Phycomyces blakesleeanus’ everyday fight: Gravitropism VS Phototropism

Phycomyces blakesleeanus’ everyday fight:

Gravitropism VS Phototropism

By Lara Narbona, Elena Calamand, Tanguy Chotel

Phycomyces blakesleeanus is a fungus (mushroom) sensible to light, gravity and way more things like touch and wind. For example, when it senses a nearby object, P. blakesleeanus will change its growth direction as well as its speed in order to avoid it.
 

In this project, we decided to focus on its reaction to light and gravity, also known as phototropism and gravitropism. Phototropism can be defined as the ability of an organism to develop towards light while gravitropism as its ability to develop according to gravity. In the case of P. blakesleeanus, it does positive phototropism and negative gravitropism, meaning that it develops towards light but against gravity.

The exact purpose of the project was to find a threshold from which phototropism would have more impact to Phycomyces than gravitropism. So, from which intensity of red light, Phycomyces would mostly grow towards the light than against gravity. We chose red light because we found that blue enhances geotropism. 

But why ? why would this fungus be attracted to light and not grow in the opposite direction of gravity ? Well from what we found and read, the "sporangiophores", the sexual parts of this organism needs to leave the ground and reach the surface to spread it's spores. In order to reach the surface, it must follow the light AND go in the opposite direction of gravity. This seems to be the most plausible explanation that we found for this mechanism.

Protocol

To test it, we made our fungi grow on different petri dishes, boxes commonly used in microbiology to grow organisms in controlled environments. We attached them to the wall and put different intensities of LED above them: gravity would make them grow up and LEDs grow down. Different intensities were 20%, 40%, 60% and 80% of total LED intensity. To be sure that they were isolated from other lights except from the LEDs, we put opaque tape all around petri dishes and LEDs and pierced it to let oxygen go inside.

To be sure that light and gravity actually had an impact on the growth of our organisms, we did what we call a ‘control’: one for the maximal exposition to the condition and another for the minimal. And you can wonder: on what does that help? It actually gives us two extreme organism responses: the one with no light (we expect no-response) and the one with 100% light (we expect the organism being completely attracted by it). From this two responses we will know if our organism reacts to the condition we are changing (what we expect will happen) or not (both controls will grow similarly). To do the controls of gravity, we put the plates vertically on the wall (we expect the fungi to grow up) and horizontally on the table (we expect them not to have a priority of growing up or down).

Results

After 2 days, we took pictures of the growth of our organism. Because we taped almost-hermetically the petri dishes, we feared that our organisms wouldn’t have enough oxygen to grow properly. Also, we didn’t let the organisms grow for enough time to develop their reproductive parts (what is actually sensible to gravity and light).

Defying our expectations we were glad to discover that many of the boxes contained beautifully grown fungi, as you can see:

 

To compare how many of the fungus had grown towards the light and how many against gravity, we drew a parallel-to-the-floor line. We supposed that what had grown under the line would have been attracted by light and what had grown over the line would have been attracted by gravity.

From this, we decided to do two different approaches to analyse the growing of our fungus:

First, we chose to compare the area under and the one over the line: this would give us an idea of which factor of attraction would have been the strongest according to light intensity. Unfortunately for us, the organisms did not grow as planned:

We cannot see any clear tendency on this graph: the areas were quite similar on the two sides of the plate (even for the controls!).

Therefore, after seeing that our first approach might not have been the best, we thought about comparing densities of development at each part of the line. We wanted to see if the density was higher when exposed to light rather than gravity. Again, we had quite a surprise :

The density of development in both zones, above and below the line seems to be the same! It looks like the development was made without taking in account the influence of either light or gravity.

Bias

How can we explain such results ? Well, obviously we had a lot of bias during our experiment: the lack of oxygen, the fact that some light could have gone through the tape, and more importantly the fact that Phycomyces blakesleeanus would only develop its reproductive part (sensor for light and gravity) after 5 days… And our experiment only lasted 2 days.

Conclusion

As a conclusion, we can say that despite a lack of conclusive results, we analyzed precisely the parts of the experiment that could have gone wrong and are pretty sure that if we were to redo the experiment, we could be almost certain that results would be as described in the literature !

