Lydia Bruno

Experimental Evolution in Daphnia magna: An Investigation into the Effects of Ammonium Nitrate Exposure

April 2, 2021   /  

Student Name: Lydia Bruno
Major(s): Biology
Minor(s): Environmental Studies
Advisor: Dr. Hilary Edgington & Second Reader: Dr. Dean Fraga

Water pollution is a major issue in the United States mostly due to agricultural pollutants entering the water systems. Agriculture leads to the increased presence of both pesticides and fertilizers in the water. One common fertilizer that is used is ammonium nitrate. Components of this chemical have been shown to lead to evolution in many different species, however this exact chemical has not been examined fully. Daphnia magna is a keystone species seen in freshwater bodies of water across the world. They provide a direct link between primary producers and other animals. They are also known to be a good indicator of pollution due to their sensitivity to pollutants. We conducted an experimental evolution study to determine the effect of ammonium nitrate on D. magna over the course of four generations. Comparisons using microsatellites and sequence data from genes of interest show no evidence of evolution. However, differences in growth rate, birth rate, and death rate were able to be seen between the treatment groups. Specifically, the low concentration treatment group experienced higher birth rates and growth rates compared to both the control and the high concentration treatment group. These higher rates could be attributed to a hormetic effect of the chemical, leading to a stimulatory effect at low concentrations but detrimental effects at high concentrations. Although evidence of evolution was not seen, changes in life history could lead to some large changes in ecosystems. At low concentrations of ammonium nitrate there could be a population boom of D. magna, while at high concentrations the population could crash. Both of these effects could lead to extensive ecosystem changes.

Lydia will be online to field comments on April 16: 4-6 pm EDT (PST 1pm-3pm, Africa/Europe: late evening).

39 thoughts on “Experimental Evolution in Daphnia magna: An Investigation into the Effects of Ammonium Nitrate Exposure”

  1. Congratulations Lydia! Very interesting work! I especially appreciated how well you explained all the work in your video.

  2. Congratulations Lydia! It would be very interesting to see if the evolutionary results would be more dramatic with the ability to research more than four generations. I am hopeful, you will have the ability to continue this research in the future. Great Job!

    1. It would definitely be possible to see different results if the research would have been able go on for more generations. I am definitely hoping to continue this type of research in the future.

  3. This was really interesting and informative. Isn’t it amazing that you can carry out an evolutionary experiment? Just think how jealous a biologist from 100 years ago would be to see what you were able to do in your IS. The Grants’ book, “Beak of the Finch,” was the first time I understood that we can see natural selection happening on the timescale of our lifetimes. Congratulations on your IS and graduation!

    1. Hi Ken! It was so cool to be able to do this type of research. I wasn’t familiar with this type of research until I was discussing potential IS topics with another professor and he mentioned another experimental evolution project that has been going on for 30+ years. Thanks so much!

  4. Congrats on the great presentation and all your hard work Lydia! Your research process and methods are so impressive – I hope you have a lot of pride for all you have accomplished with this project. I have some questions from the perspective of a non-bio major: In the beginning, what are the little black spheres in the belly of the daphnea in the picture? Also, how many daphnea total did you work with this semester? Lastly, do you think out in the wild there could be interactions between the pesticide and other agriculture-related pollutants, such as herbicides and fertilizers, that could cause different impacts for wild daphnea compared to your cultivated daphnea?

    1. Thanks Tess! The black spheres in the belly of the Daphnia are eggs! I am not entirely sure how many total daphnia I worked with this semester, but I would estimate roughly 300 total individuals over the course of the 4 generations of each treatment group. However, when taking DNA samples I only looked at 60 total individuals. Finally, it is totally possible that interactions in the wild would be totally different that what was seen in the lab. Most fertilizers in aquatic environments lead to algal blooms, which causes other types of issues such as lack of oxygen. It is also possible that algae can take up pesticides faster than animals leading to less effect. Also, pesticides seem to be more detrimental to the health of organisms so any effects seen may be contributed to pesticides because there is more research on that topic. Hope I could help!

