By Anna Petherick
Occasionally, researchers stumble across something extraordinary in a system that has been studied for decades.
Ryan Kerney of Dalhousie University in Halifax, Nova Scotia, Canada, did just that while looking closely at a clutch of emerald-green balls -- embryos of the spotted salamander (Ambystoma maculatum). He noticed that their bright green colour comes from within the embryos themselves, as well as from the jelly capsule that encases them.
This viridescence is caused by the single-celled alga Oophilia amblystomatis. This has long been understood to enjoy a symbiotic relationship with the spotted salamander, which lays its eggs in bodies of water. However, the symbiosis was thought to occur between the salamander embryo and algae living outside it -- with the embryo producing nitrogen-rich waste that is useful to algae, and the algae increasing the oxygen content of the water in the immediate vicinity of the respiring embryos.
At a presentation on 28 July at the Ninth International Congress of Vertebrate Morphology in Punta del Este, Uruguay, Kerney reported that these algae are, in fact, commonly located inside cells all over the spotted salamander's body. Moreover, there are signs that intracellular algae may be directly providing the products of photosynthesis -- oxygen and carbohydrate -- to the salamander cells that encapsulate them.
Breaking the rules
Such a close co-existence with a photosynthetic organism has previously been found in invertebrates, such as corals, but never in a vertebrate.
Because vertebrate cells have what is known as an adaptive immune system -- which destroys biological material not considered 'self' -- it was thought to be impossible for a symbiont to live stably inside them. But, in this case, the salamander cells have either turned their internal immune system off, or the algae have somehow bypassed it.
"On a lark, I decided to take a long-exposure fluorescent image of a pre-hatchling salamander embryo," says Kerney. When this revealed widely scattered dots of unstained cells fluorescing in the background -- an indicator that those cells might contain chlorophyll -- Kerney switched to transmission electron microscopy (TEM) to take a closer look.
"The surrounding salamander cells that contain the algae often have several mitochrondria bordering the algal symbiont," Kerney says, pointing to a TEM image.
Mitochondria are the powerhouses of animal cells, converting oxygen and a metabolic product of glucose into ATP, a molecule that cells use to store chemical energy. So salamander mitochondria gathered around an algal cell might be there to take advantage of the oxygen and carbohydrate generated by photosynthesis in that particular cell.
How the relationship between the two species originated is unknown. But Kerney is probing how algae enter salamander cells, and some earlier findings are proving helpful.
Lynda Goff, a molecular marine biologist at the University of California, Santa Cruz, worked on this pair of organisms about 30 years ago and demonstrated, among other things, that embryos lacking algae in their surrounding jelly are slow to hatch. "We saw a logarithmic increase in algal cells as the embryo developed," she says. And in those that did contain algae, the community was not static.
This logarithmic increase suggests that algae associated with the salamander embryos either divide rapidly as the embryo develops, or quickly enter the jelly or the embryo from outside as it grows.
So how might the algae enter the embryos? A likely moment occurs as the embryos' nervous systems begin to form. A time-lapse video made by Roger Hangarter at Indiana University in Bloomington, and presented by Kerney at the meeting, reveals a fluorescent green flash next to each embryo at that point in its development.
The flare is a bloom of algae, which is probably drawn to a release of nitrogen-rich waste from the embryo, says Kerney. If waste is released, then there must also be a way in -- and the large number of algae in the bloom increases the chances that some will make it in.
This might explain why so many researchers have failed to find algae inside spotted salamander embryos before: most of them studied embryos that had not yet reached the phase coinciding with these algal blooms, so algae inside the animals would have been scant.
However, that doesn't necessarily mean that embryos at an earlier stage contain no algae.
One of Kerney's most curious discoveries is of the presence of algae in the oviducts of adult female spotted salamanders, where the embryo-encompassing jelly sacs form -- a finding that points to the possibility that symbiotic algae are passed from mother to the offspring's jelly sacs during reproduction.
"I wonder if algae could be getting into the germ [sex] cells," says David Wake, an emeritus professor at the University of California, Berkeley, who watched Kerney's presentation. "That would really challenge the dogma [of vertebrate cells disposing of foreign biological material]. But why not?"
Both Wake and David Buckley, a researcher specializing in salamander development at the National Museum of Natural Sciences in Madrid, agree that the work might tell us more about how self-recognition is learned by vertebrate cells during development. Because salamanders can regrow limbs, almost all the cells in a grown adult retain a degree of pluripotency -- that is, the specialized cells can continue to divide and change into other cell types throughout the salamander's life.
It may be that specialized cells in these adult salamanders are able to accommodate algae inside them because the process by which they learn self-recognition is different from that of other vertebrates.
"It makes me wonder if other species of salamander that have known symbiotic relationships with algae also harbour algae inside their cells," said audience member Daniel Buchholz, a developmental biologist at the University of Cincinnati in Ohio. "I think that if people start looking we may see many more examples."