Bibliography

• « Agar papa dextrosa ». Wikipedia, la enciclopedia libre, 3 janvier 2017. https://es.wikipedia.org/w/index.php?title=Agar_papa_dextrosa&oldid=95995361.

• Barlow, P. W. « An introduction to gravity perception in plants and fungi — A multiplicity of mechanisms ». Advances in Space Research, Life and Gravity: Physiological and Morphological Responses, 17, no 6–7 (1996): 69‑72. doi:10.1016/0273-1177(95)00613-J.

• Grolig, Franz, Peter Eibel, Christine Schimek, Tanja Schapat, David S. Dennison, et Paul A. Galland. « Interaction between Gravitropism and Phototropism in Sporangiophores of Phycomyces Blakesleeanus ». Plant Physiology 123, no 2 (6 janvier 2000): 765‑76. doi:10.1104/pp.123.2.765.

• Corrochano, Luis M. « Sensory perception in the fungus Phycomyces blakesleeanus: a model organism for space research? », Vol. 41, 2016. http://adsabs.harvard.edu/abs/2016cosp...41E.376C.
• Dennison, David S. « The Effect of Light on the Geotropic Responses of Phycomyces Sporangiophores ». The Journal of General Physiology 47, no 4 (1 mars 1964): 651‑65. doi:10.1085/jgp.47.4.651.

• Galland, P. « The Sporangiophore of Phycomyces Blakesleeanus: A Tool to Investigate Fungal Gravireception and Graviresponses ». Plant Biology (Stuttgart, Germany) 16 Suppl 1 (janvier 2014): 58‑68. doi:10.1111/plb.12108.

Contact

If you guys wish to know more here are some things that could interest you !

Twitter : @Let_us_Fall                           

Github:

https://github.com/learningthruresearch/Biosensors2017/tree/master/Let%20it%20Fall            

Storify:

 https://storify.com/Let_us_Fall/the-unbelieveable-tales-of-phycomyces-blakesleeanu

How Euglena react to the intensity of the magnetic field?

Nicolas Larrouy - Nikola Zaverski - Sarah Talon Sampieri

HOW EUGLENA REACT TO THE INTENSITY OF THE MAGNETIC FIELD?

During the second week of biosensors, we studied how organisms and electronic sensors react to forces.

euglena Gracilis (Euglena gracilis) are eukaryotic organisms living in spring or salt water, and who undergo phototaxis, a property that allow them to move toward or against the light source. Nowadays some studies show they can orientate themselves following the electromagnetic field (magnetotaxis).

We decided then to investigate more on this sensitivity of euglena to react to the magnetic field, asking: “with which precision do they orientate themselves depending on the intensity of the magnetic field?”

As the aim of the week was to compare this biological sensor to an electronic one, we decided to use a magnetic sensor (mpu-9250) (part of the Movuino) to see what were the differences in catching the intensity of the magnetic field.

figure 1: Structure of Euglena. They have an eyespot and a photoreceptor to catch light. It is not clear if they have a specific receptor to catch the magnetic field lines. Source: wikipedia commons

Credits: This file is made available under the Creative Commons CC0 1.0 Universal Public Domain Dedication,

Watch this video if you want to see how Euglena use their flagellum and contract themselves to move!

To evaluate Euglena orientation, we built a setup to hold together two magnets. In that way, putting a slide with Euglena between the magnets and under a microscope would allow us to observe this orientation.

Making research, we found out that Euglena seems to orient itself perpendicularly to the magnetic field: for this reason, we needed to know the direction of the field lines between the magnets before placing our Euglena in these conditions. With some iron dust, we found out the magnetic lines were perpendicular to the position of the two magnets (look at figure 2). Keep in mind that the intensity of the magnetic field is constant because the distance between the magnets is constant!

1.                                                       B.
   
   
   Image 2: Orientation of iron Dust (fig A), that illustrate how the field lines should look like (Fig.B).

We made 10 replicates, and for our 10 slides, we also took measures used as reference: observing our populations without the magnetic field, in order to compare to the experiments with the magnetic field, and estimate a difference.

