Moina as an Epigenetic model organism

CUBE Solapur Home Lab
Moina Model

Breaking News
4 days and past 150!

Today is 4th day of the cultures prepared on 3rd Oct 2020 (250mL dechlorinated water+1 drop of milk daily) started with 5 Moina and now, 2 of the bottles have ~40, 2 have ~30 and 1 has ~20 Moina!

Here is the evidence!


The culture spanning in around five bottles. If we pause the video and observe closely in the first and fourth bottles, we can see some Moina that are fatty/bulgy. This means those are gravid ones and we can expect an increase in those bottles in a day or two, let’s say those bottles will add 15-20 more Moina to the total count!

Date and Time of Video: 7th Oct 2020, 6.57pm
Place: Solapur, MH
Data Collector: Drishtant MK.


Moina as seen in 3.8x magnification through a phone camera. A 15cm scale was placed beside a drop of the water sample. We can approximate the size of Moina by pausing the video.

Date and Time of Video: 7th Oct 2020, 7.28pm
Place: Solapur, MH
Data Collector: Drishtant MK.

Please watch the videos, people found them interesting!

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(Post shared on Sunday, 11th Oct)

Further plan of work with an objective:
To conduct an experiment to observe the colour change in Moina when Moina are kept in a low oxygen condition(s).

Requirements:
8-Plastic cups (250mL), milk, plastic dropper, Moina, 2Litres-dechlorinated water.

Design of the Experiment:
Control: 5 colourless Moina+250mL of dechlorinated water (henceforth, DC water)+1drop of milk daily.
Test 1: 5 colourless Moina+250mL of DC water+2drops of milk daily.
Test 2: 5 colourless Moina+250mL of DC water+4drops of milk on alternate days.
Test 3: 5 colourless Moina+250mL of DC water+6drops of milk on alternate days.

Hypothesis:
When the water fleas, Moina are subjected to low-oxygen conditions, they start producing extra haemoglobin because of which they appear to be coloured when compared to colourless Moina. The low-oxygen condition is created when the drops of milk are increased. Milk has nutrients (carbs, proteins, vitamins, minerals and fats as well) that can harbour bacteria present in the surroundings. These bacteria upon getting more nutrition will keep on multiplying and this multiplication costs them energy. Energy comes when respiration occurs. So bacteria will consume the dissolved oxygen in the water and will leave behind less oxygen for Moina as a result of which extra haemoglobin is being produced.

Equation:
More milk=More nutrients=More bacteria=More O2 consumed by bacteria=Less O2 for Moina=More Hb in Moina.

Expectations:
Control: Moina shouldn’t show any colour change and will remain colourless throughout the experiment. ~60 colourless Moina in 7days.
Test 1: First colour change should be seen on the 4/5th day. ~70 Moina out of which ~20-30 should be coloured in 7days.
Test 2: First colour change should be seen on the 3/4th day. ~80 Moina out of which ~20-30 should be coloured in 7days.
Test 3: First colour should be seen on the 3rd day. ~80-90 Moina out of which ~30 should be coloured in 7days.

Expectations are based on previously performed experiments in collaboration with @Zahra_R17 @saida786110.
Photos and videos will be taken as evidence.

This setup will be prepared on Wednesday, 14th October 2020.

References for Haemoglobin in Daphnia/Moina (both closely related water fleas):

  1. Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent, Thomas A. Gorr, et al, 2004; Journal of Biological Chemistry (JBC). https://www.jbc.org/content/279/34/36038.full
  2. The haemoglobin of Daphnia, Harold Munro Fox, The Royal Society Publishing; 1948. https://royalsocietypublishing.org/doi/10.1098/rspb.1948.0006
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CUBE Nerul Home Lab
Moina Model

The setup for observing a colour change in Moina due to low-oxygen has been prepared today!
Details of the setup:
Control cups C1A, C1B: 5 colourless Moina+250mL of dechlorinated water (henceforth, DC water)+ 1drop of milk daily.
Test 1 cups T2A, T2B: 5 colourless Moina+250mL of DC water+ 2drops of milk daily.
Test 2 cups T4A, T4B: 5 colourless Moina+250mL of DC water+ 4drops of milk on alternate days.
Test 3 cups T6A, T6B: 5 colourless Moina+250mL of DC water+ 6drops of milk on alternate days.