      1. Thanks for your thoughtful answers Lydia! That is very interesting, your explanation regarding algae blooms. I learn so much from you. I have a follow-up question:
        If you had 4 generations, does that make you a great-great-great-grandma, or a great-great-great-great grandma?

        1. I think that I am only a great-great-great-grandma. The actual first generation (not in this project) was like my daughters and the F1 of this generation was like my grand-daughters, so three generations after that would make me a 3X-great grandma!

  5. Great presentation! Why do you think the population of daphnia who had high concentrations of ammonium nitrate initially rose to only later crash?

    1. There was one major hypothesis that I had for that reason and it was that this concentration was still in the hormetic zone for Daphnia magna. This would have led to a stimulatory effect in the population which could have led to conditions such as the population boom. Then, there is a possibility there was a food shortage before the start of generation 3. Some research suggests that food shortages can make the effects of toxins more severe because individuals don’t have enough energy to devote to detoxification processes. I tried to keep the feeding schedule between all three treatment groups consistent, so that could have led to a period of food shortage, making the toxin more toxic to the individuals in the tank.

  6. Hi Lydia! Great job on your presentation! It was super interesting and easy to follow, and it’s super cool that you were able to research evolution in a laboratory setting. What makes a compound, specifically ammonium nitrate in daphnea, hormetic versus just being detrimental to the organism?

    1. Hi Julia! Thanks for your feedback. A hormetic compound is one that at low concentrations leads to stimulatory effects, while at high concentrations leads to detrimental effects. As for how exactly ammonium nitrate leads to stimulatory effects at low concentrations, I am not entirely sure as it needs to be further researched. Other compounds have been seen to be hormetic in Daphnia magna such as TNT (yes, the explosive). This compound is very high in energy, so it is hypothesized that at low concentrations it increases lipid metabolism in the individuals, helping them grow larger at a faster rate, while at high concentrations it leads to death because of general toxicity. I hope that answers your question!

  7. Great job, Lydia! Not being a biologist, I can’t visualize how one would extract DNA from Daphnia magna. Could you enlighten me?
    Congratulations!

    1. Hi Sanne! Thanks so much! First, the Daphnia are “homogenized,” which means the Daphnia were frozen in liquid nitrogen and then powderized. The powder was then exposed to certain chemicals that cause the DNA present in the individuals to solidify out of solution. Then I take other chemicals to resuspend that DNA in, making a liquid DNA stock that I can use for further research! I was sure to thank the Daphnia for their sacrifice in my acknowledgments.

  8. Awesome presentation, Lydia!
    What would you have done to continue the project or done differently if you had more time?

    1. Thanks Nicole! If I would have had more time and resources I definitely would have let the experiment continue for more generations. Additionally, it would have been cool to continue the research into the hormetic qualities of ammonium nitrate to definitively see if it is a hormetic toxin to Daphnia magna. This would involve exposing daphnia to many different concentrations of ammonium nitrate with replicates to see if the low populations of Daphnia magna actually perform better than those exposed to high concentrations.

  9. Brilliantly done Lydia! Very interesting, relevant, and thought provoking. Thank you for sharing and for making complicated information easy to understand. So proud of you! 🙂

  10. Congrats Lydia! This is such an interesting study (tbh I didn’t even know Daphnia existed before you took on this project.) I have a question- I know your last generation of Daphnia in the high concentration group suddenly died off. As far as you are aware, how did this impact the methods/results of your study?

    1. Great question Stephanie! It’s a little hard to say how this impacted my results because I did not have replicates in my study. Therefore, I was not able to determine if this crash was caused by chance or by the effect of the chemical or laboratory conditions. As for the methods, instead of taking DNA samples from live individuals, I took them from dead individuals in the tank. This allowed me to get similar amounts of DNA for each treatment group, but the DNA was lower quality because DNA degrades very quickly after death. I decided to allow other treatment groups to continue to evolve for as long as they could, which is why the research continued for more generations. I hope this answers your question!

