Clinking Glasses: Not Just for Toasts

Jaclyn Caruso, Salem State University

Me Aquarium

The Approach: How can sea urchins tell us whether preservatives used in cosmetics are harmful to the environment? Our approach involves the rhythmic clinking of glasses—but we’ll get to that.

As detailed in my previous post, concerns about the use of parabens as preservatives led to the introduction of new, “safer” alternatives, like phenoxyethanol and chlorphenesin. We are testing the effects of these preservatives on the early development of the purple sea urchin, Arbacia punctulata. Sea urchins are useful for laboratory studies because they are easy to rear and they have free-swimming larvae with distinct morphologies, allowing us to test for both lethal and subtle effects of these chemicals.

Because this is a first test with sea urchins and concentrations that induce a response have varied among studies, the concentrations of chemicals in seawater we are using span a large range—1000 to 0.1 parts per million (ppm). Including controls with no chemicals creates 34 jars per trial. Over the summer we will be able to complete several independent trials using different male-female pairs.

To collect sperm and eggs from male and female sea urchins, we apply a mild voltage with electrodes to the top of the sea urchin close to the gonopores, the small openings where gametes come out. Once we gather the sperm and eggs, we dilute them to specific concentrations, combine them, and let nature take its course! After an hour, we add fertilized eggs to each of the jars, which are stirred by glass paddles. The stirring allows larvae to get plenty of oxygen and to avoid settling at the bottom of the jar. The paddles have to be glass because plastics can potentially alter the results (ASTM, 2015). The whole apparatus has a pleasant clinking sound once running!

After two days, we collect the developing stages, preserve them with methanol, and keep them at ‑20°C until they are measured. Finally, the analysis begins! We load a counting chamber with the larvae and use a microscope to count the number of individuals that are in each stage of development. By this point, a larva should have 4 arms—known as the pluteus stage.

Pluteus

A preserved 4-arm pluteus larva. Scale bar = 0.1 mm. Jaclyn Caruso, 2018.

If larvae in the chemical jars are at earlier life stages compared to the controls, it suggests that the chemicals delay or stop development.

To look at more subtle effects, we are also measuring 20–25 individuals that have reached the larva stage per jar using 10 specific landmarks on the body. The microscope uses a camera lucida to project an image of the larva onto a digitizing pad that can record the location of landmarks in three dimensions. We use these data points to generate 3D models of each of the larvae to measure the size of the skeleton. This technique will allow us to test for sublethal effects of the chemicals that might not be obvious at first glance.

Workspace

The camera lucida arm of the microscope sits over the digitizing pad. Jaclyn Caruso, 2018.


Acknowledgements

Thank you to Dr. Bob Podolsky (CofC) for his mentorship, Dr. Cheryl Woodley (NOAA) for providing her procedures and resources, and Pete Meier (CofC) for teaching me the ropes of setting up aquaria. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.


References

ASTM (2015) ‘Standard Guide for Conducting Static Acute Toxicity Tests with Echninoid Embryos’, Astm, 131, pp. 1–2. doi: 10.1520/D7385-13.2.

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