Biology Research Projects 2021

Lana Giha

Advisor: Tamara Davis

Exploration of Methylation Maintenance at Imprinted Genes in Methyltransferase Mutant Mice

Our lab explores imprinted genes which are exclusively expressed from one inherited allele despite inheriting two alleles. The expression of one allele depends on its parental origin. In some cases, the maternally inherited copy may be expressed and in other cases the paternally inherited copy may be expressed. DNA methylation, the addition of a methyl (CH3) group by DNA methyltransferase, impacts the ability of the DNA to be transcribed. As a result, differential methylation of the DNA determines the expression of imprinted genes and creates differentially methylated regions commonly referred to as DMRs. There are primary DMRs which are inherited from gametes at fertilization and secondary DMRs at which differences in methylation are acquired during embryo development.

Previous work done by our lab has suggested that methylation is better maintained and more consistent at primary DMRs in comparison to secondary DMRs. DNA methyltransferase is responsible for maintaining methylation and promoting its stability, yet it appears to function differently at primary versus secondary DMRs. We would like to continue looking into methylation maintenance at imprinted and non-imprinted genes to understand why that is the case.

This project focuses on the methylation patterns of primary DMRs versus secondary DMRs in Dnmt 1 mutant mice. This will be explored using the methods of bisulfite mutagenesis, PCR, gel electrophoresis, DNA extraction, and DNA sequencing. Bisulfite mutagenesis converts unmethylated cytosines to uracil, allowing them to be distinguished from methylated cytosines which would remain unchanged. The mutagenized DNA can be used in PCR, Polymerase Chain Reaction, to amplify specific primary or secondary DMRs. The product of the PCR is analyzed by gel electrophoresis to confirm we amplified the sequence of interest. A gel extraction is performed to purify the final product. The purified final products will be pooled and submitted for Next Generation amplicon sequencing. I will then analyze the resulting sequences to determine how the Dnmt 1 mutation impacts the methylation at primary versus secondary DMRs, as well as at non-imprinted genes.  

Keywords: Imprinted genes, DNA methylation, DNA methyltransferase, primary DMR, secondary DMR, bisulfite mutagenesis

Madison Kearns

Advisor: Crystal Reynaga

Comparative Analysis Of Quadrupedal Anuran Limb Morphology and Forward Walking Locomotion

In the Reynaga Lab we are studying the changing morphology and locomotion patterns in two species of quadrupedal anurans, Phyllomedusa hypochondrialis and Phrynomantis bifasciatus. This research analyzes the ways in which varying limb dimensions affect the way a frog accelerates and decelerates to move their center of mass. Anurans have many features that enable them to jump efficiently, such as strong back legs and smaller bodies. In addition, previous work suggests walking frogs have evolutionarily altered their body composition, creating a more even ratio between fore- and hind limb length. However, in order to compensate for slight limb asymmetry some frogs maintain a more extended forelimb and a crouched hindlimb posture. In comparison to the crouched limb posture found in some walking frogs, certain species adopt a more sprawled posture when moving. However, it is unclear how sprawled or crouched postures may affect walking, as opposed to a more upright and erect posture.We use 3D high speed videography coupled with 3D ground reaction force plates to measure the forelimb and hindlimb reaction forces. The raw data is then contrasted against other species to understand the function of each limb during the stride. Using measurements of center of mass displacement, force during forward locomotion, and change in joint angles within the fore- and hindlimb we can compare the variation across species that have evolved the ability to walk quadrupedally. Overall, the research conducted this summer will continue to expand on the understanding of how quadrupedal gaits can be achieved despite the fact that the anuran morphology is specialized for jumping.

Isabelle Kuszyk

Advisor: Crystal Reynaga

Investigating How Limb Length Disparities Affect Red-Banded Rubber (Phrynomantis Bifasciatus) Frogs Walking Gait

