So there are school students as well as college students working on earthworms, so they will be talking about how do they culture the earthworms and what work they some college students who are collaborating with them, they are doing some studies on the earthworms, so studies like regeneration studies, so they will be talking about it, so I will just Abhijit Abhijit, Abhijit and your group. Myself Vedan, I am studying in 7th standard, I am from Adarsh Vidyalay. My name is Mithali, I am studying in standard 7th, I am from Adarsh Vidyalay. My name is Mithali Kamre, I am studying in 8th standard, my school name is Adarsh Vidyalay. My name is Satarva, I am from Adarsh Vidyalay school, I am studying in 7th standard. Myself, I am studying in standard 8 and my school name is Adarsh Vidyalaya. My name is Mitesh Kamre. I am studying in 8 standard and I am from Adarsh Vidyalaya School. We are from Adarsh Vidyalaya. We are working on Arthuam project. What is an Arthuam? Arthuam is an organism which we can found under the big stones, in the soil or mostly we can found in rainy season. Objective, immediate term objective is to catch Arthuam by soap or water method. Our short term objective is to culture Arthuam in different mediums like tissue paper, filter paper, magazine paper and newspaper. Our long term objective is to find which culture they live more efficiently. How to catch Arthuams? First, we have to take liquids of solution. Then we have to pour the liquids of solution on the surface of the ground. After that we have to wait for 15 minutes. Then the Arthuam will come up of the surface. We have to catch the Arthuam with the help of the paint brush. Then we have to keep the Arthuam in a tray and watch them as much time as possible. Because we have to remove the liquid soap from the body of Arthuam because they can harm the Arthuams. Now, how to make a soap solution? First, we have to take 1 liter of tap water. Then we have to add 10 ml of dishwashing liquid in it. Then we have to mix them in a conical flux. Then we have to pour it on the ground. After that Arthuam will come up on the surface. We have to catch the Arthuam with the help of the paint brush. Then we have to wash them. Thanks. To make a cup culture, first we take 2 plastic cups. Then make a hole into the bottom of the plastic cup. After that put some wet tissue paper. Then make the Arthuam and put them inside the cup culture. Then next put 15 layers of filter paper on the surface of the tray. Make it wet by some water. Then cover it with muslim coat. Your cup culture is ready. Observation. Our observation for the objective where the Arthuam which in the tissue paper were alive in excellent condition. The Arthuam which in the filter paper they are alive but one of them are dead. The Arthuam in the magazine paper they all are died because of the less weightness of the magazine paper. This is our result for the Arthuam observation. Conclusion. From this we see that the Arthuam alive in the tissue paper they are in excellent condition. Our future plan is that it is to see the Arthuam how they are regenerating. Do you all have any question? Please ask. Thank you. Yesterday we had a visitor in the lab. Her name was Vidyut. So she mentioned that it is not necessary to pour soap water in the soil to collect the Arthuam. So she suggested some alternative methods. She has been quite a geek. Though she did not actually go to any college and study biology she knows a lot of things. Possibly she is the only person in entire Mumbai who grows insectivorous plants in her kitchen garden. So very soon we are going to have insectivorous plants in cube as well. So because she is going to help us she is going to mentor us how to do it. So she told us when we told about this method of Arthuam collection she said that what you just have to do is go to the garden and put some cotton that you know you get this backing material such thick paper kind of thing. Just put it on the water and just leave it damp at the end just leave it for 2-3 days. After that when you lift the paper most of the Arthuams are already just laying just below the cotton sheet which is also already been eaten and decaying and things like that. So you will see the Arthuam already coming there and so I want you to test this. So design an experiment and try to check you know it could be let's for example just some newspaper or cotton or whatever it is. So just keep it on a wet damp soil leave it like that for few days and then disturb it only after 2 days but otherwise you know you can keep checking what happens under the thing so if this method is actually successful or not. So I am not saying that soap water is not the method because that is also widely discussed all over but suppose if something like this is done that means we don't have to spoil the soil with soap. It's much more organic and sustainable method of doing things. So let us see whether that will so that can be can you make that as another objective of your experiment next time. No no you don't have to do that. People who shop around in your vicinity will give you those packings. Otherwise you know they are thrown away anyway in the dumping ground so you don't hesitate. Wear some gloves and pick up that waste paper and then put it on the soil and use it. Reuse you don't have to do any online shopping for that. Good afternoon everyone I am from Elfiston College myself Manasvi Dekate and my colleague is Rushi Kish. So we are going to present here is developing E. Fettida earthworm as a model organism for cancer biology. So what basically what is our abstract. So how are we going to plan our model our experiment to make its cancer biology model. So we everyone knows that earthworm usually regenerates or everybody knows that earthworm regenerates. So the basic thing is when the day if we amput an earthworm what happens first is there's a bleeding. So basically to stop that bleeding first thing happens is the clotting clotting of blood. Later a group of cells we can say a group of cells come together and goes for de-differentiation. Now what do de-differentiation mean de-differentiation is a group of cells goes into its initial position or initial cell which do not know its own function. It's basically they don't know any function but they are just going and dividing. So it's de-differentiation and have a growth in a very controlled manner. So basically when we amputed our earthworm they restored their lost segments and in a very controlled manner. Whereas in cancer what happens is the cells goes into de-differentiation but they don't have any control on their cell division. So what our objective is that if we can understand a molecule which is responsible for having a growth in a controlled manner and that molecule if we induce it in a human cell as a cancer therapy then it will be a very effective therapy or a treatment for human. This is our long-term objective. So our immediate objective is to basically to maintain a cup culture of an earthworm. Now the short-term objective. So our short-term objective is to understand how the regeneration takes place. It's like if we consider an earthworm and first earthworm if I am imputing at 24th segment and the second earthworm if I am imputing at 34th segment. So basically 24th one is also going to regenerate whereas the 34th one is also going to regenerate. But the rate of restoring that segment will be different. So to understand that at what rate it's restoring or how much time it requires to restore that segment so that we can understand that okay on 24th if it's restoring faster than 34 and basically taking our time also considering our time. So we'll understand that okay the molecule or the factors which are having a huge effect for regeneration are present on this segment and not on this segment okay there will be presence of molecule but not at that level whereas it will be present on 24th if suppose like if it's restoring very fast. So this is a short-term objective to understand how the regeneration is taking part in an earthworm and the long-term objective developing an earthworm as a model organism for cancer biology. So basically I am going to explain to you the experiment what we did for this experiment. So we took three sets of earthworms and first set was for the head amputation, second was for the tail amputation and the third was for the middle amputation. What we did in head amputation we know that the earthworm like earthworm has two sides that is anterior which is head part and posterior part that is tail part.  So we took three earthworms for one set and so on for the tail amputation and middle amputation. Similarly we amputed for the tail amputation we amputed from the tail side 8th segment tail side from the 8th segment that is the smaller part and this is the bigger part and we name that set we name that set as TA1, T stands for the tail amputation, A stands for the anterior part like a TP1 like a T for tail amputation, P for the posterior part and 1 for the experiment of number of the earthworm. And for the middle amputation we actually there is actually as a goof up we actually didn't measure the exact number of segment which is present on the earthworm. So we approximately took 108 segments that we actually counted under the microscope dissecting microscope and for middle amputation we did actually half of the full segments like 108 then it become 54 segment. So we cut at the 54 segment here like 53 and 52 that is our 3 sets ok the 54 segment and we named that parts as a eKC, M stands for the middle amputation and A stands for the anterior and posterior parts like we here we can see this is the anterior part which have clientele and head and the posterior part which have the anal pore ok.  If we cut at the middle segment there will be a 2 earthworm from 1 earthworm like here you can see this part like M A like we can see that M A 1 this earthworm will regenerate all the anal segments and also gut area and this also this posterior part also regenerate clientele and head ok. The protocol for amputation what we take that we take a petri plate here you can see that we petri plate and we place a ice between 2 petri plates ok we place earthworm on the petri plate we put it under the dissecting microscope and then you have to magnify that so we can see that easily and here is the one precaution what we made is that for amputation we took a blade which is swapped with the ethanol so there can be we are going to cut earthworm like we are cutting the wire so if we so sorry that would be gently like a wire and if ethanol swap ethanol swap blade will be good for the there will be no good bacteria for on the blade and that can infect the earthworm ok. The protocol for observation you can see that is me yeah ok. The protocol for observation take a petri plate it is similar like a it is half similar like a protocol for amputation but take a petri plate take a ice in between the petri plate and here we added one like option we add a tissue paper on a like petri plate here you can see that and with the help of paint brush that is smooth smooth you can you have to use smooth paint brush ok so smooth with the help of smooth paint brush we remove the earthworm from the cup cultures and place it on a tissue tissue paper and here you have to make a measure that you keep sure the earthworm skin is wet because they respire to the skin and then observe it under the dissecting microscope so here minus we can explain the report. So report we actually took a report for 94 