PCR - Spring 2020

PCR.

the Pingry Community Research Journal. winter two thousand and twenty. a new decade for Pingry research.

CAPTURING SPECIES VARIETY ON THE PINGRY CAMPUS

join the Journal.

PCR is a community of young scientists and scholars.

We accept any like-minded individuals. Form III students are encouraged to write for the Journal, and Form IV-VI students can participate in the editorial process.

Contact: Aneesh Karuppur (V), Editor in Chief akaruppur2021@pingry.org Mr. David Maxwell, Chair of the Science Department dmaxwell@pingry.org 2

CAPTURING SPECIES VARIETY ON THE PINGRY CAMPUS

Reports GMOs: Foods of the Future Resetting the Brain: A Way to Start Over Potential Links Between Microbes and Alzheimer’s Disease

this is

PCR.

Research Fixing Hearing Loss: The JNK Signaling Pathway in Hair Cell Regeneration in the Danio rerio Lateral Line.

Classical Conditioning Through Auditory Stimuli.

Ability of Mealworms to Digest Polycarbonate Plastic.

Genetic Diversity of the Northern Pine Snake.

Generating A BCL2L12 Melanoma Zebrafish Model.

Capturing Species Variety on the Pingry Campus.

Masthead Brian SK Li (VI), Editor in Chief Aneesh Karuppur (V), Associate Editor Isabel Sheyfer (VI), Art Editor Kristin Osika (IV), Assistant Editor Noopur Bhat (VI), Ashley Lu (VI), Julia Fu (IV) , Layout Editors Anjali Kapoor (VI), Anushka Agrawal (V), Noah Bergam (V), Thomas Henry (V), Cal Mahoney (V), Mira Karande (V), Ram Doraswamy (IV), Christine Guo (IV), Brian Li (IV), Sam Wexler (IV), Copy Editors Ashley Lu and Cal Mahoney, Artists Mr. David Maxwell, Chair of The Science Department

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Reports.

GMOs - Foods of the Future

by Mirika Jambudi (III) Supermarkets have changed drastically in the past century. Bright lights decorate doorways, and big signs showcase deals. But, behind the advertisements, few know that the produce sold has also changed a lot. Now, genetically modified organisms (GMOs) are found everywhere. Rice that cures childhood blindness? An apple with a vaccine that could prevent pneumonia? Cabbage that resists caterpillar attacks? GMOs have a lot of promise, but inadequate understanding of GMOs has led to debate on the subject .

GMOs are living organisms that have had their genes modified with biotechnology. The process of genetic engineering is not found in nature and cannot be created through traditional crossbreeding methods, which humans have been utilizing for millennia to make sweeter, bigger, more nutritious, and more resilient food. Therefore, many currently think that this new technology is dangerous. The general public usually assumes that these organisms are harmful to their health, and are not suitable for the environment.

Most people believe the myth that GMOs harm the body through the genes that are inserted into the crops. That is not true. In fact, GMOs are engineered for quality. Enhanced genes allowed GMOs to produce greater amounts of beneficial nutrients. For example, an orange has a lot of Vitamin C. However, a GMO orange might provide more Vitamin C than a non-modified one. Therefore, the increased levels of vitamins in GMOs can be beneficial to malnourished children. GMOs can produce better, healthier crops. Some can even fight destructive or even fatal diseases. For instance, “In 2009, the FDA approved the first GM animal, a goat that produces an anti-clotting agent in its milk that can treat people with clotting diseases”, according to the Pennsylvania State University’s College of Agricultural Sciences.

In other words, humans have always known that a balanced diet has significant health benefits. But a century ago, we would have never imagined the amazing things that genetic engineering can do.

This goat’s milk could save lives without harming other parts of the body because they are heavily tested and regulated by the FDA and other government agencies. In addition, GMOs could fight a form of childhood blindness. A deficiency of Vitamin A leaves 500,000 blind annually.

Researcher and genetic engineer Ingo Potrykus invented “golden rice” by inserting three new genes into the plant’s DNA. The rice can now hold vitamin A in its grains, as opposed to only being able to hold the vitamin in its inedible leaves. Golden rice could prevent a quarter of million children from dying within 12 months of going blind due to Vitamin A deficiency. Even though this research was concluded about 15 years ago, fierce opposition has hindered people from accessing this innovation. But, in reality, GMOs are nutritionally beneficial and can combat illnesses. These health benefits definitely outweigh the potential drawbacks of making GMOs reasonable alternative to traditional food.

Works Cited 1. “Do Genetically Modified FoodsTend to Be More Nutritious than Non-GMO Foods?: Monsanto Hawaii.” Monsanto, www.monsantohawaii. com/do-genetically-modified-foods-tend-tobe-more-nutritious-than-non-gmo-foods/.

2. Saletan, William. “The Misleading War on GMOs: The Food Is Safe. The Rhetoric Is Dangerous.” Slate Magazine, 15 July 2015, www.slate.com/articles/ health_and_science/science/2015/07/are_ gmos_safe_yes_the_case_against_them_ is_full_of_fraud_lies_and_errors.html.

3. Naam, Ramez. “Why GMOs Matter - Especial-

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ly for the Developing World.” Grist, Grist, 30 Jan. 2014, grist.org/food/why-gmos-do-matter-and-even-more-to-the-developing-world/.

“National Geographic Freshwater 101: Food.” National Geographic Freshwater 101: Food, 18 June 2019, environment.nationalgeographic.com/environment/global-warming/food-how-altered/.

Carrington, Damian. “GM Crops Good for Environment, Study Finds.” The Guardian, Guardian News and Media, 13 June 2012, www.theguardian.com/environment/2012/ j u n / 1 3 / g m - c r o p s - e n v i r o n m e n t - s t u d y.

“Are GMO Crops Good.” Bloomberg. com, Bloomberg, www.bloomberg.com/ news/articles/2014-11-18/how-gmo-cropsc a n - b e - g o o d - f o r- t h e - e n v i r o n m e n t .

“The Science of GMOs.”The Science of GMOs

(Penn State College of Agricultural Sciences), 2015, agsci.psu.edu/magazine/articles/2015/ s p r i n g - s u m m e r / t h e - s c i e n c e - o f - g m o s.

8. “What Are GMOs and GM Foods?”

LiveScience, Purch, 2014, www.lives c i e n c e. c o m / 4 0 8 9 5 - g m o - f a c t s. h t m l .

9. Maxmen, Amy. “GMOs May Feed the World Using Fewer Pesticides.” PBS, Public Broadcasting Service, 24 July 2013, w w w. p b s. o r g / w g b h / n o v a / n e x t / n a t u r e / f ewe r- p e s t i c i d e s - f a r m i n g - w i t h - g m o s / .

10. “Are GMOs Bad for Your Health? If You’re Asking This Question, You’re Probably Missing the Point.” Precision Nutrition, 30 Aug. 2019, www.precisionnutrition.com/are-gmos-bad-for-your-health.

Resetting the Brain: A Way to Start Over

by Aanya Patel (IV) The human brain is a complex system of neu- rons convey information about the external enrons that work in perfect harmony to maintain vironment, while motor neurons control musbalance, regulate digestion, focus our cognitive cles both directly and indirectly. Interneurons abilities, among countless other critical tasks. regulate the firing of motor and sensory neuDisturbances to that system, be it trauma or neu- rons, acting as intermediaries between them. rodegenerative disease, can cause balance issues Many neurodegenerative diseases disrupt interfor a cross-country star, hand tremors for a pia- neurons’ ability to function and maintain honist, or muscle weakness that diminishes a sing- meostasis. If the frequency of neuronal signals er’s vocal cords. In most cases, today’s neurolog- is low, the brain will not accurately receive the ical treatments can only slow the progression of message; this can result in cognitive difficulties. these diseases and beget significant side effects. On the other hand, if the neuronal signals are At the University of Wisconsin Tactile Commu- too strong, the brain becomes overstimulated, nication and Neurorehabilitation Laboratory leading to hypersensitivity to sound, light, and (TCNL), a team of scientists and doctors pio- movement. Additionally, if the neural signals last neered the use of neuromodulation to reverse the too long, they create a “noisy brain,” preventing effects of these devastating neurodegenerative signals from being processed effectively as the diseases. Neuromodulation is “the alteration of brain is unable to distinguish one signal from nerve activity through targeted delivery of a stim- another. TCNL attempts to target these malfunculus, such as electrical stimulation or chemical tioning interneurons through neuromodulation. agents, to specific neurologic sites in the body.”

