PennScience Fall 2016 Issue: Synthetic Biology

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PennScience Fall 2016 Volume 15 Issue 1

PennScience is a peer-reviewed journal of undergraduate research published by the Science and Technology Wing at the University of Pennsylvania and advised by a board of faculty members. PennScience presents relevant science features, interviews, and research articles from many disciplines, including the biological sciences, chemistry, physics, mathematics, geological sciences, and computer sciences. PennScience is funded by the Student Activities Council. For additional information about the journal including submission guidelines, visit www.pennscience.org or email pennscience@gmail.com.

EDITORIAL STAFF EDITORS-IN-CHIEF Samip Sheth Jane Chuprin WRITING MANAGERS Richard Diurba Ritwik Bhatia EDITING MANAGERS Zoe Daniels Abhinav Suri DESIGN MANAGERS Emily Chen Suzanne Knop BUSINESS MANAGERS Jingyi Huang Alex Wong TECHNOLOGY MANAGER Rounak Gokhale FACULTY ADVISORS Dr. M. Krimo Bokreta Dr. Jorge Santiago-Aviles

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WRITING

EDITING

DESIGN

BUSINESS

Mia Fatuzzo Rosie Nagele Eric Teichner Hiab Teshome

Ellen Bei Sarah Fendich Natasha Gupta Rachel Levinson Christina Lin Sapna Nath Anova Sahoo Andrew Wang Kathleen Wang Lily Zekavat Aaron Zhang Brian Zhong

Chigoziri Konkwo Olivia Myer Abi Szabo Alison Weiss Grace Wu

Arjun Lal Tyler Larkworthy Olivia Medrano Felix Shen Sitara Shirol Rekha Vegesna Donna Yoo Jici Wang

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FEATURES

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8

CRISPR: The Genomic Revolution

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by Eric Teichner

Synthetic Biology and the Environment by Rosie Nagele

12 Insulin by Mia Fatuzzo

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Synthetic Metabolism by Hiab Teshome

RESEARCH

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Environmental Factors Effect on Vibrio parahaemolytics in Massachusetts Oysters Natalie Weiss Hauke Kite-Powell, PhD

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LETTER FROM THE EDITORS Dear Readers, We are thrilled to present our first issue of Volume 15 of the PennScience Journal of Undergraduate Research. The theme for this issue, Synthetic Biology, was inspired by significant attention of leading scientific journals on this rapidly advancing and diversifying field of science, engineering, and technology. For instance, research published in Nature in Fall 2016 shows that new designer cell systems hold the potential to correct high-blood glucose levels in diabetes patients. Indeed, as artificial designs are built into complex biological systems and living organisms, the promise of medical therapies, materials and biological computing continues to only grow. Our feature articles aim to explore the applications of synthetic biology in the context of recent findings while shedding some light on their role in healthcare, disease, and the living world as we understand it today. We are incredibly grateful to all our staff who collectively created this issue, undergraduate students who submitted their research findings to our Journal, and the Penn undergraduate community for engaging in scientific discourse on campus. In this issue, Mia Fatuzzo examines how scientists are using recombinant DNA techniques to produce insulin for diabetes patients. Alexandra Nagele explores how synthetic biology is playing a role in studying and clearing the environment of pollution. Hiab Teshome explains how metabolic engineering has allowed scientists to study better cellular signaling pathways in the body. Eric Teichner recaps how the new gene editing technique, CRISPR, is revolutionizing biological research. Finally, we are proud to present the original research of Natalie Weiss, who explores the relationship between bacteria levels in oysters and the environment in which the oysters are harbored, such as increasing water temperatures. We have greatly enjoyed our time leading PennScience, and we would like to welcome Richard Diurba as our new Co-Editor in Chief. As we transition to new leadership, please help us in thanking the many groups and individuals who have made PennScience possible. First, we would like to thank our incredible journal staff--our writers, editors, and design and business members--for their hard work, dedication, and enthusiasm. Our publication is entirely student run and relies on the efforts of our scientifically curious undergraduate members. We want to recognize two leading Penn scientists, Dr. Dustin Brisson and Dr. Brian Chow, and a recent PennScience alumnus, Vivek Nimgaonkar, who helped us promote scientific discourse on campus by hosting two coffee chats with and a journal club with our members. We owe our funding to the Science and Technology Wing of the King’s Court College House and Student Activities Fund, in allowing us to publish a high-quality journal every semester. We would also like to thank our faculty mentors, Krimo Bokreta and Jorge Santiago-Aviles, for their guidance and support. Finally, we want to thank you for reading PennScience--enjoy our latest issue! Sincerely, Samip Sheth C’17 and Jane Chuprin C’17 Co-Editors-in-Chief

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CALL FOR SUBMISSIONS Looking for a chance to publish your research? PennScience is accepting submissions for our upcoming Spring 2017 issue! Submit your independent study projects, senior design projects, reviews, and other original research articles to share your work with fellow undergraduates at Penn and beyond. Email submissions and any questions to pennscience@gmail.com

Research in any scientific field will be considered, including but not limited to:Â

Biochemistry Biological Sciences Biotechnology Chemistry Computer Science Engineering Geology Mathematics Medicine Physics Psychology

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Spring 2016

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FEATURES

SYNTHETIC BIOLOGY

and

the ENVIRONMENT Between the California coastline and the islands of Hawaii lies a vast area of murky, gray water, strewn with discarded plastic debris and non-biodegradable trash. This is the Eastern Pacific Garbage Patch (1), a collection of litter from across the Pacific Ocean drawn together by circular currents. This assemblage of debris poisons marine life and releases harmful toxins into the ocean that can be transferred through the food web until they eventually end up on our plates. Not only do we currently lack an effective strategy to address this issue, but the practices that have created it are still prevalent in everyday life. The Eastern Pacific Garbage Patch exemplifies the way in which many aspects of our lives, such as our reliance on plastics and fossil fuels, result in the pollution and destruction of our environment. As pollution-related diseases rise and basic resources such as clean water become harder to access, the need to change our relationship with the environment is increasingly evident.

“Scientists are now turning to synthetic biology in search of solutions. By manipulating organisms to function in new ways, research in synthetic biology can combat dangerous levels of pollution in a renewable and sustainable way.� Scientists are now turning to synthetic biology in search of solutions. By manipulating organisms to function in new 6

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By Rosie Nagele

ways, research in synthetic biology can combat dangerous levels of pollution in a renewable and sustainable way. Toxic chemicals pervade our water, soil, and air. Arsenic, for example, is a harmful contaminant of groundwater in several parts of the world such as Argentina, India, and China (2). Screening for arsenic is critical for human health in these areas, but current chemical tests are expensive, complicated, and often erroneous. In 2005, researchers successfully used an engineered version of E. Coli to detect the presence of arsenic in samples of groundwater (3). This bacteria produces bioluminescence in proportion to arsenic concentration, which both signals the presence of arsenic and gives information about its amount. The bacteria were able to detect arsenic in complex groundwater samples with higher accuracy than current tests. The simplicity, low cost, and accuracy of this method demonstrates promise for using synthetic biology to effectively detect arsenic as well as other harmful pollutants, which is the first step in restoring health to our environment. Once pollutants are detected, the next challenge is their removal. Some microorganisms are capable of removing harmful substances from the environment through a process known as bioremediation. Scientists are experimenting with manipulating this natural process to become more efficient and capable of targeting a wider range of toxins. In 2006, researchers used synthetic biology to improve the efficiency of the bioremediation of hexavalent chromium (Cr(VI)) and hexavalent uranyl (U(VI)) (4). Cr(VI), a recognized carcinogen,