We have one replicate for each concentration of milk.


A short video of the preparation of the setup. All the requirements (except Moina) can be arranged at home and are cost-effective.

Date and Time: 14th Oct, 6.54pm
Place: Nerul, Navi Mumbai
Data Collector: Vidwan and Drishtant MK.


Video showing Moina from four different cups. All the four Moina here are from the Control (first), Test1 (second), Test2 (third) and Test3 (fourth). All of them are colourless and comparatively of the same size.

Expectations:
All the 8 cups including 2 control and 6 test cups should have ~5 Moina in them and they should be colourless. No colour change should be seen tomorrow. Expectations for all the cups are the same as the added Moina are smaller than the average size of Moina i.e. 1millimetre that means they won’t reproduce.

Date and Time: 14th Oct, 7.16pm
Magnification: 4x digital zoom
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

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@drishtantmkawale This is a good detailed plan and summary! Please encourage your direct collaborator Vidwan to post. Also am looking forward to the story from your other collaborators @Zahra_R17 and @saida786110 to tell us their stories!

-DP

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CUBE Nerul Home Lab
Moina Model

Objective: To observe a colour change in Moina from colourless to coloured when Moina are subjected to low-oxygen condition(s).

Date of preparation of setup: 14th Oct 2020

Today’s observations:
Control- Number of Moina in both the control cups have decreased from 5 to 2 in both the cups.
Test1- Cup T2A has 4 light yellow coloured Moina, T2B has only 1 light yellow gravid looking Moina.
Test2- T4A has 2 light yellow coloured Moina, T4B has 3 light yellow coloured Moina.
Test3- T6A has 5 light yellow coloured Moina, T6B has 4 light yellow coloured Moina.

Interpretation:
The control cups weren’t seen as expected although Moina are colourless. An interesting observation is that a very light colour change was seen in Moina taken as sample (for video) from all the test cups.


A video showing four Moina from four different cups; control (first), test1 (second), test2 (third) and test3 (fourth).
Moina from the control cup is seen to be colourless. Though not very clearly visible, after pausing the video we can see a very slight colour difference in rest of Moina when compared with the colourless Moina.

This means the production of haemoglobin has started!!

Magnification: 4x digital zoom under white light.
Date and Time: 15th Oct, 6.17pm
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

Expectations for tomorrow:
Control cups C1A and C1B should have 2 colourless Moina each.
Moina should be ~10 and 1 in number in test cups T2A and T2B respectively and should develop a slight dark colour.
Moina should be ~10 each in test cups T4A and T4B and should develop a slight dark colour so as to be distinguished with control.
Moina should be ~15 each in test cups T6A and T6B and a distinct colour change towards pale yellow colour should be seen in ~5 of them.

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I would say this is the interesting observation. The other observations are as expected.

So, as an experimenter, you would be happy to see expected things. But we should always be vigilant of surprises. Because surprises are the places our understanding is limited.

So, why are these Moina reducing in number? Are they under fed? Are they in a different room or condition? Were the bottles different? Your expectation for this remains 2 colourless water fleas for tmrw. What makes you think they won’t further perish as happened yesterday!? Or would they revive and reach the expected population of 60 in next 5-6 days!

@Arunan @Zahra_R17 @saida786110 what do you think of this?

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Yes, correct!!

