Air date: June 15, 2013

“Mesh Traps” is the second episode of the TV series The League of the Green Hornet and first broadcast on June 15, 2013. The series started its production on June 14, 2013. After week of careful planning and scheduling, it was finally the day to ut in action the soil re-colonization project. List of supplies and specific plans were made in order for filming to happen. On June 14, the team finally started shooting under the supervision of Dr. Brett McMillan.

A total of 48 mesh traps were buried 3 inches in the soil in 6 different sites. There are two locations per site, one near the path and one away from the path.  The near the path and away from the path mesh traps are filled with topsoil. Every sample has its own control mesh trap filled with regular soil. The purpose of the Soil re-colonization experiment is to determine the rate of re-colonization of invertebrates near the path and away from the path in order to verify if the path serves as a barrier for soil invertebrates.  We hypothesize that the samples near the trail will have slower rate of re-colonization due to the disturbance of the path used by humans and horses.  An early harvest will be collected in 5 days (Wed. 19 ) to determine the rate of re-colonization.  A late harvest will be collected in 11 days (Mon. 24).

Detectives Mary Chow, Xi Huang, Shirley Mancia, and Dr. Brett McMillian had a very efficientday placing 48 traps in 5 hours.  Thanks to Mary’s muscle power with the apparatus, Xi’s courage to go through the vegetation, Shirley’s speed filling the traps, and Dr. McMillian’s broad knowledge of vegetation. After completing 4 sites, we took a lunch break and on Shirley’s way to the van she saw an eastern smooth earthsnake.  It was not terrifying but surprising as it slithered back in to the tall grass. After completing the sixth site we felt very satisfied and accomplished after a long morning in the field.

Directed by: Dr. Jacobs and Dr. McMillan

Written by: Xi Huang

Starring: Invertebrates

Mary Chow

Shirley Mancia

Xi Huang

Dr. Molly Jacobs a.k.a. Molly

Dr. Brett McMillan

Air date: June 9, 2013

From left to right. Xi Huang, Mary Chow, and Shirley Mancia.

Getting soil samples.

“A Study in Soil” is the first episode of the TV series The League of the Green Hornet and first broadcast on June 9, 2013. The series started its production on May 29, 2013. It took three days of careful planning and getting ready. List of supplies and specific plans were made in order for filming to happen. On June 6, the team finally started shooting under the supervision of Molly.

PLOT

During the summer of 2013, in an ordinary neighborhood called Hashawha, three private investigators (Mary Chow, Shirley Mancia, and Xi Huang) joined forces to solve a mysterious case: whether trails in Hashawha act as a barrier in the migration of soil invertebrate communities.

A total of 60 soil samples will be collected from eight different sites. Nine samples will be from each of the four path sites: three samples from the center of the trail, three 3 m from the creek, and three 8 m from the creek. Six samples will be collected from each of the four non-path sites: three 3 m from the creek and three 8 m from the creek. The purpose of doing this is to test if soil invertebrate communities change on a gradient away from stream.

Detective Shirley Mancia recovers her thermometer.

While the investigators were working on their first site, something unexpected happened. Detective Shirley Mancia lost her precious thermometer! After Inspector Mary Chow poked a 9-inch-deep hole in the ground with a Soil Compaction Tester, Detective Shirley Mancia accidentally dropped the thermometer that was used to measure soil temperature down the 9-inch hole. In a moment of panic, Shirley started digging frantically. Fortunately, the thermometer was recovered. So once again, the day is saved. thanks to the League of the Green Hornet. But will they be so lucky next time? Until next episode.

We’ll be joined on the blog this summer by the Becker lab (Environmental Science), and the McKenzie/McCole lab (Exercise Science & Physiology).  Stay tuned!

To insert this plasmid into our Dicty cells, we linearized the plasmid and then used an electroporator to shock the DNA into the cells. We then added these cells to media without blasticidin for a few days, and once they seemed to be growing again we started to add blasticidin to select for the Dicty DCP2 knockout. At first the results were not promising, because even our control plates seemed to be growing the same as the knockout plates. After about two weeks, there was a marked difference between the controls and the transformants. When Dicty is healthy, it will adhere to the bottom of a petri dish in liquid media. The control cells were most definitely dead because they were all floating and very small and round. The transformed cells from the DCP2 knockout had many floating and clumped cells yet areas of healthy cells growing as colonies. When we found these colonies, we were all very surprised! The presence of these colonies confirmed that blasticidin resistance had been conferred into our Dicty cells! None of us expected success in this experiment because the gene could be essential for the cells to survive and/or Dicty transformation is a notoriously difficult and long process. Needless to say, we are thrilled with our results, but much work still needs to be done to confirm our gene knockout.

