Biology

Low Contact Strategy to Measure Flutamide's Effects on Territorial Aggression in C57BL/6J Male Mice (O)

Presenter: Marissa Montandon
Faculty Project Advisor: Robert Bowen

Testosterone plays a key role in the regulation of aggression, physical activity, mating, and other behaviors influenced by the presence of an experimenter. This research explored the effects of an androgen receptor antagonist (flutamide) on territorial aggression in male C57BL/6J mice utilizing Silastic implants as a low-contact strategy for chemical delivery. A series of aggression trials were conducted on no-, high-, and low flutamide exposure groups. The time to an initial aggressive interaction and the total number of aggressive interactions with the unknown mouse were quantified. The introduction of flutamide at high and low concentrations resulted in a significant reduction in the total number of territorial aggressive interactions in the home cage 24 and 96 hours after implantation but appeared ineffective 192 hours post-implantation. This anti-androgen exposure methodology reduces confounding variables that impact mouse behavior and could prove beneficial to studying other androgen-related behaviors.

Effects of Blue Light Entrainment on Physical Activity and Behavior in Mice (O)

Presenter: Sarah Greenwood
Faculty Project Advisor: Robert Bowen

Exposure to blue light alters sleeping patterns and elicits negative behavior in humans. It is unknown if nocturnal behaviors in mice change after blue light exposure. The purpose of this study was to expose mice to blue light-emitting diode (LED) lights during the daytime restful period and evaluate nighttime activity for the potential changes to the normal participation patterns observed in mice exposed to white light. Wheel running distance, duration, and speed monitoring in C57BL/6J male mice occurred for 21 days. During the second week, a group underwent LED light exposure, and a different group experienced white LED light exposure. The lights were on a 12-hour light/dark cycle at full brightness during the exposure period. Blue LED light exposure did not appear to affect wheel running patterns. This indicates that the mechanisms adversely affected by blue light do not drive wheel running behavior.

Inhibitory Mechanisms of Herbal Extracts on Staphylococcus epidermidis Biofilm (O)

Presenter: Anna McCredy
Faculty Project Advisor: Esther Choi

Staphylococcus epidermidis is a commensal bacterium but can cause opportunistic infections by forming biofilms on medically implanted devices. Biofilms account for 80% of microbial infections and increase morbidity and mortality rates. Biofilms, multicellular communities of microbes, offer protection for microorganisms against host immunity and antibiotics. Our research aimed to determine the effectiveness of three herbal extracts in disrupting biofilms of S. epidermidis. Curcumin, Timosaponin- AIII, and O-Acetyl-L-carnitine hydrochloride showed a 33%, 30%, and 70% reduction in biofilm formation, respectively, without interfering with bacterial growth. These results demonstrate that the herbal extracts are effective at preventing biofilm formation without exerting selective growth pressure. Taken together, herbal extracts show promise as alternative and complementary measures to reduce staphylococcal biofilms, potentially providing a clinical application that could significantly enhance the effectiveness of antibiotic treatments.

Determining the Proper Dosage of MK-2206 Needed to Inhibit Akt Pathway Activity in Mice (O)

Presenter: Clara A. Kelleher
Faculty Project Advisor: Robert Bowen

Sex steroids have demonstrated biological regulation of physical activity behaviors and various chronic diseases significant to human health. The presence of human technicians while administering compounds to manipulate the physiology of sex steroids adversely affect behavioral and health outcome data in rodents. The purpose of this study was to assess the utility of administering MK-2206, a disrupter of Akt-related pathways including the insulin signaling pathway, via low-contact strategies to reduce technician presence during observations. MK-2206 was administered via gavage under anesthesia to male (n=15) C57BL/6j mice split into three groups of differing concentrations of the compound. Both mass (g) [F=1.63, p=0.220] and blood glucose (mg∙dL-1) [F=1.85, p=0.145] were unaffected by MK-2206 throughout the study period. These data present no evidence of pathway manipulation, indicating administering MK- 2206 via gavage with anesthesia is not a beneficial strategy to manipulate Akt-related mechanisms in male mice.

Using CADII and VPS4B Genes To Create Phylogeny Of The Wasp Family Rhopalosomatidae (Hymenoptera) (O)

Presenter: Ngan Kim Dong
Faculty Project Advisor: Jeremy Blaschke

Malaise-trap usage has greatly improved collection of understudied cricket-assassin wasps (Hymenoptera: Rhopalosomatidae), but many specimens have yet to be thoroughly examined for their evolutionary relationships. Twenty-eight DNA samples from four genera of Rhopalosomatidae (Rhopalosoma, Paniscomima, Liosphex, and Olixon), underwent PCR amplification, sequencing, alignment, and phylogenetic analysis using CADII and VPS4B to expand on the understudied diversity between the different genera. CADII was chosen because it is relatively free of introns, extreme nucleotide biases, and large hypervariable regions. The phylogenies showed that Rhopalosoma and Paniscomima are closely related. All four groups were recovered monophyletic, and every genus represents a unique lineage of cricket-assassin wasp.

Effects of Estrogen on the PDE5A Gene in Adult Zebrafish (O)

Presenter: Sarah McGee
Faculty Project Advisor: Faith Zamamiri-Davis

Infertility rates are on the rise, with around twenty percent of women having difficulties in conceiving. Birth control usage has also increased, especially in teens and young women at reproductive age. The PDE5A gene was studied for its role in oocyte maturation and development. 17α-Ethinylestradiol (EE2) is a form of synthetic estrogen that is found in most oral contraceptives. In this experiment, zebrafish (Danio rerio) were divided into three groups with varying exposure periods to EE2. After zebrafish were exposed to EE2, total RNA was extracted and quantified to measure the expression of the PDE5A gene using RT-qPCR. Minor behavioral and morphological changes were observed in the group exposed to EE2 for 96 hours. RT-qPCR data was inconclusive at the time of submission. However, additional trials are in progress to determine results.

Behavioral Adaptations and Interactions of Lepomis macrochirus and Amatitlania nigrofasciata (O)

Presenter: Gracie Shaddox
Faculty Project Advisors: James Kerfoot

Fish feeding habits and behavior can undergo significant adaptations contingent on species they interact with within their ecosystem, perhaps exhibiting cautious and evasive behaviors to avoid conflict. The goal of this study was to explore the interaction between tropical non-native species Amatitlania nigrofasciata (convict cichlid) and temperate native species Lepomis macrochirus (bluegill) to assess how the feeding behavior of each species changed in the presence of a conspecific, an allospecific fish, or alone. Food consumption, vigilance, and hiding behavior for both species were examined to determine how either species' behavior was influenced. A non-parametric Scheirer Ray Hare analysis was conducted and no significant differences in feeding and behavior were discovered across treatments for either species. Research treatments showed that conspecifics spent less time hiding than allospecifics and that bluegill did not change its feeding behavior or vigilance because of the invasive convict, indicating that these species are equal competitors.

Molecular Study of NFκB and Thyroid Hormone Activation in Macrophages (O)

Presenter: Gage Harrison
Faculty Project Advisors: William Thierfelder

Thyroid hormones (TH) are essential regulators of metabolism, and their activity is often upregulated during energy-requiring processes, including inflammation. Lipopolysaccharide (LPS) from gram-negative bacteria induces transcription of the TH-activating enzyme deiodinase-2 (DIO-2) in astrocytes by activating the transcription factor nuclear factor _κB (NF_κB), but it is unclear whether NF_κB also regulates DIO-2 in pro-inflammatory macrophages. In this study, a gene-edited mouse macrophage cell line was screened for successful deletion of an NF_κB subunit using a PCR-based enzymatic assay. Deletions were assessed using DNA sequencing. Cells with or without the deletion were treated with LPS and tested for expression of DIO-2 by RT-qPCR, with the expectation that cells containing the deletion would be unable to regulate it. Elucidation of this mechanism would help clarify connections between thyroid hormone regulation and inflammatory diseases.

Downstream Effects of Thrombin-Induced Par-1 Activation in Fibroblast Cells (O)

Presenter: Ben Smith
Faculty Project Advisors: Marc Lockett

An essential part of the wound healing process is the downstream activation of protease activated receptor-1 (PAR- 1), which is induced by thrombin. The following research will test thrombin's ability to initiate the wound healing process. We will test fibroblast cells in the presence and absence of thrombin. Fibroblasts are the most important cells in the dermis and help capillary growth, collagen formation, and granulation tissue formation. Then, the expression of the desired genes will be measured. Thrombin induced PAR-1 activation has been shown to increase the expression of the genes CYR61 and connective tissue growth factor (CTGF). CYR61 promotes cell adhesion and angiogenesis. CTGF is a gene that promotes fibroblast cell proliferation. It is hypothesized that the presence of thrombin will induce the expression in PAR-1 regulated genes.

Effect of Inflammatory Environments on Proliferation and Migration of the MCF-7 Breast Cancer Cell Line (O)

Presenter: Anna Hayes
Faculty Project Advisor: William Thierfelder

The Michigan Cancer Foundation 7 (MCF-7) breast cancer cell line is a widely used cell line for medical research. This research project will seek to determine how iodothyronine deiodinase 2 and 3 (DIO2 and DIO3) transcription is regulated by pro- and anti-inflammatory stimuli of MCF- 7 by cytokines such as TGF-B, PMA, IL-6. IL-10, and T3. The expression of DIO2 and DIO3 are related to cancer cell proliferation and migration. This research will seek to determine if breast cancer metastasis is partially regulated by DIO2 or DIO3. The results of this research suggest that DIO2 is not affected by the presence of cellular inflammation.

Plasmin Effect on PAR-1 Regulation and Angiogenesis (O)

Presenter: Addison Harwell
Faculty Project Advisor: Marc Lockett

Angiogenesis plays a role in cell tumor growth. During tumorigenesis there is an increase in blood vessels density to the tumor resulting in higher rates of cellular growth. Angiogenesis involves changes in gene expression, including genes affected by activation of protease activated receptor-1 (PAR-1), via proteolytic enzymes. This research examines plasmin's ability to regulate PAR-1 and its downstream effects. Fibroblast cells were grown in the presence and absence of plasmin to isolate the effects on PAR-1 signaling. Control RNA and experimental RNA from various timepoints of plasmin treated fibroblast cells have been extracted. The expression of two targeted genes have been measured. Cyr-61 mediates endothelial cell adhesion, and TNF-α promotes cell proliferation and angiogenesis. It was hypothesized that if plasmin is present, the upregulation of PAR-1 activity in 3T3-L1 cells will increase the expression Cyr-61 and TNF-α.

Transcriptional Regulation of Deiodinase 3 (O)

Presenter: Prabhavi Aghamkar
Faculty Project Advisor: William Thierfelder

Thyroid hormone triiodothyronine (T3) is a major regulatory hormone that regulates metabolic rate, protein synthesis, cell proliferation, and, for the purposes of this study, affects tumor metastasis. The deiodinase 3 (DIO3) gene codes for type 3 iodothyronine deiodinase, an enzyme that inactivates T3. DIO3 transcription is upregulated by interleukin-6 (IL-6), which is known to signal through the signal transducer and activator of transcription 3 (STAT3) pathway. In this study, MCF7 breast cancer cells were transfected with pCas9 STAT3 guide RNA constructs and assayed for deletion of STAT3 using a T7 endonuclease mutation detection kit. Results revealed a successful deletion of STAT3. To assess the proportion of IL-6-induced DIO3 expression regulated by STAT3, DIO3 mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (RT-qPCR) in cells with or without STAT3 deletion after treatment with IL-6.

Using POL and EF1α to Create a Taxa Comprehensive Phylogeny of Rhopalosomatidae (O)

Presenter: Megan Walker
Faculty Project Advisor: Jeremy Blaschke

Rhopalosomatidae are under-studied cricket-assassin wasps distributed around the world. Creating phylogenetic trees of this family increases knowledge of biodiversity. This analysis provided genetic support for the current morphologically informed classification of all four genera within Rhopalosomatidae. PCR amplification, sequencing, alignment, and phylogenetic analysis were done using two genes: RNA Polymerase II (POL) and Elongation Factor 1 alpha (EF1α). Phylogenies were constructed using Maximum- Likelihood and statistically analyzed with bootstrapping. Paniscomima was sister to Rhopalosoma, Liosphex was sister to both of these, and Olixon was most distantly related to the other three genera in the family. Future phylogenetic studies of Rhopalosomatidae should amplify and sequence more EF1α samples from all four genera to construct a phylogeny including a greater number of specimens. These results provide statistically robust representation of evolutionary history, giving greater insight into the relationship of the four genera as a monophyletic family.

Using DNA Barcoding to Identify High Priority Taxa (Hymenoptera: Ichneumonidae) in Great Smoky Mountains National Park

Presenter: Emerie Landers
Faculty Advisor: Jeremy Blaschke

The parasitoid wasp family Ichneumonidae comprises one of the largest and most diverse groups of animals in the world. Despite the positive, multifaceted effects parasitoids like ichneumonids have on their environment, they are extremely understudied. Biodiversity inventories, especially when focused on unique environments like those found in Great Smoky Mountains National Park (GSMNP), can provide invaluable data about understudied groups like Ichneumonidae. DNA barcoding has recently transformed biodiversity surveys by making species identification more efficient and accurate. To begin surveying the diversity of Ichneumonidae at GSMNP, thousands of previously collected ichneumonids were sorted to morphospecies and 95 representatives were selected for DNA barcoding. Of these, 30 specimens represented genera or species that had not been previously observed in GSMNP and therefore represent new park records for Ichneumonidae. These results demonstrate a promising future for using DNA barcoding to identify new records of understudied taxa at GSMNP.

Exploring Spider Wasp (Hymenoptera: Pompilidae) Diversity in Cypress Grove Nature Park

Presenter: Kelsey Crowley
Faculty Advisor: Jeremy Blaschke

To survey the diversity of spider wasps (pompilidae) at Cypress Grove Nature Park (Jackson, TN), Malaise and pan traps were used to collect insects which were then sorted to Order, Family, and morphospecies. Representative specimens of each pompilid morphospecies were sent to the Canadian Centre for DNA Barcoding for genetic analysis. A total of 56 pompilids representing 29 morphospecies were collected. Of these, 19 were positively identified based on >98% genetic similarity to pompilids within the Barcode of Life Database (BOLD) with confirmed species IDs. This survey was the first attempt to document pompilid diversity in CGNP and revealed 4 new genera and 6 new species, highlighting the importance of future research in pompilid systematics and the promise of future discoveries. This study emphasizes the need for additional high-quality species identifications coupled with genetic analyses in databases like BOLD to help future researchers identify species of understudied groups like pompilidae.