days after the day post amputation so this is a first report where we find out something interesting is the smaller part which we showed the first image HA2, HA3, TP2 and TP3 died after 5 days and some smaller parts were still alive after the 5 days. So basically we do not exactly know the reason why they died why they did not restore this segment because they have the property for regeneration what we thought but they did not restore and the remaining part we can say that the smaller part died and the larger part which were present the larger part or the larger segments were alive and started regenerating as we showed you can see the small mark how they started regenerating after 5 day after post amputation 5 day post amputation then on 30 10 2017 that is 38 day post amputation what we observed is the tail part the tail anterior part restored 25 segments instead of 8 segments with forming first anal pore and then the segments started regenerating so first they formed first anal pore and also in the HP2 that is head amputated part they restored first mouth and something interesting was they restored 6 segments instead of 8 segments and the third part which is the middle posterior part was alive but did not showed any regeneration and also there was no presence of mouth which was really very interesting because they did not had a mouth for feeding still they were alive for 38 after 38 day post amputation then we have 94 day post amputation that is 24 12 2017 what we observed is the as I said the middle part which did not had mouth nor he has started regeneration was still alive and also to make sure that are they feeding so we make sure that is there any fecus on our cup cultures so there was no presence of fecus so basically we do not know how are they still alive without mouth how are they taking energy or how are they feeding and they are still alive and you can also observe that the health of this and the health of this part is same so basically they are feeding on something they are not in a drought condition but we do not know how so these are the interesting question after one set of experiment the first is what is responsible for alive middle posterior part the 94th post amputation without mouth second question is what is responsible for the growth more than 8 segments in the tail amputation earthworm that is 25 segments they restore 25 segments instead of 8 segments so what is responsible and third question what we arise is is clitalin responsible for regeneration now we have shown the diagram first you can observe that the shorter part died which did not had a clitalin but the posterior part was alive which had a clitalin again in the second part tail part we observed that the posterior part which did not had a clitalin was died but this part had a clitalin and it was alive and it started regenerating and in the middle part we observe that this part started which has quite a lot, but this part didn't had any regeneration and it was alive till 94th day. So, our goof ups. So here we come with our goof ups. First, we didn't maintain our cup cultures properly like we didn't water the tray daily or the interval of three to two days. And second was is a we didn't took observation at a specific days like you have to be standardized for the observation like we we took observation randomly when we got a time and and all holidays and all and third way we didn't took any exact photos or videos for the how we did amputation. So and fourth we didn't measure exactly that what I told earlier exact number of segment and what is a approximate number of segment. Fifth is a we didn't measure that was a major goof up what I think we didn't measure exactly on which segment is clitalum is present for the all for the for the test and control part okay that can be serve that can be serve as a marker for like a measuring other segment from the from the clitalum and six was after 38 day post amputation we observed directly on the 94th amputation not 94 okay 68 day amputation. So we unable to find out the regenerator segment between this interval the coloration and pigments and all the same that what the regenerator and that what that was prior present they are they are like same color so we want some help here for the future plans what how can we identify the regenerator part after this so long time so it it like a looks like the same okay so here are the further plans. So what we thought a major question is is clitalum responsible for regeneration so what we are thinking or what we are standardizing our experiment is will take an one earthworm which will be which will be amputated at 24th segment generally clitalum starts at 25 or 26 segments and ends at 33 or 32 segment this is our own observation so will first am amput at 24th segment and will see will keep it into a respected cup cultures different different then again will take another earthworm which will be amputated at 34th segment so now this is the type anterior as well as the posterior will be having two controls for each type of amputation and experiment will be carried out for with the three earthworms so this is our future plan so our hypothesis is the posterior part of the anterior amputation should regenerate and the post the anterior part of the posterior amputation should regenerate this is our hypothesis for the further plans and this is our reference. We will start your questions for now we will take Simran and Arjun's presentation on earthworm itself a different approach towards it okay so we'll listen to them and then we can have a combined discussion on this group and Simran and Arjun's group okay. A very good afternoon to all our topic over here is earthworm as a model organism for the spinal cord injuries in humans so as we know a spinal cord in humans is a very important organ for it for the vital movement and movement of an of the body so similarly in earthworms we see there's a presence of ventral nerve cord in the earthworm which is equally functional as in humans so the function of these spinal cord remains the same there if there's any injury that may lead to paralysis as we know in humans as well any injury to the spinal cord may cause complete paralysis of the body so as we can see injury to the spinal cord of the earthworm as well which is a ventral nerve cord may cause complete paralysis of the earthworm there's no coordinated movement between the segments of the earthworm so that leads to the complete paralysis of the earthworm and the earthworm cannot move so we've seen that the regeneration of this ventral nerve cord can actually lead to the restoration of the function and can cause movement of the earthworm again so basically our immediate objective would be to standardize the process of RER which is rapid escape response so that we can conclude a proper conclusion to our experiment. Our short-term objective would be to determine the role of wind signaling pathway which is a pathway responsible for regeneration of the ventral nerve cord and our long-term objective would be to develop earthworm as a model system for injuries and spinal cord in earthworms as and then further take it to humans. So I'll be explaining what is rapid escape response that is our experiment so first I'll tell you we take our distilled water which is our control and then we take the NACL solution I guess some mistake is there so it's not able to see so first I'll talk about the control what we do so due to some technical reasons the videos are not able to play so this is the control what we do can you see here a test tube in a test tube there is 3 ml of distilled water and then in that distilled water we add we introduce our earthworm okay so we remove the cup remove the earthworm from the cup culture we wash it thoroughly so as to remove the dirt and the fishes and all that thing we wash it and then we wash it with distilled water specifically then we tap it on a folded tissue paper so that the excess water should drain out and the volume of the test tube would change okay so after doing that we introduce the earthworm and with the soft tissue paper obviously observe it for two minutes now here the distilled water is not harming the earthworm as such so the earthworm will be like okay the environment is cool and staying here I'm relaxing here okay so we observe it for two minutes and the earthworm is not escaping from that solution from that test tube okay so this is for the control now we go for the test that is what we do we take NaCl solution salt solution with the concentration of 200 millimolars okay so what we do we take the same earthworm and we introduce we sorry we introduce that earthworm in the NaCl solution like seen here okay this is for the NaCl solution we introduce it in the test tube and we dip it completely in that test tubes okay and after that now here the NaCl solution is creating a problem for earthworm like it's giving irritation for the earthworm so the earthworm will obviously try to escape from that test tube from that solution so as to know not get irritated or something like that so we do this experiment this experiment is RER assay rapid escape response assay so this first two diagrams you can see the videos are not there so this was done before lesion or before lesion is we give lesion to the ventral side so as to cut the ventral nerve cord of the earthworm and this assay was done before that to see okay to specially choose those earthworms which are fed to which are not fed and we can further use for our experiments so after done with this we do it for like five times we repeat this experiment five times with the same earthworm later on we give a lesion as Manasui and group had done the amputation in the same manner we give the lesion we give lesion in the central of the earthworm like we don't exactly count to the segments but we approximately give a lesion at the center and here while giving lesion we have two controls with us one is one the earthworm which is not lesion like no harm done to him is perfectly fit the another one is the dorsal side lesion which is again our control now the dorsal side because there is no ventral nerve cord as a name suggests so at the dorsal side there is no nerve cord sorry and the third one is that test which is a ventral nerve cord lesion okay so we give a lesion and then again after one hour we do the same experiment with the same earthworm okay and we have been observed that in distilled water it's again not harming not doing anything so the earthworm is like relaxing over there after two minutes we take it off we drain the excess water and then introduce the same earthworm in NSEL solution now what happens here as in earlier before lesion the earthworm was escaping but here the earthworm was not able to escape why now the question was like if I'm giving a cut to the ventral side and the nerve cord is very close to its skin so the nerve cord is also getting cut due to which there is no coordination in between all of his segments the starting segments are telling the people to go ahead but the people are not ready to listen to him so this is the way they are not able to coordinate the segments are not able to coordinate and hence the earthworm is not able to move out he is trying to move but is not coming completely out from the solution okay so this is what we have done this is the experimental design further goes to experimental sets like we have done four sets or not four sets we have done this experiment four times with different different earthworms now in the last