Frogs are mostly known for their magnificent jumping ability; however, some species walk quadrupedally, such as the red-banded rubber frog. A previous study analyzed the walking and running gaits of a specialized quadrupedal walking frog species and noticed depending upon which metric they used, it was hard to classify whether their gait was indeed a walk or not. The results suggested that the quadrupedal frogs could use a walking or running gait depending on what factors were studied. For instance, when the researchers calculated the kinetic and potential energies of the frogs, it showed the frogs used a running gait but when examining the footfall patterns, the frogs used a walking gait. However, previous studies have not analyzed the difference in forces applied by the fore- and hindlimbs, so it is not clear how those limbs affect propulsion. We will study how the forces applied are characterized relative to limb morphology. Other organisms, such as geckos, have developed quadrupedal gaits despite slight uneven limb lengths. Researchers have shown how the forces and limb function can differ between the fore- and hindlimb in the lateral and fore-aft direction of geckos limbs. Generally, frogs have a greater disparity between limb lengths than geckos, which is why we will test how substantially longer hindlimbs affect frogs鈥 gait. Our research will determine if quadrupedal frogs have adapted into efficient walkers based on their anatomical constraints. I hypothesize that the force exerted from the front and back limbs will be similar. To test this hypothesis, we will study frogs walking across 3D ground reaction force plates while recording them with high-speed video analysis. We will digitize videos to help us determine if the front or hind limbs exert different forces or similar amounts. Our research can help us understand how different species of frogs have developed the ability to walk independently from their ancestors.

Yeipyeng Kwa

Advisor: Gregory Davis

The Role of JH Signaling Aphid Reproduction Polymorphism

          Aphids are known to exhibit reproductive polyphenism, a type of plasticity which allows them to switch between cyclical parthenogenesis (asexual) and sexual reproduction. Female asexual viviparous aphids, called virginoparae, when exposed to short nights (and long days) and higher temperature (summer) conditions, produce female embryos that are likewise asexual and viviparous. However, when these females are exposed to long nights (and short days) and cooler temperatures (fall conditions), a special asexual and viviparous female known as the sexupara is produced, and this female produces the single generation of sexual females and males in the aphid lifecycle; these sexual females lay eggs that are able to withstand winter tempertures, an advantage for aphid species in regions known to have severe winters. Although photoperiod is known to be the environmental cue that specifies reproductive fate, received by mothers and transmitted to embryos, the nature of this maternal signal has yet to be identified.

          What we do know is that this maternal signal is an asexual promoting signal, and although the identity of this signal is not yet known, Juvenile Hormone (JH) is considered one of the leading candidates based on correlative data as well as results from tests of sufficiency: Asexual aphids express higher levels of JH than sexual aphids; JH and JH analogs, when topically applied to sexuparae, are sufficient to induce her embryos to take on an asexual fate. However, further investigations have shown that maternal JH is not necessary to specify asexual fate. My hypothesis, therefore, is that embryonic JH functions to specify fate in aphid embryos during embryogenesis and is induced by a different signal which is transmitted from the mother to the embryos.

          To better understand the role that JH plays in specifying reproductive fate, I will first be determining a reliable method for measuring JH expression in pea aphids (Acyrthosiphon pisum), which will then enable me to measure JH signaling in embryos of asexual aphids versus embryos of sexual aphids. A possible outcome is that embryos of asexual aphids express higher levels of JH than embryos of sexual aphids, which will establish JH as a signaling molecule that specifies fate in embryos during embryogenesis.

Rebecca Li

Advisor: Gregory Davis

Establishing a Neurophysiological Basis for the Reproductive Polyphenism in Pea Aphids

          The pea aphid, Acyrthosiphon pisum, exhibits a reproductive polyphenism wherein the species is cyclically parthenogenetic due to seasonal changes in photoperiod. Under summer's short-night conditions, A. pisum displays viviparous parthenogenesis (i.e. asexual females give live birth to another generation of asexual females). In the fall, in response to long nights, viviparous asexual females give birth to sexuparae, which are a form of viviparous asexual female that gives birth to sexual females and sexual males. These sexual females later reproduce by mating with males and laying fertilized eggs that can overwinter.

          We seek to investigate the nature of the maternal signal that specifies sexual versus asexual fate during the embryonic development of A. pisum. It is hypothesized that this signal is asexual-promoting and originates in Group I neurosecretory cells (Group I NSCC), which are found in the dorsal anterior region of the brain.

          Previous investigations have demonstrated that it is the photoperiod-induced state of the asexual mother, experienced pre- and post-natally, that determines the reproductive fate of her own offspring. For example, an aphid becomes a sexupara if it is exposed to long night conditions just prior to birth. Evidence indicates that the sexupara鈥檚 developing progeny then become sexual because they are not instructed to become asexual (i.e., sexual fate is the default). Neural microcautery experiments in the aphid Megoura viciae suggest that Group I cells are necessary to specify asexual offspring (i.e. in the absence of this signal, sexual offspring are produced), and thus that the signal is asexual-promoting.