Biodiversity of Velvet Ants (Hymenoptera: Mutillidae) in Cypress Grove Nature Park

Presenter: Emily Sadler
Faculty Advisor: Jeremy Blaschke

Velvet ants (Hymenoptera: Mutillidae) are parasitoid wasps known for their defense mechanisms and aposematic coloration. Mutillidae includes 4,300 described species with worldwide distribution, but little is known about their biology and systematics. To survey the biodiversity of velvet ants in a unique environment, insects were collected in Malaise traps in Cypress Grove Nature Park (Jackson, TN) from Aug. 5 – Oct. 24, 2020 and Aug. 11 – Sep. 6, 2022. Specimens were sorted to genus and morphospecies. Specimens from each morphospecies were sent to the Canadian Center for DNA Barcoding for genetic analysis. A total of 42 mutillids were collected, representing six genera and 20 morphospecies. No specimens were able to be matched genetically with known species within the Barcode of Life Data System, which underscores the importance of integrating morphological identifications with DNA barcoding for future researchers. All six genera discovered represent new park records for Cypress Grove Nature Park.

The Gator Chomp: Investigating the Influence of Prey Mobility on Feeding Kinematics of Alligator mississippiensis

Presenter: D. Victoria Seymour
Faculty Advisor: James Kerfoot

American alligators (Alligator mississippiensis) are apex, opportunistic predators. To survive, they capture both mobile and immobile prey. Little is known about how mobile versus immobile prey influences their feeding performance. This study sought to investigate the modulation in feeding kinematic behavior when feeding on mobile versus immobile prey. Six captive individuals were fed both mobile and immobile fish prey (treatments groups) and feeding events were recorded using a high-speed video camera between April and August 2023. Feeding performance was summarized by analyzing 4 kinematic feeding variables (maximum gape, duration of feeding event, approach velocity, maximum gape velocity). Results showed that none of the kinematic variables were significantly different between treatment groups and indicated that American alligators do not modulate their feeding behavior based on the mobility of the prey. Here, American alligators seem to utilize a static or constant form of prey capture behavior regarding the prey's mobility underwater.

The Effects of Urbanization on Mammalian Predators in Huntsville, Alabama

Presenter: Abigail Donham
Faculty Advisor: Andy Madison

Urbanization has impacted wildlife populations, but its effect on predator populations in Huntsville, AL, has not been examined. Camera traps were set up from mid-February to mid-May 2023 in 4 randomly selected suburban sites in the Huntsville area and 4 randomly selected sites on the Wheeler National Wildlife Refuge (which were considered rural sites). There were 2 cameras for each site, set ~30 m apart. The traps were checked periodically to ensure function and to collect data from SD cards in the cameras. A total of 15 different species were observed in the suburban sites and 11 in the rural sites. The most common independently observed species was white-tailed deer (Odocoileus virginianus) with 155 total observed in rural sites and 38 total observed in the suburban sites. Five total coyotes (Canis latrans) were observed and only 1 fox (Urocyon cinereoargenteus or Vulpes vulpes), which were the only predators photographed.

Survey and Comparison of the Bird Species of a West Tennessee Wetland Mitigation Site and Bottomland Hardwood Forest

Presenter: Brayden Paulk
Faculty Advisor: Andy Madison

Wetlands have declined significantly in North America since European settlement, which has harmed bird populations dependent on them for habitat. My research objective was to compare the bird species composition of 2 sites in Rossville, Tennessee near Memphis: the Rossville Wetland Mitigation site and a nearby bottomland hardwood forest preserve. A wetland mitigation site creates a new wetland to offset the destruction of wetland habitat elsewhere. Line transects were walked in the springs of 2022 and 2023, and the species and number of each species were recorded along with other relevant data such as sex, age, and weather conditions. Results showed that the Mitigation site had a species richness 17.6% higher than the hardwood forest site and was used primarily by grassland and wetland species. The hardwood forest was used primarily by forest interior species, especially those associated with mesic forests or swamps.

Analyzing the Immediate Physical Activity Response of Male Mice after Orchiectomy and Reintroduction of Testosterone

Presenter: Hannah Mitchell
Faculty Advisor: Robert Bowen

Analyzing the immediate physical activity response of male mice after orchiectomy and reintroduction of testosterone is important because it has been postulated that testosterone is a physical activity regulator in both rodents and humans. Most people don't meet their daily physical activity recommendations, which may be due to testosterone deficiency. The purpose of this research was to evaluate the time course (rapid or slow) by which physical activity reinvigoration occurs immediately following the reintroduction of testosterone to surgically-disrupted, testosterone-deficient mice. Wheel running speed [F (14,28) = 1.14, p = 0.372] was unaffected by exposure to testosterone. Wheel running distance [F (14,28) = 30.91, p = 0.001] and duration [F (14,28) = 27.86, p = 0.001] were affected by exposure to testosterone. While testosterone-deficiency significantly reduced wheel running distance and duration, both variables recovered to baseline immediately after testosterone reintroduction. Testosterone appears to recover wheel running vigor immediately following re-exposure.

Transcriptional Regulation of DIO-3

Presenter: Sally Howell
Faculty Advisor: William Thierfelder

Nearly 300,000 new people face breast cancer each year, and more than 10% die. This research used a breast cancer cell line to examine whether the expression of the enzyme deiodinase-3 (DIO-3) correlates with breast cancer migration and metastasis. Cells that had undergone CRISPR gene editing were tested to determine any carried deletions in DIO-3. Several clones were found that carried the deletions in DIO-3. A scratch test was performed to determine whether the cells carrying the deletion migrated differently than unmodified cells. Preliminary results suggest that deletion of DIO-3 correlates with a reduction in migration. Since DIO-3 inactivates thyroid hormone (T3), these data suggest that T3 in the cell medium supports cell migration.

A Snapshot of Flower-Visiting Insects at Cypress Grove Nature Park

Presenter: Allison Winkler
Faculty Project Advisor: Jeremy Blaschke

Pollinating insects are a vital component of healthy ecosystems, yet pollinator abundance and diversity are declining worldwide. To assess the diversity and flower color preferences of local pollinators within the unique ecosystem of Cypress Grove Nature Park, insects were collected using colored pan traps (yellow, white, blue, red) in two different habitats (forest and meadow). In just 12 days, 1,614 insects were collected, representing at least 14 orders and 25 families. Overall, yellow pan traps captured the most insects (n=736), followed by white (n=360), blue (n=334), and red (n=191). Yellow also attracted the greatest diversity of insects (109 morphospecies), followed by white (n=48), red (n=38), and blue (n=29). Insect abundance was higher in the meadow (n=1,122) while insect diversity was higher in the forest (n=102). These results demonstrate that using a variety of colors in a variety of habitats can increase the abundance and diversity of pollinators captured.

Effects of Pseudomonas fluorescens Culture Supernatant on Biofilm Formation of Staphylococcus epidermidis Strains

Presenter: Cassandra Fish
Faculty Project Advisor: Esther Choi

Staphylococcus epidermidis is a human skin commensal, but it is also one of the leading causes of nosocomial infections due to its ability to form biofilms. Biofilms are complex bacterial communities encased in exopolysaccharide matrix. Bacteria in them are hard to eradicate since they are protected from host defenses and show increased antibiotic resistance. Previously, Pseudomonas fluorescens culture supernatant (CS) was shown to effectively disturb biofilm formation S. epidermidis 1457. In this study, the CS was tested for another clinically isolated S. epidermidis strain, RP62A. Biofilm assays and quantitative PCR revealed a significant inhibitory effect of the CS on RP62A biofilm formation by downregulating icaA, which encodes for polysaccharides of the biofilm matrix. Identifying the mechanism through which P. fluorescens CS inhibits biofilm formation of clinical strains of S. epidermidis could contribute to the development of an anti-biofilm treatment for reducing S. epidermidis-mediated infections in hospitals.

Citrobacter rodentium 4280 as a Model for Pathogenic Escherichia coli Biofilm-mediated Infections in Humans

Presenter: Elizabeth Shepherd
Faculty Project Advisor: Esther Choi

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are well known human pathogens responsible for causing traveler's diarrhea. They create attaching and effacing lesions and efface the brush border microvilli of their hosts. Their propensity to form biofilms also increases their pathogenicity, as biofilms are highly antibiotic resistant. To study EPEC and EHEC safely, model organisms are used. This study used Citrobacter rodentium, a murine pathogen that shares many characteristics with E. coli. Biofilm assays were performed to discover the optimal concentrations for biofilm formation, as well as to challenge C. rodentium with other bacterial supernatants. No obvious difference was seen between trials incubated with supernatant and those without, although the biofilm that was formed was not very strong in either case. Citrobacter rodentium was also challenged by a low pH environment and results indicated that low pH was effective at reducing C. rodentium numbers.

Fish Species Survey of the Rossville Farm Mitigation Site

Presenter: Andrew Logsdon
Faculty Project Advisor: James R. Kerfoot, Jr

Ecosystem restoration is a cornerstone for biodiversity conservation. Wetland degradation reduces ecological effectiveness by influencing water quality, water movement, and the ability to support many organisms. For these reasons, wetland mitigation is tracked to ensure the restoration process is appropriately supporting its end goal. The goal of this study was to establish a baseline condition of the fish and site physio-chemical composition within the Rossville, Tennessee mitigation site. From this initial survey 14 fish native fish species, 118 total, were found in the site by seining. Notropis texanus (Weed Shiner) 32%, Lepomis macrochius (Bluegill) 26%, and Lepomis gulosus (Warmouth) 15% were the most abundant species captured. Principal component analysis of the physio-chemical data revealed potential relationships between increased temperature and depth with higher species densities. This research will be beneficial in the future by the tracking of baseline fish and physio-chemical composition changes in relation to different seasons.

The Effects of Environmental Enrichment Removal on Wheel Running Behavior in Caged Mice

Presenter: Sarah J. Ruckersfeldt
Faculty Project Advisor: Robert S. Bowen

Physical inactivity is a leading cause of death in the human population, while physical activity guards against premature mortality. Previous studies have investigated the motivational drivers of physical activity using rodent wheel running as a model of human behavior. This study evaluated the validity of such research by experimentally analyzing whether wheel running might be a purposeful behavior or a stereotypy induced by stress. C57BL/6j male and female mice were housed with running wheels for two phases of study. The experimental subjects were given environmental enrichment (EE) during Phase I, which was removed for Phase II to introduce stress. EE removal had no significant effect on wheel running distance, duration, or speed in caged mice, demonstrating that wheel running is a purposeful behavior rather than a response to stressful stimuli. We conclude that wheel running appears to be an appropriate model for assessing the motivational drivers of voluntary physical activity

Effects of Prolonged Estrogen Exposure on BRCA2 Expression in Zebrafish

Presenter: Savannah Garner
Faculty Project Advisor: Faith Zamamiri-Davis

Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer that is difficult to diagnose. In this experiment, 9 zebrafish (Danio rerio) were divided into 3 groups with varying exposure periods of estrogen to monitor the effects on BRCA2 gene expression and morphological features. The goal of this research is to analyze the effects that estrogen has on BRCA2 expression, when using the zebrafish as a model for Triple Negative Breast Cancer. After estrogen exposure, the zebrafish were harvested and there was a significant difference in expression between the 48- and 96- hour exposure times. Future research should be done focusing more intently on the 96-hour period, to see if there are any additional differences involved with this exposure time.

The Influence of Atmospheric Disturbances on Seagrass Meadows in Charlotte Harbor Aquatic Preserve in Punta Gorda, Florida

Presenter: Sarah Chhim
Faculty Project Advisors: James R. Kerfoot, Jr.

Seagrass communities provide marine ecosystems with many resources such as food, shelter, carbon storage, and nursing habitats. Seagrasses are in decline worldwide due to pollution, weather disturbances, and human activities. The research performed in Charlotte Harbor Aquatic Preserve in Punta Gorda, Florida provides insight on how to combat these disturbances. This study compared water variables to the seagrass bed community by using data collected from December 2021 and December 2022, before and after a hurricane. The results indicated that the average density of seagrass after the hurricane was significantly less than before the hurricane. Temperature and salinity decreased on average after the hurricane. Higher salinity can cause slower development in seagrasses while lower temperature can hinder germination rates. While over a short temporal scale seagrass population can decrease after a hurricane, long-term, seagrasses communities can adapt to local environmental fluctuations and stochastic events like hurricanes depending on the severity

Examining the Retention of Intraspecies Phenotypic Variation of Chinese Elm During Regeneration

Presenter: William Smith
Faculty Project Advisors: Mark Bolyard

Plant regeneration is a process where a new plant can be grown from cultured tissue of plants through use of phytohormones. Retention of intraspecies phenotypic variation through regeneration is not well studied in plant tissue culture. In this research project we examined the impacts of regeneration on the phenotype of the wildtype and Seiju variant of Chinese elm (Ulmus parvifolia). These 2 trees are the same species with extreme variation in phenotype, and we hypothesized that each would retain its phenotype through the regeneration process. We followed a procedure for regeneration developed previously, making necessary modifications. Once shoots were developed, we observed shoots for leaf development. While we saw some leaf formation of the Chinese elm, we are working to develop a proper sterilization technique for the Seiju elm. Because of this, we were unable to compare leaf phenotypes and draw definitive conclusions about retention of phenotypic variation through regeneration

Effect of PAR-1 Activation on Collagen Formation in Fibroblast Cell Lines

Presenter: Eden Rainey
Faculty Project Advisors: Marc Lockett

Collagen is a key structural component in healing wounds requiring vitamin C for proper synthesis. Fibroblasts, the cell type responsible for formation of collagen, along with the expression of protease-activated receptor — 1 (PAR-1), the primary receptor for thrombin. Thrombin, a procoagulant that plays a critical role in wound healing by generating fibrin, an essential first step. Thrombin activation of PAR-1 elicits downstream effects including regulation of collagen production. By activating PAR-1 within fibroblast cell lines, this research clarifies thrombin's role in promoting synthesis of collagen in the presence of vitamin C within injured tissue. 3T3-L1 cells were grown under standard conditions and cell lysates sample were examined for pro-collagen production via ELISA. 3T3-L1 (enriched with vitamin C) were also observed using a scratch test to observe cellular migration influences. Both ELISA and scratch test, demonstrated vitamin C influences on collagen formation via ELISA and cellular migration via scratch test.