FEATURES is a common byproduct of many industrial activities and is a dangerous contaminant of water supplies due to its high solubility (5). Bioremediation of this compound converts Cr(VI) to chromium oxide (Cr(III)), which is less toxic and less soluble. The researchers manipulated the evolution of ChrR, one of the enzymes involved in this process, to become more efficient. The new enzyme, called ChrR6, removed Cr(VI) 200 times more effectively than the natural enzyme. They were interested in the enzyme’s ability to remediate multiple contaminants and found that ChrR6 also improved the efficiency of converting (U(VI)), which is a soluble radioactive waste product, to the insoluble tetravalent uranyl (U(IV)). By transforming bacteria to contain ChrR6 and increase their permeability, the researchers created bacteria that removed Cr(VI) and U(VI) more effectively than bacteria with the unmodified gene. Not only did they create an enzyme that effectively reduced the concentration of multiple common contaminants, but also they were able to successfully transfer the enzyme into a bacteria that can be used as a medium for introducing this new biological function into the polluted environments where it is needed. This new discovery highlights the efficacy of synthetic bioremediation in helping both cleanse and sustain the environment. In addition to decontaminating the environment, synthetic biology has the potential to prevent future destruction by enabling more sustainable production of natural materials. Plastic, for example, is currently produced with materials derived from petroleum. Extracting petroleum causes severe environmental damage to the surrounding ecosystem, and the disposal of plastic contributes to the contamination of water and soil (6). Poly-3-hydroxybutyrate (PHB) is a renewable and biodegradable compound that could replace petroleum as the base for plastics if it can be produced in large enough quantities. PHB is produced naturally by bacteria through metabolic pathways which can be modified to increase efficiency. Additionally, these pathways are transferable to more complex organisms, supporting a larger scale of production. A study published in 2011 in Plant Physiology refined the technique of engineering tobacco plants with bacterial plasmids encoding for PHB production, achieving about a tenfold increase in production of PHB and fewer negative consequences on plant health (7). This is an important step toward efficiently producing plastic material that is renewable and biodegradable to reduce the negative consequences of plastic production and usage. Another major use of petroleum is in fossil fuel, so it is critical that a sustainable replacement be found. Biofuels, renewable fuel derived directly from living matter, offer an alternative method of energy production. However, although plant-based biofuels such as ethanol have been used since the early twentieth century (8), they continue to face numerous technological barriers. One major challenge is their high requirements of land and water for cultivation, which puts them in direct competition with food crops, a relationship that has grown more tense as global hunger rates rise. Consequently, researchers have begun to investigate the use of algae, which naturally produce energy-dense oils, to sustainably and effectively produce biofuels. A 2013 review paper in Current Opinion in Chemical Biology argues that recent advancements in synthetic biology have created the tools to make algal production of biofuels feasible and valuable (9). Recent efforts to sequence algal genomes have revealed immense genetic diversity, which

can be used to refine and maximize oil production using new genetic engineering techniques. Additionally, recent research in the field of metabolic engineering has the potential to modify lipid production, which could enable algae to produce the different types of lipids that are most suited for biofuels and increase the efficiency of their production. These advancements in applying synthetic biology to algal species have the potential to make a significant impact on the looming issue of the sustainable production of fuel. Although researchers have demonstrated the potential for using synthetic biology to reduce environmental damage, critical questions remain to be answered before these practices will be ready for implementation. Will the success of engineering bacteria to remove one toxic chemical from the environment translate to the removal of other harmful compounds as well? Will these techniques be able to function on a scale that will make a significant difference to our heavily contaminated environment? Do these novel techniques to manipulate biological processes have any negative side effects? These and other questions will be critical to answer before synthetic biology can make a significant impact on the environment. However, they do not pose insurmountable, and research continues to address these issues and make synthetic biology a useful tool for managing environmental issues. Although research on synthetic biology is still in its early stages, it provides hope in the face of the rapidly growing problem of environmental destruction, which has no clear solution in sight. With each day, the cycle of production and disposal continues, further damaging our ecosystems and adding waste to sites such as the Eastern Pacific Garbage Patch. By working to end this cycle, we protect not only the flora and fauna that are directly harmed by our actions, such as the marine life navigating polluted waters, but we also create an environment that is more habitable and nurturing to our own species. Techniques in synthetic biology are emerging as the powerful tools we need to make this shift. Engineering organisms for new and improved functions has the potential to change how our lives impact the natural world and create a more harmonious relationship between humans and our environment. Works Cited: (1) Turgeon, A. (2014). Great Pacific Garbage Patch. National Geographic Society. (2) Smedley, P., and Kinniburgh, D. (2001). A review of the source, behaviour and distribution of arsenic in natural waters. Applied Geochemistry 17, 517-568. (3) Trang, P., Berg, M., Mui, N., and Meer, J. (2005). Bacterial Bioassay for Rapid and Accurate Analysis of Arsenic in Highly Variable Groundwater Samples. Environ. Sci. Technol. 39, 7625-7630. (4) Costa, M. (2003). Potential hazards of hexavalent chromate in our drinking water. Toxicol. Appl. Pharmacol 188, 1-5. (5) Hexavalent Chromium. Occupational Safety and Health Administration. (6) Kharaka, Y., and Dorsey, N. (2005). Environmental issues of petroleum exploration and production: Introduction. Env. Geosciences 12, 61-63. (7) Bohmert, K., Balbo, I., Kopka, J., Mittendorf, V., Nawrath, C., et al. (2000). Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight. Planta 211, 841-845. (8) History of Biofuels. (2010). Biofuel.org.uk. (9) Gimpel, J., Specht, E., Georgianna, D., and Mayfield, S. (2013). Advances in microalgae engineering and synthetic biology application for biofuel production. Current Opinion in Chemical Biology 17, 489-495.

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CRISPR

By Eric Teichner

THE GENOMIC REVOLUTION Is it possible to cure any disease? With CRISPR, this seemingly impossible activity might be possible. CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is composed of segments of prokaryotic DNA that contain repeating base pair sequences (1). Analogous to molecular scissors, the CRISPR/Cas9 complex can cut at specific sites in the genome. Although researchers have been testing molecular engineering techniques since the 1970s, CRISPR represents a profoundly innovative, precise, and efficient method of genomic editing, providing a new outlook for humanity. Scientists have harnessed the potential of CRISPR as a controlled method to alter specific genetic sequences in vivo. In 1993, Groenen et al. from the Netherlands came across a cluster of repeating base pairs with an unknown purpose separated by spacers in the organism Mycobacterium tuberculosis (2). As a result, they named this sequence a “direct repeating” region. In the same year, Francisco Mojica, a researcher from the University of Alicante in Spain, found a similar cluster of bases in another bacteria, Haloferax mediteranii (1). With approximately 30 repeating bases separated by spacers 36 base pairs in length, the observed pattern did not resemble any known sequence (3). Eventually, this piece of DNA was given the name CRISPR. CRISPR and Cas, short for CRISPR Associated Proteins, are naturally used as a basic mechanism in a prokaryotic

immune system to prevent viral invasion (4). If a virus invades a host prokaryotic cell, viral DNA is integrated into the CRISPR system. These sequences are then transcribed into a matching RNA sequence. The Cas enzyme binds with the RNA molecules and acts as a deadly weapon against the specific virus from which the DNA was taken (5). When the Cas/RNA system recognizes genetic material that matches the CRISPR (guide) RNA, the Cas enzymes will act as “molecular scissors,” cutting that specific sequence and preventing the transcription of more viral units. As a result, CRISPR is able to target specific DNA

