We are pleased to announce we have completed our experimental trials and achieved statistical significance in our results. Please head over to our results and recommendations pages to view our findings and next steps!
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This week, we imaged what we believe to be a parasite on one of our Daphnia magna under the EVOS microscope. Alternatively, it could also be the maxillary gland that regulates oxygen and ion intake. Regardless, it is bright red in color and appears on the outside right edge of the D. magna body. This organism could not be monitored post-imaging because upon returning, its heart had stopped beating.

We also analyzed the cross-sectional areas of several control (non-estrogen) organisms as compared to that of one D. magna exposed to estrogen. In this analysis, we found that while the average ratio of heart cross-sectional area (CSA) to body CSA in a normal D. magna is 1.9%, the ratio in an estrogen-exposed D. magna was 3.4%. This is an astonishing increase and is in line with our hypothesis. Because this is only one datapoint, it is not entirely significant, but it could still be indicative of future results after spring break. The average CSA ratio of the non-estrogen D. magna was calculated using both male and female organisms, demonstrating that differences in sex are not significant when discussing the proportion of heart to body. This is especially important because female D. magna are significantly larger in both length and width than males, so it must be ensured that these differences do not cause problems.

Upon qualitative analysis of two D. magna under a standard microscope, it has also become clear that the D. magna exposed to estrogen have a significantly increased heart rate. Although we have not calculated the difference quantitatively, it is visually apparent upon comparison with previous D. magna ​videos. They also appear to be seizing at times. Again, this is not technical data because there are only two data points thus far. To view these videos, click the links below.

We've had several setbacks over the past few weeks concerning our cultures. Our starter cultures are experiencing population wipeout, with both Cultures A and B dwindling severely in numbers. We were able to determine a closer range of EE2 dosages by diluting our original HIGH (2000um/L), MEDIUM (1500um/L), and LOW (1000um/L) concentrations down to numbers between 300-900 um of EE2 per L. Unfortunately, while we were testing these concentrations, our negative concentration also experienced high mortality, indicating that there are problems with our husbandry procedures. We are currently waiting for our starter cultures to recover more before attempting another trial.

Because of shortages in time, we might not be able to complete any more pre-trials; instead, we may be forced to complete one singular experimental trial as opposed to several weeks of data collection. To collect the appropriate number of data points, we will be testing only one concentration of EE2 in several identical containers for a minimum of 120 data points.

To improve the viability of our cultures, we are experimenting with feeding them more, as research demonstrated that the color of the D. magna is indicative of the state of their food supply; if they are brightly pigmented, then they are well-fed, but if they are translucent like our current cultures, they are most likely closer to starvation.

Below are several clear images of the D. magna magnified under the EVOS microscope.

We have made extensive progress on our research in the past few weeks!

​As mentioned before, our D. magna from Carolina Biological came in in the beginning of January, so we have set up two starter cultures to prevent population wipeout.

Each starter culture is filled with two liters of stock solution - a combination of DI water and compounds recommended by Cornell University - and administered .131g of pulverized Daphnia Food (Carolina Biological) every Monday, Wednesday, and Friday. Each culture is also fed in the case of a water change, which occurs once a week, as during these cleanings, all food is removed. Finally, we have incorporated a heating pad donated by a peer advisor in order to keep our culture temperature between 20-25 degrees C.

The cultures are proliferating rapidly, with a difference of hundreds in just over a week. Pictured below is Starter Culture A on the day that we received our shipment of D. magna from Carolina Biological and then exactly seven days later, after the D. magna had time to produce offspring.

With all of these additional D. magna, we have been able to begin testing our blue light technique in order to separate the juvenile D. magna from the adults. Previous research suggested that all D. magna are attracted to blue light, particularly at a wavelength of 470 nm. Therefore, we constructed a separation tank using a nylon net with 500um holes - large enough to allow the juveniles to pass through, but small enough to stop the passage of the adults. This allows us to age-synchronize our D. magna for our experimental trials, which rely on the assumption that all of the D. magna are similar ages.

This technique has proved largely successful. Although not all juvenile D. magna chose to pass through the net to the other side, enough did travel through to supply us with sufficient D. magna for our first official pre-trial. Here, we transferred 60 juvenile D. magna from the far side of the tank into 4 containers filled with 180 mL of their appropriate concentration of EE2 (4 containers of 15 each). Due to the limits of our analytical balance, these concentrations were mixed in larger volumes by combining 997 mL of stock solution (compounds + DI) with 3 mL of DMSO and the appropriate measurement of EE2 - .001g for LOW, .002g for high, and .003g in 2L instead of 1L for MEDIUM. We then bottled the extra concentrations for later use. This trial is still running and will be analyzed for mortality rates on Tuesday.

We plan to start our experimental trials in just a few weeks or less, depending on the results of the ongoing trial. In the meantime, thanks for continuing to follow our research! Updates to follow.

 After being approved, we've begun working on our pre-trials. Because of limits in the delivery of our necessary supplies, we've started perfecting our basic husbandry using Daphnia magna from Mr. Seaquist's classroom culture instead of D. magna from Carolina Biological. Using this technique, we were able to image several D. magna. Although we were able to view their whole body, we still had difficulties imaging the heart due to time restraints and inexperience. Below are several images that we captured using the EVOS microscope.
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Additionally, we've solidified our feeding and mounting procedures. However, we are running into some problems with the viability of our culture that we are currently resolving. This may have been caused by the absence of compounds from our culture water or potentially excess light exposure. To remedy this, we are now using water from the original culture tank in Mr. Seaquist's room. We are also opting for natural window light for the time being.

Above is Peightyn, holding the original Daphnia magna culture, and Brynne, holding the lab notebook.

Wonderful news! Today, we were approved by our instructors with revisions to our pitch, meaning that we may proceed with our research under the new guidelines. This update comes after the pitch presentation we gave to nearly twenty virtual guests concerning the design and feasibility of our research. Several highlights of our presentation are included below.
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Today, we had the privilege of joining a Zoom call with our mentor, Dr. Giovannini of UCLA, and his graduate student, Phillip Richards.  We went over our tentative question, plan, and timeline using the rough visual aid linked below.  There were many uncertainties going into the call as related to our project, but the conversation with our mentors alleviated many of them; they had answers to nearly every question we posed, and we are incredibly grateful that they have agreed to help us in this process.  For example, we currently have interest in the software ImageJ, which they had worked with in the past and were able to share essential insight on. They also provided us with excellent journal articles and resources to further our understanding of our topic.  The main critique they provided was to solidify our hypothesis and justification; in other words, we need more research to back up why we were predicting what we were.  All in all, the call went extremely well, and we're very excited to have the opportunity to work under such wonderful guidance.