Factors Affecting Candida albicans Biofilm Formation

Presenter: Sloane Sellers
Faculty Project Advisor: Esther Choi

Candida albicans is a leading opportunistic pathogen in medical settings that causes serious infections through forming aggregates called biofilms. In biofilms, C. albicans evades immune detection and develops antibiotic resistance. This research aimed to determine factors that promote its biofilm formation. Different temperatures, media, and pH levels on biofilm formation were tested. When comparing biofilms at 30°C (a preferred setting) versus 37°C (a stressful setting) in complex- and chemically defined media, no significant differences were observed. However, the biofilms at 37°C were significantly higher at pH 4.2 in both media. This indicates that the yeast was stressed when exposed to two combined environmental factors (high temperature and low pH) and changed their morphology to its pseudo-hyphae form, a preferred morphology for forming thicker biofilms. By providing important biofilm promoting factors, this research may contribute to future prevention of C. albicans biofilm-mediated infection.

Production of Anti-Iodothyronine Deiodinase-1 Antibodies from Mouse Hybridomas

Presenter: Abram Adkisson
Faculty Project Advisor: William Thierfelder

Deiodinase enzymes assist in mediating the activation and inactivation of thyroid hormone. Iodothyronine deiodinase-1 (D1) produces triiodothyronine, a hormone that regulates energy usage. Commercial antibodies that currently exist for this deiodinase are of low quality, but high-quality deiodinase antibodies could be efficiently used for many laboratory techniques, like assays, and increase their overall accuracy and reproducibility. To create these antibodies, 5 laboratory mice were injected with D1 peptide every 2 weeks. After several months, the mice were sacrificed. The B cells extracted from these mice were fused with a myeloma cell, creating a hybridoma to produce monoclonal antibodies. 2 mice died. All surviving mice gave a positive immunological response and produced a polyclonal serum that recognized the antigen in an ELISA assay. The monoclonal fusion produced colonies, demonstrating the success of hybridoma production.

Zebrafish as a Pancreatic Cancer Model

Presenter: Heather Baugh
Faculty Project Advisor: Faith Zamamiri-Davis

Pancreatic cancer is the third leading cause of cancer death in the United States. Being able to research and study oncogenesis is a valuable tool in cancer diagnosis and treatment. It is possible to induce experimental pancreatic cancer with estrogen, therefore this research examined the impact of synthetic estrogen exposure on KRAS gene expression in a zebrafish model. Adult zebrafish (Danio rerio) were exposed to 500 ng/L of estrogen for varying lengths of time. After exposure, zebrafish were harvested, and RNA quantified. RT-qPCR was used to measure the KRAS gene expression in pancreatic tissue samples. Results showed no significant difference in KRAS expression values in raw data or when standardized to a common housekeeping gene. In the future, it would be beneficial for students studying estrogen exposure on the KRAS gene to experiment with longer exposure times.

Comparing Shrinkage of Tissue Types in Room Temperature and Cold Temperature Plastination

Presenter: Olivia Vermilye
Faculty Project Advisor: Micah Fern

Plastination has been used to create anatomical models that serve instrumentally in educating various health care professionals. In this study, room temperature plastination and cold temperature plastination were compared to determine if there is an ideal method for preserving different tissues. This research compared the shrinkage rates of 15 different sub-tissue types using ImageJ software for measurements before and after plastination. The data was put through an analysis of variance test to determine whether the results between the two methods were significant for any of the 15 sub-tissue types by rejecting or accepting the null hypothesis. Results pending.

Investigating Intrafamilial Relationships of Wasp Family Rhopalosomatidae

Presenter: Collette Truitt
Faculty Project Advisor: Jeremy Blaschke

Rhopalosomatidae is a family of wasps whose larvae parasitize crickets. Specimens from around the world were assembled to create the first molecular phylogeny of the taxon. A total of 30 species were included from all four extant genera (Rhopalosoma, Paniscomima, Liosphex, and Olixon), a total of 45 wasps. Genomic DNA was extracted and two genes (EF-1α and COI) were amplified and sequenced. Phylogenies were constructed using maximum likelihood and Bayesian analyses for each gene independently and on a concatenated dataset, all using two vespids as outgroups. The composite phylogeny supported several hypotheses from morphological literature: including Olixon as most genetically as well as morphologically distinct, the relationship of Rhopalosoma and Paniscomima as sister taxa, and the monophyly of all four genera. The EF-1α phylogeny was confused by intron inclusion but matched the composite phylogeny with exons only. COI required removal of the homoplasious third codon position for a strong signal.

Ichneumonid Biodiversity in Cypress Grove Nature Park

Presenter: Preston Buck
Faculty Project Advisor: Jeremy Blaschke

Ichneumonidae (Hymenoptera) are incredibly diverse parasitoid wasps which are crucial components of healthy ecosystems. Their tri-trophic interactions through pollination, host regulation, and host plant management connect food webs and help maintain healthy biodiversity. To assess ichneumonid biodiversity at Cypress Grove Nature Park, insects were collected using a Gressett-style Malaise trap and colored pan traps (yellow, white, blue, and red). Insects were stored in ethanol, identified to morphospecies, and pinned. In total, 475 ichneumonids were collected, representing 133 different morphospecies. New morphospecies records for the park included additional 12 unique morphospecies from the Malaise trap and 31 total pan specimens. Ichneumonids were most commonly captured in yellow and white pan traps (67%), but blue traps also captured unique species not found in other colors. These results suggest increasing the diversity of collecting methods will increase the number and diversity of collected ichneumonids, thus providing a more accurate estimate of true biodiversity.

Comparing Growth Sites and the Effect of Seasons on Thalassia testudinum

Presenter: Annelyse Valdivia
Faculty Project Advisor: Michael Schiebout

Thalassia testudinum (turtle grass), a seagrass of the Caribbean basin, has experienced decline in portions of its range over the last 40 years. Reasons for this decline have been difficult to pinpoint because the die-off is site-specific and variable. This field study compared percent coverage, density, and shoot height of T. testudinum from two sites off the Southern coast of Puerto Rico with two sites off the Western coast of Florida to determine which population of T. testudinum was healthier. Four transects with 20 m² quadrats were set up at each of the sites and environmental factors, community structure, and growth data were collected from them biannually in 2021-2022. An AVOVA test showed significant differences between the sites and seasons. One of the Puerto Rico sites showed higher averages in percent cover and height for T. testudinum for these years indicating a possible preference for this habitat.

Production of Antibodies Against Type 2 Deiodinase in Mice

Presenter: Elizabeth Allison
Faculty Project Advisor: William Thierfelder

Deiodinases are important in the activation and inactivation of thyroid hormones. They assist thyroid hormones in signaling to regulate homeostasis. There are 3 types of deiodinases, and this research focuses on type 2 deiodinase. Commercially produced antibodies to deiodinases are used to detect the presence of deiodinase proteins. However, the commercially produced ones exhibit poor quality, and many are not specific. To address these issues, we generated specific antibodies to type 2 deiodinase. This involved injecting mice with a peptide fragment of type 2 deiodinase, harvesting the mice's spleen cells, and fusing to create hybridomas. The hybridomas were cloned, selected, and grown out. They were then tested to through Eliza assay to show antibody production. Results from testing are still pending. Working antibodies will be useful for future detection of type 2 deiodinase protein in a variety of experiments.

The Effects of Artificial Grazing on Thalassia testudinum (Turtle Grass)

Presenter: Malachi Gorga
Faculty Project Advisor: Michael Schiebout

Turtle grass (Thalassia testudinum) is a vital part of offshore marine ecosystems. It serves as a food source for a variety of animals and helps hold substrate in place with its rhizomes. Turtle grass is currently seeing a large decrease over much of its range and causes of these declines appear to be multifaceted with certain locations responding more intensely to stressors. A factor that impacts turtle grass is herbivory. This experiment studied the effects of 2 levels of grazing on turtle grass. Heavy grazing was simulated by toping the blade and light grazing was simulated by 2 holes punched through the blade with a hole puncher. The plants that were in the control group had an average mass increase of 10.55%, the light grazing gained 9.59% and the heavy grazing gained 42.51%. The results seem to indicate that heavy grazing can be helpful to Turtle grass for gaining mass.

Loss of Testosterone Adversely Affects Physical Activity and Blood Glucose Levels in Mice

Presenter: Erik Lewis
Faculty Project Advisor: Robert S. Bowen

A sedentary lifestyle increases all-causes of mortality including some types of cancers, heart and vascular diseases, and metabolic disease in humans. Avoiding a sedentary lifestyle through routine participation in physical activity appears to be regulated by several biological factors including the sex steroids. This experiment assessed blood glucose levels and wheel-running activity in mice under physiologically-normal conditions (pre-orchidectomy), following surgically induced testosterone deficiency (post-orchidectomy), and following re-exposure to testosterone (post-testosterone-replacement). Blood glucose levels increased and remained elevated post-orchidectomy and post-testosterone-replacement, indicating a long-term effect of testosterone removal on blood glucose regulation. Daily wheel running distance (km) and duration (min) decreased significantly post-orchidectomy and returned to baseline following testosterone re-exposure, demonstrating a direct link between sex steroid levels and physical activity levels. These results indicate that low testosterone levels promote physical inactivity and might dysregulate glucose homeostasis both of which will promote the prevalence and progression of chronic diseases.

Effects of Pseudomonas fluorescens Culture Supernatant on Pre-Formed Staphylococcus epidermidis 1457 Biofilm

Presenter: Benjamin Murray
Faculty Project Advisor: Esther Choi

Staphylococcus epidermidis is a significant source of infections on indwelling medical devices. Its pathogenicity stems from its ability to form biofilms, which offer protection against host immunity and antibiotics. Thus, effective treatments to disrupt biofilms will reduce the clinical and economic burden of these infections. Our lab has previously shown that P. fluorescens culture supernatant (CS) inhibits S. epidermidis biofilm formation. The current study aimed to determine if the supernatant can disrupt existing biofilms. We treated S. epidermidis biofilms with CS at several stages of biofilm development, observing significant differences in optical density at the 12- and 16-hour time points. These disruptions were accompanied by changes in the culture's growth kinetics, but no changes in antibiotic susceptibility. Taken together, the P. fluorescens CS is promising as an alternative and complementary measure to reduce and destabilize staphylococcal biofilm within medical devices, enabling better control against S. epidermidis-mediated nosocomial infection.

Researching the Development of Deiodinase-3 Antibody in Mice

Presenter: Sarah C. Sparks
Faculty Project Advisor: William Thierfelder

The conversion of thyroid hormone (TH) into its various forms is vital for survival. Deiodinase enzymes (DIO) assist in each step of this conversion, converting TH into its most biologically needed form. Without DIO, TH would remain in a low activation state and have decreased usability in tissue. To examine the effects of DIO, laboratory techniques, such as an ELISA assay or Western Blot, require antibodies specific to the protein. Commercial antibodies that recognize deiodinases have often proved unreliable. Here, monoclonal antibodies are created to specifically recognize and bind to the DIO type 3 (DIO-3) enzyme. These antibodies will be further used in various experiments relating to the effects of the DIO-3 enzyme.

Wetland Restoration Comparison of New Channels in Rossville Mitigation Site Versus Wolf River

Presenter: Katherine Askins
Faculty Project Advisor: James R. Kerfoot, Jr.

Wetlands in the United States have been decreasing since the 16th century. Most wetland degradation is caused by agricultural practices that drain the land. In Rossville, TN previous agricultural land has been set aside for wetland mitigation. The mitigation site was engineered with tributaries off of the Wolf River that are structurally and functionally different and include different flora and fauna. The goal of this study was to form a baseline condition of the mitigation site describing the physio-chemical composition, vegetation, and presence of aquatic macroinvertebrate communities. Prominent macroinvertebrate families found within the survey were Oligochaeta 47%, Chironomidae 23%, and Cladocera 12% and vegetation included Panicum virgatum 31% and Andropogon virginicus 14%. Physio-chemical analyses between sites showed differences between flow rate, temperature, and turbidity which can affect macroinvertebrate diversity. These results will be used as a baseline of current conditions with the ability to evaluate succession at the site.

Comparing the Noose and the Lasso as Techniques of Alligator Capture

Presenter: Jacob Ramgren
Faculty Project Advisor: Micah Fern

Methods for capturing crocodilians each have disadvantages. One method of capturing crocodilians involves casting a weighted 3-pronged treble hook. The alligator is brought into the boat using a snare, a wire noose attached to a pole. While crocodilians are usually not severely harmed by this method, stress and injury to crocodilians remain significant risks to the animals. This study compares the snare to a novel lasso method in the capture of American alligators (Alligator mississippiensis). The lasso is a rope that is looped and tightened around the alligator's body. Researchers used both the snare and a lasso to pull alligators into the boat and compared the efficiency and safety of both methods. The number of attempts, success rate, and injury potential were significantly different between the two methods. The data (n=20) suggest that the snare is more efficient, but the lasso reduces injury to the alligators.

Examining between-clade incongruence within Vespoidea using Cytochrome oxidase I

Presenter: Sean Wheeler
Faculty Project Advisor: Jeremy Blaschke

The insect superfamily Vespoidea (Hymenoptera) includes two distinct wasp families: Vespidae, including hornets and their relatives; and Rhopalosomatidae, the cricket-assassin wasps. To assess the phylogenetic utility of the "barcoding gene" (COI) to accurately reconstruct relationships within Vespoidea, 133 COI sequences were downloaded from the Barcode of Life Database, representing all extant subfamilies of Vespidae, all extant genera of Rhopalosomatidae, and two outgroup taxa. Within R, the TN93+γ nucleotide substitution model was used to correct genetic distances and hierarchical clustering of the distance matrices was performed. Despite the strong evidence for the monophyly of these families, several unusual vespid subfamilies clustered with Rhopalosomatidae rather than Vespidae. To resolve this between-clade incongruence, ancestral state reconstruction (ASR) using maximum likelihood was used to estimate the ancestral sequences for clades within Rhopalosomatidae and Vespidae. Subsequent cluster analyses recovered the families as distinct groups, thus providing novel evidence for the benefit of COI for hymenopteran systematics when coupled with ASR.

Sphingosine-1-Phosphate (S1P) Effects on Gene Expression of PAR-1 and Fcγ Receptors in ARPE-19 Cells

Presenter: Tyller Paniuski
Faculty Project Advisor: Marc Lockett

Sphingosine-1-phosphate (S1P) is a member of the sphingolipid family involved in the upregulation of vascular endothelial growth factor (VEGF) and Interleukin-8 (IL-8) in retinal pigmented epithelium (RPE). This upregulation of VEGF and IL-8 in RPE aids in the development of wet age-related macular degeneration (Wet AMD) by encouraging choroidal neovascularization (CNV). The fragment crystallizable gamma receptor (FcγR) upregulates VEGF and IL-8 in RPE, whereas protease-activated receptor-1 (PAR-1) is a G-protein coupled receptor that aids in hemostasis and wound healing and is also involved in inflammatory processes. The goal of this research is to determine a potential link between the expression of Fcγ and PAR-1 in ARPE-19, a RPE cell line, in response to changes in concentration of added S1P. The RNA of the ARPE-19 cell samples have been collected, and each sample's concentrations have been quantified. The RT-qPCR results are still pending.

