Moina as an Epigenetic model organism

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CUBE Nerul, Navi Mumbai Home Lab
Update-20th June 2020

Breaking News
The fourth day has brought mixed emotions!

The Moina-Algae setup (prepared on 17th June 2020), on the fourth day, is doing well! Unexpected observations were made today. I didn’t expect to see much Moina in the control (starving) cups C1 and C2 where they aren’t being fed.
The algae bottles too are doing well!

The videos of the control and test cups and bottles below are the evidence:


Lateral/side view (with torchlight at the opposite end) of one of the test bottles A1 having ~30 Moina feeding on algae. Although the video is not much stable :grimacing::man_facepalming:, we can see the movement of small Moina and other small organisms (may be rotifers) feeding on the algae.

The expectations have been met!.
So we can say that, as seen earlier, due to onset of some unfavourable condition, Moina had died. Here, it has not happened yet but their number has doubled in 24 hours.

Expectations: ~40 Moina on the next day.

Date of Video: 20th June 2020
Data Collector: Drishtant MK


Lateral/side view (with torchlight at the opposite end) of one of the test bottles A2 having ~40 Moina feeding on algae.

Here the expectations have met.
Their number has almost tripled (from ~15 to ~40). Good to see that they are still there and reproducing. The movement of small/juvenile Moina is also seen.

Expectations: ~50 Moina on the next day.

Date of Video: 20th June 2020
Data Collector: Drishtant MK


Lateral/side view of the test cups AM2 and AM1 in which there are ~25 and ~20 Moina (negligible) respectively. feeding on the Algal water/spores.
Both the cups have met the expectations more or less.

I think that as only algal water was added, the Moina must have fed on the algae spores and now, there is a possibility that there shall be less food remaining for them so their number can decrease and they might die leaving behind dormant eggs. Here too, many small Moina are seen.

Expectations: ~35 Moina in AM1 and AM2 the next day.

Date of Video: 20th June 2020
Data Collector: Drishtant MK


Lateral/side view of the control cups C1 and C2 showing ~25 and 20 Moina (this too, negligible) respectively. Here, nothing is added as an external feed to them.
Expectations have been met more or less.

No food (given externally) from day 1 but still surviving and reproducing too! Didn’t expect them to survive till the 4th day though.

Expectations: Two possibilities; Either ~30 Moina or less than 20 Moina the next day (as nothing is being fed)

Date of Video: 20th June 2020
Data Collector: Drishtant MK


Lateral/side view of the positive control cups PC1 and PC2 showing the movement of ~50+ Moina (negligible) in both the cups.
The cups have met the expectations.

The Moina in these cups (200mL DC/W + 1 drop of milk) are showing much faster growth if we compare it with the 250mL + 1 drop of milk culture. The latter has ~40 Moina around the 4th day usually (if started with ~10).

Expectations: ~70 Moina on the next day in both the cups.

Date of Video: 20th June 2020
Data Collector: Drishtant MK

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CUBE Nerul, Navi Mumbai Home Lab
Update-22nd June 2020

Breaking News
The starved Moina are still alive!

The Moina-Algae setup (prepared on 17th June 2020), yesterday on the fifth day, is doing well! Unexpected observations were made in the control cups. I didn’t expect to see much Moina in the control (starving) cups C1 and C2 where they aren’t being fed externally since day one.
The algal bottle A1 is showing an unexpected behaviour as the number of Moina is around 10 unlike the other bottle A2 which has around 30.

The number of Moina from bottle A1 on the 5th day is same as what was the number of Moina in the same bottle when this experiment was conducted earlier. ~10 Moina were there on the 5th day.
What does this convey?
Is the same thing (that there will be no Moina the next day; 6th day) going to happen?

The videos of the control and test cups and bottles below are the evidence:


Lateral/side view (with torchlight at the opposite end) of one of the test bottles A1 having ~10 Moina feeding on algae.

I didn’t expect there to be ~10 Moina. I expected ~40 Moina as there was a steady increase (~30 Moina on 21st June) in their number.
This bottle is having the same behaviour as it was having earlier when this experiment was conducted.

Expectations: ~10 Moina on the next day as the Moina are seen to be very small as compared to the adult ones, so they won’t reproduce.

