Moina as an Epigenetic model organism

drishtantmkawale · February 6, 2020, 1:11pm

Magnification 40x→ 10x eyepiece and 4x objective

A STUDY IN THE RED

CUBE Elphinstone
Moina Group
@Zahra_R17 @saida786110

Short term Objective: To get Red Moinas

Breaking News!!
SIGNS OF RED MOINAS!!
The Red Moina setup which was started on 31 Jan 2020, with Two Control bottles and two Test bottles feeding on 1 drop and 6 drops of milk respectively now has ~50 Yellow- Orangish-Red Moinas out of the ~80 in total
Today being the 6th-day of the setup, we expected this result as we got almost the same results as earlier i.e. prominent colour change!

Both the Control bottles have ~50 colourless Moinas and the Second Test bottle has ~50 Moinas with ~30 being Colourless to Yellow

Expectations for tomorrow: Darker Orangish Red Moinas in first test bottle with ~100 being in total and ~70 in second test bottle with ~30 being Yellow in colour
And ~60 colourless Moinas in the Control bottles

dialecticalbiologist · February 7, 2020, 6:57am

So, are you sure we get Red Moina in 6 days for experiments if we set up culture bottle with colorless Moina and 6 drops of milk daily?
Suppose if we want 500 Red Moina on the 6th day for some experiment, how shall we start?
@drishtantmkawale @saida786110 @Zahra_R17 @Ram

drishtantmkawale · February 7, 2020, 5:12pm

I cannot be sure that the Red colour of the Moinas would appear on the 6th day itself.
From my experiences from the past set-ups, I had the Orangish-Red coloured Moinas on the **6th day **in a similar set-up and the proper Red colour was visible on the 7th day to be specific.

To make/get 500 Red Moinas in a week, how many Moinas are you providing to me initially?
@dialecticalbiologist

drishtantmkawale · February 8, 2020, 1:57pm

Couldn’t provide yesterday’s and today’s update as we had our college annual day yesterday followed by a holiday today as well as tomorrow.

Zahra_R17 · February 10, 2020, 5:15pm

10/02/2020
MOINA GROUP
@drishtantmkawale @saida786110
CUBE ELPHINSTONE
BREAKING NEWS
Moina are red without getting feeded for 3 days
•We had traditional day on friday and Saturday and sunday was a holiday so 3 days in total our moina weren’t fed
•Today we saw red moina in the bottle RM-M & RM-D.
Short term objective=
To change the water of hypoxic bottle RM-M & RM-D Tomorrow.

saida786110 · February 13, 2020, 5:41pm

Cube Elphinstone
Moina group
@⁨Drishtant FY⁩ @⁨Zahra Risalawala FY⁩

Today’s update -13/02/2020

We found gravid moina in CMM
Culture
Moina count
RMM - ~80 Red MOINAS
~10 colourless
RMD - ~80 red MOINAS
~10 colourless

CMM - ~40 colourless MOINAS
CMD - ~40 colourless MOINAS

CM- Control MOINAS

RM - red moinas
M- Main
C- Control

saida786110 · February 13, 2020, 5:48pm

From past 1week as we enter lab we observe that Cotton plugs are removed from the moina culture
@mayur sir suspect’s it to be Rats
Today while observing the culture we observed that there are some organism moving in one of the cultures

GN · February 14, 2020, 3:22am

This is a wonderful summary prepared. Apologies for seeing this so late.

dialecticalbiologist · February 16, 2020, 1:40pm

Don’t you think Official Holidays are great times for Cubists in get involved in long engaged discussion? @drishtantmkawale, unless we come out of the clutches of the “Mantralaya culture”…!

drishtantmkawale · February 17, 2020, 8:10am

CUBE Elphinstone
Moina Group
@saida786110 @Zahra_R17

Today, an 8L Moina tank was made
8L Dechlorinated Water + 32 drops of milk + 1 Colourless Moina

The tank used here is a plastic one.

We want to have a Single Line Culture of the Moinas to check their growth so we added only a single Moina
We are not sure whether the added one is gravid (pregnant) or not.

Objective of Making this tank is to have a Moina stock

How 32 drops of Milk?
1 drop of milk in 250mL of Dechlorinated Water
4 drops in 1000mL or 1L of DC Water
Hence, 8x4=32 drops of milk in 8000mL or 8L of Dechlorinated Water

Expectations for tomorrow: We hypothesize that
1. If not gravid, then the number would remain the same.
2. If gravid, then it can give 7-10 progenies
3. There is also a possibility that the given Moina could die.