experiment we have done with very less goof ups as I think so so the total earthworms we took was 16 out of which 10 like the size was 10 to 12 centimeter the species was if it those were clitoral earthworms and now for ventilation test we used E1V that is earthworm number one ventral so as to know don't get confused we should then for dorsal lesion E1D D for dorsal and unleashed is UN that's again our control so the results I have not mentioned it properly because our results were like so the most of the earthworms took between 1.30 seconds to two minutes to escape from that solution from NSEL solution specifically and the earthworms which took more than two minutes as I mentioned we used to consider them unfit like they were not used further for our experiments now the restoration of the functions after the VNC lesion this experiment was performed four times till now every time we used different different earthworms now the first time when we did experiments were not like too much aware so just Karliya then we saw the restoration within two days like after lesion we did it after one hour we did the same essay and then next day we did it again and then we saw that okay the earthworm was escaping from the NSEL solution so this was done in June sorry May then we moved to a second time that is the second time when we did this experiment we saw it in 10 days like we were like first it was two days and now it is taking 10 days so again there were some goof ups which we realized third time again it took 12 days which was done in November and the fourth time was done in October this year so not October yes yeah October so again it took two days so we are like quite confused where are we going wrong we are doing many problems sometime we also blame the animals due to which I don't know sir yelled at us so so right now a future prospective is to determine the role of wind signaling pathway in VNC regeneration by exposing bugger bugger bugger so minus we has told you about the differentiated and the non-differentiated cells so basically there are some cells suppose if I am put it the earthworm there are some cells so that cells might have been also like exposed out so they released some chemical XYZs chemical and the other cells which are in a proper may which are given their proper function those have some XYZ receptors this chemical gets attached to that receptors and some signaling is done so basically that signaling which tells you to regenerate so that is called as a wind signaling pathway we learned we learned it in our third year now so we need to first know more about this wind signaling pathway and then we are going to use LICL lithium chloride which is an inhibitor for the wind signaling pathway so if there is a role of wind signaling pathway we could like by using LICL if it's inhibiting so we could say that okay the wind signaling pathway is taking place so acknowledgement will be to Yusuf Mahiralli Centre as there has been providing us with the earthworms and the acube lab as well so here I'm done so any questions so the session is open for questions for this for their presentation being a very interesting model system to study a lot of things we ourselves started with earthworm in our first year in as a part of cube so first thing I would like to ask you like when you do the lesions so you make sure that the two segments are not apart like right they should be together but still it's lesion so how you make sure that whatever amount of like lesion you are putting it's just distorting the nerve cord but not basically we are not able to like know how deep the cut is but we are making sure like the nerve cord should only cut and even though the rest of the body is cutting I don't think so that should be where exactly are cutting it from from the dorsal side or the ventral side basically on the ventral side also we give a cut and on the dorsal side also we give a cut as it's our control and the ventral is are the test so we are basically on giving a lesion we are like keeping on a dissecting microscope and each a petri plate and eyes palette the scope and observing in the microscope we give a lesion directly one thing I wanted to like bring to your attention is like from there they can tell when you cut first anterior eight segments or posterior eight segments and when they are just like totally apart from the animal have you seen those small segments anterior or posterior they actually move on the petri plate they try to wriggle they try to crawl they try to move right but in case of and also I would like to suggest that whenever you guys do something on earthworm drosophila cl against or any model organisms try to find like a and dc has put a very good study on earthworms and anterior posterior segments yeah in fact they have done the identification thing and the regeneration also they have done if you see the photos and the videos it's on meta studio also yeah so my question is in in case of humans like he was saying if there's a spinal cord injury so we get paralysis so in paralysis it's not like we are not able to move that organ or the limb totally it's not like we can just move but we cannot walk but in case of earthworm if we impute it totally apart from the organism that segment stills wriggles actually it is walking so there's no concept of paralysis in earthworm right basically in each segment there is a set of ganglions like okay so there might be some signal suppose the last segment is there and the first or the third segment is there okay so now this person has to move up from the tester from the solution so obviously I have to you know drag all his body up okay you might be knowing how earthworm moves okay so there is a contraction and the relaxation of the segments so if there is no contract session and relaxation how will the earthworm will completely come out that's what I'm saying like are you hypothesizing that the whole signal to contract the whole organism and relax the whole organisms comes from one center like in human it comes from one particular center yeah so that's why if you put a lesion in between you are blocking the road and the whole organ other the rest part is paralyzed no no but in case of earthworms in case of earthworms as you said there are many ganglions spread all across the organism so and and we call them segmented organisms and that is why they are called segmented because each segment in some manner can take care of all the functions which is occurring in the other segments that's why they are called segmented so what I'm asking is even if I cut the head part the interior all the segments before clitalum still that rest part can move so where the concept of paralysis and nerve cord injury is fitting basically what happens the down part the posterior part I'm giving a lesion in the middle the posterior part will might move in some other direction like this guy wants to go up but this is not getting that signal to go up he may move down he may also move up so there is some coordination that okay you have to come in one direction not you have to go in some other direction so that would be something that coordination can only like it can come from anterior and posterior you know why we call something anterior of our organism because most of the sensory organs are present towards that side we call it anterior it moves towards that side any animal human cows buffaloes any animal you see they often move towards their anterior side because they have most of their sensory organs there so in case of earthworms they have their mouth and major six for six ganglions there so if you see go through the anatomy of Isna Fetida which we did in the cube A and D C so we'll come to know that most of the sensory parts are there in the towards the mouth part so we call it anterior that's why when it moves towards one side all the rest of the body comes so I agree with the fact that there there is a coordination like coordination comes from the anterior part but I'm asking like where does the model of spinal cord injury as a paralysis fits in because the rest of the part without an interior part is also moving it's not paralyzed right it is moving but moving in a different direction that I agree but it is moving so it's not paralyzed there's no concept of paralysis in earthworm don't you think that yeah I would say that like I never thought of that so I think I should think about that before reading the VNT I would suggest you you should read about the how earthworm actually moves and where the ganglions are actually so that you can better decide where to put a cut so that some of these segments we can study like whatever objective you are trying to study maybe you can study it but in restricted segments or through a restricted cut and how deep should be the cut because all these things forms the basis of what you're going to study yeah what if you are putting a cut but the spinal cord is not injured at all yeah yeah and you are thinking that it's moving or it's not moving and what if the earthworm is not only moving because it's injured sometimes when organisms are injured so that's because we are giving a dorsal lesion like as you are seeing like it's because of the injury he might not be moving but in dorsal like there's no ventral cord so it should move properly so for dorsal control we have seen that it is escaping from that solution so personally we don't think that injury is a major thing for that why don't you try like you put a lesion in the middle or you totally impute the earthworm in the middle and then see for the two remaining parts the anterior and the posterior what is there like do they escape from the solution have you tried this no we haven't tried if both of them escape from the solution like then you have to reframe and remodel the experiment right you should test it first cut the earthworm in the half put both the halves in a solution because both the half will wriggle okay so do they come up or not okay then we could take help of coming back to your previous question the ventral nerve cord in the earthworms quite much resemble spinal cord in the human beings so if we basically try to figure out what is the mechanism of regeneration in the earthworms we can probably correlate it to human beings and then lead to the restoration of the spinal cord in human beings as I totally agree with that but what I'm saying is in human beings the signal maybe it's the signal is coming from one center right for the locomotion the voluntary movements which we are doing except for the reflexes the signal is coming from the center the brain like if I want to lift my arm I need my brain to active but in the earthworm like she said there are different ganglions which can independently give a signal to a segment to move so where you are putting a lesion is very important if you're putting a lesion and on both sides there is a ganglion that lesion is of no use it's not discontinuing the signal right whereas I think even if they put a lesion between the two ganglions like there is a ganglion here and over here and they put a lesion in between even that will work because even in that case there won't be the coordination between the suppose the right side ganglion and the left side ganglion there won't be the coordination then neurons over there will be cut so how will this part know that where it has to move that was the basic set they don't stop moving even in their RER assay they don't stop moving at all but what we have observed from I don't know the videos