As part of my dissertation research, I spent the spring monitoring the development of wood frog eggs. Some of the eggs I brought to the lab, removed from the jelly coat and reared in incubators. I left the rest of the eggs in the ponds and checked on them daily until they hatched. Although not part of my research, I thought it would be interesting to take photos comparing the wild eggs and algae symbionts to the lab-reared eggs without. The relationship between this alga species and only a handful of amphibians is one of the most unique stories in biology. To the best of my ability, I’ve chronicled the Green Egg symbiosis story below.

Green Eggs

Folks have been fascinated by the coincidence of algae and amphibian embryos for a long time. One of the first notes was published in 1888. Orr was studying amphibian skeletal and nervous system development. He needed embryos and had collected egg masses of a few local species. He was surprised to see that the salamander eggs he procured seemed “to present a remarkable case of symbiosis.” He noticed that there were algae cells situated inside of the egg membrane.

This is surprising because that membrane, called the vitelline membrane, is impermeable to everything but ions and small molecules, which certainly excludes entire algae cells. So the only other option is that the algae entered the embryo before the vitelline membrane had formed. But this would be no less surprising because, like basically all animals, the vitelline membrane is formed in the ovaries, and in amphibians, extra layers of jelly are added in the oviducts, long before the egg is exposed to pond water.

In the figure above (from Gilbert 1942) depicting the layers surrounding a salamander egg, it is clear to see that there are many layers acting as barriers between the pond water and the egg.

Which came first–the algae or the egg?

So, Orr was left with the plaguing question: Which came first—the algae or the egg? Perplexed, he wrote, “I have not discovered how the Algae enter the membrane, nor what physiological effect they have on the respiration of the embryo, but it seems probable that in the latter respect they may have an important influence.”

It turns out that Orr was right, algae do have an important influence on the embryos, but it was not demonstrated until the 1940s.  The phenomenon continued to perplex scientists for decades. By the 1940s, algae had been reported in the eggs of multiple salamander species and also wood frogs across the continent from California to New England and Virginia. Gilbert (1942) was the first to explore how the algae came to enter the egg and also studied the importance of the algae-egg relationship.

As to the entrance of the algae into the egg, he first assumed that the algae were present in the ovitract of the female salamander. He washed the oviducts of salamanders and tried to culture the solution, but no algae developed. If algae were not present in the ovitract, then they enter from the pond after the eggs are deposited. To confirm, Gilbert took a recently inseminated female and allowed her to lay eggs in an aquarium with pond water then removed her and allowed her to lay another clutch in an aquarium with algae-free tap water. Algal cells were present in the eggs laid in pond water within hours, but no algae grew within the tap water eggs after a few days. At the end of this experiment, Gilbert put the eggs from the tap water aquarium in a natural pond. Within a few days, algae had penetrated the jelly membrane.

By closely studying the eggs, he found that the algae transformed as they penetrated the egg. When free-swimming in the pond, the algal cells are long and oval with flagella used for locomotion. As the algae penetrate the egg layers, they grow in size, become spherical in shape, and lose their flagella. It is these large, non-mobile cell-types that rest on the inner membrane and create the “verdant blanket” (as Gilbert describes it; 1942, p. 220).

To demonstrate symbiosis, he compared hatching rates in egg masses kept in light and dark to prevent photosynthesis, finding that light and algae increased hatching success. Although algalogistis (yep, that’s a thing) hadn’t officially recognized the alga species, folks who worked with amphibian eggs called it Oophila amblystoma (“Oo” and “phila” come from the Greek words “egg” and “love,” and “amblystoma” is the genus name of the salamander species the algae cohabitates with. So, the name just means “an algae that likes salamander eggs”).

Just a few years ago, researchers took a closer look at the Oophila algae. They wondered if this was really one species or a clade of algal species, and if it the latter, did different algal species associate with specific amphibian species? To answer those questions, they sequenced the genome of algae from four species across the continent.