I know that the problem of replicates will arise!
We cannot make an exact replica of this 8 Litre tank culture.

Any suggestions!!??

Zahra_R17 · February 17, 2020, 12:47pm

CUBE ELPHINSTONE
MOINA GROUP
@⁨Drishtant⁩ @⁨Sayyeda⁩
Today’s update

BREAKING NEWS

• Moinas are red in RM-M AND RM-D
there are approximately 200 moina in both the bottle and there are 50 moina in CMM & CMD and they’re are colourless.
• Bottles RM3 and RM4 - we changed the water of both the bottles becuse turbidity increased.
• We discarded M1 bottle because we found worms in the bottle and the number of moina decreased due to that.

drishtantmkawale · March 2, 2020, 1:53pm

CUBE Elphinstone, Mumbai
Moina Group
@Zahra_R17 @saida786110

Update- 02 March 20

1. The Red Moina Set-up which was made on 31 Jan 20 aimed at getting Red Moinas.
But, last week we decided to discontinue the set-up. We had taken 4 500mL bottles and had added 250mL of Dechlorinated Water in each bottle, 2 bottles were fed with 1 drop of milk as control and the other two were fed with 6 drops of milk out of which all had ~10 colourless Moinas on day 1.

The first major mistake we did is that we did not make more replicates.
The second major mistake was that in the first week itself, the control bottles were fed accidentally with 6 drops of milk instead of 1 due to which proper record could not be kept. We made 2 control bottles on the same day.

We could not feed the Moinas and take their micrographs due to holidays in between so we could not determine that when did (on what day) exactly the Moinas turned Red and when did they start turning back to colourless.

In all this, the Moinas turned Red two times but we could not isolate them and start our assay.
We need ~150 Red Moinas to start with.
Proper micrographs with the Control and Test Moinas in the same visual field, same comparative size need to be taken for their colour comparison.
We also failed to notice the gradient of colour change from Colourless to Pale Yellow to Yellow to Orangish-Red to Red.

These things will be kept in mind from the next time.

What happened to the Moinas from the set-up bottles?
On 29 Feb 20, we took the Moinas from all the set-up bottles (total ~100) and formed their single bottle.

The existing Moina bottle from which the New Cultures were made.

So, today we made 4 new bottles out of the above bottle because the Moinas were ~200 and their number would start decreasing (as observed previously) if not put into new bottles.
The bottles C1, C2 have 500mL DC/W + ~50 Moinas + 2 drops of milk
The bottles C3, C4 have 750mL DC/W + ~30 Moinas + 3 drops of milk
and the original bottle now has ~50 Moinas.

IMG_20200302_1348574160×2340 2.28 MB

The four new bottles made today.
The Moina bottles were fed today with their respective drops of milk.
The 8L tank set-up is being fed with 32 drops of milk and has ~150 Moinas

The Moina Cultures at CUBE Elphinstone.

So we can say that CUBE Elphinstone has around ~500 Moinas in total.

drishtantmkawale · March 2, 2020, 2:30pm

Research Question: How does the Low oxygen (Hypoxia) lead to the production of Extra Hb and is it due to Histone Remodeling?

Our Hypothesis:
In Normal Oxygen condition or Normoxia, when we are adding a single drop of Milk, the Moinas will appear colourless as the oxygen would be normal and there would be no need of extra Haemoglobin. But, when we will add more drops of milk, the bacteria in the culture would increase and the oxygen for the Moinas would less due to which there will be a need for the extra production of the oxygen carrier- Haemoglobin. In Daphnia (a relative of Moina), 4 genes dmhb1, dmhb2, dmhb3, dmhb4 are responsible for Hb production. In Normoxia in the cytoplasm, the Transcriptional factor indeed a protein HIF-1α due to prolyl hydroxylation gets degraded in the proteasome and synthesis of extra Hb will not occur. But, in Hypoxia, the mechanism for the production of Histone acetylases (HAT enzyme) will get activated which will acetylate the histone leading to the uncoiling of DNA and Histone. Also, the HIF-1α along with ARNT, will go into the nucleus and will bind to the Hypoxia-response elements (HREs) which are DNA segments present before the promoter region of the Hb gene. The HREs will activate and will guide RNA Polymerase to the Hb gene promoter region due to which transcription and later translation into Haemoglobin protein will occur and we will be able to see Red Moinas. But, when we will keep the Red Moinas in Normoxia, HDAC (Histone deacetylases enzyme) will deacetylate the Histone protein again, leading to the coiling of both DNA and Histone and hence Moina will turn colourless.