  11. Great presentation, this research is fascinating! I have a question: you mentioned that you sent the DNA results to an analysis facility, which facility was it? And how did you go about organizing your collaboration with them?

    1. Hi Henry! Thanks so much! I sent the DNA amplicons to the DNA Analysis Facility on Science Hill at Yale University. It wasn’t exactly a collaboration, the facility, at the time, was open to students and researchers sending their amplicons for further analysis. As of right now I don’t this this particular facility is accepting more submissions. However, there are many different facilities open to this type of help! It definitely helps with the research process.

  12. Excellent job, Lydia! I’m now aware of Daphnia magna and I learned the term “hormetic toxin”. Your project was very thorough and enlightening. Congratulations!

    1. Thanks so much! I was not aware of Daphnia or hormetic toxins prior to my research either!

  13. Nice job Lydia! This presentation was very visually appealing and informative! The concept of experimental evolution if very cool. I am curious, were there any other genes that you did not look at that you think would be interesting to study?

    1. Thanks so much Erica! Definitely, If I were to go back and add additional elements to my study, it would have been interesting to look at genetic expression as well as genetic sequences. As for additional genes, it would be interesting to look at more genes in the Terpenoid signaling pathway that is responsible for male sex-determination in Daphnia. Seeing if these genes were expressed would help to further answer my question if ammonium nitrate exposure did activate the terpenoid signaling pathway, which could lead to male-sex determination or hemoglobin expression.

  14. Great job, very proud of the scholar you have become.

    In terms of the genetics of the daphnia. Did you find a particular gene or pathway that the ammonium nitrate seemed to interfere with? I might have missed this, but how did the low levels of ammonium nitrate cause (or could cause) a population boom.

    1. Hi Mr. Monsour! I’m so glad you were able to come see my presentation. I did not actually look at genetic expression, which I think I would need to see if ammonium nitrate would interfere with the pathway or a gene. However, I hypothesized that ammonium nitrate may have impacted the terpenoid signaling pathway, which is involved in male-sex determination in Daphnia magna. The terpenoid signaling pathway may be what is associated with the cyclical hemoglobin expression I saw. This could also be evidence that the Daphnia were experiencing stress.

      As for how the ammonium nitrate could lead to a population boom, I am not too sure. Future research would be needed to determine how hormetic effects actually occur. Other hormetic toxins are seen in Daphnia magna, one of which being TNT. At low concentrations the high energy contained in the chemical leads to increased lipid metabolism. This led to increased population sizes at low concentrations, but once the concentration of TNT increased too much, it led to the death of the individuals. I hope you are doing well!

  15. Awesome presentation, Lydia! I was wondering, has this project influences your future plans at all?

    P.S. RIP Daphnia

    1. Hi Kath! It has has actually impacted my future plans. It confirmed my ideas that a Ph.D program would be right for me – specifically one in evolutionary biology!

  16. I loved this experiment! The findings of genetic variation and differences in birth rates leave enough room for additional/differing experimentation to build upon.
    One question that I had for you would be whether you have considered testing more for genetic stress that would promote genetic variation in differing generations?

    1. Hi Cameron, there is plenty of room for future experimentation if you know anyone in search of an IS project! It would definitely be a possibility to induce genetic stress that would lead to increased mutation rates. Exposing the daphnia to UV rays or other mutagenic situations could lead to increased mutation rate which could lead to an increased chance of changes in my potential genes of interest that may have led to changed in the protein leading to either beneficial or detrimental effects. This is definitely something that could be looked at in the future!

  17. Bravo, Lydia! If you were able to continue this study, would you still use Daphia magna or would you choose something else?

    1. Hi Aunt Debbie! Thanks so much! If I would continue this study I would definitely continue to use Daphnia magna. I think they are a great model organism for this study because they actually live in the environment that I was trying to simulate in the lab. Also, they are a pretty new model organism, so new research is being done on this species all the time! Making it a great system to continue to study.

  18. You did such a good job, congrats Lydia! Your project was so interesting and thought provoking!

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