Date of Video: 22nd June 2020
Data Collector: Drishtant MK


Lateral/side view (with torchlight at the opposite end) of one of the test bottles A2 having ~30 Moina feeding on algae.

Here the expectations haven’t been met.
The number of Moina in this if we compare to the above bottle A1 is less (difference of ~20 Moina).
~40 Moina were there on 21st June and now, ~30.
Will the number of Moina decrease further in this bottle (no reason though)?

Expectations: ~40 Moina on the next day.

Date of Video: 22nd June 2020
Data Collector: Drishtant MK


Lateral/side view of the test cups AM1 and AM2 in which there are ~20 Moina (in both). feeding on the Algal water/spores.
Both the cups have met the expectations more or less.

I assume that even though algae water/spores were added initially, the Moina must have fed on them and now as nothing is being fed externally, they will be relying on the decaying matter (dead Moina, bacteria growing in them) and bacteria coming from the environment, the same is happening the control cups C1 and C2.

Expectations: ~30 Moina in both the cups the next day.

Date of Video: 22nd June 2020
Data Collector: Drishtant MK


Lateral/side view of the control cups C1 and C2 showing ~20 and 25 Moina (negligible) respectively. Here, nothing is added as an external feed to them. They are spread throughout the water in the cup so not many not visible.
Expectations have been met more or less.

No food (given externally) from day 1 but still surviving and reproducing too! Didn’t expect them to survive till the 5th day!

Expectations: Two possibilities; Either ~35 Moina or less than 20 Moina the next day (as nothing is being fed)

Date of Video: 22nd June 2020
Data Collector: Drishtant MK


Lateral/side view of the positive control cups PC1 and PC2 showing the movement of ~80 and ~20 Moina respectively in the given cups.
The cup C1 has met the expectations but the cup C2 hasn’t.

The Moina in the cup C1 are showing good growth (adults as well as juveniles are seen) but cup C2 is just sustaining ~20 Moina although both the cups have 200mL of dechlorinated water + 1 drop of milk is fed to them daily and ~10 Moina were added initially.

Expectations: ~100 Moina in C1 and ~30 in C2 on the next day.

Date of Video: 22nd June 2020
Data Collector: Drishtant MK

This experiment is being done to check, do the Moina survive by feeding on algae or not?

Initially, it is known that they feed on algae in ponds, lakes and freshwater bodies.

Then why am I doing this?
In the first week of June 2020 (5th of June) when I had transferred ~10 Moina in the two bottles in which there was algal growth, the number of Moina went to a peak of ~100 through a week and then exactly on the 7th day (12th June), there were no Moina in both the bottles.
Both the bottles were more or less showing the same growth curve.
There is a possible that the Moina which were dying must have layed dormant eggs or ephippia.

Why did this happen?
Some toxicity?

So, to check if this happens the second time too, I again started this experiment (on 17th June) this time with Controls (along with replicates of it).

So far till the 5th day, the algae bottles are sustaining Moina with ~10 Moina in A1 and ~30 Moina in bottle A2 both with 500mL of dechlorinated water and algae.

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Hi Drishtant,

Your story and the way you have narrated makes it exciting! :smiley: :smiley:

From a quick glance through the last few days, you seem to have mixed observations related to the algae and Moina.

Before we continue the conversation and get into the details of the contents of bottles, their culture trajectories etc., can you very briefly state what is the expected relation, and the basis for this expectation?

-DP

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CUBE Solapur (earlier Nerul), Home Lab
Update of the Moina culture-12th August 2020

Breaking News
Moina have travelled 400kms down the south!

Yes, due to some reasons, the CUBE Nerul, Navi Mumbai Home Lab is now shifted to Solapur, MH ( (a city near MH-Karnataka Border, famous for textiles).
And the Moina too have travelled 400kms and are surviving in the bottle cultures.

There are around 250 Moina in five-500mL transparent plastic bottles having 250mL of dechlorinated water and are fed with one drop of milk.

