Learning Thru Research: Deflagellation and regeneration : When green algae becomes a mystery…

If you ever took samples from a stagnant water and tried to observe the microscopic world swarming inside, you probably encountered Chlamydomonas. They are very common unicellular algae, mostly known for being a good case of study for flagellar motility.

Here you can view a video showing some little Chlamydomonas and Euglena.

As you might have seen on the video, Chlamydomonas have a pair of flagella, tubes extruding from the cell body, allowing them to move. More surprisingly, Chlamydomonas are able to lose their flagella, a process named deflagellation, and regenerate them during what is called the flagellar regeneration in less than two hours !

These processes are observed under specific conditions : When Chlamydomonas are placed under stressful conditions, such as high temperatures, unbearable salinity or acidity of their environment, complex pathways of molecules and proteins are activated and lead to the loss of flagella. When Chlamydomonas are reintroduced in normal conditions, the regeneration starts.

Keep in mind that these processes are far away from a complete comprehension and people are still studying about them. Our project was inspired by online protocols from different Universities. You can access one of these through this link. We saw that deflagellation is usually done by the mean of a pH shock (a sudden variation of the environment’s acidity) and we wanted to pursue in this way, experimenting on the stress induced by the acidity of the environment. Therefore the main question of our experiment was : Down to what pH Chlamydomonas can keep their flagella ?

We wanted to see which value of acidity will trigger the loss of flagella for Chlamydomonas.

For that, we decided to add different amounts of acetic acid to modify the pH of the environment.

Here is a visual representation of our experiment (Figure 1 & 2) :

Figure 1 : Schematic of the first part of our experiment : the Preparation

Step 1 : We made 3 samples of 400µL from our Chlamydomonas culture. Each sample will be exposed to a different value of acidic shock.

Step 2 : Different volumes from a solution of acetic acid were added to each sample.

Step 3 : We waited for 1 minute, letting the time for Chlamydomonas to lose their flagella.

Step 4 : Finally, we checked the acidity of the obtained solutions with pH paper. Just to be sure that the pH shock occurred. One drop of the solution is enough to color the paper depending on the pH.

Figure 2 : Schematic of the second part of our experiment : the Observation

Step 5 : Immediately after the 1 minute waiting for the pH shock, the samples of Chlamydomonas were fixed in a Lugol solution. This solution kills and stains all the cells, allowing us to better observe them.

Step 6 : 5 µL of the solution were put on microscopic slides for later observation. We also placed a coverslip on the top.

Step 7 : We observed Chlamydomonas under a fluorescence microscope at x630 magnification and we counted the flagellated and deflagellated Chlamydomonas on each slides.

The observations were made manually : it means that, we looked at the sample under the microscope, swept along, and noted down every deflagellated and flagellated Chlamydomonas. Image analysis and programming could help for further experimentations by counting more rapidly. Obvious observation biases come with this protocol. Indeed, due to imprecisions for some Chlamydomonas, we were not able to determine if they were flagellated or deflagellated without any doubt. However it allows us to make some statistics and graphs, and be able to compare the influence of acidic shock on Chlamydomonas (Figure 3) :

Figure 3 : The average proportion of flagellated Chlamydomonas made with 3 measurements for each experiment. We made 3 repetitions of our experiment for each pH shock value.

The proportion of Chlamydomonas with flagella has been represented (figure 3) according to the pH. A slight tendency is visible : the number of flagellated Chlamydomonas is more important at pH 7. However, between pH 4 and 5.5, our results don’t allow use to conclude to a clear influence of a more acid medium to Chlamydomonas deflagellation.

But what are the advantages of losing their mobility’s structure only for reconstructing them afterwards ? Particular elements on these filaments allow Chlamydomonas to encounter and fix with potential mates for sexual reproduction. Living organisms put a lot of effort in reproduction and in granting good chances of survival for their progeny. Knowing that, we made an assumption : What if this behaviour was selected through evolution, seeing deflagellation as a mechanism disabling reproduction between Chlamydomonas during hard times ? Chlamydomonas able to lose their flagella would not waste their energy for uncertain reproduction compared to those who will reproduce anyway. The offspring of the “patient” Chlamydomonas would proliferate more because introduced in favorable conditions and thus more competitive.

If you want to know more about our precise protocol, data, coding elements, go on our GitHub page. During all our one week project, we also used Tweeter to inform people about our updates of the day. We made this storify to resume our adventure !

Hope you will like it and start your own ;)