Let me first clarify that Moina that were added on 14th Oct in all the cups were smaller than any average Moina that has a size around 1millimetre. By size, we can take into consideration one thing that they had not attained a reproductive age. Reproductive age means an age from when the organism can give rise to offsprings.
This is the reason why the expectations for 15th Oct were that no increase in the number of Moina will be seen in all the cups.

Not all Moina can survive in the conditions in which they are kept. Out of 5 Moina, only 2 remained in both the control cups on 15th Oct, that tells us remaining Moina must’ve died because the condition wasn’t suitable for them. Others survived!
Moina cannot be underfed because we can see that not all Moina have died.

No, they are not in a different room or subjected to different conditions. All the 8 cups have been kept at a single place, the difference is of the number of drops of milk.

In the preparation video, we can see there are two stock culture bottles from where Moina have been transferred in the cups. Out of the 8 cups, Moina in the first four cups, C1A, C1B, T2A and T2B are from one bottle and the rest of the cups have Moina from the other bottle. But all Moina are Moina macrocopa only.

Yes, the expectation for today is that no increase will be seen owing to their size (reproductive age not attained) and Moina will remain colourless.

Umm, I thought of thinking towards the positive side. There is an equal chance that they may flourish or they may perish. But now that these water fleas in the controls cups have survived in these conditions for a couple of days, we can expect them to survive in the future (and flourish at the same time)!!

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CUBE Nerul Home Lab
Moina Model

Breaking News
It’s the third day and still no colour change seen!!

Today’s observations:
Control: 1 gravid colourless Moina each in cup C1A and C1B. Expectations of seeing 2 Moina in both cups not met.
Test1: T2A has 12 colourless Moina out of which 7 are juvenile and T2B has only one colourless Moina. Expectations of both cups T2A (of having ~10 Moina), as well as T2B (of having just 1 Moina), have been met.
Test2: T4A and T4B both have just 1 colourless gravid Moina. Expectations of both the cups of observing ~10 Moina (in total) out of which 3-4 would be coloured haven’t met.
Test3: T6A and T6B both have 3 colourless gravid Moina. Here too, expectations regarding the first colour change and their number getting increased (till ~15 in both the cups) haven’t met.

Even though it’s the third day, no colour change was seen in Moina from the cups having 4 drops and 6 drops of milk in 250mL of dechlorinated water and also, the expectations regarding the numbers have also not been met in cups T4A/B as well as T6A/B.


A short video showing the colourless Moina from the control (first) followed by Moina from Test1/2/3 respectively showing lack of colour even though its the third day since they were introduced in this condition.

Magnification: 3x via digital zoom.
Date and Time: 16th Oct, 7.31pm
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

Expectations for tomorrow:
Control: Both the control cups should have ~10 colourless Moina as the water fleas observed on 16th Oct were gravid.
Test1: Moina in T2A should be around 20 in number and ~5 Moina should have light yellow colour. Cup T2B should have ~10 Moina (as one Moina was seen to be gravid) out of which the parent Moina should be of light yellow colour.
Test2: Moina in T4A as well as T4B should be ~10 in number as here too, the single Moina was gravid. The parent Moina should show a colour change.
Test3: Cups T6A and T6B should have ~20 Moina (as currently, all Moina in the cups are gravid) and again, the parent Moina should show a distinct colour change towards a light yellow colour.

In the above expectations, the parent Moina should show a colour change and not the offsprings (at the initial stage) because the offsprings do not inherit the colour same as of their parent, an exception is when the parent Moina is colourless too.

As per the design of the experiment, milk in the control (1drop of milk in 250mL DC water) and test1 (2drops of milk) cups will be added daily whereas, in the test2 (4drops) and test3 (6drops of milk in 250mL DC water) cups, milk will be added on alternate days.
So, all the control and test cups were added and also as it was the third day on 16th Oct, test3 and test4 cups were added with their respective drops of milk too.