An appeal to all the CUBist Collaborators working on various model systems: Let us not give up on posting updates!
Updates will ensure that we all are proceeding ahead together, learning from the mistakes and most importantly, it will maintain the rhythm. We all learn from each other, so when we put updates (not as a ritual) new CUBists will ask questions, clear doubts, add their own points from which there is a possibility that we will come to know something which was not known to us. There are many things like these.

Let us continue the legacy of CUBE by remembering the three C’s: Culture, Collaboration and Continuity!

URGENT…
Let’s take up this initiative everyone.

Carton Lab
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This is my home lab!
Yes, a carton with few culture bottles, plastic cups, droppers and stickers (along with a bottle of dechlorinated water not shown in this photograph)! This is all I have carried from Navi Mumbai to Solapur.
This shows the simplicity with which we CUBists work.

Date and Time of the Photograph: 12th August 2020, 1am
Place: Solapur, Maharashtra
Data Collector: Drishtant MK


Around 100 Moina seen in this bottle L3 which is one of the five culture bottles having Moina. This culture has juvenile as well as adult Moina (which can be differentiated on the basis of their size, although may not be clearly visible in the video).

Date and Time of the Video: 12th August 2020, 1am
Place: Solapur, Maharashtra
Data Collector: Drishtant MK

86

Apologies for reverting back late!

I am quite confused about the question though. At first, I didn’t realise the actual question. Then I read it once again.
Anyways

I hope here, we are talking about the expected relation between Moina and Algae.
I am answering for this.

So, talking about the relation between Moina and Algae, both are freshwater organisms. It means they are found in ponds, lakes, rivers, streams (some algae are marinewater too). There are many types of algae which are harmful as well as good for the organism (here, Moina) which are consuming the Moina. In the above-mentioned habitats, algae (phytoplankton) are found everywhere, attached to the rocks and freely floating on the surface. Moina are filter-feeders, which means that they strain the suspended matter and food particles from the water by passing them through the filtering units in their bodies (in the carapace; a hard covering). So, while taking-in water, the algae too comes inside which gets metabolised and then provides energy to the organism. And this is how Moina survive by feeding on algae. One variable is that we don’t know whether the algae which Moina is consuming is good or harmful to the Moina.

In my case, I had seen a greenish growth in one of my bottles. The greenish growth was suspected to be algae as I kept the bottle (having 500mL dechlorinated water + yeast (from the earlier experiment) + dead Moina debris) in the window and as days passed, the greenish growth started increasing. Below is the collage depicting the increasing algal growth.

https://sci-hub.tw/https://doi.org/10.1007/BF00017499

In this paper titled Fecundity, reproduction, and growth of Moina macrocopa fed different
algae, the three scientists have given three different algae as a feed to Moina macrocopa and have checked the effect of these algae (as a diet) on the fertility, reproduction and growth of the organism.

Yeah, it is indeed exciting!

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More algae=less O_2. Does lower O_2 level affect Moina?

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@jtd, how did we arrive at this conclusion?

Yes, the low oxygen level does affect the Moina.

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Read somewhere that algae causes oxygen depleted dead zones in the oceans. Am I mixing up things here? If algae are plants they should be emitting O_2 right? which would make the oxygen content higher.

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Yes, you read it right. Algae do cause dead zones in the oceans.

Mixing up things is alright! And yes, this is where we get confused when we hear that algae consume oxygen as well as emit O_2!

“Algae produce oxygen during the day through photosynthesis but also quickly consume oxygen at night during respiration. Bacteria decompose the algae after the bloom dies, using a significant amount of oxygen in the process.”
“The result is a lack of available oxygen for other plants and animals that need it, possibly causing a fish kill.”

Reference: https://www.michiganseagrant.org/lessons/lessons/by-broad-concept/earth-science/water-quality/oxygen-and-carbon-dioxide/#:~:text=Algae%20produce%20oxygen%20during%20the,of%20oxygen%20in%20the%20process.

How do we counter this?
Do Algae consume more oxygen (at night while respiration) than they produce (in the day through photosynthesis)?

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I made a search on the internet on the above-mentioned question and got this.

“Like most plants, many algae produce oxygen during the daylight as a by-product of photosynthesis. At night these algae consume oxygen, but usually much less than was produced during the daylight.”

Reference: Louisiana Fisheries - Fact Sheets.

