Final Thoughts


This semester, I initially wanted to explore the incidence of cloning in brittle star larvae, specifically in the daisy brittlestar, Ophiopholis aculeata. Larval cloning is a phenomenon found in many echinoderm species, but is least studied in brittle stars. My goal was to learn more about what induces cloning in brittle stars, to help better understand why they clone. Due to complications with spawning the adult brittle stars, I ended up changing projects to focus on the effects of different fluorescent stains on brood star larvae from the species Leptasterias tenera. 



In the summer I began writing a grant proposal for my proposed research on brittle star larvae. In the fall, I learned the basics of the lab as a volunteer and mostly focused on tank care and editing my proposal. By the start of the spring semester, my proposal was submitted and I spent some time reading over the background literature on cloning in echinoderms.


Brittle Stars:

From the end of January to February 20th, when the adult brittle stars arrived, I planned out my experimental design in more detail. This included deciding the temperatures I was going to use, the amount of lab space I would need, and how I wanted to schedule working on the larvae. I also solidified ideas such as separating any clones I found and looking out for broken vs. full larval arms. Right before the adults were set to arrive, I set up stirring racks for the larvae. I reconfigured the stirring rack at least 3 times to make the stirring motion as precise as possible.

On February 21st,  I attempted to get the adult brittle stars to spawn by placing them on a push cart and driving it around the ISC. I took 2 laps around the ISC, waited for any spawning to occur, then took another 2 laps around. No spawning occurred that night, and by Wednesday, February 27th,  the adults were still not reproductive. At this point I changed the focus of my research to brood star development.


Brood Stars:

Two female brood stars were included in the shipment of bat stars the lab had recently ordered. Both of them were holding broods of larvae, but died soon after their arrival. The broods remained mostly intact, and could potentially be used for a project. Danie and I sorted the brood star larvae and isolated all clumps into different bowls, and any individuals that had left those clumps into well plates. By March 13th, only 10 juveniles remained, from 1 female. On the 14th, I stained the 10 juveniles, submerging five in calcein and five in calcein blue. I left the juveniles in the stains for 24 hours, then checked them under different wavelengths of light to see if they had absorbed the stains. The calcein absorbed well, and the juveniles were stained a bright yellow color. The calcein blue created a more faint, blue stain.

On March 20th, I began taking photos of the juveniles. I took two photos of each individual, one using regular light and one using polarized light. I then measured the brood stars using these pictures in imageJ software. I measured the circumference of each juvenile, as well as the size of each of their internal disks. In April, I began feeding the juveniles frozen brine shrimp every other day. I photographed the juveniles two more times on April 12th and 22nd to determine if any growth was occurring. On April 22nd, one of the calcein stained juveniles died, and by the 24th a second calcein stained juvenile had died as well.


New Broods:

On April 12th, another order of six brood stars shipped from Maine. These were much healthier than the first two, and were still holding their broods. I isolated each brood star and her brood, as well as any extra clumps of larvae not attached to a female. My new goal was to isolate 25 juveniles from each female to create five treatment groups of four different stains and one control. On April 15th, I isolated 25 healthy looking juveniles from broods 1, 2, and 4 and put them in well plates. By the 18th, I was able to isolate 25 juveniles from five out of the six females. On April 22nd, I stained the new brood stars with four different stains: calcein, calcein blue, tetracycline, and alizarin. On May 1st, I checked the juveniles under different wavelengths and determined that the stains hadn’t been absorbed well, so I put the stars back in the stains for another 24 hours. On May 7th, Jon checked the brood stars again to see if the stains were absorbed. The calcein stained juveniles were much brighter, the alizarin stained juveniles were slightly brighter, and there was no improvement in the calcein blue and tetracycline stains.

Since the semester is nearly over and I graduate in 3 days, I will have to leave my project in this stage. Although I was not able to collect a significant amount of data this semester, I was able to conclude that calcein is a viable stain to use in larval brood stars, and calcein blue may be viable under the right conditions. Hopefully my work this semester can serve as a baseline for using stains in the future.



Works Cited:

Johnson, A. S., Salyers, J. M., Alcorn, N. J., Ellers, O., & Allen, J. D. (2013). Externally visible fluorochrome marks and allometries of growing sea urchins. Invertebrate Biology.

Eaves, A. A., & Richard Palmer, A. (2003). Widespread cloning in echinoderm larvae. Nature.