TCNL developed the Portable Neuromodulation The brain is made up of three types of neurons: Stimulator (PoNS), a device measuring the size sensory, motor, and interneurons. Sensory neu- of a stick of gum that has 143 electrodes and is

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placed on the patient’s tongue. The tongue is ideal as it is served by two major cranial nerves that connect it to the brainstem, through which the brain’s motor and sensory systems communicate with the peripheral nervous system. The PoNS device sends electrical signals through the brainstem, stimulating broken interneurons with the goal of inducing neuroplasticity. Neuroplasticity describes the brain’s ability to build new neural pathways to bypass those that have been destroyed.

The PoNS device is typically worn for twenty minutes at a time, up to six times a day. Patients work through rigorous mental and physical rehabilitation while wearing the device. The particular course of rehabilitation is specifically tailored to the patient’s deficiencies and includes activities such as balance practice, gait training, and meditation.

In “How the Brain Heals,” Norman Doidge shares the success story of Broadway singer Ron Husmann. At age 44, Husmann developed multiple sclerosis (MS), which caused him to lose his mobility, bladder control and, most devastating to him, the ability to sing. MS results in the formation of plaques on the nervous system that ultimately prevents neurons from firing correctly.

Desperate for relief, Ron traveled to TCNL where he spent two weeks using the PoNS device and partaking in intensive speech therapy. In the end, Ron was able to sing and dance again. While the exact mechanism is not understood, Dr. Yuri

Danilov of TCNL believes that PoNS generates electrical “spikes” in interneurons that have been disabled by disease or injury. The spike “activates” these interneurons and restores their ability to maintain the nervous system’s homeostasis.

The PoNS device is in the process of being commercialized by Helius Medical as a treatment for traumatic brain injuries and a variety of neurodegenerative illnesses. The treatment has been approved by Canada and is under review by the EU. If approved, it will be able to help people like Ron get their lives back and alleviate the problems faced by countless others.

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Works Cited Doidge, Norman, MD. The Brain’s Way of Healing.

“FDA Rejects Helius Medical Technologies’ PoNS Device.” Verdict Medical Devices, www. medicaldevice-network.com/news/fda-rejects-helius-medical-pons-device/.

“The International Neuromodulation Society.” International Neurological Society, https://www.neuromodulation.com.

“The Portable Neuromodulation Stimulator (PoNS®) Device.” Helius Medical Technologies, heliusmedical.com/index.php/divisions/ heliusmedical/the-pons-device.

“WHAT IS NEUROPLASTICITY?” Brain Works Neurotherapy, brainworksneurotherapy.com/what-neuroplasticity.

Potential Links Between Microbes and Alzheimer’s Disease

by Zoe Wang (IV) Alzheimer’s disease is the most common form of and removed, but, amyloid plaques are hard, indementia, which affects over 5.5 million Ameri- soluble accumulations of beta-amyloid proteins cans. The brains of people affected by Alzheimer’s that clump together between the nerve cells have an abnormal buildup of proteins known as (neurons) in the brains of Alzheimer’s disease amyloid plaques and tau tangles. Amyloids are patients. Tau tangles, also known as neurofibrilprotein fragments that the body normally pro- lary tangles, are insoluble twisted fibers inside duces, and beta-amyloid is a protein fragment the brain’s cells, thought to be a result of besnipped from a much larger protein, known as ta-amyloid buildup. The main protein in the tau the amyloid precursor protein (APP). In a healthy tangle, as the name suggests, is the tau protein, brain, these protein fragments are broken down which forms microtubules that help transport

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nutrients and other important substances from one part of the nerve cell to another. However, in Alzheimer’s disease, the tau protein is abnormal and the microtubule structures do not function properly. Overall, amyloid proteins and tau tangles damage neurons, which lead to Alzheimer’s.

tion is a forest fire,” says Tanzi. “Microbes are what’s lighting the match.” With this data, treatments and clinical trials of drugs already approved for other uses have begun. An NIA-funded proof-of-concept trial at Columbia University has randomized 130 HSV-positive people with Alzheimer’s to receive the antiviral valacycloEarlier this year in January 2019, a paper by Ste- vir or placebo. Participants will be followed for phen S. Dominy was published which suggested 18 months, with tests of cognitive function, and that Porphyromonas gingivalis, the bacteria that PET scans and cerebrospinal-fluid (CSF) studcauses a common type of gum disease, may also ies to assess amyloid and tau accumulation. play a role in Alzheimer’s. Most of the experimentation was conducted with mice, but the researchers Although scientists are getting closer to findfound that they could stop this damage in mouse ing a cure, Ruth Itzhaki of the University of brains by targeting toxic enzymes produced by Manchester would like to see a trial of vaccinathe bacteria. Although the study was helpful, mice tion. “The earlier the treatment the better,” she brains are not the exact same as human brains. says, “and prevention is much better than cure.”

The microbes most likely to have a major role in Alzheimer’s are herpesviruses. In June 2018, a journal was published that found the brains of deceased people who had Alzheimer’s had higher levels of herpesviruses than those who did not.

Although the virus may contribute to the development of Alzheimer’s, it is likely not the only factor. In one paper, researchers at Icahn School of Medicine at Mt. Sinai and Arizona State University analyzed data from postmortem brains of people with signs of Alzheimer’s disease and others without. They found that human herpesviruses 6 and 7 (HHV6, 7) were more common in brains that had signs of Alzheimer’s disease than in non-Alzheimer’s disease brains. The viral levels also corresponded with the severity of dementia. These viruses appeared to have an effect on molecular processes in neurons. They altered the expression of genes that had been affected in Alzheimer’s, and pathways involved in amyloid production. With this data, HHV6 and 7 may become new drug targets for Alzheimer’s treatment.

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Moir of Harvard Medical School published a study that talked about the antimicrobial protection hypothesis. They found that the beta-amyloid protein is actually an antimicrobial peptide 7.

(AMP). The hypothesis suggests that the presence of microbes triggers the generation of beta amyloid as a defensive measure. “Amyloid is the match, tangles are brush fires, neuroinflamma-

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Works Cited Adams, J. U. (2017, September 1). Do Microbes Trigger Alzheimer’s Disease? Retrieved from https://www.the-scientist.com/features/do-microbes-trigger-alzheimers-disease-30999 Amyloid Plaques and Neurofibrillary Tangles. (n.d.). Retrieved from https://www.brightfocus.org/alzheimers-disease/infographic/amyloid-plaques-and-neurofibrillary-tangles Dominy, S. S. (2019). Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Science Advances, 5. https://doi.org/10.1126/sciadv.aau3333 Moir, R. D., Tanzi, R. E., & Lathe, R. (2018). The antimicrobial protection hypothesis of Alzheimer’s disease. Alzheimer’s Association, 14(12), 1602-1614. https://doi.org/10.1016/j.jalz.2018.06.3040 Robertson, S. (n.d.). What are Amyloid Plaques? Retrieved August 23, 2018, from https://www.news-medical.net/ health/What-are-Amyloid-Plaques.aspx Saplakoglu, Y. (2019, January 29). What Causes Alzheimer’s? We Don’t Really Know Yet. Retrieved from https://www.livescience. com/64597-causes-alzheimers-disease.html Sherman, C. (2019, June 26). Finding Links Between Microbes and Alzheimer’s Disease. Retrieved from https:// www.dana.org/article/finding-links-between-microbes-and-alzheimers-disease/

Research.

Fixing Hearing Loss: The JNK Signaling Pathway in Hair Cell Regeneration in the Danio rerio Lateral Line

by Dylan Anidjar (VI), Ashna Kumar (VI) AP Biology will examine the stained zebrafish under a fluAbstract According to 2007 data from the Centers for orescent scope. Our research has focused on Disease Control and Prevention (CDC), approx- breeding and raising zebrafish as well as pracimately 36 million American adults report some ticing staining to get a baseline hair cell count.