are not playing but what we have observed is that they do move but it is not very coordinated movement both the parts move but it is not coordinated in order to help the earthworm escape from the test tube so you have seen that right yeah so if I just remove the mouth part it is is it same as I am imputing it from the middle because it is moving to feed right exactly what I am saying is but in this case it's not moving to feed right it's mouth is not there mouth is not even in the earlier in this experiment it is moving just to escape from that irritation which is for that I suggest I don't think I don't think even for that it should move towards the mouth only it can move in any direction just to escape from that irritation that will hold for the interior segment as well right the interior portion exactly that's what I said you check the two halves do they escape like in amount of time or not yeah this we should try obviously you can think of like where to put a lesion so that the other part is not moving at all and if we can restore it through like you can collaborate with the regeneration team that whether it regenerates those ganglions and can move now okay that's it okay we have my question to Manasvi didi and Rishikesh dada why you're putting keeping eyes on petri plate yeah that's a nice question I don't know the proper reference to this but I think that if we put eyes on our hand for a for a long time so the skin will numb numb and numbness and we are already comparing the earthworm with the like humans and human cells so why can't we compare this criteria to the earthworm only so that and we tried that and it's gone successful like they stand they they like a did they stop crawling on the eyes so we did that experiment for them they are just doing that to stop earthworm is moving vigorously so while imputing you have to they have to count the segment and impute so they don't want earthworm to move continuously so it's just to like I want to ask the question to a first group for cutting the earthworm why only ethanol was taken as the antiseptic that for so you can use any decent victim so basically we want a blade free from bacteria's and microorganism so we you can add you can use any disinfectant so is easy that I have a question you said yeah yeah so our 38-day observation in one case it happened like 25 segments were regenerated and in other case only six right of the eight segments you cut so I don't think it is means the regeneration is as you want it to be specific so are you still considering this as a model for regeneration where you want it to specifically regenerate only eight segments the camp for cancer biology right you don't want it to regenerate more than the eight segments so you're still considering it as a model yeah as you earlier said that there has to be a controlled growth you mentioned in the very beginning that there has to be there is a control growth in regeneration but what we are seeing from your observation that it is not controlled but it's growing beyond the amputated parts that's what they had said that they had they observed that they cut eight and it grew into 25 segments so among those three the one you said it grew 25 it regenerated 25 what about the rest two so the tail part which we showed it restored the last segments like 25 segments so the remaining two so the remaining two earthworms to restore 10 and some restored 12 okay so this is the anterior part of the tail amputation so the first what was the question was that they restored 25 segments from here the anterior part the bigger part so they remaining two we expected it but we got 25 we have two more replicates yeah it was not yeah it was not specific 25 but it was more than it that is 10 and 12 okay so what you are claiming is that more than what was amputated the regeneration took place regeneration not only restored but added more segments than what has been amputated yeah when the tail piece was amputated right okay so that's the only generalization you can take not 25 out of this thing but more than what is more than what is amputated was restored and regenerated in addition to restoration if you read about it why it happened like so I'm not going to give you the answer but it there's a very interesting thing about regeneration like you know about plenaria it's the most studied organism for regeneration models so in plenaria there's like whatever from whatever orientation you cut we came through this in cube by a goof up what we did when you used to impute the earthworm used to impute it like horizontally with respect to the segment okay if you somehow by mistake impute it diagonally like some say three four segments are being cut so do it intentionally and see what happens so what do you expect is if the earthworm is like what you're drawing it's in a horizontal plane here so if you impute vertically it goes it grows in a horizontal plane right but if you impute it diagonally intentionally do it for three form oblique oblique okay so you will see the rest of the body is not growing horizontally it is growing to an angle to the earthworm have you done that no we did it by mistake and we also posted the picture so then we go back because that doesn't that does not have any advantage to the earthworm because normally for the regeneration is when earthworms are normally feeded and preyed upon by the birds and crows so what happens is if they are half in soil and half outside the bird clutches it from the any part and tries to like push it pull it out from the soil so it gets detached it gets cut so if the cut is oblique the rest of the body starts regenerating to the oblique angle okay so then we went back and we read so there are two types of regeneration basically homogenous and heterogeneous regeneration patterns so in planaria you cut it any angle it will again grow into the same orientation the body plan remains same but in earthworm it's not so so why is that you can read it and you can like address address more like beautiful questions about it so we so the tail restored more than the last segment and the head restored lesser than the last segment three times three we have three earthworms three actually in the head part we got six segments restored instead of eight and we use three earthworms so basically the three earthworms give us the same result that is six segments that was constant but at the tail it was not constant when restored 25 another restored 10 and another one restored 12 and the middle part is not restoring posterior part middle posterior is not restoring any okay so what do you want to do now so next is the basic question is clitalum responsible for this so you are you saying that this was all these were all clitalar animals that you used yes we use clitalar so then what what's the question of clitalum so if you used all clitalar animals and you are getting this restoration not exact and stuff like that why would you say about the clitalum involved or not because what we observed is this part the posterior part restored again here the anterior part also started restoring and in the middle again no no no it's not clear to us at all you are saying that it more than restored that's what you are telling yeah is it not so what what do you mean by that restored now now you are saying it started regeneration regeneration can you explain a little more and see it's a very complicated thing for us you you may be knowing it but we are not able to understand at least I am not able to understand yeah yeah the part which is smaller 8 to 10 segments any part of the earthworm if I take 8 segments or 10 segments or 12 segments with or without clitalum it will not regenerate so then why is this have you seen any any posterior part or any anterior part regenerating any segment have you seen that the smaller parts the smaller part but again this is the this is also the main question right here you are seeing both the parts regenerating in the when you impute in the middle the anterior part starting regeneration and it's restored all the segments but whereas the posterior part is not restoring the this anterior not even a single segment no not even a mouth they can be two ways of doing it you use non-clitaler earthworms the very young earthworms okay and you see whether it regenerates or not so there is also previous data from a and dc itself if you see so they have done both on clitaler as well as a non-clitaler and also one more thing the answer can also be in like energy right for to regenerate lots of protein and a lot of things needs to be produced within a cell and for all that thing you need like energy right so can it be something to do with if the smaller segment it does not have that amount of resources with it to regenerate into a whole organism because first thing it is not getting substantial amount of energy from outside it's not feeding okay so that's why it's clotting its wound so you so you see clotting of wound in all the segments right yeah so it means it is capable of initiating the the process the process is you clot then there's a closure then there's a bird and there's a segment right so they are capable of closing the wound but they are not regenerating it right and then we went on to check the survival like for how many days that smaller segment anterior or posterior survives so it it is a it can be a possibility i'm just suggesting that it closes its wound but it chooses not to regenerate but to survive on whatever energy it has for whatever time it can so for how many days you saw the survival of the smaller ones one was the remaining two wells we showed in the report see these all smaller parts died and this part you had to tell when did it die when did you observe it dying you know so please please tell us some details yeah so the anterior as well as the posterior part the shorter segments died before fifth day post amputation before fifth day all of them yeah this is when the the amputation was done within six segments or eight segments or what is it please give us some details very clearly these are the eighth segment because you were telling about a posterior segment there uh didn't it it healed and it survived for long is it not earlier in all one of the cartoons you have shown that in your figure that one of the figures earlier in the earlier slide he has shown that the larger posterior piece they survived for long is it not yes so what so you had to tell us what is the number of the segments which didn't survive when the number of segments where this it survived for long you only have the answers in our data if you just look what is the difference between a posterior segment which is amputed in the middle it is it just have more number of segments without mouth without chlitalum and another posterior segment which is just eight segments without mouth without chlitalum so they are same in other respects except for the number of segments is it right yeah and for one you are seeing the survival is just five days for the other you are seeing the survival is 94 94 days right and both of them are not capable of feeding from the soil is it something else for this bigger posterior segment which is making it survive for 94 days whereas the smaller eight segments that also posterior cannot survive for five days right there is definitely there is something right both are capable of closing their wound okay but the survival is different but do you agree with that what what he is concluding do you agree with that so to me there is a that is very