Image 3:  We considered the orientation of each euglena by seeing what angle was formed between the direction of the euglena and the line of the magnetic field. For the negative control, we looked at this same angle, even if there was no magnetic field, to see if there was a real difference in orientation in the two conditions.

With the same technics, we tested out our Movuino in the same conditions, in order to compare our biological values to our electronic ones.

The distances set for the electronic sensor were 34mm. We measured how effectively the MPU-9250 is able to measure the orientation of magnetic fields. For that we placed the y-axis of the magnetometer in parallel with the field lines. 

Image 4 : This is a vector symbolizing the orientation of the magnetic field compared to the orientation of the y-axis. Here you can see that the arrow is pointing in the same direction of the Y-axis. After some calculations, we observed that the angle between the two is near 0°. Conclusion: the magnetometer is really precise at sensing magnetic field’s orientation

After that, we compared our control data (without magnetic field) with our test. It appeared a difference in the mean values between those 2 data, and a statistical test has proven that it was significant. However, we have to be careful, because in both case, the values were far from the mean, what can be explained by the noise we had, conducting the experiments.

The results are interesting because it shows that you can obtain statistically relevant results whereas in reality you cannot conclude. Indeed, while you’re making an experiment, a lot of errors and biases can impact your results. Just to explicit one among many: we had to measure 30 Euglena per picture in order to determine the average orientation but we might, unconsciously, chose the ones that were orientated in a specific way. It is called a bias of measurement. The only thing you can do about that is to acknowledge them and design a follow-up protocol avoiding them.

Anyway, there is a lot to discover about Euglena and their interaction with magnetic fields :)

If you want to know more:

• Look at our Design Plan, our Laboratory Notebook and our codes and programmes on Github to reproduce the experiments.  
• Follow @Pelforceandwine on the Twitter page and take a glance at the story of our posts in our twitter storify!
• additional material:
• Ascoli, C., et al. "Effects of electromagnetic fields on the motion of Euglena gracilis." Biophysical journal 24.3 (1978): 601-612.
• Nice video of locomotion of euglena
• Euglena soccer game

What is the reaction time variation of Daphnia depending on changes in light stimulus location ?

What is the reaction time variation of Daphnia depending on changes in light stimulus location ?

For this first week of Biosensors, the theme is LIGHT! We’re going to compare biological and electronical sensors properties with light!

We chose to study the variation of reaction time of Daphnia individuals when they are exposed to increasing color-alternating frequency.

But after a little research, we observed that information about phototaxis (behavior under light conditions) with Daphnia is unclear and sometimes sources contradict others sources.

“Animals reacted to ultraviolet light (260±380 nm) with negative phototaxis, whereas visible light (420±600 nm) caused positive phototaxis.” U. C. Storz and R. J. Paul in “Phototaxis in water fleas (Daphnia magna) is differently influenced by visible and UV light” in 1998.

But we can see in this video, and in other sites that the positive phototaxis (reaction to light) appears with blue light, and red light has no visible effect…

Preparation of our experiment

For our experiment, we tested the reaction of Daphnia to the red and blue light. We designed an arduino code to be able to alternate blue and red light during at a given frequency. We also designed a code to be able to write data collected by an RGB sensor (TSC 3200). The totality of our codes are on Github.

Wednesday (18/01/2017), we bought Daphnia at Truffaut, but were disappointed when we saw that they’d almost all died. We must wait until Thursday to begin the experiment with new organisms.

First experiment

Thursday morning, we saw only juvenile daphnia move in the water. We collected about 20 of them, put in a beaker of de-chlorinated water. During all the experiment we observed no vertical migration like we had hoped.

Thanks the group “Photons Unchained” who brought new Daphnia, we were able to do the experiment during the afternoon with adult and healthy Daphnia ! But we observed no vertical migration with adults either… But after all, this experiment showed that Daphnia are attracted by light ! They don’t migrate vertically, but horizontally !

Final experiment: Friday (20/01/2017)

We put twenty adult Daphnia in a large petri-dish with a diameter of 13,7cm and we traced areas on a piece of paper at the bottom of the container. We alternately turned on blue LED that were placed on opposite sides of the plates and timed how long it took for five daphnia to swim into our target areas underneath the lights.