Introduction degree of hearing loss. In mammals, hearing loss is most commonly caused by the impairment of Hearing loss in humans is caused by the damage mechanosensory hair cells (5). The damage and or death of cochlear inner ear hair cells in hudeath of hair cells is often permanent in mam- mans. This is because human sensory hair cells mals; however, nonmammalian vertebrates have do not regenerate upon damage; they are permathe ability to regenerate hair cells. The basis of nently lost. However, in zebrafish (D. rerio), this is this difference in regenerative ability remains not the case. The sensory hair cells of zebrafish, largely unexplored (4,5). We plan to study hair which are structurally and functionally homolcell regeneration in Danio rerio (zebrafish) with a ogous to human sensory hair cells, are located specific focus on the role of c-JunN-terminal ki- along the lateral line system and are capable of nase (JNK), a protein in the MAPK developmen- regeneration if killed or injured. The lateral line tal signaling pathway (1). The investigation of the is composed of several receptor organs called role of the JNK signaling pathway in Danio re- neuromasts. Within neuromasts, there are sensorio hair cell regeneration is a potential approach ry hair cells surrounded by support and mantle to targeting hearing loss in humans. In order to cells. Mantle cells can differentiate into mechastudy the role of JNK in hair cell regeneration nosensory hair cells when specific death signals in the D. rerio lateral line, we will take a base- from surrounding cells are detected. In zebrafish, line count of the hair cells on a fish as our con- these hair cells help them detect changes in prestrol, induce hair cell death in experimental fish sure and vibrations in water currents to help them groups using the drug neomycin, and recount the navigate. By studying the mechanisms of mechhair cells to ensure regeneration has occurred anosensory hair cell regeneration in a zebrafish and that the drug was effective. We will then ad- model, scientists can hope to implement their minister a JNK inhibitor, SP600125, shortly after knowledge to remedy human hearing loss (3,5). a dose of neomycin. This will test if the activity of JNK is essential to hair cell regeneration. We are focused on JNK, a protein kinase in the mitogen-activated protein kinase (MAP-K) sigWe will quantify the hair cells in all these naling pathway heavily involved in proliferation, scenarios using a 2-[4-(dimethylamino)sty- apoptosis, embryonic development, and organryl]-N-ethylpyridinium iodide (DASPEI) flu- ogenesis in zebrafish. This protein is crucial for orescent stain to highlight the sensory struc- the initial development of hair cells in zebraftures containing hair cells in zebrafish, and we ish embryos. With these things in mind, we will

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be investigating the role that JNK plays in hair cell regeneration, and to what extent it will affect hair cell growth when inhibited. Though experimentation, we hope to observe if when these hair cells die, the neighboring cells initiate the regenerative process. We have researched other factors, such as retinoic acid, to find their effect on hair cell regeneration (4). We hope to measure the effect of JNK inhibition on regeneration. We will first induce hair cell death in the zebrafish and later inhibit JNK with a drug, SP600125. We hypothesize that when JNK is inhibited, it will activate p53 and p21, which will then induce cell cycle arrest and therefore cell death. Without functional JNK, the differentiation of mantle cells and development of hair cells may not occur: this has been observed in larval zebrafish (1).

In our experiment, we will first stain the zebrafish using DASPEI stain, and then we will take quantitative data under a fluorescent scope to get the baseline hair cell count prior to treatment.

We will use this as our positive control. DASPEI works by labeling the mitochondria of hair cells, so we can easily label these cell’s changes throughout. We will then induce hair cell death using an antibiotic called Neomycin that targets and kills sensory hair cells (3). DASPEI is a live stain, so once originally stained, we can again image the fish and quantify the hair cells. To knock out JNK, we will use a JNK1, 2, and 3 inhibitor called SP600125. We will have three experimental scenarios: in our positive control, we will quantify an untreated fish. In our second control, we will administer Neomycin to kill hair cells, but leave JNK uninhibited. Finally, we will kill off the hair cells with Neomycin and inhibit JNK. Around 4 hours after administering Neomycin, when most hair cells should be killed, we will administer SP600125 before the regrowth process occurs. After each process, we will recount the number of hair cells following the initial stain.

Methods Breeding and Raising Embryos To successfully breed the fish so that they produce progeny for accurate data and imaging, we place one female and one male in the breeding chamber with a plastic divider between them.

Once placed, they remain there overnight, and the divider is removed the following morning.

The fish will have had several hours to acclimate in the dark prior to their optimal breeding

time in the morning. The breeding chambers are checked periodically throughout the day for eggs in the bottom of the chamber. If there are no eggs present, the same pair will be left in the breeding chamber overnight again. The following day, if eggs are present, we will collect them. If the pair does not breed successfully, we will remove the pair and replace the fish. They will be placed into the “Recently Bred” tanks to allow them a recovery period. This process is repeated until an adequate number of progeny is produced.

If eggs are found, we will take both male and female fish and place them into respective gender-separated “successful breeding pair” tanks.

We will remove the sieve from the bottom of the breeding chamber and tap out any remaining eggs. We will prepare beakers filled with 250 mL system water in the incubator to acclimate. Once preheated, we will then pipette 15-20 eggs from the chamber per beaker. The beakers will then be placed into the incubator, and we will wait for the eggs to hatch (about 2-4 days post-fertilization (dpf)). Once hatched, we will feed the embryos live paramecia twice a day, in the morning and afternoon, by pipetting approximately five drops of our paramecium cultures into the beakers. At nine days, we will begin to feed both paramecia and brine shrimp by pipetting equal parts into the beakers. We will gradually increase the proportion of brine shrimp and phase out the paramecia in the following 7 days. At around 21 days, fish will be moved into tanks and added to the system, while still being fed baby brine shrimp. We will feed adult food to the progeny fish as soon as they are big enough to consume it. This process is performed for all the embryos purchased.

Culturing Paramecium One tablespoon of autoclaved whole-wheat berries is brought to a rolling boil for 10 minutes using Nanopure water. After boiling, the wheat berries are removed from the hot plate, and excess liquid is poured off. Six culture dishes are filled half of the way up with Nanopure and labeled with the date. A light sprinkle of powdered brewer’s yeast and approximately five wheat berries are added to each dish. The dishes are covered in plastic wrap to avoid contamination. This paramecium is cultured for embryonic zebrafish which are too small to search for food themselves. We use paramecium to feed these fish, because par-

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amecium can swim into the zebrafishes’ mouths.

Reculturing and Inoculating Paramecium Each paramecia culture is poured through sieves to create a concentrated paramecium solution.

The cultures are first poured through a 100 μm filter to remove large particle debris and wastewater. The collected water is then poured through another 10 μm filter. The 10 μm sieve retains the paramecia and the flow-through wastewater is discarded. When the majority of the wastewater has been removed, a squirt bottle with Nanopure is used to rinse the paramecia out of the sieve and into a smaller beaker. The concentrated solution is equally separated into the previously prepared culture dishes using a pipette and is set aside to reculture. This protocol is repeated every two weeks to keep the larvae food source viable.

DASPEI Staining The DASPEI solution is stored in a cold, dark location to prevent the activation of the dye. Once ready for use, the dye can be used quickly and in a dark place.We will use 3-5 dpf embryos for optimal size and development. We will transfer the living fish into a DASPEI solution made up of 100 mL Nanopure H2O to 12.5 mL DASPEI. The mixed solution will then be warmed to the temperature of the fish to prevent death via shock. The fish will swim in the solution for ~45-55 minutes in the stain, and will then be transferred to a wash of system water. The fish will swim in the system water for 10 minutes, then be transferred to an additional system water wash for another 10 minutes.

After both washes, the fish will be transferred to a microscope slide to be imaged and viewed under the fluorescent scope. Once visible, we can quantify and capture images of the hair cells before returning the fish back to the normal fish system.

Administering SP600125 SP600125 will be dissolved in dimethyl sulfoxide at a stock concentration of 50 mM and further diluted to the desired concentrations in fresh system water (trial dependent). Four hours following the first Neomycin exposure, the fish will be incubated in this SP600125 solution for 2 days. The water will be changed daily and washed in fresh system water. Ideally, JNK1, 2, and 3 should be inhibited within the fish.

Results Throughout the year, we have been trying to breed offspring from the adult fish we have. Because breeding with our own fish was not effec-

tive, we recently turned to using previously determined breeding pairs to produce more promising results. We have also purchased 100 zebrafish embryos. We sustained them in beakers filled with regularly replaced system water. We fed them paramecium soon after hatching, but we slowly integrated brine shrimp into their diet as they grew older and could move independently. Currently, we have 16 surviving baby fish from the embryos that have been added into larger tanks in our fish system and are being fed flake food like the adults. We have begun practicing DASPEI stains on adult fish. In the future, our team will perform DASPEI stains on our young fish to perfect our technique and collect quantitative data. We will also induce hair cell death in some hatched embryos using neomycin to make sure the drug properly targets the neuromasts to kill hair cells.

Discussion From these procedures, we expect to see that the neomycin has killed off zebrafish hair cells.

To analyze the results, we will conduct a fluorescent DASPEI stain to take the hair cell count of the zebrafish in question before the treatment.