Habitat Suitability Model for Seagrass (Thalassia testudinum) Off the Southwest Coast of Florida

Presenter: Jasmine Kirchner
Faculty Project Advisor: James R. Kerfoot, Jr.

Seagrass beds are important ecosystems that provide goods and services and have been rapidly declining due to various anthropogenic pressures. Geographic Information System technology has been used to document the decline in seagrass habitat and aid in developing habitat suitability models for restoration. Models can be used to predict the presence or absence of seagrass and maximize conservation efforts by identifying areas to protect. Using water quality data collected from study sites in Punta Gorda and Naples, Florida, and species preference data collected from other studies, a habitat suitability model for Thalassia testudinum was created. Important drivers of population growth were determined to be increased light penetration/reduced depth along with reduced turbidity. Over a larger spatial scale, flow dynamics also related to the presence or absence of T. testudinum. Regionally the model was precise on the variability of T. testudinum density levels, which can aid in future restoration efforts.

Creation of a GFP-based Model to Test Gene Editing in Eukaryotic Cells

Presenter: Jaxon Via
Faculty Project Advisor: William Thierfelder

Gene editing is a revolutionary tool that can be used for a myriad of purposes. Clustered regularly interspaced palindromic repeats (CRISPR) is a form of gene editing that many researchers and lab workers have been utilizing for genetic experiments. It is a tool that researchers at Union have been attempting to use with limited success. Green fluorescent protein (GFP) is a protein that fluoresces when exposed to ultraviolet light. We incorporated GFP into a breast cancer cell line and applied CRISPR to delete it. A stable clone of the human breast cancer cell line MCF7 was created which expresses eGFP constitutively. This cell line is being subjected to CRISPR gene editing to test whether GFP expression can be successfully suppressed.

Developing a Fully Validated Edna Assay for the Detection of Alligator mississippiensis Presence in West Tennessee

Presenter: Jake Phelps
Faculty Project Advisor: Micah Fern

The Tennessee Wildlife Resource Agency has confirmed several sightings of the American Alligator (Alligator mississippiensis) in West Tennessee. Previous research has developed a primer that is species specific to A. mississippiensis and allows for the detection of A. mississippiensis DNA in high concentrations from synthetic DNA or DNA extracted directly from an alligator. This research will test this primer's sensitivity to ultimately allow for A. mississippiensis DNA detection in minute quantities from environmental DNA samples. Samples were collected from a laboratory holding tank that houses A. mississippiensis as well as in the field from the Rockefeller Wildlife Refuge in Louisianna where alligators are known to be present. The Assay has been able to detect minute quantities of DNA from environmental samples, however, definitive results are pending. Ultimately this assay will be used to study the presence and range expansion of A. mississippiensis to West Tennessee from its established southern range.

Immune Response Variance Induced by Planktonic Versus Biofilm Forming Staphylococcus epidermidis

Presenter: Sammy Bakeer
Faculty Project Advisor: Esther Choi

Biofilm formation is the main mode of pathogenicity of Staphylococcus epidermidis, human skin bacteria. In biofilms they stick to each other and attenuate the host defense system. This study aimed to determine the different cytokines produced by the host in response to two types of S. epidermis, the wild type (wt) biofilm forming and the Δica mutant non-biofilm forming S. epidermidis. The quantitative PCR was used to measure the pro- and anti-inflammatory cytokine levels produced by A431 cells, the human skin epithelial cells. A431 challenged with Δica mutants showed greater levels of pro-inflammatory cytokines than the cells challenged with wt. This indicates that host cells do not detect biofilm forming S. epidermidis effectively and fail to communicate with other cells to remove pathogens. Elucidating the mechanism by which biofilms avoid host detection will give greater insight on how to eliminate biofilm-mediated infections.

Phylogeny of the Cricket-assassin Wasps (Hymenoptera: Rhopalosomatidae) Using POL2 and VSP4B

Presenter: Benjamin Scott
Faculty Project Advisor: Jeremy Blaschke

The understudied cricket-assassin wasp family Rhopalosomatidae includes four extant genera: Rhopalosoma, Paniscomima, Liosphex, and Olixon. To generate the first genetically-informed phylogeny of this understudied family, 45 specimens representing 30 species and all four genera were gathered from collaborators and collections around the world. Two wasps from Vespidae were included as outgroup taxa. The two nuclear coding genes POL2 and VSP4B were extracted, amplified, and sequenced. Maximum Likelihood and Bayesian phylogenies were constructed and confirmed several longstanding morphological hypotheses, including 1) the monophyly of all four rhopalosomatid genera, 2) the close affinity of Rhopalosoma and Paniscomima, and 3) the basal position of the unique brachypterous taxon Olixon. Additionally, VSP4B was used here for the first time in rhopalosomatid phylogenetics and was effective at accurately resolving deep nodes in the phylogeny, indicating broad phylogenetic utility of this gene among the Hymenoptera.

Gene Expression of Par-1 in Cancer Cells in the Presence of Thrombin

Presenter: Kaitlyn Moss
Faculty Advisor: Marc Lockett

Proteinase-activated receptor-1 (PAR-1) not only regulates platelet function but has a role in the development of multiple cancers. PAR-1 is activated by thrombin, which is the key enzyme of blood coagulation and when overproduced, can promote tumor growth. This project explores the importance of the PAR-1 activation in response to varying concentrations of thrombin using the cancer cell line CCF- STTG1. To investigate this response, three different concentrations of thrombin were used: 2.5 units, 5 units, and 10 units. Results showed that the higher concentration of thrombin, the higher cell count of CCF-STTG1. A PAR-1 antagonist was then used to confirm if thrombin activity could be inhibited to decrease the activation of CCF-STTG1 cells. Finally, to determine downstream effects of PAR-1 activation, a RT-PCR was performed (results pending).

Observing the Differences in Mammal Species Across Suburban and Rural Wooded Areas Using Camera Traps

Presenter: Charisse Walker
Faculty Advisor: Andy Madison

My research objective was to observe mammal species' differences across suburban and rural wooded areas using trail camera traps. I assumed that rural areas would have higher species richness and abundance. Camera traps were placed from 2 January - 30 July, 2022 and all mammals observed were documented. Two sites in Chattanooga, a site in Jackson, and a site in McKenzie, TN, were used for observations. Two different statistical analyses were used to compare data from the different sites. Tentative data for the Chattanooga sites show the suburban site has higher richness and abundance than the rural site, contrary to my hypothesis. This could be random chance (only one site was chosen) or because the mammals were concentrated into the only available habitat. Chattanooga is more industrialized and larger than Jackson, so that could also be a factor in the final results.

Myotis septentrionalis and Myotis sodalist Habitat Suitability Model Analysis Using ArcGIS on a Wetland Mitigation Site in West Tennessee

Presenter: Emma Straley
Faculty Advisor: Michael Schiebout

The Rossville Farm Mitigation Site, near Memphis, TN, was acoustically surveyed for 2 federally protected Myotis species, Myotis septentrionalis (northern long-ear bat) and Myotis sodalis (Indiana bat). Bat presence can be an indicator of habitat health. Because both Myotis species utilize the same feeding grounds and there is indication the Mitigation Site might harbor them, we used ArcGIS to create a habitat suitability model for the 2 bat species. Bat calls were recorded with Wildlife Acoustics Echo Meter Touch Pro. The recordings were analyzed using Wildlife Acoustics' Kaleidoscope software and analyzed. Three bat species were detected, although not our target species. The presence of these other bats suggests the reclamation plan being implemented for the Rossville Farm Mitigation Bank is successfully establishing functional wildlife habitats.

Six in One: Cryptic Species and a New Host Record for Olixon testaceum (Vespoidea: Rhopalosomatidae) Revealed by DNA Barcoding

Presenter: Allaina Armstrong
Faculty Advisor: Jeremy Blaschke

New discoveries have increased our knowledge of the systematics and behavior of Olixon Cameron, an understudied genus of parasitoid cricket-assassin wasps (Vespoidea: Rhopalosomatidae). Little is known about the biology of these brachypterous wasps. Olixon testaceum is one of the most widespread of rhopalosomatid species, occurring in Central and South America. The widespread distribution and habitat variation of O. testaceum makes it an excellent candidate for genetic species delimitation study using data from the Barcode of Life Database (BOLD). Here we report Anaxipha cf. calusa as the first confirmed host of O. testaceum, as well as genetic evidence of 6 cryptic species within the nominal species O. testaceum.

Comparison of Cold and Room Temperature Plastination

Presenter: Lauren Gaggley
Faculty Advisor: Micah Fern

The use of plastinated specimens for anatomical studies is a growing field. Plastination is a method of preservation that replaces water in living tissues with plastic polymers. The current standard for this method of preservation is cold temperature plastination, but specimen shrinkage, equipment cost, and long procedure duration have been problematic. Room temperature plastination is an alternative method to the standard cold temperature plastination. Cat (Felis catus) and pig (Sus scrofa domesticus) brains were plastinated using cold temperature (CT) and room temperature (RT) plastination methods effect on specimen shrinkage. Criteria used to measure all plastinates included mass, length, width, and height. Equipment dependence, length of processes and costs were also calculated for CT and RT plastination methods. Results of this study are pending.

Downstream Effects of Thrombin-Induced Par-1 Activation in Fibroblast Cells

Presenter: Sydney Sample
Faculty Advisor: Marc Lockett

Clotting factors regulate several processes during wound healing, with angiogenesis, new blood vessel formation, being crucial. In addition, fibroblasts and endothelial cells mediate physiological functions of angiogenesis. The clotting factor, thrombin, has been implicated in mediating angiogenesis in endothelial cells. However, the effect of thrombin on fibroblast function is less well characterized. This research is being conducted to examine if thrombin mediates angiogenesis in fibroblast cells through protease- activated receptor-1 (PAR-1) activation. It was hypothesized that thrombin will induce angiogenesis through PAR-1. This was determined by culturing 3T3-L1 fibroblast cells in varying conditions: control group, thrombin, PAR-1 agonist, and PAR-1 antagonist with thrombin. Cell cultures were performed for various time periods to examine the time frame in which thrombin and PAR-1 mediate gene expression. RT-qPCR was then used to analyze expression of angiogenesis genes, cathepsin-S and vascular endothelial growth factor (results pending).

Effect of Pseudomonas Supernatant on Biofilm Formation and Gene Expression of Staphylococcus epidermidis

Presenter: Gabi Mirabella
Faculty Project Advisor: Esther Choi

Staphylococcus epidermidis is an opportunistic pathogen and an important cause of nosocomial infections by forming biofilms in medical devices. Biofilms impair penetration of antibiotics and evade immune surveillance. Our lab has observed that Pseudomonas fluorescens supernatant disrupted S. epidermidis biofilms due to competition between bacteria. Polysaccharide intercellular adhesin is the main component of the S. epidermidis biofilm matrix and is synthesized by the products of icaADBC genes. Quantitative RT-PCR was performed to observe differences in biofilm- related gene expression. All icaADBC gene expression was significantly downregulated in supernatant-treated S. epidermidis compared to controls. These results indicate that polysaccharides are the component of S. epidermidis targeted by inhibitors in Pseudomonas supernatant. The inhibitory effect of the P. fluorescens supernatant has potential to be used for destabilizing and removing S. epidermidis biofilms from medical devices when combined with antibiotics.

Differentiating Clades of Cricket Assassin Wasps

Presenter: Charis Murrey
Faculty Project Advisor: Jeremy Blaschke

Two genetically distinct clades of cricket-assassin wasps (Rhopalosoma) occur as sympatric cryptic species in eastern North America. To investigate potential morphological traits that can reliably distinguish clades, specimens were collected from Cypress Grove Nature Park, Jackson, TN from July to September 2020 using malaise traps. Specimens were preserved in 95% ethanol and genetically barcoded to identify clades. Pictures were taken of the hindwings of ten specimens including both males and females (six total from Clade 1, four total from Clade 2). Nine length measurements and seven vein angle measurements were calculated and analyzed with ImageJ. The length of the Rs and M-Cu vein and three angles showed significant statistical differences. These morphological differences should be confirmed with additional specimens, but preliminarily analyses indicate that this may be a reliable way to differentiate the cryptic species of cricket-assassin wasps.

Chemical and Physical Factors Contributing to Staphylococcus epidermidis Biofilm Formation

Presenter: Alana Parkey
Faculty Project Advisor: Esther Choi

Nosocomial infections are often caused by the microorganism Staphylococcus epidermidis. Typically, S. epidermidis is a part of normal skin microbiota causing no harm, but environmental factors can cause it to become pathogenic. The major mode of pathogenicity is what is known as biofilm. My research focused on analyzing how chemical and physical factors contribute to S. epidermidis biofilm formation. The factors tested included temperature, surface types, and competitive bacteria supernatant. Serial dilutions were performed for S. epidermidis, allowing the biofilm to grow in the appropriate conditions; the biofilm was then stained and quantified. When comparing S. epidermidis biofilm growth between 2 temperatures as well as plastic and silicon surfaces, no significant differences were observed. When comparing competitive bacteria supernatant, P. fluorescens and B. subtilis supernatant significantly inhibited biofilm formation. These observations provide a new perspective to destabilize S. epidermidis biofilm accumulated on medical devices.

Biofilm Inhibitory Effects of Bacterial Supernatant on Pseudomonas fluorescens and Candida albicans

Presenter: Melissa Aguirre
Faculty Project Advisor: Esther Choi

Biofilms are an accumulation of bacteria enclosed in an extracellular matrix. By forming communities, microorganisms exhibit antibiotic resistance and evade immune surveillance. The supernatant of one bacterial species may inhibit biofilm growth of another species, due to competition and sensing mechanisms. Previously, our lab has shown growth and biofilm formation of Staphylococcus epidermidis were inhibited by Pseudomonas fluorescens culture supernatant. This research examined inhibitory effects of various bacterial culture supernatants on different types of microorganisms. Candida albicans was not inhibited by P. fluorescens supernatant. P. fluorescens bacterial growth and biofilm formation was also not affected by S. epidermidis culture supernatant. These results indicate that P. fluorescens, a Gram-negative bacteria, produces inhibitors specifically targeting Gram-positive bacteria such as S. epidermidis.