CRISPR is composed of segments of prokaryotic DNA that contain repeating base pair sequences. Analogous to molecular scissors, the CRISPR/Cas9 complex can specifically cut at certain sites in the genome.

target DNA

PAM:NGG

Cas9 RNA for gene modification

crRNA

tracrRNA

8

sequences at precise locations as short as 20 base pairs long. By giving Cas the right RNA sequence guide, scientists are able to precisely cut and paste DNA sequences into the genome (3). The applications of CRISPR offer molecular biologists the ability to study and target particular DNA sequences within billions of genomic base pairs. Although the excitement has focused

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double strand break by Cas9

insertion gene correction deletion


FEATURES

on the largescale applications, studies have found that making small changes in the genome can cause large phenotypic changes in organisms such as plants, fruit flies, and human cells. Scientists can also investigate the effects of CRISPR when it does not function properly, specifically by breaking these “molecular scissors.” Stanley Qi, a synthetic biologist from Stanford University, mutated the Cas9 enzyme so that it can still attach to the target DNA sequence, but no longer degrade it. This resulted in blocked transcription of specific DNA sequences. A major result of this procedure is to determine the phenotypic effects of turning on or off specific genes (6). These new molecular applications are extremely useful for scientists exploring the emerging field of genomic editing. CRISPR can be used for medical therapy, since it can target deleterious genes, thereby curbing disease incidence. The first clinical trial of CRISPR in the United States was preliminarily approved this year. Researchers at the University of Pennsylvania hope to use CRISPR to genetically modify T-cells in patients with multiple myeloma, melanoma, and sarcoma so the cells can target and destroy tumor cells when they are infused back into a patient. Furthermore, researchers at the Massachusetts Institute of Technology have used CRISPR to ameliorate symptoms stemming from a rare liver disorder (type I tyrosinemia) in mice. In this experiment, researchers injected adult mice carrying a mutated form of the enzyme FAH, an enzyme that breaks down tyrosine in the liver, with guide RNAs and a DNA template for the correct sequence of the FAH gene. Ultimately, it resulted in ~0.4% liver cells initially expressing the correct FAH gene (7). The applications of CRISPR in the clinical setting have shown promise in improving the prognosis of diseases and prolonging the lifespan of many patients. CRISPR technology has already proven successful, and it has the potential to achieve even more. For example, CRISPR could eradicate diseases such as malaria. Malaria is deadly—in 2015 alone, there were over 200 million people infected and over 500,000 people dead (8). In order to help fix this major problem, CRISPR could be used to edit the genome of malaria-causing mosquitoes. Normally, when a malaria-infected mosquito bites a host, the sporozoites, the infective agents inserted into the host, immediately travel to the liver, causing flu-like symptoms.

When another mosquito bites the infected human, the viral particles transfer to the mosquito, rapidly causing an endemic outbreak. CRISPR can add a new antibody gene to mosquitoes that targets the malaria parasite. However, merely changing a gene in one mosquito would not be helpful. Using a method called gene drive, the CRISPR inserted gene would become dominant in the mosquito population (9). This procedure could go far beyond malaria, treating other similar diseases such as yellow fever. While CRISPR is clearly promising, there are ethical considerations to consider before full implementation as the creation of organisms devoid of genetic defects may reduce biodiversity. Undoubtedly, CRISPR is the leading technology for genetic alterations by changing what has previously been fantasy into a potential reality. Further investigation regarding CRISPR’s precision and efficiency is needed to perfect this inchoate technology, yet it is an exciting time for biologists wishing to create advances in clinical care. CRISPR technology allows humans to become closer than ever to editing the molecular basis for life, which will change how scientists attempt to solve the world’s greatest biological problems. Works Cited: (1) Questions and Answers about CRISPR (2016). Broad Institute. (2) Groenen, P., Bunschoten, A., van Soolingen, D. and van Embden, J. (1993). Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by novel typing method. Mol. Microbiol. 10, 105765. (3) Lander, E. (2016). The Heroes of CRISPR. Cell 164, 18-28. (4) Jinkek, M. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821. (5) Doudna, J., and Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science 346. (6) Ledford, H. (2016). CRISPR: gene editing is just the beginning. Nature 531. (7) CRISPR Reverses Disease Symptoms in Living Animals for First Time (2014). Genetic Engineering & Biotechnology News. (8) Gates, B. (2014). The Deadliest Animal in the World. Gates Notes. (9) Regalado, A. (2016). The Extinction Invention. MIT Technology Review.

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Synthetic Metabolism By Hiab Teshome

The human body is an intricate and purposeful machine. Every action that occurs is an accumulation of numerous biochemical reactions operating simultaneously. These reactions allow us to live by creating structurally organized, specialized compounds. Metabolism is the process of generating chemical products that will later be used by the body to sustain life. However, sometimes the production of these molecules is inefficient and can contain damaging errors. Metabolic engineering, a popular field of biotechnology, is the process of optimizing genetic and regulatory processes within a cell to more efficiently increase cell production and to solve issues pertaining to metabolic dysfunction. By providing a mechanism to transform biological tools and compounds on a large scale, metabolic engineering has paved the way for new forms of pharmaceutical use and biological analysis. Metabolic engineering has been used by scientists since the 1940s; however, this field took a larger presence in the 1990s with the successful practice of recombinant DNA technology, particularly in the mass production of human insulin. This discovery catalyzed a new, driven attempt to genetically engineer cells to produce chemicals that can be readily processed by humans. There are many different approaches to develop desirable biochemicals. For example, some scientists mutate genes to create new proteins that alter the cell’s function. Other scientists overexpress the genes of the product they are trying to create, thereby overproducing the product of a given pathway. Both of these approaches, and many others, efficiently produce valuable products (1). Recently in the scientific community, there has been a shift in the mechanisms used for metabolic engineering. Instead of artificially introducing foreign DNA into the cell through recombinant DNA technology, which requires many expensive resources, scientists are now able to directly and efficiently produce their desired products by simply altering metabolic pathways. New metabolic engineering techniques, which utilize creating mutations to alter the amino acid sequence of enzymes, have been used for the mass production of important proteins in

the body. This technique is used for the production of a nonnatural amino acid, L-homoalanine (2). L-homoalanine is an important precursor for many antiepileptic drugs. Today, scientists are able to completely biosynthesize L-homoalanine by modifying glycolysis, a natural step in cellular respiration. This artificial metabolic pathway directly converts glucose to L-homoalanine, reducing the environmental impact of its once complex production cycle and decreases the costs of antiepileptic drugs. Instead of artificially introducing foreign DNA into the cell through recombinant DNA technology, which requires many expensive resources, scientists are now able to directly and efficiently produce their desired products by simply altering metabolic pathways. Using discoveries from this new method to generate L-homoalanine, scientists continue to bio-synthesize chemicals, thereby adapting natural human processes into microbes for mass production. In addition to pharmaceutical production, biologists are now attempting to use metabolic engineering attempting to develop cells that mirror human metabolic pathways. According to Dr. Eytan Ruppin, a professor of computer science at Tel Aviv

“This discovery catalyzed a new driven attempt to genetically engineer cells.“

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University in Israel, the development of such a simulated cell would help biologists understand elusive metabolic processes and the unknown microscopic universe inside of a cell (3). This project has been in development since the 1950s; currently scientists are working to improve an in silico model of E. coli