DASPEI highlights the neuromasts of the zebrafish, allowing us to count the number present on the lateral line of the fish. By taking a quantitative measure of this, we will be able to tell if the Neomycin is functioning as expected. After inducing Neomycin death, we will again examine the fish under a microscope to see whether hair cell regeneration has occurred. If the hair cell count is greater than the previous count, we will be able to conclude that hair cell regeneration does occur following Neomycin treatment. Down the line, we will repeat this experiment with the JNK inhibitor SP600125 and take the same numeric measurements to analyze our results. We encountered some problems when executing our procedures. We had trouble yielding embryos from breeding our mature fish. High levels of algae growth in the fish tanks served as a deterrent to breeding and was detrimental to the health of some fish. We realized the issue lay in the ultraviolet filter, which needed to be replaced to effectively eliminate pathogens and algae from the system water. Now, algae buildup, while still present, is not as severe. In addition, our first DASPEI stain on an adult fish was unclear; the neuromasts were not brightly illuminated. This can be attributed to the excessive amount of time we

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kept the fish in the DASPEI solution. We reevaluated our protocol, and we are working on finding the ideal amount of time needed for the fish to swim in the DASPEI stain solution, and we are continuing to practice staining on our baby fish.

Works Cited 1. Cai, C., Lin, J., Sun, S., & He, Y. (2016). JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development. Frontiers in Cellular Neuroscience,10. doi:10.3389/fncel.2016.00019 2. Harris, J. A., Cheng, A. G., Cunningham, L. L., Macdonald, G., Raible, D. W., & Rubel, E. W. (2003). Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish ( Danio rerio ). JARO - Journal of the Association for Research in Otolaryngology,4(2), 219-234. doi:10.1007/s10162-002-3022-x 3. Kniss, J. S., Jiang, L., & Piotrowski, T. (2016). Insights into sensory hair cell regeneration from the zebrafish lateral line.

Current Opinion in Genetics & Develop-

ment,40, 32-40. doi:10.1016/j.gde.2016.05.012 4. Rubbini, D., Robert-Moreno, À, Hoijman, E., & Alsina, B. (2015). Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27 kip and sox2 in Supporting Cells. The Journal of Neuroscience,35(47), 15752-15766. doi:10.1523/jneurosci.1099-15.2015 5. Steiner, A. B., Kim, T., Cabot, V., & Hudspeth, A. J. (2014). Dynamic gene expression by putative hair-cell progenitors during regeneration in the zebrafish lateral line. Proceedings of the National Academy of Sciences,111(14). doi:10.1073/pnas.1318692111 Acknowledgments Dr. Colleen Kirkhart, Olivia Tandon, David Maxwell

Classical Conditioning through auditory stimuli of Drosophila melanogaster

by Gabrielle Billington (VI), Praesanna Danner ‘19, Nina Srikanth (VI), and Olivia Tandon AP Biology Abstract Introduction In previous studies, Drosophila melanogaster The auditory system of D. melanogaster has been have been successfully classically conditioned studied and extensively researched (1). D. melanothrough electric shock and taste (6), but there gaster detect sound through their antenni rather has been difficulty in the success of auditori- than through the use of ear-like structures. They ly conditioning D. melanogaster. In our experi- can detect sound at various frequencies and can ment, we used appetite conditioning by pairing even amplify faint noises. Due to these qualities, proboscis extension with an auditory stimulus. D. melanogaster’s auditory system is a reliable sysWe placed the D. melanogaster on their backs, tem to base an experiment off of. Additionally starved them, and presented them with a su- D. melanogaster can be classically conditioned, crose solution in accordance with a 200 Hz tone. as shown by T. Tully (6) who used electric shock Our results confirmed D. melanogaster could be stimuli to classically condition D. melanogaster to classically conditioned auditorily, which opens associate various odors with the shock. Fruit flies up the possibility of future studies investigat- have the longest memory retention when it comes ing addiction triggered by classical conditioning. to appetitive conditioning; up to 12 hours after the conditioning occurs, fruit flies will still be able

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to have an automatic reflex to the conditioning.

Because of this, we used appetitive conditioning in our experiment (3). Previous research has also expressed the potential flexibility of fly behavior through the use of conditioning (5). Therefore, our proof of conditioning could open up the potential to more experiments on behavior with D. melanogaster. Lastly, the process of classically conditioning D. melanogaster through the use of auditory stimuli has been tested in previous studies (4). Just as previous researchers (4) have used proboscis extension reflex (PER) as a vital facet of their studies, this experiment also used PER in order to determine the success of classical conditioning. However, Menda’s study was inconclusive because it attempted to compare different sound intensities. In our experiment. we are attempting to see if D. melanogaster can be successfully classically conditioned to associate a 400 Hz tone with sucrose. We believe that the D. melanogaster will be successfully classically conditioned due to the reliability of the D. melanogaster auditory system and the research done on successfully classically conditioning D. melanogaster.

Methods The D. melanogaster are glued onto their backs after being starved for 24 hours, and knocked out with CO2 gas. Once the flies regain consciousness, usually about three hours later, a 400 Hz tone was played while a small bubble of sucrose solution is placed on their legs, ensuring that each fly was only exposed to the solution for three seconds. The PER was then observed and recorded. After the first training session was completed, the tone and sucrose routine was repeated five times for a total of six training sessions. After finishing the sessions, there was a final test to examine whether or not the D. melanogaster was successfully classical conditioned which occurs after a 15 minute rest period. In this final session, the tone would be played and the D. melanogaster would be presented with water instead of sucrose. Proboscis extension will then be recorded. If the proboscis extends, this repeated action throughout multiple testing sessions will show an association of the tone and sucrose. In order to ensure that the results were unaffected by other variables, a number of controlled experiments were performed, such as including sound and water, just the tone, and just the sucrose.

Results When testing the flies for classical conditioning, seven flies died throughout the sessions plus the rest period between the session 6 and the final test (Figure 1). In all of the sessions, more than 80% of the flies extended their proboscis.

The lowest percent extension was in session 5 where 81% of the flies extended their proboscis, and the highest percent extension was in session 1 where 93% of the flies extended their proboscis. In the final test, which proved if the flies were classically conditioned or not, 63.15% or 12/19 of the flies extended their proboscis.

In the control that paired water and sound, only about 5% or 1/19 of the flies extended their proboscis during the final test (Figure 2). The water was used instead of the sucrose solution to eliminate the reward from the procedure.

The control group that displayed sucrose without the sound was done in order to ensure that sucrose was a reliable medium to use eliciting PER. Sucrose is shown to be a reliable medium in the results of final test with 100% of the flies extending their proboscises when exposed to sucrose (Fig. 2). Lastly, a chi-squared test was performed in order to determine the significance of the data.

Discussion The chi square test relates expected results and observed results to determine a p-value. The p-value is the probability that we would receive our results assuming proboscis extension and exposure to a 400 Hz noise are completely independent. Our chi square value of 127.2 (Fig. 3) with 18 degrees of freedom translates to a p-value below 0.0001. Since this p-value is well below the traditional 0.05 benchmark, we therefore conclude that our data is statistically significant, meaning the flies extended their proboscis, not due to random chance, but because they were classically conditioned to. This was determined as only one of 19 flies extended their proboscis in response to only water + sound (control), while on average 12 out of 19 experimental flies extended their proboscis after being trained to pair the sound with sucrose (exerpimental).

However, it is possible that some of the flies ate the sucrose solution early in the experiment and were no longer hungry during the rest of the trials. This would result in a lack of proboscis extension in later sessions and therefore the flies would

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not be successfully classically conditioned. Other factors like the nail polish of the researchers, the heat from the microscope light, or general lack of hunger could have contributed to a lack of proboscis extension and death in the training or testing sessions. But since these errors were unlikely to occur, our research supports the hypothesis that D. melanogaster can be successfully classically conditioning through the use of auditory stimuli.

Other researchers looking to conduct a similar test should use a different statistical analysis methods such as Fisher’s exact test, which is commonly used when evaluating the significance of the results regarding categorical data.

Additionally, it is recommended to use a smaller amount flies on each microscope slide in order to expose all of them to the sucrose within the 10 second duration that the sound is played, which would improve overall reliability.

Works Cited 1. Boekhoff-Falk, G., & Eberl, D. F. (2013). The Drosophila auditory system. Wiley interdisciplinary reviews. Developmental biology, 3(2), 179-91.

2. Galili, D. S., Lüdke, A., Galizia, C. G., Szyszka, P., & Tanimoto, H. (2011, May 18). Olfactory Trace Conditioning in Drosophila. Retrieved February 15, 2019, from http://www.jneurosci. org/content/31/20/7240

3. Kim, Y., Lee, H., & Han, K. (2006, June 01).

Classical reward conditioning in Drosophila melanogaster. Retrieved February 15, 2019, from https://onlinelibrary.wiley.com/doi/ full/10.1111/j.1601-183X.2006.00241.x 4. Menda, G., Bar, H. Y., Arthur, B. J., Rivlin, P. K., Wyttenbach, R. A., Strawderman, R. L., & Hoy, R. R. (2011). Classical conditioning through auditory stimuli in Drosophila: methods and models. The Journal of experimental biology, 214(Pt 17), 2864-70.

