Bad Neighbors: How the Collateral Damage Hypothesis Modulates Predation and Parasitism Pressure in Neo-Tropical Frog Species.

Our Project:

A calling Tungara frog.

Each year, Professor Trillo brings X-SIG students to conduct research at the Smithsonian Tropical Research Institute in Gamboa, Panama. One of Dr Trillo’s research projects in Panama focuses on the role of eavesdroppers on sexual signaling in frogs. This summer, the labs’ main focus is to understand how collateral damage can influence predation and parasitism risk within a single species. Collateral damage, a concept pioneered by Dr. Trillo, refers to the dangers associated with calling near highly attractive heterospecific species. Previously, Dr. Trillo found an increase in midge parasitism for hourglass treefrogs when they called near Túngara frogs, which are highly attractive to predatory bats and parasitic midges. Dr Trillo’s hypothesis is that attractive signalers can inadvertently increase predation or parasitism on heterospecific neighbors by increasing the number of these enemies that are attracted to the aggregation as a whole.

Trachops cirrhosus, a frog eating bat.

This same idea of collateral damage across species could also be applicable within a single species, if individuals within that species use more than one type of advertisement signal, and if one of those signals is more attractive to predators and parasites than the other. This is the case for Túngara frogs. 

Corethrella, a genus of parasitic midges. These midges tend to siphon off blood through the frog’s nostrils – ouch!

When attracting a mate, Túngara frogs can modulate the complexity of their mating calls. A “simple call” (see Figure 1), which involves a whine only, is less attractive to eavesdropping predators and parasites. However, simple calls also are less attractive to potential female mates.

Conversely, Túngara frogs can produce complex calls (see Figure 1), where extra “chucks”, made of broadband sound with a greater frequency range, are produced in addition to the tonal whine. These calls are highly attractive to females, but they also increase the number of predators and parasites to the calling frog. With these ideas in mind, we hypothesize that the highly attractive complex calling Túngaras will increase the risk of predation and parasitism of their simple calling neighbors. In order to test this hypothesis, we are currently performing playbacks of simple and complex Túngara frog calls to investigate how signaling patterns of neighboring frogs change the risk of attracting eavesdropping bats and midges.

Figure 1: Comparison of frequency ranges between Tungara simple and complex calls.
Setting up a nocturnal playback trial along the Pipeline trail.

To test our hypothesis, we conduct acoustic field playback experiments to quantify bat and midge visits to complex and simple Túngara frog calls. Each night, we set up, two speakers with a combination of simple or complex calls. Our four treatments consist of simple call-simple call, complex call-complex call, simple call-complex call, or a single speaker with only one simple call. We set up camera traps to record bats visiting each speaker and we place fly traps above each speaker to collect the number of parasitic midges attracted to each type of calls. After the experiment is done, we count the number of flies present and carefully upload the bat videos taken at each site in order to score them in the future. 

Each day, after the field playbacks, we identify and count upwards of a thousand Corethrella midges.

Although we expect each experiment to run smoothly, troubleshooting is almost always necessary. Performing field work in the tropics poses unique difficulties. The humidity and heat of the tropics force us to take extra precautions with our electrical equipment. To prevent water damage due to condensation, equipment needs to be carefully stored in a heated box each night after trials. Battery charge, which could degrade quickly due to corrosion, is carefully monitored each day. Each night entails a long hike from site to site through the tropical jungle. Although collecting data in the field can be grueling, our lab is eager to see our results, and the potential effects of collateral damage on túngara frogs. 

Research off Campus

The Smithsonian Tropical Research Institute New Lab in Gamboa, Panama.

Conducting research off campus at the Smithsonian Tropical Research Institute, or STRI, in Panama offers opportunities to gain a unique perspective on academia, global research, culture and nature. STRI is one of the leading tropical research organizations where scientists come from around the world conduct field work next to the Panama canal. In Gamboa, we work alongside a closely-knit community of organismal biologists, geologists, ecologists, and other scientists dedicated to understanding and sharing information about tropical ecosystems.

Dr. Warkentin presenting at a weekly frog talk.

Each week, we go to a “frog talk” or a “Tupper talk” depending on our work schedule. These seminars are held to allow researchers in Panama to share their work and ideas, and to receive feedback from their colleagues. Frog talks, one of our lab’s favorite events, occur each Wednesday in the casual setting of a neighbor’s living room. Every week researchers of universities from a variety of different countries gather in a living room, with refreshments in hand, to listen to a speaker present their latest results. These presentations are an awesome display of cross-disciplinary and cross-cultural work: often, presentations examine biology from an interdisciplinary perspective. For instance, Dr. Warkentin, a professor in Biology and Women and Gender Studies at Boston university, gave a lecture on “queering herpetology,” which examined social biases that affected how animal behavior had been previously studied. Other talks range from examining the molecular costs of insect pheromones, to demystifying perplexing female coloration in the white-necked Jacobin hummingbirds. In all, while many assume that working in a lab as an undergraduate entails hours of repetitive tasks and uninspiring work, working at the Smithsonian Tropical Research Institute has given our lab an incredibly diversified insight into research.

Nature and Culture:

A 3-toed sloth hanging out as we made our way to the La Chunga site to set up a playback trial.
A common basilisk out and about during late-night hours.

            Aside from the questions our lab aims to answer, our surroundings have also impacted our understanding of current environmental issues. Simply put, Panama has a wealth of biodiversity. It isn’t uncommon to wake up to the raucous chorus of parrot calls each morning, to see monogamous pairs of toucans flying from tree to tree, nor to encounter a caiman during our night excursions on the pipeline hiking trail. For some of the lab members, the idea of “tropical diversity” became a tangible concept – a concept that prior to this trip had vaguely existed on the glossy pages of a National Geographic magazine.

Keel-billed toucan in Professor Trillo’ backyard.
A troop of howler monkeys passed overhead during one of our weekend hikes on the Pipeline trail.

Working in the field has also been an inherently immersive learning experience. During our first few outings, the frog chorus at experimental pond was a cacophony of croaks and whines. However, after a few weeks of nightly visits to the pond we could begin to identify the approximate number of frogs calling at the pond, their species, and the locations from which they called. We began to understand when frogs started and stopped calling or how the rain that day might affect the density of frogs that night. Likewise, as we continued our daily hiking trips through the tropical forest, we slowly began to recognize different flora and fauna, and understand how the wild plants interacted with surrounding organisms. It was as if we were slowly learning a wild language through our immersion in the tropical forest, and understanding this language helped us empathize with our surroundings.

           As we continue our project with Dr. Trillo, we are eager to analyze our data to find previously unrecognized patterns that improve our understanding of the links between predator and prey. While these last few weeks will be a whirlwind of hiking, playbacks, video viewing and fly counting, we will continue to take full advantage of our unique and incredibly beautiful surroundings.