FEATURES cells. This external, computerized model of E. coli can perform normal metabolic processes of the prokaryote. In the future, researchers plan to replicate human processes in E. coli by observing and recreating the vast metabolic connections between human and E. coli cells. Through the use of in silico models this process, scientists will quickly and easily have the resources to observe entire cellular systems. These processes not only regulate the body’s metabolism, but also play a critical role in the efficacy of the immune system. Recently, scientists have successfully recreated T cell receptor signalling pathways, providing new insight into protein signalling in complex cellular processes. T cells are the soldiers of the immune system. They are white blood cells that circulate the body in order to destroy foreign antigens. The biosynthetic pathways for immunity are directly dependent on signal transduction pathways. When T cell extracellular receptors recognize specific antigens, signalling pathways are activated in T cells to destroy foreign invaders. However, if the functionality of even one receptor is compromised, the entire pathway will become inactive. One can view this system as a chain reaction, with one event directly impacting a succession of similar events. Like a row of falling dominoes, if one T cell receptor is inhibited, succeeding T cell receptors can not function. Scientists at the Marine Biology Lab at the University of California, San Francisco have recently used synthetic biology to reproduce these T cell receptor pathways, which led to the discovery of their spatial organization and response mechanism to antigen attacks. This is a breakthrough for many autoimmune diseases because it opens up the possibility of altering malfunctioning T cell receptor pathways, such as in Type 1 diabetes, which would halt the disease progression. Dr. Ron Vale, the principal investigator at the aforementioned lab, describes the experiment as a long process that focused on one T cell receptor signalling pathway. The team biochemically reconstituted the 12-component signaling pathway on model membranes, creating a biosynthetic cell that produced various other products. By focusing on a single T cell, his team was able to discover other important signal transduction pathways in the body, such as protein differentiation, that depended on T cell pathways to activate this function (4). Studying the intricate methods of T cell regulation can lead to the understanding of many other regulatory signalling pathways and chemical processes of the body. Metabolic processes provide a wide range of intricate and accurate chain reactions. Naturally occurring cellular pathways are being harnessed for our benefit to improve our lives. Current studies in synthetic metabolism are not only giving scientists a better and clearer understanding of metabolic processes in the

the body, but by attempting to control and manipulate these processes, scientists can also mass produce large quantities of drugs and reconfigure malfunctioning metabolic pathways that are present in disease. This can significantly improve the lives of many individuals by providing high- demand drugs at a lower price. The research of manipulating and studying diseased pathways can also save the lives of many individuals through

“Metabolic processes are the fundamental building blocks of the human body...Current studies in synthetic metabolism are not only giving scientists an enhanced understanding of metabolic processes in the body.“ harnessing these discoveries to treat and halt diseases. This biosynthetic approach to medicine allows for new methods of understanding the world and opens up a wide range of possibilities in the scientific community. Works Cited: (1) Kim, H., Charusanti, P., Lee, S., and Weber, T. (2016). Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites. Nat. Prod. Rep. 33, 933-941. (2) Zhang, K., Li, H., Cho, K., and Liao, J. (2010). Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proc Natl Acad Sci USA 107, 6234-6239. (3) Rehmeyer, J. (2008). Simulated Metabolism -- A First Step Toward Simulated Cells. Biomedical Computational Review 14, 17-24. (4) Su, X., Ditlev, J., Hui, E., Xing, W., et al. (2016). Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595-599.

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Insulin By Mia Fatuzzo

A century ago, diabetes was essentially a death sentence. The disease rendered patients unable to produce insulin, a hormone necessary to regulate blood sugar. Patients were prescribed a “starvation diet” of just fat and protein and doctors offered only opium to ease the pain of the condition. Then, in 1922, a fourteen-year-old boy suffering from diabetes and on the brink of death, was given an experimental injection of insulin purified from the pancreas of a cow. Doctors knew that injections of the hormone, purified from the pancreas of pigs and rabbits, lowered blood sugar in animals, but they had never successfully administered insulin to a human. Their patient’s blood sugar, dangerously high before treatment, dropped to a normal level almost immediately (1). Within a year, the pharmaceutical firm Eli Lilly and Company was massproducing the hormone. The scientists who extracted and purified the insulin, Frederick Banting and John MacLeod, were awarded a Nobel Prize for their work. Banting and MacLeod were not the first scientists to attempt to purify insulin. Other researchers also succeeded in isolating insulin effective in the animal from which the compound was derived. The animal insulin protein itself differs from its human analogue by only one or two residues. However, these animal-derived compounds contained impurities that produced immune responses in humans that ultimately rendered the compounds intolerable. Early commercial insulins, derived from either pigs or cows, continued to produce (non-lethal) allergic reactions in patients. The insulin, derived from either pigs or cows, often induced swelling and pain at the site of injection. Some patients even developed antibody-mediated insulin resistance. MacLeod were the first to produce a compound pure enough to safely administer to humans (2). Their first patient, the fourteen-year-old boy, experienced swelling at his sites of injection, but ultimately tolerated the extract. The quality of the insulin did improve over time; scientists developed additional purification steps and, by the 1970s, had succeeded in producing essentially nontoxic animal insulins (3). Still, the question remained – would human insulin work even better than the best animal insulins? Early attempts to manufacture human insulin had some drawbacks. The protein could be extracted from cadavers but only in small quantities. Animal insulin, derived from pancreases shipped from commercial pork and beef operations, 12 PENNSCIENCE JOURNAL | FALL 2016

could be produced in much larger quantities. Then, in the 1970s, as our understanding of genetics and molecular biology solidified, scientists began experimenting with the production of synthetic human insulin. Several techniques allowed scientists to begin this project. First, scientists discovered the existence of restriction enzymes – molecular “scissors” that could cut DNA at specific sites (4). This discovery allowed scientists to pioneer the technique of “recombinant DNA.” First, scientists used restriction enzymes to “cut” a small piece of DNA – a gene. Then, scientists inserted this gene into a bacterial chromosome. The bacteria, usually E. coli, were allowed to absorb the modified chromosome. Then, using their own mechanisms, the bacteria transcribed and translated the new gene. This process allowed scientists to isolate interesting genes and mass-produce their respective proteins cheaply and efficiently.

“This process allowed scientists to isolate interesting genes and massproduce their respective proteins cheaply and efficiently.” This technique had immediate and obvious applications for insulin production. If scientists could find the gene responsible for the production for insulin, they could use recombinant DNA technology to employ billions of E. coli to cheaply and easily produce synthetic human insulin. Scientists were able to link the production of insulin to a small chunk of DNA on chromosome eleven (5). At this point, methods diverged. Some researchers tried to synthesize the insulin gene artificially. Others attempted to isolate the gene by testing random pieces of DNA for insulin functionality. In 1978, scientists working with Eli Lily and the pharmaceutical startup Genentech succeeded in expressing genetically-engineered human insulin from E. coli using the latter method (6). Genentech then worked to develop a safe and efficient method of commercially produce the compound and their drug entered the market in 1982. Over the past three decades, animal insulin has been


FEATURES completely supplanted by synthetic human insulin and is no longer available for patients within the United States. This begs the question: is synthetic human insulin superior to its

“One hundred years ago, scientists were injecting their patients with semi-toxic and impure animal insulins. Now, millions of diabetics across the United States manage their condition with a variety of safe synthetic animal insulins. animal-derived analogues? A series of recent studies examined the differences between human insulin and animal insulin. Cochrane, an independent research organization, recently produced a review on the subject that found no “clinically relevant” differences between the two types of insulin (7). Essentially animal insulin, if purified to the best of our abilities, is not significantly inferior to human insulin. That being said, our access to animal insulin is limited. Production within the United States has long since been discontinued and the FDA only occasionally allows Americans to import personal supplies of pork or beef derived insulin (8). Today, Americans can pick from a multitude of synthetic human insulins engineered to act for specific durations of time and at specific rates. However, only three pharmaceutical firms, Eli Lily, Aventis (acquired by Safoni in 2004) and Novo Nordisk, hold the patents to manufacture these drugs. Patient advocates have accused the firms of unfairly increasing the price of insulin, considered

an “essential medicine” by the World Health Organization, to prohibitively high levels. The pharmaceuticals, on the other hand, frame these price hikes as the necessary cost of continual, if incremental, improvements to their drugs (9). Hopefully, as the patents on these synthetic human insulins begin to expire, other companies will intervene and produce cheaper “generic” versions of this essential drug. In summary, the treatment of diabetes has progressed insurmountably in the last century. One hundred years ago, scientists were injecting their patients with semi-toxic and impure animal insulins. Now, millions of diabetics across the United States manage their condition with a variety of safe synthetic human insulins. While there may be some flaws with the current state of insulin production, it has benefited countless lives and will do so for the foreseeable future.