5. Tabone, C. J., & Stephen de Belle, J. (1970, January 01). Christopher J. Tabone. Retrieved February 15, 2019, from http://learnmem.cshlp.org/content/18/4/250.full 6. Tully, T., & Quill, W. G. (1985, September).

Classical conditioning and retention in normal and mutant Drosophila melanogaster. Retrieved February 15, 2019, from https:// www.ncbi.nlm.nih.gov/pubmed/3939242 Acknowledgements Thank you to Dr. Kirkhart for helping us create the procedure and training us.

Figure 1: This table shows the proboscis extension from all 4 trials. The total number of flies decrease because of death over the six sessions.

Figure 2: This table shows the average proboscis extension from the two controls. One fly died during the testing session.

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Figure 3: This is the chi square analysis of our data, which determines its significance.

Ability of Mealworms to Digest Polycarbonate Plastic

by Armani Davidson ’19, Caroline Friend ‘19, Sonia Talarek (VI), and Olivia Tandon Abstract In a search to find more eco-friendly methods of plastic disposal, scientists discovered that mealworms could digest styrofoam, a type 6 plastic notorious for its inability to decompose quickly (6). We wanted to test if mealworms could also digest type 7 plastic, specifically polycarbonate, as it also decomposes slowly and harms the environment. Our experiment aimed to find out if mealworms would be able to survive on a polycarbonate diet the way they could with styrofoam.

Our experimental design included four groups of mealworms: a negative control that was not given food, a positive control that was fed a normal diet of bran and fruit, a control group that was fed styrofoam, and our experimental group that was fed polycarbonate. Throughout our first trial we recorded the average masses and lengths of the mealworms in each group as well as noted their overall behavior. We hypothesized that those fed the polycarbonate would decline in health and not survive as readily as the styrofoam group.

Our hypothesis was supported by our polycarbonate group exhibiting the same results as the mealworms in our negative control group, suggesting that polycarbonate type 7 plastic was not a sufficient energy source for the mealworms. Although our length and mass measurements ended up being inconclusive, we noticed patterns in life cycles and survival rates indicating that the mealworms could not survive on a diet purely composed of polycarbonate. Throughout our process, we realized that we should have used different methods to collect data and collected

numerical data on their survival rates and behaviors instead of their lengths and masses.

Introduction Plastic makes up a significant portion of waste and plays a significant role in polluting the Earth, motivating scientists to look for new methods to dispose of plastic in a sustainable fashion (7).

Research has shown that mealworms, the larvae of Tenebrio molitor, are able to eat, chew, and digest various forms of polystyrene, a type 6 plastic, which is considered to be resistant to biodegradation. Mealworms are able to break down polystyrene due to the microorganisms in their gut.

This bacteria is known as Bacillus. Strain Y1 and is capable of degrading polystyrene and using it as a source of carbon (6). In a study where this gut bacteria was inhibited by gentamicin, an antibiotic to treat infections, the mealworms were no longer able to consume polystyrene and mineralize it into carbon dioxide (6). Many studies have also monitored the health and behavior of mealworms that are fed styrofoam, a type of polystyrene, over the span of one month (5). It has been observed that these mealworms spend longer periods of time in the pupae stage compared to those on a regular bran diet. This is because styrofoam does not provide mealworms with a sufficient amount of nutrition for proper growth and development (4). There is limited research, however, on mealworms consuming polycarbonate, a type 7 plastic.

Our group decided to create a project comparing the health and behavior of mealworms digesting a normal bran diet, polystyrene, and polycarbonate. We aimed to answer the following question:

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are mealworms able to digest type 7 polycarbonate plastic as they would when they break down type 6 polystyrene plastic? If so, how does this affect their overall health? We hypothesized that mealworms fed with bran and fruit would exhibit the highest growth rates in length and mass, while those fed with type 7 plastic would decline in health and have a shorter lifespan in comparison.

Methods For our research, we compared the overall health, behavior, and development of mealworms on different diets. We sorted the mealworms into the following groups: negative control, positive control, control, and experimental (depicted in Figure 1).

Results According to Table 1, the average mass of the mealworms in the positive control group was the greatest on Day 0. On Day 30, however, the average mass of the mealworms in the negative control group was the greatest.

This was not expected since the mealworms in the negative control group were starved.

Although the styrofoam group in Figure 2 has the largest numerical value for the average length of the mealworms, there is not a large difference between this group and the other groups.

Similarly, the average masses of the mealworms shown in Figure 3 do not greatly differ.

One discrepancy we found is that the negative control group has a greater mass than the negative control group on the second day of data collection.

Analysis and Conclusion We collected data on the average mass of the mealworms in each group on two separate dates. The results are given in Table 1. The mealworms in the polycarbonate group had less mass than the mealworms in the negative control group and the mealworms in the positive control group. A result that deviated from our expectations was that the average mass of the negative control group taken on 12 March 2019 was greater than the mass of the positive control group and the polycarbonate group. Additional results regarding the length and mass of

the mealworms from each group are given in Figure 2 and Figure 3.

When recording the length and mass of the mealworms from each cup, we also noted their behavior. The two most interesting observations were from the positive control and the polycarbonate group. The mealworms in the positive control group squirmed when we attempted to pick them up and even crawled away. The polycarbonate group, however, was generally slow-moving, lethargic, and did not squirm.

We expected the negative control group to have the smallest average mass because they had no source of food. This result could be explained by our limited sample size of four mealworms from each cup (twelve from each group), which makes our data statistically insignificant. We were unable to measure the mass of the mealworms in the styrofoam-fed group because they burrowed themselves deep in the cup. We also concluded that the polycarbonate group was lethargic and slow-moving since polycarbonate powder was not a sufficient energy source for the mealworms.

After various methods of collecting data, we realized that our experimental design was both inefficient and inaccurate. The process of measuring the mealworm lengths and individually massing them was far too tedious and possibly flawed, so we gradually collected less data. Additionally, we had several problems with data collection from the styrofoam group as the styrofoam was sticking to the mealworms and spilling. When selecting the mealworms from the cups we noticed additional inaccuracies in our procedure. We realized that our data collection could be potentially biased and the numbers we were using were not statistically significant. For example, we only measured the lengths of nine mealworms and the mass of twelve mealworms from each group.

There also could have been a bias because it was easier to pick up the larger mealworms; the smaller ones are better at squirming away and hiding at the bottom.

Given another chance at this trial, we would have put a more manageable amount of styrofoam in the cups, made sure the mealworms had enough

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Figure 1: Set-up of the experiment The negative control group was only given water and no food while the other groups were given various food sources. The diets were comprised of equal masses of bran and fruit, styrofoam (polystyrene), and polycarbonate powder. We observed both the lengths and masses of the worms and took notes on their behavior, including how much they moved and which stage of the life cycle they were in. water, and tried to use a computer program to measure the lengths of the mealworms instead of measuring by hand. We would also focus more on the behavioral patterns and life cycles of the mealworms.

References 1) Yang, Y., Yang, J., Wu, W.-M., Zhao, J., Song, Y., Gao, L., … Jiang, L. (2015). Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 1. chemical and physical characterization and isotopic tests. Environmental Science & Technology, 49(20), 12080–12086.

2) Wu, Q., Tao, H., & Wong, M. (2018). Feeding and metabolism effects of three common microplastics on Tenebrio molitor L. Environmental Geochemistry and Health, 40.

3) Anja Malawi Brandon, Shu-Hong Gao, Renmao Tian, Daliang Ning, Shan-Shan Yang, Jizhong Zhou, Wei-Min Wu, & Craig S. Criddle (2018) Biodegradation of polyethylene and plastic mixtures in mealworms (Larvae of tenebrio molitor) and effects on the gut microbiome, Environmental Science & Technology, 52(11), 6526-6533.

4) Nukes, N., Umar, S., Amanda, S. P., & Kanedi, M. (2018). Effect of styrofoam waste feeds on the growth, development and fecundity of mealworms (tenebrious molitor). Online Journal of Biological Sciences, 18(1), 24-28.

5) Yang, Y., Yang, J., Wu, W.-M., Zhao, J., Song, Y., Gao, L., . . . Jiang, L. (2015). Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. role of gut microorganisms.

Environmental Science and Technology, 49(20),

Table 1: Average mass of mealworms recorded on two separate dates.

Figure 2: Average length of mealworms

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2087-2093.

6) Yang, Y,; Chen, J,; Wu, W, M; Zhao, J,; Yang, J. (2015). Complete genome sequence of Bacillus sp. YP1, a polyethylene-degrading bacterium from waxworm’s gut. Journal of Biotechnology, (200), 77-78.