Thrombin Involvement in Tissue Remodeling and Chemotaxis of 3T3-L1 Fibroblasts

Presenter: Anders Rider
Faculty Project Advisor: Marc Lockett

When injured, blood coagulation factors aid in the clotting response. Research was conducted to test if a factor, thrombin, leads to wound healing. It was hypothesized that thrombin and protease-activated receptors would contribute to fibroblast function after an injury. This was determined by chemotaxis assays, cell counts and collagen deposition using 3T3-L1 fibroblast cell lines. Cell counts were performed on 3T3-L1 cells with and without the presence of thrombin and an inhibitor. A Transwell assay was used to determine chemotaxis ability. To assess collagen deposition, an ELISA was performed. Cell plates with higher concentrations of thrombin contained larger numbers of cells than cell plates without thrombin. It was determined cells in the presence of thrombin were more mobile than cells without thrombin. The cells incubated with thrombin produced collagen, however the results of the collagen assay were inconclusive. This study could prove beneficial in fibrosis research.

Extraction of Degraded Genomes from Cricket Assassin Wasps (Rhopalosomatidae) Using Non-Destructive Enzymatic Techniques

Presenter: Nicholas Lewis
Faculty Project Advisor: Jeremy Blaschke

Rhopalosomatidae are a taxonomically understudied group of cricket-assassin wasps. Specimens are rarely collected and consequently, no family-level molecular phylogeny has been completed. For rhopalosomatid samples stored in ethanol for >10 years, typical DNA extraction methods that keep the valuable specimens intact are unsuccessful at consistently extracting high-quality genomic DNA. To address this difficulty and create the first family-level phylogeny for Rhopalosomatidae, a new non-destructive enzymatic technique was applied to sixteen rhopalosomatids of >10 years storage age whose DNA failed to amplify using normal extraction methods. Seven specimens, representing all four genera of Rhopalosomatidae, were successfully amplified (43.75%), three genes were sequenced (COI, EF1-α, POL), and a phylogeny was assembled using the concatenated data from all three genes. Our results indicate that non- destructive enzymatic techniques are effective at extracting amplifiable DNA from degraded genomes and can be used as an additional tool to solve persistent difficulties related to understudied taxa.

Novel Specific Primer Design for Alligator mississippiensis in Preparation of Environmental DNA (eDNA) Survey Research

Presenter: Sydney Zemke
Faculty Project Advisors: Micah Fern and William Thierfelder

In recent years there has been an increase in the number of sightings of Alligator mississippiensis in West Tennessee. Environmental DNA surveying is a newer management tool that can be used to identify which waters have A. mississippiensis in them. A common problem with previous eDNA research is using a poor primer that produces false positives for genetically similar species. This research evaluated and tested six primers for A. mississippiensis, using polymerase chain reaction to test the primers' specificity with isolated DNA from A. mississippiensis specimens and an outgroup that included representatives from five different caiman species to measure specificity and false positive rates. Assessment of the correct annealing temperatures and refinement of the PCR procedure was also conducted. Gel electrophoresis was used to see if the primers were effective. Results are still pending, but there is one primer that could be used under the right annealing temperature in PCR.

Analysis of the Effects of Glucose on HuC Protein Expression in Zebrafish

Presenter: Carly Bryant
Faculty Project Advisors: Faith Zamamiri-Davis and Hannah Henson

Diabetes Mellitus is a glucose intolerance identified by abnormal blood glucose levels due to defects involving insulin. Pregnant women that develop gestational diabetes are not only at risk for developing Type II diabetes but also for having a miscarriage, stillbirth, or premature birth. Infants born to mothers with gestational diabetes are often diagnosed with hyperglycemia, although long-term effects of hyperglycemia are unknown. To investigate the effects of hyperglycemia on brain and neuron development, we used zebrafish (Danio rerio) embryos subjected to a 4% glucose solution to observe changes in HuC expression levels. HuC is a protein in the nervous system used as a marker for neuron development. After using reverse transcription quantitative real-time polymerase chain reaction, results indicated that glucose has an inhibitory effect on HuC protein expression during embryonic development, which could be a potential model for the effects of gestational diabetes.

Effect of Herbicide Exposure on Zebrafish Skull Morphometrics

Presenter: Alyssa Hughes
Faculty Project Advisors: James R. Kerfoot and Hannah Henson

Glyphosate (Roundup) production has increased drastically over time and is the most widely used herbicide in the United States. Studies have shown a link between glyphosate and autism spectrum disorders due to the increasing prevalence of both. With autistic individuals, there tends to be brain enlargement which causes skull structures to expand. Using zebrafish (Danio rerio) as a vertebrate developmental model, the objective of this study was to investigate the effects of glyphosate on cranial development. The experimental group was exposed to glyphosate and the control group to normal conditions with each group containing approximately 150 embryos. Once reaching the larval stage, they were cleared and stained to measure 8 cranial elements with a compound microscope. A Multivariate Analysis of Variance revealed significant differences in cranial elements between treated and untreated individuals. The data suggests that glyphosate exposure causes an increase in cranial development and size.

Effects of Exposure to 17a-Ethinylestradiol on Developing Zebrafish Behavior, Morphology, and the Brain Transcriptome

Presenter: Alexis Tinsley
Faculty Project Advisor: Faith Zamamiri-Davis

17a-Ethinylestradiol (EE2) is a form of synthetic estrogen, commonly found in oral forms of birth control. Studies have shown that this endocrine-disrupting chemical is linked to harmful changes in aquatic life development. Because there is a noteworthy similarity between hormone receptor systems in humans and zebrafish, it raises the question: could this synthetic hormone be negatively affecting humans? Zebrafish (Danio rerio) embryos were exposed to high levels of EE2 for 5 days while their development was monitored alongside the control group. Following exposure, the embryos were harvested, and RNA quantified. Vtg1, and esr2 (genes found to be changed with endocrine disruption) were assessed via RT-qPCR and compared with elfa, a housekeeping gene. Developmental differences were observed but there was no significant difference in vtg1 or esr2 gene expression. Future studies will explore adjusting exposure time and minimizing the variability of our housekeeping gene.

Diversity and Seasonal Abundance of Ichneumonid Parasitoids at Cypress Grove Nature Park

Presenter: Hannah Juliussen
Faculty Project Advisor: Jeremy Blaschke

Ichneumonidae are extremely diverse and ecologically important parasitoid wasps. Due to tri-trophic interactions between parasitoid, host, and host plant, a high diversity of ichneumonids in an ecosystem can indicate a healthy diversity of host species and host plants. The abundance and diversity of ichneumonid wasps were surveyed at Cypress Grove Nature Park in Jackson, TN from 5 August-7 October 2020 using a Gressit-style Malaise Trap. Specimens were collected every ~48.75 hours, stored in 95% ethanol, and sorted to morphospecies. The number of new species over time was plotted to create a species accumulation curve. Four hundred and sixty-two specimens representing 133 morphospecies were identified. Common genera included the lepidopteran parasitoids Enicospilus, and Baryceros. This incredible diversity indicates Cypress Grove is a diverse and healthy ecosystem, and the species accumulation curve suggests there are far more species of Ichneumonidae still awaiting discovery.

Analyzing Relative Condition of Local Fish Populations and Determining Differences in Samples Affected by Varying Degrees of Parasitism

Presenter: Noah Reid
Faculty Project Advisor: James R. Kerfoot

West Tennessee hosts a variety of baitfish commonly affected by parasitism. In a previous study documenting the prevalence of Eustrongylides spp. parasitism in populations of Gambusia affinis in three west Tennessee sites, fish parasitism was found in varying frequencies between sites. It is thought that parasitism can affect the physiological condition of fish populations. The goal of this study was to investigate the link between physiological condition and prevalence of parasites at sites. Fish preserved from a previous study were weighed and measured from lip to caudal fin to calculate the species- specific physiological condition. The fish specimens were grouped based on prevalence of infection at each site as high, moderate, and low. A series of Sheirer-Ray-Hare and Kruskal- Wallis analyses indicated a significant difference in condition between Western Mosquitofish (Gambusia affinis) and other collected species and a significant difference between sites. In areas with moderate parasitism, condition was nearest to 1.0.

Examining the Effects of Synthetic Estrogen on HPT Gene Expression in a Zebrafish Model

Presenter: Alexis Hightower
Faculty Project Advisor: Faith Zamamiri-Davis

Endocrine disruptors are chemicals that interfere with hormonal systems and found within many products, including detergents, plastics, and pesticides. 17-ethinylestradiol (E2) is a synthetic estrogen, an endocrine disruptor, and a potent environmental toxin with high resistance to degradation by wastewater treatments. E2 has the potential to impact the secretion of thyroid hormones from the hypothalamic-pituitary-thyroid (HPT) axis, which plays a significant role in development of fetal nervous systems, and thyroid neoplasms, hypothyroidism, or cardiovascular disease in adults. To observe how E2 alters the mechanism of the HPT axis, zebrafish (Danio rerio) were exposed to varying E2 concentrations, harvested, and analyzed by RT-qPCR to measure expression of HPT genes, dio3, trh, and tshβ. E2 did not lead to differences in gene expression when compared to non-treated controls. Future research will utilize higher levels of E2 and perform ELISA to evaluate the production of thyroid hormone, T4 in our samples.

Plasmin Involvement on 3T3-L1 Fibroblast Replication

Presenter: Isaias Leon
Faculty Project Advisor: Marc Lockett

This research is being conducted to examine the relationship between wound healing and degradation of clotting. It was hypothesized that plasmin would increase cell 3T3-L1 fibroblast replication and protease-activated receptor-1 (PAR- 1) would inhibit replication. This was determined with 3T3-L1 fibroblasts, derived from mouse fibroblast cells, and varying concentrations of plasmin, cell counts, and PAR-1 antagonist. Cell count was performed on 3T3-L1 fibroblast cell plates with and without the presence of an inhibitor. An ELISA was performed on the plasmin buffer to ensure it was viable. Using an analysis of variance (ANOVA) we were able to find a statistical difference in 3T3-L1 cell plates with plasmin compared to that of cell plates either without plasmin or with inhibitors and plasmin. This research could prove beneficial to plasmin research.

Tracking Production of Secondary Metabolites in Wormwood (Artemisia absinthium) after Introducing Bacterial Extracts

Presenter: Adna Alihodzic
Faculty Project Advisors: Mark Bolyard and Jimmy H. Davis

Southern wormwood (Artemisia abrotanum) is one of the largest and most widely distributed plants from the family of Asteraceae. This species is widely recognized due to its distinctive aroma, herbaceous flavor, but most importantly, human health benefits. Wormwood volatile oils, such as borneol, camphene, camphor, 1,8-cineole, and limonene, represent the basis of application of these benefits to humans. This research explores the production of the secondary metabolites in Southern wormwood by extracting and comparing the essential oils from specimens grown in the greenhouse, a control group, compared with the wormwood regenerated from tissue culture. In vitro cultures were also tested with heat treated Gram + or Gram — bacterial extracts. Analysis of the essential oils was performed by gas chromatography/mass-spectrometry (GC/MS). The results are still pending, but a significant difference between the control and the regenerated plant is expected to be seen.

An Initial Analysis of Plant Composition of a Wetland Mitigation Site in Fayette County Tennessee

Presenter: Shelby Swaby
Faculty Advisor: Michael Schiebout

A plant survey was done at the Rossville Mitigation Site in Rossville, TN. This site has been selected as a wetland mitigation site for the state of TN and is currently being converted from cropland into a wetland system. The objective of the study was to establish a flora baseline and to determine wetland status of current plants as well as to evaluate species composition compared to a bottomland reference site. This baseline data will help determine the success of the conversion process. The survey was conducted from April–July 2021 and represents vegetation composition for the first year of transition. During this 4- month study, 45 families, 99 genera, and 79 species including infraspecific taxa were identified. The mitigation site and reference site had 32 duplicate plants. The mitigation site had more invasive species than the reference site based off of survey size. This was expected for a site this early in the process of being re-establishing as a wetland area.

The Effects of Temperature on the Feeding Kinematics of Convict Cichlids

Presenter: Gavin Woodring
Faculty Advisor: James R. Kerfoot

Temperature regulates the metabolism of ectotherms, such as fish, so it should influence how invasive fish feed as they move into areas that experience cooler temperatures. We used convict cichlids for this experiment, as they are a known invasive species in Florida, and theorized that changes in temperature would affect their feeding. We recorded fish feeding at 30°C, 24°C, and 20°C using a highspeed camera. We analyzed the videos, determined the average duration of feeding, time to max gape, and attack velocity. Our results showed no statistical difference between the different temperatures at any parameters. This could indicate convict cichlids adapt easily to temperature change, although we only tested 4 fish, and they were quite small. Future tests should include greater fish numbers to verify our results.

The Effects of Glyphosate on Pax6 Expression in Zebrafish

Presenter: Callie Wofford
Faculty Advisor: Hannah Henson

The pax6 gene is a crucial gene for brain and eye development in zebrafish. The gene is so complex that it has been classified into pax6a and pax6b. When this gene is altered, it results in ocular diseases and malformations. Glyphosate, a chemical commonly used in pesticides, has been hypothesized to cause some of these, and this study examined if glyphosate altered pax6 gene expression in zebrafish (Danio rerio) after exposure. Zebrafish were bred, embryos were collected, and exposed to glyphosate until they were five days old. The gene was then assessed by way of cDNA synthesis by PCR and an unpaired t test. Although not statistically significant, an increase in pax6a expression and a decrease in pax6b expression were found.

Applying a Novel Technique to Quantify Corticosterone Levels in American Alligators (Alligator mississippiensis)

Presenter: Grant Jacobs
Faculty Advisor: James R. Kerfoot

Corticosterone (a stress hormone) levels of American alligators from conditioned water samples in various feeding trials, with or without a conspecific, were measured. Studies have found it possible to measure corticosterone levels of amphibians by using water samples, however, it has never been tried in American alligators. Using 6 blood samples as a baseline, 38 water samples were collected from 6 alligators. These samples were collected before and after individuals were presented with food alone and then paired with a conspecific, to see if competition induced stress. Water and blood samples were filtered, and corticosterone was extracted for each treatment. An ELISA assay was used to quantify corticosterone concentration and levels in both blood and water samples were higher after the treatment with a conspecific, however the difference was not significant

Using Mice as a Model to Study the Effects of a Ketogenic Diet on Psoriasis-Like Skin Inflammation

Presenter: Amanda Gilbert
Faculty Advisor: William Thierfelder

Psoriasis is a chronic, inflammatory, autoimmune disease that causes itchiness, erythema, painful lesions, and plaques on the skin. This study used mice as a model to determine whether a ketogenic diet helps relieve inflammation caused by a psoriasis-like skin condition. Mice were shaved and treated with imiquimod cream on their backs for one week and fed a ketogenic diet for one week while continuing treatment. Systemic inflammation was quantified by isolating spleen lymphocytes and using PCR to determine expression of pro-inflammatory cytokines IL-17 and IL-23 and anti-inflammatory cytokines IL-10 and TGFβ. Local inflammation was analyzed by harvesting lymphocytes from the affected skin area and using PCR to quantify their levels of the same pro- and anti-inflammatory cytokines. The results of this study showed that a ketogenic diet increased systemic and local inflammation in mice. Therefore, a short-term ketogenic diet would not be a recommended treatment for patients suffering from psoriasis.