A conclusion can be drawn here, relatively, more O_2 is produced in the daytime than is consumed at night by the algae.

But what about the algae that cause dead ocean zones?
@jtd

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I guess, the take home point here @drishtantmkawale @jtd is that after too many algae grow, there is a stage that these algae start dying and the dead algae will be feast for bacteria and these bacteria consume oxygen and deplete oxygen in the region of algal growth/bloom…Does it make sense?

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One of the possibilities can be this. And yes, it does make sense.
But then why will the algae die if it has enough oxygen as well as sunlight?

Do we have something such as an algal growth curve for algae as we have a bacterial growth curve for bacteria?
@Arunan @jtd @Lydia @yash_sheregare @KiranKalakotiR

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Does any cubist experiment with yeast as food for Moina, Apart from algae, bacteria culture of milk? Is it possible to culture both Moina and yeast in the same bottle? Moina may not like fermenting conditions? Does yeast grow in the oxygen-rich environment also?

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Yes, CUBist Abhijith Vinod from CUBE Kolenchery, Kerala has been maintaining Moina by feeding them with Yeast (inactive yeast, activated when put in water) granules. It has been almost a year since the Moina were taken from CUBE HBCSE, Mumbai to CUBE Kolenchery, Kerala by @Arunan and they are still being maintained.

Talking about culturing Moina and Yeast in the same bottle, we need to discuss the requirements of each organism in order to grow in the medium.
Moina requires bacteria, algae, yeast, decayed matter to feed upon.
Yeast on the other hand requires Sugars in the form of carbohydrates to break them down and give acid/alcohol.

So the only link here is milk!
Milk has sugar (lactose) for Yeast and this sugar also acts as a nutrient source for water as well as milk bacteria to grow.

What are the fermenting conditions here?

Some Yeast require oxygen for growth and some are facultative anaerobes which means that they have to switch to aerobic method for energy-production.

Here, a question arises, whether aerobic or anaerobic or both, will the end-products of fermentation differ in both the cases?

This is possible as of now from the above conclusion.
By using milk, “ek teer se do nishane”!

But before trying this out, what is the real question that we are addressing here?

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CUBE Solapur, MH Home Lab
Moina Model 6th September 2020

Breaking News
New Cultures prepared!

Although one day late (5th Sept 2020 was mentioned in the plan of work), Moina Culture Bottles have been prepared today.
The species of Moina being cultured in home labs in Ranchi, Kerala and Mumbai, Kolkata: Moina macrocopa JSK1.

moina
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The five bottles L1 to L5.
All of them having 250mL of dechlorinated water+5 Moina and 1 drop of milk will be added daily.
The Moina used here is from the previous culture. While transferring, they had been transferred into a transparent plastic cup.

Expectations: Around 60 Moina in all of the bottles after 7days.

Date and Time of Photo: 6th September 2020, 7.40pm
Place: Solapur, MH
Data Collector: Drishtant MK

Video
A self-explanatory video showing the procedure to prepare bottle cultures by using things available at home (labs) except Moina.
It tells us about the steps such as transfer of Moina from old bottle to a stock → washing the bottles clean → picking Moina from the stock culture → transferring them to the newly prepared culture medium.

Date and Time of Video: 6th September 2020, 7.15pm to 7.50pm
Place: Solapur, MH
Data Collector: Drishtant MK

Isn’t the culturing and maintenance of Moina easy and affordable?
We have this model that can be used to address the frontier-level questions in the field of science and especially the Nobel Prize-winning study can be understood!

Our plan is to design an experiment to go ahead with our research question and objectives.
Before that, the thing which stands first in the line is updates about the culture in the home lab with photos and videos!

Let’s look forward to it.

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CUBE Solapur, MH Home Lab
Moina Model Update-11th September 2020

Breaking News
Expectations have been met before time!

New culture medium for Moina was prepared on 6th September 2020 in five bottles.
5 Moina were added in each of the five bottles having 250mL of dechlorinated water and 1 drop of milk.
After adding the Moina, ~60-70 Moina were expected after 7 days or on 13th September 2020.


