Ryan Kerney on discovery of algae inside salamander embryos

Ryan Kerney’s team of biologists has found green algae living inside salamander embryo cells, the first cellular-level symbiosis ever seen in a vertebrate.

For more than 100 years, scientists have known about a close association between spotted salamander eggs and green algae. Now a recent study has revealed surprising new details about this relationship. Algae were not just growing around the eggs. They were also found inside the salamander embryo capsules, growing between embryo tissue cells, and even inside the embryo tissue cells. This remarkable discovery came from a team of scientists led by Ryan Kerney of Dalhousie University in Halifax, Nova Scotia.

EarthSky asked Kerney how algae finds its way to the salamander egg masses. Is that algae species abundant in the salamander’s habitat, or do the adult spotted salamanders transfer it to the embryos? He suspects it’s a bit of both, that the algae is obtained from the environment and passed from breeding adults to the embryos. He said:

We have amplified an algal gene from the reproductive tracts of some adult salamanders, suggesting that they may be carrying the algae from one generation to the next.

Adult spotted salamander. Image Credit: Ryan Kerney

Spotted salamanders, that bear the taxonomic name Ambystoma maculatum, are found in hardwood forests throughout eastern North America. These amphibians spend much of their lives underground, living in moist areas under logs and stones, in leaf litter, or burrowed in the ground. In spring, they gather in ponds to breed, depositing fertilized eggs in the water where the embryos develop in the eggs over one to two months. They hatch as salamander larvae with external gills. Over the next two to four months, the larvae gradually metamorphosize – losing their gills in the process – into juvenile salamanders that leave the water for a life on land.

Ryan Kerney. Find his homepage here: https://web.mac.com/ryankerney/Site/Welcome.html

Symbiotic associations at the cellular level, where one organism lives inside the cells of another in a mutually beneficial relationship, is called endosymbiosis. It was thought that vertebrate immune systems would not tolerate a foreign organism as a symbiont – that is, until the surprising discovery made by Kerney’s team.

The algae species that’s been long associated with spotted salamander eggs in the wild is called Oophila amblystomatis. Its genus name, Oophila, is Latin for “loves eggs.” The average algal cell is about 5 microns (0.005 mm or 0.0002 inches) in diameter, while the salamander cells are roughly 50 to 100 microns (0.05 to 0.1 mm, or 0.002 to 0.004 inches) in diameter.

Salamander embryos growing inside egg capsules covered with and often infiltrated by Oophila algae. Image Credit: Roger Hangarter.

The association between Oophila algae and spotted salamander eggs has been known for some 120 years. In the 1980s, laboratory experiments revealed more about the intriguing relationship between the two species. Oophila algae were found to grow more robustly in water that had been exposed to salamander embryos. When grown with Oophila algae, salamander embryos were healthier, developed faster, and suffered lower mortality rates compared to those grown without the algae. But it was difficult to see algae in embryo cells using conventional microscopes, so the true extent of the algae-salamander embryo relationship remained hidden until new microscopy techniques were used to look at the cells in a different way.

As in plants, green algae need chlorophyll for photosynthesis. Chlorophyll, the green energy-producing component in chloroplasts, has a property known as fluorescence. When electrons in chloroplasts are exposed to light, they temporarily gain a higher energy state, then fall back to a lower energy state while emitting light (the fluorescence effect). If there’s chlorophyll in a cell, it can be detected using a fluorescence microscope.

Black-and-white image of a salamander embryo head, with an overlay showing red spots that are fluorescing chloroplasts from the algae. Image Credit: Ryan Kerney.

Kerney told EarthSky how he found the algae in the salamander embryos, first using fluorescence microscopy, then with an electron microscope to see inside the salamander embryo cells.

The fluorescent microscopy revealed that the algae are within the embryonic tissues. We used transmission electron microscopy (TEM) to determine that the algae were also inside the salamander cells (a big surprise). Some of the intracellular algae eventually degrade, while others appear to be forming cysts.  There is no sign of the cysts harming the embryo. We have not seen any of these cysts degrade inside the embryonic tissues.

Electron microscope image of an algal cell inside the cell of a salamander embryo. Image Credit: Ryan Kerney.

Kerney also verified the identity of the algae inside the embryos and egg capsules. Like all cells, Oophila algae has a molecule called ribosomal RNA (rRNA) that uses amino acids to form proteins. Its rRNA has a unique form that can be identified with a chemical marker – a short string of nucleic acids that targets and binds to it, thus making it detectable. This analysis confirmed that Oophila amblystomatis is indeed the algal species inside the embryo cells.

A spotted salamander embryo. Image Credit: Roger Hangarter.

How did Oophila algae infiltrate the spotted salamander embryos? Could it be that their immune system wasn’t mature enough to recognize the intruder? Said Kerney:

That may be how the intracellular symbiosis is established. Jawed vertebrates have an adaptive immune system, which is programmed during their lifespan. This immune system is what differentiates “self” from “non-self.” The initial algal invasion precedes the maturation of the adaptive immune system in our salamanders.

Is the algae present at all stages of the embryo growth? Does this endosymbiotic relationship continue in adult salamanders?

There have been earlier studies that show nitrogenous wastes released by the embryos are stored by the algae as proteins. The combination of carbon dioxide, nitrogenous wastes, and protected habitat (the egg capsule) all appear to benefit the algae.

We first see the algae bloom outside a hole in the embryo called the blastopore [an opening to the embryo’s primitive gut]. This occurs during a period when the central nervous system has just begun to form. After the bloom, the algae penetrate the salamander’s tissues and cells. The bloom coincides with a period of increased nitrogenous waste within the egg capsule. We hypothesize that this nitrogenous waste is the cue for the bloom. Algae invasion occurs after the bloom.

We don’t really know the extent to which [algae] may persist into the adult [cells]. We have some evidence from amplifying algal genes from adult tissues, which indicates that the algae can persist into the adult stages.

A movie of time-lapse images (below), created by Roger Hangarter at Indiana University in Bloomington, was taken over a 16.6 hour period, with one image every minute. It shows the early stages of spotted salamander embryo development, as the algae first becomes established in it.  Each flash represents an algae bloom, when algae growth increases significantly as it comes in contact with nitrogen waste in the embryo capsule.

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What, then, does the salamander embryo get in return for hosting the Oophila algae? Oxygen. Salamander eggs have a jelly coating that prevents the eggs from drying, making it difficult for oxygen in the water to diffuse into the embryo capsule. Photosynthesis by Oophila algae provides the growing embryo with oxygen.

Could other endosymbiotic relationships, like that found between Oophila algae and salamander embryos, exist in other vertebrates? Kerney told EarthSky that his team will continue studying spotted salamanders, and plan to extend their investigations to Northwestern salamanders.

Ryan Kerney of Dalhousie University and his team reported on their discovery of algae inside salamander embryo capsules, growing between embryo tissue cells, and even inside the embryo tissue cells in the Proceedings of the National Academy of Sciences.

Adult spotted salamander in leaf litter. Image Credit: Roger Hangarter.

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