DRIVING HIS red, decrepit, Ford pick-up truck on a narrow country road that winds through endless farms, rivers, and villages, Dr. Kerney finally took Liz and I to our familiar-enough destination, Michaux State Forest, on a chilly spring day. Here, thirteen miles west of Gettysburg College, is where we were authorized to collect spotted salamander embryos under our 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, chances are you could easily encounter spotted salamander embryos in a local pond after the first warm rain in the spring.
These beautiful tiny salamander eggs, which look like marble beads sprayed with green glitters, are each surrounded by an egg capsule. Coating these eggs 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 Dr. Ryan Kerney, biology professor at Gettysburg and my research advisor who is 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 was 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 apparently offers nitrogen-rich waste products to the algae as nutrients. According to Dr. John A. Burns, postdoctoral researcher in the Division of Invertebrate Zoology at American Museum of Natural History and our research collaborator, 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 John.
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? The “salamander guy” decided 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
When Dr. Kerney observed the salamander embryos under fluorescent microscope, he found something very exciting. “ 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,” Dr. Kerney says, “and it was totally surprising to see there is algal cells embedded inside the embryo itself.”
To further investigate what is happening between the salamander embryo and algae, Dr. Kerney took a step further by observing the specimen under a transmission electron microscope—microscope that have high enough resolution and can magnify objects for 10,000,000 times. Seeing the image from the electron microscope, Dr. Kerney realized he had 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 were 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 our collaborator Eunsoo Kim, who is the assistant curator of microbial diversity and systematics in the Division of Invertebrate Zoology in American Natural History.
So far, our research team has compared mRNA (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. The goal for this RNA comparison was to discover the differences of gene expression for both algal and salamander cells when they have come together.
Things started to become clearer with more and more investigations being done for this unique endosymbiosis relationship. We have discovered that several genes in both algae and salamander have changed their expressions to adapt to their host or symbiont. 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
“We are learning that the algal cells and salamander cells are dramatically changing each other to adopt each other,” Eunsoo says, “This change may be relevant for other symbiotic systems including human and parasitic bacteria relationships.” In fact, we, the humans, also have trillions of microorganisms living in or on or bodies. Therefore, our project is also hoping to shed light on the secret of how microbes can interact with vertebrates—like us—and affect our physiology.
One afternoon in the forest, we have collected enough embryos for the next round of experiment. Though exhausted, I am very proud of myself for helping propel science by dressing in the waders and trekking through the cold, muddy ponds to collect the embryos, I told myself. Here we are again—in Dr. Kerney’s red, decrepit, Ford pick-up truck—heading back to the campus. Awaits us are more excitements and more unknowns to unveil!