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Hi @drishtantmkawale,

Firstly, relieved to see that in control case you have gravid moina, so today I expect they would have reproduced. :smiley:

And, as you say you have not met the expectation in Test 2 and test 3, you have not found enough number of colored Moina. Are you really worried, or is it possible to have this much of buffer for the variations?

Other than, only now I noticed that in

  • Test-1 you have 2 drops/day, and
  • Test 2 you have 4 drops/2days

Aren’t they are effectively same? What is the rationale behind this?

-DP

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Yes, this is a matter of worry because in the previous experiments, a distinct colour change from colourless to (pale-yellow) coloured Moina was seen on the 3rd day itself when 6drops of milk were added in 250mL of dechlorinated water on alternate days.

Apologies for not describing this clearly in the writeups.

In Test-1/2/3 cups, 2 drops (everyday), 4 drops and 6 drops (both on alternate days) of milk respectively are being added. In Test-2 and Test-3, milk is being added on alternate days because in the initial stages, as the number of Moina are less (say 5-10), adding 4 and 6 drops daily will result in a nutritional load in the cups. There will be more nutrients in the cups which will have the ability to harbour more bacteria. This may result in a sudden decrease in the levels of dissolved oxygen in water in the cups and Moina won’t be able to produce enough of Haemoglobin in this short span of time and can die. If there are enough of Moina (say ~30-40) and then if they are exposed to the condition of adding milk daily, then we can expect atleast 50% of them to survive.

This is being said based on the earlier experience of adding 6 drops of milk in 250mL of DC water daily wherein the result was that Moina died on the second day without showing any colour change.
Although this was observed just a couple of times (which means there is a possibility of this as a chance event), further experimentation on the same parameter (6drops daily) can be done to see the results this time.

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CUBE Nerul Home Lab
Moina Model
Update for 17th Oct, Saturday (apologies for the delay)

Breaking News
Still no prominent colour change!!

It was the fourth day (made on 14th Oct) of the setup and still no colour change in Moina from the test cups (having 4 and 6 drops of milk) was seen!
Moina in the control cups having 1 drop of milk in 250mL of DC water are seen to be colourless.


Video evidence showing Moina from the control, test1, test2 and test3 cups respectively.
The control Moina is colourless (as expected) but the rest of the Moina that were subjected to 2 (daily), 4 and 6 drops (on alternate days) of milk are colourless too! All of them are gravid. The expectations were to see a colour change in Moina from the test cups.

Magnification: 5.5x via digital zoom.
Date and Time: 18th Oct, 1.10am
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

I used a new method for taking this video of Moina samples.
In this, a phone’s flashlight was kept at the bottom above which there was a transparent writing pad above which I kept a ruled-paper and above that there was a glass slide on which Moina were kept in a drop of water (from the cups).

There was no light directed from above.

What could be a possible reason(s) (the devil) for not seeing a colour change even after 4days?
Let me mention that test1 is added with 2drops of milk daily and cups test2 and test3 are added with 4 and 6drops of milk on alternate days. All of this is added in 250mL of dechlorinated water.

Expectations for 18th Oct:
Control: To see ~20 colourless Moina in both the cups C1A and C2B.

Test1: ~20 Moina should be there in T2A out of which ~5 should have developed a very light yellow colour and the rest being colourless. The cup T2B will have no Moina in it.

Test2: ~7 Moina should be there in cup T4A out of which 1 (parent Moina) should have a pale yellow colour and rest colourless. As there is just a single gravid Moina in cup T4B, ~10 Moina are expected out of which 1 (the parent) should have a pale yellow colour.

Test3: ~20 Moina should be there in both the cups T6A and T6B out of which ~5 should have a distinct yellow colour.

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CUBE Nerul Home Lab
Moina Model
Update for 19th Oct

Breaking News
Moina are flourishing but no colour change still!

Moina in six (out of the eight) cups have increased in number but there is no colour change seen in Moina from the test cups.
GC
Above is the growth curve of Moina starting from 14th Oct till 19th Oct.
It is indicating the growth of Moina as the days are passing.