Components of Staphylococcus epidermidis Biofilm and Inhibiting Factor in Pseudomonas fluorescens Supernatant

Presenter: Bethany Wells
Faculty Advisor: Esther Choi

Staphylococcus epidermidis is of medical importance because it causes nosocomial infections on inserted medical devices. We investigated the composition of Staphylococcus epidermidis wt 1457 biofilm. We first examined which biomolecules compose the biofilm by inhibition with supernatant and inhibitors. Since biofilm are composed of lipids, polysaccharides, proteins, and nucleic acids, we looked at the polysaccharide and protein portions using inhibitors such as Proteinase K and NaIO4. The second aim focused on what component in the supernatant inhibited biofilm development. Lipase treated supernatant was added to biofilm and compared to non-lipase treated supernatant biofilm development. Our results indicated that polysaccharides were in the biofilm, but the results were inconclusive regarding the presence of proteins. Likewise, lipase-treated supernatants did not consistently affect the biofilms in a different way than the untreated supernatants. We are hopeful that further investigation of Staphylococcus epidermidis biofilm components will lead to treatments for Staphylococcus epidermidis nosocomial infections.

Effects of Glyphosate Exposure on HuC Expression in Developing Zebrafish Embryo

Presenter: Charley Kate Barcroft
Faculty Advisor: Hannah Henson

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by atypical behaviors such as social impairment and repetitive behaviors. Those who suffer from ASD struggle to communicate effectively, and some need assistance in daily tasks. ASD is common, but there is no definitive cause. Pesticide exposure contributes to neurodevelopmental disorders. HuC is a protein in the central nervous system and is a marker in developing zebrafish (Danio rerio). In this study, zebrafish embryos were exposed to 50 µg/mL glyphosate, a compound commonly found in pesticides, for 4 days. At 4 days, a whole mount immunohistochemistry was performed to determine differences in HuC protein expressions. HuC expressions were calculated using corrected total cell fluorescence (CTCF), and a 2-tailed t-test was performed at β=0.05. Glyphosate-treated embryos showed less CTCF than the controls, but there was no difference in HuC expressions between the control and the treatment (p=0.087)

Identifying Ecosystem Drivers of Thalassia testudinum Communities in Puerto Rico and Southwestern Florida

Presenter: Allison J. Marsch
Faculty Advisor: James R. Kerfoot

Populations of Thalassia testudinum, an important seagrass species found throughout the Caribbean Sea and Gulf of Mexico, have experienced decline. Collaborative research efforts have increased, but decline is variable and dynamic, requiring site-specific research. We measured the impact of environmental and community variables on T. testudinum growth in four sites: two in Jobos Bay, Puerto Rico; one in Charlotte Harbor Aquatic Preserve, Florida; and one in Rookery Bay, Florida. Community structure and environmental variables were measured in 20 quadrats across four transects at each site. Akaike Information Criterion (AIC) models evaluated the interaction of community and environmental variables on T. testudinum cover, density, and height. The interaction between light and community structure best explained cover, light and environment best explained density, and no variables best explained height. Our research may suggest which factors regulate and influence T. testudinum populations in these sites and across its range.

A Preliminary Study of Avian Populations on Agriculture Land Being Converted to Bottomland Hardwood Forest

Presenter: Elyssa Smith
Faculty Advisor: Andy Madison

The continued destruction of wetlands for agricultural or urban development has resulted in habitat fragmentation of local flora and fauna. In 2020, the Pictsweet Company created a conservation easement on 238.69 ha of agriculture land to be converted into a bottomland, hardwood forest, located in Rossville, Tennessee. A preliminary study of avian populations was conducted using 500m transects within 3 different habitats (woods, grassland, and edge) across 5 sites located throughout the area. Results showed no differences between grassland and woods habitat regarding species richness, but the woods habitat did have more bird abundance than grassland habitat, although it was not significant. Edge habitat had the greatest species richness and abundance, which was expected since it is the mixture of the 2 habitats. We suggest that research should be conducted again in ~10 years to determine mitigation site progress.

The Biotic and Abiotic Correlates of the Abundance and Distribution of Seagrasses in Lake Worth Lagoon, Palm Beach County, Florida

Presenter: Samantha Jones
Faculty Advisor: James Kerfoot, Jr.

Seagrass beds have undergone massive declines, and the goal of this study was to model potential patterns of environmental parameters and community structure that may correlate with its decline in Lake Worth Lagoon (LWL) over time. Nine transects and 4 polygonal areas throughout the LWL were visited annually (2007-2019) and environmental parameters (e.g., temperature), seagrass and macroalgae Braun-Blanquet (BB) scores, and community structure were recorded. Akaike Information Criterion (AIC) assessed the models developed to explain seagrass abundance for the transect data and community structure for polygonal areas. The transect AIC indicated that an interaction between year and macroalgal abundance best explained seagrass abundance. Interestingly, seagrass BB score showed a decline in 2013, with seagrass and macroalgae remaining very low. The polygonal AIC showed an interaction between year and site best explained the shift in community structure over time. Only certain sites experienced a shift in community structure correlating with 2013.

Investigating a Cricket's Worst Nightmare: Description of a New Species of Parasitoid Wasp (Vespoidea)

Presenter: Darius Mullin
Faculty Advisor: Jeremy Blaschke

The genus Rhopalosoma (Cresson) (Hymenoptera: Rhopalosomatidae) contains rarely encountered wasps with the unusual lifestyle of cricket ectoparasitism. Only one Rhopalosoma species, R. nearcticum (Brues), has been described from America north of Mexico. However, molecular evidence from rhopalosomatid larvae collected in Cypress Grove Nature Park (Jackson, TN) has revealed the presence of two genetic clades — at least one of which must represent a new species record. Adult Rhopalosoma specimens were subsequently collected from Cypress Grove Nature Park using a Gressitt-style Malaise trap from Aug. 5 – Sep. 17, 2021. A total of eighty-three specimens were collected and their morphology was compared with known species of Rhopalosoma. As a result of this analysis, we report a new species, Rhopalosoma cupressi, and include a morphological description of key traits, including ocellar characteristics, wing venation, and its distinction from the closely related R. nearcticum.

Interstate Effects on a Woodlot's Bird Population

Presenter: Sarah Childress
Faculty Advisor: Andy Madison

This project compared bird abundance and diversity in the Union woods and a Union-owned woodlot adjacent to I-40. This project provided a baseline for birds living in the I-40 woodlot and to determine if interstate noise negatively affects bird abundance and diversity. I performed 10 min. point counts at 3 sites in each location from September to October and recorded all birds seen or heard. Point counts were conducted at all 3 sites 12 times for each location. No significant difference in diversity or abundance between the locations was found. Additionally, there were no species found significantly more in one location than the other. This indicates that bird diversity and abundance were similar between the Union woods and the I-40 woodlot, and that I-40's noise does not affect birds more than noise from roads adjacent to the Union woods.

Investigation of the Role of Deiodinase-3 in MCF-7 Cancer Cells Using CRISPR

Presenter: Leigh Walker
Faculty Advisor: William Thierfelder

Clustered Regularly Interspaced Palindromic Repeats (CRISPR) technology can be used to knock out a targeted gene within a cell using the endonuclease Cas9. Recent studies have recognized the significance of deiodinase expression in cancer cells. In the human breast carcinoma cell line MCF-7, activity of iodothyronine deiodinase-3 (DIO-3), a deactivator of the hormone triiodothyronine (T3), was shown to increase in certain conditions. This project explores the importance of DIO-3 expression for the growth of MCF-7 cells, as well as how these cells respond to pro-inflammatory molecules (IL-6) and anti-inflammatory molecules (IL-10) when no longer expressing DIO-3. To investigate this response, 4 different guide RNAs targeting human deiodinase-3 and 2 non-targeting guide RNAs, were successfully cloned into a Cas9 expression plasmid and stably transfected into MCF7 cells, generating 6 lines containing the plasmids. Experiments to determine whether targeting and Cas9-mediated knockout of DIO3 were achieved are in progress.

Development of Plant Regeneration Protocols for the Globally Rare Crataegus harbisonii

Presenter: Grace Ingram
Faculty Advisor: Michael Schiebout

This research focused on developing plant regeneration protocols for the globally rare Crataegus harbisonii (Harbison hawthorn). The research began in the field, with the objective of verifying previously identified C. harbisonii's populations and collecting cuttings that were used in a bud regeneration protocol. A second phase used 222 seeds harvested from a cultivated C. harbisonii specimen and investigated the effects of cold stratification, scarification, and light quality/shade tolerance on seed germination. Finally, we investigated stem grafting and leaf tissue culture protocols on 2 commercial Crataegus species (C. viridis and C. phaenopyrum) with the goal of developing procedures in these species that could be transferred to C. harbisonii. Wild specimens were found, but the bud regeneration procedure was unsuccessful. For phase 2, none of the collected seeds germinated. For phase 3, 33% of the C. viridis stems developed roots. Progress has been made developing a standard leaf tissue culture for hawthorn.

Validation of Environmental DNA Surveillance to Detect the American Alligator (Alligator mississippiensis) in Controlled Experiments

Presenter: Dylan Parmely
Faculty Advisors: Micah Fern and William Thierfelder

American alligator (Alligator mississippiensis) sightings have become more common recently in West Tennessee. The re-expansion of this cryptic species has led to the need of further investigation. As alligators dwell in the environment, they slough off DNA that can be used for environmental DNA (eDNA) detection, through a series of tests. Polymerase chain reaction (PCR) was used to detect eDNA by a primer specifically developed for the alligator. Alligator tissue samples were taken to test the primer's specificity to alligator DNA. Gel electrophoresis showed positive reactions by a band at roughly 100 bp. To further confirm the correct gene was amplified, the gel band was sent to Saint Jude for sequencing (results pending). Water samples were taken from alligator display tanks at Bass Pro Shops (Memphis) and DNA will be extracted to be used in PCR for a positive control (results pending).

Optimal Root Structure for Thalassia testudinum

Presenter: Amber Rhodes
Faculty Advisor: Michael Schiebout

Turtlegrass (Thalassia testudinum) is a dominant seagrass species in the Atlantic that provides habitat, food, and carbon storage for many ocean communities. Turtlegrass communities are decreasing in many areas of its range. This lab-based study investigated the best root structure of T. testudinum for growth in culture with the goal of using these cultures for reintroduction or for additional lab studies. The 3 root structures were bare root, grow bag, and attached via rhizomes. All plants within grow bags died while both of the other treatments (rhizome and bare root) yielded leaf growth, a T-test indicated that this growth was not significantly different (p=0.8). Additionally, a field portion of the study investigated the effects of varying Photosynthetically Active Radiation (PAR) ratios on T. testudinum growth in Jobos Bay Puerto Rico. Preliminary calculations suggest a weak relationship between PAR ratios and seagrass density.

Chemical and Physical Factors Promoting Biofilm Formation of Candida albicans

Presenter: Hayden Rash
Faculty Advisor: Esther Choi

• Introduction: Oral Candidiasis is a result of the overgrowth of Candida albicans within the oral cavity. The fungi, a commensal organism, can become pathogenic when forming biofilm. The goal of this study was to determine what chemical and physical factors promote its biofilm formation.
• Methods: C. albicanswere plated on microtiter plates, incubated for 48 hours. After removing planktonic C. albicans, firmly attached biofilm was stained with crystal violet.
• Results: Biofilm formation of C. albicans was significantly less in synthetic media when compared with complex media, and less in 37 °C than in 30°C.
• Discussion: Taken together, these results indicate that C. albicans biofilm formation requires optimum concentration of carbon and its natural growth temperature rather than human body temperature. Our results can be used to prevent C. albicans biofilm-mediated infection in immunocompromised patients.

Detection of Environmental DNA (eDNA) for Identification of Freshwater Turtles in West Tennessee Ponds

Presenter: Kristen Bukowsky
Faculty Advisor: Micah Fern

Use of Environmental DNA (eDNA) through polymerase chain reaction (PCR) analysis is a valuable molecular ecology research method for identifying species indirectly within a given ecosystem. This study aims to establish a procedure for the identification and validation of freshwater turtle eDNA within the water of a closed pond. Specifically, it investigates whether a species' eDNA can be identified from water within a tank of known organisms and a pond of relatively unknown organisms using species-specific primers designed to target the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Results are currently pending.

Effect of CRISPR Knockout of NF-kB in Mouse Macrophages on Deiodinase-2 Expression during Inflammation

Presenter: Luke Spivey
Faculty Advisor: William Thierfelder

The inflammatory response is essential for human survival. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a complex of transcription factors responsible for expression of many inflammatory genes. NF-kB is thought to regulate the thyroid hormone activating type 2 iodothyronine deiodinase (DIO2) in astrocytes, but its role in mammalian macrophages is still unknown. To investigate DIO2 regulation by NF-kB, we used intracellular clustered regularly interspaced palindromic repeats (CRISPR) technology to disrupt the NF-kB p50 subunit in a mouse macrophage cell line. We plan to induce the cells with lipopolysaccharide (LPS) and test for upregulation of DIO2 by qRT-PCR. We predict that non-transformed cells will upregulate DIO2, while transfected cells will not. If this is the case, one can infer NF-kB indeed regulates DIO2 in mammalian macrophages.

Analysis of Cytokinins for Callus Production in Araucaria araucana

Presenter: Jordan Crawford
Faculty Advisor: Mark Bolyard

The purpose of this project was to observe Araucaria araucanacallus formation and growth. Three different cytokinins, 6-(γ,γ-Dimethylallylamino) purine, phloroglucinol, and meta-topolin, along with the auxin naphthapenacetic acid (NAA), were used to treat preexisting calluses which had been formed on media containing NAA and a variety of other cytokinins. Results indicated that callus growth continued in all the media treatments, but no shoot formation was noted. Callus treated with meta-topolin (55.6%; n=9) and phlorogluconol with NAA (47.6%; n=21) had the highest percentage of leaves with over 50% green callus, although the meta-topolin had a smaller sample size.