Probing lights finite travel speed in large scale structures

In Cosmological research, data is derived from two main sources, observation based, and simulation based. Each type of data has its pros and cons. In observational data, you see real, physical situations allowing better insight into how the universe works, but getting the information is time consuming and often a large portion of the information is unable to be retrieved. In simulation data, you create pseudo-physical situations and have access to all the data and parameters for an object. The downside of this is it’s based on our interpretation of physical situations. A major issue arises when trying to compare these two forms of data, the finite nature of the speed of light.

 In simulated models, light speed is assumed to be infinite, all times are centralized and there is no delay when looking at a distant object in a simulation. That is not the case in observational data. Light takes time to travel, when you look up into the sky you are gazing into the past. This becomes an issue across the largest structures currently formed in the universe, like galaxy clusters. Objects that are further away take longer for time to reach us than objects that are closer. For a galaxy, rotating rather slowly when compared to the speed of light, this effect would probably be negligible.  For a galaxy cluster, who’s size scale is on the range of Mega-Parsecs (3-30 million light years), and the velocities of its constituent galaxies are on the order of a percent the speed of light, this effect becomes much more meaningful.

The issue that arises with observational data is that it is projected down to 2 dimensions on the sky. A 3-dimensional object in the plane of the sky loses all of its dimension of depth. Since Galaxy Clusters have a dimeter of 3–30 million light years, the light emitted from objects at the far edge of the cluster takes 3-30 million years to reach the front. So, when an observation is made of a galaxy cluster, further objects are 3-30 million years back in time when compared to objects in the front, by the time light from both objects reach the plane of the sky.

There have been many statistics I’ve had to use to try and probe this issue. The first being seeing how velocity dispersion profiles were changed when created with a time offset or not. A velocity dispersion profile maps how the distance a galaxy is radially from the center of the cluster. We ran into challenges with this method, while we saw a minor difference, we came to determine that the statistic wasn’t sensitive enough to small changes in position (relative to the size of the cluster) to provide a meaningful result.

 Our next statistic to probe was the Lee Statistic. The Lee Statistic is a bivariate analysis judging the likelihood that a collection of objects on the sky make up one group, or more. The statistic takes your data and projects it down to a one-dimensional line, it then compares each point and judges the dispersion of points to the left of the point, the right of the point, and the total dispersion. It is highly sensitive to changes in position, so we judged to it be a solid follow up. Our first method was the 2-dimensional lee statistic. I took the simulated 6-dimensional data (3-dimensional position, and 3-dimensional velocity), and project it into 2 dimensions of position (in the plane of the sky). I also copy the data and insert an artificial time offset, I then project the data sets onto a line with varying angle of phi with respect to the axis. After probing this with a single cluster, we started work with a merging cluster. There was a larger difference between the data with the artificial time offset and the one without it due to the larger distance light must travel across the merger. We then moved onto the 3-dimensional Lee Statistic which also considers a dimension of velocity along the line of sight (perpendicular to the plane of the sky) along with the 2 dimensions of position. We found this to be our most sensitive statistic to time offsets as measured so far. The goal of this research will be to determine some way to decide how necessary it is to make a correction with statistic.

Perhaps what was unequivocally the most important aspect of my summer was my daily hackey sack sessions with my research group. There’s a new person you discover inside of yourself when you are in the arena with your fellow comrades, and I’ve come to get to know, and love, that person. All struggles, big or small, seem to be insignificant when you achieve the state of hack. I have yet to forsake the hack, and I never will, as is commandment number one for hack sack master and chief – Jedi Johnson.  

Throughout this project I have experienced a myriad of problems that I needed to think critically to solve. There were times where I needed to totally adapt large portions of my code to allow different simulations to be tested and being able to just have access to various data bases and finding ways to download it is rather difficult. Other issues I faced were minor errors in my code that started early but went unnoticed that I had to spend hours debugging. Over the past two years I had to completely reinvent my thought process when it comes to coding, and it made me become a better programmer, and a better scientist.

Figure 1 – Illustris data set primarily used this summer for data collection

Figure 2 – pseudo merging cluster created from illustris data set. Shown from two separate viewpoints

Figure 2 – various 2D projections of a merging cluster at different angles from the merger axis.  This is done on data similar to the set in figure two and projected down to two dimensions at different inclination angles.

  Figure 3 – Various 3D Lee Statistic images 1000 iterations with 100 members in each group. Separation between the red and blue lines shows a statistically significant different between time corrected and non-time corrected data.

Observational Astronomy Crew: Meet the Team and Check out Our Work

This is our observational astronomy group. Pictured above left is Sheldon Johnson.  Sheldon is a rising sophomore and a physics major, with plans to be an aerospace engineer. You can normally hear him laughing at some ridiculous thing he or someone else did and normally he has his headphones on singing out loud to his heart’s content.

Pictured above right is Brian Taylor. Brian is a rising junior and physics major. He has an interest in astrophysics and would like to continue to study it in graduate school.

Pictured above middle is Autumn Tripet. She is a rising sophomore and an aspiring physicist who wishes to continue astronomy in graduate school.  On a typical workday you can find her in Master’s listening to music while simultaneously trying to sort through and reduce data from our recent trip to the Lowell Observatory in Flagstaff, Arizona.

Behind the camera is Dr. Jacquelynne Milingo. She is our faculty mentor for the work we’re doing this summer.

We started off our research by reading through the basics of telescopes and trying to get an understanding of the world around us. We spent a good amount of time becoming familiar with the material and concepts we would be working with, as well as reading through an abundance of software manuals. As our beloved professor Dr. Milingo would say… “R.T.F.M. !” (if you’re curious as to what that means, ask us in person).

After getting up to speed, we officially started working on a cap design for the baffle on our campus telescope (a baffle helps eliminate stray light).   This cap replaced the plastic bag that was covering the baffle at the Gettysburg College Observatory.  Seriously, we’ve had a plastic bag covering the baffle of our telescope for more than 15 years, so you could say it was time for a change. Pictured below is the infamous plastic bag.

After a week of measuring and tinkering around with dimensions on the OnShape CAD program, each team member was able to come up with a unique design to replace the trash bag. The programming and tinkering with the cap models looked a little something like this:

We printed our designs on a 3D printer and completed a prototype to finally switch out the bag. Here’s that glorious moment for everyone to bask in … 

Aside from working on new designs for the Gettysburg College Observatory, we also put our new small telescopes together. This allowed us to better understand the material we read on telescopes prior, letting us see first hand how the different parts work together in creating a magnified image of the sky. At the end of July we will use these telescopes during a public outreach event with girl scouts at Camp Happy Valley in Fairfield, PA. Our hope with this event is to educate kids on the possibilities in science, especially the influence of women in science. One of the telescopes we assembled is pictured below.