Observations made on 19th Oct 2020:

Control: ~70 and ~60 colourless Moina in cups C1A and C1B. As there is an increase (from ~40 to ~60-70) in the number of Moina, ~40 in both cups are young ones. Expectations for both the cups have been met.
Test1: ~60 colourless Moina in cup T2A with ~30 being young Moina. No colour change seen in Moina. Cup T2B has no Moina.
Test2: ~50 colourless Moina in cup T4A with ~30 young Moina. Cup T4B has no Moina.
Test3: Both the cups T6A and T6B have ~50 Moina with none of them showing a colour change.

In all the cups except the control C1A and C1B and two of the test cups, expectations for numbers have met but the expectations for colour change haven’t been met.


Video showing Moina from the control, test1, test2 and test3 cups respectively. All of them seen to be colourless.
Now that we have figured out the possible reason for not seeing a colour change, I think we should not expect the Moina to be coloured.

Magnification: 2.1x via digital zoom. No external light has been flashed so as to avoid interference with the actual colour of the Moina.
Date and Time: 20th Oct, 1.38am
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

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I think the problem is in the design of the experiment.
The expectations were to observe the first colour change on the third day.
This is for Moina from the test cups in which 4drops of milk and 6drops of milk is added on alternate days.

But later I realised that these expectations were seen when the same experiment was done using 500mL plastic bottles instead of plastic cups.

The design should not have included the expectations of the plastic bottles as they are not being used here.
Using plastic cups in place of plastic bottles is showing a major difference.

And this is possibly a GOOFUP!
Expecting an observation that is true for another design and not really true for the current design!
Missing a small detail (bottles–>cups) has changed the outcome of the experiment.

The bottles that were used were in earlier experiments were of 500mL volume.
If we visualise a typical bottle, it will have a narrow mouth. The bottles were covered with a cotton plug.
Having a narrow mouth as well as covering the mouth with cotton plug minimises the entry of air.

And that is what we want!!
As a low oxygen condition is supposed to get created after addition of 6drops of milk (in 250mL DC water), we will not want oxygen (air) to enter the bottle because if oxygen is entering inside the bottle, it will get dissolved in the water and the low-oxygen condition won’t be low oxygen anymore.
But in the cups, the opposite thing is happening altogether.
The plastic cups have a broad mouth and are not covered with cotton plug but just a cotton cloth is being used to cover the cups.

As a result, oxygen through the air will be entering the cups and will be dissolving in the water, because of which the low-oxygen condition doesn’t persist!

And if a low-oxygen condition isn’t created in the water, then Moina will be having oxygen and Moina won’t produce extra-haemoglobin!

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Cool!
So basically you claim that hypoxia (low oxygen) condition is not created, as the containers were broad mouthed and not plugged. btw, I believe cotton plugs don’t make it air tight, and still allow oxygen transfer! That is the reason fruit fly culture use them.

Is there a way to estimate the effect of this? Does it depend on the area of water surface exposed or area of the mouth of the container!

Does anyone any idea?

-DP

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@drishtantmkawale Does this thread have an answer? The title suggests so! You too seem to have taken part in it.

-DP

Correct! And I agree to you too that cotton plugs do not make it airtight and will allow oxygen transfer. That is why it is preferred to wear cotton clothes in summer! To allow passage of air! :sweat_smile:

If we think logically or even visualise the broad mouth of the cups and the narrow mouth of the bottles, there is a considerable difference in both.
Even though cotton-plugged, can we not say that air-passage in the bottles will be comparatively less than that of the cups?
In addition to this, 4 and 6drops of milk was added on alternate days in two of the test cups. A ~24hour time period would be enough for the oxygen-level rise.

A way to estimate the effect of what @karnamdpdurga? (Sorry for missing it)

It will depend on both; the area of the water surface exposed as well as the area of the mouth of the container.
The first point of entry of air will be the mouth of the container and then the surface of the water.

