Investigating Resistance Development in Paramecium tetraurelia to Carminic Acid

Introduction

This study examines whether Paramecium tetraurelia can develop resistance to substances initially fatal to them by incrementally increasing the substance's concentration every 48 hours. The focus was on Carminic acid A, a naturally derived red food dye known for its antimicrobial properties but potentially lethal to P. tetraurelia.

Carminic Acid

Carminic acid (C22H20O13), also known as E120 or Carminic acid, is a colorant (most probably) produced (and extracted) by Cochineal insects, which are also known as D. coccus. As Cochineal insects live with other microorganisms (symbiosis) it cannot be said with certainty that D.

coccus produce the Carminic acid themselves. They use the Carminic acid to deter predators. Mature Females contain the highest percentage of Carminic acid, so just before they lay eggs, they are killed through boiling, drying or steaming. At the end they are then defatted and purified through alcohol. It has been used for colouring clothes and today it is mainly used as a food dye and in lipsticks.

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Synthetic food dyes are found to be harmful to human health hence the upward trend for use of natural food dyes like Carminic acid. It has been found to have antimicrobial activity against unicellular organisms like S. Aureus. It has no significant effect on humans as long as the person doesn’t have a Cochineal allergy. If the person has an allergy it can cause asthma and other symptoms.

Carminic acid is a derivate of α- hydroxyanthraquinone. It is soluble in water, alcohol, ether and alkaline and just stays the way it is.

It only changes its molecular structure when heated or when put in different pH concentrations. Carminic acid is well suited for binding with metal ions due to the location of the hydroxyl group.

Only one source indicates the existence of more than one type of Carminic acid, Carminic acid A and B , while others indicate the existence of only one. The difference between the two is that Carminic acid A is more lethal to Paramecia than Carminic acid B. The only way I could find out if my substance was Carminic acid A or B was to add E120 (in very high concentrations) to the Paramecia and wait for the effects to show. In the first 2 minutes most of the Paramecia tetraurelia died and after 3 minutes, all the Paramecia tetraurelia were dead. As in Carminic acid B no death could be observed for the first 20 minutes, I came to the conclusion that my substance was Carminic acid A.

C22H20O13

Paramecia Tetraurelia

Paramecium is a unicellular Eukaryote which belongs to the genus of ciliates and lives in stagnant fresh water (ponds, lakes etc.) that are rich in organic matter (decaying) . They are widely spread and are also used very often in research (genetic research) . Their cell form resembles the sole of a shoe and they are ~120 μm long. They move with the help of cilia (hairlike structures that are located on the surface of the P. tetraurelia) that whip and move the P. tetraurelia in the correct direction. They can cover up to 4 times their body length in one second. They are found to be able to distinguish between different levels of lighting (up to 6.5 volts). An interesting aspect of P. tetraurelia is that they have the ability to “learn”. This is referred to as cell memory or epigenetic learning.

Genetic Modification

Epigenetics are changes in the phenotype that don’t necessarily involve modifications in the Genotype (DNA). Epigenetics most often involves activating and deactivating some of the genes which then affect which amino acids are produced and which ones aren’t. Epigenetic modifications are controlled by surroundings, age, diseases etc.

On the contrary mutations are alterations in the gene sequences. This results in the synthesis of completely new protein types. Mutations are also caused by environmental changes but unlike epigenetics mutations happen more randomly, are usually unpredictable and aren’t always beneficial for the organism (just like in Epigenetics). But if the organism produces offspring, chances for a mutation that is beneficial for the organism (usually unicellular organisms) happen more often than for example in humans. This is also the reason why there has to be a constant research in rat poison because rats develop a mutation or become epigenetically modified to make them resistant against the rat poison.

This is why I waited for 2 days to give them enough “recovery time” and enough time to accommodate to their new surroundings and eventually develop a mutation or epigenetic modification so that I can increase the concentration.

Materials and Methods

Before starting the experiment, I chose turnip as food for the Paramecia. Bought a turnip, cut it into small cubes, dried them in the sun until no water was left. Paramecia tetraurelia were initially cultured in small bottles till they were numerous enough to be transferred into the bigger Flasks for my experiment.

First I took 10 bigger flasks of around 500ml capacity each, put 2 grams of dried turnip pieces in them and poured 200ml hot water. Once the water is cooled down, I transferred around 20ml of water containing Paramecia and let them multiply even more for 5 days. Out of the 10 flasks, 5 flasks were used as “carmine/experiment” group and the rest 5 as “control” group, which were kept aside without adding anything to them during the whole experiment(22days). All these flasks were kept at room temperature 25°C (±3°C). Throughout the experiment, before counting the number of Paramecia, I always stirred the flasks once to make sure that the P. tetraurelia aren’t in one place (e.g. where there was most food).

