Viable Ancient Choanoflagellates from Siberian Permafrost

  Choanoflagellates are the closest unicellular relatives of the animals (Metazoa).

  They seem like very simple organisms. They have a flagellum, a collar like sponge choanocytes which they use to capture bacteria, and some species have other external structures like basket-looking loricas and thecas.
 
Read more about choanoflagellates here and here.

  Choanoflagellates are very common in freshwater and marine environments and they look very gentle and fragile. Yet they managed to survive for around 28-50 thousand years in one of the most extreme environments on Earth. Part of my research was about studying these amazing protists that we found in Siberian permafrost.
   Many groups of organisms are known to have this remarkable ability to stay viable for long time, some bacteria are found in permafrost samples that are several million years old. Some of them stay metabolically active, some form resting stages.

  The choanoflagellates that I was working with are not that old but the fact itself is very interesting.

A choanoflagellate (Codosiga botrytis) cell feeding on bacteria

  Basically a lot of various protists were found in permafrost: amoebae, ciliates, heliozoa and flagellates. Most of observed protists form resting stages - cysts, however for some species cysts are not described.

  Surviving desiccation and freezing is totally normal for microorganisms, in a lot of environments they have to tolerate all these inimical conditions periodically.
However thousands years under extreme conditions with the temperature of -13 degrees Celsius, lack of free water, light, oxygen and food resemble being in space, like on a planet without an atmosphere.

  Do they have metabolic activity or not? How do they "see" if the environment is suitable for life and what triggers their excystment? Can they actually survive in space? Those are just few questions that arise when you deal with ancient fauna.

The ciliate on the left is dyed with acridine orange, match animation phase contrast-fluorescence. The animation requires flash. On the right one of the flagellates observed in permafrost - Anisonema ovale.

  Codosiga botrytis is the most common choanoflagellates species observed, however when it comes to identifying species in microorganisms, traditional approaches fail. The most common definition of species involves sex: if two organisms can give a viable offspring that can reproduce they considered to belong to the same species. But even when protists do have some sort of horizontal gene transfer, classical approach does not always work. Two protists can be morphologically identical and yet phylogenetic analysis reveals differences. At the same time, molecular approaches cab show identity of morphologically different cultures. Species problem overall is a very serious issue in protistology and microbiology.   In my case all Codosiga culteres from permafrost and same morphospeceis isolated from various regions around the world are not identical based on rRNA genes. Not only they have a lot of cryptic species, they also form a phylogenetic clade standing aside from other known choanoflagellates. This fact is making comparison of ancient and modern species very difficult.

  One of the most interesting things to look at were resting cysts that have not been described for the morphospecies. It is the very same stage they use to tolerate permafrost environment. Cysts can be different in terms of shape and cell wall.   The following animations show cysts under different focus planes. The method used is high resolution DIC with digital enhancement (AVEC-DIC), which allows getting resolution of around 10000x of a live specimen.   Interesting thing that you can notice from the first cyst animation is ornamentation on top. The cell wall is very thick when it's present.

  In the second animation another type of cysts is visible. This kind of resting stage does not form any thick walls. Both kinds of cysts can be produced even in the same culture.   Aside from the differences in wall, cysts can differ in terms of shape and size, some of which are even bigger than the active stages.

Two conjoined cysts visible through the microscope. 100x, phase contrast, noise reduction applied.

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  Just to make it clear, the choanoflagellates are normally 4-15 micrones in length.    This is a small part of the work that I am about to publish. Currently we don't know anything about the mechanisms the protists use to survive thousands of years of cryoconservation, protists are very tricky object to work with and we know so little about them. Like bacteria, there are a lot of protists that totally even refuse to grow in cultures making their study nearly impossible.   Choanoflagellates are becoming a popular object to work with mainly due to their relation to multicellular animals and favorite object for those who study origin of multicellularity. For two species full genome has been already sequenced.

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Citation:

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Anastassia V. Shatilovich, Lubov A. Shmakova, Alexander P. Mylnikov and David A. Gilichinsky (2009). Ancient Protozoa Isolated from Permafrost Permafrost Soils, 16 (II), 97-115 : 10.1007/978-3-540-69371-0_8 High resolution DIC images and animation created together with Aron Keve Kiss Added: Our article came out.

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