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Sir please send the link of today’s class

CUBE Nerul Home Lab
Moina Model

Summary of the experiment with Moina.
Objective: To observe a colour change in Moina when they are kept in low-oxygen conditions.
Date of Setup preparation: 14th Oct 2020
Result: The objective wasn’t achieved according to the expectations. The expectations were to observe the first colour change in Moina [kept in the cups having 250mL of dechlorinated water+6drops of milk (on alternate days)] on the third day i.e. on 17th Oct. But no colour change was seen in Moina.
Below is the link of the video from the 3rd day.

image
Two of the test cups (with one replicate each) having 4drops and 6drops of milk (milk added on alternate days) were added with milk on 14th, 16th, 18th, 20th Oct whereas the remaining test cup(s) (one replica) were added with 2drops of milk daily. Moina in none of the test cups showed a colour change from colourless to coloured.
The control Moina (in cups) were added with 1drop of milk daily. Moina in control cups remained colourless throughout and are still colourless.

No colour change was seen in Moina from all the test cups and the control Moina remained colourless.
Meanwhile no colour change was seen, number of Moina in the cups increased as well as decreased i.e. they followed a growth curve.
The above growth curve [X-Axis: number of days from 14th to 28th Oct, Y-Axis: number of Moina in all (8) the cups aggregate] tells us about different phases of growth of Moina.

From 14th to 16th Oct, the number of Moina remained constant more or less–> Lag Phase.
From 17th to 23rd Oct, the number of Moina increased–> Exponential Phase.
From 23rd to 25th Oct, the number of Moina remained constant–> Stationary Phase.
From 25th Oct till now, the number of Moina has been decreasing that marks the onset of death phase.

This growth curve is more or less similar to a bacterial growth curve.

Explanation of the observations:
As there was no colour change seen in Moina from any of the test cups at any point (totally opposite of what was expected), I think there are two reasons for this both because of an improper design of the experiment.
i) The design had 8 cups in total that were divided into 4 sets of two each. Control and Test1 cups were added with 1 and 2drops of milk daily. The Test2 and Test3 cups were added were 4 and 6drops of milk on ALTERNATE DAYS.

This is where the devil was, I think. As the expectations were to observe the first colour change in 6drops of milk set on the 3rd day (17th Oct), milk should’ve been added everyday so that a low-oxygen condition would’ve been maintained. But as 4 and 6drops of milk were added on alternate days, the low-oxygen condition didn’t sustain. In addition to this, the cups have a broad mouth and were covered with a cotton cloth, oxygen kept on diffusing in the water due to which the dissolved oxygen level of the water didn’t really decrease till such an extent, where Moina would’ve started the production of extra-haemoglobin.
That is why the test Moina appeared colourless throughout.

ii) The expectations of observing a colour change in Moina (from the test cups) on the third day were not of this experiment. This can be expected when 6drops of milk are added in 250mL of DC water daily and not on alternate days. Also, in the same experiment, plastic bottles having a narrow mouth plugged with cotton were used. Plastic bottles having a narrow mouth and plastic cups having a broad mouth do make a difference in the giving entry to oxygen inside. Here, the difference that was seen was the bottles having Moina+water+6drops of milk daily, colour change was seen on the third day whereas we saw no colour change in this experiment.

GOOFUP:
Not designing an experiment with proper test sets.
This improper design of the experiment with expectations from another experiment led to an unexpected outcome of this experiment!

I learned that while designing an experiment like this, we should keep in mind the smallest of things such as the size of the container, the drops of milk that are being added and whether expectations are according to the setup…

Celebration:
Learning from the above goofup, another design of the experiment has been planned, this time with proper test sets.
Keeping in mind the expectation of observing a colour change in Moina, this experiment will give results.
The design will be shared later tomorrow.

Till then, everyone please suggest improvisations on the design.

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