          Given that these experiments were performed over four decades ago 鈥 and in a different aphid species 鈥 we aim to establish the existence of an asexual-promoting maternal signal in A. pisum through functional disruption of the signal. A variety of techniques will be explored, including electrocautery and surgical ablation of the Group I NSC, and blocking light to the head capsule.

Penelope Northing

Advisor: Thomas Mozdzer

Mapping Functional Traits to Genotypes of P. australis in a Common Garden

Coastal wetlands are important to study in the context of global change because they sequester more carbon than any other ecosystem while simultaneously being influenced by sea level rise, species invasion, and other effects. Recent advancements in the field of coastal wetland ecology indicate that the invasive Eurasian perennial grass, Phragmites australis, may experience selective pressure from different global change factors, causing rapid evolution. This finding indicates that rapid evolution on a population-level could be an important process to consider when employing restoration and management strategies to remove P. australis in the coastal wetlands of eastern North America. In order to determine whether or not populations of P. australis are evolving due to global change, it is necessary to determine the relative plasticity and heritability of functional traits. This summer, my project seeks to answer the question of how different genotypes of P. australis correspond to functional traits by mapping the relationship between traits and genotype, the data from which will inform the long-term heritability study that Dr. Mozdzer is conducting over the next few years. This project utilizes an experimental common garden set-up with 300 unique genotypes of P. australis harvested from the Smithsonian Environmental Research Center, Parker鈥檚 Creek, and the Global Change Research Wetland in Edgewater, Maryland. I will be measuring the height, basal diameter, photosynthetic rate, nitrogen and carbon content, specific leaf area, and other functional traits of the P. australis plants in the garden. Ultimately, the results of this project will help contribute to a growing body of knowledge about P. australis' impact as an invasive species in the marshes of eastern United States, which could aid scientists and policymakers in improving practices and policies for restoring and managing our precious wetlands. Proper management of P. australis has the potential to not only save coastal wetland ecosystems, but help fight climate change by making the most of their carbon capturing abilities and other vital ecosystem services.

Camille Pastrana

Advisor: Sydne Record

Host-Parasite Dynamics of the Mistletoe Viscum alba

Parasitism is a form of species-species interaction in which one species benefits from the other species at the expense of the other species. Certain plants are considered parasitic based on their ability to feed directly on the nutrients and water from either the roots or the shoots, the xylem, of a host plant. By observing the population dynamics of the parasitic plant, one can gain general knowledge about the host plant and its relationship with the parasite. In this study, we specifically look at a species of the parasitic plant that is mistletoe, Viscum alba, in a research forest located in Silwood Park, England. We ask how the positioning of V. alba on the host plant influences the mistletoe survival, growth, and reproduction. We hypothesize that the lower the mistletoe is on the host plant, the better access it would have to the water and nutrients in the host plant based on hydraulic constraints. From 2014 to 2021, digital images were taken of various trees containing V. alba on their branches and each image was digitized, taking measurements on the number of individuals on each tree, the size of each individual, whether or not individuals were reproductive, and how high up on the tree each individual was. From this information, integral projection models will be made to look at population projections of mistletoe. Through these models, we can learn about the varying population dynamics of V. alba, and gain general knowledge of host-parasite dynamics.

Clementine Payne

Advisor: Tamara Davis

Comparison of Methylation at Imprinted and Non-Imprinted Gene

          During development, both maternal and paternal DNA contributes to an embryo's genome. Much of the development of an organism is regulated by both the maternal and paternal copies of these genes. However, when a gene is imprinted, only one of these two parental copies is expressed. Imprinted expression is regulated through epigenetic modification or DNA methylation, the addition of a methyl group (-CH3) to cytosine in a CpG dinucleotide. This change in DNA chemistry affects the expression of the gene. The methylated copy is typically silent while the unmethylated copy is expressed. Parent of origin-specific differential methylation patterns acquired during gametogenesis, primary differentially methylated regions (DMRs), are maintained throughout development by DNA methyltransferase 1 and have proven to be stable across development. In contrast, methylation patterns are less stable and asymmetric at secondary DMRs, sites at which methylation is acquired during embryonic development. These results suggest that epigenetic modifications acquired during post-implantation development are maintained differently suggesting DNA methyltransferase 1 works less consistently at these sites.

