By Cheyenne Moore, Team SPAW:
Week seven was one of the most productive weeks yet! On Tuesday, Libby and I extracted more DNA from the snails we had collected from Fennon Slough a couple weeks ago. Last week, Ryan and Libby had pulled the remaining snails from their shells and froze them all together in one container. This frozen blob of snails is what Libby and I started with on Tuesday. Opening the container was probably the worst part of the whole process because the snails stunk so badly! Once we got the frozen chunk out of the tube, we had to break it into smaller chunks and put it on ice so it wouldn’t melt. We used a mortar and pestle to grind the snails up with dry ice and placed the ground up snails into a container. We put all of the ground up snail into one container, hoping that it would be homogenized enough that we could detect the cercariae using PCR if there were any in any of the snails.
The next step was using the DNeasy Plant Kit to extract the DNA. Libby and I each did five samples each using roughly 100 mg of snail, for a total of ten samples. We thought that ten samples was a pretty good amount of samples to test, and that’s all we had enough tubes from the plant kit to do. Adding different reagents, centrifuging, and repeating is how the rest of the DNA extraction went. The smell from two week old, rotten snails is definitely not very pleasant. Amy, one of the girls who works in the lab, said she was going to move a table outside and make us work outside because of the smell.
On Wednesday, Ryan and I ran gels of the snail DNA that Libby and I extracted the day before. The reason we ran a gel was to make sure that DNA had been extracted. With the many practice runs Libby had us do with other DNA, Ryan and I figured out how to set up our samples for our gel on our own with success. It may not seem like much, but when you can do something on your own in the lab with success it’s like a small victory. From our gels, we could see that the DNA had been degraded because the snails sat for a few days rotting before they were frozen; nonetheless, we had DNA.
The next step with the snail DNA was running PCR with the primers we had chosen. Ryan and I chose three samples total, two from the first snail DNA extraction and one from the snail DNA extracted the day before. We chose our samples based on the concentration of DNA in the sample; the higher the amount of DNA, the better is what we thought. Using previous knowledge, we set up our samples with the primers we designed and primers for a repeat sequence in T. Ocellata. We also used Lambda DNA spiked with snail DNA as a positive control and each set up a no DNA tube. We programmed the computer to use the same two step cycling we have been using and let it work its magic. We had three samples total amplify, two we had picked and the one spiked Lambda DNA. In order for them to amplify, the DNA has to bond with the primers, so we thought that we had managed to get cercariae DNA along with the snail DNA.
On Thursday, using the same DNA as the two samples that amplified in the earlier PCR, we set up another PCR reaction. This time, we doubled the amount of everything we used to double the reaction size. We did everything exactly the same using the same primers, only doubled the amounts. The PCR had completely different results though. Only one of the samples amplified and it had a goofy looking curve compared to the curve shown on the last PCR. Libby suggested that we should purify the DNA and send it to the University of Montana for sequencing so we can look at the sequence and see if the primers actually could bond to the sequence or if it was a fluke. We used a PCR purification kit and purified only the sample that had amplified in both PCR runs.
As of right now, we don’t have the results we would like to have for our project, but that’s all a part of research. We have lots of stuff that we can use for our paper and poster about our tried and failed attempts, but hopefully next week we can get more snails and get some positive results. After spending so much time working on this, I am really anxious to see if what we have been doing will be successful.
By Jacob Feistner, Team SPAW:
Week 7 was an action packed week. With time running out on the field work phase of this project, I needed to get to two more lakes. Getting to and sampling Courville and Lucifer Lakes would take some planning, a lot of work, and I would need some help from others. We would also need the cooperation of the weather, our gear, and our instruments.
On Monday morning we had our usual team meeting. This week ours was at the Berthelote home since the school was closed. We had pancakes and bacon, Shawna did a primo job on the breakfast. After our meeting and discussion Ryan Gustafson, Sam Wall, and myself packed up and were ready to head for the mountains. As usual we were in for some hot weather and bugs. This trip is long enough and far enough from home that it would require us to stay the night at Courville Lake. To get to Courville Lake we take the same trail that gets us to Mud Lake 1, 2 and 3. This is a pretty good trek in itself, just getting to the third Mud Lake. Once at this lake I needed to retrieve my raft which I had cached in a tree two weeks earlier. After retrieving my raft and taking a short break we changed our direction from hiking due east to hiking due south. This took us off trail and over a pass that would drop us into another sub watershed where we find Courville Lake. We arrived at Courville Lake at about 5:30 in the evening, tired, hungry, and bug bitten.