It turned out that the Oophila are closely related, close enough to be considered a single clade or species, but within the group there are subclades that tend to associate with each host. However, the researchers also found evidence that different subclades of the algae occasionally defect and switch to other host species.

Friends or just roommates?

Since Gilbert’s era, we’ve learned a lot about the economy of the biological transactions between the algae and the embryo. Using microelectrodes inserted within the egg membrane, Bachmann et al. (1986) measured oxygen concentration within the egg in contrast to the outside water in both light and dark. In the light, the algae worked overtime, producing so much oxygen through photosynthesis that the oxygen production exceeded that needed by the embryo and the environment inside of the egg became super-oxygenated, even when the surrounding water was anoxic (oxygen-poor).

This extra photosynthetic boost is more than just a little helpful. Most amphibian embryos get oxygen by diffusion across the egg membrane from surrounding water or air. Rinder and Friet (1994) wondered if diffusion of oxygen from the surrounding water alone could support amphibian species that had evolved with algal symbionts. They measured oxygen gradients across the cell membrane and injected dye in egg masses to see how much water would be able to flow past the eggs. In wood frogs, they found that the egg mass was loose enough and contained enough channels that water diffusion alone could support respiration. However, spotted salamander egg masses are dense and water does not penetrate to the interior eggs. In their case, the innermost eggs would suffocate without their personal algal oxygen factories. (Note: Hutchinson and Hammen (1958) inferred this relationship many decades before through experimentation rather than directly measuring O² within the eggs.)

This plant-animal relationship is a multifaceted exchange. The reciprocity extends past simple O² and CO² cycling. The algae also benefit from the nitrogen waste produced by the embryo, which limits the ammonia buildup in the egg which is toxic to embryos (Goff and Stein 1978).

Friends or more-than-just-friends?

This symbiotic relationship is certainly interesting, but not altogether surprising. After all, lots of animals host symbiotic microbes, even humans house a panoply in our guts and all over our skin (Gilbert et al. 2018). These types of ectosymbionts, commensal organisms that live in and on other organims’ bodies but outside of the tissue are common.

But the green egg story gets even more fascinating. In 2011, Kerney et al. used a technique that binds only to algae DNA and causes it to glow under fluorescent lights (fluorescent in situ hybridization (FISH)). In doing so, they could see algae cells inside(!) of the salamander tissue and cells. This makes this salamander-algae relationship the only example of endosymbiosis in a vertebrate ever seen! Previously, this type of animal-plant union was thought to only be possible in simpler organisms like coral and mollusks.

Friends or frenemies?

So, not only do these free-swimming algae manage to invade the egg membrane, but they even make it all the way inside of the embryonic cells. To some extent, this makes sense for the algae. Why would you want to live out in the pond water with variable temperatures and predators when you could live inside a nice cozy egg with abundant CO2 and nutrients? But once inside the egg, why would algae invade inside an animal cell where there is less access to light for photosynthesis?

A team of researchers (Burns et al. 2017), including Kerney, wanted to figure out the riddle. They isolated and sequenced the genes expressed by salamander cells with and without algae cells from inside and outside of the animal cell. Inside the animal cells, algae seemed to be under more stress, to the point that they switch from photosynthesis to fermentative energy production, something that would normally only happen in highly unfavorable environments. On the other hand, salamander cells with algae were unfazed and even seemed to suppress their immune response which would ordinarily attack foreign cells, perhaps to keep the algae within. For algae, then, there appears to be a risk-benefit balance that drives them inside of the egg where conditions are favorable, but risk being captured inside of the embryonic animal cells.

Better than Seuss

After over a century of investigation, the story of the Green Eggs has gotten more and more interesting with finer and finer resolution. The saga goes to show how the naturalist’s intrigue can inspire a cascade of illuminating research.