Works Cited: Banting, F., et al. (1922). Pancreatic Extracts in the Treatment of Diabetes Mellitus. Canadian Medical Association Journal 12, 141-146. Rosenfeld, L. (2008). Insulin: Discovery and Controversy. Clinical Chemistry 48, 22702288. Holt, R. (2010). Textbook of Diabetes. Wiley-Blackwell. Arber, W., and Linn, S. (1969). DNA modification and restriction. Annual review of Biochemistry 38, 467-500. Bell, G., et al. (1980). Sequence of the human insulin gene. Nature 284, 26-32. Johnson, I. (1983). Human insulin from recombinant DNA technology. Science 219, 632-637. Richter, B., Neises, G. (2005). ‘Human’ insulin versus animal insulin in people with diabetes mellitus. Cochrane Database of Systematic Reviews 2005. Federal Drug Administration (2005). Questions and Answers on Importing Beef and Pork Insulin for Human Use. Lipska, K. (2016). Break Up the Insulin Racket. New York Times.

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On the Half Shell: Environmental Factors Effect on Vibrio parahaemolytics in Massachusetts Oysters Natalie Weiss1, Hauke Kite-Powell, PhD2 1University of Pennsylvania, Philadelphia, PA 19104 2Marine Policy Center at Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Oyster production makes up about 95% of Massachusetts’ shellfish industry, which was valued at $25.4 million in 2013. Bacterial outbreaks in oysters of Vibrio parahaemolyticus, a bacterium found in estuarine environments, pose a public health threat to consumers and an economic threat to the oyster industry. Past studies have shown an association between increasing water temperatures and rising V. parahaemolyticus concentrations within oysters. Our results show that V. parahaemolyticus had a strong positive correlation with water temperature and that the relationship with turbidity was more complex and dependent on site-specific turbidity. The harvesting technique of dredging and then placing oysters in trays within the water column of the subtidal growing area depurated some of the total and pathogenic bacterial load. Lastly, we found that the concentration of environmental V. parahaemolyticus does not reflect the level of bacteria within oysters. Further research should be completed on the relationship between environmental V. parahaemolyticus and concentrations within oysters.

Introduction Massachusetts is famous for its shellfish industry, which produces oysters, hard-shell clams, soft-shell clams, and scallops. The industry was valued at $25.4 million in 2013 and employed around 800 people directly (Massachusetts Shellfish Aquaculture Economic Impact Study, 2015). Oysters accounted for around 95% of the shellfish production in 2013. Due to the scale of the oyster industry, Massachusetts is considered one of the major players on the Atlantic coast; however, Vibrio parahaemolyticus bacterial outbreaks in oysters negatively impact the oyster industry and consumers. V. parahaemolyticus is a gram-negative, halophilic bacterium that is found naturally in estuarine environments (DePaola et. al, 1990; Cox and Gomez-Chiarri, 2012; Zimmerman et. al, 2007; Julie et. al, 2010; Johnson, 2010). Increasing water temperatures are closely associated with the increase of V. parahaemolyticus levels in oysters. The V. parahaemolyticus bacteria move from an undetectable, non-culturable state in the sediment during the winter months, to a colony-forming bacteria in the water column, existing as free-swimming cells or attached to particles in the summer. This is partially temperature-dependent, as the non-culturable state exists at 13-15oC and the colony-forming state thrives in water temperatures above 18-19oC (Kaneko and Colwell, 1973; FDA, 2005; Anses; Parveen et. al, 2008). The oysters filter feed particles and phytoplankton colonized by the bacteria from the water column, which facilitates V. parahaemolyticus to enter and concentrate within the oysters (Oliver, 2014; Johnson et. al, 2012). The bacteria are not harmful to the oysters, but can cause illness in humans who consume raw oysters concentrated with the bacteria. The illness severity can range from mild diarrhea to gastroenteritis (DePaola et. al, 1990; Cox and Gomez-Chiarri, 2012; Zimmerman et. al, 2007; Julie et. al, 2010; Johnson, 2010). In the context of rising water tem14 PENNSCIENCE JOURNAL | FALL 2016

peratures due to climate change, predicting and mitigating risks of V. parahaemolyticus outbreaks has become an important public health issue (DePaola et. al, 1990; Cox and Gomez-Chiarri, 2012; Zimmerman et. al, 2007; Julie et. al, 2010; Johnson, 2010). Currently, the Massachusetts Vibrio Control Plan designates mid-May to mid-October as high-risk months for Vibrio outbreaks (Massachusetts Vibrio parahaemolyticus (Vp) Control Plan, 2015). Because water temperatures are above 18oC for most of the summer, it is difficult to know when peaks could occur on a smaller scale during this season. More accurate predictions of health risk would better protect consumer health and industry performance. Not all V. parahaemolyticus bacteria are pathogenic; and certain strains are considered more pathogenic than others. The presence of tdh and trh genes in V. parahaemolyticus has been linked to pathogenicity (FDA, 2005; Johnson, 2012; DePaolo, 2003). Past studies have shown that pathogenic V. parahaemolyticus responded to environmental factors differently than total V. parahaemolyticus and was variable over time and between seasons (DePaola, 2003; Paraveen et. al, 2008). Risk assessments of V. parahaemolyticus used the presence of tdh gene as an indication of pathogenicity, even though some strains without this gene have been known to be pathogenic and some strains with this gene have been found to be not pathogenic (FDA, 2005; Nishibuchi and Kaper, 1995). Although the tdh gene is not a foolproof method for testing for pathogenicity, this paper will define pathogenic V. parahaemolyticus as positive tdh presence, in concurrence with other risk assessments (FDA, 2005). This study has three main parts. First, we investigate whether various harvesting techniques could reduce V. parahaemolyticus burden. Growing the oysters out in deeper, cooler water would safeguard the oysters from experiencing high-risk temperatures that can aid bacterial growth. Another potential method for reducing bacterial


