New insight from the wonderful world of radiolaria

The postdoc Anders K. Krabberød at the University of Oslo's Department of Biosciences has a long list of arguments for studying the tiny radiolarians, one of the reasons being that they are beautiful and somewhat alien.

Their complex exoskeletons are built from silica or strontium sulfate and can take on a myriad of strange forms. The German zoologist Ernst Haeckel's detailed drawings of radiolarian exoskeletons from the 1870s is as much art as science.

Radiolarians are really tiny creatures, usually between 0.1 and 0.2 millimetres in size. Most of the time, they are drifting around in the oceans as zooplankton, eating for instance bacteria or other species of plankton even smaller than themselves.

Dead radiolarians sink to the bottom of the ocean and are transformed into a siliceous ooze, covering a large part of the ocean floor.

Geologists appreciate the radiolarian ooze very much, because the alien-looking creatures have lived on our planets for approximately 500 million years. Therefore, fossil radiolarians are very useful for dating geological structures.

Shedding new light on tumors

Another and more important reason for studying radiolarians, is that Krabberød had to make himself an expert on a novel DNA technology in order to analyse the genetic relationship between different species in the group.

In the old days, scientists needed access to hundreds or maybe thousands of individual cells in order to obtain enough DNA to analyse such kinship.

Krabberød has instead used a new method for transcriptome analysis of single cells. The method was developed by the Italian scientist Simone Picelli at the Sandberg Lab at the medical university Karolinska Institutet in Sweden.

"The method can start with tiny amounts of RNA, even from only one cell, and transform it to DNA before running it through a genetic "copy machine." Thereafter, we can use well-known technology in order to analyse the DNA sequences in the material. The copying in itself is surprisingly fast, the most time-consuming part of the job is actually to find the actual radiolarian cell and extract RNA from it," explains Krabberød, who was a PhD student when the study was made.

The new technology was originally developed for use in healthcare, where personalized medicine  or precision medicine is the new buzzword. Personalized medicine is about analysing the genes of patients in order to find the best treatment for their diseases.

When Krabberød has used the technique for analysing the genes of radiolarians, he is only a small step away from analysing also the genes of for instance cancer tumors or cells in the human immune system.

Rewriting the radiolarian genealogy

Until now, it has not been possible to establish the genetic kinship between the ca. 4000 known species of radiolarians, because nobody has managed to grow them in the laboratory. Thus, it was impossible to acquire the hundreds or thousands of cells necessary to analyse their DNA.

But Anders Krabberød has been able to use the new transcriptome method in analysing more radiolarians genes than any other scientist has done before him, and nobody else has worked with such a big and representative selection of species.

This led to discoveries that suggest the genealogy of the little creatures should be rewritten.

"Ernst Haeckel and other former natural scientists classified the radiolarians according to their morphology, that is their appearance in a broad sense. But it is not always true that organisms are genetically related even if they have a similar appearance," Krabberød explains.

Has proved that morphology falls short

For example, it might be tempting to assume that a big, round radiolarian with two spikes is more closely related to a small, round radiolarian with two spikes, than with a cone-shaped species with no spikes. In any case, that's what Haeckel imagined, and Krabberød's DNA studies have proven the old German right in some of these cases.

But in other cases, Krabberød has proved that morphology falls short.

"Haeckel divided radiolarians into four groups, but it has later been shown that one of his groups, thePhaeodaria, are not radiolarian at all. In addition, I have found that also the species Sticholonche zancleais not a radiolarian. Instead, it seems to be the common ancestor for both extant radiolarians and another group known as foraminifera," says Krabberød.

In order to determine the genetic kinship between different radiolarian species, and between radiolarians and other living creatures, you need a lot of computing power. Anders Krabberød used nearly 350 CPU years on the University of Oslo's computational resource, the Abel computer cluster, in order to perform his calculations.

"I was actually the largest university user on Abel for a period. They were just about to lose their patience with me," Krabberød admits.

Hunting for Sticholonche

Anders Krabberød has lost count of all the hours he spent stooping over his microscope, using a home-made tool for delicately turning radiolarians and other creatures around in the petri dish. The tool was made from a chopstick and had a tiny thread from a Japanese toothbrush glued to its tip.

"I needed something very thin and fine for picking at the radiolarians, and Japanese toothbrushes are made with much thinner threads than the Norwegian ones. You can learn a lot from having colleagues from other parts of the world!"

The hunt for Sticholonche proved to be especially exhausting. The approach for catching radiolarians is to pull a fine-meshed scoop net with a mesh size of 50 micrometres through the waters of the ocean. This causes the radiolarians to collect at the bottom of the net, together with myriads of other strange things.

Anders Krabberød had acquired the porridge-like result of one such expedition in the Oslo Fjord, and was looking for Sticholonche in his microscope. He needed this particular species in order to use the new transcriptome method, and he was almost certain that some individuals should be there, somewhere in the porridge.

But they wouldn't show themselves. Krabberød's back was aching, his eyes were very tired, and he was staring in fiasco's face after hours of searching that only gave him tiny creatures he wasn't looking for.

In his own time, Krabberød plays the electric bass in the progrock band Panzerpappa. In the end, his interest in music came to the rescue.

"I was listening to the Peter Gabriel album "So" in my headphones, and was close to quitting. But then, Kate Bush suddenly started to sing "Don't give up!." The song inspired me to continue, and from then, it didn't take long before I finally found Sticholonche."

The remaining mysteries

Already around 1870, Ernst Haeckel discovered a photosynthesizing algae living in colonies together with radiolarians, in a common gel secreted by the radiolarian hosts. Organisms living together like this are calledsymbionts.

Anders Krabberød has, by using DNA analysing techniques, shown that several marine species in the protistan alveolate group also seem to be living as symbionts with radiolarians, but these are hiding themselves inside the siliceous exoskeletons of certain species.

The alveolates are probably not performing photosynthesis, it is instead more probable that they are parasites.

Even if Krabberød has revealed some of the radiolarians' secrets, there is a lot that remains to be explained.

"Among other things, we just don't know how they procreate. Ernst Haeckel observed that if you keep them in a petri dish long enough, they come to a point where the cells start sending out thousands of very tiny "swarmers" in a cloud. The hypothesis is that these swarmers constitute the reproductive phase, but we don't know if they are spores that can grow asexually into a new individual. Maybe they instead are sexual gametes who must meet another gamete to mate with before they can start growing," explains Krabberød.

Set to outlive humans

Radiolarians are exclusive salt water species and can be found in every ocean on Earth. Some of them have pseudopodia -- "false feet" -- that can be used for different purposes.

"One Japanese colleague has filmed a species with a pseudopodium several times the length of the cell itself, and it is being used as a fishing rod. But the ecology of radiolarians is pretty much uncharted territory, we don't even know if they have any natural enemies. So there is a lot of interesting science just waiting to happen."

"Radiolarians have been around for hundreds of millions of years, and it is a safe bet to predict that they are going to outlive us humans by a wide margin. They are namely very robust creatures -- you would be surprised to know how much I struggled to break their exoskeletons in order to get at the cell material with DNA inside them," admits Krabberød.