Understanding this complex relationship can also help us understand threats to amphibian populations. Herbicides can kill the symbiotic algae and result in lower hatching success and reduced developmental rates. Olivier and Moon (2009) tested the effect of the herbicide atrazine on spotted salamander egg masses. They found that even low concentrations of the chemical killed the algae. In addition to the direct effects of the chemical, the loss of the symbiont had negative repercussions.

References:

Bachmann, M. D., Carlton, R. G., Burkholder, J. M., and Wetzel, R. G. (1986). Symbiosis between salamander eggs and green algae: microelectrode measurements inside eggs demonstrate effect of photosynthesis on oxygen concentration. Can. J. Zool. 64, 1586–1588.

Burns, J. A., Zhang, H., Hill, E., Kim, E., and Kerney, R. (2017). Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis. Elife 6. doi:10.7554/eLife.22054.

Gilbert, J. A., Blaser, M. J., Caporaso, J. G., Jansson, J. K., Lynch, S. V., and Knight, R. (2018). Current understanding of the human microbiome. Nat. Med. 24, 392–400.

Gilbert, P. W. (1942). Observations on the Eggs of Ambystoma Maculatum with Especial Reference to the Green Algae Found Within the Egg Envelopes. Ecology 23, 215–227.

Goff, L. J., and Stein, J. R. (1978). Ammonia: basis for algal symbiosis in salamander egg masses. Life Sci. 22, 1463–1468.

Hutchison, V. H., and Hammen, C. S. (1958). Oxygen utilization in the symbiosis of embryos of the salamander, Ambystoma maculatum and the alga, Oophila amblystomatis. Biol. Bull. 115, 483–489.

Kim, E., Lin, Y., Kerney, R., Blumenberg, L., and Bishop, C. (2014). Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses. PLoS One 9, e108915.

Olivier, H. M., and Moon, B. R. (2010). The effects of atrazine on spotted salamander embryos and their symbiotic alga. Ecotoxicology 19, 654–661.

Orr, H. (1888). Memoirs: Note on the Development of Amphibians, chiefly concerning the Central Nervous System; with Additional Observations on the Hypophysis, Mouth, and the Appendages and Skeleton of the Head. J. Cell Sci. s2-29, 295–324.

Pinder, A., and Friet, S. (1994). Oxygen transport in egg masses of the amphibians Rana sylvatica and Ambystoma maculatum: convection, diffusion and oxygen production by algae. J. Exp. Biol. 197, 17–30.

29 March 2018

My first wood frog of the season, hopping slowly from one snowy bank to the other across the mist-dampened road. This is the first warm night of the spring. The air has condensed against the cold ground into eerie sheets of mists that look like they are getting tangled in the trees and spilling out over the more open ponds.

I pick up this first little frog caught in my headlights to move her on toward her destination and away from tires. I can feel the gritty snow crystals on her skin, which I imagine must be terribly uncomfortable on her soft underbelly. She is sluggish but clear-eyed. Females generally are redder in coloration, but she seems brighter and more sanguine than any wood frog I’ve ever seen. Maybe her ruddy complexion is due to the profusion of blood just recently mobilized and coursing through the capillaries of her skin—blood that until just a few hours ago had been frozen in her cells.

30 March 2018

Noon – The ponds are mostly quiet. A few bold peepers chirp in the warmer ponds with more open canopies. I see wood frogs listlessly floating at the surface and racing for the leaves at the pond bottom when I step into the water, but none of them are vocalizing, yet.

5 PM – The warm day and persistent drizzle this evening were a clear call for the lethargic amphibians to join the early wood frogs in the ponds. Spotted salamanders and wood frogs are out in waves, but other species are on the move, too. I saw one crayfish scuttling across the road from forest to a large tussock-filled pond and two four-toed salamanders apparently racing side by side.