DNA Sequencing and Barcoding of Rhopalosomatidae Wasps and Its Phylogenetic Consequences

Presenter: Christopher Johnson
Faculty Advisor: Jeremy Blaschke

The cricket-assassin wasps of family Rhopalosomatidae are one of the few remaining taxa within Hymenoptera lacking a comprehensive family-level molecular analysis. To remedy this, rhopalosomatid specimens from around the world were gathered for DNA extraction and sequencing. The genes elongation factor 1 (EF-1a) and RNA polymerase II (POL II) were chosen based on their high number of phylogenetically informative sites. Rhopalosomatidae specimens from each genus (Olixon, Liosphex, Rhopalosoma, and Paniscomima) were included, with Vespula (Hymenoptera: Vespidae) as an outgroup. Maximum likelihood and Bayesian analyses were performed and results from each gene were compared individually and together against current morphological classifications of the family. The traditional classification of Rhopalosomatidae was recovered with the unusual taxon Olixon as sister to the rest. In contrast, the new-world genus Rhopalosoma was recovered paraphyletic with respect to the old-world Paniscomima, potentially indicating common ancestry and the need to synonymize these two taxa into a single genus.

Tracking Autism Risk Gene Mutations in Zebrafish after Exposure to Glyphosate

Presenter: Lisa Hamilton
Faculty Advisor: Hannah Henson

The purpose of this research was to determine the effects of glyphosate, a component of Roundup, on developing zebrafish (Danio rerio) embryos. Residue from glyphosate can enter the food chain through crops and water sources, and previous research has shown that exposure to glyphosate may disrupt neurodevelopment. Shank3 is a popular gene associated with neurodevelopmental disorders and autism. Zebrafish possess homologues of this gene, shank3a and shank3b. Demonstrating the effects of glyphosate on shank3a/b expression in zebrafish embryos can model the dangers these pesticides may pose to aquatic life and humans, specifically in early development. After exposing zebrafish embryos to glyphosate for 96 hours, their RNA was extracted, and quantitative PCR was used to detect changes in shank3a and shank3b expression. Results are pending

Molecular Phylogenetics of the Cricket-Assassin Wasp (Hymenoptera: Rhopalosomatidae)

Presenter: Bailey Krebs
Faculty Advisor: Jeremy Blaschke

Rhopalosomatidae are unusual wasps whose larvae parasitize crickets. The family is composed of 4 genera: Olixon, Liosphex, Paniscomima, and Rhopalosoma. Currently, the systematics of this family are based on morphology alone without the use of molecular data. To investigate intergeneric relationships within Rhopalosomatidae, DNA from 30 specimens representing the worldwide diversity of rhopalosomatids was extracted, amplified, and sequenced. Added to this data matrix were 35 COI barcode sequences from the Barcode of Life Database. The resultant maximum likelihood phylogeny showed high statistical support throughout, providing molecular evidence that largely matches previous morphological evidence (e.g. Olixon sister to the other genera) with one exception: Paniscomima was found to be nested within Rhopalosoma, indicating a possible need to merge these two genera into a single monophyletic genus. Future studies including additional genetic evidence and taxa are required to resolve this question.

Investigating the Effects of Pseudomonas fluorescens on the Intestinal Barrier in Zebrafish

Presenter: Cooper Bullough
Faculty Advisor: Hannah Henson

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, causes chronic inflammation of the intestinal tract. Mechanisms behind the pathology of the disease are not fully understood, however, previous studies indicate that certain strains of bacteria residing in the intestines may contribute to the disease. Additionally, no cure exists and treatments simply manage symptoms. This research used zebrafish (Danio rerio), as a model organism to investigate the effects of the bacteria Pseudomonas fluorescens on the intestinal barrier. Zebrafish larvae were grown for five days and then exposed to P. fluorescens for two days. The fish were then treated with a small fluorescent tracer, and were imaged using fluorescence microscopy to determine if the intestinal barrier had been disrupted by the bacteria. Complete results remain to be determined, but our preliminary data suggests that fish treated with bacteria show more fluorescence outside of the intestinal tract than control fish.

Creating DNA Constructs to Make a Transgenic Zebrafish Line

Presenter: Jordan Crawford
Faculty Advisor: Hannah Henson

Clusterin is a glycoprotein that is used in many physiological pathways and is thought to contribute to many different diseases including cancers and Alzheimer's disease. Specifically, it is involved in cell death pathways and oxidative stress. Because of Clusterin's involvement in Alzheimer's disease, a better understanding of its role in this pathology is needed. This project used the zebrafish (Danio rerio) as a model system to determine Clusterin's role in physiological and disease pathways. Using the Tol2 transposon system, DNA constructs containing the clusterin promoter and the green fluorescent protein (GFP) were generated. These constructs will allow GFP to act as a fluorescent marker for clusterin expression. Injecting these constructs into zebrafish embryos will allow the fluorescence from the GFP to be tracked throughout zebrafish development, thereby allowing clusterin expression to be traced as well. The results of this experiment are pending.

Effects of Vitamin D on Deiodinase 3 Levels in Adult Zebrafish (Danio rerio)

Presenter: Cordelia Moss Hoverson
Faculty Advisor: Faith Zamamiri-Davis

This experiment examined a potential link between vitamin D and type III iodothyronine deiodinase 3 (DIO3). Elevated DIO3 can be indicative of thyroid disease, as DIO3 catalyzes conversion of active thyroid prohormones and hormones into inactive forms. Zebrafish (Danio rerio) are a good model for thyroid disease studies due to similar gene patterning for the thyroid between zebrafish and mammals. Zebrafish were divided into control and experimental groups, and the experimental group was exposed to exogenous vitamin D at 5 μM for 1 week. RNA was extracted, and quantitative reverse transcription PCR (RT-qPCR) was used to measure DIO3 expression. Initial analysis of data shows no significant difference in DIO3 expression in treated zebrafish when normalized to 2 reference genes. Limitations of this study, including small sample size and unstable reference genes, suggest a need for further research using adult zebrafish to explore the relationship between vitamin D and DIO3.

Meleagris gallopava Assessment at the Milan Army Ammunition Plant

Presenter: Samuel Ross
Faculty Advisor: Andy Madison

Wild turkeys (Meleagris gallopava) are an abundant game species throughout Tennessee. To prevent over-harvesting, population assessments are needed to determine the status of individual populations. This project provides an assessment of the wild turkey population on the Milan Army Ammunition Plant. Beginning in late winter/early spring of 2020 we collected turkey population data from all areas of the MAAP. Randomly selected road strip-transects were conducted throughout the study area beginning in the afternoon hours of late March/early April. Transitioning to late spring, gobbler point-counts were conducted beginning 30 minutes prior to sunrise, lasting for three hours. Statistical analyses of these data are pending, but we expect to observe more wild turkeys than we hear. Upon reviewing results, we will have an updated, concise account of the wild turkey population on the MAAP, thus allowing us to manage the study area appropriately for future wild turkey populations.

Chemical Factors Promoting Pseudomonas fluorescence Pf0-1 Biofilm Formation

Student Scholarship: Kristen Holley
Faculty Advisor: Esther Choi

Biofilms consists of bacteria that use a self-produced extracellular polymeric substance (EPS) to adhere to a surface and communicate with each other. EPS provides extremely efficient protection against toxins and phagocytes, permitting bacteria in biofilms to survive antimicrobial treatments and host cell immune responses. Pseudomonas aeruginosa biofilms are known to cause nosocomial infections and increase mortality rates of cystic fibrosis patients. This experiment investigated chemical factors promoting biofilm formation by using Pseudomonas fluorescence Pf0-1. Pf0-1 displayed increased biofilm levels in minimal media supplemented with glucose versus complex media, but this effect was negated with additions of amino acids and zinc chloride. Taken together, it is concluded that Pf0-1 differentially regulates its amino acid metabolism pathways from P. aeruginosa, but they have similar metal ion uptake. Therefore, zinc could be a preventative agent for P. fluorescence biofilm accumulation in food industries.

Constructing a Transgenic Line of Zebrafish that Express GFP when Clusterin is Expressed

Student Scholarship: Olivia Coffman Cooper
Faculty Advisor: Hannah Henson

Clusterin is an important glycoprotein linked with many diseases and chronic injuries. Further research on clusterinmay reveal pathways to diagnose or cure diseases like Alzheimer’s or Parkinson’s. Zebrafish (Danio rerio) are an excellent model organism and their translucent nature allow the green fluorescent protein (GFP) to be visualized inside the zebrafish during development. This experiment attempted to create a transgenic line of zebrafish that express GFP when clusterin is expressed. Tracking clusterinusing GFP as a marker for its expression would provide a better understanding of clusterin’s normal function and its role in disease, especially in neurogenic diseases. A Tol2kit was used to create the DNA constructs for this transgenic zebrafish line. A polymerase chain reaction (PCR) was used to amplify zebrafish clusterin promoter, which was then ligated into the Tol2 DNA construct. Results are pending on this method’s success and creation of the transgenic zebrafish line.

The Effect of a Ketogenic Diet on an Induced Mouse Model of Plaque Psoriasis

Student Scholarship: Parker Lewis
Faculty Advisor: William Thierfelder

Psoriasis is an inflammatory condition which presents with dry, itchy plaques across the patient’s epithelia. In this experiment, an induced mouse model of psoriasis using topical Imiquimod cream was employed. After 1 week of treatment resulting in plaque formation, mice were given a ketogenic diet for 1 additional week. To determine the level of systemic inflammation, spleen cells were isolated from the mice. The erythrocytes were lysed, followed by incubation of the lymphocytes with anti-CD3. The RNA was then isolated from the lymphocytes, followed by DNase treatment of the nucleic acid to destroy residual DNA. To quantify systemic inflammation, RT-PCR was carried out to measure expression of the pro- and anti-inflammatory cytokines IL-23, IL-17, TGF-β, and IL-10. These data implied that the ketogenic diet increased systemic inflammation through an increase in transcription of IL-23 and IL-17, along with a decrease in transcription of TGF-β and IL-10.

Inhibition of Biofilm Formation of Staphylococcus epidermidis and Differential Gene Expression of THP-1 Macrophages

Student Scholarship: Emily Ebert
Faculty Advisor: Esther Choi

Staphylococcus epidermidis is a human skin commensal that limits the colonization of other pathogens. Adversely, they can form biofilms or aggregation of microorganisms to evade phagocytic cells and become resistant to many antibiotics. In order to combat nosocomial infection associated with S. epidermidis biofilm, this research aimed to determine factors contributing to biofilm formation of S. epidermidis and investigate how the biofilm forming bacteria changes the profiles of pro-inflammatory cytokines of macrophages. The results showed that the biofilm formation was inhibited in the presence of Pseudomonas quorum sensing molecules. It is currently under investigation how differently THP-1 macrophage-like cells react against bacteria when challenged with biofilm forming verse biofilm negative mutant S. epidermidis. Taken together, the results suggest biofilm formation and immune evasion of S. epidermidis can be prevented with small inhibitory molecules that interfere inter-species bacterial communication.

Innate Color Preference in the Blue Bottle Fly

Student Scholarship: Alexis Enriquez-Jones
Faculty Advisor: Jeremy Blaschke

Blue bottle flies (Calliphora vomitoria) are well-known for their contribution to forensic science and as household pests. These flies use both olfactory and visual cues to locate mates, food, and oviposition sites. In this experiment, the innate color preference and associative learning ability of blue bottle flies was tested using a sucrose/yeast solution as bait and yellow, black, white, or blue colored discs as landing platforms. The results indicate that when looking for food, blue bottle flies are strongly attracted to blue and white and almost completely ignore black. Regardless of food/color association, the innate attraction of the flies to the blue and white discs overshadowed any possible associative learning. Their preference for white and their apparent inability to learn may indicate that, in contrast to honeybees, associative learning may not provide a selective advantage for these short-lived insects.

Mechanical Antibiotic Effect of Blue Dasher Dragonfly (Pachydiplax longipennis) Wings on Fluorescently Transformed E. Coli

Student Scholarship: Claire Hamblen
Faculty Advisor: Jeremy Blaschke

Bacterial antibiotic resistance is a growing concern in the healthcare community, resulting in increased research into the development of novel chemical and mechanical bactericides. This research examined the mechanical antibiotic effects of blue dasher dragonfly (Pachydiplax longipennis) wings on fluorescently transformed E. coli. Wings were cut into 6 mm circles and placed in nutrient broth with transformed bacteria in a 96-well plate. Growth of bacteria was monitored for 24 hours using a fluorescence microplate reader. Dragonfly wings as a substrate for bacterial growth were compared to wells containing either no substrate, glue only, polypropylene plastic, or contact lenses. Wings inhibited bacteria growth compared to wells with no substrate, glue only, and polypropylene plastic, but there was not a significant difference between the bacterial growth in wells containing dragonfly wings versus contacts. More trials are needed before blue dasher dragonfly wings can serve as a model for mechanical bactericidal surfaces.

Investigating Anuran Species Composition in Madison County

Student Scholarship: Madison Morris
Faculty Advisor: James Kerfoot

Nearly 60% of amphibian species worldwide are either threatened with extinction or species data is lacking. Eighteen anuran species are reported in Madison County, Tennessee, however, studies on the link between microhabitats and anuran community structure are lacking. The objective of this study was to investigate anuran populations in Madison County and to determine how environmental factors play a role in structuring their communities. To determine species presence/absence at four sites in Madison County, manual calling surveys were conducted during peak calling times from May 2019-March 2020. Calls were recorded for all but one reported species, the pickerel frog (Lithobates palustris). Preliminary results of a principle components analysis indicate two sites have wider species breadth, whereas the other two have a smaller species composition. This variation in species composition may be related to slow moving water and this microhabitat characteristic may confer success of all life history stages for species.

Evaluation of Growth Regulator Concentrations Necessary for Monkey Puzzle Tree Regeneration by Plant Tissue Culture

Student Scholarship: Jordan Meadors
Faculty Advisor: Mark Bolyard

The purpose of this study was to use plant tissue culture to induce callus of Araucaria araucana leaves and observe possible shooting after auxin removal from regeneration media. Concentrations of phytohormones 6-Benzyladenine (BA) and 1-Naphthaleneacetic acid (NAA) in the media were 0.1 mM and 1 mM, respectively, and induced callus production. In the second part of this project, NAA was eliminated from 6 groups at different time periods. All leaves were originally plated on basal media and then regeneration media containing both NAA and BA. A new group was transferred to BA-only media every 2 weeks until the end of the study. Calluses were evaluated according to color and size. Results showed that removal of auxin did not effectively allow for shoot morphogenesis or further callus growth.