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Eventually, we found ourselves on an airplane flying to Arizona to do some observing at the National Undergraduate Research Observatory (NURO) which is part of Lowell Observatory in Flagstaff, AZ. NURO is a 31 inch telescope. We spent 6 days in the southwest, with 4 nights of observing. Our goal was to take images of the open cluster NGC 6866 for future studies of spotted stars in the cluster. We also took images of standard stars for calibration. A picture of the open cluster can be seen below.

NGC 6866 open star cluster

Image courtesy of the Aladin Sky Atlas: https://aladin.u-strasbg.fr/AladinLite/

 While we were observing, a typical day would start at about 6 in the evening. We made our way to the observatory around that time to setup and run startup routines for calibration frames before sunset.  Once it was dark enough (after nautical twilight) we could focus the telescope and begin making observations of our target stars. The night usually didn’t end until about 4 in the morning if Mother Nature was kind enough to give us a clear night.

During the night, we took turns on each of the computers, each working and controlling a different aspect of the telescope and camera. The dual monitor shown below left allowed us to control the camera, the filter wheel, and to analyze images as they came in.  The older style computer shown below right controlled the telescope directly giving it coordinates to move to, objects to look at, etc.  The third job was keeping a log for the night. This consisted of writing down our course of action, system failures, coordinates, air masses, filters, exposure times, and any other useful notes for that night.

Our workstation in the warm room is shown below.  Below is is the telescope control computer monitor as well as the auto guider system monitor.  The auto guider allowed us to lock on certain objects in the sky for longer exposures.

Telescope control and star auto guider

Trials and Tribulations While Observing

Observational Astronomy is not all clear weather and good data. Our team found this out while working at NURO. System failures, software crashes, and clouds were a big bump in the road while we were trying to gather data. We soon realized that this was a somewhat normal occurrence when doing observational work and dealing with complicated interconnected equipment.  Add in the mild sleep exhaustion due to our nightly runs and it’s all part of the experience of astronomical observing.

Fun things We Did While in Arizona

Even though we spent our nights at the observatory (instead of sleeping), our team managed to get up at a reasonable time every day to explore the great views of Arizona. Staying in Flagstaff, we were about a 30 minute drive to scenic Sedona, Arizona. We were lucky enough to hike the West Fork of Oak Creek Canyon, visit the Grand Canyon, explore Sedona, and be submerged into the Flagstaff community. A big part of the observing experience was being able to explore and learn about the land around us, and specifically how the certain climates and elevation affects our observational work. Some of our favorite scenic moments are highlighted in the pictures shown below.

Group Picture at Oak Creek Canyon
West Fork of Oak Creek Canyon
The Grand Canyon
Sedona, Arizona
Group Picture at The Grand Canyon
West Fork of Oak Creek Canyon, Sedona, Arizona

Good Vibrations in the Caldwell Lab

            When many people think of sound, they think of it traveling through the air to a listener’s ear for interpretation. From a broader perspective, sound is vibrational energy that can take many paths on the way to a receiver – through the air, through ground, through plants, etc. Sometimes, these vibrations are interpreted in the ear, but animal species vary in the physiological structures they use to interpret this energy. In the Caldwell lab, we are especially interested in learning how animals interpret vibrations traveling through multiple substrates to inform behavioral interactions between animals of the same or different species.

            The first thing we did was jump right into science… Just not exactly how we had expected. Fieldwork brings about a whole set of challenges that do not occur in typical controlled lab settings. Our first experiment is investigating vibrotaxis, or the movement of an organism in response to vibrations, in foraging snakes. In addition to airborne sound, calling frogs produce vibrations in the plants they sit on. If snakes are able to detect these vibrations, then they may be able to use them to hunt frogs. In order to conduct this experiment, we needed an arena that would allow us to observe how snakes use vibrations in their foraging behavior. This arena had to fit in the little hut that we had access to that doubled as a storage building, but also had to be large enough to fit a Y-shaped branch of some sort that was about two meters tall. With these guidelines, Professor Caldwell took us shopping to get a bunch of materials and then left us to figure out how to build it. 

            We started with the basics- measuring the space and making the frame. We accomplished this without any major catastrophes. Then we had to determine the best material to wrap around the sides, top, and bottom to prevent a snake from escaping the arena. We planned to use a black mesh material for most of the arena, but were unsure if a camera with an infrared light could see through it to monitor the snake. Following a trial, and after adding a large infrared light attached by wire to the ceiling, we found that the mesh worked well.

            The next step was, in theory, simple. We needed a small mesh door that could both close with Velcro and prevent a snake from escaping. What this boiled down to consisted of sewing sticky adhesive Velcro. This entailed quite a bit of frustration at the beginning due to the adhesive component of the Velcro and the repeated knotting of the thread as the material was stitched together. Eventually, however, we developed the technique and made the door.

            After several more experiences including sewing mesh together that was already stapled to the wooden frame, having screws snap in the wood as we made cross beams, and hot gluing across every seam to be doubly sure that the snake wouldn’t get out, our arena was done. Unfortunately, on our second trial a snake escaped the arena within five minutes and we were back to sewing.

            Once our arena was truly complete, it was onto the more typical science. We captured a few chunk-headed snakes and set up small terraria for them complete with a water tray, Nalgene bottle with some water and plants, and a branch that they could climb on. We named these snakes and recorded notes on their care. We ran three preliminary trials per snake in our arena with no vibrational stimulus. This consisted of attaching two Red-eyed treefrog egg clutches to different sides of the Y-shaped branch in our arena and waiting for five hours between typically 8PM and 1AM while the snakes foraged. If they ate both egg clutches before the five hours, we ended the trial. In each of these trials, the snake foraged, meaning that we could commence the experimental trials.

            To prepare for the experimental trials, we had to get raw material for the stimulus. This meant going out to the Experimental Pond nearby and recording frog calls and the vibrations produced in the plants they call from. We used a shotgun microphone to record frog calls with as few background sounds as possible, as well as an accelerometer to capture the plant-based vibrations. An accelerometer looks like a thin wire with a little cylindrical head on one side. It works by measuring the acceleration at which the plant moves, therefore detecting vibration information.

            The only issue with this was that in order to attach the accelerometer to the plant, we had to tie it on with flagging tape. If we tried to do this while a frog was on the plant, which is ideal because we want to get the vibrations produce by a calling individual, we would disturb the frog and sometimes cause it to jump away to another plant, making the accelerometer position ineffective. To remedy this, we would often attach the accelerometer to a plant and then move one or more frogs onto that plant, then quickly walk away for a few minutes, and come back cautiously to see if they stayed and started calling. If they did, we could record. If not, we had to try again. This required retrieving a lot of frogs and moving them to adequate positions. If they were not on the edge of the pond where we could reach and were instead in the center, which happened more often than not, we would have to wade into the pond and catch them, bringing them back out to the plant with our recording set up. This resulted in a lot of flooded boots and wet feet.