          To assess whether the variability in DNA methylation patterns at secondary DMRs are a unique feature of these loci, it is important to assess the methylation at both imprinted and non-imprinted loci.  The aim for this study is to analyze DNA methylation at non-imprinted sequences to be compared to the profiles which have been observed at imprinted loci. Our hypothesis is that these non-imprinted sequences will have similar methylation profiles to the primary, gametic, DMRs, consistent with what is known about DNA methyltransferase activity, while looking different from the secondary, post implantation, DMRs.

          We are characterizing the methylation dynamics at non-imprinted genes. Of the three genes analyzed thus far, two, ZFP553 and Hnf4a, have methylation patterns consistent with our hypothesis; both have very stable methylation patterns similar to primary DMRs associated with imprinted genes. In contrast, Glut3 exhibited methylation patterns which were less consistent than primary DMRs while being more consistent than secondary DMRs. These data suggest the need for broader analysis therefore we are analyzing additional loci to further inform our study.

Lili Randolph

Advisor: Thomas Mozdzer

Quantifying Genotypic Recovery of a Foundational Wetland Plant Species

Wetlands are one of the most effective biomes for carbon sequestration and provide valuable ecosystem services that make their welfare valuable to the environment and its inhabitants. Biodiversity of different species within these wetland ecosystems and within the species itself (intraspecific diversity) promotes a healthy biome and fluctuates greatly depending on external factors like climate change, introduction of invasive species, and nutrient enrichment. In 2004, researchers working on the TIDE project began adding nitrogen to each incoming tide of two field sites to simulate coastal eutrophication. In 2009 an additional field site was added. Samples were collected from two additional unfertilized sites to act as controls. During this process of eutrophication, researchers expected to see phenotypic responses in the Spartina such as a shallower root structure and increased height above ground. In addition to these phenotypic responses, researchers also noticed that the genotypic diversity of these plants decreased. Researchers have since stopped adding additional nitrogen to these sites with the expectation of stopping the eutrophication process, allowing Spartina鈥檚 genetic diversity to increase in the affected areas. I will be quantifying how much the genetic diversity of Spartina alterniflora has shifted in our field sites during the recovery from excessive nitrogen enrichment using microsatellites, analyzed in Geneious, to identify Spartina genotypes, and using GenAlEx calculate genetic relatedness of all Spartina samples. We will then use ANOVAs to compare genotypic diversity and genetic relatedness across fertilized and unfertilized creeks. We will also compare genetic diversity and relatedness before fertilization, at the end of fertilization, and after three years of recovery without nitrogen addition. By doing this, we can quantify the rate and level at which Spartina recovers from nitrogen excess and how to proceed with the management of nutrient enrichment in wetland areas.

Frances Romero

Advisor: Thomas Mozdzer

Re-evaluating Phragmites and Phragmites management in terms of coastal resilience

The question of my SSR research project is how management of the invasive marsh grass Phragmites australis affects coastal marsh resilience. Marshes are some of the most effective ecosystems at storing carbon. Climate change is increasing sea level rise at a rapid pace, and marshes need to keep pace with this rise in order to continue functioning as a carbon sink.  Phragmites australis is a grass species that is invasive to North America and has been linked to biodiversity loss in marshes as well as changes in biogeochemical cycling. The United States spends over $4.6 million on Phragmites management yearly, primarily via the spraying of glyphosate. While there are many studies on the role that Phragmites plays in marsh ecosystems, there is little known about the effectiveness of spraying glyphosate for Phragmites management and how it affects marsh resilience to sea level rise. The focus of this study is to assess the management practices of Phragmites australis in terms of coastal marsh resilience to determine if we need to re-evaluate when and where we treat Phragmites. My hypothesis is that Phragmites will increase blue carbon pools as well as ecosystem resilience in comparison to the native grass species. In addition, we hypothesize that Phragmites invasion will increase decomposition rates and methane emissions which could offset these predicted carbon gains. Taking both of these predictions into account, I hypothesize that management of Phragmites will decrease soil carbon pools and that restored wetlands will have the lowest resilience to sea level rise and lowest blue carbon pools. The broader impacts of this research are crucial because we spend millions of dollars a year on phragmites management, and re-evaluating how we manage Phragmites in a way that effectively restores marsh resilience, is crucial for maintaining marshes ability to store carbon in the face of climate change.