interesting thing first thing I have a complaint to you because all these things that you have been right you have been getting it you people have not been reporting it at all so all this thing in the last day for 20 minutes or something we will have to understand it but otherwise everybody has been communicating and telling so that's my complaint so the best thing is that you could have reported it every time regularly so that all of us would be privy to it we would be knowing about it so we don't have to break our head over this you know you would have been okay so the my point is this so if you got that the middle amputation what you wrote as middle amputation the PT something that you have the longer piece the longer posterior piece which you claim that it is survived that's what I understand it survived for 94 days or something yeah don't you think that's a very exciting thing though there's a rider he says that the other didn't survive maybe because so you had to ask that why did it survive now the point that's the first question but the second point is much more interesting if it survives for 90 days nine that is three months you can do hell of a lot of things on that because it's a it's a tissue culture system it's a it's an organ system which is surviving without anything you had only poor water and that said it's surviving and as they said without all without all the anterior signals without coordination without anything yes the point I'm trying to say is that you can study a lot of things in that in 90 days immune system studies you can do it metabolism studies you can do it fat metabolism you can do it if the fat is what is being used they preserved the fat is what is being used in order to in order to maintain the metabolism so that they can survive and these so this this piece is a very very extra interesting piece so normal behavior as is can also be done like the things which are what is the opposite of attractive repulsive the things the chemicals which are repulsive to an earthworm an earthworm like when to a normal earthworm with the head and all those things how it behave how it behaves and how this segment which is surviving for 94 days how it behaves without the entire okay the last half a half a half a question so how many such longer posterior pieces survived at your hand for 90 days and things like that only like a only one piece survive for them up till 94 day one out of three one out of three see the point is that that is 33 percent okay even even if with all your casual treatment that one third of the animal survived so that means you can actually develop that into a win interesting model system yeah you can see if you do in 10 earthworms does three or four does like you should increase yeah you should increase your ends to get something substantial out of it right maybe it's just the super super earthworm okay others don't yeah maybe all of them survive yeah I mean I have a suggestion the suggestion is you know I am also been hearing about the same experiment for several years but you know there is something interesting happening now which is the survival of a part of the body which doesn't feed yes and it looks so nice it has life in it and it is undergoing cell division as you can make out and I think most of the people everywhere in the world must be discarding that yes we cubists will not discard we'll convert that into a model system okay forget about regeneration come to come to you know to that part then that will be like a biggest contribution of the cube to the rest of the world and seek the attention of all the big biologists to this piece I mean when we looked at the pictures and the videos which are sent from A&D college last time and then it is getting repeated again that means it's a replicatable study it's happening in your petri dishes it's happening in Delhi it must be happening in all the other places so if it happens that would be great so forget about that so replicate the pieces and how long do they survive 96 days I mean my goodness this is real life I mean what's the life of an earthworm by the way it is around a year it is two and a half years and three years earthworms can live for years so which means that this fellow is still a major amount of its life it can live without a head and then the question is I'm pretty sure that it must be feeding on something else yeah right is there any alternative is there any alternate thing you know is it just diffusing then the question would be like start adding some nutrients in the medium yourself and see if you can you know prolong it absolutely yeah A&D started doing this by putting some glucose solution 10 to 20 like from different concentration of glucose solution in the media in the tissue itself and to see whether these small eight segments is their life prolonged yeah if it if there is diffusion some kind of so they convert this into a major experiment yeah because regeneration everyone has seen as you can see living is not enough you know living happens only when you fight against the environmental perturbations which means bacterial infections and things like that that means immunity question comes up right you know it would possibly become and turn it out into a fascinating thing you know like one without a whole surviving without a mouth without an anus no feces but then surviving that would be a fantastic system and also to check whether the segmentation number of segments are increasing or decreasing in that piece so there's a lot of experimental possibilities out here and getting excited about this yeah okay I think we are running out of the time so we will call the next group that is a drosophila group so basically there is a lot of you know the evolution is major topic in the biology you know whole biology you know doesn't make any sense except in the light of evolution so they are studying actually evolution aspects of evolution in the drosophila so I will call Harshita and Ruchi yeah okay yeah okay they will be called okay good afternoon everyone we are from Chambo Naka MPH school our project name is drosophila so we were do in drosophila the catch the flies catch the fly and after that we study the activity pattern yes so our objective of the like we started doing the experiment the like how how should we trap the fruit fly we started doing it so the method of fruit fly trapping so we trap fruit flies so how how we trap fruit fly I'm just going to tell you about that so we have to take the like transparent bottle if I handed ML bottle or big bottle we have to take and then we have to take the cotton and rotten banana or peel and then we have to take the label so we we take a transparent bottle and in that bottle we put the banana peel inside the bottle and then we put the label like time what is the time we put that on like we label it the bottle and then we keep that bottle near dustbin or kitchen and then we see like how many flies has been come or not in that bottle so like this we have done like this is this is the matter this is the method of trapping fruit fly we used we like to we we always trap like this only so we have trapped to like we have trapped to the fruit flies one year so we have seen lots of like like we have seen lots of things in the fruit fly while we're trapping so that after we were trapping a fruit fly we get some data and we are conclude our objective was like we trap the fruit fly like this so our main objective was that like we wanted to see that activity rhythm out of activity rhythm of a fruit fly like at what time they're most active and active so we wanted to see that so for that we for for that seeing that activity rhythm of a fruit fly we have drawn a we have drawn a graph like so we we put the bottle near kitchen or dustbin so we keep the bottle at 7 a.m. to 9 a.m. in the morning and like 7 a.m. we keep the bottle and then at 9 a.m. we come and check the bottle like how many flies have been come in that bottle or not and then similarly we keep and then we take that bottle and count the number of flies how many flies is in that and then we can count and we write in our log book and then 9 9 to 11 again we keep two bottle and then see after 11 a.m. we see like this we do this experiment till 9 p.m. and so we have come like we have observed that like the flies are more more active like here so like jen july october jen to march we have seen that flies are more active at 11 a.m. to 1 p.m. and in the evening 5 p.m. 5 p.m. to 7 p.m. they are more active and in july october we have seen that like peak has been shifted little earlier like 9 a.m. to 11 a.m. and of 3 to 5 p.m. to 5 p.m. so we have the reason also behind this so in 2017 we got the same data like that march jen to march 11 a.m. to 1 p.m. and evening 5 p.m. to 7 p.m. So as this one an aksana told that peak was shifting there in lab we came and discussed we came to know that in this all there is called equinox also happened so that here we can see jen and fab 3 we can see december so in december the night is longer and day is shorter so because of that our peak was coming 11 a.m. to 1 p.m. because the night is longer and day is shorter that's why sunday is later that's why we got 11 a.m. and 1 p.m. and then after july to october we saw that our peak was shifted little bit earlier then then again we came to see equinox so here we can see in june day day is longer and night is shorter then we conclude that in june july to october our peak was little earlier because of the equinox so 9 a.m. to 11 a.m. then again we see our peak was then november to december we can again see that our peak was same 11 a.m. to 1 p.m. because of the equinox so like that time is shifted earlier or later so that have that say have some problems like jetlates or sad disease in summers in winter in winter seasons some like many people's go in depressions because of because of that there is climate happening and they are can't survive in the seasons and that so we were studied so we were studied we will study about take a fruit fly and then to what is the problem and how to how to change this disease and how to conclude thank you yeah but are you connecting because this year's Nobel Prize is has gone for actually this work means the this is a part of that work you know that the activity so you can talk about something about yeah so this work actually has got so the Nobel Prize of this year that the activity just I wanted to add that you know the they are actually deciphering that know what kind of why this type of activity is there in the fruit flies like why do they sleep at particular time why do they are active at at particular time so there are some there are many hormones in our body which are activated at that time at which means there is also there are some gene regulations which are happening at that particular time all those things are so this is a very beautiful model for this studies which can be so this kind of work is know very much of is a frontier area anyone has any immediate question yeah yeah how you can identify it the drosophila is male or female through the abdomen of the fruit fly we came to know that it's a male or female so like so if the if the abdomen of a fruit fly it's if it's a fruit fly have a spot or like black spot on abdomen then it's a male and if the like female have the white abdomen so through it through this week we identify say male and female yeah so so every day we were do experiment and return data in log book and then when we plot a graph everyone's data take it and every