At Courville Lake I wanted to accomplish everything that I had accomplished at the other lakes, such as a physical profile, several different on-site titrations, and testing different parameters and different depths. I also wanted to collect water samples to be filtered and then brought back with us to the lab, and I wanted to catch fish that I could fillet and bring back to check for mercury content. This would be too much to try and do the next day so I would need to get some of it done the evening that we arrived. So while Sam and Ryan are getting some of their things out and organized I started pumping up the raft and getting gear ready to take out on the water. When I pumped up the raft I noticed that a hole I had patched the last time I used the raft looked like it was leaking again, and I noticed another hole that I had not seen before. I attempted to patch these holes and we headed out onto the lake. Ryan and I paddled out to the middle of the lake and collected 1800 mL of water .5 m below the surface. We also completed an alkalinity test, cl test, and turbidity. Then we returned to the shore, I moved on to filtering the water while Ryan grabbed his fishing pole and went to work trying to catch fish. I was successful in filtering the water and I got it on ice to keep it cool until our return home the next day. Ryan returned to camp with several fine looking cutthroat trout. We were hungry, so I looked in my pack for the butter and spices that I had brought specifically for this occasion. By the time we were done eating fish it was dark and time to hit the sack. We hoped the fishing would be just as good the next day so that we would have some good samples to bring back to the lab and test for mercury.
On Tuesday we woke up at the lake and were ready to get some work done. Ryan and I would head out on the water and Sam was going to catch the fish that we needed. Ryan and I paddled out onto the water, but as we neared the opposite end of the lake the patch that I had fixed on the raft came loose. When the patch came loose the air in the main chamber didn’t waste any time getting out of the raft. This gave us the opportunity to show anybody watching how fast we could paddle this raft to the nearest shore. Our rafting on Courville Lake had come to an end for this trip. I called to Sam to bring Ryan’s boots and I started walking the deflated raft around the perimeter of the lake back to our camp. Along the way I found a good rock to work off of and obtained some more readings with the hydrolab, and DO kit. It took all three of us the rest of the morning to catch the three fish that we would take back to the lab. After catching them I filleted them, and packaged them to get them back to the lab. Next, we packed up camp and hit the trail for home. I would need to get home at a decent hour so that I could get rested up to hit the trail again on Wednesday.
Wednesday morning I met Shandin in St. Ignatius and we hit the trail on our way to Lucifer Lake. Lucifer Lake is a beautiful lake, but the trail is a real grunt to get up. This lake is of special interest because of its location and the geology that surrounds it. This lake is set in a limestone geology which we assume will affect the alkalinity, pH, and the lake’s overall ability to buffer against atmospheric pollution. This was a very nice and enjoyable day. Along the way I was able to talk to Shandin about my project, the report that I am working on, and also some of the cultural significance of the plants, flowers, and rocks that we passed by on the way to the lake. Here again at Lucifer Lake I obtained the filtered water samples and the fish samples that I needed to take back with me to the lab. Fortunately the fishing was a lot better at this lake than at some of the previous lakes. I was able to get the fish I needed in just a few minutes. My raft is out of commission so I left it at home this trip. I collected my water samples from the center of the lake using an inflatable mattress, and the rest of the tests that I carried out were done off of a rock along the shore of the lake. We were able to accomplish everything that we had set out to do and we returned home happy to have been able to include Lucifer Lake in our study.
On Thursday I was able to get down to the University of Montana and take all of my water samples from the three lakes to the chemistry lab there. Heiko Langer and Matt Young had agreed to run an ion balance, and a metals test on the water samples I collected. I got a brief tour of their lab and then I headed for home to get to work on other things. On Friday I had planned on getting into the lab and cleaning all the equipment that I had been using. The lab was closed and there was nobody around to open it up. I was able to get the fish samples into the freezer but it looks like the sampling will have to wait until next week.