            After getting several recordings, we could create our stimulus for the playback experiments. We listened to the recordings and looked at the signal to noise ratio especially in the vibration data to determine which recordings would be best to use. Once that was done, Professor Caldwell created the stimuli for the experiment by first removing all extraneous sounds and vibrations. Then it was time to equalize the stimulus to the branch in our arena so that we could play back call vibrations with similar acoustic properties to those of the original call.

            Playback experiments rely heavily on the stimulus being played back sounding like the stimulus that was recorded. However, recordings never really sound exactly how they did when they were recorded, partially because the equipment acts as a filter. Audio equipment is generally good enough to avoid any serious alterations. Substrate vibrations are usually altered more drastically because they attenuate differently than sound in the air. The substrate itself filters vibrations and each substrate does so differently. For these reasons, equalization is necessary for vibration playbacks. This involves playing white noise, consisting of equal amplitudes at all frequencies, through the substrate/system being used, in our case a branch, and then rerecording the white noise. The branch will filter this noise, resulting in the new recording having altered amplitudes across some frequencies. The measure for how the system filtered the noise is a transfer function. To account for this, the inverse of the transfer function is added to the system, playing the lowered frequencies relatively louder than the unaltered frequencies. After running this process a few times to make the transfer function accurate, we apply it to our recorded stimulus to make a more natural playback.

            After making the stimulus and equalizing it, we started our experimental trials. To focus on the snake’s response to vibrations specifically, the audio portion of the stimulus was played above the center of the arena instead of on one side of the branch like the vibrations. We introduced the snake to the arena and recorded it on a camera for up to three hours. Once the snake had passed the “Y” in the branch, we recorded it for one more hour to observe its foraging behavior. Eventually, these videos will have to be analyzed thoroughly, taking note of which side or sides the snakes went to as well as the amount of time spent there.

            Our experiences in the Caldwell lab have shown us that not all aspects of science are actually collecting data. Lots of it, especially in field work, is trouble shooting, and making what we have available work for what we need to do… And troubleshooting again. Every day is engaging and although some aspects can be frustrating when things don’t work, it is highly satisfying when we come up with a good idea and solution to a problem. With more experimental trials with our snakes in the near future and ABR studies on the horizon to determine what frequencies of sound and substrate vibration snakes and frogs can sense, we are going to have a busy remainder of the summer.

Social Media, Self-Compassion, and Symptoms of Disordered Eating

The idealization of thinness is prominent in Western cultures, and much research has shown that women’s exposure to images promoting the desire to look thin in television, movies, and magazines is associated with their body dissatisfaction and symptoms of disordered eating (including excessive dieting, and binge/purge behaviors). Over the last decade, the increase in the availability of smartphones and social networking platforms has made media exposure more accessible and more constant in women’s daily lives. Now, though young adults and adolescents are less likely to be flipping through the pages of the latest Cosmopolitan magazine, over a billion active users are exposed to countless images and videos on their Instagram feed every day. It is therefore important to examine the effects of exposure to social media content on women’s well-being.

The Personality Lab has been studying a number of common so-called “positive” cultural trends that are actually detrimental to mental health because of the toll they take on self-compassion. One such trend that the lab has been investigating is  ‘fitspiration.’

Fitspiration (also known as fitspo), is an online trend focused on promoting a healthy lifestyle by motivating viewers to exercise, eat well, and take care of their bodies. Images under the fitspiration hashtag usually depict images of females who have generally skinny, toned, perfectly built bodies. Despite its good intentions, research suggests that fitspiration photos have detrimental effects on body image, body satisfaction, and self-compassion, perhaps because they transmit guilt-inducing messages and unrealistic portrayals of the ideal beauty, similar to those of pro-anorexia websites.

Self-compassion is defined as being kind to oneself, being mindfully aware of one’s flaws and accepting them as part of being human (Neff, 2003). Self-compassion is important for body image because it acts as a protective factor against body shaming, body dissatisfaction, and disordered eating behaviors. Because of this, research conducted in the personality lab has been examining how exposure to different kinds of Instagram content affects self-compassion and mood. We have been especially interested in whether the effects of exposure to different kinds of images may depend on the individual’s level of disordered eating symptoms. That is, exposure to fitspiration images may be even worse for some people than others.

Earlier this summer, we analyzed the results of an experiment conducted in a sample of Gettysburg College women and found that as predicted, looking at fitspiration images lowered participants’ self-compassion and mood more than looking at images related to body transformation (successful weight loss), healthy eating tips, and travel (control). Moreover, the effects of looking at fitspiration and body transformation images on self-compassion were even worse for individuals with higher levels of disordered eating symptoms. Interestingly, all body-related images (even those with healthy eating tips) had negative effects on participants’ mood, relative to the travel images.  

To extend this research, we have been developing a follow-up study to examine whether these effects are also true of another common online body-image trend: body positivity. Because body positivity promotes the acceptance of all body types and treating our perceived body inadequacies with care and support, it may be more compatible with self-compassion (i.e. self-kindness, mindfulness, common humanity). On the other hand, it could be that all body-related images, including those presented with good intentions, are triggering for individuals with disordered eating symptoms. We plan to research whether or not viewing images related to body positivity may be a healthier alternative to viewing images of fitspiration, and particularly in those with higher eating disorder symptoms. This research is important in today’s age, especially as we begin to rely more and more on social media as a source of what is deemed acceptable or trendy in society.

– Cindy and Thao

We look at bugs underground

Hello it’s Georgia, Owen, and Professor Urcuyo coming at you from the great caves of Pennsylvania. In our lab we are working on creating an inventory of the invertebrates inhabiting local caves in Franklin County. We collect the samples ourselves during our caving trips and then bring them back to the lab to sort, photograph, and identify.

Before our first caving trip at Carnegie Cave…

and after!

With low levels of light, coldness, and lack of cycling nutrients, caves are a difficult environment to inhabit. Besides the entrance, you soon find the only source of light is the one from your headlamp. Caves remain at a constant temperature and are typically around 52˚F in PA. There is no presence of primary productivity, so the only available nutrients come from anything entering the cave and the water that resides. Some of the passages in the cave can drop as short as a foot tall and are quite difficult to get through. There can also be pretty deep water that accumulates inside. Some passageways can become flooded sectioning off portions of the cave.

Flooded passage

Along with trapping invertebrates inside the caves, we also collect data on temperature and humidity in the back, middle, and entrance of the cave.