After 5 days, I first counted the Paramecia in all the 10 flasks. I did that by always putting the same amount of fluid from the flask on the slide (in my case 1 ml) and then counting the paramecia with the help of a microscope. I put cotton fibres so that the paramecia had no room to move too much which facilitated the counting on the slide significantly. For the Carmine group, initially I added around 1 gram of Carminic acid (During the whole experiment, I used around 1 gram of Carmine at a time to the carmine group flasks), waited for 48 hours (±2 hours). Again after counting the number of P. tetraurelia in both groups again(carmine and control group), I added Carminic acid to carmine group, mixed till it was completely dissolved in the solution. Repeated this process 11 times by waiting for 48 hours between each time and counted them. I chose to wait for 2 days to give them enough “recovery time” and enough time to accommodate to their new surroundings (eventually develop a mutation or epigenetic modification).

At the end of my experiment I took some Paramecia tetraurelia from the Carminic acid group and put them back into their usual habitat (Water) in a separate small bottle and around half of them survive but the others did not after 5 minutes.

As I wanted to observe the behavior of Paramecia when exposed to carminic acid directly, I put the carminic acid powder on the slide along with the paramecia. I could not mix the powder evenly on the slide because of its small size and so the concentration was higher in some places than in others. I observed that the Paramecia tetraurelia migrated from the high concentrated areas to the lower. I tried this experiment on both carminic and control group Paramecia.

Materials: Light Microscope, 10 big flasks, Paramecia tetraurelia, dried turnip cubes, Carminic acid in powder form, teaspoon, weighing scale, water, water boiler.

Table 1: Experimental Setup

Method for controlled variables: I changed the location of the flasks containing Paramecia tetraurelia when the air temperature was too hot. When I change the location to a place where there is no direct sunlight, the speed of Paramecia tetraurelia also changed. There is a significant difference in speed when the there is light and when there is no light. The speed of the cilia beating is increased at high light intensities and decreased when there is no light or less light. These reactions happen in the course of 30 seconds which means that there is no effect on the data whatsoever, as I took much longer than 30 seconds to prepare the slide. All the flasks were on the same table in front of the window, so the light intensity was almost the same (minor differences) for all the Paramecia. I weighed the food (turnip cubes) in grams and distributed it evenly to all the 10 flasks.

Method for collecting data: I always put the same amount of liquid, which was 1 ml, on the slide with some cotton fibres (control group, Carminic acid group) and then counted the Paramecia tetraurelia on the slide. The cotton fibres facilitated the counting, as they restricted the movement of Paramecia tetraurelia. And after counting, I removed the cotton fibres to see how fast they cross the section which I could see with the help of microscope.

Ethical, Safety and Environmental Considerations

In this experiment some of the Paramecia tetraurelia were killed, due to the inhibition of leucine aminopeptidase and acid phosphatase. As for humans, Homo sapiens does have the same enzymes that are inhibited in P. tetraurelia, but as humans have much more of these enzymes, it is not very dangerous if some of the enzymes are inhibited. Carminic acid has virtually no effect on humans, except when the person is allergic to it and so could cause asthma and other symptoms.

This led me to making a small side experiment. I took some Paramecia tetraurelia from the Carminic acid concentration and then I separated them from the rest of the group, gave them some food, and then left them for ±5 days in the same concentration to see how they react. And then afterthe 5 days, they regained their “original” speed and took around 1-3 seconds to cross the 150. It may have taken less time for that, but I was away so I could not check on them every day. But they regained their speed, and after that I put them in a higher concentration and treated them the same way I treated the other groups just separate from the other groups, but I did not make the usual 5 repetitions. Generally speaking they became slower so speed decreased but they were still significantly faster than the ones that did not have the “recovery time”, but I could not go into it as deeply as I maybe should have. I treated it more like a fascinating side experiment that would not have been needed.

This side experiment showed that the Paramecia tetraurelia do not need that much time to adapt themselves to the unusual conditions unlike multicellular organisms as they reproduce faster and the chances for mutations increase the more an organism divides.

After the transfer of the Carminic acid group back to their normal habitat, some died, others survived (approximately ½ died).