particular time to calculate then a plot it take the bottle first we keep the bottle near the kitchen then we plug the bottle with a cotton and then you pick it up the bottle and we count like without naked eye yeah like 9 a.m. we keep at the bottle and then at 11 a.m. we come and you pick up and then we count and then we write yeah place of yeah like this we do till 9 p.m. so yeah so this is yeah so like month month data we have plot like month all every total every once and ready a question so at some point like we were we will conclude male or female but at some point at some point we can't conclude because at some point of flies coming in more than 20 or 20 20 or 25 or 20 so that we can't able to conclude in female or male or how to that we keep the flies in the bottle only like for two three days it's in the bottle only and then after two three days we like wash the bottle and reuse the bottle so we leave them okay so good afternoon everyone I am Ruchi Modgekar from Elphinstone College and we are working on the model organism drosophila the title suggests that we are trying to develop drosophila as a model system for starting aspects of evolution so as we all know drosophila has all faction as its primary sensory modality for finding food and hence used for studying various core some of the core neurological aspects along with these it is also used for studying evolution that we have a special line called csvz flies those are the canton special benza flies as mentioned and these flies were trapped by sima benza benza in canton for and these these flies are cultured in this media bottles that is a ready made media that is provided to these flies so for approximately past 2000 generations these flies are cultured in a this particular way so these flies the csvz flies these were later identified and they happened to be drosophila milano gastro like the complete identification is done of these flies now talking about evolution the phenomenon of natural selection it's like there are demilano gastros in wild as well as the csvz they are the csvz and if you consider the phenomenon of natural selection it's seen this selection is seen prominently in native that is the wild drosophila milano gastro meaning only the flies which have the better ability to smell even the lowest concentration are able to survive whereas if you consider the csvz this selection is absent because these flies are provided with readymade nutrition this this difference we are considering that this difference of providing artificial environment would would be a quantifying reason for difference in the olfaction behavior of these two flies thus larval olfactory assay provides us evidence for behavioral differences in these flies larvae's due to the selection of natural due to the phenomenon of natural selection the objectives will be explained by my colleague okay so as the abstract says that the csvz flies have been cultured for past 2000 generations so you know the csvz are being kept in an artificial environment an artificial environment means we are ready made there is a readymade environment which we are providing them and it is very easy for the flies to survive in that environment because of this there are certain changes like in environment there is something very natural but here when you provide the artificial environment that is the readymade culture media in which we are keeping the flies there are certain conditions which can lead to the difference in the olfaction behavior as she said that drosophila have their primary sensory modality as olfaction so for the survival of any fly a fly has to sense the food has to smell the food and that's the that's how it goes to the food it eats and that's how it survives so there are some difference in the olfaction behavior of a fly that are carried out at the genetic or the molecular level which are random which are natural so we need to study what the what these certain changes lead to also we need to develop drosophila as a model organism for evolution and to study what are the natural selections occurring now to study these long-term objectives we need to carry out the experiments which lead to my immediate term objective that is maintain a single line and the csvz cultures now for comparison of the csvz flies with any other fly we need to have a single line culture so for that we have our culture that is rslv4 and rslv3 it's ruqi single line wasai because the flies were trapped in wasais and ruqi was the person who catch the flies so those were our single line cultures so we need to maintain them also we need to standardize the olfactor meter now we are mainly studying olfaction in these flies so we need to carry out the experiment that is olfactor meter and standardization of that experiment is important to get proper results also the short term objective is identification of the single line flies now if my single line flies that my native flies are not drosophila melanogaster as the csvz flies are so there is no point of comparison comparison between these two flies hence identification is very important also we need to carry out the olfactory assay in more replicates as in if i'm just carrying one or two sets of runs with the olfactor in the olfactor meter then i cannot conclude my hypothesis or my long term objective which will either be very close to my hypothesis or it can be absolutely wrong so what my hypotheses are now that my csvz flies are being kept in a very special environment there are certain changes in their olfaction now we it's been known that the fly has maxillary pulp and antennae through which it sends it senses the molecules and that's how it goes towards the food so for example here it's written that we expect that our native line cultures should have more should be more sensitive than the csvz culture now what okay so now we are keeping the csvz flies in culture media that is in glass bottles while are kept in the natural environment for so many generations right yeah so that's the second image is the fly is the bottle wherein the bottles are been kept flies sorry so there are many cases in this now my csvz is being kept in the culture and they are not that sensitive as in the concentration of the smell in the bottle is very high like if i am talking about the receptors which are responsible for you know sensing the molecule so in csvz the molecules around one receptor will be huge amount so they can easily get bind and they can smell whereas in native if there is shortage of food in the environment the native ones which have got a better olfaction and the flies whose receptors are very sensitive or else like when i say that there is only one or two molecule in the environment and the fly has to sense it so the receptor has to be that much good enough to sense that even one or two molecule so the fly can go towards the smell so that's the reason why i say that my hypothesis states that my native flies should have a greater olfaction rather than the csvz flies also along with that we can say that these csvz flies have been you know cultured from 50 years 50 to 60 years so like there are approximately more than 2000 generations in that bottle itself so there can be some mutations in the genes which can lead to the which can lead to the you know gene mutations which can lead to the synthesis of which can you know prohibit the synthesis of the receptors because of which the flies might not sense the molecule and might not go towards the smell so this is what my hypothesis states mutation in the olfaction receptors of csvz yes our chances like that's what our hypothesis is so as afsana and raswana explained how we do the trapping so we i'll just brief up brief up with the protocol we take a dry plastic bottle and add a banana to it as it acts as an odorant or attractant you can call and we keep this we also wipe the mouth after adding the banana so that the flies go inside the bottle and not just stick around to the mouth also there is one another alternative that you cannot take any plastic sheet something which is too thick you know that we used to cover our books like if you can make a funnel out of it and you can keep on the mouth and you can add the banana peels and then we place these bottles in the areas where the chances of getting the getting the rosapilas is more like somewhere near the dustbins and after particular intervals of time we can just go and observe there is presence of fly and if we have flies just plug it with the cotton and get them now as she said we need a single line culture to compare the olfaction what we do is we have a mixed culture and there are gravid gravid that is pregnant females normal females and male flies the gravid females are those which would lay eggs and give rise to the new progenies so we need to transfer one single gravid female from this mixed culture into a fresh media bottle so how we do is we take the media the bottle with the mixed culture and place it on top of it on top of the bottle with the mixed culture so that and allow some flies first we need to check if there are flies which are gravid which are going up as well and as they are placed inverted some flies will go up whereas some flies will stay in the mixed culture bottle itself and by doing this two to three times we isolate a single gravid fly and then transfer it to our media bottles so the table explains the composition of the media bottles that we use for the drosophila this composition is for 1000 ml coming back to the life cycle the metamorphosis of the fly takes place for it takes place in a span of 8 to 10 days so when the fertilization takes place and a female gravid fly lays an egg it takes 24 hours for the egg to turn into first instar larvae first instar larvae is the first staged larva another 24 hours for the larva to convert into the second instar larvae and another 24 hours to transform into a third instar larvae so by day four we have a third instar larvae the transformation of third instar larvae to pupa takes approximately one to two days and the pupal stage is for around three to four days again so it's like a span of 8 to 10 days as I mentioned before identification by her ship okay so now we need to identify what my single line cultures are so we have got rslv three and rslv four two single line cultures we had rslv one and rslv two but we lost those cultures and we also had not identified them okay so the identification of the single line cultures is like first we need to find out which kingdom it is so it needs to be animalia because animalia are heterotrophs they are eukaryotes and they are multicellular so a drosophila definitely is a heterotroph and also eukaryote next comes the phylum phylum is arthropoda that means they have joint appendages means they have the paired appendages so even the drosophila have paired appendages and next comes class class is insecta insecta it means that the body of the organism needs to be divided into three segments three parts that is head thorax and abdomen and also it needs to have three pairs of legs that is and all the three pairs of legs should arise from the thorax so that's how class insecta has been identified in our rslv three and rslv four cultures next comes the order order is diptera diptera diptera die means two and tera means wings that means there needs to be two pairs of wings one one which is used for flying and the other one which is reduced to hall tiers so even we observed our culture single lines and they were having the big hall tiers as well as the