This is what has been discovered by the 2019 Nobel Prize Winners in Physiology and Medicine: Gregg Semenza, Peter Ratcliffe and William Kaelin. We have tried to relate the same discovery.

A graphic from the Nobel Prize-winning thesis showing the proteasomal degradation of the HIF-1 alpha protein in Normal Oxygen or Normoxia and binding of the same HIF-1 alpha protein to the Hypoxia Response Elements (HREs) in Low Oxygen or Hypoxia leading to the activation of the genes.

According to our earlier Valproic Acid experiment and as per the current hypothesis, the Red Moinas in the Colourless Control Bottle had turned colourless on the 4th day as there was no requirement for extra Hb to be produced. The Moinas in the Red Control Bottle remained Red throughout the experiment as 6 drops of milk was continuously fed to them and there remained a hypoxic condition which made the Hb essential to be there throughout. In the 5ug concentration, on the effect of VPA, the Red Moinas remained Red till the 4th day and then onwards, they started turning colourless and in the 10ug concentration, they remained Red till the 6th day and started turning colourless then onwards. This shows that there was an effect of VPA till the 6th day.
Further, we propose to perform a similar experiment and prove our hypothesis of Histone remodeling due to acetylation and deacetylation.

This is the Acetylation and Deacetylation {of Histone protein due to Histone acetyltransferases (HATs) and Histone deacetylases (HDACs)} I am talking about.
Source of image: Google Images

The design of the experiment would be shared soon!

drishtantmkawale · March 7, 2020, 5:47pm

While discussing in our College, we came to know that

HREs are DNA Segments where the transcription factor HIF-1 alpha binds and starts the transcription.

HATs are enzymes (proteins) which acetylate (add -ve charge) the Histone protein so that it repels the -vely charged DNA because of which it is open for transcription and then RNA Polymerase transcribes DNA to mRNA.

So, our question is What signals the HATs to acetylate the Histone?
OR
What is the link between Hypoxia Response Elements (HREs) and Histone acetyltransferases (HATs)?

Please help!!

drishtantmkawale · March 14, 2020, 9:39am

ALERT!!!
REALLY LOOOONG MESSAGE AHEAD

CUBE Elphinstone
Moina Group
@saida786110 @Zahra_R17

Research Question: How does the Low oxygen (Hypoxia) lead to the production of Extra Hb and is it due to Histone Remodeling?

Long term Objective: *To prove histone remodeling in Moinas using Valproic Acid as an HDAC Inhibitor

Short term Objective: To get Red and using them for the Valproic Acid Experiment.

Design for getting Colourless to Red
*Requirements:

~80 Colourless Moinas
6-500mL Plastic bottles
Milk
2 Litres Dechlorinated Water
~7 Plastic droppers or Pasteur Pipettes
Cotton for Plugging

We plan to make 6 bottles out of which 3 would be of Control and 3 Test.

Control bottles are the ones which would be having Colourless Moinas throughout the experiment.
Test bottles are the ones in which the colour change of the Moinas from Colourless to Red would happen.

Control
3-500mL bottles + 250mL DC Water + ~5 Colourless Moinas + 1 drop of milk in each bottle.

Expectations: Based on our previous observations, we expect the Moinas in all Control bottles to remain Colourless after a week too with them being ~70-100 in number.

Test
3-500mL bottles + 250mL DC Water + ~5 Colourless Moinas + 6 drops of milk in each bottle.

Expectations: Here too, based on our previous observations, we expect the Moinas in Test bottles to turn Red in colour on the 7th day with ~60-80 being Red and rest ~30-40 colourless.

As we are feeding them with 6 drops of milk, the water+milk bacteria would consume the dissolved oxygen (to be known yet) and comparatively less oxygen would be remaining for the Moinas to consume so they would start producing extra Hb (the reason for their Red Colour) for surviving the low oxygen conditions.

