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Look for four new faces in the Islands and Lakes gallery: a quartet of western pond turtles. These endangered natives of Washington state are ambassadors for a long-term collaborative conservation project, part of it being carried out in Shedd’s microbiome lab. In addition to rescuing and restoring a species, the project has spurred innovations in how hatchling turtles are raised for release.

A turtle swims up toward the surface

The western pond turtle, Actinemys marmorata, is a focus species in the Association of Zoos and Aquariums’ Saving Animals From Extinction (SAFE) initiative. AZA’s action plan for the species complements a long-term recovery program established and led by the Washington Department of Fish and Wildlife, Seattle’s Woodland Park Zoo, the Oregon Department of Fish and Wildlife and the Oregon Zoo.

Once abundant in Pacific Coast states, the Washington population bottomed out at about 150 animals in 1991, decimated by overhunting, habitat loss and predation by introduced animals. Washington state listed the species as endangered in 1993. Through a head-start program, in which zoo-reared yearlings are released into the wild, the turtles’ numbers have gradually grown to more than 1,000 thriving at six sites. Without this long-term collaborative effort, western pond turtles would most likely be extinct in Washington state today.

A few years ago, biologists spotted mysterious lesions on the plastrons, or bottom shells, of some of the adult released head-started turtles. The fungal-associated infection, which can eat through the bony shell, threatened the pond turtles in a new, potentially lethal way. To combat this, part of AZA’s conservation action plan for western pond turtles included the Unidentified Shell Disease Project. Shedd, with its microbiome lab, came on as a partner.

Two experts in the field look at a turtle's shell, held between them

At a monitoring site in Washington state, Dr. Matt O’Connor, senior staff veterinarian, examines the plastron of an adult western pond turtle with a biologist from the Washington Department of Fish and Wildlife.

Dr. Matt O'Connor holds a turtle upside-down for a shell swabbing

At the same site, Chrissy Cabay, who oversees Shedd’s microbiome laboratory, takes a cloacal swab from an adult western pond turtle held by Dr. Matt.

Teamwork

Joining project partners at western pond turtle habitats in Washington and Oregon, Chrissy Cabay, Shedd’s scientific research director, and Dr. Matt O’Connor, senior staff veterinarian, collected water and other environmental samples and took shell and cloacal (tail) swabs from adult turtles. Back at Shedd, DNA analysis of the samples provided a baseline for the microbiomes of the turtles and their environments.

Then, thanks to a grant for a graduate partnership with the University of Illinois, in January 2018 Shedd brought on Monique Hazemi, a master’s student in microbial ecology at the Urbana-Champaign campus, to begin a two-year study to answer these questions:

  • Do the turtle-associated and/or habitat microbiomes differ between head-started animals and wild ones?
  • Can the head-starting environment and turtle microbiomes be altered to resemble the natural environment?
  • And if so, what effect does that have on the suspected shell disease fungus?
Monique Hazemi holds up two turtles for a photo in the field

Researcher Monique Hazemi holds two adult head-started western pond turtles at a study site in Washington state.

Photo by: © Angela Kent, courtesy Monique Hazemi

A thousand samples

To answer those questions, Monique needed more samples—about 1,000 total—from the turtles and from three distinct rearing environments.

(While Monique was pursuing her field and lab work, project partners at the University of Illinois College of Veterinary Medicine named the fungus, identifying it as a new genus and species, Emydomyces testavorans, literally “shell-devouring freshwater turtle fungus.”)

At natural woodland sites in Washington and Oregon, Monique collected pond water, biofilm—slimy clusters of microbial communities growing on rocks, logs and other surfaces—and nesting material. She also took shell and cloacal swabs of free-ranging wild turtles in Oregon, released head-started adults in Washington and hatchlings in the zoo head-start programs.

At both zoos, the hatchlings were divided between two rearing “treatments.” The first was the traditional intensively managed “superhygienic” method, with regular water changes and disinfection with antimicrobials to kill bacteria and other microbes that could harm the turtles. With recent discoveries about the importance of microbiomes, biologists wondered if what they used to kill bad microbes might also be killing good microbes needed to prime the turtles’ immune systems and help them resist infections. The turtles in this treatment were the control group.

The second treatment was less managed. Instead of sanitation with antimicrobials, environmental quality was maintained by recirculating water through a biofiltration system containing a community of beneficial microbes to process waste products. Both treatments were set up inside zoo buildings.

A researcher holds a small turtle in a blue-gloved hand

One of the head-started western pond turtle hatchlings

Photo by: © Angela Kent, courtesy Monique Hazemi

Back in Shedd’s microbiome lab, Monique extracted, sequenced and analyzed the DNA from the samples. She explained, “The purpose of all that is to get an idea of which microbes are present in each sample and how the microbial communities are structured—for example, how similar are the highly managed microbiomes to the less intensively managed microbiomes, and how similar are the two head-starting environments to the wild.”