flies as well as the wings sorry and next comes the family drosophila day family drosophila day is something from where the different drosophila and different species of drosophila are coming out so we did the identification only till drosophila day and we have not yet finalized the genus of our single line cultures so for drosophila day family they need to have a coastal vein a subcoastal vein now in coastal vein then they need magnifying the image that's not possible yeah so there are two breaks in the coastal vein the first vein of the wing has to have two breaks yeah so we first started with the csbz either the csbz is having the two breaks or not so it was having and it was also seen in rslv 3 and the rslv four cultures this and the two breaks this first break and the second break next characteristic of drosophila day is that it needs to have a incomplete subcoastal vein now just below the coastal vein there is a subcoastal vein so these are just you know terms which have been used as the characteristics so the csbz had a coastal vein just below the subcoastal vein just below the coastal vein and also the rslv 3 and rslv 4 had next is the post usila bristels on the frontier of their head and it was seen in the csbz we do not have proper images about it but it was seen in rslv 4 and rslv 3 as well so this concludes that all the characteristics of drosophila day family are there which you know are similar to carry out the olfactory assay with the csbz flies so what our experiment exactly is for we are carrying out the olfactory assay with second instaur larvae and not the flies what we do is as i mentioned the life cycle of the fly previously it takes 24 hours to transform into a new stage of larvae so what we do on a certain day if we put some number of flies say approximately for about 100 flies into a media bottle we need to make sure that we transform that transfer them back to some other bottle after 24 hours which will as in will be confirmed that the like the flies are given only 24 hours to lay eggs and after every 24 hours there will be a transformation from one larval stage to the other larval stage so on day one we transfer the flies to the media bottles on day two we transfer them back to some other or the same media bottles so now the the the flies have laid the eggs now these eggs will take 24 hours to get transformed to first instaur larvae and more 24 hours to get transformed to second instaur larvae so on day three i'll be getting my second instaur larvae so that's how i'm obtaining stage larvae for my olfactory assay now we need to harvest this larvae as well what we do is in the media bottles that contain the larvae we add one test tube that is 10 to 12 ml of tap water and with a help of a brush we get give a swirl to the topmost layer of the bottle as the larvae are feeding in the media the layer is quite soft as compared to the solid media and it's easy to scrape off the larvae from the layer once the larvae are in the liquid medium we transfer the liquid into a strainer as i have mentioned is shown in the picture we are putting all the liquid so the strainer will drain off the fluid and what will remain back in the strainer is larvae and some media particles that might stick to it then under a gentle flow of tap water we just give a gentle wash to these larvae and these larvae and they are then transferred to 25 percent sucrose solution now there is necessary that the contact of brush and the larvae should be minimum as it might damage the larvae and we might not get the desired result that we are expecting from olfactory assay okay so now we have the larvae the staged larvae in the strainer so what we do is take a brush and take the larvae from the strainer and put it into a sucrose solution we put 25 percent sucrose solution 25 percent sucrose solution in 100 in 100 ml that is 25 grams in 100 ml of tap water or distilled water it does not matter because it does not matter which water are you taking most probably because you are just putting them for some period of time for around two to five minutes so after taking the larvae in the cylinder you just need to wait for five minutes for the media particles to settle down because what happens is when you put the larvae along with the fly along with the media into the cylinder the media particles clump up and settle down because of their high density and the larvae float on the sucrose solution yes yes okay so for in the first image you can see the I don't know if you can see but majority of the media particles have settled down and the topmost layer has the staged larvae so in next step what we use is there were two you know you can use a strainer to carry out the further experiment or a muslin cloth mayu sir was mentoring us and he said that muslin cloth can be a good idea and you can try it and it worked for us so we take a muslin cloth gently wash it gently wash it and make it wet and the cylinder which has the larvae we just tilt it up on the muslin cloth so that the larvae come out on the muslin cloth and the media particle is just down in the cylinder itself now again we give that muslin cloth with the larvae a gentle wash of tap water so that the excess sucrose solution that is on the body of the larvae gets drained off and in next step the first image you can see the maximum number of larvae are on the muslin cloth and now we need to put this larvae in some solution somewhere where we can keep them properly and then we can carry out the experiment so for that we use ringer solution ringer solution is a composition of mini salts so it consists of kcl nacl tris hcl and tris base and hcl so we directly use the tris sorry the ringer solution that was present in the cube lab and we just take around 5 ml in a petri plate and tilt and just flip the muslin cloth on the petri plate also if the larvae are not just sticking to the muslin cloth we can pour some on the top of the muslin cloth so that the larvae can easily come off and we get the staged larvae for our experiment okay ringer solution is a salt solution salt now as you see the skin of the larvae the larvae are basically very delicate and you need to provide proper osmotic conditions for the larvae to survive if we put the larvae in normal water there are high chances that the fluid inside the larvae body might come come out so ringer solution is a solution that properly matches to the in our salt concentrations of the larvae you can cancel yeah yeah so next comes the olfactory acid and the sanitization of the olfactory meter now for olfactory acid we need to have a agar petri plate plain agar petri plate and the percentage of agar used is 1.5 percent so what we need to do is first take a petri plate and along with that you need a marker so that you need to know where you are keeping your larvae so a marker is a you know exact size of circle of the petri plate and on that you put the marks for this around around one centimeter in the center where you can keep your staged larvae and exact opposite two ends of that you can put the sample on the control so this diagram is of the control one now we need to keep this keep the petri plate along with the marker in a black box black box is used because you just want because we want to eliminate the chances so that you I because I do not want that my I do not know that my larvae are having their that they have properly developed their eyes or not so I do not want them to use their other senses to like visuality so that they can see the sample where it is kept and they just crawl towards it so to eliminate that I use a black box and cover it with a lid so that they just use their olfaction and go towards the sample yes yes because okay now along we need to also standardize the olfactor meter because so for standardizing standardization of olfactor meter this is the control plate where we just add on both the sides we add two drops of distilled water like one on each side and as she mentioned the procedure we keep it in the black box so what if the larvae what we expect is equal distribution of larvae throughout the petri plate but what if the larvae are just moving as there is presence of some miscellaneous smell and not by using their olfaction so for this what we tried was like for the marker we are just dividing the petri plate into horizontal two horizontal sections but after carrying the carrying out the olfactory acid to check whether the petri plate is properly standardized what we can do is just divide it vertically and find the number of larvae present on both the sides of the vertical halves and similarly it can be done by dividing the petri plate diagonally and checking the number of larvae on each of the sides so this can tell us the that there is no any other miscellaneous smell that is interfering with my olfactory acid so now how do we calculate our olfactory response index it is basically calculated on basis of the distribution of larvae on the petri plate for control what we do is put distilled water both the sides one side as you all can see it's labeled C1 and C2 so it's the distilled water and we we place the petri plate in the black box with the petri plate lead and the black box cover and give larvae one minute of time to do that get the olfactory run after one minute of time we remove the black box and click the picture and count the number of larvae on both the sides so C1 is the number of larvae towards the C1 or distilled water side and C2 is on to the C2 distilled water side similar is for sample sample here we use isomile acetate this solute this is the chemical which smells exactly like banana like we are trapping the dross filler and placing banana as an odorant so isomile acetate is what why we use as an attractant yeah we actually just we started the olfactory assays just few months ago and so we don't have a very solid data too but this is good so what we did there are many goof ups we we did while performing the assay which i mentioned later the these are the control slide as runs and the sample runs sample yeah yeah we we have used are the dilutions of isomile acetate which are 10 raised to minus 1 minus 2 and minus 3 10 raised to minus 1 being 10 times diluted minus 2 being 100 times diluted and minus 3 being 1000 times diluted so what what do we hype what our hypothesis says that for for a control petri plate we need to have an equal distribution on both the sides so the olfactory response index for control should be 0 yeah and that for sample should be 1 as what we expect is the larvae should all the larvae in the petri plate should move towards the sample side and what there are differences in results because there were some of the goof ups that we did like using same larvae for dilutions and for control whereas carrying out control controls later and doing the dilutions first and using the same larvae again and these are some of the results that we got what i would like to conclude from this result is more replicates that are positive towards our hypothesis will will give us the threshold that we are trying to find out the threshold is the smelling capacity that the csbz can smell and the versus the native flies can smell and it will also tell us how better olfactometer is standardized so these are some of the goof ups the two of the goof ups i have mentioned earlier and two more are there it's like when we started first preparing the media we were ignorant and we changed the concentration of orthophosphoric and propionic acids the orthophosphoric acid acts as an