“If you see a raccoon in the tunnel, just get on your feet and hands and let it crawl under you”

–Ish

All of us are working together to identify our samples to the lowest taxonomic level, we’ve split up our preliminary research but otherwise we all have the same role in the lab.

Hey it’s Georgia and I’m focusing my research on Collembola. Collembola, otherwise known as springtails, are small jumping hexapods estimated to have 50,000-65,000 species worldwide. They typically range from 1-5mm long which makes them difficult to sort from sediment and to photograph under the microscope. I also control the lab music which is super important (default to Big Booty Mix 15 if we really need to focus). I really enjoyed my first experience caving and I can’t wait to go back. I’ve done some pretty intense hikes with climbing and cave like passages but I’ve never experienced something like this before. Professor Urcuyo had us lay down and turn off our headlamps at one point in the back of the caves and it was surprisingly peaceful. It was crazy to experience darkness like that, I couldn’t even see my fingers an inch from my face.

The largest collembola on the left is around 6mm long. The two to the left are in the suborder Arthropleona while the one to the right is in Symphypleona.

Owen here, I am focusing his research on the order Coleoptera (beetles), in particular the superfamily Staphylinoidea which describes over 70,000 species. The size of these rove beetles make them much easier to find and study, however the little details on morphology can be hard to discern. Based on our time slaving over dichotomy keys, we think we have found the genus Philonthina and Quedius and we’re hoping to send off a few of our samples to entomologists to analyze our findings. You’ll often see me modeling in the cave passages, when not distracted by the beautiful iridescence of rove beetles. Our first experience into the caves was quite something…cold, wet, muddy, and cramped. As a bigger individual I was often hunched over and I struggled to get through small crevices, it was quite exhausting. Regardless, all of these factors were worth it. Having to explore an area that has not been touched by many people is exhilarating. In the dark you’ll never know what to run into or what’s around the corner, and that’s the best part.

A beetle found in Carnegie Cave that we identified down to the genus Bisnius. This sample is around 1.5cm. Notice the iridescence on the abdomen and elytra.

Figuring out what we have collected is quite the project. Owen and Georgia have gotten very good at finding articles with original descriptions (some dating back to the 1800’s) and keys that allow us to identify our organism. The keys can be very particular and the genus differentiation can come down to something seemingly meaningless like whether or not the beetle has hairs between its tarsal claws. We have to go to our maximum magnification to look at certain morphological features and so for the collembola (which are much smaller than the beetles) we are considering using electron microscopy.

Along with the beetles and springtails we also have plenty of yet to be identified millipedes, gnats, flies, crickets, and other invertebrates.  So, the rest of the our research summer is going to be busy.

Ion Acoustic Waves and Double Layer studies in a Double Plasma Device

This summer we’ve been busy working on the 4th state of matter, plasma.  While plasma isn’t on the forefront of most brains, plasma is the most common state of matter in the universe, photo on the left. Crab-nebula-combined-300x300.jpg Our sun is entirely made of plasma. By studying how plasma behaves we can understand more about our very own sun. The exact mechanism for solar flares is currently being investigated and theorized by studying plasma.  One possible mechanism that is believed to accelerate solar flares is the double layer. The double layer is a phenomenon in plasma that causes ions to accelerate, but first let’s understand what plasma is before we go into the double layer.

Plasma can be understood as “hot” or high energy gas.  This gas is ionized, which is what gives the plasma its unique glow.  This mixture of electrons, and ions make up the plasma.  These charged particles arrange themselves that it becomes quasi-neutral overall, except with the double layer.  The double layer is an area of the plasma where this quasi-neutrality is violated. In this area we get two sheets of charge, one positive and one negative.  This creates an electric field between the two sheets. This electric field is the force that accelerates the ions.

Acceleration of ions has been suspected to be responsible for creating the auroras in the northern and southern hemisphere.  The acceleration ionizes gas particles in the air causing them to glow in the sky. The various colors come from the various types of gases that exist in our atmosphere.download.jpg

Our research is part of a larger research group that is investigating how the double layer and ion acceleration can be used as a method of jet propulsion in space.  These engines would behave exactly as a normal jet engine. The Engine propels material backwards and that propels the ship forward. Except instead of just normal gas it’s plasma.

.In our plasma chamber, Pickett’s Charge Plasma Device (PCPD), we haven’t yet observed the double layer.  But we continue to test various parameters and explore other options in order to see the double layer.  The DL is characterized by a change in electric potential, measurable via our Langmuir probes.  Nonetheless the search continues and remains hopeful. 

The ion acoustic wave (IAW) is a type of longitudinal wave that propagate in plasma. The ion acoustic wave, is very similar to an acoustic or sound wave.  In the same techniques that you would use to identify the speed of a sound wave, the relative temperature and density of the gas, you can calculate the “ion sound speed”. However, while sound waves travel through a pressure wave, the IAW travels through a disturbance in the electric field that propagates the wave. Similar to the sound wave, the IAW also dampens over time. Besides its velocity, we are also interested in how the surrounding plasma might affect the wave as it travels.Untitled.png

Two different sources will cause the dampening we observe in the wave. Ion-neutral collision will cause a weak damping. What we expected to see is that the wave velocity decreases within an exponential curve. Another damping source is from “particle surfing”, called Landau damping. When the ion velocity is larger than wave velocity, the energy of ions’ will add to wave. So the wave amplitude would grow. In the same manner, when the ion velocity is smaller than the wave velocity, then the waves will lose energy to ions. Ions travel faster than the wave, therefore Landau damping is our main source that slows the wave down. Normally we only need to deal with the Landau damping in the interferometer method of wave analysis.

Now with 2 weeks left in our research we have successfully measured the Ion Acoustic Wave at multiple frequencies.  While signs and possible versions of the DL have been observed, a complete study on the double layer is still elusive.  Our sights now move to the laser in order to become LIF capable in order to add a whole new depth to our data.

From Neng and Rikard from Good’s Lab!Image-3 Have a great summer

A Phage Tail

By: The Phage Pham

    Despite having the largest XSIG lab this year, we, the Delesalle Lab, work with some of the smallest creatures on the planet! Phage are viruses that infect bacteria and are the most numerous organisms in the world (an estimated 10^31 phage on Earth!). Phage infect bacteria by injecting their DNA into the host bacteria, causing the bacteria to reproduce more phage particles. Phage’s dependence on bacteria to reproduce has labeled phage as “non-living,” however, our lab disagrees since phage have the ability to evolve.  The process of infection ends with numerous phage being produced and released into the environment, but the bacterial cell is killed.

     Phage are used today as antibacterial solutions on our lettuce and beef.  In the Soviet Union, phage therapy was used extensively to treat bacterial infections, and America is starting to investigate the medical applications of phage as well. Our lab is interested in lots of different aspects of phage research; how they evolve against bacteria, whether they impact biofilm formation, why host ranges differ, and how phage can work together to fight bacteria. But first, let’s meet the lab!