RESEARCH burden is depuration. Depuration allows the oysters to filter fresh seawater or a saline solution to flush out bacteria that has been concentrated in the tissues. The study tests these hypotheses by comparing V. parahaemolyticus levels throughout the summer of 2015 at two oyster farms in Duxbury, MA and two in Wellfleet, MA, which employ different harvesting techniques. Second, we explore the effects of water temperature and turbidity on total and pathogenic V. parahaemolyticus levels. Turbidity may fine-tune our understanding of when V. parahaemolyticus concentrations peak during periods of optimal temperatures for bacterial growth. High turbidity increases the amount of suspended particulate matter in the water column that can be colonized by V. parahaemolyticus, and eventually filtered by the oysters. High turbidity also increases nutrients in the water that promote bacterial growth (Parveen et. al, 2008; Johnson, 2012; Julie et. al, 2010). Turbidity changes with the daily and monthly tidal cycles; turbidity is highest before low tide and during spring tides (Atkinson et. al, 2015). Due to the predictability of turbidity based on tides and weather, a strong relationship between turbidity and V. parahaemolyticus levels could provide insight on when V. parahaemolyticus concentration increase during the summer season. Third, the study examines the relationship between the concentration of V. parahaemolyticus in the water column and sediment, and bacteria within the oyster. Understanding how the oysters’ V. parahaemolyticus levels compare to environmental bacterial concentrations at the time of harvest could show when the concentration of V. parahaemolyticus is highest in oysters, based on measurable environmental conditions. Overall, the study aims to elucidate how harvesting techniques, environmental conditions, and environmental V. parahaemolyticus concentration affect V. parahaemolyticus levels within oysters in order to mitigate health risk to consumers and economic risk to industry from bacterial outbreaks. Methods For this study, two oyster farms in Duxbury, MA and two farms in Wellfleet, MA were sampled at two-week intervals between April and October of 2015. Ten oysters, water samples, and sediment samples were collected at each site and date. The tissues and mantle fluid of the ten oysters taken at each date were blended together until they were uniform, and then the mixture was tested using qPCR for total V. parahaemolyticus concentrations per gram, presence of the tdh and trh genes, and concentrations of each gene. Trh gene presence will not be discussed in this paper because pathogenicity will be defined by tdh presence only. Most studies utilized the most-probable number (MPN) method and measured V. parahaemolyticus in colony-forming units (CFU). Due to the MPN’s variability in accuracy, this study used the qPCR method of estimating bacterial concentrations and measured in cell concentration per gram. Bacterial concentrations measured using the MPN method cannot be directly converted into qPCR measurements (FDA, 2005; Johnson, 2012; DePaolo,

2003). The water and sediment samples were only tested for total V. parahaemolyticus. Water samples were filtered using 0.22, 0.45, 0.8 micrometer filters, and V. parahaemolyticus concentrations at each filtration were measured. The total V. parahaemolyticus concentration was found by adding these measurements together since oysters are able to filter particles from the water column less than 1 micrometer (Sarasota Water Atlas). Particles larger than 1 micrometer are largely rejected as pseudofeces from the oyster and V. parahaemolyticus attached to this class size of particle would not concentrate within the oyster. The two oyster growing areas, Duxbury and Wellfleet, differ geologically. The Duxbury growing area is subtidal, whereas Wellfleet is intertidal. Subtidally grown oysters stay submerged under water, in contrast to intertidally grown oysters growing areas, which are exposed to air during tidal cycles. Two farms in Duxbury, Goose and Hunts, were sampled along with two farms in Wellfleet, Cummings and Wallace. The water in Wellfleet was generally warmer than Duxbury and was significantly more turbid. At the Duxbury locations, Goose and Hunts, two different harvesting techniques were employed. The Goose farm’s oysters grew on top of the sediment and were harvested using a dredging technique. The Hunts oyster samples were initially harvested using a dredge and then grown in trays within the water column of the subtidal flats. The oysters did not come into contact with sediment unless it was suspended in the water column; during extreme tidal periods, the oysters were exposed to ambient air. Growing out the oysters in trays in the water column allowed the oysters to filter seawater, acting as a depuration process, before they were harvested for consumption. At the Wellfleet locations, Cummings and Wallace, two different harvesting methods were employed. The Cummings farm grew oysters in trays on the bottom of the water column, whereas Wallace grew oysters in trays within the water column. Wallace’s oysters were more frequently exposed to air, and Cummings oysters were exposed to air more intermittently, varying with the intensity of the tidal cycle. Direct sunlight could heat the inside of the oyster, creating better conditions for bacterial growth, or the oysters could experience evaporative cooling, lowering internal temperature. The two harvesting techniques will test these theories. Temperature and turbidity data were measured in 15-minute intervals during the entire study period by sensors attached to buoys in Duxbury Bay and Wellfleet Harbor. Turbidity was not measured for the week of September 13, 2015 in Wellfleet, and corresponding data points for the Wellfleet locations were taken out of the turbidity analyses. Environmental conditions were considered uniform throughout each growing area and the same temperature and turbidity measurements were used for both farms in Duxbury and in Wellfleet.

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RESEARCH Non-parametric t-tests for V. parahaemolyticus concentrations within oysters—both total and pathogenic, within water column, and within sediment—were performed in order to measure any significant differences in mean concentrations between locations, and between farms within a certain location. Correlations and significance of correlations between factors were tested using R version 3.3.0. All concentrations were normalized during statistical analyses. Results

Figure 1. Boxplots of V. parahaemolyticus concentrations. Differences in V. parahaemolyticus concentrations. Between Duxbury locations, Hunts’ oysters that were initially dredged then stored in the water column experienced lower V. parahaemolyticus burden than Goose’s oysters that were only dredged, although this difference was not statistically significant (p=0.25). The Duxbury locations peaked in concentration on the same date, yet Goose experienced a higher absolute concentration (Figure 2). Because there was no significant difference between V. parahaemolyticus levels in Duxbury locations and there were homogeneous environmental conditions, the later analyses will consider pooled Duxbury data, in addition to individual farm data. Between Cummings and Wallace in Wellfleet, Wallace’s oysters that were grown out in the middle of the water column experienced a significantly higher V. parahaemolyticus burden within oysters than Cummings’ oysters that were grown out on the bottom (p<0.01). We will not consider pooled Wellfleet data for the rest of the study due to this significant difference in total V. parahaemolyticus 16 PENNSCIENCE JOURNAL | FALL 2016


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Figure 2. Concentrations of total V. parahaemolyticus in Duxbury over the study period.

Figure 3. Concentrations of total V. parahaemolyticus concentrations in Wellfleet over the study period. concentrations. The Duxbury locations’ bacterial concentrations shared the same pattern of growth over the study, where Wellfleet’s locations did not (Figures 2 and 3). Differences in V. parahaemolyticus pathogenic concentrations. The tdh gene was present in 26 out of 49 samples (53%). 13 out of 25 samples (52%) were pathogenic in Duxbury and 16 out of 23 (69.5%) were pathogenic in Wellfleet. Goose had a higher bacterial burden than Hunts in Duxbury, yet the difference was not significant (p=0.09). Wallace had a significantly higher pathogenic load than Cummings in Wellfleet (p< 0.01). For later analyses, we will pool Duxbury data, but not Wellfleet because of significance results. Despite the differences in absolute concentrations, the changes in concentrations at Duxbury farms and at Well-

fleet farms coincided throughout the study period (Figure 5). The same trends as the total V. parahaemolyticus concentrations were reflected in the pathogenic V. parahaemolyticus concentrations. Wellfleet Cummings showed the lowest total V. parahaemolyticus and pathogenic V. parahaemolyticus concentration. Duxbury Goose and Wellfleet Wallace had higher burdens of total V. parahaemolyticus and pathogenic V. parahaemolyticus concentrations than their counterparts.