For the most part, she discovered, not very similar. Habitats from each head-starting treatment had distinct bacterial and fungal communities compared with the natural sites. The juvenile head-started turtles also had distinct bacterial and fungal communities from their counterparts in the wild environments. But between hatchlings in the two treatments, over the time period when samples were collected, those raised in less intensively managed biofilter habitats seemed to be exposed to a fungal microbiome that more closely resembled the diversity and structure of that in the natural environment.

Fungus found

One of the most noteworthy results was that in the highly managed head-starting environment, more than 60 percent of the fungal population in the habitat water and nearly 100 percent of the fungal population on the turtles’ plastrons was E. testavorans—the suspected shell-eating pathogen. By comparison, E. testavorans represented less than 1 percent of the fungal populations present in both the water and on the plastrons of the turtles in the biofilter head-starting environment. This mirrored levels observed in the natural environment as well as on wild-reared and disease-free turtles.

While Monique’s research showed the relative abundance of E. tesetavorans within the natural and the head-starting environments, it did not investigate where or when the turtles get colonized by the fungus. (Other collaborators on the project are tracking where the pathogen is coming from.) And while the fungus is present on the turtles that have the shell disease, researchers still have not determined if it actually causes the disease.

Three small turtles sit on a rubber mat in a rub filled with water

Head-start program turtles bask in an indoor habitat at Seattle’s Woodland Park Zoo.

Photo by: © Monique Hazemi

Early results

Adult turtles sampled in the wild that did show symptoms associated with the shell disease were almost exclusively from the head-started population. Monique said, “Our theory is that the traditional intensive management practices used in the head-starting envrionment prevent turtles from developing as diverse microbiomes as those turtles in a natural setting.”

Based on the preliminary findings, Woodland Park Zoo and Oregon Zoo switched exclusively to biofiltration systems on their western pond turtle head-starting habitats. Washington Department of Fish and Wildlife biologists note that continued data collection is needed to confirm that biofiltration systems will reduce the prevalence of E. testavorans on turtles long term.

It’s also the practice at both zoos to put all the turtles in outdoor pools a few weeks before their release to help them acclimate to real-life weather conditions. This has the secondary benefit of exposing them to the ambient microbiome, which could help support the health of the turtles’ microbiomes and, by extension, their overall health.

A turtle walks towards the camera

At Shedd

A total of eight western pond turtles reside at Shedd. The siblings, hatched in 2016 for the head-start program, were made available to the aquarium because they have a genetic defect—a slight indentation on the edge of the carapace, or top shell—that disqualified them for reintroduction.

Aside from that, Chrissy Cabay, Shedd’s scientific research director, said, “They were all clinically normal when they arrived. But since then, some have developed the fungal disease and are undergoing treatment.” All were initially raised in the traditional highly managed setup. Chrissy continued, “Short-term data from Monique’s research show that the biofilter head-starting environment is most effective if done from day one.”

As the turtles grew—and outgrew their original accommodations in the animal healthcare center—four were moved to a large habitat in Islands and Lakes. Chrissy said the turtles will probably rotate between the gallery habitat and the display habitat outside the animal hospital—where they are a highlight of behind-the-scenes tours. “They are great ambassadors for the work we do in the microbiome lab,” Chrissy said. All eight continue to be a source of data for the shell disease project.

A western pond turtle stretches its neck and stares at the camera
A turtle stretches its neck comically long

Research to reinforce recovery

Monique said, “It will be important to continue following the microbiomes of head-started hatchlings sampled during this study into the future, especially until they are old enough to potentially start showing disease symptoms.” The mobilization by program partners, original and new, to identify and combat the fungal infection will make this recovery effort even stronger and perhaps benefit other head-starting efforts.

Head-starting is the keystone to western pond turtles’ continued survival. It’s a tool biologists need until they can reduce mortality from other threats and increase survivorship at all life stages in the wild—almost always a long-term proposition, and especially so with slow-to-mature reptiles. The young turtles, smaller than a nickel when they hatch, are extremely vulnerable to predators such as the non-native American bullfrog, the largest frog species on the continent, which preys on them right out of the nest.

Monique said, “The goal of the head-starting program is to enhance survival and population recruitment by rearing hatchlings to a size large enough to avoid predators. In a nutshell, they are about 1-year-old turtles that are the size of 3-year-old turtles.”

She continued, “With head-starting programs, we have been very successful in helping the wild populations recover their numbers. But we can’t always give the young animals exactly what they need if we don’t know what it is that they need.

“Research like the microbiome investigation is how we find out. We try to move one step closer in the direction of caring for them in the best way possible.”

Karen Furnweger, web editor

Funding for the graduate partnership with the University of Illinois came from the Arthur L. and Elaine V. Johnson Foundation.