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                                                                          Phamily Photo                                                                                          From Left to Right: Dr. Delesalle, Rachel W, Madeleine, Leigh, Bacillus subtilis T89-06, B. subtilis 1A1, Rachel L, B. subtilis T88-12, Sam, Matt, Will

 

A Meme A Day Keeps The Contamination Away!!!

Madeleine:                                                 Will:

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Rachel L:                                         Sam:

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Leigh:                                                                      Rachel W:

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                                              Matt:

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Evolution and Passaging:

     Phage must infect bacteria in order to survive, and bacteria must evolve resistance to phage in order to survive. They go back and forth in a cycle of bacteria evolving phage resistance and phage evolving enhanced infectivity. We term this interaction an “arms race.” This evolution occurs incredibly quickly because bacterial life spans are quite short. This speed allows us to look at evolutionary dynamics over hundreds of generations in a short time. 

     This summer we are studying Bacillus subtilis, a species of bacteria found in soil, and its interaction with various phage. We are looking at combinations of three strains of bacteria (T8906, 1A1, T8812) with three types of wild phage (000TH010, 049ML001, 049ML003), and one domesticated phage (SPP1). Previous studies have looked at the dynamics between bacteria and domestic phage, but these results are not indicative of natural dynamics. Domesticated phage have shorter genomes and have had sometimes decades adapting to their host bacteria. 

    We’re interested in how phage can evolve against bacteria over time. In order to investigate this, we’re allowing phage and bacteria to grow together in flasks of media for the summer. However, bacteria eat up the nutrients in the media pretty quickly, so every 2 days we have to “passage.” Passaging is the process of removing samples of phage from the old flasks and adding them to new flasks with fresh bacteria from stock. This means that we can replenish the nutrients available to the cells, and we can keep phage evolving against an unchanging bacteria strain. At the end of our experiment, we can observe the mutations that arose over the course of the phage’s evolution.

     So far this summer we’ve passaged for 6 weeks, and we have 2 left to go! When the passaging is finished, phage will be isolated from each sample, and the DNA will be sequenced. We can compare the sequence of the pre- and post-experiment phage to see where mutations arose. With this data, we can identify which genes acquire mutations, what kinds of proteins those genes encode, and whether these mutations are similar across replicates and phage types.  We expect that the domestic phage will produce fewer mutations due to its acclimation to the host strain whereas the wild phage should produce more mutations in their genomes. These mutations will result in an increase in fitness, but possibly a decrease in host range.

Host Range:

     Our previously isolated novel phages of Bacillus subtilis represent a diversity of genetic clusters, groups of phages with nucleotide similarity over 50% of their genomes. This diverse collection of phages varies in host ranges, their ability to eat a narrow or broad range of bacteria, amongst other characteristics. Based on the sequencing of 27 novel phages, they belong to eight clusters, including three new clusters, based on <5% nucleotide similarity compared to phages in NCBI. Finally, unlike phages isolated on “simplified” bacterial laboratory strains which possess surface features and defense mechanisms modified for standard growth media without phages, these phages were isolated on wild strains of B. subtilis.

     The impact of phages on bacteria can be determined by comparing bacterial growth in the absence or presence of phage. By measuring optical density as a proxy for bacteria population size, we can ascertain whether the phage can lyse a particular bacteria strain and how efficiently it does that (ie drive bacteria extinction or lower population density). Through the use of a cell plate reader and measuring the growth of bacteria through a 24-hour growth period, we can determine the optimal phage to bacteria ratio to understand the full impact of growing the two together.

Biofilm:

     Biofilms are a slimy layer of extracellular matrix comprised of extracellular polymeric substances secreted by bacteria growing in a colony. Dental plaques are a common example of a bacterial biofilm in the human body and the protection from chemical agents they afford to bacteria growing in your mouth is a major reason why brushing your teeth is important. The action of physically brushing your teeth breaks up those biofilms allowing your toothpaste to more effectively kill bacteria on your teeth. 

     In this lab, we are looking into the interactions of Bacillus biofilms and phage, specifically asking if phage have differential success in infecting different Bacillus strains growing with biofilms. Studies of the interactions between phage and biofilms have both clinical and industrial relevance.  Knowledge about what allows phage to better kill bacteria in a biofilm is applicable to the sanitation of bacterial biofilms growing on things like medical implants or in the development of more powerful phage-based industrial cleaning agents able to penetrate bacterial biofilms better than existing chemical cleaning agents. 

 

Ice Age: The Clathrate Drift

Who’s who in the herd?

Suvrajit “Manny” Sengupta, in addition to being the leader of the herd, is an assistant chemistry professor just finishing his first-year teaching at Gettysburg College. His academic interests lie primarily in physical and biophysical chemistry. Like any woolly mammoth, he has a wealth of knowledge (though, don’t ask him what ‘yeet’ means). When he’s not polishing his tusks or yelling at Sid and Diego, he enjoys making fun of astrology, drinking coffee, and wandering around the science center.

“Uhhh, I’m not sure how much of that you could hear…?”

Professor Sengupta’s Favorite Manny Quote from “Ice Age”

Sarah “Diego” Kotchey is a rising junior Environmental Studies major and Chemistry minor in the Teacher Certification program from Binghamton, New York. This is her first summer working in Professor Sengupta’s lab but second summer in the X-SIG program. In her free time she loves doing yoga, walking around the battlefields, visiting her friends from home, and cheering on the Bullets Football team.

“Save your breath, Sid. You know humans can’t talk.”

Sarah’s Favorite Diego Quote from “Ice Age”

Sarah’s role in the herd is to investigate clathrate hydrates, which are crystalline water-based solids that resemble ice. They are non-stoichiometric compounds composed of a host water network and another molecule (usually a gas). Clathrate is essentially another word for cage, so clathrate hydrates are formed when gases get trapped inside “cages” of hydrogen bonded, frozen water molecules. What we refer to as “ice,” that we see in our drinks daily, is structurally hexagonal. This structure allows small gas molecules to pass through the ice. In clathrate hydrates, however, the structure is a lattice where gas molecules cannot pass through, but instead get trapped inside a water cage. The gas trapped inside the ice is typically referred to as the “guest” molecule. The guest preferences come from the size of gas. Meaning certain gases only fit in certain structure types. It is important to note that the guest molecules are typically hydrophilic, yet they somehow stabilize the clathrate structure which is thermodynamically less stable than the ice structure.

Figure 1: The image on the left illustrates the hexagonal lattice structure of typical ice. The image on the right illustrates the various cage structures of clathrate hydrates. 