antibacterial and ortho propionic acid acts as an antifungal agent so we had just interchange their concentrations and the recent goof up was we didn't have sucrose available and so we added glucose extra equal to the amount of sucrose while making the media so actually we haven't got the results yet we have transferred flies that's the latest media that we have made so we'll be getting back to the results but we are considering it that's some mistakes yeah okay so now for now for the plans are to standardize the olfactometer and to carry out good results that are either you know exactly similar to what we hypothesize or are exactly opposite to uh to it we need to carry out more replicates of the olfactory acid that is every time i'm doing a control it needs to be proper and i need to do many resolutions of the isomal acidate to get a proper threshold and get the results along with that we need along with that also uh i need to study about what the receptors play important role in their olfaction so this is what we want to study about and this is our future plans these are some of the references these are the references for the media that we used and the identifications as well as last slide of acknowledgement so that's it any questions anybody has questions please yeah so for the olfaction acid did you use the csvz flies or the rslv flies yeah as i had mentioned the olfactory run was done by just csvz flies we did mention it we used csvz larvae and not flies okay so i'm planning to do it with the rslv yes okay yeah yeah so uh yes yes no that's not the point that we want yeah actually uh currently what we are no no no the point is that is there a reason why you worked with the csvz flies for olfactory assay i think that's the question that that was say she's a very cunning person no she wanted to trap you and you people escaped so i don't allow you to escape that you can ask but but this is an you see i think that's what she told you were asking you know is it not why it did what did you which because i think i will give you a cue i think uh you were going to standardize the olfactory meter is it not so i think she was meaning that if you are standardizing the olfactory meter you should be using some standard flies to start with though you are questioning this standard nature of that standard fly at the end that's what i i think that's another question that we'll have to to come to am i making it clear you used csvz flies for some particular reason or that just became it became handy so used it that's what her question is yeah okay i think so is it she's yeah what i have got the question is like we started the standardization a bit later but csvz flies we were done with little identification like we checked for the characteristics that were present and we had a sufficient number of larvae that were needed for the olfactory assay whereas if you consider the single line cultures they were not that uh well not well developed but the ruji the point is that csvz fly has got some value is it not what does that value what is the value of csvz fly what is the csvz fly in comparison to how our avat birar ka fly or something uh yeah the csvz flies they have it cultured in the media bottles so there is a difference where does it come from where what is the origin of csvz flies where did you get it from now do you want to really see that whether this is a csvz fly is actually a drosophila melanogaster or not you understand what i am asking drosophila melanogaster is a species that has been studied for a very long time and most of the genetic and molecular uh uh bases are the information or the content about the species has been known to humans but what is the connection between csvz fly and drosophila melanogaster they are same it's drosophila the csvz are the uh single line or the wild type of the drosophila melanogaster what she's trying to see now what is the origin of csvz fly you have any idea about it or you are just saying any anyhow you are trying to say that csvz fly is like any arm uh drosophila fly from uh bangal or kerala or uh bombay or maharashtra or where am i no unless you you you have that why should we bother about this you know you take one fly from bangal and one fly from kerala one fly from maharashtra and to study it or is there something special about csvz flies okay so your first slide has it it seems could you just go back to your first slide do you understand the question that we are asking is there anything special about csvz flies samar wanted to study behavioral genetics samar wanted to study behavioral genetics no you explained it once in the cube lab itself no no you see you had to tell the whole audience in canton samar wanted to study behavioral genetics so he chose drosophila and he started culturing it since then so the fly itself drosophila melanogaster it is being cultured since then in the bottoms so so it's an it's an identified drosophila melanogaster yes which is cultured by samur benzer in 1960s 60s or something and then the progeny of that and its progeny progeny handed down to several people and it came to you yeah so which essentially means it's a very very well defined fly stock yes of drosophila melanogaster now unless we got get that type of a emphasis on that why should we bother about your comparison with uh uh birer fly or uh elf instant college fly so what is the importance of that that's what she was trying to say could you do you want to once again repeat it so samar wanted to study behavioral genetics around in 1960s so he trapped those flies after he started culturing them he identified them as drosophila melanogaster and since then those flies are still being cultured in such environment like bottle conditions media bottle conditions so we have that going on yeah so you have a fly that has come from that has come from samur benzer's lab which was raised in to in 1960s or something and cultivated cultured in bottles after bottles and it has handed down to you so you know that it is a stock which is which has got the antecedents in benzer's lab which is an identified fly as drosophila melanogaster what you are questioning is that when so many generations 60s to today that is uh 40 57 years 57 years it has been transferred from one drosophila medium to the other drosophila medium which is a cooked up medium whereas your fly what you are talking about is uh is uh caught from the wild so its antecedents its predecessors in 1960s who would have been them the native drosophila melanogaster so you may be comparing this is it not one that has been reared for several generations several several generations how many generations you think that one year a drosophila fly will give in a bottle how many days is the life cycle 10 days so 365 days means 36 36 generation let's say 30 generation you calculate 30 generation to 60 okay 2000 generations so one is 2000 generations eating the ready made food the other is somehow they are managing from generation to generation came to virah came to virah you got it from virah is it okay so that's the comparison that you are trying to do so you are claiming i think what you what you said is that you are claiming that these fellows in virah must have been going through several problems of finding food and things like that so the only the best one who could find out best one in the sense the one who could really find out the food in so many generations once in so many generations would be able to survive and once also would have duct several predators because in the bottle there are no predators so duct so many predators so they must have been having a jail body probably there's a possibility and and they probably would be having a very good immune system because they must have been surviving in favorable conditions so these are the type of changes probably you would be anticipating in a local one which has not been fed in the bottle whereas this one so so the point is though what i forgot your name what naina okay so what naina was talking about was that so many generations it has been fed ready made food whereas in your case it is coming out coming from the from out so is there is there a change that you are you are trying to observe so you are actually trying to observe the change in that no so you expect that they they cooked up they ready made food eat and fellows so that is the question i think i think she was worried about that that's the question we'll have to ask one is that what do you think will be happening in that bottles you said that there will be mutations that is something something something you mentioned to her as an example so i was asking exactly mutation at the olfactory level olfactory level as i mean now we not know but i agree that concept that you said the flies but i think it is easy it is assumed that in olfactory level it must have there must be some mutation that mutation or some change must have taken place that's what their assumption is is it not in that cultured one in the cultured one in the cultured one okay okay that will that you'll come to but what their assumption is when from their suggestion their assumption is that mutation must have taken place in several cases several genes in in 2000 generations out of which some could be some could be even in in olfactory sensing mechanism that's what their assumption is do you doubt in that assumption that's the question that you'll have to ask i have a silly question that i i mean when i heard this i thought about transposons so the the gene it could be transfer mutation can be by transposons or anything you know there could be several ways of uh mutations mutation can't be stopping now over there it can change also the mutation can reverse also i mean but the mutation can happen is it not yeah so that's that's all what they were they were asking for so if i think the idea is that you should be making it very clear about it so mutation can be by spontaneous mutation or by transposon mediated mutation any any type of mutated it may not happen in only in the sensory this thing it may be happening everywhere the point is that any in in efficient mutant mutant that is coming out of that will be weeded out in the natural population whereas this fellow will not be weeded out here that's the that's the point so the so your first question is that could would there be any mutations that is taking place and that will be stabilized there that's that's the question do you have any any doubt about it you are you yourself are suggesting that there could be a transposon mediated mutation so you don't have any doubts about it can we just conclude in this yeah so do you have some suggestion on that see she has a very genuine question so you have your assumption is that there will be there will be mutations and that mutation will be remaining there because they are not being weeded out in the natural population they will be weeded out so because i was worried because the cv suppose there is a mutation in cvz and suppose vahi mutation will happen in wild type so how can you compare oh that's what the point i think you didn't hear the last point i was telling in the why in the native ones native ones if such mutation happens that they cannot smell will not find food and they will die that's what their argument is okay is is that yes exactly yeah we have to break for the lunch so lunch is actually outside in the canteen so you have to come around uh in half uh 45 minutes okay 45 minutes yeah 45 good photo


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