BUT TILL 2-3 DAYS THAT IS, TILL THEIR NUMBER IS LESS, WE WILL FEED THE TEST BOTTLE MOINAS 6 DROPS OF MILK ON ALTERNATE DAYS BECAUSE LOGICALLY THINKING, IF WE FEED 5-10 MOINAS WITH 6 DROPS OF MILK REGULARLY TILL 7 DAYS, THEY WOULD EXPERIENCE VERY LESS OXYGEN AND THEY MIGHT DIE. WE HAVE OBSERVED THIS EARLIER TOO. SO TILL THEIR NUMBER INCREASES FROM 5-10 TILL 30-40, WHICH TAKE AROUND 3 DAYS, WE WILL FEED THEM ON ALTERNATIVE DAY AND HENCEFORTH EVERYDAY.

They follow a colour gradient from turning Colourless to Red.
Colourless (till 2nd day)
Pale yellow (3rd day)
Yellow (4-5th day)
Orangish Red (6th day)
Red (7/8th day)

Design for keeping the Red Moinas in Red colour using Valproic Acid
Requirements:

~200 Red Moinas
12-50mL Sugar tubes (test tubes)
Milk
500mL Dechlorinated Water
~10 Plastic droppers or Pasteur Pipettes
Cotton for Plugging
Valproic Acid tablets

The concentration of the Valproic Acid tablets available commercially is 200mg.

So we will make a stock solution of the tablets by taking the tablets, crushing them and dissolving them in 200mL of Dechlorinated Water so it would become 1mg/mL.

We are planning to use 2 concentrations which are 5ug (microgram) and 10ug.

The concentration stock solution preparation:
For 5ug: 995mL DC Water + 5mL VPA (1mg/mL) stock solution.
For 10ug: 990mL DC Water + 10mL VPA stock solution.

12 test tubes would be prepared out of which 6 would be of Control and 6 would be of Test.

Here, we will need two types of Controls:

Colourless Control which would consist of Red Moinas not being treated with VPA and they would be fed with one drop of milk (until the experiment ends) so that their colour change from Red to Colourless would happen and we would be able to compare the Red with Colourless.
Constituents:
3-50mL test tubes + 30mL DC Water in each + ~5 Red Moinas + 1 drop of 1:5 dilution of milk (1 drop of milk diluted with 5 drops of DC Water)

Expectations: Moinas should turn colourless till the 4th day

Why dilution?
If we add directly milk drops in low volume of milk, the oxygen level would drop rapidly as compared to when we add 1 drop in 250mL DC Water.

How 1:5? (There’s some Maths in there… that’s why it’s STEM!!)
We add 1 drop of milk in 250mL DC Water as a feed for the Moinas.
So, 1:10 dilution (1 drop of milk diluted in 20 drops of DC Water) for 25mL of DC Water
1:5 dilution for 30mL of DC Water.

The concentration of milk and water or the dilution is inversely proportional to the volume of total Water.

Red Control which would consist of Red Moinas (these too, not being treated with VPA) fed with 6 drops of milk throughout the experiment so that they will be Red in colour throughout the experiment, their colour would not change and they would be easily comparable to the VPA treated Red Moinas for a colour change (if any occurs).
Constituents:
3-50mL test tubes + 30mL DC Water in each + ~5 Red Moinas + 6 drops of 1:5 dilution of milk (1 drop of milk diluted with 5 drops of DC Water)

Expectations: The Moinas should remain Red till the end of the experiment as we are giving them the same condition i.e. 6 drops of milk throughout so Extra Hb would be produced throughout

Test
5ug (microgram): 3-50mL test tubes + 30mL DC Water in them + ~5 Red Moinas + 1 drop of 1:5 dilution of milk + 1 drop of 5ug stock of VPA.

10ug (microgram): 3-50mL test tubes + 30mL DC Water in them + ~5 Red Moinas + 1 drop of 1:5 dilution of milk + 1 drop of 10ug stock of VPA.

Expectations: The Moinas treated with this concentration should remain Red for some days (we will find out that😬)

Regular Micrographs would be sent along with the Data.

The 500mL plastic bottles which we’ll be using for the experiment

drishtantmkawale · March 19, 2020, 7:11pm

CUBE Elphinstone, HBCSE
Moina Group
@Zahra_R17 @saida786110

On 16 March 2020, i.e. on Monday, due to the COVID-19 outbreak, we had brought our Moina Culture bottles CUBE Elphinstone to CUBE HBCSE.
But now, the situation is getting worse day by day so we decided to bring the bottles home as we won’t be able to travel t HBCSE regularly.

Although our Short-term Objective was to get Red Moinas
For that, we had prepared bottles on 14 March.
But now, we had to bring them home.
So, we can’t take the micrographs at home.