Clathrate hydrates tend to have a “memory effect,” which basically means a sample that has been formed, then completely melted, and then placed back at a low temperature will form faster than a freshly made one. In general, this effect can be diminished by placing the sample in a higher temperature or for an extended time. Exactly how clathrate hydrates form and decompose is not yet fully understood at a molecular level. At a fundamental level, it requires the reorganization of the hydrogen bonding network, propagation of defect structures, and the stabilization of thermodynamically unfavorable structures through non-covalent interactions. The surface of ice is covered by a thin layer of disordered “liquid-like” water called the quasi-liquid layer (QLL) or premelting layer over a range of temperatures below the melting point of ice. This QLL is thought to play an important role in the formation of hydrates from ice at low temperatures. Our lab is interested in characterizing the role of the QLL in hydrate formation in more detail.

Outside of the lab, clathrate hydrates form in oil pipelines leading to blockages, which can be dangerous and have severe environmental implications. In terms of size, they can form on a scale from nanometers to kilometers. Clathrate hydrates bring about a potential new energy source, as amounts of hydrate deposits naturally found are at least twice that of fossil fuel reserves available. However, this would not fit with trend towards renewable energy sources, as these are typically formed from methane, which is up to twenty times more of a potent greenhouse gas than carbon dioxide. But, clathrate hydrates are found in sediments under the permafrost as well as in sediments along continental margins, so climate change may lead to them being released whether we like it or not. They can also be used as a gas storage system. For example, clathrate hydrates can be made with hydrogen gas as a safer way to transport it from one place to another.

Julia “Sid” Sharapi is a rising sophomore from Greenbelt, Maryland. This is her first summer in the X-SIG program and her first time working in a lab. Like Sarah, she’s an Environmental Studies major and Chemistry minor. On campus, Julia is involved with the Honor Commission, works in the Office of Student Scholarly Engagement, and is an upcoming RA. In her spare time, she enjoys reading, eating bread, and going to Target. Though her sloth like nature does make her accident prone, she has yet to drop anything especially important in lab.

“Ah, you know me, I’m too lazy to hold a grudge.”

Julia’s favorite Sid Quote from “Ice Age”

Julia’s role in the herd is to explore antifreeze proteins (AFPs), which are found in fish, plants, insects, and soil bacteria. These proteins restrict the growth of ice by adsorbing to ice planes which induces a curvature on the ice growth-front, making it thermodynamically unfavorable for ice formation and therefore depressing the freezing point. This creates thermal hysteresis, which is the difference between the melting point and this new non-colligative, non-equilibrium freezing point. This lowering of the freezing point allows fish and other organisms containing these proteins to survive at temperatures they would not be able to tolerate previously. 

AFP’s are unique because they are very structurally diverse, despite the fact they perform the same function. The exact reason for this is unknown, but their diversity may be because they share no common ancestor due to their recent evolution (20 to 40 million years) in multiple phylogenetic kingdoms. So far, researchers have identified five types of antifreeze proteins in fish, two types in insects, and three types in plants. Typically, insect AFPs are more active than fish AFPs.  Higher AFP activity is correlated with multiple ice plane binding and higher shape complementarity between the interacting AFP and ice planes. There are two broad categories of antifreeze proteins: glycoproteins (AFGPs) and nonglycoproteins. In our lab, we focus on nonglycoproteins, of which there are 4 classifications in fish: Type I AFP, Type II AFP, Type III AFP, and Type IV AFP. The most extensively studied is the Type I AFP, which is popular because of it simplistic structure. In our lab, we will be using a Type I antifreeze from winter flounder and expressing it in E. coli. This AFP is alanine rich, with alanine being 23 of its 37 amino acids. It is also repetitious, with threonine repeating every 11 amino acids.

Figure 2: Winter Flounder Type I AFP HPLC6 (PDB ID: 1WFB).

AFP’s are very appealing to developments in a variety of fields, as improvement to freeze hardiness in various organisms, pharmaceutical products, and other goods. Two major uses are in the food industry and in cryotechnology. In the food industry, AFPs can help improve food texture by reducing cellular damage and decrease food’s nutrient depletion, especially in fruits, vegetables, and meat. With cryotechnology, AFP’s could help improve cryopreservation, not just in humans, but also lab materials, like other proteins. Additionally, it might have a role in cryosurgery, which is an emerging way to treat cancer and tumors. They could be particularly helpful with cancer that forms in the prostate, liver, and other organs that are deep in the body. 

Currently, the petroleum industry primarily uses a commercially available kinetic hydrate inhibitor (KHI) poly-N-vinylpyrrolidone (PVP) solution to avoid a build up of clathrate hydrates; but, AFPs may be more effective at inhibiting hydrate formation. Additionally, PVP solution is shown to have no effect on the “memory effect” that clathrate hydrates exhibit, but certain AFPs have been observed to eliminate or lessen the effect. 

Current Research

This summer, after rescuing a baby, avoiding the meltdown, and stealing a dinosaur egg, we decided to take a break from adventuring and focus on forming our own hydrates. Using vials containing a solution of THF-Water with a 1:15 molar ratio, we have successfully created THF hydrates two ways. The first, dubbed the needle method, which involves inserting a copper wire, cooled via an ethanol dry ice bath and threaded through a hypodermic needle, into a vial containing THF solution at 1.5°C, so the cooled wire and solution are in contact. The second way, dubbed the seed method, involves extracting a small crystal from a previously made needle crystal with a cooled metal spatula and placing it into a vial containing THF solution in 1.5°C. The vials are kept at 1.5°C using a Thermo Scientific NESLAB RTE Series Refrigerated Bath. We decided to form them at 1.5°C instead of 0°C to ensure that hydrates are forming, and not ice. Freshly prepared THF solutions did not form clathrate hydrates without being subjected to either of these methods. This shows the importance of establishing nucleation sites.

Figure 3: Set up for synthesizing clathrate hydrates through the needle method. The image on the left shows a dry ice bath containing a copper wire and needle threaded through a vial, placed in a Refrigerated Bath. The image on the right shows a formed clathrate hydrate with the needle inside the vial. 

In addition to forming clathrate hydrates, we are also testing the memory effect. So far, experiments have used over-temperatures of 5.0°C, 7.0°C, and 10.0°C. Using several samples of hydrates grown by the needle method and seed method, vials are placed at the over-temperature and once completely melted, incubated for 5 minutes, then transferred back to the 1.5°C bath. Samples are then observed in approximately 5 minute increments to see if clathrate hydrate reformation occurs. If there is reformation, the process is repeated with increasing incubation time at the over-temperature after melting.

Figure 4: Images of formed and unformed clathrate hydrates from a memory effect experiment. Unformed hydrates appear to be melted and liquid, while formed hydrates are frozen solid. 