We next wanted to isolate clonal populations of Dicty that had the DCP2 gene knocked out.  To do this, we diluted the transformed cells and started to grow them on LP agar plates with B/r, a strain of E. coli bacteria. Dictyostelium eats bacteria in its natural habitat, the forest floor, so once our Dicty cells begin to consume the bacteria Dicty plaques will form on the plates.  A plaque is a hole in the bacteria that is assumed to have been initiated by one Dicty cell, thereby producing a clonal knockout population. When the cells have eaten all of the available bacteria in the area, fruiting bodies will form which contain spores. Then we will isolate the sporeheads and begin growing them in regular media with blasticidin.  Next, we will need to confirm the gene knockout by either PCR or Northern Blot. There is a possibility that the blasticidin resistance gene was put somewhere else into the genome rather than where the DCP2 gene is located, so while our cells seem to be blasticidin resistant, we cannot be completely certain of the knockout until further screening is done. Unfortunately I will not have time to screen these cells further, so Dr. Parrish will generously be coming in for the rest of the summer to finish the experiment. We all hope that the knockout is confirmed, but then we need to assess the consequence of the loss of this gene for Dicty cells.   We will look at the effect on mRNA turnover in these knockout cells, since DCP2 encodes a putative mRNA decapping enzyme.

In the last week of research we performed a Northern Blot, which is a method to measure levels of gene expression during different times of development. As you learned in Catherine’s post, we performed a time course last week and harvested the cells at different stages in their development. After extracting RNA from the cells we ran a gel to resolve the RNA. The blotting part of the experiment involved placing a membrane under the gel and a weight so that the RNA was transferred onto the membrane. After this was finished, we treated the membrane with luminescent probes for the gene of interest and visualized the results by film development with autoradiography paper.

Unfortunately, our film did not turn out as desired but this is most likely due to a problem with the blotting procedure. There was a lot of background on the X-ray film, making the results hard to interpret. There may have been problems with the actual transfer of the RNA to the membrane or possible RNA degradation, yet in either instance the experiment must be repeated. Dr. Parrish and future research students will troubleshoot the experiment to receive better results.

Summer research was an invaluable experience that I will remember for the rest of my life. Although Catherine and I worked hard (most of the time), we had a ton of fun in the lab and kindled a new friendship. Dr. Parrish was wonderful with helping us get a better grasp of our projects and a better understanding of molecular biology in general. We hope that you enjoyed our posts and enjoyed learning more about Dictyostelium!

~Kirsten

Last Friday was the last day for Kirsten and Catherine in the Dicty lab at McDaniel College. Although the summer was extremely busy for all three of us, I was still extremely sad to say goodbye to these bright and talented young ladies. As I sit in my office today, I miss hearing their giggles from my lab next-door, listening to their entertaining stories, and watching them grow as young scientists.  I think this means that I am in the right profession!

I referred to Catherine and Kirsten as my “miracle workers” this summer.  They were extremely successful with their experiments and accomplished more than I even anticipated.  I will let them tell you about their exciting results in their own blog posts coming soon.  However, to understand how much work they accomplished, here is a sampling of some of the techniques they mastered this summer:  Dicty cell propagation (both on media and bacteria), Dicty development, Dicty genomic DNA extraction, the polymerase chain reaction, agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation and selection, plasmid purification, Dicty transformation and selection, Dicty RNA extraction, Northern blotting, and autoradiography.  Perhaps most impressive of all, they learned how to do science, including how to plan experiments, perform the proper controls, and interpret their results.  They plan on presenting their work at a conference in the fall and this research will also be the focus of their senior capstone poster and paper next spring.