So, the only thing which can be done at home is feeding them milk and discussing about the experiment via online causerie.

The bottles brought by me from CUBE HBCSE.
And 3 other bottles have been carried by @Zahra_R17 .

Rest all the Cultures are kept at CUBE HBCSE itself which are going to be fed by Nagarjuna Sir. @GN

drishtantmkawale · April 20, 2020, 3:50am

CUBE Nerul, Navi Mumbai Home Locality Lab
Moina Model System

20 April 2020

Lockdown Time!

Research Question: How does the low-oxygen condition (hypoxia) change the colour of the Moinas?
Short-term Objective: To prepare a Red-Moina Setup at home during the lockdown period.
Long-term Objective: To prove Histone modification due to low oxygen in Moina using Valproic Acid.

To some, science at home during the lockdown period seems impossible!
But for me, it is not so difficult to maintain my Moina culture atleast.
So, planning to prepare a Red-Moina Setup!

Design

Design of the Red-Moina Set-up
8 Plastic Cups, Milk, ~2L Dechlorinated Water, Cloth pieces, dropper, Moinas!

Thought of taking 8 cups.
4 for control and 4 for the test.

*Control?
200mL Dechlorinated Water + ~10 colourless Moinas + 1 drop of milk daily.
4 Controls so that each would be a replicate of one another.

Test?
200mL Dechlorinated Water + ~10 colourless Moinas + 6 drops of milk (not daily but on the alternate days till their number increases. If I add 6 drops of milk in 200mL of DC Water daily and as their number would be less, the water will become turbid, more bacterial growth will be there, very less oxygen would also be there as the bacteria will multiply. So, less Moinas won’t be able to survive. So till the 4th day when their number will become ~50-60, I will add 6 drops of milk daily.)
Here too, 4 test cups will be replicates of one another.

Dechlorinated Water?
Half a bucket of tap water kept open for a day, we will get DC Water!
As the chlorine would evaporate, that Water would be Dechlorinated the next day!

Why 200mL Dechlorinated Water?
The cups are of more or less 200mL in volume.
Although the method which I and other CUBists follow is 250mL of DC Water + 1 and 6 drop(s) of milk, I will use 200mL of DC Water + 1 and 6 drop(s) of milk for maintaining Normal Oxygen Concentration or Normoxia and Low Oxygen Concentration or hypoxia respectively.

Why am I doing this?
I thought of giving the Moinas some work and see that how does it go in my home lab.
Earlier, I have tried this in CUBE Elphinstone, Mumbai as well as CUBE HBCSE, Mumbai

What will I prove after this experiment?
I will prove that the Red Moinas adapt to low oxygen condition (hypoxia-provided by me by adding more milk) by turning into Red Moinas.
This Red Colour would be due to the extra production oxygen-carrying protein pigment Haemoglobin.

How is adding more milk inducing hypoxia?
Milk contains bacteria as well as Carbohydrates, proteins, fats, vitamins and minerals which will act as a nutrient source for the bacteria.
We should note that the Dechlorinated Water which I will be using will also have some bacteria which will feed on the milk (as their nutrient source) and replicate.
So Milk bacteria + Culture Medium Bacteria will consume more oxygen as compared to the Moinas due to which less oxygen would be remaining for the Moinas to consume.
And now, to consume this less oxygen, the Moinas will start synthesizing the extra-haemoglobin through its four globin genes due to which they will appear red.
Because I am adding 6 drops of milk alternatively/daily, the hypoxia would be maintained throughout.

According to a 2004 Research Paper on Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent by Thomas Gorr and team in Journal of Biological Chemistry, it says that there are 4 globin genes present in Daphnia magna (a relative of Moina, that is why we are assuming the same in Moina too).

All four will be actively synthesising Haemoglobin in Hypoxia and in Normoxia, only a single gene would be active.

The quote from the Paper
“Protein products of the hb3 and hb2 genes are strongly induced by hypoxia, whereas the up-regulation of the hb1 gene is only moderate…”
Which to me, it means that only a single gene would be active in Normoxia!

This in-turn relates this to us to the 2019-Nobel Prize-winning Work by William G. Kaelin, Jr., Peter J. Ratcliffe and Gregg L. Semenza for their discoveries of hypoxia and how cells sense and adapt to oxygen availability.

You can correct me if I am wrong (I should be😬)!!

The link for the Paper:
https://www.jbc.org/content/279/34/36038.full

My expectations at the end of the 7days or a week?

