Driving his decrepit red Ford pick-up truck on a narrow country road that winds through farms, hills, and creeks, Dr. Ryan Kerney finally takes Liz and I to Michaux State Forest, on a chilly spring day. Here, thirteen miles west of Gettysburg, PA, is where we are authorized to collect spotted salamander embryos under a Pennsylvania state permit.

Michaux State Forest, of course, is not the only place to find spotted salamanders embryos. If you live in New Brunswick, Nova Scotia, Ontario, Québec of Canada, or Alabama, Arkansas, Connecticut, Georgia, Illinois, Indiana, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, New Hampshire, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, Vermont, Virginia, West Virginia, Wisconsin of America (It’s a long list…isn’t it?), chances are you could easily encounter spotted salamander embryos in a local pond after the first warm rain in the spring.

Egg mass of spotted salamander: they can easily be found in ponds during the spring across the East Coast of the United States.

Surrounded by their egg capsules, these tiny dark spotted salamander embryos look like marble beads garnished with green glitters. Outside the egg capsules is a thick layer of jelly coat, forming a big egg mass amid the vegetation in water. “Deep inside these egg masses, there are cells that are interacting between the world of algae and the world of salamander,” says my mentor Dr. Kerney, Associate Professor of Biology at Gettysburg College, who is also known as the “salamander guy” for his dedication of salamander studies.

It takes two to tango

The association between spotted salamander embryos and green algae is first discovered by naturalist-scientist Henry Orr in 1888. Orr’s discovery offers a great demonstration of a mutualistic symbiosis—the harmonious living together of two species: algae provides oxygen to salamander host by photosynthesis, while the salamander offers nitrogen-rich waste products to the algae as nutrients. According to our collaborator Dr. John A. Burns, postdoctoral researcher in the Division of Invertebrate Zoology at American Museum of Natural History, the symbiosis between algae and spotted salamander is more than beneficial but rather necessary.

“Salamander embryos tend to be smaller and have a lower chance of survival if the algae stops supplying oxygen by photosynthesis or if the algae is taken away from the salamander,” says Burns.

Spotted salamander embryo observed under fluorescent microscope: right dots shown are the light emitted by algae under fluorescent light.

Questions, however, arise around how these beneficial algae get through the thick jelly surrounding the embryos, and how these algae can break through the egg capsule that further protects the salamander embryos from the environment? To answer these questions, Kerney decides to observe these salamander embryos under a microscope to find some hints. Fortunately, the photosynthetic property of algae offers some convenience to be easily observed—because algae are photosynthetic, if you shine one wavelength of light on them, they will emit another wavelength of light back, making them easily to be observed under a fluorescent microscope (microscope that can emit certain wavelength of fluorescent light to the object).

The High-Five Moment

The high-five moment comes when Kerney observed the salamander embryos under fluorescent microscope.

“ I looked at a later stage of embryo with a fluorescent microscope to see if there is any sign of algae persisting near the time of hatching,” Kerney says, “and it was totally surprising to see there is algal cells embedded inside the embryo itself.”

Transmission electron microscope of a spotted salamander cell containing endosymbiotic algal cells.

To further investigate what is happening between the salamander embryo and algae, Kerney takes a step further by observing the specimen under a transmission electron microscope, a microscope that have high enough resolution and can magnify objects for 10,000,000 times. Seeing the image from the electron microscope, he realizes he have just discovered something totally unexpected and astonishing: the algal cells not only live around salamanders, but also they go inside the individual cells of the salamander embryo. Indeed, this cell living within a cell relationship, which scientists refer to as “endosymbiosis” that occurs between algae and spotted salamander embryo—like a Russian matryoshka doll—is the first known example of a symbiont entering into the host cells of a vertebrate.

A Learning Process

After the exciting moment of revealing the first known example of vertebrate endosymbiosis between algae and spotted salamander, more questions regarding this unique intimate relationships are apparent.

“The question that we are currently tackling is what kind of molecular change is happening when these salamander and green algal cells are together,” says Dr. Eunsoo Kim, who is the assistant curator of microbial diversity and systematics in the Division of Invertebrate Zoology in American Natural History, and our collaborator.

To unveil the molecular changes that may lead to this unique phenomenon, Kerney, Liz and I decide to compared messenger RNA (the middle-step information code between DNA and protein) from four different groups of cells: salamander cells with algae endosymbiont, salamander cells without algae edosymbiont, algal cells live within salamander host, and algal cells outside the salamander hosts. Our goal is to discover the differences of gene expression for both algal and salamander cells when they have come together.

Explanations to this unique endosymbiosis relationship start to emerge with more and more investigations being done. We have recently discovered that both the algae and the salamander have changed their gene expressions (segments of DNA that encodes useful genetic information) in order to adapt to each other. Genes that are responsible for importing inorganic nutrients for algal cells, for instances, have been turned off when algal cells are inside the salamander cells, since inside the salamander cells, there are enough organic nutrients for algae. (Will you still do grocery shopping if there is already enough food for you?)

Uncovering the bigger secrets

Like how spotted salamanders are closely associated with microbes, humans also have trillions of microbes (called microbiome) living on and inside our bodies. Illustration from American Natural History Museum.

“We are learning that the algal cells and salamander cells are dramatically changing each other to adopt each other,” says Kim. “This change may be relevant for other symbiotic systems including human and parasitic bacteria relationships.”

We, as humans, also have trillions of microorganisms living in or on or bodies. Therefore, based on our investigations on spotted salamanders and their algal symbionts, we are also hoping to shed light on the secrets behind how human microbes interact with their human host—us—and alter our physiology.

One afternoon in the forest, dressing in the waders and trekking through the cold, muddy ponds, we have collected enough embryos for the next round of experiment. With exhaustion, I, once again, find myself on Kerney’s decrepit red Ford pick-up truck zig-zagging along the narrow country road that leads us back to town.