I want to leave by saying that everyone always says that the young have much to learn from the “old”.  While this is certainly true, I continue to learn so much from my students.  They remind me not to take myself so seriously and that humor can be found in the every day experience; I just have to change my frame of reference.  For example, while exiting the darkroom, the three of us became trapped in the dark room revolving door, basically a small enclosed tube that is in complete darkness.  After several harrowing minutes of being trapped, we finally managed to get out of the door.  My students stayed much more calm than me, and our perilous situation became the running joke of the week because of their light-hearted way of seeing the situation.  The door, post-incident:

Another example of their refreshing attitude related to our experience with a temperamental autoclave (basically a big pressure cooker) that we use to sterilize media and equipment.  On the last day, Kirsten and Catherine gave me a hot glove for taking hot items out of the autoclave.  On it they had written “ Autoclave Survivors 2012” “Living in Peril to Keep Things Sterile”.  (Don’t worry; we weren’t really in danger from the autoclave!)

I also received as gift a CD that had a picture of our shaking incubator as the cover.  All of the songs on the CD have the word “Shake” in the lyrics.   The gift bag also had a picture of Catherine and Kirsten embracing the autoclave.  How did I get so lucky to have such creative, kind, and bright students?

Our final day and a visit to “The Cow” for a frozen treat.

This picture shows the gel resulting from our restriction digest. This digest demonstrates that the bacterial plasmid DNA contains our insert. It was very exciting to see that all our work paid off.

We then used this plasmid to knock-out the Dictyostelium DDB_G0278957 gene by homologous recombination. The knock-out plasmid was engineered to confer blasticidin resistance and we were able to obtain D. discoideum colonies that were blasticidin resistant, suggesting that we were successfulin knocking out the DDB_G0278957 gene!. The next step is to obtain clonal isolates of the knock-out strain by diluting the cells and growing them on bacteria. The entire process of a Dicty transformation can take over a month! Once we confirm that the DDB_G0278957 is knocked out, we will analyze the biochemical consequences of the absence of the DDB_G0278957 gene and protein.

One of the coolest things we have done was to look at developing D. discoideum cells. When starved for nutrients, Dicty cells aggregate together to form a multicellular fruiting body containing spores that will be released when conditions are more favorable. The process of development takes 24 hours and we were able to induce development and see six developmental stages. We harvested these different stages to isolate mRNA and examine the expression pattern of DDB_G0278957 during development by Northern blotting. We started work early and plated the cells on non-nutritive filters. Throughout the day (and night), we looked at the plates under a dissecting microscope and observed the different developmental stages.. The first stage we observed was ripples. The cells gave the filters a shiny brown color and only four hours into development, waves could be seen forming on the plate. Four hours after that we saw loose mounds forming. By 11:30 at night these mounds had grown tips. It was really cool to see the cells changing. The next morning the cells had formed the final fruiting body stage containing the spores. Unfortunately, we did not have a camera set up to take pictures of the different stages as seen from a microscope but I have found this picture for you:

By Tijmen Stam, IIVQ (SVG conversion) – user:Hideshi (original version) (en:Image:Dicty Life Cycle H01.png) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC-BY-SA-2.5 (http://creativecommons.org/licenses/by-sa/2.5)], via Wikimedia Commons

Below you can see Dicty growing on plates containing bacteria. The clear regions are the regions where the Dicty cells have eaten the bacteria. As they deplete the bacteria, the cells aggregate and undergo development. The “fuzz” seen on the plates are the fruiting body containing spores.

This summer I have learned so much about molecular biology and have had a great time working with Kirsten and Dr. Parrish. Thank you Dr. Parrish and McDaniel for giving me such a great opportunity!

As a recent graduate, I was fortunate enough to receive a full-paid internship with the National Oceanic and Atmospheric Administration (NOAA) aboard the Okeanos Explorer to participate in sonar mapping of the Northeast Canyons in the Atlantic Ocean. The Okeanos is an educational research vessel whose main mission is ocean exploration (Okeanos-primary Greek water deity; Explorer-one who travels in search of scientific information). We left Norfolk, VA on May 27^th and arrived back in port in Davisville, Rhode Island on June 13^th.

A nervous intern, about to board the Okeanos Explorer in Norfolk, VA

As excited as I was to be selected as an intern, I was super nervous about living at sea for three weeks! How would I react to not seeing land for that long a time? Would I get seasick? What if the food isn’t good? What if I run out of shampoo? All of these questions lingered in my head as I lugged my overstuffed duffel bag up the gangplank.