The 4 Control cups having 200mL of DC Water + 1 drop of milk daily + ~10 colourless Moinas (initially) will have ~60-70 Moinas with all the Moinas being colourless.
The 4 Test cups having 200mL of DC Water + 6 drop(s) of milk alternatively/daily + ~10 colourless Moinas (initially) should have ~100-120 Moinas in total out of which 60-70 should be orangish-red/red and remaining would be colourless/yellow.

Expectations for 21 April 2020?
Control as well as Test: ~20 Colourless Moinas in all the cups.

All these expectations are on the basis of the data which I got previously.

Photos, as well as Videos along with the updates, will be posted!

drishtantmkawale · April 20, 2020, 8:12am

The 4 Control Cup cultures C1, C2, C3 and C4 with 200mL Dechlorinated Water + 1 drop of milk + ~10 colourless Moinas each.

The 4 Test Cup cultures T1, T2, T3 and T4 with 200mL Dechlorinated Water + 6 drops of milk + ~10 colourless Moinas each.

Video showing the movement of the Control Cup Moinas towards light.

Video showing the (very less😬) movement of the Test Cup Moinas towards light.

These cup cultures are covered with a cloth.

drishtantmkawale · April 21, 2020, 10:33am

CUBE Nerul, Navi Mumbai Home Locality Lab
Moina Model System

21 April 2020

Update of the Red-Moina Set-up prepared on 20 April 2020
Breaking News!

The Moinas in the Test cup T2 have turned into Pale Yellow on the second day itself where it takes ~3 days to see a colour change!
The Moinas in the Test cups T1 and T4 (containing 200mL of Dechlorinated Water + ~10 colourless Moinas + 6 drops of milk fed yesterday) have died.

Rest all the remaining 6 cup cultures have Moinas.
Control: C1- 12 colourless C2- 13 colourless C3- 15 colourless C4- 8 colourless
I expected there to be around ~15 colourless Moinas in each cup.
These expectations have been met more or less.

Test: T1- No Moinas T2- 10 Pale Yellow coloured Moinas T3- 2 colourless T4- No Moinas
Here too, I expected the number of Moinas in each cup to be around 15 with all being colourless.
But, the cups T1 and T4 do not have any Moinas and T2 and T3 have not met the expectations.

Expectations for 22 April 2020
Control Cups: ~20 colourless Moinas in each cup.
Test Cups: ~20 Moinas in total out of which 10-15 should be pale yellow and rest colourless in cup T2 and ~10 colourless Moinas in cup T3.

Video showing ~15 colourless Moinas in the Control Cup

Video showing ~10 Pale-Yellow Coloured Moinas in Test Cup T2.

Video showing a single Pale-Yellow coloured Moina from the Test Cup T2

drishtantmkawale · April 22, 2020, 10:23am

CUBE Nerul, Navi Mumbai Home Locality Lab
Moina Model System

22 April 2020

Update of the Red-Moina Set-up prepared on 20 April 2020
Breaking News!

There were ~15 Moinas in the Control Cup (200mL DC Water + 1 drop of milk) yesterday, but today there are almost 30!
The Test cups T2 and T3 having 200mL Dechlorinated Water + 6 drops of milk have just 2 pale-yellow coloured Moinas and Test cup T2 had 10 pale-yellow coloured Moinas yesterday, but today there are only two.

Control: C1- ~25 colourless C2- ~15 colourless C3- ~30 colourless C4- ~25 colourless
I expected there to be around ~25 colourless Moinas in the Control cups.
These expectations have been met more or less with Cup C3 having ~30 colourless Moinas! which is more than my expectation!

Test: T1- No Moinas T2- 2 Pale Yellow coloured Moinas T3- 2 Pale Yellow coloured Moinas T4- No Moinas
Here, the number of Moinas have come down 2 from 10 in cup T2 and the Moinas are same in number in cup T3.

But, the cups T1 and T4 do not have any Moinas so I haven’t discarded them but I have kept them to look for Moinas if there might have been some resting eggs (epiphium).

Expectations for 23 April 2020
Control Cups: ~35 colourless Moinas in each cup.
Test Cups: ~10 Moinas in total out of which ~2-3 should be pale yellow and rest colourless in cup T2 and T3.

The Moina on the left side is pale-yellow coloured (from the Test cup) and right side one is colourless (from the control cup).

~30 colourless Moinas from the Control cup C3