7) PlasticsEurope, Plastics The Facts 2014/2015 An Analysis of European Latest Plastics Production, Demand and Waste Data; PlasticsEurope, Belgium, 2014; www.plasticseurope.org/documents/ document/20150227150049-final_plastics_the_ facts_2014_2015_260215.pdf Acknowledgments Special thanks to David Maxwell for helping with our initial proposal.

Figure 3: Average mass of mealworms (styrofoam group not included)

Genetic Diversity of the Northern Pine Snake

by Isabel Devito‘19 Abstract The Northern pine snake faces severe habitat fragmentation as a result of human development. There has been annual research tracking the movement and population of pine snakes, as well as extensive behavioral studies, but no genetic work has been done on the species before the development of this project. Using snake blood to obtain DNA samples and microsatellites as genetic markers, our project aims to establish techniques and protocols for preliminary genetic analysis of the species to inform future ecology research and conservation efforts.

Introduction The Northern pine snake (Pituophis melanoleucus melanoleucus) is a threatened subspecies of pine snake that is largely found in the NJ Pine Barrens. Human development of the Pine Barrens breaks up previously contiguous habitats with roads and highways, where snakes are often killed while crossing or basking on roads. This impacts natural movement and population interaction; if snakes cannot move between these fragmented regions, breeding across the larger population is limited. Without genetic variation, a population

cannot evolve in response to a changing environment and, as a result, faces increased risk of extinction. Our project aims to determine if isolated pockets of the Pine Barrens population are exhibiting inbreeding or a decrease in genetic variety.

Preliminary genetic analysis on the Pine Barrens population of pine snakes is useful for assessing the overall health of the New Jersey pine snake population as well as informing conservation research and projects. An understanding of how genetically varied snakes in areas that have been the subject of long term conservation projects and decades of behavioral and population research can provide context for ecological data. Genetic analysis also makes it possible to identify areas of critical ecological importance by mapping out where pockets with a lack of variety and inbreeding may be. Knowing what parts of the Pine Barrens are the most fragmented helps inform preservation and land acquisition by conservation groups, and could serve as evidence for a specific habitat’s value when fighting development plans.

Methods Fieldwork Extensive research has been done on the behav-

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ior, movement and ecology of the New Jersey pine snake population, including the longest running annual study of a reptile population in New Jersey. Multiple snakes are radio-tracked throughout the year, usually for a period of four or five years.

Researchers routinely track in the field to gather consistent data on the location of transmitter snakes, and to look for new individuals that snakes with transmitters may lead them to. Every spring, at the “snake dig” event, about 10 hibernaculums at several different sites are dug up before warm temperatures hit so snakes are still in their winter den. For every snake removed from the excavated den, we record data on weight, length, sex, age, health, pit tag ID, as well as behavioral data immediately after removal from the den. Behavioral data collection involves marking any movement, aggression (hissing or striking), tongue flicks, and response to provocation over the course of several minutes. Ground and air temperature are also recorded, as temperature drastically impacts the activity of snakes coming out of the den. In recent years, we have also taken swabs of snakes’ skin to test for fungus, and blood samples, which our project is utilizing to extract DNA. Microsatellite Loci Microsatellite repeats are short, repeated segments of DNA found in the non-coding region of the genome. Microsatellite repeat sections have a high rate of mutation, but the sequences that flank the repeats are usually conserved, so the flanking sequence can be used to identify and isolate microsatellite loci. We use primers developed for microsatellite loci in the Louisiana pine snake, Pituophis ruthveni. The project uses microsatellite repeats (short repeated segments of DNA in a non-coding region of the genome) as a quantifier of genetic diversity. Individual snakes have different numbers of repeated segments, but all have common flanking regions on either side of the repeat. These flanking regions can be used as a ‘tag’ section by running a polymerase chain reaction (PCR) with primers complementary to the flanking region sequence.

After isolating microsatellite repeat sections in DNA extracted from snake blood samples, we can sequence the repeat sections. By assessing how many times the microsatellite sequences are repeated and if they are present in the genome sequence in each snake, we can examine

and quantify the difference in microsatellites between and among populations of pine snakes.

Lab The current lab procedures being run in our project are DNA extraction and PCR. We use the Qiagen DNEasy Blood and Tissue kit protocol to extract genomic DNA, and use a modified protocol for PCR from Kwiatkowski et al. that developed primers for the Louisiana pine snake (Pituophis ruthveni), which is in the same genus as the Northern pine snake. We run PCR on test primers provided by our partners at Drexel, to confirm our ability to correctly run PCR on genomic snake DNA and legitimize all data going forward. If the test primers are present in our DNA samples, our DNA extraction and PCR protocols are working correctly. We are also finding the snake identity for the blood samples in our catalogue, and mapping out where individual snakes have been identified by examining archives of data from snake digs, radio tracking, and lists of pit tag ID numbers.

Results After modifying and correcting the Qiagen protocol, we have successfully perfected our DNA extraction technique, yielding high concentrations of DNA. A Qiagen protocol for making one of the buffers necessary for extraction using spin columns contained a mistake on the amount of salt used in the phosphate-buffered saline (PBS).

After recalculation, we found that the Qiagen sheet required 10 times the necessary quantity, and our unsuccessful extractions were due to overly salty buffers degrading the DNA. After correcting our PBS buffer, DNA extraction yielded high quality DNA samples. Our partners at Drexel provided us with primers Eub13 and Eub16, which are known to work for the pine snakes, as a check for our PCR. Our previous PCR protocol produced results that showed the expected loci amplified for test primers in only some samples, and our results differed from PCR on the same samples at Drexel. The first samples we ran with our new PCR protocol show the expected primers amplified, and we have confirmed that the protocol we designed is working correctly.

The designed PCR protocol aligns with protocols used successfully in other snake genetic experiments, including studies on Crotalus horridus (timber rattlesnake), and Elaphe obsoleta (black rat

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snake). We created a touchdown thermal cycling program, since most ecologists utilizing microsatellite markers have found that touchdown cycling is better suited to using a variety of primers that have different annealing temperatures. The PCR cocktail was modified from other reptile studies to suit the resources in our lab and minimize potential sources of error.

Discussion and Conclusion After running PCR with the modified protocol, we have confirmed that our modified PCR is working properly, and we have gotten the expected results for the test primers. We expect more consistency from the updated protocol, but Eub13 and Eub16 not being amplified in every sample in the catalogue does not necessarily indicate it is failing. If multiple samples showed amplification on either all or none of the primers being used, it may have meant that the protocol was not working correctly, or that contamination occurred. However, every sample but PM 15 amplified for Eub16, and duplicates from PCR ran with Eub16 at Drexel were consistent with our samples.

The new PCR was run with four samples from each of two nearby populations, Warren Grove and Ed’s Place, both of which have hibernaculum included in the snake dig study. Although looking at only four snakes each for one microsatellite loci is not enough to make any conclusions about the variety between these populations, the microsatellite loci corresponding to Eub16 was amplified for seven of the eight tests, so Eub16 may be a useful loci to sequence PCR products for quantifying the number of repeats in the individuals. After running PCR on a large number of samples, we can screen primers to decide which loci are relevant for further analysis. Screening includes the Hardy-Weinberg equilibrium check and a check for null alleles that do not amplify in PCR. After screening our primers, we will send the PCR products for loci that we are investigating out for sequencing. After sequencing the microsatellite repeats, we will know the number of repeats for each sample and can start assessing the level of genetic variety. We want to look into various softwares that will assist with modeling the population info to help put our data in context with the ecological and be-

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Figure 1: DNA gel of PM 424 and PM 723 after PCR with Eub1, Eub13, and Eub16 (left to right: 424-1, 424-13, 424-16, 723-1, 723-13, 723-16).

Figure 2: DNA gel of PM 384 and PM 387 after PCR with Eub1, Eub13, and Eub16 (left to right: 384-1, 384-13, 384-16, 387-1, 387-13, 387-16).

havioral research records we are examining.

Works Cited Blouin‐Demers, G. and Gibbs, H. L. (2003), Isolation and characterization of microsatellite loci in the black rat snake (Elaphe obsoleta). Molecular Ecology Notes, 3: 98-99.

Clark, Rulon W., William S. Brown, Randy Stechert, and Kelly R. Zamudio. “Roads, Interrupted Dispersal, and Genetic Diversity in Timber Rattlesnakes.” Conservation Biology 24.4 (2010): 1059-069 Glenn, Travis C., and Nancy A. Schable. “Isolating Microsatellite DNA Loci.” Methods in Enzymology Molecular Evolution: Producing the Biochemical Data (2005): 202-22.