You’ll be happy to know that I adapted extremely fast to living at sea. Before we left port, I stocked up on the necessary toiletries and snacks, just to be safe. Upon meeting the other members of the science team, I quickly realized I was the “baby on board”- the youngest and most inexperienced member! Being the newbie has its perks, however. Mapping took place 24 hours a day, which meant team members had to be in the control room around the clock. I was lucky to receive the 8am-4pm shift. My fellow intern, a graduate student at Old Dominion University, was not so lucky, receiving the 8pm-4am shift.

Learning the ropes of multibeam sonar mapping was smoother than I could have imagined. The science team was extremely patient in explaining how multibeam sonar works, and what my duties were during my shift. From 8am to noon, I kept log books updated and watched over the multibeam monitor to make sure the data we were collecting was usable. While mapping these canyons, the water depth changes drastically, and if the multibeam is not compensated for these changes, the data will be useless. From 12:30 to 4pm, it was my turn to clean the data we were receiving in a program called CARIS. A picture of what canyon data looks like in CARIS lies above (terrasond.com). My primary job was to search for and remove “outliers” in the data-points that were simply out of place and were clearly not a part of the data set (a school of fish for example). Being able to view the topography of the ocean floor, literally as you float above it, was an incredible experience.

One member of the science team, David Packer, planned on using the data we collected in order to identify deep sea coral habitat within the canyons. Working for NOAA’s Northeast Fisheries Science Center, he hopes to conclude that these canyons serve as prime habitat for deep sea corals, and to begin the process of naming these canyons as marine protected areas. I interviewed Packer for the Okeanos Explorer website. The full story of his mission can be found here: http://oceanexplorer.noaa.gov/okeanos/explorations/acumen12/1204_interview/welcome.html

While onboard, I had to learn the nautical lingo. Starboard side was the right side, port side was the left, the stern was the back of the boat and bow was the front, forward meant toward the front, and aft meant toward the back. Frequently we had to contact the bridge during our shifts in order for NOAA Corp officers to position the ship where we needed to map. We had to don a headset with a microphone, and call the bridge using a switch board saying “Bridge, this is survey…” (survey is lingo for control room). We also had to use phrases like “Copy that”, “Roger”, and “back deck is secured”. At first I felt silly, like I was in the military, but after the first few days these phrases became second nature.

So that was my job during my shifts! During my time off was a different story!

I shared a stateroom with another member of the science team, who recently received her PhD from the University of Delaware. We each had a bunk and a large closet, and our room was complete with our own bathroom. Additionally, our room was equipped with Direct TV, so we were never without our favorite TV shows. Our evenings consisted of Friends, Seinfeld, and Family Feud.

Meals onboard the ship were absolutely incredible! Three cooks served us hearty breakfasts, lunches, and dinners every day. Breakfast consisted of French toast, pancakes, or Belgian waffles, bacon, sausage, ham, and hash browns. If you wanted eggs, you simply took your plate to the kitchen door, informed the cook how many and how you wanted them cooked, and waited for your name to be called when they were ready. Lunches varied-pizzas, burgers and hotdogs, Reuben’s, tacos, and Swedish meatballs (one of the chef’s favorites). Dinners varied as well. Italian night consisted of ravioli and veal parmesan with homemade garlic bread, Chinese night with homemade fried rice and low mien, and our last meal was steaks and King crab legs. Homemade chocolate chip cookies were always on hand, and a freezer in the pantry was fully stocked with ice cream whenever you wanted.

Once a week, we had to participate in emergency drills. The first drill would be a fire drill, followed by an abandon ship drill. An officer would come over the intercom saying, “This is a drill, this is a drill, this an abandon ship drill”. We were required to grab a ball cap, a long sleeved shirt, long pants, our life jacket, and our survival suit (nicknamed Gumby suit because you literally look like Gumby). Once on deck, roll was called and we had to prove to the NOAA Corp officers that we could don our Gumby suits in 1 minute-easier said than done! Once the drill was complete, we could return to our regularly scheduled programming.

The weather was beautiful for most of the trip. Sunny skies and mid 60 degree temperatures made for comfortable outings on deck. Dolphin sightings in the morning and afternoon became the norm, and we were lucky enough to catch a humpback whale one morning. Occasionally, we would set up camp chairs on the fantail (back deck) and eat dinner. On Tuesday and Friday nights, we would drape a sheet over the ROV hanger on the fantail and watch a movie (The Muppets was my favorite!). One night we discovered the sea had turned neon green due to the bioluminescence of the plankton.