The Efficacy of Santalum album as a Natural Preservative in Cosmetic and Dermatological Applications

Student Scholarship: Steven A. Hutcherson
Faculty Advisor: Esther Choi

Atopic dermatitis, a growing condition in both medical and veterinary fields, is often caused by opportunistic pathogens from the Staphylococcus genus of bacteria. Due to consumer demand, product ingredients are rapidly shifting from synthetic chemicals to natural sources. This study aimed to determine the efficacy of Sandalwood Essential Oil (SAO) as an antibacterial preservative against Staphylococcus epidermidis 1457, one of the normal skin microbiotas. SAO showed strong growth inhibition at low concentrations (<0.2% by volume). The interactions between SAO and human cells such as a non-cancerous epithelial cell line (A-431) and a cancerous breast cell line (MCF-7) are under investigation. The interesting results of these studies indicate that SAO can be an effective natural product not only as a preservative, but as a medicinal component in treating more serious skin-related illnesses outside of the cosmetic profession.

Survey of Mammal Biodiversity in Urbanized Areas in West Tennessee

Student Scholarship: Elizabeth Gibson
Faculty Advisor: Andy Madison

Urbanization is globally increasing at a rapid rate. Habitat fragmentation, vehicle and animal collisions, and habitat destruction are all leading to a decline in mammalian populations. Examining the biodiversity in mammals in West Tennessee will provide important data about how mammal populations differ across varying levels of urbanization. This study was conducted January-May 2019. Two cameras were placed at nine study sites: three were in downtown Jackson, three were on the edge of Jackson, and three were in rural areas of Jackson, Tennessee. Species and numbers were recorded. Results are pending.

Scent Preference in Bobcats (Lynx rufus)

Student Scholarship: Sophie Ledes
Faculty Advisor: Andy Madison

Surveying animal populations in their natural habitats is one of the most important first steps in wildlife conservation and management. Estimating numbers of cryptic animals, such as the bobcat (Lynx rufus) can be difficult. The objective of this study was to test different baits for assessing bobcat numbers. Twenty trail cameras were distributed at the Milan Army Ammunition Plant near Milan, Tennessee, five cameras for each of the four treatments: wet cat food, dry cat food, sardines, and the control. New treatments were put out every week for five weeks and recorded the species that visited. Wet and dry cat foods attract the most bobcats.

Effect of Cosmetic Preservatives on the Growth, Biofilm Formation, and Gene Expression of Staphylococcus epidermidis 1457

Presenter: Audrie Campbell
Faculty Advisor: Esther Choi

There are many beneficial microorganisms in our skin; however, our skin is frequently subjected to treatments and stresses that change the type and prevalence of skin microbiota. Growing concerns of synthetic preservatives in cosmetics have shifted our attention to natural preservatives, like seed oils and plant extracts. To provide a guideline for using natural preservatives in skin products, we aimed to compare effects of synthetic versus natural cosmetic preservatives on a skin bacteria, Staphyloccocus epidermidis 1457. The bacteria was treated with propylene glycol, propyl paraben, and tea tree oil and the effect was determined on its bacterial growth, biofilm formation, and gene expression. A-431, a skin epithelial cell line, was also treated with these compounds to detect changes in proteins and cytokines involved in skin innate immunity. Our results can provide important guidelines in choosing effective cosmetic preservatives with safety while minimizing unwanted changes in skin microbiota.

Examining the Antibacterial Surface Properties of Blue Dasher (Pachydiplax Longipennis) Dragonfly Wings

Presenter: Taylor Lewelling
Faculty Advisor: Jeremy Blaschke

The remarkable hydrophobic properties of dragonfly wings may also function as natural antibacterial surfaces. Here, we investigate the ability of Blue Dasher wings (Pachydiplax longipennis) to inhibit the growth of the Gram negative bacteria Escherichia coli. Bacteria were genetically transformed to express Green Fluorescent Protein and a microplate reader was used to measure the increase of fluorescence over time as the bacteria colony grew. Dragonfly wings were cut and placed in individual wells of a 96-well plate and covered with nutrient broth. Double-sided sticky tape and polypropylene plastic were used as control surfaces. When compared to controls, dragonfly wings exhibited significantly less E. coli growth (p<0.05 ) as evidenced by lower expressed fluorescence over time. These results indicate that dragonfly wings may naturally inhibit the formation of biofilms by reducing the growth of Gram negative bacteria. Mimicking the physical structure of these wings could lead to the production of novel synthetic surfaces with antimicrobial properties.

Determining the Antibiotic Properties of Artemisia abrotanum Essential Oil using Spectrum Analysis and a Disk Diffusion Protocol

Presenter: Christopher Roberts
Faculty Advisor: Mark Bolyard

The southern wormwood (Artemisia abrotanum) is a fragrant, perennial herb native to Asia and the Mediterranean; this species has been well-regarded by southern Europeans for its sharp citrus scent and medicinal potential, both of which are due to volatile oils found within the plant, such as borneol, camphor, and 1,8-cineole. The objective of this research is to attempt extraction of essential oils from wild-type A. abrotanum and from A. abrotanum clones grown in tissue culture; the compounds in the oils are to be examined and compared using gas chromatography/mass spectrometry (GC/MS), and their antibiotic properties assessed using a disk diffusion protocol. Results of GC/MS and disk diffusion are yet to be determined.

Investigating the Effects of Pseudomonas Fluorescens on Intestinal Barrier Formation using Fluorescent Tracers

Presenter: Ashley Scott
Faculty Advisor: Hannah Henson

Crohn's disease is an incurable intestinal disease caused by a disruption of the intestinal barrier. In this research, zebrafish (Danio rerio) was used as the model organism to investigate intestinal barrier integrity after the ingestion of the bacteria Pseudomonas fluorescens. The prediction was that the intestinal barrier will be compromised after ingestion of the bacteria. A 3-K Dalton Rhodamine dextran was used to trace the effects of the bacteria on the intestinal barrier epithelial cells. Tracer leakage would indicate that tight junctions in the intestinal barrier epithelial cells have been compromised. After measuring fluorescence intensity to detect tracer leakage outside of the intestine, no significant difference was found between those not treated with bacteria and the fish treated with P. fluorescens. Future studies will show if this is due to the size of the tracer or the amount of time the fish were exposed to the bacteria.

The First Recorded Host for the Assassin Fly (Xanthomelanodes atripennis) and a Phylogenetic Analysis of the Genus

Presenter: Edward Sickle
Faculty Advisor: Jeremy Blaschke

Xanthomelanodes are rarely encountered endoparasitoid flies that attack assassin bugs. Only 4 species are currently recorded in the US, and only 2 of these have known hosts. Here, we report the sundew assassin bug (Zelus luridus) as the first documented host of Xanthomelanodes atripennis and include the first molecular phylogeny of the genus as a whole. Forty-five specimens representing all 4 US species and encompassing diverse geographic populations were included. Available barcodes (COI) were downloaded from the Barcode of Life Database and added to our newly generated sequences of COI. These sequences were concatenated with those from the nuclear coding gene MCS to reconstruct the final phylogeny. In agreement with morphological hypotheses, the molecular relationships between species were reconstructed as X. atripennis + (X. flavipes + (X. arcuatus + X. californicus)). An analysis of host-parasite evolution did not reveal any evidence of co-evolution with their assassin bug hosts.

In Vitro Culture of Araucaria Araucana

Presenter: Joshua Moody
Faculty Advisor: Mark Bolyard

The goal of this project is to induce callus formation for Araucaria araucana and potentially observe shoot regeneration. The 3 main phytohormones that were used in this project were naphthaleneacetic acid (NAA), thidiazuron (TDZ), and 2,4-dichlorophenoxyacetic acid (2,4-D). For the first experiment, media was produced that contained NAA and TDZ in every combination of 0.1 μM, 1.0 μM, and 10.0 μM. In the second experiment, regeneration media was produced that contained 2,4-D and TDZ in every possible combination of 0.1 μM, 1.0 μM, and 10.0 μM. Results indicate successful callus production with both types of regeneration media. Calluses were ranked from 5 (good quality) to 0 (poor quality) for color and size. This project determined there was no significant difference between using NAA and 2,4-D as auxins in regeneration media for Araucaria araucana.

A Study on the Application of Pigments on Tissues Throughout the Plastination Process

Presenter: Rachel Lewis
Faculty Advisor: James Huggins

Plastination, a process of tissue preservation developed by Dr. Von Hagens in 1978, has become a valuable tool to the anatomy and physiology field for teaching and learning. Plastinated tissues have gained popularity due to the retained structural integrity and the ease in handling and storing of the plastinates when compared to other methods of preservation. However, specimens often lose natural coloration throughout the sample. To test restoration of this natural coloration, 3 pigments were added at 4 separate points during the 5-point plastination process to squirrel leg muscle samples. Each of the 12 experimental groups were compared back to fresh squirrel leg muscle samples to determine whether any of the dyes combined with the addition points can restore natural coloration without disrupting the plastination process in any way. This research concluded with promising results for the future of plastination coloration at Union University.

The Effect of Phosphate and other Agricultural Influences on Invertebrate Communities in Riparian Zones Adjacent to Agricultural Lands in Tennessee

Presenter: Kristin Smith
Faculty Advisor: J.R. Kerfoot

Riparian zones are important ecotones that act as natural corridors, chemical buffers, and habitat for wildlife. Riparian zones occur adjacent to agricultural lands, making them susceptible to run off from agricultural fertilizers. Potentially, riparian zones may buffer those pollutants from entering the waterway. Aquatic macroinvertebrate community composition is known to be impacted by pollution and the goal of this study was to investigate correlations between macroinvertebrate communities and physicochemical properties and riparian zone size. It was hypothesized that chemical pollution correlates with a decrease in macroinvertebrate diversity. Samples and measurements were taken at three riparian zone sites throughout Madison County in Jackson, TN. Samples were collected at 3 sites in winter 2017 and in summer 2018. Preliminary results of a Principal Components Analysis indicate significant separation of sites based on physiochemical parameters and estimates of macroinvertebrate communities are different between sites, suggesting, initially, a correlation between the two variables.

The Effect of Soil Moisture Content on Bottomland Oak Growth

Presenter: Josh Bowden
Faculty Advisor: Andy Madison

Wetland habitats are important ecosystems, providing numerous ecosystem services and, when managed appropriately, valuable timber. This research investigates overall growth and survivorship as well as growth rates of 8 oak species planted in a bottomland wetland of West TN at the UT AgResearch Center in Jackson to determine which species would grow best in this environment. Specifically, it compares growth rates over the last 7 years to initial growth rates measured for the first 5 years. The survival and diameter at breast height (DBH) of 136 trees for each species was recorded from February to early March 2018 and compared to data collected from the same trees in 2010. Nuttall oak had the largest mean DBH (13.7cm) while Shumard oak had the lowest (7.88cm). Swamp White Oak has the highest survival percentage (99%) while Shumard oak had the lowest (55%).

Analysis Investigating the Effects of Staphylococcus epidermidis on Mmp-14 Expression in Zebrafish Retinas

Presenter: Matt Tucker
Faculty Advisor: Hannah Henson

Staphylococcus epidermidis represents a common source of bacterial infections on indwelling medical devices including those used for retinal procedures. These infections can cause an inflammatory response resulting in the activation of matrix metalloproteinases (MMPs), which degrade the extracellular matrix around the cell. For this study, I examined this relationship between S. epidermidis infections and MMP activation using zebrafish (Danio rerio). Interestingly, zebrafish retinas possess a robust regenerative ability in response to damage or inflammation. Specifically, I examined changes in MMP-14 expression which is localized to the zebrafish retina. After injecting the bacteria into the zebrafish eye, I collected the zebrafish and used quantitative PCR to look for changes in MMP-14 expression.

Life History Notes on the Rare Ectoparasitoid Wasp Rhopalosoma nearticum

Presenter: Sarah Lounsbury
Faculty Advisor: Jeremy Blaschke

Rhopalosoma nearticum is an uncommonly collected ectoparasitoid wasp whose known hosts include the restless bush cricket (Hapithus agitator) and loud-singing bush crickets (Orocharis saltator). Little is known regarding the biology and life-cycle of this wasp. We investigated the presence of R. nearticum at 2 study sites (Shenandoah National Park, VA and Cypress Grove Nature Park, TN) from April-October 2018, and attempted to rear larvae to adulthood. The larva and their crickets hosts were kept in a controlled laboratory environment for observation, pupation, and eventual emergence of adults. Anaxipha exigua represents a new host species for R. nearticum. Two cocoons and one pre-pupa were observed and described to provide more information on this cryptic species. The species was previously not known to utilize a pre-pupa stage, and the two cocoons are predicted to emerge in March 2019.

Measuring Glucose Uptake of Zebrafish

Presenter: Gray Hamilton
Faculty Advisor: Hannah Henson

Premature infants are commonly diagnosed with hyperglycemia due to insulin resistance or glucose infusions. However, long-term effects of hyperglycemia during early development, specifically on the brain, are largely unknown. One structure that may be affected by hyperglycemia is the choroid plexus. The choroid plexus transfers blood into the cerebrospinal fluid through the blood-cerebrospinal fluid barrier. We examined whether hyperglycemia affects the development of this barrier using zebrafish (Danio rerio). Zebrafish are transparent during early development, making visualization of internal structures easy. The purpose of this project is to develop methods to induce hyperglycemia in zebrafish and to measure their glucose levels. Multiple trials were performed using different concentrations of glucose to determine what concentrations zebrafish can tolerate. One trial used dexamethasone treatments, a steroid known to promote elevated glucose levels. However, results indicated dexamethasone treatments had an adverse effect on the concentration of glucose present in zebrafish embryos.

Feeding Behavior of Lepomis macrochirusin Different Light Intensities and Turbidities of Water

Presenter: Brandon Johnson
Faculty Advisor: J.R. Kerfoot

Bluegill (Lepomis macrochirus) are found throughout North America in different light intensities and turbidity levels. Even though this species is found throughout many different light intensities and turbidities of water, it is unknown how these factors influence their feeding behaviors. The objective of this study was to be test light intensity and turbidity effects on feeding kinematics. This was done by filming a bluegill 3 times in different turbidities of water, using a slow-motion camera. First, starting with no turbidity, then adding soil to slowly make the water turbid.