Amara Saha

Advisor: Adam Williamson

Molecular Insight into the Initiation of Phagocytosis

          Transmembranal engulfment receptor proteins such as Draper in Drosophila melanogaster and its mammalian homolog Megf10 act as molecular bridges which connect phagocytes with their targets through ligands. The activation of these receptors initiates a cytosolic signaling program that ultimately leads to engulfment of the target cells. However, the molecular geometry of these receptors is not well understood. Such knowledge of receptor configurations may provide critical insights into their function and enhance our understanding of how they interact with other proteins to initiate phagocytosis. In order to define the geometry of the receptors, I will conduct a dimerization experiment to understand whether Draper/Megf10 molecules interact with each other in cis, i.e. on the same cell. To study this protein-protein interaction, I will perform a co-immunoprecipitation of modified Draper-GFP and Draper-FLAG proteins using GFP and FLAG specific antibodies. If Draper dimerizes in cis, the anti-GFP western blot would show a band for FLAG and an anti-FLAG blot would show one for GFP.

          Studies in the field suggest that Draper is phosphorylated by kinases such as Src42A to become activated and initiate phagocytosis. I will use biochemical assays to understand aforementioned receptor interactions that can further give insight into events such as post-ligation microclustering that favor the activation. In addition, other molecules such as Shark, a non-receptor Tyrosine kinase, can recognize activated Draper to commence downstream signaling processes and complete phagocytosis. The initial dimerization study can enable room for experiments to understand Draper鈥檚 interaction with Shark and provide a platform to understand their collaboration in initiating phagocytosis. Collectively, my work will provide molecular insight into ancient mechanisms underlying receptor-mediated immunity

Katrina Sparks

Advisor: Adam Williamson

Mechanical control of phagocytosis

          Mechanobiology is a field that bridges physics and biology to understand how mechanical cues instruct and regulate living systems. Phagocytosis is the process by which immune cells called phagocytes engulf their targets, including dead cells and pathogens. Well-characterized 鈥渆at me鈥 signals on the surface of dead cells serve as chemical ingestion signals for phagocytes, but how the dynamic forces and mechanical properties of targets and the environment control phagocyte behavior remains unclear. My guiding question this summer is whether or not phagocytic eating behavior is determined by the stiffness ratio between the target and environment. Recent work points to phagocytes鈥 enthusiasm toward engulfing denser particles (e.g., dead cells that are denser than living cells), but I hope to learn how phagocytic proficiency changes over a range of target-to-environment stiffness ratios.

          I hypothesize that larger ratios of target-to-environment stiffness will facilitate higher levels of engulfment, whereas lower ratios will inhibit engulfment. I will vary target stiffness by feeding phagocytes soft round beads with different, known densities. I will vary environmental stiffness by growing the phagocytes on substrata of different densities made from polydimethylsiloxane (PDMS), a soft elastomer. This cell biology experiment, which I will image on a confocal microscope at the University of Delaware, may provide insight into the fundamentals of how phagocytes interact with their environment. Specifically, my work will illuminate whether phagocytes discriminate between possible targets based on measurements of target stiffness alone, or, rather, the stiffness ratio between a target and their environment. This work is a collaboration between the Cheng and Williamson labs. In addition to providing insight into the mechanical control of phagocytosis, my work will also help foster a growing cross-disciplinary collaboration at Bryn Mawr.

Nuan Zhang

Advisor: Crystal Reynaga

Investigating How Compliant Substrates Affect the Biomechanical Properties in Hopping Cane Toads

Cane toads, Rhinella marina, use hopping as their primary mode of movement. They can hop shorter distances over longer bouts of time. There are another species called Osteopilus septentrionalis, also known as Cuban tree frogs, that jump fast and far distances. Previous work has shown that the Cuban tree frogs do not actively change muscle activation patterns jumping off springy substrates, and can utilize the elastic energy stored in their tendons. In Dr. Reynaga鈥檚 lab, we are interested in investigating the biomechanical differences between hoppers (cane toads) vs. jumpers (Cuban tree frogs) while jumping off compliant substrates. Extensive work has shown that cane toads can modulate and tune their landing motor patterns relative to their environment. So for this project we hypothesize that: 1) cane toads may be able to adjust muscle activation patterns when jumping off different springy substrates and 2) cane toads may not be able to utilize the elastic energy stored in the substrate as effectively as the tree frogs. This research will help us to understand how different frog species utilize different muscle recruitment strategies when facing perturbations in the environment. High-speed videography is used to film the jumping performances of cane toads on a physical compliant substrate. The videos will be digitized and analyzed to characterize the crucial parameters including joint angles, the velocity of limb extension, and energy output. Moving on we will observe the muscle activation patterns and compare the results of the two species to determine the impact of perturbations in the environment.