Sleeping onboard was never a problem either! The gentle heave and roll of the ship on the waves rocked you to sleep. In fact, the first night back in port I slept horrible because I was simply stationary. Tropical storm Beryl did cause the team one miserable night. The waves were so rough it was impossible to sleep. You were literally airborne while lying in your bunk, your personal items falling off your dresser, and your door flying open and slamming shut again in the middle of the night. Needless to say, the data collected that night were useless. But one night out of nineteen isn’t bad!

The day we pulled into port was a depressing day. I had loved living at sea. Being away from the hustle and stress of the real world was liberating, and being on the ocean is definitely where a marine biologist belongs. Shortly before we docked, our leader Meme told me I should go up on deck because land was insight. Disheartened, I declined saying I’d rather be back at sea. That night we went out to dinner and toasted our successful cruise, and sadly said our goodbyes to each other the next morning.

Now, as I search for graduate schools in the coming future, I am so thankful I was given the opportunity to participate in this expedition. It opened a whole new door and is shaping my visions for grad school and career paths. I realized I would be happy and content to be at sea frequently, and this conclusion was sealed when tears fell as I left the ship. I am know considering deep sea biology as an option for grad school, as well as earning my certificate in ocean floor mapping, in order to participate in future expeditions.

Pheww…you’re probably exhausted from reading this entry. I guess when you’re passionate about something it’s easy to get carried away. NOW McDaniel students: I challenge YOU to find your passion. Study hard, work hard, persevere! Nothing important ever comes easy. Try new things, especially the things that scare you, and take chances! That’s where I found my passion-on the open ocean.

“We lose ourselves in the things we love. We find ourselves there too.”

The science team of the EX1204 on the fantail. I'm in the "L"!!

Full picture of the Okeanos Explorer at sea! (moc.noaa.gov)

From left to right: Joe "Noctiluca" Odierno, Steve "Ophlitaspongia" Hein, Deanna "Assassin Snail" Campell, Molly "Internal Wave" Jacobs

An explanation of the costumes:

Joe: Noctiluca is a free-living marine dinoflagellate famous for bioluminescence – these tiny single-celled organisms release brilliant green flashes of light when they are disturbed.  During a bloom at night, boats leave green glowing wakes and swimmers are surrounded by sparkles.  The outside of Joe’s cape was a nondescript gray, allowing him to flash the luminescent interior at appropriate moments.  He won a “people’s choice” award for this costume!

Steve: Ophlitaspongia is a genus of marine sponges, often found in the local intertidal.  They are bright orange in color.  Most predators and grazers leave them alone due to their chemical defenses, but if you look closely you can often see a small specialist nudibranch predator (Rostanga sp.)

Deanna: The assassin snail Clea helena is a small tropical freshwater snail, known as a deadly predator of other snails.  You can’t see the back of her costume in this photo, but she had a shell displaying the typical black and yellow stripes.

Molly: An internal wave is an oceanographic phenomenon – we’re all used to waves that propagate on the surface, but waves can also propagate under the surface, usually along interfaces between upper and lower bodies of water (for example, along a thermocline or halocline).  Small planktonic organisms (shown in the pictures on the box and my shirt) often collect at these interfaces.  My costume showed these organisms along an interface, and I also waved internally (from inside the box).

I did figure out how to include the S.E.M. by looking to see how morphological aspects of juvenile Oregonia gracilis affect their behavior. I am also interested in characterizing the different morphological features of the very young juveniles, as it has not been done before. Finally I am looking to see if there are any differences in setal density between different periods of O. gracilis’ life.

I learned how to use the S.E.M. last week. It is amazingly easy to use. If you can use a camera you can learn how to use the S.E.M. It only took about 5 minutes to master. The theory on how it works, on the other hand, is not so easy. Well, here are some pictures I took with it.

A carapace of an O.gracilis megalopa, the final larval stage of a crab before metamorphosis into a juvenile crab. They can look quite different between species. There are a few here that look similar but the way the spines are laid out distinguishes between them.

Compound eyeballs look tight.

A carapace of a few day old juvenile O. gracilis. All the hair like projections are called hooked setae. These are how decorator crabs attach decoration to themselves. It is a mechanical attachment much like Velcro. Hooked setae are unique to the spider crab family Majoidae.

Here is a juvenile eye ball because it looks cool. It kind of has an eye brow of hooked setae. Pretty sweet.

A close up of some of the setae. These ones are on the base of the rostrum.

Cheers,
Steve