We do the same experiment for each frequency, and we repeat the light changes 5 times for each frequency.

We choose to make lights switch every 1,30min, 2min, 3min, 3.20min, 4min and 4,20min.

For our first panel of daphnia, we observed a small area and we tried with shorter times than 1.30, but the daphnia did not have the time to migrate fast enough, so we chose to extend our target area of observation and decrease our alternance frequencies.

We performed controls by having one experiment where both light sources were on simultaneously and another where no light sources were on. We then observed how the Daphnia were placed throughout the Petri dish and confirmed that the light did have a noticeable effect on migration behavior.

During the afternoon, we did not have access to the laboratory, so we did an experiment with the electronic sensor in a room with a low quantity of light, and we put the sensor and the LED installation in a box.

We took 5 measurements for each of the different frequencies of lighting.

Here, we changed the light's color rather than its location to observe the captors reaction.

We recorded the measurements just before the changing of light to be able to have measurements according to time and see values stabilisation of the sensor.

We performed positive and negative controls with the sensor by conducting the experiment once with only red light, once with only blue light and once without.

This confirmed that the reaction we were observing was truly due to the changes in light during other experiments.

We could then determine the time needed for the stabilisation of the sensor’s data.

We determined that the sensors’ reaction time was between 10 and 20 milliseconds and was a constant. We obtained these results with the sensor:

 

For the experiment with Daphnia, we obtained these results:

Results received from experiments with Daphnias showed highly variable reaction times, but no clear correlation between these variations and the frequency of the alternating lights. This is perhaps because our method of measuring individuals was very imprecise and that the organisms did not always behave as expected, perhaps due to wear or other factors that were not taken into account.

If you want to learn more :
Github
Storify 
Twitter

Sources : 
Ebert, Dieter. Introduction to Daphnia Biology. National Center for Biotechnology Information (US), 2005. https://www.ncbi.nlm.nih.gov/books/NBK2042/.

Cellier, S., M. Rehaïlia, J.-L. Berthon, et B. Buisson. « Le rôle de l’œil, dans les rythmes migratoires de Daphnia magna et Daphnia longispina (Cladocères) ». Annales de Limnologie - International Journal of Limnology 34, no 2 (1 juin 1998): 159‑64. doi:10.1051/limn/1998015.

KN, Zhang L. and Baer. « The influence of feeding, photoperiod and selected solvents on the reproductive strategies of the water flea, Daphnia magna. - PubMed - NCBI ». Consulté le 20 janvier 2017. https://www.ncbi.nlm.nih.gov/pubmed/15092821.

Storz, U. C., et R. J. Paul. « Phototaxis in Water Fleas (Daphnia Magna) Is Differently Influenced by Visible and UV Light ». Journal of Comparative Physiology A 183, no 6 (1 décembre 1998): 709‑17. doi:10.1007/s003590050293.

« Check out the course “Tableaux et jeux de lumière avec plusieurs LED” on OpenClassrooms ». OpenClassrooms. Consulté le 20 janvier 2017. https://openclassrooms.com/courses/programmez-vos-premiers-montages-avec-arduino/jeux-de-lumiere-et-tableaux-avec-plusieurs-led.

« Sequential_blinking.ino ». Dropbox. Consulté le 20 janvier 2017. https://www.dropbox.com/s/bivwdnehp7ln1iq/Sequential_blinking.ino?dl=0.

« Éliminer le chlore de l’eau du robinet gratuitement ». consoGlobe, 7 mai 2016. http://www.consoglobe.com/eliminer-chlore-eau-robinet-gratuitement-2895-cg.

« Anatomy of a Mini Breadboard - Pimoroni Yarr-niversity ». Consulté le 20 janvier 2017. https://learn.pimoroni.com/tutorial/170pt-projects/anatomy-of-a-mini-breadboard.

« Arduino Color Sensing Tutorial - TCS230 TCS3200 Color Sensor - HowToMechatronics ». Consulté le 23 janvier 2017. http://howtomechatronics.com/tutorials/arduino/arduino-color-sensing-tutorial-tcs230-tcs3200-color-sensor/.