Bugs’ Breath

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Greetings! This is Allison Lutz, a Juniata College student researcher for the Grant lab. Recently I’ve been working on a research project that involves aquatic insects (macroinvertebrates/macros) and their gills. I’ll be telling you all a little bit of what’s happening with the project currently and how it all got started.

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A net-spinning caddisfly (roughly 9-12 millimeters)

So, this project started about a year ago when I read a scientific paper about macroinvertebrate gill deformities that occur in mercury enriched streams. I then thought it would be really cool to look at macros from streams that are considered ‘fracked’ and streams that are considered pristine to see if there were any deformities in the gills. After deciding what to research, a couple of my friends and I went to collect the macros from two of the sites (one pristine and one fracked) that we usually sample during the summer. We all kick-netted for macros at the sites and put the macros in ethanol to preserve them for later identification. At the pristine site we even kick-netted a small brook trout, which pretty much never happens due to the trout being evasive. After collecting the macros we brought everything back to the lab where the macros were identified to the family level.

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I decided to use the family Hydropsychidae (net-spinning caddisflies) because of the gills on their abdomen that are easy (relatively) to remove. Along the way, I made sure to keep gills from each individual macro separate and once I removed all the gills from one individual using a pair of forceps, I mounted the gills on slides. It took me multiple tries to find a way to mount the gills; I just couldn’t get the mounting medium right until I noticed some clear nail polish. It seemed like too simple a fix, but I tried it anyway and it worked almost perfectly. I used a pipette to put the gills onto the slide then I waited until the ethanol dried, and finally I put a drop of clear nail polish on top of the gills and covered it with a cover slip. In total it takes about two hours to make ten slides. The next part of the process is using a light microscope.

I carefully carried the slides up to the microscopy lab and tried to decide on how to view the gills with the microscope. I finally settled on viewing the slides using dark phase which has a black background and the item on the slide is white/opaque. After viewing a few of the slides, I realized that trying to use statistical software to analyze percent deformities would be difficult and maybe there was a better way. I decided to take pictures of all of the slides and use a program to measure the widths of the gills and determine if there were any differences between the two sites. I took as many measurements possible on each slide at the midpoint of the gills visible on the slides. This process probably took the longest since I took pictures of the slides wherever I saw gills, so each slide had up to twenty pictures and each picture required multiple measurements.

Finally I got to the final part of the process: the data analysis. I used statistical software to analyze if there were differences between the two sample sites. There was a difference between the streams! The gills of the macros at the fracked site had significantly wider gills than macros from the pristine stream. From this point it was a lot of searching the internet for research papers in my search as to why the gills could be wider. I found a few papers that mentioned dissolved oxygen and its effects on gills. Everything I found seemed to point to dissolved oxygen (oxygen that is in the water), since lower dissolved oxygen levels can cause phenotypic plasticity (i.e. gills becoming wider) due to the organisms not receiving enough oxygen from the environment. Well that would be a slight problem because dissolved oxygen measurements weren’t taken from either site so there was no way to know if that was the cause of the wider gills. The finding was still very exciting though, because there was a difference between the fracked and pristine stream.

The initial study was meant to see if it was worth looking into macro gills and it was so the project moved forward. Macros and dissolved oxygen readings were collected from all of the sites we visited this past summer. Analyzing what was collected this past summer will be the majority of my work this semester and I am really excited to get this data analyzed to see if dissolved oxygen levels are playing a part in the gills of macros. There will be a few changes to the project and a major one would be using the same genus to compare between sites instead of the same family. The genus level is a lower level of classification and therefore it can’t be argued that the differences in gill sizes are just differences between the genuses that fall under the net-spinning caddisfly family. I also hope to measure the macros themselves to account for body length when I measure gill widths and use similar sized macros from each stream. I just to make sure I didn’t use larger macros from the fracked site and smaller ones from the pristine site because that could explain the difference (macros that are larger need larger gills to receive enough oxygen). I am very hopeful that this project will determine whether or not fracking practices might be affecting dissolved oxygen in streams. I can’t wait to see the results of this project!

-Allison Lutz

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