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RESEARCH whereas Wallace showed only a slight positive correlation with water temperature measurements. This could be because the trays within the water column at Wallace’s farm were frequently exposed to air during tidal cycles. The other locations were more consistently submerged in water and showed a stronger positive correlation with water temperature. The scatterplots show that although there was a strong significant relationship between V. parahaemolyticus concentrations and water temperature, high concentrations can occur at lower temperatures (14-17C); the temperature range when V. parahaemolyticus starts to move from a non-culturable state in the sediment to freely-existing in the water column (Kaneko and Colwell, 1973). The correlation underestimated concentrations at low temperatures and overestimated concentrations at high temperatures. Relationship with water temperature for pathogenic V. parahaemolyticus. Pathogenic V. parahaemolyticus did not show as consistent, strong, positive correlation with water temperature at the individual farm level as total V. parahaemolyticus. The Duxbury Goose and Duxbury Hunts pathogenic bacteria levels showed a weak, positive correlation with both water temperature measurements and the Wellfleet Cummings farm had no correlation with water temperature. The Duxbury pooled data had a strong, positive correlation with average temperature the week and day before harvest (p<0.01), but the scatterplots show that concentrations of zero bacteria existed at the same temperatures as the highest concentrations. There was a wide range of concentrations at high temperatures, suggesting that even a strong correlation with temperature may not accurately capture pathogenic concentrations.

Figure 4. Scatterplots of pooled Duxbury V. parahaemolyticus concentrations (left) and total pooled pathogenic concentrations (right) vs. weekly temperature. Relationship with water temperature for V. parahaemolyticus. Both the average weekly temperature and the average temperature the day before harvests were used in the correlation analyses in order to see whether one is more closely correlated with levels of V. parahaemolyticus. The change in temperature between the beginning of the week and end of the week was not significantly correlated with V. parahaemolyticus concentrations for any farm. Goose, Hunts and Cummings showed significant positive correlations (p<0.05) for both water temperature measurements, 18 PENNSCIENCE JOURNAL | FALL 2016

Relationship with turbidity and V. parahaemolyticus. Turbidity measurements at both locations reflected the cyclical pattern of turbidity, with higher turbidity before low tide and during spring tides. Additionally, both Duxbury and Wellfleet experienced higher turbidity during the spring season. The intertidal flats of Wellfleet experienced extreme tidal cycles, causing a larger range of turbidity (0.012-125 NTU) than Duxbury (0.03-39.97 NTU). Wellfleet’s turbidity was significantly higher than Duxbury (p <0.05) for both the average turbidity 24 hours before harvesting and the average turbidity the week leading up to harvest. The data was trimmed to include only values that occurred during Vibrio season (mid-May to mid-October) as outlined by Massachusetts Vibrio control plan. Trimming the data for points during the designated Vibrio season, when water temperatures were high enough for the V. parahaemolyticus to exist in the colony forming state, helped expose whether turbidity explains when and why a peak occurred when water temperatures were within optimal range. In Duxbury, both Goose and Hunts showed significant, negative relationships between total V. parahaemolyti-


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Figure 5. Log pathogenic V. parahaemolyticus and average weekly water temperature over time at each location. cus concentrations and average turbidity the day before (p<0.01). Each farm also showed a negative relationship between bacteria levels and turbidity the week before, although the relationship was not significant. For pathogenic V. parahaemolyticus levels, both Duxbury farms showed negative relationships with both turbidity measurements. In Wellfleet, there was no consistent relationship between individual farms total or pathogenic V. parahaemolyticus and turbidity. From looking at the scatterplots (Figure 6), a pattern emerges between concentrations of pathogenic V. parahaemolyticus and the two measurements of turbidity. High levels of pathogenic V. parahaemolyticus occurred when turbidity the day before was low and turbidity the

week before was high. Differences in concentrations of V. parahaemolyticus in the water and sediment. There was no significant difference in amount of V. parahaemolyticus in the water or in sediment between Duxbury and Wellfleet or between Duxbury farms. Wellfleet Wallace showed significantly higher amounts of V. parahaemolyticus found in the water (p<0.05), but not in the sediment. This showed that V. parahaemolyticus in the water was not homogeneous throughout Wellfleet’s growing area and could contribute to differences in V. parahaemolyticus levels in the oysters.

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Figure 6. Scatterplots of pathogenic V. parahaemolyticus (Log conc/g) vs. turbidity (NTU) at Wellfleet locations. Relationship between V. parahaemolyticus in oyster and V. parahaemolyticus in the water and sediment. Pooled Duxbury data showed a positive relationship between the V. parahaemolyticus concentration in the water and V. parahaemolyticus concentration within oyster. Both Wellfleet farms showed no relationship with V. parahaemolyticus concentrations within the oyster and within the water. The stronger, positive correlation of Duxbury locations (r=0.38) with concentration of V. parahaemolyticus in water could be because Duxbury locations were subtidally harvested and constantly submerged in the water. The concentration of V. parahaemolyticus in the sediment and the V. parahaemolyticus within an oyster was not statistically significant for any farm; however it is notable that both Duxbury farm’s oysters showed a positive relationship with V. parahaemolyticus concentration in the sediment and could be due to the fact that both oysters at Goose and Hunts were initially dredged from the mud. Although there was a positive relationship for both total concentration of V. parahaemolyticus in the oyster and to20 PENNSCIENCE JOURNAL | FALL 2016

tal concentration in the environment, none of these relationships are statistically significant and more evidence is necessary before conclusions can be made. Discussion Our analyses aligned with past research that rising water temperature contributes to higher concentrations of total V. parahaemolyticus and pathogenic V. parahaemolyticus (DePaola et. al, 1990; Cox and Gomex-Chiarri, 2012; Zimmerman et. al, 2007; Julie et. al, 2010; Johnson, 2010). Because total V. parahaemolyticus and pathogenic V. parahaemolyticus were closely correlated with water temperature, one would assume that they would both reach their maximum concentrations around the same time, when the water temperature is the warmest. However, this is not the case. Duxbury locations peaked in total bacteria in middle of July and pathogenic bacteria in late August, whereas Wellfleet locations peaked in total bacteria late June and early July and pathogenic bacteria in early June (Figures 2, 3, 4). Due to peaks in pathogenic V. parahaemolyticus and


RESEARCH total V. parahaemolyticus concentrations occurring at different times, the ratio of pathogenic to total V. parahaemolyticus changed throughout the study period and should not assumed to be constant when assessing pathogenic risk of consuming raw oysters. Although both total and pathogenic V. parahaemolyticus concentrations showed strong, positive relationships with water temperature, pathogenic bacteria had a stronger relationship with turbidity than normal V. parahaemolyticus. Pathogenic bacteria had a strong, negative relationship with average turbidity the day and week before in Duxbury locations. Other studies have cited a strong, positive relationship with turbidity due to the increased filtering rate of suspended material and high nutrient levels of turbid waters (Johnson, 2012; Parveen et. al, 2008); however, oysters’ filter feeding can also justify a negative relationship with turbidity. In high turbidity, species of Crassostrea may close their valves to filter feeding, reducing the intake of particulate matter, or they may eject large amounts of pseudofeces (Sarasota Water Atlas). The rate of water pumping in an oyster decreases as turbidity increases and water pumping can be sensitive to changes in turbidity. Water pumping can even decline to zero in highly turbid waters (Sarasota Water Atlas). The negative correlations between V. parahaemolyticus concentrations and average turbidity the week and day before harvest for the Duxbury locations suggest that pathogenic V. parahaemolyticus peaked when the temperatures were optimal and when turbidity were low. Turbidity was higher during spring tides, low tides, and in the spring in Duxbury, so pathogenic V. parahaemolyticus concentrations were more likely not to peak in the spring season or during spring tides. Pathogenic V. parahaemolyticus also had a clearer relationship with turbidity than total V. parahaemolyticus in Wellfleet. Total V. parahaemolyticus concentrations had a negative relationship with average turbidity the week before and a negative relationship with average turbidity the day before. In contrast, pathogenic V. parahaemolyticus levels showed a positive relationship with weekly turbidity and negative relationship with turbidity the day before harvest. This suggests that pathogenic V. parahaemolyticus peaked after a period of high turbidity, but after the turbidity has subsided. This type of scenario could occur at the end of a spring tide or at the end of the spring season when turbidity decreased. Local adaptations for filter feeding in turbid conditions could contribute to the different relationships between bacterial concentrations and turbidity at each location. Duxbury had a narrower range of turbidity than Wellfleet, so the oysters in Duxbury may have been more sensitive to changes in turbidity compared to Wellfleet oysters. The specific relationships with turbidity at each location provide insights into when pathogenic bacteria may peak once within optimal water temperatures. More research into the relationship between turbidity and pathogenic V.

