her findings at the Annual Island Fox Conservation Working Group Meeting in May of this year, everyone was intrigued. A healthy microbial biome is vital to healthy digestion, immune response to disease, behavior, and even development. Because island foxes on five islands have been through population bottlenecks, where the number of surviving individuals was very low, there is a potential that island foxes on other islands may have disrupted microbial communities as well.

As island foxes are counted and given health checks across the islands this year, they are also getting swabbed for microbes in their ear canals and at their anuses. DeCandia describes the process as "similar to cleaning your ears with a cotton swab, except you don't throw away the swab afterwards." The swab samples will be sent to DeCandia at the Smithsonian Conservation Biology Institute in Washington D.C.

 

 

DeCandia in the lab

FIF Research Grant funding will be used to extract DNA samples and process DNA sequencing to identify the various microbes in island fox ear canals and digestive systems. 

This investigation provides a unique opportunity to do comparative analyses between subspecies of island foxes on different islands. DeCandia hopes to:

1. survey variation of microbes within island foxes on each island
2. characterize the differences between islands
3. identify the drivers of ear canal tumors on Santa Catalina Island
   

This work is at the cutting edge of science and may have important consequences for the long term survival of the island fox.

If you are an FIF donor, 

you are making this important work possible.

 

If you haven't donated yet, Please Donate 

This is Science, For Fox Sake!

 

Mites, Microbes, and Cancer in Santa Catalina Island Foxes by Alexandra DeCandia

(Thank you to our guest blogger Alexandra DeCandia a doctoral candidate at Princeton University)

Over the last few decades, we've realized that organisms are far more complicated than they initially appear. What may look like an individual fox is actually an ecosystem containing trillions of microorganisms on every square inch. [Figure 1]

Despite their tiny size, microbes influence important host functions, such as development, digestion, stress tolerance, behavior, and even immunity. Therefore learning more about these hidden actors can inform wildlife conservation of at-risk species in the modern molecular era.

Looking into the ear canal of an island fox.

Microbes may be particularly important to species that lack genetic diversity, such as Channel Island foxes, especially where disease threatens long-term persistence. On Santa Catalina Island, scientists discovered extremely high rates of ear canal tumors, where roughly half of adult foxes have growths in their ears. Although the exact cause is unknown, researchers linked ear mite infection to tumor growth and development. The most prominent hypothesis states that infection with ear mites leads to inflammation and rampant cell growth in the ear canal, which in turn leads to tumors. Thankfully, treating foxes with the acaricide Ivermectin has already decreased mite burdens and tumor rates in these foxes.

However, there's more to this story. We still don't fully understand how mite infection leads to tumor growth. In particular, my collaborators and I wondered whether microbes play a role in this system. For example, do mites disrupt healthy microbes and cause secondary bacterial infections? And do those infections then contribute to the chronic inflammation that precedes tumor growth?

Figure 2: Island fox is swabbed during health check

To address these questions, my collaborators at the Catalina Island Conservancy collected microbe samples by swabbing ear canals (and a few other body sites) of healthy and mite-infected foxes. [Figure 2] (This process is similar to cleaning your ears with a cotton swab, except you don't throw away the swab afterwards.) Once a bunch of foxes were swabbed, all samples were sent to New Jersey, where I extracted DNA, collected genetic sequences, and analyzed the data.

 The results came back loud and clear: microbes differed between mite-infected and uninfected ear canals. Rather than a rich community of diverse microbes (as seen in healthy ears), mite-infected ear canals had fewer microbial species present. We further found that the balance of microbes (know as "relative abundance") differed between infection groups.  [Figure 3]

Figure 3: Classes of bacteria found in swab samples

As it turned out, this pattern was almost entirely driven by an overabundance of one bacterial species: Staphylococcus pseudintermedius (Class: Bacilli, shown in brown in Figure 3).

Even though this microbe is commonly found on canid species (such as domestic dogs and foxes), it can become an opportunistic pathogen when healthy communities are disrupted. Once it proliferates, it can be incredibly difficult for the immune system or even antibiotics to eradicate, leading to chronic inflammation.

We now hypothesize that mite infection and secondary bacterial infection with Staphylococcus pseudintermedius contribute to chronic inflammation and tumor growth in Santa Catalina Island foxes. 

Photo courtesy of Glenn Jensen

Although further tests are needed to definitively establish causation, these insights into the microbial dynamics of mite infection can help us monitor the population for antibiotic resistant forms of Staphylococcus pseudintermedius that could cause a disease outbreak. They can further help us explore other open questions, such as why Santa Catalina Island foxes are the only subspecies with ear canal tumors, despite ear mites on other islands. As always in science, answers lead to more questions. But at least one thing is clear: there's more to this story (and indeed, to all organisms) than what initially meets the eye.  

Alexandra DeCandia, Department of Ecology & Evolutionary Biology, Princeton University, Princeton, New Jersey.

Read the full paper: Ear mite infection is associated with altered microbial communities in genetically depauperate Santa Catalina Island foxes (Urocyon littoralis catalinae)

More Research Regarding Island Foxes:

Origins of the Island Fox

New Findings on Island Fox Diet

Epidemic Disease Threats to the Island Fox

More