The video showing Moina in bottles L1 to L5. The astonishing observation is that the number of Moina have increased in a shorter time than expected. As mentioned above, ~70 Moina were expected in 7days but we can see that 4 out of 5 bottles have around 70 Moina in them on the fifth day itself!
25 Moina to 300+ Moina in 5days!!

Date and Time of Video: 11th September 2020, 10.18pm
Place: Solapur, MH
Data Collector: Drishtant MK

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CUBE Solapur Home Lab
Moina Model

Breaking News
Moina follow a growth curve!

The Moina cultures consisting of five bottles made on 6th Sept 2020 are now having around 100 Moina (in all five bottles combined).

Today 30th Sept 2020 being the 24th day, we can see that the Moina in the bottles have decreased.
On 6th Sept, 5 Moina were added in 250mL of dechlorinated water and 1 drop of milk was added daily.

As per the data (on the number of Moina) of these five bottles L1 to L5, after a week i.e. on 12th Sept, there were ~80 Moina in all the bottles. This is where the increase started!

Again on 19th Sept (after 14days), two of the bottles had ~200 Moina* and the rest three bottles had ~150 Moina.

The data for the third week i.e. from 20th Sept till 26th Sept wasn’t recorded. The decrease in the number of Moina from triple figures (200s) to double figures (40s, 50s) must have happened in this time.

On 27th Sept, (22days past) three bottles had around 40 Moina and the rest had ~10.

And yesterday, 29th Sept 2020, three bottles had ~25 Moina and two had ~10.

From this, we can see that Moina too follow a growth curve that lasts around four weeks or 30days.

Video showing Moina culture spanning in five bottles. There is debris at the bottom of the bottle. The colour of water (culture medium) also changes. Earlier, the water was much transparent but now, the colour has changed to faint yellow.

Date and Time of Video: 30th Sept 2020, 12.55pm
Place: Solapur, MH
Data Collector: Drishtant MK.

Further plan: On 2nd Oct 2020, new culture medium for Moina will be prepared so that the existing Moina will be transferred in this medium. Preparation will involve transferring the Moina from the old culture medium to a cup which will be the stock. And then the bottles will be washed after which water will be added and then Moina will be transferred in those bottles followed by the addition of milk.

A detailed setup will be shared after the preparation.

Further plan towards the research question and objective:

Our research question is to find how does a low-oxygen condition induce a colour change in the Moina and is it due to chromosomal modifications?
For this, the objective will be to conduct an experiment in which the Moina will be kept in a low-oxygen condition until the first colour change (from a colourless Moina to a coloured Moina, showing the production of Haemoglobin protein) is observed and then again, the coloured Moina will be kept in normal condition (till they turn back to colourless from coloured).

The date (of the start) and design of the experiment will shared.

Collaborators: Zahra, Saida and Priyanka from Mumbai.
Abhijith and Arunima from Kerala.
Rechel, Man and Ram Deepak from Ranchi.
Batul from Kolkata.

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GC
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A growth curve of Moina that includes the data from 6th September 2020 to 30th September 2020 i.e. 25days.
This graph is plotted by taking average readings of the number of Moina in five bottles.
New cultures (five bottles) were made on 6th Sept 2020 by adding 250mL of dechlorinated water+5 Moina+1 drop of milk.

This Moina growth curve has a rough resemblance (with some variations) with the bacterial growth curve.
The phases are Lag, Log/Exponential, Stationary and Death phase.

Bacteria
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A bacterial growth curve taken as a reference for comparison with Moina growth curve.
This graph was sent by @Jaikishan during one of the ChatShaala’s last week.

In the Lag Phase, the organism is getting used to the conditions provided to it.
Log Phase indicates the multiplication of the organism rapidly, thus an increase in the number is seen.
In the Stationary Phase, the number of organisms remain constant even though some continue to multiply, others begin to die.
The Death Phase shows a sharp decline in the number of organisms.

All of these phases are common between both of the growth curves shown above.

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@drishtantmkawale how did you make these plots?

Do you have any more data that we can see as plots?

-DP

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Yes, I actually have data from the month of May. Wherein we can see recurring graphs showing a pattern. A pattern will be seen because new cultures have been made 5-6 times.
@karnamdpdurga