Kwiatkowski, Matthew A., Christopher M. Somers, Ray G. Poulin, D. Craig Rudolph, Jessica Martino, Tracey D. Tuberville, Cris Hagen, and Stacey L. Lance. “Development and Characterization of 16 Microsatellite Markers for the Louisiana Pine Snake, Pituophis Ruthveni, and Two Congeners of Conservation Concern.” Conservation Genetic Resource (2010): 163-66. 11 Mar. 2010.

Figure 3: DNA gel of PM 3, PM 4, PM 5, PM 12, PM 13, PM 15, PM 34 and PM 45 after PCR with primer Eub16.

Selkoe, K. A. and Toonen, R. J. (2006), Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters, 9: 615-629.

Smith. R. M, Ward, D.C., and Bien, W. F. (2016) Habitat buffering as a conservation strategy for single point dispersal animals: Case study of the northern pine snake (Pituophis melanoleucus melanoleucus) in the New Jersey Pinelands. Journal of Herpetology. In review Figure 4: Map of Warren Grove bombing range and Ed’s Place, major roads and highways outlined in blue.

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Generating A BCL2L12 Melanoma Zebrafish Model

by Annette Jones’19, Caroline Knorn (VI), Luc Francis (IV), Ms. Huang-Hobbs AP Biology / IRT Abstract This project is focused on studying the role of BCL2L12 in Melanoma in a zebrafish model. For the past three years, we have been attempting to create a plasmid that contains our candidate oncogene through a process known as Gateway cloning. Gateway cloning will allow us to integrate the plasmid into the genome of our zebrafish and enable us to study the impact of BCL1212 on cell growth and development. These Zebrafish models are prone to melanoma due to specific tumor suppressor knockouts, including p53 (a crucial tumor suppressor), allowing us to ensure that melanoma development will occur.

We have run the Gateway cloning process and are currently confirming the results of our final plasmid. Once the final plasmid is completed, we will inject the plasmid into the zebrafish embryo via microinjections. Once the fish develop, we will be able to study the impact of BCL2L12 on cell growth.

Introduction Melanoma is the most lethal form of skin cancer, originating in melanocytes. Melanoma makes up less than 1% of skin cancer cases but results in the majority of skin cancer deaths. Genomic studies of melanoma tumors exhibit upregulation of BCL2L12 in ~50% of human melanomas tested (6). BCL2L12 is a protein within an apoptotic protein family, but its specific function has never been studied or defined. The project’s goal is to investigate the function of an upregulated BCL2L12 in melanoma tumor development, since the role of this mutated gene has not been studied before in isolation. As a result, researchers are unsure how BCL2L12 directly impacts melanoma independently, specifically apoptosis.

We predict that the upregulation of BCL2L12 will inhibit apoptosis in tumor cells, and as a result, our experimental group will have more

melanoma growth compared to our control group.

To test this, we are expressing BCL2L12 using a miniCoopR system and an existing zebrafish model that contains all components for melanoma genesis (Tg(mitfa:BRAFV600E); p53(-/-)).

We will integrate the BCL2L12 gene into the zebrafish genome and observe the differences in tumor development between zebrafish with standard melanoma and zebrafish with melanoma with BCL2L12 present (ie: differing levels of apoptosis, tumor cell viability, etc).

Methods In order to induce melanoma in zebrafish, we will modify two genes, BRAF and MITFA, that control cell growth and transcription of proteins in melanocytes respectively.

1. BRAFV600E BRAF is an oncogenic protein that is involved in growth signaling. BRAF is the most commonly mutated gene in melanoma. A specific mutation, V600E, where the 600th amino acid, valine (V), is switched out for glutamic acid (E), is the most common mutation. This puts the protein in a state where it consistently promotes cell growth.

2. MITFA MITFA is the “master” melanocyte transcription factor. It is involved in the specification and development of melanocytes. Without MITFA, melanocytes will not develop.

3. Zebrafish Model Previous research by Zon Lab found that transgenic BRAFV600E in zebrafish caused nevi to develop (Fig. 2). However, when p53, an important tumor suppressor gene, was knocked out, those nevi could develop into malignant mela-

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noma (Fig. 3). Photos below provided courtesy of Zon Lab (1).

4. MiniCoopr System We are using the miniCoopR system, which gives us control over melanoma genesis. Our zebrafish model has all the necessary ingredients to develop melanoma, but due to a lack of MITFA, melanoma will not grow. However, a MITFA minigene will be added through the miniCoopR vector, sallowing for melanocytes and melanoma to develop again. In addition, our candidate oncogene (BCL2L12) can be added to the miniCoopR vector, which will be expressed in melanocytes if promoted by MITFA. 5. Gateway Cloning To construct our MiniCoopR plasmid, we are using Gateway cloning, which combines components between different cloning vectors. We have three entry vector components as listed previously. The Tol2 miniCoopR Backbone Vector serves as the destination vector. The Gateway reaction will end in the four plasmids combining all the necessary sequences into one final plasmid (Fig. 4). The result should be a plasmid that contains the BCL2L12 gene that can be inserted into the zebrafish genome to create our experimental fish that we plan to observe.

Results and Analysis The Gateway cloning process has proven difFigure 1: Wild Type Zebrafish

Figure 2: Zebrafish with mitfa:BRAF V600E

ficult. Our latest attempt produced colonies during the transformation step (Figure 5), indicating the cloning process may have been successful, and we are currently in the process of confirming the success of the cloning process.

We sent this plasmid to be sequenced, and the results confirmed the presence of a portion of the final gateway reaction plasmid (Fig 6).

This sequence, however, is present in both the miniCoopR Backbone (one component of the reaction) and in the final reaction. Therefore, the sequencing was not sufficient to confirm the reaction was a success.

Next, we looked to confirm the length of the plasmid through a restriction digest of the plasmid using enzymes BST1107I and ECORI. This should have resulted in two DNA sequences of around ~5500 bp and ~4500 bp. The resulting gel (Fig 7) produced one DNA segment of around ~10,000 bp.

This indicates that the restriction digest did not work, which could either be a result of an unsuccessful combination or a restriction site that is not present because the reaction did not complete.

Future Directions Currently, we are redoing the DNA gel and redoing the restriction digest by repeating the original reaction as well as completing a reaction with ECORI and SalI (SalI is a restriction site not found on the final plasmid but is found on the miniCoopR Backbone). From this reaction, we will know why the first DNA gel returned the results it did, whether the plasmid we have is incorrect, or if there is simply a problem in the previous restriction enzyme reaction. Once the final plasmid is obtained, we will practice our microinjection and proceed with integrating

Figure 3: Zebrafish with mitfa:BRAF V600E, p53 - / shown from two sides Figure 4: An example LR reaction

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Figure 5: E. Coli plates with the final plasmid DNA. Small colonies can be identified the final plasmid into the DNA of the zebrafish, promoting melanoma growth. Once the fish develop, we will be able to compare the melanoma growth with BCl2L12 present versus without BCl2L12, clearly showing BCl2L12’s impact on cell growth and death.

References 1. Patton, E. E., Widlund, H. R., Kutok, J. L., Lee, C., Fisher, D. E., & Zon, L.I. (2005). BRAF Mutations Are Sufficient to Promote Nevi Formation and Cooperate with p53 in the Genesis of Melanoma. Current Biology. https://doi.org/10.1016/j. cub.2005.01.031 2. Scorilas, A., Kyriakopoulou, L., Yousef, G. M., Ashworth, L. K., Kwamie, A., & Diamandis, E. P. (2001). Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family.

Science Direct, 72(2). Retrieved from https://

Figure 6: Plasmid map of the proposed final plasmid after Gateway Cloning and part picked up after sequencing. A: T3 promoter where sequencing began, B: Range of plasmid confirmed through sequencing.

www.sciencedirect.com/science/article/pii/ S0888754300964553?via%3Dihub 3. Nakajima, A., Nishimura, K., Nakaima, Y., Oh, T., Noguchi, S., Taniguchi, T., & Tamura, T. (2009). Cell type-dependent proapoptotic role of Bcl2L12 revealed by a mutation concomitant with the disruption of the juxtaposed Irf3 gene. Proceedings of the National Academy of Sciences of the United States of America, 106(30), 12448–12452. http://doi.org/10.1073/ pnas.0905702106 4. Stegh, A. H., & DePinho, R. A. (2011). Beyond effector caspase inhibition: Bcl2L12 neutralizes p53 signaling in glioblastoma. Cell Cycle, 10(1), 33–38. http://doi.org/10.4161/cc.10.1.14365 5. Kong, B. Y., Carlino, M. S., & Menzies, A. M. (2016). Biology and treatment of BRAF mutant metastatic melanoma. Melanoma Management, 3(1), 33–45. http://doi.org/10.2217/mmt.15.38

Figure 7: DNA gel showing DNA length between 1015 kilobase pairs.