parahaemolyticus should be done at specific growing sites due to seasonal changes in turbidity, tidal cycle effects, and local adaptations of the oysters to turbidity. Coinciding peaks and troughs of total and pathogenic V. parahaemolyticus concentrations over the study period between farms in the same growing area suggest that the mechanism of concentrating total and pathogenic V. parahaemolyticus within oysters worked in the same way. In Duxbury, both farms experienced the same environmental conditions and environmental V. parahaemolyticus exposure and also showed similar peaks and valleys. Yet on average, Hunts experienced 80% of Goose’s total V. parahaemolyticus concentration and 27% of Goose’s pathogenic V. parahaemolyticus concentrations. The reduction in V. parahaemolyticus burden at Duxbury Hunts was likely attributed to the different harvesting methods. After initial dredging, Hunts oysters were placed in trays within the water column, which seems to have successfully depurated some of the bacterial load, specifically pathogenic bacterial load. Most depuration studies only analyze clinical strains of Vibrio and clinically inoculated oysters. These studies showed successful reduction in Vibrio reductions through depuration methods using refrigerated seawater and UV light (Yi Cheng et. al, 2010; Ramos et. al, 2012). Our results suggest that depuration of environmental V. parahaemolyticus strains in oysters may also be possible. Allowing the oysters to flush out some of the bacteria in trays within the water column after dredging reduced some of the bacterial load and further research on depuration of environmentally inoculated oysters should be performed to validate these results. In Wellfleet, Cummings experienced 26% of the total bacterial load and 0.1% of the pathogenic burden of Wallace. The peaks in total V. parahaemolyticus at Wallace did not coincide with Cummings (Figure 3). Although each farm experienced the same turbidity and temperature, the load of total V. parahaemolyticus in the water and sediment was significantly different and the Wallace oysters were more frequently exposed to air, which can contribute to the variability and difference in total V. parahaemolyticus concentrations. The pathogenic V. parahaemolyticus peaks at Cummings coincided with the peaks at Wallace (Figure 4), suggesting that mechanisms of concentrating pathogenic V. parahaemolyticus were the same, and Cummings’ harvesting method may be more effective in reducing the load of pathogenic V. parahaemolyticus. There is not enough convincing evidence to suggest a linear relationship between V. parahaemolyticus concentrations in the water or sediment and V. parahaemolyticus concentrations within oysters. The concentration of V. parahaemolyticus in the environment at the time of harvest was not indicative of the concentration within the oyster at harvest. The concentration and density of V. parahaemolyticus within an oyster did not directly reflect the composition of the water column or sediment (Froelich and Noble, 2014). V. parahaemolyticus concentrations within the sediment and water were not reliable proxies for the

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RESEARCH concentration of V. parahaemolyticus within the oyster. Conclusion Our results show that V. parahaemolyticus has a strong positive correlation with water temperature and that the relationship with turbidity is more complex and dependent on site-specific turbidity. Dredge harvesting and then placing oysters in trays within the water column of the subtidal growing area depurated some of the total and pathogenic bacterial load. In the intertidal farms, growing out the oysters in trays along the bottom of the flat had reduced bacterial burdens; however, due to significantly different concentrations of V. parahaemolyticus in the water and in the sediment between farms, the reduced levels of bacteria within the oysters cannot be fully credited to harvesting method. Acknowledgments I would like to thank Hauke Kite-Powell, for mentorship on the research project, the Marine Policy Center at Woods Hole Oceanographic Institution and OAR/NOAA Sea grant aquaculture research program, for funding the research, and Woods Hole Oceanographic Institution. I would also like to thank Roxanne Smolowitz and her lab at Roger Williams University and John Brawley. Lastly, I would like to acknowledge Andy Beet and PennScience editors. References Anses (2012) Vibrio parahaemolyticus: data sheet on foodborne biological hazards. Atkinson, I. et. al. (2015). Turbidity. OzCoasts, Australian Online Coastal Information. Retrieved from http://www.ozcoasts.gov.au/indicators/turbidity.jsp Chae, MJ., Cheney, D., Su, YC (2009). Temperature effects on the depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American oyster (Crassostrea virginica). Journal of Food Science, 74(2): Cox, A. and Gomez-Chiarri, M. (2012) .Vibrio parahaemolyticus in Rhode Island Coastal Ponds and the Estuarine Environment of Narragansett Bay. Applied and Environmental Microbiology, 78(8):2996-2999. DePaola, A., et. al. (1990). Incidence of Vibrio parahaemolyticus in U.S. Coastal Waters and Oysters. Applied and Environmental Microbiology, 56(8): 2299-2302 DePaola, A. et. al. (2003). Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters. Applied and Environmental Microbiology, 69(3): 1521-1526. Froelich, B. and Noble, R. (2014). Factors Affecting the Uptake and Retention of Vibrio vulnificus in Oysters. Applied and Environmental Microbiology, 80(24):74547459. 22 PENNSCIENCE JOURNAL | FALL 2016

Johnson, C. et. al. (2012). Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States). American Society for Microbiology. 78(20): 7249-7257. Johnson, C. et.al. (2010). Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico. Applied and Environmental Microbiology, 76(21): 7076-7084. Julie, D. et. al. (2010). Ecology of pathogenic and nonpathogenic Vibrio parahaemolyticus on the French Atlantic coast. Effects of temperature, salinity, turbidity and chlorophyll a. Environmental Microbiology, 12(4):929937. Kaneko, T and Colwell, RR. (1973). Ecology of Vibrio parahaemolyticus in Chesapeake Bay. Journal of Bacteriology. 113(1): 24-32. Massachusetts Shellfish Aquaculture Economic Impact Study. UMASS report prepared for Cape Cod Cooperative Extension, Woods Hole Sea Grant & SEMAC, Winter 2015. Massachusetts Department of Fish & Game (2015). Massachusetts 2015 Vibrio parahaemolyticus (Vp) Control Plan, 322 CMR 16.00. Mass.gov. Parveen, S., et. al. (2008). Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters. International Journal of Food Microbiology, 128: 354-361. Ramos, RJ, et. al. 2012. Refrigerated Seawater Depuration for Reducing Vibrio parahaemolyticus Contamination in Pacific Oyster (Crassostrea gigas). Journal of Food Protection, 8:1366-1541 Sarasota Water Atlas. Biology of the oyster. Retrieved from http://www.sarasota.wateratlas.usf.edu/upload/documents/Biology%20of%20the%20Oyster.pdf. Su, YC., Yang, Q, Häse, C. (2010). Refrigerated Seawater Depuration for Reducing Vibrio parahaemolyticus Contamination in Pacific Oyster (Crassostrea gigas). Journal of Food Protection, 6:1111-1115. U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition. (2005). Quantitative Risk Assessment on the Public Health Impact of Pathogenic Vibrio parahaemolyticus In Raw Oysters. Zimmerman, A. M., et. al. (2007). Variability of Total and Pathogenic Vibrio parahaemolyticus Densisities in Northern Gulf of Mexico Water and Oysters. Applied and Environmental Microbiology. 73(23): 7589-7596.


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