Exploring the nature of the memory effect helps inform us further about this complex property of clathrate hydrates and can establish baseline data which we can compare with future research. This is especially important when we begin to introduce AFPs and wish to evaluate their effectiveness. 


Figure 5: Memory effect data collected using an over-temperature of 5.0°C and vials containing clathrate hydrates formed using a THF-Water solution with a 1:15 molar ratio. Note that reformation stopped after 50 minutes in the over-temperature. 


Figure 6: Memory effect data collected using an over-temperature of 7.0°C and vials containing clathrate hydrates formed using a 0.5 mL THF-Water solution with a 1:15 molar ratio. A total of 32 vials were used, with 16 created using the needle method (blue) and 16 created using the seed method (orange). Note that the needle vial that reformed at 7.5 minutes and at 10 minutes is the same sample. 

At 5.0°C, hydrate reformation is relatively standard, with an increasing reformation time until hydrates stop reforming at 50 minutes in the over-temperature. From the data collected thus far, we believe that the reformation of hydrates at 7.0°C is random, as spanning the three sets of experiments we have conducted, 6 of the 32 hydrates have not reformed at all. Further, they may be random in the time they take to reform; while most samples reform standard to the group, there are a few outliers that have taken longer. The only clathrate hydrate that has reformed after 5 minutes at the over-temperature of 7.0°C was one needle crystal. Future trials are necessary for both over-temperatures to confirm our current findings. These experiments were done in conjunction with several control vials of THF solution, which failed to form clathrate hydrates even when kept at 1.5°C for over 24 hours.

Thanks for dropping by!

The FUNgi/FUNgal Lab!

Hi, its Katie, Matt, and Cailin from Dr. James’ lab! We used to have a fifth member, Morgan, but she has moved on to bigger (but not better) things in the real world. Katie has been working in Dr. James’ lab for two years and Matt and Cailin started this summer. You can tell when you’re near the James lab when you hear the fungal jams playlist in the hallways as we jam while working. 

Matt and Cailin’s Projects

We’ve spent the summer so far studying gene WD-1, a gene that is novel to fungi and unstudied. Prior research in the James lab demonstrated that mutants with deleted copies of gene WD-1 (referred to as ΔWD-1) had interesting effects on colony morphology and survivability. 

Here, we see two strains of A. nidulans, with one possessing an active copy of WD-1 (top) and the other in which the WD-1 gene has been deleted (bottom). The strains were grown at three different temperature conditions. 37°C is the optimal growth temperature for many strains of A. nidulans, but ΔWD-1 mutants grow slower and smaller than their wildtype counterparts. As the temperature decreases to 29°C and 21°C ΔWD-1 strains struggle to grow and fails to grow entirely, respectively. The mutants can be classified as having a “cold-sensitive lethal” phenotype.

In a search for additional phenotypes of ΔWD-1 mutants we examined the hyphae (filamentous cells) of the fungus under an inverted microscope. Strains having normal WD-1 activity (“Wildtype”) have hyphae that extend in straight branches. At lower, or “restrictive” temperatures, strains where WD-1 has been knocked out show atypically curled and totally gnarly™ hyphae.

Together, these observations led to the development of our running hypothesis, which is that the WD-1 protein product stabilizes the cytoskeletal structures known as microtubules.

In order to better understand gene WD-1 we have been playing matchmaker with the fungi and crossing ΔWD-1 strains with strains of Aspergillus containing deletions of microtubule associated proteins (MAPs). If we observe new phenotypes in strains doubly deleted for WD-1 and one or more MAPs we can better understand what role WD-1 plays in the organism. Creating these double mutants is about a four-week process so progress in the genetic area of Aspergillus involves a lot of waiting.

We also have utilized our good friend the Nikon Ti-U inverted epifluorescence microscope to examine proteins of interest tagged with fluorescence markers. This has yielded interesting but inconclusive results. One phenotype that we observed using fluorescence markers of microtubule proteins is that ΔWD-1 strains have a high mitotic index. This indicates that cells are unable to exit mitosis and could be one of the reasons that ΔWD-1 strains grow poorly.

The final way that the James lab has been investigating gene WD-1 is through S-tag protein affinity purification. This is a protein biochemistry technique that involves tagging protein WD-1 with an extra amino acid sequence, then incubating the strain with the tagged protein with S-peptide beads that bind very tightly to the extra amino acid sequence on protein WD-1. This means that when the beads are washed, releasing all unbound proteins, only WD-1 and the proteins attached to it will remain. These proteins can be analyzed to determine the binding partners of WD-1.

  Katie’s Project

I am currently working on a functional dissection of the C-terminus of WD-1, the same gene Matt and Cailin are working on. This means that I am cutting off the end of the gene in multiple locations to see how much is needed for function. We are interested in the C-terminus because it is unusually acidic and conserved throughout fungi. To do this we use a technique called Polymerase Chain Reaction (PCR) to truncate the WD-1 gene and attach the truncation to the promoter of the alcA alcohol dehydrogenase gene. This promoter allows us to control the expression of the truncation by simply varying the carbon source in the growth media. For example, glucose (table sugar) represses alcA, preventing it from being active, whereas alcohols such as ethanol and threonine strongly activate alcA activity. The alcA-controlled WD-1 truncations are inserted  into a strain of Aspergillus that is deleted for WD-1 (ΔWD-1).  

Between last fall and this summer I have been able to create three truncations and transform them into Aspergillus. In the image below is an example of the results from the truncation experiments. you can see three control at the top of the plate (right: wild type, ΔWD-1, and an alcA-driven WD-1) and all the other colonies are transformants. These plates were grown at 23° C and as you can see ΔWD-1 did not grow, therefore all the other colonies that did grow rescued the mutation, indicating that we can cut away the C-terminus of WD-1 without disrupting its function. In fact, these experiments show that this gene remains fully functional with as much as 11% of the C-terminal end of the gene missing. This was unexpected but super exciting! As a result, I now am creating three additional and more severe truncations to determine the minimal gene length required for functionality.

I have also done a suppressor screen of WD-1. This means that I mutagenized two ΔWD-1 strains of Aspergillus, plated the mutagenized spores, and put them at the restrictive temperature (21° C) to grow (image below). Then I left them in the chamber, crossed my fingers, wished on a shooting star, and hoped that colonies grew. My wish came true and I found suppressors of WD-1, now I have to perform a lot of crossing to learn more about each suppressor.

Images of the plates containing original mutagenized spores, each red circle is a colony that to grown out on a new plate and screened to determine if is suppressed the ΔWD-1 

This has been a very exciting summer, filled with really interesting discoveries and I am very excited to continue my research and learning more about both my truncations and my suppressors!

Bonus exhibit: Interpretive pipette tip art