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6. Iyengar, S., Houvras, Y., & Ceol, C. J. (2012).

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model. Journal of Visualized Experiments,(69). doi:10.3791/50086 7. Gartner, J.J, et al. Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma. (2013). Proceedings of the National Academy of Sciences, 111(1). doi:10.1073/pnas.1323160111 8. Kwan, K. M. et al. (2007). The Tol2kit: A multisite gateway‐based construction kit for Tol2 transposon transgenesis constructs. Wiley Online Library. Retrieved from https://onlineli-

brary.wiley.com/doi/abs/10.1002/dvdy.21343 9. Cantwell-Dorris, E. R., O’Leary, J. J., & Sheils, O. M. (2011). BRAFV600E: Implications for Carcinogenesis and Molecular Therapy. AACR Journals. https://doi.org/10.1158/1535-7163.MCT10-0799 10. Kawakami K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biology.

2007;8(Suppl 1):S7. doi:10.1186/gb-2007-8-s1-s7.

Acknowledgements Zon Lab, Lateral Line Team, Dr. Kirhart, Mr. Maxwell, Ms. Tandon, Raymond Chen

Capturing Species Variety on the Pingry Campus

by Nicole Kloss ‘19, Isabelle Sheyfer (V), David Maxwell, Olivia Tandon AP Biology

Abstract The purpose of our project was to measure the biodiversity of medium-sized mammals on the Pingry campus. We used camera traps to capture the movement and frequency of said mammals at three different locations on campus. In our experiment, we found that while species richness was high, evenness in species distribution could be improved; our data shows that Pingry’s diversity index is at a low 0.29 as compared to the 0.49 diversity index from a similar study done a few years ago. In conclusion, we found that Pingry has a low biodiversity of wildlife on its campus. We hope that future students will continue this research to better our conservation efforts of the species we observed, as well as take their habitats into account when developing.

Introduction Due to factors such as climate change and poaching, animal species are rapidly diminishing. In many environments, “...mammalian abundance is unknown despite their importance to ecosystems” (Tape). It is important for methods to be developed to conserve these species by monitoring their migration patterns and biodiversities in various environments; one of these is camera

traps. Camera traps are a non-invasive tool to observe wildlife. They allow the observation of changing species variety in all types of environments and they help the improvement of conservation efforts. “Camera-trap techniques can be a vital tool to confirm the presence of large mammals that might be under pressure from exploitation, habitat loss, and fragmentation” (Tape). One promising study utilizing camera traps, conducted in the Río Plátano Biosphere Reserve of Eastern Honduras in 2008, was highly effective in the observation of the variety of species in the area.

The research done was “...especially important in biosphere reserves, national parks, and multiple-use reserves where human settlement coexists within a mosaic of tropical forest and agricultural landscapes” (Tape). This interesting study inspired aspects of our project, including how cameras can be utilized further encouraged the importance of discovering species variety. A group of Pingry AP Biology students conducted a similar study a few years ago, focusing mainly on larger mammals around the Pingry campus. While we also made sure to include large mammals in our study, we felt that if birds and smaller mammals were omitted from the study, a large piece of important data would be neglected.

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With the data from the cameras that were placed around our campus, we calculated a diversity index, which allowed us to answer the question: what is the species diversity in our community?

We hypothesized that we would observe deer and larger forest-dwelling mammals the most out of any other species. We believe that this is a first step towards preserving our wildlife at Pingry.

Methods Throughout the winter months, three different locations on the Pingry campus were equipped with camera traps: the deer enclosure, the pond, and the trail. We chose these areas because we deduced that these were the most common areas where deer and other wildlife often congregate.

Four cameras were initially set up, however, one camera was not working properly and could not collect the data needed to move forward with the project. The cameras have night vision capabilities and snap pictures when movement is detected. We did a trial run the week before our study officially began, and tested different angles to get the best vantage point possible. The cameras were placed about ¾ of the way up a small/medium sized tree in each of our locations. When the cameras detected motion, pictures were taken, accumulating data on wildlife variety on our campus. After having the cameras out for a few months, we retrieved them to collect and analyze the final data.

Specifically, we wanted to see whether deer would be the most prevalent species out of the observed animals. We also calculated the Simpson diversity index, which measures the species diversity, for our community. The index takes into account the number of species present, as well as the rela-

Figure 1: Pie chart of final species diversity data coagulated from 3 different camera traps.

tive abundance of each species. Diversity increases as species richness and evenness increase.

Results We recorded 2 racoons, 85 deer, 6 fox, and 3 squirrel by the deer enclosure; 1 racoon, 52 deer, 3 fox, 2 squirrel, and 10 crows by the pond; 3 deer by the cross-country trail; inconclusive data by the chicken coop.

Discussion From our data, we concluded that our mammal species in order of largest to smallest population size was deer, crows, foxes, squirrels, and finally, racoons. Deer were most prevalent throughout the different locations on the Pingry campus.

However, as observed through the Simpson diversity index, our data yielded a low diversity rate of 0.29. Although there is variety in the types of species present on campus, this population numbers are not even between species. Therefore, we concluded that there is a lower biodiversity of wildlife in our area. A similar research study was conducted on the Pingry campus last year that counted 69 deer and 41 foxes, yielding a diversity index of 0.45. Despite this higher diversity, a smaller range of wildlife was observed. Using this information, we know that we need to create more even population distribution and cater to other species besides deer, an animal of which Pingry currently has an overpopulation. It is important to make sure that the fitness of the other species we observed increases, and that we make the Pingry campus a more suitable environment for them so that we can have a more even biodiversity ratio. Students who end up doing a future study will be able to utilize the data from the past two years to continue observing the species variety and calculating diversity indexes in order to determine how species variety changes. The more information in species diversity, the better our future conservation efforts.

Works Cited 1. Ken D. Tape, David D. Gustine; Capturing Migration Phenology of Terrestrial Wildlife Using Camera Traps, BioScience, Volume 64, Issue 2, 1 February 2014, Pages 117–124, https:// doi.org/10.1093/biosci/bit018 2. David J. Gonthier, Franklin E. Costañeda; Large- and Medium-Sized Mammal Survey Using Camera Traps in the Sikre River in the Río Plátano Biosphere Re-

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Figure 2: Examples of pictures taken via camera traps (Upper Left: Crows, Upper Right: Squirrel, Lower Left: Red Fox, Lower Right: White-Tailed Deer)

3.

4.

5.

6.

serve, Honduras, Tropical Conservation Science, 1 September 2013, https://doi. org/10.1177/194008291300600409 Caravaggi, A. , Banks, P. B., Burton, A. C., Finlay, C. M., Haswell, P. M., Hayward, M. W., Rowcliffe, M. J., Wood, M. D., Pettorelli, N. and Sollmann, R. (2017), A review of camera trapping for conservation behaviour research. Remote Sens Ecol Conserv, 3: 109-122. doi:10.1002/rse2.48 Howe, E. J., Buckland, S. T., Després‐Einspenner, M. , Kühl, H. S. and Matthiopoulos, J. (2017), Distance sampling with camera traps. Methods Ecol Evol, 8: 1558-1565. doi:10.1111/2041-210X.12790 Randler, C., & Kalb, N. (2018). Distance and size matters: A comparison of six wildlife camera traps and their usefulness for wild birds. Ecology and Evolution, 8(14), 7151–7163. http://doi.org/10.1002/ece3.4240 Sunarto, Sunarto & Sollman, Rahel & Mohamed, Azlan & Kelly, Marcella. (2012). Camera trapping for the study and conservation of tropical carnivores. The Raffles bulletin of zoology. 28. 21-42.

7. Jumeau, J., Petrod, L., & Handrich, Y. (2017).

A comparison of camera trap and permanent recording video camera efficiency in wildlife underpasses. Ecology and Evolution, 7(18), 7399–7407. http://doi.org/10.1002/ece3.3149 8. Burton, A. C., Neilson, E., Moreira, D., Ladle, A., Steenweg, R., Fisher, J. T., ... & Boutin, S. (2015). Wildlife camera trapping: a review and recommendations for linking surveys to ecological processes. Journal of Applied Ecology, 52(3), 675-685.

9. Wegge, P., Pokheral, C. P., & Jnawali, S. R. (2004, August). Effects of trapping effort and trap shyness on estimates of tiger abundance from camera trap studies. In Animal Conservation forum (Vol. 7, No. 3, pp. 251-256). Cambridge University Press.

Acknowledgements Mr. Maxwell, Mrs. Tandon, The Pingry School

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