Results

Carminic acid is a food dye deadly to Paramecium. The first effects were shown immediately after 1-2 minutes: They moved around much more slowly and after 3-5 minutes, around half of the Paramecia tetraurelia died, while others were moving even slower than the first few minutes. In normal habitat P. tetraurelia took 1-3 seconds to cross 300 μm, while in the Carminic acid solution they took 5-8 seconds.

Table 2: Average Number of P. tetraurelia per ml Over Time

We can see that both groups started with a same average number of Paramecia tetraurelia. But after the first addition of Carminic acid, the average number of Paramecia tetraurelia that survived was less than half of the initial number. The number in the carmine group is fluctuating slightly but there is a slight upward trend, while the control group initially increased and then stayed more or less constant. This can be seen more clearly in Figure 1. The significance of the difference of my data is determined by the Students unpaired TTest. For the TTest I used table 5. The result turned out to be far below than 0.05 (2,29*10-26), which means that the difference between the control and carmine group is significant.

Table 3: Speed Assessment of P. tetraurelia Across 300 μm

We can see that both groups had the same initial average speed. After adding Carminic acid for the first time, the speed of the carmine group became 3 times slower and continued to get slower over each addition. The control group on the other hand had a more constant speed. This is shown very clearly in Figure 2. There is significant difference in the speed of Paramecia in both the groups which could be seen in the TTest (Table 4) where the result is below 0.05 as in the average number of paramecia per ml.

When they were moving slowly, it was as if like they were moving through some sort of gel, but when I checked the consistency of the substance, it appeared to be having the same consistency as water. Maybe there was some very small difference that could not be seen with the bare eye.

When the Carminic acid Paramecia tetraurelia were transferred back to their normal habitat, some of them died after 1-2 minutes (approximately ½ of them), the others regained their “normal” speed after 3-4 minutes and they were much more comfortable in their initial environment than in the Carminic acid solution. The distinction between the two groups was that the Carminic acid group was slightly pinker/redder compared to the control group.

Discussion

Adding Carminic acid to paramecia tetraurelia inhibits four enzymes, namely leucine aminopeptidase, acid phosphatase, esterase and γ-glutamyl transpeptidase. Leucine aminopeptidase is an enzyme that breaks down Leucine, which is an essential amino acid, into amino acids which are then used in the biosynthesis of new proteins. The inhibition results in some proteins not being produced as the essential leucine can not be broken down for new proteins. Acid phosphatase is responsible for the hydrolysis of phosphate. But when phosphate can not be hydrolysed, then the production of ATP will be impeded. The inhibition of esterase is probably not relevant for the slow movement.

Most probably the inhibition of these both vital enzymes cause the Paramecia tetraurelia to move slowly or even die. The uptake of Carminic acid happened through the intake of food, as the food I gave them turned red (because it’s food dye) and through that the Carminic acid could enter the organism and inhibit the respective enzymes.

As some Paramecia survived the higher concentrations of Carminic acid they must have adapted to the newer and more hostile environment. One possible explanation for this would be epigenetic modification, where some genes were activated while others were deactivated, like the new active gene sequence codes for enzymes that have similar purpose like leucine aminopeptidase and acid phosphatase. Or the other possibility is that this could have happened through various mutations. Both explanations are very plausible, as chances for a mutation are very high, as Paramecia tetraurelia reproduce relatively fast (all 10-20 minutes (in optimal conditions) which means that one Paramecium could have hypothetically divided itself more than 95000 times during my whole experiment). This makes the later more possible than the epigenetic modification, as the Carminic acid is an extract from insects that live in extremely dry areas while P. tetraurelia live in water.

Due to this difference in habitat P. tetraurelia could near to impossible have come in contact with Carminic acid in the past. This hints that they could not have evolved a Genetic sequence to be resistant to Carminic acid and as they didn’t need it anymore, they just shut that gene off. That is why a mutation is more plausible than an epigenetic modification.

Conclusion

With this Extended Essay, I can accept my hypothesis that Paramecia tetraurelia can be made resistant to lethal substances through regular and gradual increase of concentration. I basically answered my research question, but I couldn’t determine how they could adapt to their new, hostile environment as both groups did not have the same speed at the end, nonetheless they survived the high carminic acid concentration, which they initially did not. If I would have given them even more time, unlike the 48 hours which I gave, to adapt to the new conditions, they could have regained their original speed. This was shown in my side experiment where they had ±5 days to adapt to the concentration. But the following questions could not be answered from my experiment. What is the highest concentration of Carminic acid they can survive (with this procedure)? Would this experiment also work with only 24 hours of recovery time? Is it a mutation or an epigenetic modification that made them survive the high concentrations?