9 PM – Driving around tonight as slowly as possible yet still swerving to miss late-recognized salamanders, I realized that spotted salamanders are a great object lesson against teleological evolution. Evolution is obviously not forward-looking if it resulted in a slow-moving, soft-bodied animal that so perfectly matched the color or wet pavement. Even the bright yellow spots that one might think would stand out in the headlights are indistinguishable from the bright flecks in the chip-and-seal road and just act as further camouflage.

The frogs have found their voices and the choruses are erupting in vernal pockets across the forest. In one pond, I caught a mass of six or more wood frogs clamoring together in a tangle of splayed hindlegs. It is clear that these frogs are driven by a severe case of FOMO incomparable to even the most angst-ridden teenager—all are too concerning with missing out on the chance for breeding none have realized that they’ve engage not a gravid female wood frog, but a male spotted salamander! The poor salamander is helpless in the trap-like bear hugs of the frogs.

31 March 2018

A handful of early clutches were generated in last night’s excitement. I collected a few eggs from each clutch, carefully recorded the water chemistry, and installed a temperature logger at the oviposition site. As the eggs develop, I’ll be back to check their progress and monitor the water conditions, comparing the wild embryos to their brethren back in the lab.

1 April 2018

It is a cloudy day but dry and breezy. A few ponds are chorusing, but they are skittish and clam-up any time I get near. Only a few new egg masses appeared overnight. The forecast is calling for snow tonight, so it may remain quiet for the next couple of days.

Twitty, Victor Chandler. 1966. Of Scientists and Salamanders. W. H. Freeman and Company. San Fransisco. 176p.

From a childhood growing up in southern Indiana to a PhD at Yale, the details of Victor Twitty’s autobiography are remarkably similar to my own (albeit much less auspicious). Also like Twitty, my work entails about equal hourage behind a dissecting microscope as in the field wearing waders. And, this is probably why I enjoyed this book.

I have to admit that I had no expectations when I began reading and no previous knowledge of Twitty or his work. This volume just happened to be on the discount shelf at my favorite used book barn, and I have a rule of buying anything amphibian-related for under $3.

At the beginning of his career, while at Yale, Twitty worked under the embryologist Ross Harrison. Using a shard of glass as a scalpel, he performed “microsurgery,” excising all manner of body parts from embryos at different developmental stages and transplanting them to others. The team even made reciprocal tissue transplants across species, between embryos of the large tiger salamander and the much smaller spotted salamander. The resultant chimeras developed following the body plan like that of the species from which the tissue originate. So, tiger salamander with a spotted salamander’s forelimbs grafted on looks like a T-rex, and a spotted with eyes from a tiger salamander looks like a boggle-eyed anime cartoon. The creatures that arose from these studies contributed enormously toward advancing how we understand vertebrate development and stem cells.

Twitty’s career eventually outgrew the lab and he gravitated to field ecology. He moved to Stanford where he was granted tenure and established a field site in the California hills studying newts of the Taricha genus. In the book, he details the circuitous research path that seemed to have spiraled off into more side projects than forward progress. In the process of understanding hybrid compatibility in the newts, he managed to accidentally produce seminal research in the life history, longevity, dispersal, and (most impressively) newt homing ability using ingeniously concocted and laboriously actuated field experiments.

Looking at Twitty’s career topics from a twenty-first century perspective, his studies were ripe for molecular techniques. If only sequencing were available in his time, he almost certainly would have been a whirlwind of novel discoveries in molecular ecology.

Just a year after this autobiography was published, Twitty committed suicide by cyanide poisoning in his Stanford lab. I wasn’t aware of this fact until I had finished the book. I reread the final chapter and noted many instances in which he reference future studies and results. He seems especially enthused in the future of his long-term hybrid introgression and dispersal experiments that were only just coming to fruition at the time of writing Of Scientists and Salamanders.

It saddens me to consider the ground breaking, but now unrealized, research that may have come from Twitty. But I also take this book as a lesson that our science and lab work, even if it is exemplary science and lab work, is only a small component of a sustainable lifestyle. We all need to be watching out for ourselves and our collaborators and lab mates.