Sperm and egg variety show

Cecilia Bueno, Lewis & Clark College

This summer my research sought to look at the effects of low levels of salinity on the sperm activity and fertilization in squirrel treefrogs (Hyla squirella). In particular, we were interested in whether there was variation in how much sperm were slowed down by saltwater and whether this variation would correlate with the percentage of eggs fertilized by a particular male.

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Squirrel treefrogs have a wide variety of colors from brown to green. Preliminary results suggest they also vary in sperm response and fertilization success at elevated salinity. (photo credit)

In order to answer these questions, I performed two experiments- one looking at sperm activity in response to salinity, the other looking at fertilization at 6ppt salinity.

Our sperm experiment produced videos of sperm at different salinity levels (~0ppt, 4ppt, 5ppt, 6ppt, 7ppt, and 8ppt) from 30 different males. I then ran the videos through an updated Computer Assisted sperm Analysis (CASA) software on Imagej in order to find out how many sperm were moving in each video (percent motile) and how fast they were moving (average velocity). The percent motile and average sperm velocities were calculated for each male across trials.

Our preliminary results suggest that there are significantly lower levels of sperm activity at an increased level of salinity than the control- both with lower average velocity and lower percent motile. However, there does not appear to be a clear relationship between increasing salinity and sperm activity when comparing between 4 and 8ppt.

Since we used sperm from 30 males, we were able to look at variation in these males as a sample of their population. Our preliminary results suggest that there is significant variation in how different males’ sperm responds to increasing salinity. This is important because variation is needed if the population could to adapt, though future experiments need to be done to determine if this variation is heritable and can be passed down from parent to offspring.

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Male 2 (H. squirella) from my experiment where he was collected from at Dixie Plantation, Hollywood, SC.

The fertility experiment looked at fertilization success at 6ppt with 32 males and females. We found a large amount of variation in fertilization success overall within the population, from near 0% too near 100%, with many in-between. Preliminary results suggest that a significant amount of this variation can be attributed to both male and female influence.

When comparing sperm activity and fertilization success at 6ppt salintiy, our preliminary results do not find a significant relationship between the two. This proposes future questions to be studied about what could be causing variation among the two factors.

Overall, our preliminary results do suggest that salinity has a negative effect on sperm activity and fertilization. However, results also suggest that there is variation in the severity of these effects. There are many more questions to be answered about the effects of salinity on gametes (sperm and egg) and fertilization in frogs- from what could be causing variation, to effects on unfertilized eggs, to variation between different species and more. As freshwater systems continue to be threatened by increased salinity, answers to these questions will be increasingly important.

I would like to thank the National Science Foundation for funding this REU program, and the Grice Marine Lab of the College of Charleston for hosting us. In particular, I would like to thank my mentor Dr. Allison Welch for her help and support.

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